<%BANNER%>

Inheritance of Morphological Characters of Pickerelweed (Pontederia cordata L.)


PAGE 1

INHERITANCE OF MORPHOLOGICAL CHARACTERS OF PICKERELWEED ( Pontederia cordata L.) By LYN ANNE GETTYS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

PAGE 2

Copyright 2005 by Lyn Anne Gettys

PAGE 3

iii ACKNOWLEDGMENTS I would like to thank Dr. David Wofford for his guidance, encouragement and support throughout my course of study. Dr. Wofford provided me with laboratory and greenhouse space, technical and financial support, copious amounts of coffee and valuable insight regarding how to survive life in academia. I would also like to thank Dr. David Sutton for his advice and support throughout my program. Dr. Sutton supplied plant material, computer resources, career advice, financial su pport and a long-coveted copy of Grays Manual of Botany Special thanks are in order for my advi sory committee. Dr. Paul Pfahler was an invaluable resource and provided me with la boratory equipment and supplies, technical advice and more lunches at th e Swamp than I can count. Dr Michael Kane contributed samples of his extensive collection of divers e genotypes of pickerelweed to my program. Dr. Paul Lyrene generously allowed me to take up residence in his greenhouse when my plants threatened to overt ake all of Gainesville. My program could not have been a success without the help of Dr. Van Waddill, who provided significant financia l support to my project. I ap preciate the generosity of Dr. Kim Moore and Dr. Tim Broschat, who a llowed me to use thei r large screenhouses during my tenure at the Fort Lauderdale Resear ch and Education Center. Thanks also go to my friends and advocates at the FLREC: Nancy Gaynor, Joanne Korvick, Luci Fisher and Susan Thor for technical and greenhous e assistance; Bill and Sarah Kern for identifying greenhouse critters; Bill Latham for providing me with access to the

PAGE 4

iv chemistry lab; and Bridge Desoran and his cr ew for building anything I asked them to in a completely unreasonable timeframe. I exte nd my sincere appreciation to Mr. Eric Ostmark and Mr. Doug Manning for providing technical support to my program in Gainesville. Thanks are also in order to Dr Eastonce Gwata and Ms. Gabriela Luciani for helping out when I was in a bind and to the faculty and graduate students of the Agronomy Department for providing mora l support throughout my tenure at the University of Florida. I truly appreciate the assistance provided by Ki m, Paula, Sandy and Nancy (in Gainesville) and Cherie, Sarah, Ve ronica and Sue (in Fort Lauderdale), who helped navigate the labyri nth of paperwork to ensure that I got paid on time. I am eternally grateful to my family for their unflagging support of this and all of my endeavors. My parents, Mykel and Jody have been and conti nue to be my most enthusiastic cheerleaders; w ithout their support I would proba bly be running a print shop somewhere. Thanks also go to Mr. Paul J. Best II, who provided unwavering friendship, moral support and a nice swampy home for my culled plants and to Dr. Ed Duke for being a fantastic mentor, advi sor, inspiration and friend. Finally, I would like to thank the Univers ity of Florida Alumni Association for providing me with a 4-year Outstanding Alumni Fellowship and the Crop Science Society of America for awarding me a Gerald O. Mott Scholarship.

PAGE 5

v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES...........................................................................................................viii LIST OF FIGURES.........................................................................................................xiii ABSTRACT.....................................................................................................................xvi CHAPTER 1 INTRODUCTION AND OVERVIEW........................................................................1 2 A REVIEW OF THE LITERATURE..........................................................................3 Economic Importance...................................................................................................3 Classification, Origin and Distribution.........................................................................4 Morphology..................................................................................................................6 Culture........................................................................................................................ ..8 Propagation and Dormancy..........................................................................................8 General Heteromorphi c Incompatibility.....................................................................12 General Tristyly..........................................................................................................12 Morph Inheritance in Tristylous Species....................................................................14 Population Structure of Tristylous Species.................................................................15 Breakdown of Tristyly................................................................................................15 Cryptic Self-incompatibility.......................................................................................17 Prevalence of Tristyly in Sp ecies of the Pontederiaceae............................................19 Self-incompatibility in Speci es of the Pontederiaceae...............................................20 Morph Inheritance in Species of the Pontederiaceae..................................................20 Morph Inheritance in Pickerelweed............................................................................21 Pollen Diameter Trimorphism a nd Production in Pickerelweed................................21 Floral Structure and Reproductive Or gan Arrangement in Pickerelweed..................22 Pollen Physiology and Male Fitness in Pickerelweed................................................25 Self, Intramorph and Intermorph Co mpatibility in Pickerelweed..............................26 Pollen Growth in vivo.................................................................................................27 Population Structure of Pickerelweed........................................................................28 Impact of Pollinator Behavior....................................................................................28 Stigmatic Pollen Loads in Pickerelweed....................................................................31 Greenhouse Production vs. Natural P opulations of Pickerelweed.............................32

PAGE 6

vi 3 POLLEN GRAIN DIAMETER, IN VITRO POLLEN GERMINATION AND REGRESSION BETWEEN GRAIN DIAMETER AND IN VITRO GERMINATION........................................................................................................42 Introduction.................................................................................................................42 Materials and Methods...............................................................................................46 Results and Discussion...............................................................................................50 Conclusions.................................................................................................................52 4 DEVELOPMENT OF NOVEL POLLINATION TECHNIQUES TO REDUCE SELF-INCOMPATIBILITY RESULTING FROM HERKOGAMY........................59 Introduction.................................................................................................................59 Materials and Methods...............................................................................................62 Results and Discussion...............................................................................................66 Conclusions.................................................................................................................69 5 OPTIMUM SEED STORAGE AN D GERMINATION CONDITIONS...................79 Introduction.................................................................................................................79 Materials and Methods...............................................................................................81 Results and Discussion...............................................................................................85 Conclusions.................................................................................................................90 6 INHERITANCE AND GENETIC CONTROL OF ALBINISM.............................104 Introduction...............................................................................................................104 Materials and Methods.............................................................................................105 Results and Discussion.............................................................................................105 Conclusions...............................................................................................................115 7 INHERITANCE AND GENETIC C ONTROL OF FLOWER COLOR..................143 Introduction...............................................................................................................143 Materials and Methods.............................................................................................144 Results and Discussion.............................................................................................145 Conclusions...............................................................................................................150 8 INHERITANCE AND GENETIC C ONTROL OF FLORAL MORPH..................162 Introduction...............................................................................................................162 Materials and Methods.............................................................................................164 Results and Discussion.............................................................................................164 Conclusions...............................................................................................................172

PAGE 7

vii 9 LINKAGE RELATIONSHIP BETWEEN THE LOCI CONTROLLING FLOWER COLOR AND FLORAL MORPH..........................................................185 Introduction...............................................................................................................185 Materials and Methods.............................................................................................186 Results and Discussion.............................................................................................187 Conclusions...............................................................................................................222 10 INHERITANCE AND GE NETIC CONTROL OF SCAPE PUBESCENCE..........244 Introduction...............................................................................................................244 Materials and Methods.............................................................................................244 Results and Discussion.............................................................................................248 Conclusions...............................................................................................................252 11 INHERITANCE AND GENETIC C ONTROL OF A SECOND LOCUS INFLUENCING FLOWER COLOR.......................................................................260 Introduction...............................................................................................................260 Materials and Methods.............................................................................................260 Results and Discussion.............................................................................................262 Conclusions...............................................................................................................266 12 SUMMARY AND CONCLUSIONS.......................................................................271 APPENDIX A POPULATION DEVELOPMENT FO R INHERITANCE STUDIES.....................276 Introduction...............................................................................................................276 Materials and Methods.............................................................................................277 Descriptions of the Families.....................................................................................280 Results and Discussion.............................................................................................281 B OBSERVATIONS....................................................................................................295 Introduction...............................................................................................................295 Plant Care and Maintenance in the Greenhouse.......................................................295 Greenhouse Pests (a.k.a. Ever ybody Loves Pickerelweed)..................................296 Response to Colchicine.............................................................................................297 Variegation...............................................................................................................298 Leaf Shape................................................................................................................298 Provenance and Dormancy.......................................................................................299 LITERATURE CITED....................................................................................................311 BIOGRAPHICAL SKETCH...........................................................................................320

PAGE 8

viii LIST OF TABLES Table page 3.1. Analysis of variance of pollen grain di ameter in microns of s-pollen, m-pollen and l-pollen of pickerelweed....................................................................................54 3.2. Analysis of variance of pollen tube length in microns produced in vitro by s-pollen, m-pollen and l-pollen of pickerelweed......................................................55 4.1. Seed set after self -pollination of L-morph plants of pickerelweed subjected to control and stylar surger y pollination treatments.....................................................72 4.2. Analysis of variance of arcsine transforme d percent seed set in control and stylar surgery pollinations of L-morph plants of pickerelweed.........................................73 4.3. Seed set after self -pollination of S-morph plants of pickerelweed subjected to control and corolla remova l pollination treatments..................................................74 4.4. Analysis of variance of arcsine transf ormed percent seed set in control and corolla removal pollinations of Smorph plants of pickerelweed............................76 5.1. Analysis of variance of ar csine-transformed data for germination of fresh fruits and seeds of pickerelweed........................................................................................92 5.2. Analysis of variance of ar csine-transformed data for germination of fruits and seeds of pickerelweed stored for 3 mo.....................................................................93 5.3. Analysis of variance of ar csine-transformed data for germination of fruits and seeds of pickerelweed stored for 6 mo.....................................................................95 5.4. Analysis of variance of arcsine-transf ormed data for germination of seeds of pickerelweed germinated under water......................................................................97 6.1. Number of green and albino seedlings from F1 and S1 families of pickerelweed..117 6.2. Goodness-of-fit tests for F1 and S1 families of pickerelweed segregating for albinism..................................................................................................................118 6.3. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism and derived from the initial cros s/reciprocal set WMBL (genotypes P1P2AaBb x P1P2AaBB )..............................................................................................................119

PAGE 9

ix 6.4. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism and derived from the initial cros s/reciprocal set WSBL (genotypes P1P2aaBb x P1P2AaBB )..............................................................................................................121 6.5. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism and derived from the initial cros s/reciprocal set WSBM (genotypes P1P2aaBb x P1P1aaBB or P1P1AAbb or P1P1AABB )..................................................................124 6.6. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism and derived from the initial cros s/reciprocal set WMBS (genotypes P1P2AaBb x P1P1aaBB or P1P1AAbb or P1P1AABB )..................................................................126 6.7. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism and derived from the initial cros s/reciprocal set BSBL (genotypes P1P1aaBB or P1P1AAbb or P1P1AABB x P1P2AaBB )...................................................................128 6.8. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism and derived from the initial cros s/reciprocal set BMBL (genotypes P1P1aaBB or P1P1AAbb or P1P1AABB x P1P2AaBB )...................................................................129 7.1. Number of blue-flowered and white-flowered F1 and S1 progeny of pickerelweed...........................................................................................................152 7.2. Goodness-of-fit tests for F1 and S1 families of pickerelweed segregating for blue and white flower color............................................................................................153 7.3. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived from the initial cross/reciprocal set WMBL (genotypes ww x Ww ).............................................................................................154 7.4. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived from the initial cross/reciprocal set WSBL (genotypes ww x Ww ).............................................................................................155 7.5. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived from the initial cross/reciprocal set WSBM (genotypes ww x Ww ).............................................................................................156 7.6. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived from the initial cross/reciprocal set WMBS (genotypes ww x WW )............................................................................................157 7.7. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived from the initial cross/reciprocal set BSBM (genotypes WW x Ww )...........................................................................................158

PAGE 10

x 7.8. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived from the initial cross/reciprocal set BSBL (genotypes WW x Ww )...........................................................................................159 7.9. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived from the initial cross/reciprocal set BMBL (genotypes Ww x Ww )............................................................................................160 8.1. Number of S-morph, M-mor ph and L-morph progeny in F1 and S1 families of pickerelweed...........................................................................................................175 8.2. Goodness-of-fit tests for F1 and S1 families of pickerelweed segregating for short, mid and long floral morphs..........................................................................176 8.3. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set WMBL (genotypes ssMm x ssmm )...................................................................................................................177 8.4. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set WSBL (genotypes Ssmm x ssmm )...................................................................................................................178 8.5. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set WSBM (genotypes Ssmm x ssMm )..................................................................................................................179 8.6. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set WMBS (genotypes ssMm x SsMM ).................................................................................................................180 8.7. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set BSBM (genotypes SsMM x ssMm )..................................................................................................................181 8.8. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set BSBL (genotypes SsMM x ssmm )...................................................................................................................182 8.9. Goodness-of-fit tests for F2 populations of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set BMBL (genotypes ssMm x ssmm )...................................................................................................................183 9.1. Segregation of progeny and goodness-of-fit tests for F1 families of pickerelweed segregating for flower color and floral morph.......................................................224 9.2. Goodness-of-fit test for the F1 family WSBM segregating for flower color and floral morph............................................................................................................225

PAGE 11

xi 9.3. Goodness-of-fit test for the F1 family BMBL segregating for flower color and floral morph............................................................................................................226 9.4. Goodness-of-fit tests for the F1 families WSBM and BMBL segregating for flower color and floral morph................................................................................227 9.5. Summary of goodness-of-fit tests for gene tic distances ranging from 4.4 m.u. to 22 m.u. for the linked W and M loci in the F1 families WSBM and BMBL..........228 9.6. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family WMBL segregating for flower color and floral morph.......................................................229 9.7. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family WSBL segregating for flower color and floral morph.......................................................231 9.8. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family WSBM segregating for flower color and floral morph.......................................................232 9.9. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family WMBS segregating for flower color and floral morph.......................................................234 9.10. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family BSBM segregating for flower color and floral morph.......................................................237 9.11. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family BSBL segregating for flower color and floral morph.......................................................240 9.12. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family BMBL segregating for flower color and floral morph.......................................................242 10.1. Segregation and goodness-of-fit tests for F2 families of pickerelweed segregating for scape pubescence..........................................................................254 10.2. Segregation and goodness-of-fit tests for F2 families of pickerelweed segregating for scape pubescence..........................................................................255 10.3. Segregation and goodness-of-fit tests for F2 families of pickerelweed segregating for scape pubescence..........................................................................256 10.4. Segregation and goodness-of-fit tests for F2 families of pickerelweed segregating for scape pubescence..........................................................................257 11.1. Flower color of progeny in S1, F1 and F2 families of pickerelweed derived from the parents WL and WM........................................................................................268 11.2. Segregation for flower colo r and goodness-of-fit tests for F2 families of pickerelweed...........................................................................................................269

PAGE 12

xii A.1. F1 progeny of pickerelweed derived from the parents WS and WM, and S1 progeny derived from self-pollinations of the parental lines WS, WM, BS, BM and BL.............................................................................................................282 A.2. F1 progeny of pickerelweed derived from the parents WS and BM, and F2 progeny derived from self-pollination of F1 progeny........................................283 A.3. F1 progeny of pickerelweed derived from the parents WS and BL, and F2 progeny derived from self-pollination of F1 progeny........................................284 A.4. F1 progeny of pickerelweed derived from the parents WM and BS, and F2 progeny derived from self-pollination of F1 progeny........................................285 A.5. F1 progeny of pickerelweed derived from the parents WM and BL, and F2 progeny derived from self-pollination of F1 progeny........................................286 A.6. F1 progeny of pickerelweed derived from the parents BS and BM, and F2 progeny derived from self-pollination of F1 progeny........................................287 A.7. F1 progeny of pickerelweed derived from the parents BS and BL, and F2 progeny derived from self-pollination of F1 progeny........................................288 A.8. F1 progeny of pickerelweed derived from the parents BM and BL, and F2 progeny derived from self-pollination of F1 progeny........................................289

PAGE 13

xiii LIST OF FIGURES Figure page 2.1. Pre-packaged water garden kit with bare-root pickerelweed plants, damp sphagnum moss, soilless planting substrate, fertilizer tablets and planting basket..33 2.2. Pickerelweed growing along the margin of Roberts Pond in Bainbridge, New York..........................................................................................................................34 2.3. Inflorescence of white-flowered pickerelweed........................................................35 2.4. Yellow nectar guides (eyespots) on ba nner tepal of pickerelweed flower...........36 2.5. Fresh fruit, seed and dried fruit of pickerelweed.....................................................37 2.6. Flowers of Singapore Pink pickerelweed..............................................................38 2.7. L-morph flowers of pickerelweed............................................................................39 2.8. M-morph flowers of pickerelweed...........................................................................40 2.9. S-morph flowers of pickerelweed............................................................................41 3.1. Grain diameter in microns of s-polle n, m-pollen and l-pollen of pickerelweed......56 3.2. Mean length of pollen tubes in micr ons produced in vitro by s-pollen, m-pollen and l-pollen of pickerelweed 30, 60, 120 and 240 min after germination...............57 3.3. Regression between pollen grain diamet er and in vitro tube length 30, 60, 120 and 240 min after germination.................................................................................58 4.1. Stylar surgery of an L-mo rph flower of pickerelweed.............................................77 4.2. Pollination of an S-morph flower of pickerelweed after corolla removal................78 5.1. Fruits and seeds of pickerelweed germ inated under water in half-pint (250 mL) bottles.......................................................................................................................98 5.2. Fruits and seeds of pickerelweed germin ated on or 0.5 cm below the soil surface under mist irrigation.................................................................................................99 5.3. Percent germination of fresh fr uits and seeds of pickerelweed..............................100

PAGE 14

xiv 5.4. Percent germination of fruits and seed s of pickerelweed stored for 3 months......101 5.5. Percent germination of fruits and seed s of pickerelweed stored for 6 months......102 5.6. Percent germination of seeds of pi ckerelweed germinated under water................103 6.1. Albino seedling of pickerelweed............................................................................130 6.2. Punnett square of expected albinism genotypes of F1 populations of pickerelweed derived from the initial cross/reciprocal set WMWS (genotypes P1P2AaBb and P1P2aaBb ) and expected segregation of F2 progeny for albinism.131 6.3. Punnett square of expected albinism genotypes of F1 populations of pickerelweed derived from the initial cross/reciprocal set WMBL (genotypes P1P2AaBb and P1P2AaBB ) and expected segregation of F2 progeny for albinism.132 6.4. Punnett square of expected albinism genotypes of F1 populations of pickerelweed derived from the initial cr oss/reciprocal set WSBL (genotypes P1P2aaBb and P1P2AaBB ) and expected segregation of F2 progeny for albinism.133 6.5. Punnett square of expected albinism genotypes of F1 populations of pickerelweed derived from the initial cross/reciprocal set WSBM (genotypes P1P2aaBb and P1P1aaBB or P1P1AAbb or P1P1AABB or P1P1aabb ) and expected segregation of F2 progeny for albinism..................................................................134 6.6. Punnett square of expected albinism genotypes of F1 populations of pickerelweed derived from the initial cross/reciprocal set WMBS (genotypes P1P2AaBb and P1P1aaBB or P1P1AAbb or P1P1AABB or P1P1aabb ) and expected segregation of F2 progeny for albinism..................................................................135 6.7. Punnett square of expected albinism genotypes of F1 populations of pickerelweed derived from the initial cross/reciprocal set BSBM (genotypes P1P1aaBB or P1P1AAbb or P1P1AABB or P1P1aabb and P1P1aaBB or P1P1AAbb or P1P1AABB or P1P1aabb ) and expected segregation of F2 progeny for albinism136 6.8. Punnett square of expected albinism genotypes of F1 populations of pickerelweed derived from the initial cr oss/reciprocal set BSBL (genotypes P1P1aaBB or P1P1AAbb or P1P1AABB or P1P1aabb and P1P2AaBB ) and expected segregation of F2 progeny for albinism...................................................137 6.9. Punnett square of expected albinism genotypes of F1 populations of pickerelweed derived from the initial cross/reciprocal set BMBL (genotypes P1P1aaBB or P1P1AAbb or P1P1AABB or P1P1aabb and P1P2AaBB ) and expected segregation of F2 progeny for albinism...................................................138 6.10. Punnett square of expected albinism genotypes of S1 progeny of pickerelweed from the parent WS (genotype P1P2aaBb ).............................................................139

PAGE 15

xv 6.11. Punnett square of expected albinism genotypes of S1 progeny of pickerelweed from the parent WM (genotype P1P2AaBb )...........................................................140 6.12. Punnett square of expected albinism genotypes of S1 progeny of pickerelweed from the parent BS (genotype P1P1aaBB P1P1AAbb P1P1AABB or P1P1aabb )...141 6.13. Punnett square of expected albinism genotypes of S1 progeny of pickerelweed from the parent BM (genotype P1P1aaBB P1P1AAbb P1P1AABB or P1P1aabb )..142 7.1. Flowers of pickerelweed........................................................................................161 8.1. The three floral morphs of pickerelweed...............................................................184 10.1. Glabrous inflorescence scape of pickerelweed......................................................258 10.2. Pubescent inflorescence scape of pickerelweed.....................................................259 A.1. Fruits of pickerelweed............................................................................................290 A.2. Seeds of pickerelweed germinating in jars of water in the greenhouse.................291 A.3. Newly transplanted F1 seedling of pickerelw eed in 612 cell pack.........................292 A.4. F2 seedlings of pickerelweed in the greenhouse.....................................................293 A.5. F2 plants in the greenhouse.....................................................................................294 B.1. Growth of pickerel weed in the greenhouse............................................................301 B.2. Lepidopteran pests of pick erelweed and feeding damage......................................302 B.3. Spider mites on leaves and inflorescence of pickerelweed....................................303 B.4. Slug and aphids on pickerelweed...........................................................................304 B.5. Screen enclosures designed to excl ude lepidopteran pests from seedlings............305 B.6. Flowers of Moonglow and wild-type plant............................................................306 B.7. Mean area in mm2 of flowers of pickerelweed......................................................307 B.8. Variegated leaves of pickerelweed.........................................................................308 B.9. Leaf shapes of pickerelweed..................................................................................309 B.10 Growth habit of dormant and nondorma nt plants during winter in southern Florida....................................................................................................................310

PAGE 16

xvi Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INHERITANCE OF MORPHOLOGICAL CHARACTERS OF PICKERELWEED ( Pontederia cordata L.) By Lyn Anne Gettys August 2005 Chair: David S. Wofford Cochair: David L. Sutton Major Department: Agronomy Pickerelweed ( Pontederia cordata L.) is a diploid (2n=2x=16) perennial aquaphyte used in wetland mitigation and restoration a nd in ornamental aquascapes. There have been no reports of the inherita nce of characters of pickerel weed so the primary goal of this work was to provide information regard ing the genetic control of albinism, flower color, floral morph and scap e pubescence in pickerelweed. Pickerelweed and other heterostylous species often exhibit some degree of self-incompatibility due to st ructural differences among the heteromorphic flowers. One factor thought to contribute to self-incompatib ility in pickerelweed is differential pollen tube growth among the three types of pollen pr oduced by this species. A study of in vitro pollen germination showed that pollen tubes fr om l-pollen and m-pollen were longer than those from s-pollen 240 min after germinati on. Pollen diameter measurements revealed that l-pollen was larger than m-pollen or s-pollen and that m-pollen was larger than

PAGE 17

xvii s-pollen. A significant positive regression between pollen grain diameter and pollen tube length 240 min after germination was identified as well. Only the M-morph of pickerelweed is se lf-fertile; therefore, novel pollination techniques (corolla removal and stylar surgery) were developed to circumvent self-incompatibility in S-morph and L-morph flowers, respectively. The effect of seed storage intervals, conditions and germination environments were studied; this experiment revealed that best germination occurred when seeds stored for less than 6 months were germinated under water. Albinism in pickerelweed was conditioned by three diallelic loci with expression influenced by epistasis. The P locus was epistatic, while the A and B loci were hypostatic and functioned as duplicate factors with dominan t gene action at each locus. Flower color was usually controlled by a single diallelic locus ( W ), with blue flowers dominant and white flowers recessive, but a second locus influencing flower color was identified as well. Floral morph was conditioned by two diallelic loci ( S and M ), with dominant gene action; expression was influenced by epis tasis. Scape pubescence was controlled by duplicate gene loci, with pubescence do minant to a glabrous condition. The W locus controlling flower color and the M locus involved in floral morph were linked by 16 m.u.

PAGE 18

1 CHAPTER 1 INTRODUCTION AND OVERVIEW Pickerelweed ( Pontederia cordata L.) is a diploid (2n=2x=16) perennial native aquatic species that is frequently used in wetland mitigation and restoration and in ornamental aquascapes. There have been no reports describing the inheritance of morphological characters in pickerelweed, so the primary goal of this work was to provide information regarding th e genetic control of albinism, flower color, floral morph and scape pubescence in pickerelweed. A review of the current literature is pres ented in Chapter 2. This describes the work that has been reported to date regard ing the taxonomy, culture, propagation and reproductive biology of pickerelweed. Chapter 2 also includes a review of heterostyly and provides a body of knowledge to draw from throughout this dissertation. Heterostylous species often exhibit at leas t some degree of self-incompatibility due to anatomical, structural or physiological differences among the heteromorphic flowers; these differences encourage insect-media ted cross-pollinations between disparate members of the same species and also act as barriers against self-fertilization. One of the factors thought to contribute to self-incompa tibility in pickerelw eed is the different lengths of pollen tubes produced in vivo by the three types of pollen borne by this species. Chapter 3 examines the in vitro germin ation of pollen in an effort to determine whether differences in pollen tube growth ar e evident in an in vitro system as well. An objective of this work was to de sign a breeding program and develop a population of pickerelweed that would yi eld the information needed to conduct

PAGE 19

2 inheritance studies. Most plant breeding and genetics experiments benefit greatly when self-produced progeny can be analyzed; however only one of the three floral morphs of pickerelweed is self-fertile. Novel pollinati on techniques were developed to increase seed production after self-pollination; these techniques are described in Chapter 4 and can be used by future investigators to bypass or ove rcome self-incompatibility in pickerelweed. Population development requires the production not only of seed s, but also of seedlings; therefore, Chapter 5 examines the effect of various seed storage intervals, conditions and germination environments on the seeds of pickerelweed to determine optimum storage and germination conditions. Chapters 6 through 11 explore the inherita nce of multiple morphological traits in pickerelweed. Albinism is a lethal trait and seedlings exhibiting this condition do not survive for more than 3 weeks after germination; the genetic control and inheritance of this deleterious trait is described in Ch apter 6. The inheritance and genetic systems controlling flower color and floral morph ar e examined in Chapters 7 and 8, while the linkage relationship between these two traits is elucidated in Chapter 9. The genetic control of scape pubescence is described in Chapter 10 and the inheritance of non-allelic or complementary flower color is discussed in Chapter 11. This work is offered as a contribution to the body of scientific knowledge available for pickerelweed. It is by no means comprehe nsive, but I hope that other investigators will find this dissertation to be a useful resource for further studies.

PAGE 20

3 CHAPTER 2 A REVIEW OF THE LITERATURE Economic Importance Pickerelweed ( Pontederia cordata L.) is an attractive shoreline aquatic species that is frequently used in wetland mitigation and restoration and in ornamental aquascapes. The showy purplish-blue or white inflores cences of this herbaceous perennial make pickerelweed a prime candidate for inclusion in water gardens and its status as a native plant provides many opportunities for use in proj ects where ecosystem fidelity is critical. Pickerelweed is classified as an obligate wetland species (Garbisc h and McIninch 1992), but the species tolerates a wi de range of moisture levels and may be useful in less saturated areas as well. Many nurseries that produce aquatic pl ants offer pickerelweed for use in ornamental water gardens and aquascapes. Pi ckerelweed may even be purchased at many of the big box retail stor es (e.g., Wal-Mart, Lowes); in fact, Wal-Mart offers pickerelweed with either purplish-blue or wh ite flowers for sale in a pre-packaged kit with two plants, fertilizer, a planting cont ainer and growing substrate for a very reasonable price (Figure 2.1). Pl ants are bare-root specimen s packed in damp sphagnum and recover quickly after planting. When used in wetland mitigation or restor ation, pickerelweed pr ovides a refuge and habitat for many types of fauna. The flow ers attract butterfli es, skippers and hummingbirds (Larson 1995; Speichert and Speichert 2001). Florida apple snails ( Pomacea paludosa ) frequently lay their eggs on the sturdy emergent stems (Turner

PAGE 21

4 1996), while dragonflies and damselflies use the upright stems as perches to shed their final larval stage before reaching adulthood (Speichert and Speichert 2001). The fruit of pickerelweed is an important food source for ducks and small animals (Tobe et al. 1998); Speichert and Speichert (2001) and Taylor ( 1992) also state that humans may safely eat fruits, leaves and stems. Classification, Origin and Distribution Pickerelweed was described by Linnaeus (1753) and named in honor of Italian botanist Giulio Pontedera (1688-175 7), an Italian professor of botany at the University of Padua (Bailey 1949; Turner and Wasson 1998). The type specimen used by Linnaeus for the original description of the species was found in Virginia and was most likely supplied to him by Gronovius (Lowden 1973). The genus Pontederia is namesake for the monocotyledonous family Pontederiaceae, which encompasses ca. nine genera and thirty species (Zomlefer 1994). The Pontederiaceae is of New World origin and includes the invasive genus Eichhornia whose member species E. crassipes (waterhyacinth) is infamous for its ability to dominate aquatic habitats and to render waterways impassible through prodigious production of floating vege tative mats. Other genera in the family include Heteranthera Monochoria Eurystemon Hydrothrix Scholleropsis Reussia and Zosterella Species in the genus Pontederia include P. cordata L., P. rotundifolia L., P. subovata (Seub.) Lowd., P. parviflora Alex. and P. sagittata Presl.; however, only P. cordata is commonly found in the eastern United States (Lowden 1973; Zomlefer 1994). There also exists a form commonly referred to as lance-leaf pi ckerelweed that is classified as either P. cordata var. lanceolata or as P. lanceolata (Muhl.) Torrey. The primary characteristics used to distinguish between P. cordata and lance-leaf

PAGE 22

5 pickerelweed are lance-shaped leaves and pers istent pubescence in the mature floral tube. There is a great amount of natural varia tion in these characters and many taxonomists lack confidence in the classi fication of this type as a separate species (Godfrey and Wooten 1979). The taxonomic hierarchy for pickerelweed is Kingdom Plantae, Subkingdom Tracheobionta, Superdivision Spermatophyta, Division Magnoliophyta, Class Liliopsida, Subclass Liliidae, Order Liliales, Family Pontederiaceae, Genus Pontederia L., Species Pontederia cordata L. (USDA 20021). Multiple synonyms exist for P. cordata and include species of Narukila and Unisema ; these genera no longer exist and all members previously placed in these genera have been reclassified as forms of P. cordata (USDA 2002; Wunderlin and Hansen 2000). Pickerelweed is hardy in USDA Zones 4 through 11 and has a North American geographic range that extends from Prince Ed ward Island to the Florida Keys (Godfrey and Wooten 1979). The species is also found in Central America, Brazil, the West Indies and Argentina (Godfrey and Wooten 1979; Lowd en 1973). The center of diversity for the family is thought to be lowland South Am erica (Kohn et al. 1996). Emergent vegetation dies back in colder regions during the winter, but rhizomes maintained below the frozen epilimnion will overwinter and produce new s hoots in spring. In Ontario and eastern Connecticut, new shoots begin to emerge from rhizomes in April and many well-developed leaves have formed by June. Flowering commences in June and peaks in July, with maximum fruit disp ersal occurring in August. Shoots begin to die back by autumn and most aboveground biomass is dead by December (Heisey and Damman 1 Retrieved 12 July 2002 from the Integrated Taxonomic Information System online database (http://www.it is.usda.gov)

PAGE 23

6 1982; Price and Barrett 1984). Plants in more hospitable climes like southern Florida remain green throughout the winter and will flower for up to 10 months of the year. Morphology Pickerelweed is a diploid (2n=2x=16), erect, emergent, herbaceous aquatic perennial and produces vegetative growth up to 1.5 m tall. The species is found in marshes, swamps, streams, ditches and the shallow water along the margins of lakes and ponds (Figure 2.2) (Bell and Taylor 1982; G odfrey and Wooten 1979; Tobe et al. 1998). Pickerelweed is most common along shorelines and in flooded areas th at are fairly still and shallow (less than 30 cm in depth); se asonal fluctuations in water levels do not adversely impact growth of pickerelweed (Heisey and Damman 1982). Pickerelweed reproduces using both sexual and vegetative strategies. Singleseeded fruits are produced in large amounts and allow fo r dispersion of the species, while creeping rhizomes rooted in the substrate encourage the formation of large, extensive, clonal colonies. Leaves are glabrous, entire, basal, erec t and borne individually on long petioles. Blade size and shape is highly variable a nd ranges from cordate to lanceolate. The racemose inflorescence is borne at the distal end of a stem and is subtended by a single leaf-like bract. Each inflorescence measures from 5 to 20 cm in length and bears up to 250 individual flowers [although Ornduff (1966) noted more than 450 individual flowers on a single inflorescence] (Figure 2.3). Anth esis begins in the morning and flowers remain open for up to 12 h; an average of 20 individual flowers are open on any given day on a single inflorescence. The perianth is composed of six petalo id tepals arranged in two whorls of three; tepals range in color fr om violet-blue to lilac to rarely white, with yellow nectar guides (eye spots) marking the median upper tepal of the floral envelope (Figure 2.4). Each flower is zygomorphic, ba sally connate and perfect, bearing one style

PAGE 24

7 and two sets of three stamens. Concentrated nectar with up to 55% sucrose equivalents is produced during anthesis. Fruits of picker elweed are buoyant a nd surrounded by light aeriferous tissue and may float for up to 15 d (S chultz 1942). The fruit has been described as a nutlet (Richards and Barre tt 1987) or utricle (Bailey 1949); the difference between the two classifications lies in the degree of at tachment of the ovary wall to the seed. The wall of the fruit is formed from the floral tube and is ridged with a dentate crest (Figure 2.5). The seed contained within the fruit is filled with starchy endosperm and contains a linear embryo that traverses th e entire length of the seed (Martin 1946). Garbisch and McIninch (1992) stated that 1 kg contained ca. 11,000 moist seeds (seeds were stored in water but all excess water wa s removed before weights were recorded). Pickerelweed produces flowers fr om mid-June to mid-August in the northern extremes of the species range, while flowering is almost continuous in southern Florida (Bell and Taylor 1982; Godfrey and Wooten 1979; Price and Barrett 1984; Tobe et al. 1998; Wolfe and Barrett 1989; Zomlefer 1994). Roots of pickerelweed are white, purplis h or fuchsia when young and may turn rusty red due to accumulation of oxidized iron. Roots darken as they age and become brown or black as they decay and die (Heisey and Damman 1982). In addition to the species, another type of pickerelweed is available from large aquatic plant nurseries. This form is referred to as Singapore Pink ; the original plant was reportedly found in Thailand and is pr opagated through clonal tissue culture or micropropagation techniques. Some sources list Singapore Pink as a cultivar of P. cordata while other sources list the clone as Pontederia sp.. Several characters suggest that Singapore Pink may genetically distinct from the species. Flowers are pink

PAGE 25

8 (as opposed to the wild-type blue-violet found in natura l habitats) and lack the anthocyanins produced at the throat of both blue-violet and white flowers (Figure 2.6). Anecdotal reports suggest that Singapore Pi nk is sterile and much less tolerant of temperature extremes (high or low) than th e species; in addition, this form is often shorter, more compact and less vigorous than the species and produc es uniformly hastate leaves. Culture Vegetative growth of pickerelweed is affected by substrate moisture level and nutritional status. Heisey and Damman (1982) stated that greatest biomass accumulation occurred at sites high in nutrients; maximum biomass was achieved 100 to 150 d after significant growth was first noted in spring. Ge ttys et al. (2001) found that plants being cultured in nursery containers grew most vi gorously when the rooting substrate provided high water-holding capacity. Barbieri and Esteves (1991) found that 1.50, 2.49 and 0.15% of total plant dry mass was attributable to N, K and P, respectively. Barbieri and Esteves (1991) also stated that Ca contribu ted 1.10% of total plant dry mass, while Mg, Na and C (ash) were responsible for 0.27, 0.12 and 10.7% of total plant dry mass, respectively. Growth of picker elweed is also influenced by light availability during plant growth. Heisey and Damman (1982) showed that net photosynthetic efficiency during the growing period was 1.5% based on peak biom ass values and 1.3% when seasonal net production was considered. Plants grown under full sun are usually more compact than plants produced under shade. Propagation and Dormancy The environmental conditions that mimic a drawdown (i.e., the reduction in water depth that occurs during the dry season) of ten induce germination in wetland plants.

PAGE 26

9 These conditions include cold stratification in the light with an alternation of temperatures (i.e., 20 C / 30 C) (Shipley and Parent 1991) Cold stratification is a common requirement to ensure the perpetua tion and survival of many seed-propagated temperate species. Seeds that ge rminate soon after being shed by the parent plant in late summer or early fall produce plantlets that will most likely be killed by winter conditions. Chilling requirements ensure that seeds remain dormant and will not germinate until freezing temperatures have passed. Galinato and van der Valk (1986) and Leck and Simpson (1993) noted that stratification was necessary for germination of seeds of most aquatic species. Shipley and Parent (1991) studied germination of stratified seeds (9 mo in moist sand at 4C) of 64 wetland species; seeds from 10 of the species experienced poor ge rmination (less than 10%), while seeds from the remaining 54 speci es had adequate germination (60 to 80% on average). Muenscher (1936), Speichert and Speichert (2001) and Whigham and Simpson (1982) stated that seeds of pickerelweed re quired a cold, moist period of stratification before germination. Whigham and Simpson (1982) showed that less than 5% of freshly collected unstratified seeds of pickerelweed germinated 16 wks after being placed in Petri plates lined with moistened filter paper and that 8 wks of moist stratification at 4C was adequate to initiate germination. Whi gham and Simpson (1982) found that best germination of stratified seeds of picker elweed occurred when a minimum constant temperature of 20C to 30C was maintain ed or when a regime of alternating temperatures (>10C / >20C with 12 h ther moperiods) was used. Leck (1996) stated that freshly collected seeds of pickerelweed from Delaware or New Jersey stored in jars of

PAGE 27

10 water at 5C for 7.5 mo germinated only wh en moved to an alternating temperature regime of 25C / 15C with 12 h thermoperiods. Salisbury (1970) and Grime et al. (1981) found that most mudflat and wetland species germinated better or faster in light than in dark; Galinato and van der Valk (1986) also noted better germination was realized in the presence of light than in dark, but further stated that dark germination wa s improved by stratification. Whigham and Simpson (1982) stated that presence or abse nce of light did not affect germination of seeds of pickerelweed. Several authors (Berjak et al. 1990; Leck 1996; Robe rts and King 1980; Simpson 1966) noted that seeds of aquatic species te nd to be recalcitrant (i.e., desiccation sensitive); in fact, as little as 2 week s of dry conditions can negatively impact germination in sensitive species (e.g., Zizania aquatica ) (Simpson 1966). Muenscher (1936) found that germination of seeds of 40 a quatic species (seeds stored dried for 2 to 7 mo at 1C to 3 C) was only 8%; germination of s eeds of 45 aquatic species (seeds stored at room temperature) was only 13%. Grime et al. ( 1981) found that seeds from 37 of 45 wetland species were capable of germin ation after being stored for 1 yr at 5 C. Whigham and Simpson (1982) suggested that seed s of pickerelweed lost viability within 1 yr of being shed. Garbisch and McIninch (1992) found that seeds of pickerelweed collected in Maryland remained viable for more than 3 yrs and had no dormancy requirement; however, seeds were stored in water at 1.1C to 4.4C and should be considered stratified. Williges and Harris (1995) conducted greenhouse experiments under natural conditions and stated that germination was significantly higher in inundated treatments

PAGE 28

11 than in non-flooded treatments. All materi al used by Williges and Harris (1995) was collected in the area around Lake Okeechobee as part of a seed-bank density sampling experiment and was refrigerated for an unsp ecified length of time before germination experiments commenced. Galinato and van der Valk (1986) found that seed burial reduced germination percentage, while Leck ( 1996) stated that seeds would not germinate in Petri plates. Barrett et al. (1983) reported that seeds germin ated poorly in water at 30C to 40C; only 76 seedlings were produced from 15 inflorescences, which theoretically could have produced up to 3,000 seeds. Rhizomes and roots account for a large pe rcentage of total plant biomass at all times. The ratio of new living below-ground biomass to new above-ground biomass ranges from 0 in spring to 1.71 in autumn as new rhizomes and roots develop. Energy is stored in rhizomes as compounds that can be metabolized to support new growth or as structural compounds that remain in the rh izome. Most rhizomes produced during the course of one growing season overwinter, then produce new shoots and rhizomes when growth commences in spring. Structural compounds remain in the original rhizome, which usually dies by autumn (Heisey and Damman 1982). Rhizomes narrow when growth slows in autumn and become wider when growth resumes in spring. These differing growth hab its form a constriction and the rhizome is easily fragmented at the juncti on. Energy fixed during the previ ous year is stored in the rhizome and subsidizes new growth when ac tive growth begins in spring. Heisey and Damman (1982) estimated that as much as 30% of the biomass present in live overwintered rhizomes and roots at the begi nning of spring will be used to produce new tissue when active growth commences. Rhizomes and rootstocks of pickerelweed do not

PAGE 29

12 require stratification to overcome dormancy, as plants begin to grow as soon as soil temperatures exceed freezing. Whigham and Si mpson (1982) stated that rhizomes of dormant plants maintained in cold storage (2C to 4C) for 8 to 16 wks produced new growth within 15 d after being removed fr om cold storage and being placed in a greenhouse with a temperature of 20C to 30 C. Large-scale producti on of pickerelweed is frequently accomplished using micropropa gation and tissue-culture techniques (Kane and Philman 1992, 1997). General Heteromorphic Incompatibility Heteromorphic incompatibility is found in 24 angiosperm families and refers to a reproductive system where differences among fl oral morphs, or heterostyly, determine incompatibility types. Heterostylous species are considered simultaneous hermaphrodites, but unequal fitness of pollen and ovule cont ributions is common in many heteromorphic species, especially those that exhibit di styly (Wyatt 1983). Heterostyly promotes disassortative mating among floral mor phs and encourages insect-mediated cross-pollination betw een different morphs (Crowe 1964; Darwin 1877; Ganders 1979; Vuillenmier 1967). Species with floral heteromo rphisms are typically associated with a self-incompatibility system that operates under sporophytic control to regulate mating patterns in populations (Barrett 1977; Ganders 1979; Ordnuff 1966). Heteromorphic sporophytic incompatibility is not known in any monocotyl edonous plant family other than the Pontederiaceae (Kohn et al. 1996). General Tristyly Tristyly is likely the most complex breed ing system in plants; the system has an elaborate developmental basis and is rare, sugg esting that evolution of the trait is difficult (Kohn et al. 1996; Richards and Barrett 1987). Tristyly is a type of heteromorphic

PAGE 30

13 incompatibility and is only found in f our angiosperm families: Amaryllidaceae, Lythraceae, Oxalidaceae and P ontederiaceae. Some tristylous species are self-compatible, while others have degrees of self-inc ompatibility (Barrett 1988, 1993; Barrett and Anderson 1985; Darwin 1877; Eckert and Ba rrett 1994; ONeill 1994). Populations of species that employ tristylous incompatibility systems have three distinct floral morphs, each with a unique set of characters. Floral mo rph differences include length of stigmatic papillae, style coloration and pollen exine sculpturing (Barrett 1988), but the most obvious visible difference among the morphs is style length. There are three positions within each flower, with each pos ition occupied by either a single style or one of two sets of stamens. Floral morph designation is dete rmined by style length; flowers with long styles are L-morphs (Figure 2.7), while thos e with mid styles and short styles are classified as M-morphs (Figur e 2.8) and S-morphs (Figure 2. 9), respectively. Reciprocal positioning of anthers and stigmas occurs so that each plant produces flowers with anthers borne at the same level as the stig mas of the other morphs. This arrangement promotes insect-mediated cross-pollination between anthers and stigmas of equivalent height, resulting in seed set. Darwin (1877) referred to this as legitimate pollination, while illegitimate pollination between anthers and stigmas at different levels results in little or no seed production. Style color in some species may be an indicator of floral morph. For example, long styles of Eichhornia paniculata and E. crassipes are purple, while mid or short styles are lilac/lavende r or white, respectiv ely (Barrett 1985, 1988). Stigma and pollen polymorphisms can lead to correlations between th e relative size or spacing of stigmatic papillae and the diamet er of pollen grains; these structural

PAGE 31

14 polymorphisms may function as a lock and key mechanism to encourage and facilitate legitimate pollination (Dulberger 1981). Self-pollinations with pollen from anther s positioned closer to the stigma may result in increased seed production, as the structural differences typically found in heteromorphic flowers (i.e., stigmatic papi llae density, pollen heteromorphism) may be less pronounced in reproductive structures with reduced herkogamy. Pollen produced by anthers borne on mid-length filaments is cla ssified as m-pollen, while pollen produced by anthers borne on long or short filaments is cl assified as l-pollen or s-pollen, respectively. Morph Inheritance in Tristylous Species Inheritance of style length in tristylous systems was controlled by two diallelic loci in the species studied thus far [e.g., species of Lythrum (Anderson and Ascher 1995), Eichhornia (Barrett 1988) and Oxalis suksdorfi (Ordnuff 1964)]. The L-morph (long style) phenotype was produced by the completely recessive genotype ssmm while the M-morph (mid style) phenotype was due to a recessive condition at the S locus and the presence of at least one dominant allele at the M locus (genotype ssMM or ssMm ). The dominant S allele was present only in plants wi th S-morph (short style) flowers, which have the genotype SSMM SSMm SSmm SsMM SsMm or Ssmm The sporophytic incompatibility system present in thes e species drastically reduced successful self-pollination, so homozygosity of the S and M alleles was unlikely. The S locus was epistatic to the M locus and prevented expres sion of alleles at the M locus (Anderson and Ascher 1995; Barrett 1988; Char lesworth 1979). L-morph plants were true-breeding for floral morph so self-pollination produced only L-morph progeny. Segr egation ratios of progeny resulting from the self-pollination of S-morph and M-morph plants would be dependent on the genotype of the parent plant. Populations of a tris tylous species that

PAGE 32

15 include plants bearing S-morph flowers could only produce this phenotype in one of three ways: the founder population had at least one S-morph plant, pollen from an S-morph plant in a nearby population was transported by pollinators, or a chance mutation arose to change an s to an S (Anderson and Ascher 1995). Population Structure of Tristylous Species If all three morphs are of equal fitness and if the genes controlling tristyly are independent from one another, the only possi ble condition in large tristylous populations is isoplethic equilibrium (1:1:1) (Barre tt et al. 1983). Members of the species Eichhornia and Pontederia are exceptions and exhibit anisople thic population structures. The gene pool of a population may be influenced by tristy ly, as there is unequa l representation of the alleles. The S-morph is most often pr esent in low frequencies or may be lost altogether from populations; this may be at tributable to the low frequency of the S allele, as the S allele is present only in the S-morph. Successful development of an isoplethic population would require the pr esence of all three floral morphs and the production of pollen and nectar rewards necessary to attr act bees and other sp ecialized long-tongued pollinators (Barrett 1988; Charlesworth 1979). Ordnuff (1964, 1966) suggested that populations of tristylous species may have anisoplethic stru ctures with unequal representation of floral morphs due to low numbers of founders in colonizing populations, weak recruitment in later genera tions and intensive clonal propagation as a means to increase population size. Breakdown of Tristyly Self-pollinating populations of normally cr oss-pollinated species usually occur on the fringe of a species geographic range or at marginal sites within the species range. Self-compatible individuals often have a sel ective advantage in low-density situations

PAGE 33

16 and may be favored in pioneer habitats or under conditions associated with the development of population bottlenecks. Self-c ompatibility is an im portant strategy if specialized pollinators typically required by the species are not present to facilitate cross-pollination (Arroyo 1974; Baker 1955, 1967; Barrett 1985; Moore and Lewis 1965; Stebbins 1957). The breakdown of tristyly and resulting development of predominantly self-pollinating populations with semi-homostylous forms has been documented in three species of Eichhornia ( E. paniculata, E. azurea and E. crassipes ). The S allele is lost, resulting in the disappearance of th e S-morph, while the loss of the m allele causes production of the L-morph to cease. The loss of these alleles and morphs may be due to the lower frequency of the S allele, or may be due to a reduction or loss of long-tongued bees and other specialized pollinators that facilitate cross-pollination (Barrett 1988). Members of the Lythraceae and Oxalidaceae ma y form semi-homostylous populations in addition to stable distylous populations (Ba rrett 1988; Charlesworth 1979; Lewis and Rao 1971; Mulcahy 1964; Ordnuff 1972; Weller 1976). Newly formed populations serviced by unspecialized generalist pollinators pr ovide a selective advantage that favors semi-homostylous variants, as autonomous se lf-pollination and self-compatibility results in reproductive assu rance (Barrett 1988). Pollen diameter overlap increases in m onomorphous and dimorphous populations of species of Eichhornia Pollen borne by same-level anth ers may differ in diameter based on the presenting floral morph. In E. azurea l-pollen produced by anthers borne on long filaments of M-morph flowers may be si gnificantly larger than l-pollen produced by anthers borne on long filaments of S-morph flowers (Barrett 1978). This condition also

PAGE 34

17 occurs in E. paniculata but considerable pollen diameter overlap is evident in this species. This is most likely due to relaxed selection pressure and random accumulation of small mutations that influence pollen diameter (Barrett 1988). Other morphological differences have been noted in semi-homostylous populations of E. azurea The size and prominence of yellow nect ar guides and the degree of perianth limb extension is greatly reduced and all fl owers in an inflorescence open and senesce within 1 or 2 days. In addition, the axes of inflorescences in semi-homostylous populations are shorter and produ ce more condensed inflorescences that are enclosed in a sheath. These modifications suggest a reduced need to attract pollinators, as pollen transfer from an individual with a differe nt genotype is no longe r necessary (Barrett 1978). Members of monomorphous populations of E. paniculata in Jamaica uniformly bear mid-length styles and up to three sets of anthers (most commonly one or two sets) adjacent to the stigmas; in addition, plants are self-compatible a nd autogamous (Barrett 1985). Brazilian monomorphous populations of E. paniculata have small flowers borne in reduced numbers on shorter plants than Br azilian tristylous popul ations (Barrett 1985). A host of floral abnormalities are associated with developmental instability of floral morph expression. In E. paniculata these abnormalities include the formation of five tepals instead of the typical six, collapsed pe rianth limbs, twisted or asymmetric perianth parts, male sterility in pollen produced by an thers borne on long filaments, uniform pale flowers and weakly developed nectar guides (Barrett 1985). Cryptic Self-incompatibility Cryptic self-incompatibility (also called weak self-incompatibility) describes the production of primarily out-crossed progeny by a self-compatible species after pollination

PAGE 35

18 with a mix of self-produced and externally-p roduced pollen. Pollen competition results in fewer selfand intramorph matings and an ex cess of intermorph matings when mixtures of all three pollen types (e.g., self, intram orph and intermorph) are deposited in amounts in excess of the number of receptive ovules avai lable. Legitimate pollen grew faster and outcompeted illegitimate pollen in all three floral morphs of E. paniculata (Cruzan and Barrett 1993). This permits a flexible mating strategy that leads to the genesis of out-crossed progeny during pollinator abundan ce and the production of self-produced progeny during pollinator scarcity (Batem an 1956; Becerra and Lloyd 1992; Bowman 1987; Cruzan and Barrett 1993). The system is referred to as cryptic since it is difficult to detect without the us e of genetic markers. Case Study Lythrum salicaria L. Lythrum salicaria L. (purple loosestrife) uses heteromorphic incompatibility in the form of tristyly to reduce the likelihood of self-produced progeny. Mal et al. (1999) found that legitimate pollination events resulted in greater fruit and seed set than illegitimate intermorph, intramorph and self-pollinations. In addition, seeds produced from legitimate pollinations had increased germination rates when compared to seeds produced from any illegitimate pollination (81.4 and 72.7%, respectiv ely). Mal et al. (1999) also noted that the floral morphs differed in maternal fitn ess and in level of incompatibility. L-morph flowers of purple loosestrife produced more seeds after legitimate pollination than did S-morph flowers; incompatibili ty (as measured by seed se t) was strongest in S-morph flowers and weakest in M-morph flowers. Ma l et al. (1999) descri bed differences in siring ability of pollen borne by different anther whorls within the same flower.

PAGE 36

19 Self-pollinations of L-morph a nd M-morph flowers with l-polle n resulted in significantly more seed set than self-pollinations of th ese morphs with s-pollen (Mal et al. 1999). Hermann et al. (1999) found that the m ean diameter of L-morph stigmas of L. salicaria was significantly larger than the mean diameters of stigmas of M-morph and S-morph flowers; in addition, the stig matic papillae on L-morph stigmas were significantly larger and less de nse than papillae on M-morph and S-morph stigmas. There was no difference in the number of pa pillae on stigmas of all three morphs. Studies by Eckert et al. (1996) provided th e first evidence of frequency-dependent selection on morph ratios in natural populations of L. salicaria and found that morph evenness and the frequency of rare morphs in creased significantly over a 5-year period. Eckert et al. (1996) suggested that thei r study was successful because the species self-incompatibility system re sulted in disassortative matings among morphs, rare morphs were present in moderate (0.05 to 0.15) frequencies, high recruitment occurred and populations were large and re sistant to the short-term effects of genetic drift. Prevalence of Tristyly in Species of the Pontederiaceae Solms-Laubach (1883a) characterized the floral morphology of the Pontederiaceae and reported that Pontederia (with the exception of P. parviflora ), Reussia and E. paniculata (Spreng.) Solms were trimorphic, while E. crassipes (Mart.) Solms was dimorphic (L-morph and M-morph only). Solm s-Laubach (1883a, b) also stated that E. natans (P. Beauv.) Solms and Heteranthera were monomorphic. The species E. paradoxa (Mart.) Solms was described by Solms-Laubach (1883a) as possibly trimorphic and by Schwartz (1930) as homom orphic. The heteromorphic composition of E. azurea (Sw.) Kuntz has also been debated; East (1940) and Solms-Laubach (1883a)

PAGE 37

20 referred to the species as trimorphic, while others (Johnson 1924; Mller 1883; Schultz 1942) stated that E. azurea was dimorphic. Self-incompatibility in Sp ecies of the Pontederiaceae Workers have described moderate self-compatibility in the M-morphs of several species of Eichhornia similar to that found in Pontederia ; however, species of Eichhornia lack the pollen trimorphism evident in Pontederia Agharkar and Banerji (1930) and Francois (1964) found that the M-morph of E. crassipes was moderately self-compatible; Agharkar and Banerji (1930) also noted that pollen from the upper set of anthers was more productive in self-pollina tions than pollen from the lower set of anthers. Johnson (1924) witnessed the same w eak self-incompatibility in the M-morph in E. paniculata Barrett (1985) found illegitimate pollinations of the S-morph of E. paniculata to be much less productive than illegitimate pollinations in the other morphs, as flowers with the S-morph produ ced little or no seeds when undisturbed; Barrett (1985) suggested that herkogamy might have been sufficient to prevent autogamy in the S-morph of the species. The M-morphs of both P. rotundifolia and P. sagittata were moderately self-compatible when pollinated with l-pollen, while S-morphs and L-morphs exhibited much stronger self-incompatibility (Ba rrett 1977, 1988; Barrett and Anderson 1985; Glover and Barrett 1983; Ordnuff 1966). Morph Inheritance in Species of the Pontederiaceae Francois (1964) analyzed progeny resu lting from illegitimate pollinations of dimorphic E. crassipes and theorized that a single diallelic locus was responsible for inheritance of style le ngth in the species, with the M-morph dominant and the L-morph recessive. The loss of the S-morph (and therefore the S allele) from the population would

PAGE 38

21 result in a homozygous recessive state at th e S-locus; therefore, the genotypes in the population under investiga tion were most likely ssmm (L-morph) and ssMm (M-morph) rather than mm (L-morph) and Mm (M-morph). If this is the ca se, the inheritance of style length in E. crassipes is probably similar to the syst em proposed for other tristylous species (e.g., Lythrum and Oxalis ); segregation for all three morphs would occur if the S-morph and S allele were present. Morph Inheritance in Pickerelweed Barrett and Anderson (1985) assessed a small set of S1 progeny (20 seedlings) produced from controlled self-pollinations. Tw o of the 4 observed populations segregated for style length and self-pollination of S1 plants yielded self-compatibility rates similar to those of their parents. Barrett and Anders on (1985) thought these data suggested that self-incompatibility was associated with st yle length; however, th e small population size precludes serious speculation about the inhe ritance of style lengt h in pickerelweed. Pollen Diameter Trimorphism and Production in Pickerelweed Pollen production in pickerelweed varies based on the position of the anthers bearing the pollen. Anthers borne by long filaments produce small amounts of large pollen grains; anthers borne by short filaments produce larg e quantities of small pollen grains and anthers held by mid-length f ilaments produce an intermediate amount of medium pollen grains (Barrett 1985, 1988; Price and Barrett 1982, 1984). Two distinct classes of anthers have been id entified. Large anthers measure 1.02 0.06 mm and are borne by long filaments of M-morph and Smorph flowers and mid-length filaments of L-morph flowers. Small anthers meas ure 0.85 0.05 mm and are borne by short filaments of M-morph and L-morph flower s and mid-length filaments of S-morph flowers (Price and Barrett 1982). Barrett et al (1983) and Price and Barrett (1982) found

PAGE 39

22 that anthers borne by mid-leng th filaments of the S-morph produce nearly twice as much m-pollen as anthers borne by mid-length filaments of the L-morph. Several workers have described differen ces in the diameters of pollen grains produced by the three sets of anthers. Three distinct pollen diameter classes are evident, with the largest pollen produ ced by anthers borne on long filaments and the smallest pollen produced by anthers borne on short fi laments (Barrett a nd Glover 1985; Halsted 1889; Hazen 1918; Leggett 1875a,b; Ordnuff 196 6; Price and Barrett 1982, 1984). There is no overlap in pollen diameter so pollen origin may be identified without ambiguity (Price and Barrett 1982, 1984). Diam eter classes are preserve d regardless of whether pollen is fresh or acetolyzed (Barrett and Glover 1985; Price and Barrett 1984). Grains of l-pollen (produced by anthers borne on long filaments) are largest and measure 65.65 3.22 m in diameter when fresh and 43.82 2.43 m in diameter when acetolyzed. Pollen grains from anthers borne on short filaments are smallest; freshly collected grains of s-pollen are 34.52 2.57 m in diameter, while acetolyzed grains are 25.59 1.43 m in diameter. Anthers borne on midlength filaments generate m-pollen that is intermediate in size, w ith fresh pollen grains measuring 53.95 3.60 m in diameter and acetolyzed grains measuring 36.93 1.78 m in diameter (Barrett and Glover 1985). Harder and Barrett (1993) found no difference in pollen release times among the three morphs. Floral Structure and Reproductive Organ Arrangement in Pickerelweed Flowers of pickerelweed are tr imerous with two series of three tepals, two series of three stamens and a tricarpellate ovary with a single ovule (Richards and Barrett 1987). The tepals are joined to form a perianth tube into which stamens are inserted. Individual

PAGE 40

23 flowers are 12 to 16 mm long, zygomorphic an d marked with a bright yellow nectar guide (eyespot) on the large upper (banner) tepal. There is a close relationship between stigma and anther height in organs with reciprocal positions (Price and Barrett 1982). Richards and Barrett (1987) theorized that the rate of development of reproductive organs could be affected by the diallelic system that is thought to control inheritance of floral morph. Richards and Barrett (1987) suggest ed, for example, that the presence of an M allele could alter reproductive growth rates to increase filament length and decrease style length. Differences in anther heights are attribut able to the position of insertion of the filament on the floral tube a nd to differences in filament length. Filaments bearing long anthers grow quickly, while filaments beari ng mid anthers elongate more slowly. Length differences between filaments bearing long a nd short anthers may be due primarily to growth rate. Filaments of s hort anthers experience rapid early growth but elongation is sharply reduced when floral bud length ex ceeds 3 mm; in addition, filaments of short anthers are inserted farther down in the flor al tube than filament s bearing mid or long anthers (Richards and Barrett 1987). The filaments bearing the long anthers of both M-morph and Smorph flowers are inserted at the same point in the floral t ube. The filaments beari ng the short anthers of both L-morph and M-morph flowers are also in serted at the same point but further down the floral tube than filaments bearing the long anthers. Mid anther s of S-morph flowers constitute the lower anther set of the morph and filaments are inserted on the adaxial side of the perianth tube, while mid anthers of Lmorph flowers are the upper anther set of the

PAGE 41

24 morph and filaments are inserted on the abaxial side of the perianth tube (Barrett et al. 1983; Price and Barrett 1982; Ri chards and Barrett 1987). Stamens of the same level within a flower are produced in different ways. Short anthers of L-morph and M-morph flowers and mid anthers of S-morph flowers are classified as upper shorter stamen level; within this level the central stamen is longest and is inserted on one of the inner tepal series, while the outer two stamens are shorter and are inserted on the outer tepal series. The long anthers of M-morph and S-morph and the mid anthers of L-morph flowers are classified as lower longer stamen leve ls; within this level the central stamen is shortest and is insert ed on one of the outer tepal series, while the outer two stamens are longer and are inserted on the inner tepal series. As a result, each stamen level within a flower has members that represent both inner a nd outer tepal series (Richards and Barrett 1987). Stigmatic height variation is attributable to style length, as ovar y length is similar in all three morphs. Styles of L-morphs M-morphs and S-morphs measure 12.6 0.7, 7.6 0.3 and 2.7 0.1 mm, respectively (Richa rds and Barrett 1987). Price and Barrett (1982) recorded similar measurements. Price and Barrett (1982) found that styl es of L-morph, M-morph and S-morph flowers of pickerelweed were purple, lil ac and pink, respectivel y. Price and Barrett (1982) also noted differences in the dens ity of stigmatic papillae among the morphs. Stigmas of L-morphs had lowdensity papillae and thus larg e interstitial spaces, while stigmas of S-morphs had very dense papill ae and small interstitial spaces. Price and Barrett (1982) speculated that these differe nces corresponded to the pollen diameter

PAGE 42

25 trimorphisms noted in pickerelweed and ma y present a physical barrier to exclude incompatible pollen. There are no significant differences among the morphs in regard to flower and inflorescence production, fecundity or ability to produce seed, fruit weight or germination (Barrett and Anderson 1985; Price and Barrett 1982). Pollen Physiology and Male Fitness in Pickerelweed The pollen of pickerelweed is binu cleate (Brewbaker 1967). Ordnuff (1966) suggested that pollen grains produced by anthers occupying corresponding positions in different morphs (e.g., l-pollen from M-mo rphs and l-pollen from S-morphs) were physiologically equivalent when utilized in legitimate pollination events. However, later work by Barrett et al. (1983) and Price and Barrett (1982) found th at anthers borne by mid-length filaments of the S-morph produced nearly twice as many grains of m-pollen as anthers borne by mid-length filaments of the L-morph. This difference in pollen production is attributable to th e larger size and therefore vol ume of mid anthers produced in the S-morph as compared to the mid anthers of the L-morph. Barrett et al. (1983) proposed a differential male fertil ity hypothesis, which stated that this differential poll en production rendered m/S pollen (m-pollen from a S-morph) more fit than m/L pollen (m-pollen from a L-morph). Simulation models derived by Barrett et al. (1983) suggeste d that the anisople thic population structures found in many populations of pickerelweed could be due to the two-fold difference in m-pollen production between the S-morphs and L-morphs Barrett et al. (1983) examined a small number of flowering progeny (76 seedlings ) collected from fifteen open-pollinated M-morph plants in a mixed S-morph-dominat ed population of pickerelweed and assessed segregation of S-morphs versus non-S-morphs ; these ratios were id entical to expected

PAGE 43

26 values calculated using their hypothesis, but the small sample size precluded the level of confidence needed to ensure that results were not skewed. Field studies of natural populations by Barrett et al (1983) provided limited support for their hypothesis. Self, Intramorph and Intermorph Compatibility in Pickerelweed The different floral morphs of pick erelweed exhibit varying levels of self-incompatibility, but all morphs produce mo re seeds after legitimate pollination than after illegitimate pollinati on (Barrett and Anderson 1985; Barrett and Glover 1985; Ordnuff 1966). Ordnuff (1966) found that own-form (e.g., M x l/M or M x s/M) and other-form (e.g., M x l/S or M x s/L) illegi timate pollinations were less successful than legitimate pollinations (e.g., M x m/S or M x m/L). Ordnuff (1966) also noted that illegitimate pollinations of L-morphs a nd S-morphs were most productive when self-produced m-pollen was used, while ill egitimate pollinations of M-morphs had greatest seed yield when self-produced l-polle n was utilized. Ordnuff (1966) stated that differential self-incompatibility response among morphs may have been largely attributable to carpellary (s porophytic) factors and cited the following example: l-pollen from M-morph plants was highly productive when used in self-pollination (M x l/M) but poorly productive when applied to S-morph plants (S x l/M). Self-incompatibility is strongest in S-mo rphs. Own-form illegitimate pollinations resulted in an average of 2.8% (S x l/S) and 12.7% (S x m/S) seed set, while other-form illegitimate pollinations yielde d 3.1% (S x l/M) and 2.7% (S x m/L) seed set. Legitimate pollinations of S-morphs (S x s/M and S x s/ L) produced an average of 61.3% seed set (Ordnuff 1966).

PAGE 44

27 Self-incompatibility is slightly weaker in L-morphs. Own-form illegitimate pollinations L-morphs resulted in an average of 7.1% (L x s/L) and 18.7% (L x m/L) seed set, while other-form illegitimate pollinations yielded 11.7% (L x m/S) and 7.0% (L x s/M) seed set. Legitimate pollinations of L-morphs (L x l/M and L x l/S) produced an average of 71.1% seed set (Ordnuff 1966). Self-incompatibility is very weak in M-morphs. Own-form illegitimate pollinations of M-morphs resulted in an average of 21.3% (M x s/M) and 53.9% (M x l/M) seed set, while other-form ille gitimate pollinations yielded 39.8% (M x l/S) and 27.6% (M x s/L) seed set. Legitimate pollinations (M x m/S and M x m/L) of M-morphs produced an average of 82.7% seed set (Ordnuff 1966). Pollen Growth in vivo The growth rate of compatible and in compatible pollen grain tubes is of importance, as flowers of pickerelweed ha ve an anthesis period of only 6 to 8 h. Anderson and Barrett (1986) found that bot h compatible and incompatible grains germinated readily on stigmas, which suggested that incompatibility in the species was not due to strong stigmatic inhibition. Incompa tible pollen tubes that reached the base of the style enlarged, curled or lost direction, bu t frequently were able to enter the ovary. Compatible pollen grew more quickly in vivo than incompatible pollen. Pollinations by Anderson and Barrett (1986) sh owed that compatible pollen grains reached the base of the style more often than incompatible pollen grains; exceptions were noted in pollinations of the S-morph, wh ere all grains of a ll three pollen types successfully reached the ovary. All compatib le pollinations resulted in pollen tubes reaching the base of the ovary within 2 h after pollination, while incompatible pollinations took 4 h or longer to reach the ba se of the ovary. Pollinations of M-morphs

PAGE 45

28 and L-morphs with s-pollen ra rely resulted in pol len tubes reaching the ovary and growth of the pollen tubes from s-pollen cea sed after 8 h (Anderson and Barrett 1986). Anderson and Barrett (1986) found a correla tion between pollen gr ain diameter and pollen tube growth. Pollen t ubes from s-pollen, m-pollen and l-pollen reached 4 to 7 mm, 7 to 9 mm and 14 mm in length, respectivel y. These results sugge sted that storage reserves played a role in compatibility of some combinations, but Anderson and Barrett (1986) stated that some form of ovarian in hibition was most likel y responsible for many incompatibility reactions as well. Reduced seed set in illegitimate pollinations in spite of ovule penetration suggests the pr esence of an ovarian inhibito ry system to retard seed production after self-pollinati on (Anderson a nd Barrett 1986). Population Structure of Pickerelweed All three morphs are usually present in na tural populations of pickerelweed (Barrett et al. 1983; Price and Barrett 1982, 1984), but S-morphs are often over-represented while L-morphs are frequently under-represented (B arrett et al. 1983; Morg an and Barrett 1988; Wolfe and Barrett 1989). Pri ce and Barrett (1982) speculate d that the abundance of S-morphs may be due to the increased produc tion of m-pollen by S-morphs as compared to production of m-pollen by L-morphs. Morgan and Barrett (1988) st ated that population structure was strongly influenced by the genot ypic constitutions of the founders and that historical factors played an important role in determining population structure. Impact of Pollinator Behavior Price and Barrett (1984) st udied four natural populati ons of pickerelweed and found that total stigmatic po llen loads decreased in conj unction with a decrease in inflorescence density and pollinator activ ity. Barrett and Glover (1985) found that stigmatic pollen loads were typically 13.6% l-pollen, 22% m-pollen and 64.4% s-pollen.

PAGE 46

29 The proportion of legitimate pollen present in total stigmatic polle n loads on L-morphs and M-morphs remained constant thr oughout the season, but the legitimate pollen component increased as the season progressed. Legitimate pollination was inhibited and illegitimate geitonogamous pollination was favored by non-random foraging behavior of local pollinat ors and by non-random distribution of morphs attribut able to the clonal nature of the species. Price and Barrett (1984) found that 75% of flights by Bombus spp. were among the five nearest neighbors; in addition, the probability that the thr ee nearest neighbors posse ssed the same morph was greater than 70%. Legitimate pollination was found in spite of these constraints, which suggested substantial pollen carryove r by pollinators. Legitimate pollination was highest in L-morph flowers a nd lowest in S-morph flowers, while legitimate pollination of M-morph flowers was intermediate. Price a nd Barrett (1984) suggest ed that this might have been due to partitioning of pollen on th e bodies of pollinators. Northern populations of pickerelweed are serviced by non-specialized bumblebee pollinators (e.g., Bombus spp.) with broad foraging pr eferences that are not highly co-adapted for the floral arrangement found in P. cordata while long-tongued solitary bees ( Melissodes apicata ) visit populations in southern regions. The z ygomorphic flowers of pickerelweed present pollinators with a limited number of orientati ons for floral entry (Faegri and van der Pilj 1979). Laberge (1956) suggest ed that species of Melissodes were specialized pollinators of Pontederia spp. and had hairs on their probosc is to allow collection of pollen concealed in the short anthers. The abdo mens of the pollinators came into intimate contact with l-pollen and the stigmas of L-morphs, while the face and head (proboscis

PAGE 47

30 base) contacted m-pollen and the stigmas of M-morphs. The proboscis tip was aligned with s-pollen and the stigmas of S-morphs (Laberge 1956). Wolfe and Barrett (1989) st ated that pollinators ( Bombus spp., Apis mellifera and Melissodes apicata ) typically visited less than 10 flowers on a single inflorescence and removed 45% of total pollen production duri ng single visits to previously unvisited flowers. Each anther level retained less than 40% of its original pollen complement within 90 min after dehiscence. The largest number of total pollen grains was removed from anthers borne by short filaments, while lesser amounts were removed from anthers borne by mid and long filaments. Rapid polle n depletion occurred, with 69% of l-pollen, 50% of m-pollen and 38% of spollen removed in a single visit (Wolfe and Barrett 1989). Harder and Barrett (1993) found slightly di fferent results, with pollinators removing 39% of l-pollen, 24% of m-pollen a nd 28% of s-pollen during a firs t visit. Pollen distribution on the bodies of pollinators was non-random; l-pollen and m-pollen was found at greatest concentrations where it was initially deposited (i.e., the abdomen and head), while most s-pollen was displaced to the exterior of the pollinator, probably due to grooming activity. M-morph flowers captured the grea test total pollen load (mean = 68 pollen grains per stigma) and S-morph flowers received the smallest total pollen load (mean = 18 pollen grains per stigma). The la rgest proportion of compatible pollen grains was found on stigmas of L-morphs (59% of to tal stigmatic load); stigmas of M-morphs and S-morphs had much smaller proportions of compatible pollen grains (22% and 25% of total stigmatic load, respectively). Many pollinator visits failed to deposit compatible pollen; 40% of visits to S-mo rphs, 21% of visits to M-mo rphs and 26% of visits to L-morphs did not result in deposition of co mpatible pollen on stigmatic surfaces (Wolfe

PAGE 48

31 and Barrett 1989). Pollinators showed no prefer ence for any of the three morphs (Price and Barrett 1982). Stigmatic Pollen Loads in Pickerelweed Stigmas of M-morphs received highest pol len loads and intact flowers captured more pollen grains than flow ers that had been emasculated. Emasculation caused some contamination with self-produced pollen, but the mean number of self-produced pollen grains on unpollinated stigmas borne by emasculated flowers was only 4.14% of the total stigmatic pollen load found on intact flowers (Barrett and Glover 1985). The difference in the number of legitimate pollen grains captured by intact vs. emasculated flowers was insignificant, which provided evidence that stamens did not obstruct the transfer and receipt of legitimate out-cro ssed pollen (Barrett and Glover 1985). Barrett and Glover (1985) also found that even large amounts of illegitimate pollen did not cause stigmatic clogging and did not interfere with growth of legitimate pollen tubes. Pollinator visitation was unaffected by emasculation (Barrett and Glover 1985). Stigmas of S-morphs and L-morphs received only small amounts of illegitimate pollen, with 13% (S-morph) and 14.4% (L-morph) of stigmatic pollen attributable to self-produced or geitonogamous pollen. The amount of illegitimate pollen on stigmas of M-morphs was considerably higher, as self -produced or geitonogamous pollen accounted for 64.3% of total stigmatic po llen load (Barrett and Glover 1 985). L-morphs received the largest amount of legitimate pollen, while pollen deposition on stigmatic surfaces of M-morphs and S-morphs was characteristic of random pollination (Glover and Barrett 1986). Flowers of pickerelweed have only one functional ovule and typical pollen loads have copious amounts of compatible pollen grains ; as a result, seed set in the species in rarely pollen-limited (Barrett and Glover 1985; Glover and Barrett 1986).

PAGE 49

32 Greenhouse Production vs. Natura l Populations of Pickerelweed Ordnuff (1966) noted that seed production in a greenhouse environment was reduced (especially in L-morphs and S-mor phs) when compared to natural populations. Legitimate pollinations of S-morphs in greenhouse populations and field populations produced an average of 61.3 and 88.9% seed set, respectively. Legitimate pollinations of L-morphs in greenhouse populations and fiel d populations produced an average of 71.7 and 94.2% seed set, respectively. Le gitimate pollinations of M-morphs in greenhouse populations and field populations produced an av erage of 82.7% and 89.0% seed set, respectively (Ordnuff 1966). Ordnuff (1966) speculated that differential seed production among the morphs in the greenhous e was eliminated under field conditions, where all three morphs were equa lly capable of seed production.

PAGE 50

33 Figure 2.1. Pre-packaged water garden kit wi th bare-root pickerelweed plants, damp sphagnum moss, soilless planting substrat e, fertilizer tablets and planting basket. A kit with blue-flowered plan ts is shown, but a kit with whiteflowered plants is also available.

PAGE 51

34 Figure 2.2. Pickerelweed growing along the marg in of Roberts Pond in Bainbridge, New York.

PAGE 52

35 Figure 2.3. Inflorescence of white-flowered pickerelweed.

PAGE 53

36 Figure 2.4. Yellow nectar guides (eyespots) on banner tepal of pickerelweed flower.

PAGE 54

37 Figure 2.5. Fresh fruit, seed and dried fruit of pickerelweed.

PAGE 55

38 Figure 2.6. Flowers of Singapore Pink pickerelw eed. Note pale floral throat due to lack of anthocyanins.

PAGE 56

39 Figure 2.7. L-morph flowers of pickerelweed. Note the singl e style in the long position, three anthers in the mid position and the barely-visible three anthers in the short position.

PAGE 57

40 Figure 2.8. M-morph flowers of pi ckerelweed. Note the three anthers visible in the long position and the single style in the mid position; anthers in the short position are barely visible in the lowest flower of this inflorescence.

PAGE 58

41 Figure 2.9. S-morph flowers of pi ckerelweed. Note the three anthers in the long position and the three anthers in the mid position; the single style in the short position is obscured by the throat of th e flower and is not visible.

PAGE 59

42 CHAPTER 3 POLLEN GRAIN DIAMETER, IN VITRO POLLEN GERMINATION AND REGRESSION BETWEEN GRAIN DIAMET ER AND IN VITRO GERMINATION Introduction Pickerelweed ( Pontederia cordata L.) is a naturally outcrossed tristylous species that relies on heteromorphic incompatibility to reduce or prevent self-pollination. Some tristylous species are self-compatible, while others have degrees of self-incompatibility (Barrett 1988, 1993; Barrett and Anderson 1985; Darwin 1877; Eckert and Barrett 1994; ONeill 1994). Three distinct floral morphs ar e produced by tristylous species, but each plant always produces flowers of the same morph. Floral morphs may differ from one another in characters includi ng length or density of stigma tic papillae, style coloration and pollen exine sculpturing (Barrett 1988), but the most obvious visible difference among the floral morphs is style length. Stigmatic height variation is attributable to style length, as ovary length is similar in all thr ee floral morphs (Richards and Barrett 1987). There are three positions within each flow er, with each position occupied by either a single style or one of two sets of stamen s. Floral morph designation is determined by style length; flowers with long styles are L-morphs, while thos e with mid styles and short styles are classified as M-morphs and S-mo rphs, respectively. Reciprocal positioning of anthers and stigmas occurs so that each plan t produces flowers with anthers borne at the same level as the stigmas of the othe r morphs. This arrangement promotes insect-mediated cross-pollination between anthers and stigmas of equivalent height, resulting in seed set. Darwin (1877) referred to this as legitimate pollination, while

PAGE 60

43 illegitimate pollinations between anthers and st igmas at different levels result in little or no seed production. Several workers have described differen ces in the diameter of pollen grains produced by the three sets of anthers in pickerelweed. Three distinct pollen diameter classes are evident; anthers in the long pos ition (borne by long filaments) produce the largest pollen and anthers in the short posit ion (borne by short f ilaments) produce the smallest pollen, while anthers in the mi d position (borne by mid-length filaments) produce pollen that is intermediate in diam eter (Barrett and Glover 1985; Halsted 1889; Hazen 1918; Leggett 1875a,b; Ordnuff 1966; Price and Barrett 1982, 1984). Pollen produced by anthers borne on mid-length filame nts is classified as m-pollen, while pollen produced by anthers borne on long or short filame nts is classified as l-pollen or s-pollen, respectively. Pollen is further identified as s/M or s/L (s-pollen originating from M-morph plants or L-morph plants, respectiv ely), m/S or m/L (m-pollen derived from S-morph plants or L-morph plants, respectivel y) and l/S or l/M (l-pollen from produced by S-morph plants or M-mo rph plants, respectively). There is no overlap in pollen diameter so pollen origin (i.e., anther level) may be identified without ambiguity (Price and Barrett 1982, 1984). Diameter classes are preserved regardless of whether pollen is fresh or acetolyzed (B arrett and Glover 1985; Price and Barrett 1984). Barrett and Glover (1985) reported that fresh grains of l-pollen measured 65.65 3.22 m in diameter, while fresh grains of s-pollen and m-pollen were 34.52 2.57 m and 53.95 3.60 m in diameter, respectively. There are no differences among the floral morphs in regard to flower and inflorescence production, fecundity or ability to produce seed after cross-pollination, fruit

PAGE 61

44 weight or seed germination (Barrett a nd Anderson 1985; Price and Barrett 1982). The different floral morphs of pickerelweed exhi bit varying levels of self-incompatibility but all morphs produce more seeds after legitimate pollination than after illegitimate pollination (Barrett and Anderson 1985; Barrett and Glover 1985; Ordnuff 1966). Ordnuff (1966) found that ille gitimate pollinations (e.g., L x m or s, M x l or s, S x l or m) were less successful than legitim ate pollinations (e.g., L x l, M x m, S x s). Ordnuff (1966) also noted that illegitimate pollinations of L-morphs and S-morphs were most productive when self-produced m-pollen wa s used, while illegitimate pollinations of M-morphs had greatest seed yield when self-produced l-pollen was utilized. Self-incompatibility was strongest in S-morphs. Illegitimate pollinations resulted in an average of 2.7 to 12.7% seed set, while legitimate pollinations produced an average of 61.3% seed set. Self-incompatibility was sli ghtly weaker in L-morphs. Illegitimate pollinations produced from 7.0 to 18.7% seed set, while legitimate pollinations averaged 71.1% seed set. Self-incompatibility was very weak in M-morphs. Illegitimate pollinations resulted in an average of 21.3 to 53.9% seed set, while legitimate pollinations produced an average of 82.7% seed set (Ordnuff 1966). Anderson and Barrett (1986) found that bot h compatible and incompatible pollen grains germinated readily on stigmas, wh ich suggested that incompatibility in pickerelweed was not sporophytic (i.e., due to strong stigmatic inhibition). Incompatible pollen tubes that reached the base of the st yle enlarged, curled or lost direction, but frequently were able to enter the ovar y. Anderson and Barrett (1986) showed that compatible pollen grains grew more quickly an d reached the base of the style more often than incompatible pollen grains; exceptions we re noted in pollinations of the S-morph,

PAGE 62

45 where all grains successfully reached the ovary. Compatible pollination of all floral morphs resulted in pollen tubes that reached the base of the ov ary within 2 h after pollination, while incompatible pollinations took 4 h or longer to reach the base of the ovary. Pollinations of M-morphs and L-mor phs with s-pollen rare ly resulted in pollen tubes reaching the ovary and gr owth of the pollen tubes ce ased after 8 h (Anderson and Barrett 1986). Anderson and Barrett (1986) noted a corre lation between pollen grain diameter and in vivo pollen tube growth. Pollen tubes from s-pollen, m-pollen and l-pollen reached 4 to 7 mm, 7 to 9 mm and 14 mm, respectively. Richards and Barrett (1987) found that stigmatic heights of S-morphs, M-mor phs and L-morphs measured 2.7 0.1 mm, 7.6 0.3 mm and 12.6 0.7 mm, respectively, with similar measurements recorded by Price and Barrett (1982). Thes e results suggested that polle n storage reserves played a role in compatibility of some combinati ons, but Anderson and Ba rrett (1986) stated reduced seed set in illegitimate pollinations in spite of ovule penetration suggested the presence of an ovarian inhib itory system that may have re tarded seed production after self-pollination. The objectives of this experiment were threefold. The first objective was to compare pollen grain diameter of same-level pollen produced by different floral morphs (s/M vs. s/L, m/S vs. m/L and l/S vs. l/M) and to identify differences in grain diameter among pollen produced by the three different an ther levels (s-pollen vs. m-pollen vs. l-pollen). The second objective was to compare in vitro pollen tube growth of same-level pollen produced by different floral morphs (s/M vs. s/L, m/S vs. m/L and l/S vs. l/M) and to determine whether the differences in pollen tube growth in vivo described by

PAGE 63

46 Anderson and Barrett (1986) also exist in an in vitro system The final objective of this experiment was to define the relationship between pollen grain diameter and in vitro pollen tube length 240 min after germination. Materials and Methods Pollen grains from both anther levels of twelve plants were examined in this experiment; these comprised four each of L-morph plants (BL2, HWL, PBL and WL1), M-morph plants (BM2, PBM, PWM and WM1) and S-morph plants (BS1, BS2, BS5 and PWS). Plants were grown in a greenhouse in 1-L nursery containers filled with Metro-Mix 5001, a commercially available growing substrate that contains 40 to 50% composted pine bark, 20 to 35% horticultural grade vermiculite and 12 to 22% Canadian sphagnum peat moss by volume with a nutrient ch arge and pH adjustment (Scotts-Sierra, Marysville OH). Nutrition was supplied by the incorporation of 10 g of Osmocote Plus 15-9-12 (Scotts-Sierra, Marysville OH) per container. Plants were sub-irrigated and kept in a pollinator-free glasshouse with air temperature maintained at 27 C (day) and 16 C (night). During earlier experiments, we observed that some genotypes were more floriferous when grown under long days; ther efore, supplemental lighting was employed to artificially extend daylength to 16 h for the duration of this study. Grains of s-pollen from 8 plants were st udied; 4 of these plan ts (BM2, PBM, PWM and WM1) were M-morphs and the remaining 4 plants (BL2, HWL, PBL and WL1) were L-morphs. Grains of m-pollen from 8 plants were studied; 4 of thes e plants (BL2, HWL, PBL and WL1) were L-morphs and the rema ining 4 plants (BS1, BS2, BS5 and PWS) 1 Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the product by the Florida Agricultural Experiment Station and does not imply its approval to the exclusion of other products that may be suitable.

PAGE 64

47 were S-morphs. Grains of lpollen from 8 plants were studi ed; 4 of these plants (BS1, BS2, BS5 and PWS) were S-morphs and the remaining 4 plants (BM2, PBM, P0WM and WM1) were M-morphs. Pollen grain diameter. Dehisced anthers were rem oved from open flowers with fine forceps and placed in ca. 2 mL of pollen killing and fixing solution in 6-well culture plates (BD Falcon Multiwell Cell Culture Plates #353046, BD Biosciences, Bedford MA; well volume 15 mL, well surface area 9.6 cm2). The pollen killing and fixing solution consisted of 5 parts formaldehyde, 3 parts glacial acetic acid, 20 parts glycerin and 72 parts deionized water and allowed hydr ation of the pollen grains but prevented germination. Three anthers bearing the same t ype of pollen from an individual plant were placed in each well. Each plat e contained 6 wells, so an i ndividual plate contained pollen from both anther levels of 3 different plants. Each plate assembly included a fitted cover, which was labeled with the source of the po llen in each well (donor identity and anther level). Grain diameter was measured for 50 polle n grains from each plant/anther level combination. Grains were magnified and visualized using a Bausch and Lomb microprojector (Leica Microsystems, Wetzlar, Germany). Diameters of magnified pollen grains were recorded in millimeters then c onverted to actual size in microns with a multiplier appropriate for the magnification used to visualize the sample. Means of converted values were calculated for each plant/anther level combination and these values were subjected to st andard analysis of varian ce procedures. The model was constructed to identify differences among sa me-level pollen produced by plants with different floral morphs (s/M pollen vs. s/ L pollen, m/S pollen vs. m/L pollen, l/S pollen

PAGE 65

48 vs. l/M pollen). The model also tested for differences among diameters of pollen grains produced by the three anther le vels (s-pollen vs. m-pollen vs l-pollen). Morph was nested within anther level, as each anther level was present in only two of the three morphs (s-pollen from M-morphs and L-morphs, m-pollen from S-morphs and L-morphs, l-pollen from S-morphs and M-morphs). Mean s were separated usi ng t-tests to detect least significant differences. In vitro pollen germination. This experiment utilized an agarose germination medium consisting of 10% sucrose, 0.6% agar, 0.02% Ca3NO4 and 0.01% boric acid dissolved in deionized water. This mixture was boiled for 5 min then allowed to cool slightly before being tr ansferred to the 6-well culture plat es. Prepared culture plates were cooled to room temperature then stored at 4 C for up to 72 h before pollen collection. Pollen was collected from dehisced anthers at ca. 10:00 am. Fine forceps were used to remove anthers from open flowers and pol len was transferred to the surface of the germination medium by gently dragging the anthers across the surface of the medium. Each well was dusted with pollen from three an thers collected from the same level of an individual plant. Each plate contained 6 we lls, so each plate contained pollen from both anther levels of three different plants. Each plate assembly included a fitted cover, which was labeled with the source of the pollen in each well (donor identity and anther level) and with the collection time. Plates were pl aced in a germination chamber maintained at ca. 30 C and treated with killing and fixing solution (components described in the previous section) at specified time intervals. Pollen grains from each anther level of each plant were killed at one of four time intervals: 30, 60, 120 and 240 min. All pollen

PAGE 66

49 samples in a single plate were killed at th e same time interval by adding ca. 2 mL of killing and fixing solution to each well. Pollen tube length was measured for 200 germinated pollen grains of each plant/anther level/interval combination. Polle n tubes were magnified and visualized using the Bausch and Lomb microprojector described above and tube length data were obtained by utilizing digital calipers to measure pollen tubes from the point of emergence from the pollen grain to the distal end of the pollen tube. These data for magnified pollen tubes were recorded in millimeters then converted to actual size in microns using a multiplier appropriate for the magnification used to visu alize the sample. Means of converted values were calculated for each plant/anther level/in terval combination and these values were subjected to standard analysis of varian ce procedures. The mode l was constructed to identify differences in lengths of pollen tube s from same-level pollen produced by plants with different floral morphs (s/M poll en vs. s/L pollen, m/S pollen vs. m/L pollen, l/S pollen vs. l/M pollen) at al l four intervals. The model also tested for differences among lengths of pollen tubes from pollen produc ed by the three anther levels (s-pollen vs. m-pollen vs. l-pollen) at all four intervals. Morph was nested within anther level, as each anther level was present in only two of the three morphs (s-pollen from M-morphs and L-morphs, m-pollen from S-morphs and L-morphs, l-pollen from S-morphs and M-morphs). Means were separated using t-te sts to detect least significant differences. Relationship between pollen grain diameter and tube length. The relationship between pollen grain diameter and tube lengt h 240 min after germination was examined by computing a regression coefficient between the two variables. The Pearson product-moment correlation formula was used for this analysis.

PAGE 67

50 Results and Discussion Pollen grain diameter. There was no difference in same-level pollen grain diameter produced by the different floral mo rphs (Table 3.1), so further discussion of grain diameter will refer to pollen only by the anther level producing the pollen (i.e., s-pollen instead of s/M and s/L, m-pollen inst ead of m/S and m/L, l-pollen instead of l/S and l/M). Significant differences were ev ident among grain diameters of s-pollen, m-pollen and l-pollen (Table 3.1). Grai ns of l-pollen averaged 44.97 0.30 m in diameter, while grains of mpollen and s-pollen were 35.04 0.49 m and 20.46 0.34 m in diameter, respectively (Figure 3.1). In vitro pollen germination. There was no difference in the lengths of pollen tubes generated by same-level pollen produced by the different floral morphs 30, 60, 120 or 240 min after germination (Table 3.2), so data for all pollen produced by the same anther level were pooled prior to comparisons among the three anther levels. Since floral morph did not have a significan t impact on pollen tube lengt h, further discussion of grain size will refer to pollen only by the anther level producing the pollen (i.e., s-pollen, m-pollen and l-pollen). Significant differences in pollen tube lengths were evident during in vitro germination of s-pollen, m-pollen a nd l-pollen (Table 3.2). Pollen tubes from l-pollen and m-pollen were longer than tube s from s-pollen at all time intervals under investigation (Figure 3.2). Tubes from l-pollen and m-pollen were longer than tubes from s-pollen 30 min after germination, but there was no differen ce between tubes from l-pollen and tubes from m-pollen during the same time interval Pollen tubes from s-pollen averaged 80.49 m in length 30 min after germination, while pollen tubes from m-pollen and

PAGE 68

51 l-pollen reached aver age lengths of 106.26 m and 118.16 m, respectively, 30 min after germination (least significant difference 15.41 m) (Figure 3.2). Pollen tubes from l-pollen and m-pollen we re longer than tubes from s-pollen 60 min after germination; in addition, pollen tubes from l-pollen were longer than pollen tubes from m-pollen during the same time interval. Pollen tubes from s-pollen reached an average length of 137.27 m 60 min after germination; po llen tubes from m-pollen grew to 175.18 m in length and pollen tubes from lpollen reached an average length of 195.07 m 60 min after germination (least significant difference 17.34 m) (Figure 3.2). Pollen tubes from l-pollen and m-pollen were longer than pollen tubes from s-pollen 120 min after germination, but th ere was no difference between pollen tubes from l-pollen and pollen tubes from m-pollen during the same time interval. Pollen tubes from s-pollen averaged 187.77 m in length 120 min after germination, while pollen tubes from m-pollen and l-pollen reached average lengths of 306.34 m and 313.74 m, respectively, 120 min after germinati on (least significant difference 63.62 m) (Figure 3.2). Pollen tubes from l-pollen and m-pollen were longer than pollen tubes from s-pollen 240 min after germination, but th ere was no difference between pollen tubes from l-pollen and pollen tubes from m-pollen during the same time interval. Pollen tubes from s-pollen reached an average length of 265.57 m 240 min after germination; pollen tubes from m-pollen grew to 431.14 m in length and pollen tubes from l-pollen reached an average length of 486.43 m 240 min after germination (l east significant difference 64.27 m) (Figure 3.2).

PAGE 69

52 Relationship between pollen grain diameter and pollen tube length. There was a highly significant regression between polle n grain diameter and pollen tube length 240 min after germination (Figure 3.3). The result of this regression was an increase of ca. 9.13 m in in vitro pollen tube length fo r each micron increase in pollen grain diameter 240 min after germination, with simila r trends noted at the three other intervals as well. These results suggested that polle n grain diameter had a significant positive impact on pollen tube growth in an in vitro system. Conclusions This experiment confirmed the results of Barrett and Glover (1985) and Price and Barrett (1982, 1984) and showed that diamet ers of pollen grains produced by the three anther levels of pickerelweed were signi ficantly different from one another with no overlap in grain diameter among the classes. Measurements of pollen in this experiment differed from those reported by Barrett and Glover (1985) a nd Price and Barrett (1982, 1984); however, this was most likely a fu nction of the disparate methods used to collect data as opposed to a true difference in grain diameter. In vitro pollen germination did not produce the same results as those reported in vivo by Anderson and Barrett (1986). Pollen tubes germinated in vitro did not generate the impressive growth repor ted in vivo, but this is not unexpected since pollen germination in vitro is often less vigor ous than pollen germination in vivo. The relationships among pollen tubes from the th ree pollen grain diamet er classes differed from those described by Anderson and Barrett (1986) as well; these workers reported significant differences among all three classes but this experiment showed no significant difference between pollen tubes produced by l-pollen and those produced by m-pollen. The reason for these conflicting results is unknow n but it is possible th at factors such as

PAGE 70

53 stylar interaction (e.g., the pr esence of inhibitory or stimulatory substances) with the germinating pollen grain influence in vivo germination. This experiment also detected a highly significant regression between pollen grain diameter and in vitro pollen t ube length; these results were similar to those described by Anderson and Barrett (1986) for in vivo po llen germination and s uggested that pollen diameter had a positive impact on the growth of pollen tubes produced as a result of in vitro germination. These results provided an explanation for Ordnuffs (1966) findings that self-pollinations of M-morphs and L-mo rphs were most fruitful when pollen from upper-level anthers was used; as grain diamet er conditions pollen tube length, larger pollen is more likely to produce a pollen t ube long enough to travel down the length of the style and reach the ovary. These results also supported Anderson and Barretts (1986) hypothesis that storage reserves played a role in compatibility of some combinations; however, Anderson and Barrett (1986) also pointed out that reduced seed set in illegitimate pollinations in spit e of ovule penetration suggested the presence of an ovarian inhibitory system (i.e., somatoplastic inco mpatibility) that may have retarded seed production after self-pollination.

PAGE 71

54 Table 3.1. Analysis of variance of pollen grai n diameter in microns of s-pollen, m-pollen and l-pollen of pickerelweed. Data an alyzed were the means of 50 pollen grains from both anther levels of 12 plants. Source DF MS F-value Pr > F Anther 2 1215.68 7902.82 <.0001 Morph(anther) 3 0.25 1.63 0.2185 Error 18 0.15 Total 23 DF: Degrees of freedom MS: Mean square Anther: Anther level (s-pollen, m-pollen, l-pollen) Morph(anther): Anther level nested within morph (s/M vs. s/L, m/S vs. m/L, l/S vs. l/M)

PAGE 72

55 Table 3.2. Analysis of variance of pollen t ube length in microns produced in vitro by s-pollen, m-pollen and l-pollen of pick erelweed. Data analyzed were the means of 200 pollen tubes from each po llen diameter class of 12 plants at 4 intervals. Source DF MS F-value Pr > F Pollen 2 103457.94 43.06 <.0001 Morph(pollen) 3 2070.26 0.86 0.4650 Time# 3 359125.88 149.49 <.0001 Pollen*time 6 16675.24 6.94 <.0001 Morph(pollen)*time 9 2352.19 0.98 0.4646 Error 72 2587.88 Total 95 DF: Degrees of freedom MS: Mean square Pollen: Pollen diameter clas s (s-pollen, m-pollen, l-pollen) Morph(pollen): Morph nested within pollen diameter class (s/M vs. s/L, m/S vs. m/L, l/S vs. l/M) # Time: In vitro germination inte rval (30 min, 60 min, 120 min, 240 min) Pollen*time: Interaction between pollen gr ain diameter and in vitro germination interval Morph(pollen)*Time: Interaction between mo rph nested within pollen grain diameter and in vitro germination interval

PAGE 73

56 Grain diameter (microns) 0 10 20 30 40 50 s-pollen m-pollen l-pollen 44.97a 35.04b 20.46c Figure 3.1. Grain diameter in mi crons of s-pollen, m-pollen a nd l-pollen of pickerelweed. Bars represent the mean diameter of 400 grains per pollen class. Means were separated using a t-test to detect leas t significant differences. Grain diameters coded with different letters are significantly different at p=0.05.

PAGE 74

57 Figure 3.2. Mean length of pollen tubes in microns produced in vitro by s-pollen, m-pollen and l-pollen of pickerelweed 30, 60, 120 and 240 min after germination. Symbols represent the mean length of 2,000 pollen tubes for each anther level/interval combination. 0 100 200 300 400 500 600 30 minutes60 minutes120 minutes240 minutesAverage tube length in microns Short Mid Long

PAGE 75

58 Pollen grain diameter in microns 01020304050 Pollen tube length in microns 0 100 200 300 400 500 600 0 100 200 300 400 500 600 30 min: y = 1.398x + 51.246, r 2 = 0.519, r xy = 0.7204 60 min: y = 2.228x + 97.430, r 2 = 0.6002, r xy = 0.7749 120 min: y = 5.574x + 93.555, r 2 = 0.406, r xy = 0.6372 240 min: y = 9.134x + 92.167, r 2 = 0.660, r xy = 0.8124 Figure 3.3. Regression between pollen grain di ameter and in vitro tube length 30, 60, 120 and 240 min after germination. Regressions computed using 24 XY pairs with the mean diameter of 50 pollen grains (X) and the mean length of 200 pollen tubes (Y) for each plant/anther level/interval combination.

PAGE 76

59 CHAPTER 4 DEVELOPMENT OF NOVEL POLLINATION TECHNIQUES TO REDUCE SELF-INCOMPATIBILITY RESULTING FROM HERKOGAMY Introduction Genetic studies designed to investigate the type of gene action and mode of inheritance of a given trait of ten examine several generations of the organism of interest, including progeny derived from se lf-pollination. Pickerelweed ( Pontederia cordata L.) is a naturally out-crossed tristylous species th at relies on herkogamy (spatial separation of reproductive organs) and heteromorphic incompatibility to reduce or prevent self-pollination. Some tristylous species are se lf-compatible, while othe rs have degrees of self-incompatibility (Barre tt 1988, 1993; Barrett and Anderson 1985; Darwin 1877; Eckert and Barrett 1994; ONeill 1994). Three distinct floral morphs are produ ced by tristylous species, but each plant always produces flowers of the same morph. Floral morphs may differ from one another in characters including length or density of stigmatic papillae, styl e coloration and pollen exine sculpturing (Barrett 1988), but the most obvious visible difference among the floral morphs is style length. Stigmatic height variat ion is attributable to style length, as ovary length is similar in all three morphs (R ichards and Barrett 1987). There are three positions within each flower, with each positi on occupied by either a single style or one of two sets of stamens. Floral morph desi gnation is determined by style length; flowers with long styles are L-morphs, while those with mid styles and short styles are classified as M-morphs and S-morphs, respectively. Reciprocal positioning of anthers and stigmas

PAGE 77

60 occurs so that each plant produces flowers w ith anthers borne at the same level as the styles of the other morphs. This arrangemen t promotes insect-mediated cross-pollination between anthers and stigmas of equivalent he ight, resulting in seed set. Darwin (1877) referred to this as legitimate pollination while illegitimate pollination between anthers and stigmas at different levels results in little or no seed production. Several workers have described differen ces in the diameter of pollen grains produced among the three lengths of filament s in pickerelweed. Three distinct pollen diameter classes are evident, with the larg est diameter pollen produced by anthers borne on long filaments and the smallest diameter pollen produced by anthers borne on short filaments (Barrett and Glover 1985; Halsted 1889; Hazen 1918; Leggett 1875a,b; Ordnuff 1966; Price and Barrett 1982, 1984). Pollen grains produced by anthers with mid-length filaments are classified as m-pollen, while pollen grains produced by anthers borne on long or short filaments are cl assified as l-pollen or s-pol len, respectively. There is no overlap in pollen diameter so pollen origin (i .e., anther level or filament length) may be identified without ambiguity (Price and Barrett 1982, 1984). Diameter classes are preserved regardless of whether pollen is fresh or acetolyzed (B arrett and Glover 1985; Price and Barrett 1984). Fresh gr ains of l-pollen measured 65.65 3.22 m in diameter, while the diameters of fresh grains of s-pollen and m-pollen averaged 34.52 2.57 m and 53.95 3.60 m, respectively (Barrett and Glover 1985). There are no significant differences among the morphs in regard to flower and inflorescence production, fecundity or ability to produce seed after cross-pollination, fruit weight or seed germination (Barrett a nd Anderson 1985; Price and Barrett 1982). The different floral morphs of pickerelweed exhi bit varying levels of self-incompatibility but

PAGE 78

61 all morphs produce more seeds after legitimate pollination than after illegitimate pollination (Barrett and Anderson 1985; Barrett and Glover 1985; Ordnuff 1966). Ordnuff (1966) found that ille gitimate pollinations (e.g., L x m or s, M x l or s, S x l or m) were less successful than legitim ate pollinations (e.g., L x l, M x m, S x s). Ordnuff (1966) also noted that illegitimate pollinations of L-morphs and S-morphs were most productive when self-produced m-pollen wa s used, while illegitimate pollinations of M-morphs had greatest seed yields when self-produced l-po llen was utilized. Self-incompatibility was strongest in S-morphs. Illegitimate pollinations resulted in an average of 2.7 to 12.7% seed set, while legitimate pollinations produced an average of 61.3% seed set. Self-incompatibility was sli ghtly weaker in L-morphs. Illegitimate pollinations produced from 7.0 to 18.7% seed set, while legitimate pollinations averaged 71.1% seed set. Self-incompatibility was very weak in M-morphs. Illegitimate pollinations resulted in an average of 21.3 to 53.9% seed set, while legitimate pollinations produced an average of 82.7% seed set (Ordnuff 1966). Anderson and Barrett (1986) found that bot h compatible and incompatible pollen grains germinated readily on stigmas in vi vo, which suggested that incompatibility in pickerelweed was not sporophytic (i.e., due to strong stigmatic inhibition). Incompatible pollen tubes that reached the base of the st yle enlarged, curled or lost direction, but frequently were able to enter the ovar y. Anderson and Barrett (1986) showed that compatible pollen grains grew more quickly an d reached the base of the style more often than incompatible pollen grains; exceptions were noted in pollinations of S-morphs, where all grains successfully reached the ovary. Compatible pollination of all morphs resulted in pollen tubes that reached th e base of the ovary within 120 min after

PAGE 79

62 pollination, while incompatible pollinations t ook 240 min or longer to reach the base of the ovary. Pollinations of M-morphs and L-morp hs with s-pollen rarely resulted in pollen tubes reaching the ovary and growth of the pollen tubes ceased after 480 min (Anderson and Barrett 1986). Anderson and Barrett (1986) hypothesized that a correla tion existed between pollen grain diameter and in vivo pollen tube gr owth. Pollen tubes from s-pollen, m-pollen and l-pollen reached 4 to 7 mm, 7 to 9 mm and 14 mm in length, respectively. Richards and Barrett (1987) found that stigmatic height s of S-morphs, M-morphs and L-morphs measured 2.7 0.1, 7.6 0.3 and 12.6 0.7 mm, respectively, with similar measurements recorded by Price and Barrett (1982). These results suggested that pollen storage reserves may have played a role in compatibility of some combinations; however, Anderson and Barrett (1986) also stated that reduced seed set after illegitimate pollinations in spite of ovul e penetration suggested the presence of somatoplastic incompatibility or an ovarian inhibitory system that may ha ve retarded seed production after self-pollination. The objectives of this experiment were twofold. The first objective was to assess the level of self-incompatibility among members of a greenhouse population of pickerelweed and to assign a designation of se lf-compatible or self-incompatible to each member of the population. The second objectiv e was to develop methods to overcome or avoid self-incompatibility mechanisms and to improve seed set after self-pollination in members of the population classi fied as self-incompatible. Materials and Methods Plants used in this experiment were from experimental F1 greenhouse populations of pickerelweed created and maintained at the University of Florida in Gainesville. Plants

PAGE 80

63 were grown in 1-L nursery containers filled with Metro-Mix 5001, a commercially available growing substrate that contains 40 to 50% composted pine bark, 20 to 35% horticultural grade vermiculite and 12 to 22% Canadian sphagnum peat moss by volume with a nutrient charge and pH adjustment (Scotts-Sierra, Marysville OH). Nutrition was supplied by the incorporation of 10 g of Osmocote Plus 15-9-12 (Scotts-Sierra, Marysville OH) per container. Plants were sub-irrigated a nd kept in a pollinator-free glasshouse with air temperature maintained at 27 C (day) and 16 C (night). During earlier experiments, we obser ved that some genotypes were more floriferous when grown under long days; therefore, supplemental light ing was employed to artificially extend daylength to 16 h for the duration of this study. All plants were pollinated using sel f-produced pollen from anthers borne on filament lengths described by Ordnuff (1966) as being most productive (i.e., L-morphs were pollinated with m/L pollen, M-morphs with l/M pollen and S-morphs with m/S pollen). Anthers were removed from flower s with fine forceps and pollen transfer was accomplished by brushing the stigma with an anther (Land M-morph flowers) or by depositing a whole anther deep in the throat of the flower (S-morph flowers). Magnifying headgear was worn during all pollinations to allow visual confirmation of successful transfer and adhesion of polle n grains to the stigma. Forceps were flame-sterilized between pollinations of different plants to prevent contamination with foreign pollen. Pollinations commenced with the opening of th e first flowers of an inflorescence and continued until all flowers on the inflorescence had been pollinated (ca. 7 to 12 d). All 1 Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the product by the Florida Agricultural Experiment Station and does not imply its approval to the exclusion of other products that may be suitable.

PAGE 81

64 pollinations were performed between 10 am and 2 pm daily and all flowers in each inflorescence were pollinated using the same method. Daily pollination data were recorded on jewelry tags placed on each inflor escence. Each completed inflorescence was enclosed in a small mesh bag and secured with a plastic-covered twist-tie until fruits were ripe. Fruits were considered ripe when the bearing infructescence sh attered (usually 23 to 30 d after completion of pollinations). Fruits were collected in their mesh bag a nd air-dried for ca. 7 d, then fruits were de-husked using a rubber-covered rub board. Th e use of the rub board allowed removal of the outer husk of the fruit without scarification of the en closed seed. Percent seed set was calculated by dividing the total number of seeds by the total number of flowers pollinated on each inflorescence. Genotypes that produced less than 10% seed set using the methods described above were classified as self-incompatible and were subjected to one of two novel pollination techniques. L-morph genotypes deemed self-incompatible were treated with stylar surgery. A thumbnail was placed under the style of the fl ower; the style was then shortened with a surgical blade held between the thumb and fo refinger of the other hand. The style was cut so that the tip of the style was at ca. the same level as the mid anthers, then m-pollen from the same flower was immediately transfer red to the cut tip of the style (Figure 4.1). All flowers on each inflorescence were pollinat ed using stylar surgery and completed inflorescences were treated as described above. The floral envelope was completely re moved from self-incompatible S-morph genotypes to increase access to the stigma Removal of the floral envelope was accomplished by firmly grasping the center of the open flower with forceps and gently

PAGE 82

65 pulling to remove the coro lla. This protocol effectively exposed the stigma in most cases; however, some plants required further mani pulation to remove te pal tissue obstructing access to the stigma. Removal of the floral envelope resulted in emasculation of the flower, so m-pollen was taken from the remove d portion of the flower and transferred to the exposed stigma (Figure 4.2). All flowers on each inflorescence were pollinated in this manner and completed inflorescences were treated as described above. The seeds produced during the course of this experiment were used for inheritance studies (presented in later chapters of this dissert ation). Multiple inflorescences were pollinated on most plants to ensure production of sufficient quantities of seeds. Most statistical tests require that the sa mples under investigati on meet three basic conditions in order for the analyses to be va lid. Data for each group to be studied must be drawn at random from a normally distribute d population and sampled populations must have homogeneity of variances; in addition, any factor or treatment effect must be additive (or linear) in nature (Zar 1996). The use of percenta ge or proportion data such as those utilized in this experiment violates the requirement of population normality, as percentages form a binomial distribution; however, the deviation from normality presented by these data can be corrected by transformation of the percentage data (Snedecor 1946; Steel et al. 1997; Zar 1996). Several types of transforma tion may be employed; howev er, the most appropriate for percentage or proportion data is arcsine tr ansformation (also referre d to as angular or inverse sine transformation). Ar csine transformation is very us eful for the modification of percentage data where all data points fall between 1.0 (100%) and 0.0 (0%), as the mathematical manipulation of the original bi nomially distributed dataset results in the

PAGE 83

66 creation of a transformed dataset that is normally distributed. The equation for arcsine transformation is p p arcsin where p represents the transformed percentage and p symbolizes the original percentage data (Snedecor 1946; Steel et al. 1997; Zar 1996) While this equation is useful in most circumstances, it is less accurate when data fall at the extreme ends of the range (between 0 to 30% and 70 to 100%). Zar (1996) suggest ed that more accurate results could be obtained if actual proportions were used in the modified equation 1 1 arcsin 1 arcsin 2 1 n x n x p where x / n symbolizes the actual prop ortion data. Proportion data were recorded for this experiment, so statistical analyses were c onducted on data transformed using the latter, more accurate equation. These tran sformed data were then subjected to standard analysis of variance procedures, with means of contro l and treatment pollinations separated using t-tests to detect least significant differences. Results and Discussion M-morph plants. A total of 14,425 flowers were self-pollinated on 53 M-morph plants. Seed set ranged from 37.3 to 98.8% with 10,979 seeds produced. All M-morph genotypes studied in this experiment were cla ssified as self-compatible, so further data regarding this group of plants will not be presented. This high level of seed production after self-pollination corresponde d well with previous reports (i.e., Barrett and Anderson 1985; Barrett and Glover 1985; Ordnuff 1966) that self-incompatibility was weakest in M-morph flowers of pickerelweed.

PAGE 84

67 L-morph plants. There were 51 L-morph plants self -pollinated in this experiment; 28 of these plants were classi fied as self-compatible. Self -pollination of 13,412 flowers in the self-compatible group produced a total of 4,429 s eeds. Seed set ranged from 15.3 to 66.3%. Stylar surgery was not perfor med on these self-compatible plants and further data regarding this group will not be presented. The 23 remaining L-morph plants were cl assified as self-incompatible. Control (normal) self-pollination of 9,237 flowers in th is group resulted in the production of only 262 seeds, with seed set ranging from 0 to 9.8%. Stylar surgery and subsequent self-pollination of 11,049 flowers in the se lf-incompatible group resulted in the production of 3,248 seeds, with seed set ranging from 14.7 to 61.6% (Table 4.1). Data from L-morph plants subjected to c ontrol and stylar surgery pollinations were transformed using the arcsine transformati on procedure described above. Analysis of variance revealed that mean seed set in se lf-incompatible L-morph plants subjected to stylar surgery differed from mean seed set in the same plants after control pollinations (Table 4.2). Mean seed set of all self-incom patible L-morph plants after stylar surgery was 29.93% (arcsine transforme d mean 33.17), while mean seed set of the same plants after control pollinations was 1.97% (arcsine transformed mean 8.06). T-tests showed that mean seed set in self-incompatible L-mo rph plants subjected to stylar surgery was significantly greater than mean seed set in the same plants after cont rol pollinations (least significant difference between arcsine transf ormed means 3.57). These results suggested that stylar surgery effectively improved s eed set in L-morph plants that had been classified as self-incompatible when pollinated using control techniques.

PAGE 85

68 S-morph plants. There were 72 S-morph plants se lf-pollinated in this study. A total of 32 plants were pollinated using only one of the methods (i.e., either control or corolla removal). A group of 12 S-morph plants flowered early in the experiment and had already produced sufficient qua ntities of seeds before the corolla removal technique was fully developed; as a result, these plants re ceived only the control treatment. A total of 1,741 seeds were produced from control pollinat ion of 5,730 flowers and seed set ranged from 22.6 to 41.2%. Corolla removal was a r easonably simple manipulation and early results showed that seed set was greatly increased following removal of the corolla; therefore, all S-morph plants pollinated duri ng the last few months of this experiment were subjected to this protocol. A group of 20 plants were pollinated using only the corolla removal treatment. A total of 3,380 s eeds were produced af ter corolla removal pollination of 7,627 flowers and seed set ranged from 13.6 to 78.1%. Since the plants described above were subjected to only one of the two pollination tec hniques, further data obtained from these groups will not be presented. The 40 remaining S-morph plants were pol linated using both control and corolla removal techniques. Control self-pollination of 11,144 flowers in this group resulted in the production of 2,199 seeds, w ith seed set ranging from 4.6 to 42.9%. Corolla removal and subsequent self-pollination of 11,644 flowers of the same plants produced 5,012 seeds, with seed set ranging from 18.0 to 86.0% (Table 4.3). Data from S-morph plants subjected to control and corolla removal pollinations were transformed using the arcsine transfor mation procedure described above. Analysis of variance revealed that mean seed set in S-morph plants subjected to corolla removal differed from mean seed set in the same pl ants after control pollinations (Table 4.4).

PAGE 86

69 Mean seed set of all S-morph plants after corolla removal was 45.53% (arcsine transformed mean 42.44), while mean seed set of the same plants after control pollinations was 19.76% (arcsine transforme d mean 26.39). T-tests showed that mean seed set in S-morph plants subjected to co rolla removal was significantly greater than mean seed set in the same plants after cont rol pollinations (least significant difference between arcsine transformed means 3.90). Thes e results suggested that corolla removal effectively improved seed set in S-morph plants when compared to seed set after control techniques. Conclusions Self-incompatibility and the resultant poor seed set of some floral morphs of pickerelweed may be overcome with th e use of the novel pollination techniques developed, tested and described in this experiment. Some workers (e.g., Anderson and Barrett 1986) have stated that poor seed set after self-pol lination may be due to the presence of somatoplastic incompatibility or an ovarian inhibitory system that retards seed production after self-pollination. This study suggested that physical constraints (e.g., style length in L-morphs and stigma access in S-morphs) played an important role in the prevention of self-pollination in pi ckerelweed and could be bypassed to effect adequate production of seed s after self-pollination. It is likely that the polle n tube growth limitations described by Anderson and Barrett (1986) are at least partly responsible for self-i ncompatibility and poor seed production after normal self-pollination of th e L-morph of pickerelweed. The largest diameter pollen grain produced by the L-mor ph is m-pollen; Anderson and Barrett (1986) found that m-pollen formed a pollen tube th at was 7 to 9 mm in length, but Price and Barrett (1982) and Richards a nd Barrett (1987) stated that st yles of L-morphs measured

PAGE 87

70 12.6 0.7 mm in length. The stylar surgery t echnique employed in this experiment artificially shortened the style and lowere d the pollen reception surface, which reduced the travel distance required for a pollen tube to reach the ovule and effect fertilization. Roggen and van Dijk (1972) used a steel brush to simultaneously mutilate and pollinate the stigma of Brassica oleracea L. in order to increase seed set, but this species is self-incompatible due to sporophytic factors (i .e., the stigma is the site of inhibition), so the goal of their experiment was to eliminate the stigmatic barriers responsible for incompatibility. Stylar surgery is more similar to the stump pollination technique employed by Davies (1957) to facilitate se ed set in interspecific crosses between members of the genus Lathyrus The two species investigated by Davies, L. odoratus and L. hirsutus produced styles of different lengt hs; the length of the style of L. odoratus was 10 mm, while the length of the style of L. hirsutus was 4 mm. The interspecific cross-pollination of L. hirsutus x L. odoratus resulted in fertiliza tion, but the reciprocal event normally failed to produce seeds. Davies (1957) removed the stigma and part of the style of L. odoratus and applied pollen from L. hirsutus to the cut end of the style; this resulted in fertilization as the pollen had to travel a shorter distan ce to reach the ovary. It is likely that reduced a ccess to the stigma is at l east partly responsible for self-incompatibility and poor seed production after normal self-pollination of the S-morph of pickerelweed. Price and Barrett (1982) and Richards and Barrett (1987) stated that styles of S-morph flowers m easured only 2.7 0.1 mm in length; also, gross visual observation revealed that the reproductive structure wa s ensconced deep within the throat of the flower. Barrett and Anderson (1985 ) used forceps to spl it the floral perianth of S-morph flowers to increase access to the s tigma, but stated that it was still difficult to

PAGE 88

71 conduct pollinations using S-mor ph flowers as seed parents. We found that removal of the corolla to allow increased access to the stigma was a reasonably simple manipulation if done properly and the increased seed pr oduction resulting from utilization of the technique was well worth the small effort required. This information will be helpful for plan t breeders and geneticists interested in studying this and other tristylous species. Genetic studies designe d to investigate the inheritance and genetic control of a given trait often examine several generations of the organism of interest, including progeny deri ved from self-pollination; therefore, geneticists can use this information to im prove seed set after self-pollination. Plant breeders may employ these techniques to deve lop inbred lines of tristylous species, barring the presence of seve re inbreeding depression in the species of interest.

PAGE 89

72 Table 4.1. Seed set after self-pol lination of L-morph plants of pickerelweed subjected to control and stylar surgery pollination treatments. Plant Control CPCT Treatment TPCT# Diff L1 0/150 0.00 146/482 30.29 30.29 L2 1/272 0.37 117/479 24.43 24.06 L3 3/280 1.07 111/604 18.38 17.31 L4 32/522 6.13 85/369 23.04 16.91 L5 6/500 1.20 98/457 21.44 20.24 L6 1/313 0.32 124/565 21.95 21.63 L7 0/47 0.00 76/518 14.67 14.67 L8 19/437 4.35 69/364 18.96 14.61 L9 3/476 0.63 354/875 40.46 39.83 L10 26/372 6.99 156/412 37.86 30.87 L11 30/958 3.13 176/527 33.40 30.27 L12 0/385 0.00 137/512 26.76 26.76 L13 0/501 0.00 189/713 26.51 26.51 L14 8/408 1.96 129/488 26.43 24.47 L15 17/230 7.39 149/242 61.57 54.18 L16 22/421 5.23 148/446 33.18 27.95 L17 2/238 0.84 131/409 32.03 31.19 L18 2/489 0.41 101/452 22.35 21.94 L19 1/537 0.19 119/465 25.59 25.40 L20 50/512 9.77 201/471 42.68 32.91 L21 19/319 5.96 111/226 49.12 43.16 L22 1/360 0.28 148/439 33.71 33.43 L23 19/510 3.73 173/534 32.40 28.67 Plant: Identification code of plant subjected to control and stylar surgery pollinations Control: Number of seeds produced using control method / number of flowers pollinated CPCT: Percent seed set after control pollination TS/P: Number of seeds produced using styl ar surgery / number of flowers pollinated # TPCT: Percent seed set after stylar surgery Diff: Difference in percen t seed set; TPCT CPCT

PAGE 90

73 Table 4.2. Analysis of variance of arcsine tr ansformed percent seed set in control and stylar surgery pollinations of L-morph plants of pickerelweed. Source DF MS F-value Pr > F Treatment 1 7246.32 201.37 <.0001 Error 44 35.99 Total 45 DF: Degrees of freedom MS: Mean square Treatment: Pollination type (control, stylar surgery)

PAGE 91

74 Table 4.3. Seed set after self-pol lination of S-morph plants of pickerelweed subjected to control and corolla remova l pollination treatments. Plant CS/P CPCT TS/P TPCT# Diff S1 24/111 21.62 110/325 33.85 12.23 S2 40/264 15.15 252/485 51.96 36.81 S3 10/53 18.87 161/444 36.26 17.39 S4 18/138 13.04 81/451 17.96 4.92 S5 36/220 16.36 113/467 24.20 7.84 S6 75/428 17.52 61/230 26.52 9.00 S7 59/384 15.36 116/250 46.40 31.04 S8 35/130 26.92 148/262 56.49 29.57 S9 106/286 37.06 54/118 45.76 8.70 S10 77/315 24.44 102/147 69.39 44.95 S11 19/96 19.79 117/332 35.24 15.45 S12 70/186 37.63 161/228 70.61 32.98 S13 67/185 36.22 47/100 47.00 10.78 S14 157/366 42.90 99/160 61.88 18.98 S15 61/418 14.59 131/353 37.11 22.52 S16 30/333 9.01 92/192 47.92 38.91 S17 78/374 20.86 173/270 64.07 43.21 S18 19/183 10.38 197/426 46.24 35.86 S19 71/558 12.72 86/188 45.74 33.02 S20 100/572 17.48 65/224 29.02 11.54 S21 66/169 39.05 56/109 51.38 12.33 S22 117/625 18.72 64/211 30.33 11.61 S23 48/189 25.40 334/473 70.61 45.21 S24 36/372 9.68 92/512 18.97 9.29 S25 63/329 19.15 127/272 46.69 27.54 S26 11/99 11.11 134/247 54.25 43.14 S27 35/325 10.77 65/287 22.65 11.88 S28 13/124 10.48 160/326 49.08 38.60 S29 43/308 13.96 114/396 28.79 14.83 S30 34/168 20.24 173/417 41.49 21.25 S31 5/85 5.88 143/417 34.29 28.41 S32 19/149 12.75 243/551 44.10 31.35 S33 83/408 20.34 117/263 44.49 24.15 S34 17/371 4.58 131/451 29.05 24.47 S35 84/385 21.82 92/155 59.35 37.53 S36 74/412 17.96 66/133 49.62 31.66 S37 96/322 29.81 87/149 58.39 28.58 S38 80/356 22.47 96/200 48.00 25.53 S39 42/117 35.90 260/316 82.28 46.38 S40 81/201 40.30 92/107 85.98 45.68

PAGE 92

75 Table 4.3. Continued Plant: Identification co de of plant subjected to contro l and corolla removal pollinations Control: Number of seeds produced using control method / number of flowers pollinated CPCT: Percent seed set after control pollination TS/P: Number of seeds produced using coro lla removal / number of flowers pollinated # TPCT: Percent seed set after corolla removal Diff: Difference in percen t seed set; TPCT CPCT

PAGE 93

76 Table 4.4. Analysis of variance of arcsine tr ansformed percent seed set in control and corolla removal pollinations of Smorph plants of pickerelweed. Source DF MS F-value Pr > F Treatment 1 5149.33 67.06 <.0001 Error 78 76.79 Total 79 DF: Degrees of freedom MS: Mean square Treatment: Pollination type (control, corolla removal)

PAGE 94

77 Figure 4.1. Stylar surgery of an L-morph flower of pickerelweed. Note pollen on cut surface of surgically shortened style.

PAGE 95

78 Figure 4.2. Pollination of an S-morph flower of pickerelweed after corolla removal. A) Exposed stigma. B) Pollen on the exposed stigma. B A

PAGE 96

79 CHAPTER 5 OPTIMUM SEED STORAGE AN D GERMINATION CONDITIONS Introduction Pickerelweed ( Pontederia cordata L.) is an attractive shoreline aquatic species that is frequently used in wetland mitigation and restoration and in ornamental aquascapes. Pickerelweed reproduces utilizing both sexual and vegetative strategi es, but dispersion of the species is accomplished primarily thr ough the production of copious amounts of single-seeded fruits. The fruit has been desc ribed as a nutlet (Richards and Barrett 1987) or utricle (Bailey 1949); the difference between the two classifications lies in the degree of attachment of the ovary wall to the seed. The wall of the fruit is formed from the floral tube and is ridged with a dentate crest. Fr uits of pickerelweed are buoyant, surrounded by light aeriferous tissue and may float for up to 15 d (Ba rrett 1978; Schultz 1942). Garbisch and McIninch (1992) stated that 1 kg contai ned ca. 11,000 moist seeds; seeds were stored in water but all excess water was removed before weights were recorded. The seed contained within the fruit is filled with starchy endosperm and contains a linear embryo that traverses the entire lengt h of the seed (Martin 1946). Several authors (Berjak et al. 1990; Leck 1996; Robe rts and King 1980; Simpson 1966) noted that seeds of aquatic species were recalcitrant (i.e., desicc ation sensitive); in fact, as little as 2 wks of dry conditions negatively impact ed germination in sensitive species (e.g., Zizania aquatica ) (Simpson 1966). Muenscher (1936) found that germination occurred in only 8% of seeds of 40 aquatic species stored dried for 2 to 7 mo at 1 to 3 C and 13% of seeds of 45 species stored at room temperature, but Grime et al.

PAGE 97

80 (1981) found that seeds from 37 of 45 wetland species were capable of germinating after being stored for 1 yr at 5 C. Whigham and Simpson (1982) stated that presence or absence of light did not affect germination of seeds of pickerelwee d, but Salisbury (1970) a nd Grime et al. (1981) found that most mudflat and wetland species germin ated better or faster in light than in dark. Galinato and van der Valk (1986) also noted better germination was realized in the presence of light than in dark, but further stated that dark germination was improved by stratification. Muenscher (1936), Speichert and Speichert (2001) and Whigham and Simpson (1982) stated that seeds of pickerelweed re quired a cold, moist period of stratification prior to germination. Whigham and Simpson (19 82) showed that less than 5% of freshly collected unstratified seeds germinated 16 wks after being placed in Petri plates lined with moistened filter paper and that 8 wks of moist stratification at 4C was adequate to initiate germination; however, Leck (1996) stat ed that seeds would not germinate in Petri plates. Whigham and Simpson (1982) found that best germination of stratified seeds occurred when a minimum constant temperature of 20 to 30C was maintained or when a regime of alternating temper atures (>10C / >20C; 12 h thermoperiods) was utilized. Leck (1996) stated that freshly collected seed s from Delaware or New Jersey stored in jars of water at 5C for 7.5 mo germinat ed only when moved to an alternating temperature regime of 25C / 15C (12 h thermoperiods). Garbisch and McIninch (1992) found that seeds of pickerelweed collected in Maryland remained viable for more than 3 yrs and had no dormancy requirement; however, seeds were stored in water at 1.1C to 4.4C and should be considered stratified.

PAGE 98

81 Whigham and Simpson (1982) suggested that seed s of pickerelweed lost viability within 1 yr of being shed. Williges and Harris (1995) conducted greenhouse germination experiments and stated that germination of pickerelweed was si gnificantly higher in inundated treatments than in non-flooded trea tments. All material utilized by Williges and Harris (1995) was collected in the ar ea around Lake Okeechobee as part of a seed-bank density sampling experiment and was refrigerated for an unspecified length of time before germination experiments comme nced. Galinato and van der Valk (1986) found that seed burial reduced germination pe rcentage. Barrett et al. (1983) found seeds germinated poorly in water at 30C to 40 C; only 76 seedlings were produced from 15 inflorescences, which theoretically could have produced up to 3,000 seeds. The fulfillment of conditions required for germination determines the success of long-distance dispersal of pickerelweed but also plays a critical role in inheritance experiments. Genetic studies require the evaluation of sexually derived progeny to determine the mode of transmission of the tra it of interest from parents to offspring, so the ability to efficiently produce seed-borne progeny is of great importance. Several workers have studied storage and germinati on of seeds of picker elweed; however, as shown above, the literature is conflicting a nd does not conclusively define optimum storage and germination conditions for the spec ies. The objective of this experiment was to determine the storage and germination cond itions that induced op timum germination in seeds of this population of pickerelweed. Materials and Methods Open-pollinated fruits were collected in December 2003 from a heterozygous and heterogenous population of plants being grown outdoors at the University of Florida Fort Lauderdale Research and Education Center a nd were air-dried for ca. 7 d. Dried fruits

PAGE 99

82 were counted into lots of 100 fruits; each lot was placed in a small zip-lock bag and sealed. Some fruits were used immediately af ter the 7 d drying peri od to assess the effect of germination conditions in fresh fruits and s eeds, while others were stored at either RT (room temperature ca. 25 C) or at 4 C for 3 or 6 mo before being moved to a germination environment. Two pre-germination treatments were studied in this experiment. Before placement in a germination environment, some dried fr uits were cleaned to remove the husk surrounding the seed; this was accomplished using a rubber-covered rub board. De-husked fruits were classified as seeds, while intact fruits were cl assified as fruits. Three germination environments were ex amined: under water, on the soil surface and 0.5 cm below the soil surfac e. Fruits and seeds to be germinated under water were placed in glass half-pint (250 mL) bottles and covered with ca. 5 cm of water (Figure 5.1); additional water was added as needed throughout the course of the experiment to maintain a constant depth. Frui ts and seeds to be germinated on or below the soil surface were placed in propagation flats filled with Metro-Mix 5001 (Scotts-Sierra, Marysville, OH) and maintained under a mist irrigation system (duration 5 sec, interval 10 min, 24 h / d) (Figure 5.2). This experiment studied three storage peri ods (fresh, 3 mo and 6 mo), two storage temperature regimes (RT and 4 C), two pre-germination treatments (fruits and seeds) and three germination environments (under wate r, on the soil surface and 0.5 cm below the soil surface). Fresh fruits and seeds were pl anted immediately after the 7 d drying period; 1 Note: Mention of a trademark or a proprietary produc t does not constitute a guarantee or warranty of the product by the Florida Agricultural Experiment Station and does not imply its approval to the exclusion of other products that may be suitable.

PAGE 100

83 therefore, no storage temperature regime wa s applied to fresh fruits and seeds. Each storage period/storage temperature regi me/pre-germination treatment/germination environment combination was replicated four tim es, with 100 fruits or seeds per replicate. The first part of this experiment utilized fresh fruits and seeds and was started on 12 January 2004; fruits being stored for 3 or 6 mo were moved to the appropriate storage temperature regime on this date as well. Th e second part of this experiment commenced on 14 April 2004 and utilized fruits and seeds that had been stored for 3 mo, while the final part of this experiment began on 12 Ju ly 2004 and used fruits and seeds that had been stored for 6 mo. All treatments were monitored on a weekly basis for 12 wks after being moved to the appropriate germinat ion environments. Radicle emergence was considered evidence of germination and germin ated fruits and seeds were removed from the experiment after data were recorded to eliminate redundant data collection. Most statistical tests require that the sa mples under investigati on meet three basic conditions in order for the analyses to be va lid. Data for each group to be studied must be drawn at random from a normally distribu ted population, and sampled populations must have homogeneity of variances. In addition, any factor or treatment effect must be additive (or linear) in nature (Zar 1996). The use of percenta ge or proportion data such as those utilized in this experiment violates the requirement of population normality, as percentages form a binomial distribution; however, the deviation from normality presented by these data can be corrected by transformation of the percentage data (Snedecor 1946; Steel et al. 1997; Zar 1996). While several types of transformation may be employed, the most appropriate for percentage or proportion data is arcsine transformation (also referred to as angular or inverse si ne transformation). Arcsine

PAGE 101

84 transformation is very useful fo r the modification of percentage data where all data points fall between 1.0 (100%) and 0.0 (0%), as the ma thematical manipulation of the original binomially distributed dataset results in the creation of a transformed dataset that is normally distributed. The equation for arcsine transformation is p p arcsin where p represents the transformed percentage and p symbolizes the original percentage data (Snedecor 1946; Steel et al. 1997; Zar 1996) All data collected during the course of this experiment were transformed prior to statistical analysis. These transformed data were then subjected to standard analysis of variance procedures, with treatment means separated using t-tests to dete ct least significant differences. These data can be analyzed in several di fferent ways. It is possible to consider storage period as a factor and to treat this study as one experiment with a 5 x 2 x 3 factorial arrangement with five stor age treatments (fresh, 3 mo at 4 C or RT and 6 mo at 4 C or RT), two pre-germination treatments (fruits and seeds) and three germination environments (under water, on the soil surface and 0.5 cm below the soil surface). Another method would be to consider each stor age period as a separate experiment (fresh fruits and seeds, fruits and seeds stored for 3 mo and fruits and seeds stored for 6 mo); this would result in three sepa rate experiments. The first experiment would be limited to fresh fruits and seeds and would have a 2 x 3 factorial arrangement (fruits and seeds = two pre-germination treatments; under water, on the soil surface and 0.5 cm below the soil surface = three germinati on environments). The remaining two experiments one using fruits and seeds stored for 3 mo and one using fruits and seeds stored for 6 mo would each have a 2 x 2 x 3 factorial arrangement (fruits and seeds = two

PAGE 102

85 pre-germination treatments; RT and 4 C = two storage temperature regimes; under water, on the soil surface and 0.5 cm below the soil surface = three germination environments). This latter method seems a more reasonable way to approach this study; as noted in this chapters introduction, many seeds of aquatic pl ants are recalcitrant and lose viability quickly when kept in dry conditions, so this analysis would test the effect of the germination condition factors on frui ts and seeds of the same age. As related in the introduction of this chapter, the informa tion regarding the effect of age on viability of seeds of pickerelweed is conflicting. A secondary analysis will be performed to determine the effect of seed age across storage periods. Results and Discussion Data for each storage time will be discusse d separately. Germination of fresh fruits and seeds are presented in Experiment 1, while germination of fruits and seeds stored for 3 and 6 mo will be presented in Experiments 2 and 3, respectively. As previously stated, data were collected weekly for twelve weeks after seeding; however, in the interest of brevity only data recorded during weeks 4, 8 and 12 will be discussed. Experiment 1 (fresh fruits and seeds) Pre-germination treatment (fruits or seeds) had a highly significant impact on germination (Table 5.1). The average number of seeds per replicate germinated 4 wks after seed ing was 23.30, while average fruit germination during the same period was 3.44% (Figure 5.3). An average of 46.40 and 55.18 seeds per replicate had germinated 8 and 12 wks afte r seeding, respectively; during the same periods, average fruit germination was 23.38% and 42.92%. Germination of seeds was significantly greater th an germination of fruits at 4, 8 and 12 wks after seeding.

PAGE 103

86 Germination environment (under water, on the soil surface or 0.5 cm below the soil surface) also had a highly significant impact on germination (Table 5.1). The average number of fruits or seeds per replicate th at had germinated 4 wks after seeding under water was 49.84, while average germination on the soil surface and below the soil during the same period was 2.34% and 0.96%, resp ectively (Figure 5.3). Germination under water 8 wks after seeding av eraged 87.70%; during the same period, an average of 15.83 and 6.58 fruits or seeds per replicate germin ated on the soil surface and under the soil, respectively. An average of 89.96 fruits or seeds per replicate had germinated under water 12 wks after seeding, while average ge rmination on the soil su rface and below the soil during the same period was 27.09% and 25.7 0%, respectively. Germination of fruits or seeds under water was significantly greater than germination on the soil surface or below the soil 4, 8 and 12 wks after seeding. Germination on the soil surface was greater than germination below the soil 4 and 8 wks after seeding, but this difference was no longer evident 12 wks after seeding. A significant interaction between pre-germination treatment and germination environment was evident in fresh fruits and seeds of pickerelweed 4 wks after germination (Table 5.1). Seed s under water germinated at a significantly higher rate than any other pre-germination treatment/germina tion environment combination during this time period; however, the interaction between pre-germination treatment and germination environment was no longer eviden t 8 and 12 wks after seeding. Experiment 2 (fruits and seeds stored for 3 mo). Storage temperature regime did not have a significant impact on germinati on of fruits and seeds stored for 3 mo (Table 5.2).

PAGE 104

87 Pre-germination treatment (fruits or s eeds) had a highly significant impact on germination of fruits or seeds stored for 3 mo (Table 5.2). The average number of seeds per replicate germinated 4 wks after seed ing was 34.94, while average fruit germination during the same period was 19.43% (Figure 5.4). An average of 46.64 and 51.99 seeds per replicate had germinated 8 and 12 wks after seeding, respectiv ely; during the same periods, average fruit germination was 35.39% and 38.95%, respectively. Germination of seeds was significantly greater than germinati on of fruits at 4, 8 and 12 wks after seeding. Germination environment (under water, on the soil surface or 0.5 cm below the soil surface) also had a highly significant impact on germination of fruits or seeds stored for 3 mo (Table 5.2). The average number of fruits or seeds per replicate that had germinated 4 wks after seeding under water was 78.85, while average fruit or seed germination on the soil surface and below the soil during the same period was 20.67% and 0.46%, respectively (Figure 5.4). Germ ination of fruits or seeds under water 8 wks after seeding averaged 84.55%; during the same period, an average of 36.66 and 6.91 fruits or seeds per replicate germinated on the soil surface and under the soil, respectively. An average of 85.00 fruits or seeds per replicate had germinated unde r water 12 wks after seeding, while average fruit or seed germination on the soil surface and below the soil during the same period was 43.05% and 10.51%, respectively. Germination of fruits or seeds under water was significantly greater than germinati on of fruits or seeds on the soil surface or below the soil at 4, 8 and 12 wks after seeding. In addition, germination of fruits or seeds on the soil surface was greater than germinati on of fruits or seeds below the soil 4, 8 and 12 wks after seeding.

PAGE 105

88 A significant interaction between pre-germination treatment and germination environment was evident in fruits and seeds of pickerelweed stored for 3 mo (Table 5.2). Seeds under water germinated at a signi ficantly higher rate than any other pre-germination treatment/germination e nvironment combination. A significant interaction among all three factor s in this experiment was also noted at 4, 8 and 12 wks after seeding. Highest germination percentage s occurred in seeds stored at RT and germinated under water. No other signi ficant interactions were detected. Experiment 3 (fruits and seeds stored for 6 mo). Storage temperature regime (RT or 4 C) had a highly significant impact on the germination of fruits or seeds of pickerelweed stored for 6 mo (Table 5.3). Averag e germination of fruits or seeds stored at RT was 31.37% 4 wks after seeding, while averag e germination of fruits or seeds stored at 4 C during the same period was 19.70% (F igure 5.5). An average of 35.91 and 37.10 fruits or seeds stored at RT per replicate had germinated 8 and 12 wks after seeding, respectively; during the same periods, average ge rmination of fruits or seeds stored at 4 C was 24.68% and 25.32%. Germination of fruits or seeds stored at RT was significantly greater than germination of fr uits or seeds stored at 4 C at 4, 8 and 12 wks after seeding. Pre-germination treatment (fruits or seed s) also had a highl y significant impact on germination of fruits or seeds stored for 6 mo (Table 5.3). The average number of seeds per replicate germinated 4 wks after seed ing was 28.79, while average fruit germination during the same period was 21.98% (Figure 5.5). An average of 33.13 and 34.25 seeds per replicate had germinated 8 and 12 wks after seeding, respectiv ely; during the same periods, average fruit germination was 27.25% and 27.94%. Germination of seeds was significantly greater th an germination of fruits at 4, 8 and 12 wks after seeding.

PAGE 106

89 Germination environment (under water, on the soil surface or 0.5 cm below the soil surface) had a highly significant impact on germin ation of fruits or seeds of pickerelweed stored for 6 mo (Table 5.3). The average numbe r of fruits or seeds per replicate that had germinated 4 wks after seeding under wate r was 52.15, while average fruit or seed germination on the soil surface and below the soil during the same period was 38.87% and 1.03%, respectively (Figure 5.5). Germinati on of fruits or seeds under water 8 wks after seeding averaged 5 2.46%; during the same period, an average of 45.56 and 3.67 fruits or seeds per replicate germinated on the soil surface and under the soil, respectively. An average of 52.46 fruits or seeds per replicate had germinated under water 12 wks after seeding, while average frui t or seed germinati on on the so il surface and below the soil during the same period was 47.40% and 4.10%, respectively. Germination of fruits or seeds under water wa s significantly greater than germination on the soil surface 4 and 8 wks after seeding a nd germination below the soil at 4, 8 and 12 wks after seeding. In addition, germina tion on the soil surface was greater than germination below the soil 4, 8 and 12 wks after seeding. There was no difference in germination of fruits or seeds under water and on the soil surface 12 wks after seeding. All interactions in this experiment were significant (Table 5.3). A significant interaction between storage temperature re gime and pre-germination treatment was revealed when seeds stored at RT germinated at a significantly higher rate than any other storage temperature regime/pre-germina tion treatment combination. A significant interaction between storage temperature regi me and germination environment was also evident, as fruits or seeds stored at RT and germinated under water germinated at a significantly higher rate than any other storage temperature regime/germination

PAGE 107

90 environment combination. A significant inte raction between pre-ge rmination treatment and germination environment resulted in hi gher germination percentage in seeds under water than in any other pre-germination tr eatment/germination environment combination. Lastly, an interaction among a ll three factors was evident; seeds stored at RT and germinated under water achieved the highest germination percentage of any storage temperature regime/pre-germination treatm ent/germination environment combination. Effect of seed age on viability. Seeds germinated under water resulted in the highest percent germination in all three storage periods tested ; therefore, this secondary analysis will be limited to examine the effect of storage period or seed age on viability of seeds germinated under water. Germination ra te of seeds under water was influenced by seed age (fresh, stored for 3 mo or stored for 6 mo) (Table 5.4). Seed age did not have a significant effect on viability 4 wks after seeding, but had a significant impact on viability 8 and 12 wks after seeding. The average number of fresh seeds per replicate that had germinated 8 wks after seeding was 93.81 and no additional germination occurred by week 12 (Figure 5.6). Average germination of seeds stored for 3 mo was 87.66% after 8 wks and 88.03% after 12 wks. Average germ ination of seeds stored for 6 mo was 65.15% after 8 wks and no additional germina tion occurred by week 12. There was no difference between fresh seeds and seeds stored for 3 mo at either 8 or 12 wks after seeding, but both of these clas ses produced significantly hi gher germination than seeds stored for 6 mo. Conclusions This study showed that best germination occurred when seeds were germinated under water. These results supported the work of Williges and Harris (1995), who stated that germination of pickerelweed was signifi cantly higher in inundated treatments than in

PAGE 108

91 non-flooded treatments, and the work of Galin ato and van der Valk (1986), who found that seed burial reduced germination percentage. Germination percentage decrea sed with seed age, but de leterious effects were not detected in seeds stored for 3 mo. This supported the work of Whigham and Simpson (1982), who suggested that seeds of pickerelweed lost viabilit y within 1 yr of being shed. These data contradicted the findings of Garbisch and McIn inch (1992), who stated that seeds of pickerelweed collected in Maryla nd remained viable for more than 3 yrs; however, seeds used in their study were stored in water at 1.1C to 4.4C, while seeds in this experiment were stored in a dried condition. The effects of stratification were not ex amined in this experiment; however, the reasonably high germination rates achieved in this study without stratification suggested that stratification was not necessary to i nduce germination of seeds of pickerelweed. Seeds of pickerelweed experienced reduced viability when stored for more than 3 mo; therefore, fresh seeds should be produ ced for breeding and ge netic studies and for seed propagation of the species. Seeds should also be germinated under water, as this environment resulted in higher germination ra tes than seeds germinated on or below the soil surface. The information reported in this study can be useful to plant breeders and seed producers to efficiently and effectively store and germinate seeds of pickerelweed.

PAGE 109

92 Table 5.1. Analysis of variance of arcsine-tran sformed data for germination of fresh fruits and seeds of pickerelweed. Separate analyses were performed for data collected 4, 8 and 12 wks after germination. 4 wks Source DF MS F-value Pr > F PT 1 1981.38 276.88 <.0001 GE 2 3809.49 532.35 <.0001 PT*GE# 2 203.21 28.40 <.0001 Error 18 7.16 Total 23 8 wks Source DF MS F-value Pr > F PT 1 1179.68 42.01 <.0001 GE 2 6898.22 245.63 <.0001 PT*GE 2 4.74 0.17 0.8459 Error 18 28.08 Total 23 12 wks Source DF MS F-value Pr > F PT 1 297.36 15.59 0.0009 GE 2 4400.71 230.68 <.0001 PT*GE 2 62.81 3.29 0.0605 Error 18 19.08 Total 23 DF: Degrees of freedom MS: Mean square PT: Pre-germination treatment (fruits, seeds) GE: Germination environment (under wate r, on the soil surface, below the soil) # PT*GE: Interaction between pre-germina tion treatment and germination environment

PAGE 110

93 Table 5.2. Analysis of variance of arcsine-tran sformed data for germination of fruits and seeds of pickerelweed stored for 3 mo. Separate analyses were performed for data collected 4, 8 and 12 wks after germination. 4 wks Source DF MS F-value Pr > F SR 1 11.28 0.28 0.6008 PT 1 1219.49 30.12 <.0001 GE# 2 13993.20 345.64 <.0001 SR*PT 1 61.38 1.52 0.2262 SR*GE 2 63.40 1.57 0.2228 PT*GE 2 402.30 9.94 0.0004 SR*PT*GE 2 169.87 4.20 0.0230 Error 36 40.48 Total 47 8 wks Source DF MS F-value Pr > F SR 1 0.14 0.00 0.9615 PT 1 517.90 8.76 0.0054 GE 2 10732.36 181.43 <.0001 SR*PT 1 50.49 0.85 0.3617 SR*GE 2 58.24 0.98 0.3835 PT*GE 2 355.59 6.01 0.0056 SR*PT*GE 2 281.49 4.76 0.0147 Error 36 59.15 Total 47 12 wks Source DF MS F-value Pr > F SR 1 0.13 0.00 0.9612 PT 1 678.66 12.81 0.0010 GE 2 9352.98 176.58 <.0001 SR*PT 1 84.05 1.59 0.2159 SR*GE 2 47.31 0.89 0.4182 PT*GE 2 434.89 8.21 0.0012 SR*PT*GE 2 236.45 4.46 0.0185 Error 36 52.97 Total 47 DF: Degrees of freedom MS: Mean square SR: Storage temperature regime (room temperature, 4 C) PT: Pre-germination treatment (fruits, seeds) # GE: Germination environment (under wate r, on the soil surface, below the soil) SR*PT: Interaction between storage te mperature regime and pre-germination treatment SR*GE: Interaction between storage temperature regime and germination environment PT*GE: Interaction between pre-germina tion treatment and germination environment

PAGE 111

94 Table 5.2. Continued SR*PT*GE: Interaction among storage temper ature regime, pre-germination treatment and germination environment

PAGE 112

95 Table 5.3. Analysis of variance of arcsine-tran sformed data for germination of fruits and seeds of pickerelweed stored for 6 mo. Separate analyses were performed for data collected 4, 8 and 12 wks after germination. 4 wks Source DF MS F-value Pr > F SR 1 713.25 30.04 <.0001 PT 1 242.70 10.22 0.0029 GE# 2 7374.25 310.56 <.0001 SR*PT 1 278.96 11.75 0.0015 SR*GE 2 192.32 8.10 0.0012 PT*GE 2 319.57 13.46 <.0001 SR*PT*GE 2 269.89 11.37 0.0001 Error 36 23.74 Total 47 8 wks Source DF MS F-value Pr > F SR 1 592.32 25.36 <.0001 PT 1 161.66 6.92 0.0125 GE 2 6006.78 257.17 <.0001 SR*PT 1 306.02 13.10 0.0009 SR*GE 2 221.96 9.50 0.0005 PT*GE 2 373.73 16.00 <.0001 SR*PT*GE 2 433.06 18.54 <.0001 Error 36 23.36 Total 47 12 wks Source DF MS F-value Pr > F SR 1 641.50 24.48 <.0001 PT 1 183.43 7.00 0.0120 GE 2 5941.51 226.69 <.0001 SR*PT 1 377.33 14.40 0.0005 SR*GE 2 201.90 7.70 0.0016 PT*GE 2 358.76 13.69 <.0001 SR*PT*GE 2 406.22 15.50 <.0001 Error 36 26.21 Total 47 DF: Degrees of freedom MS: Mean square SR: Storage temperature regime (room temperature, 4 C) PT: Pre-germination treatment (fruits, seeds) # GE: Germination environment (under wate r, on the soil surface, below the soil) SR*PT: Interaction between storage te mperature regime and pre-germination treatment SR*GE: Interaction between storage temperature regime and germination environment PT*GE: Interaction between pre-germina tion treatment and germination environment

PAGE 113

96 Table 5.3. Continued SR*PT*GE: Interaction among storage temper ature regime, pre-germination treatment and germination environment

PAGE 114

97 Table 5.4. Analysis of variance of arcsine-tr ansformed data for germination of seeds of pickerelweed germinated under water. S eeds were fresh, stored for 3 mo or stored for 6 mo. Separate analyses were performed for data collected 4, 8 and 12 wks after germination. 4 wks Source DF MS F-value Pr > F SA 2 344.96 3.35 0.0594 Error 17 103.04 Total 19 8 wks Source DF MS F-value Pr > F SA 2 800.25 6.83 0.0066 Error 17 117.15 Total 19 12 wks Source DF MS F-value Pr > F SA 2 813.57 6.95 0.0062 Error 17 117.05 Total 19 DF: Degrees of freedom MS: Mean square SA: Seed age (fresh, stored for 3 mo, stored for 6 mo)

PAGE 115

98 Figure 5.1. Fruits and seeds of pickerelweed germinated under water in half-pint (250 mL) bottles.

PAGE 116

99 Figure 5.2. Fruits and seeds of pickerelweed germinated on or 0.5 cm below the soil surface under mist irrigation.

PAGE 117

100 Percent germination 0 20 40 60 80 100 Fruits germinated under water Fruits germinated on soil surface Fruits germinated below soil Seeds germinated under water Seeds germinated on soil surface Seeds germinated below soil a. 4 weeks b. 8 weeksc. 12 weeks a b c d e e a b c d d e a b c cd d d Figure 5.3. Percent germination of fresh fruits and seeds of pickerelweed. Bars represent the mean of 4 replicates (100 fruits or seeds per replicate) for each treatment. Means were separated using a t-test to detect least significant differences. Treatments coded with the same letter are not significantly different at p=0.05 from other treatments evaluated at the sa me time (i.e., separate analyses were performed for data recorded 4, 8 and 12 wks after seeding). a. 4 weeks. b. 8 weeks. c. 12 weeks.

PAGE 118

101 Percent germination 0 20 40 60 80 100 a a b a c c d de ef ef ff ef f ab cd f bc e f a d f de e ab c e bc d e a c e a. 4 weeks Stored at 4C Stored at RTb. 8 weeks Stored at 4C Stored at RTc. 12 weeks Stored at 4C Stored at RT a a Fruits germinated under water Fruits germinated on soil surface Fruits germinated below soil Seeds germinated under water Seeds germinated on soil surface Seeds germinated below soil Figure 5.4. Percent germination of fruits and se eds of pickerelweed stored for 3 months. Bars represent the mean of 4 replicates (100 fruits or seeds per replicate) for each treatment. Means were separated using a t-test to detect least significant differences. Treatments coded with th e same letter are not significantly different at p=0.05 from other treatments evaluated at the same time (i.e., separate analyses were performed for data recorded 4, 8 and 12 wks after seeding). a. 4 weeks. b. 8 weeks. c. 12 weeks.

PAGE 119

102 Percent germination 0 20 40 60 80 100 a bc bcd bc d b b cd e e e e cd cd fg cd e f de bc f a b g cde cd fg cde e fg de bc f a b g a. 4 weeks Stored at 4C Stored at RTb. 8 weeks Stored at 4C Stored at RTc. 12 weeks Stored at 4C Stored at RT Fruits germinated under water Fruits germinated on soil surface Fruits germinated below soil Seeds germinated under water Seeds germinated on soil surface Seeds germinated below soil Figure 5.5. Percent germination of fruits and se eds of pickerelweed stored for 6 months. Bars represent the mean of 4 replicates (100 fruits or seeds per replicate) for each treatment. Means were separated using a t-test to detect least significant differences. Treatments coded with th e same letter are not significantly different at p=0.05 from other treatments evaluated at the same time (i.e., separate analyses were performed for data recorded 4, 8 and 12 wks after seeding). a. 4 weeks. b. 8 weeks. c. 12 weeks.

PAGE 120

103 Percent germination 0 20 40 60 80 100 Fresh seeds Seeds stored for 3 months Seeds stored for 6 months a a a a a b a a b a. 4 weeks b. 8 weeks c. 12 weeks Figure 5.6. Percent germination of seeds of pi ckerelweed germinated under water. Bars represent the mean of 4 replicates (100 seeds per replicate) for each treatment. Means were separated using a t-test to detect least significant differences. Treatments coded with the same letter are not significantly different at p=0.05 from other treatments evaluated at the sa me time (i.e., separate analyses were performed for data recorded 4, 8 and 12 wks after seeding). a. 4 weeks. b. 8 weeks. c. 12 weeks.

PAGE 121

104 CHAPTER 6 INHERITANCE AND GENETIC CONTROL OF ALBINISM Introduction Photosynthesis occurs in green plants when chlor ophyll-bearing chloroplasts capture light energy by fixing and conve rting atmospheric carbon dioxide into carbohydrates (Hopkins 1995). Plants that l ack chlorophyll are described as being non-photosynthetic or albino and are unable to generate the energy required to sustain life; as a result, albinism is a lethal tra it in non-parasitic plant species. Albinism is uncommon but the genetic control of the trait has been descri bed in a handful of species. Shull (1915) described two types of albini sm in maize one producing pure white seedlings and the other resulting in yellowish-w hite (chlorina) seedlings. Both types of albinism were recessive, simply inherited and controlled by a single diallelic locus. Ortiz and Vuylsteke (1994) studied albinism in plantain-banana hybrids and found that albinism in Musa spp. was controlled by at least tw o independent recessive loci with complementary gene action. Coffelt and Hamm ons (1971, 1973) reported that albinism in peanut was conditioned by thr ee diallelic loci whose expression was influenced by epistasis. Preliminary studies of pickerelweed ( Pontederia cordata L.) revealed that albino seedlings were regularly produced by a gr oup of plants maintained for breeding and inheritance studies. The objective of this expe riment was to determine the type of gene action and number of loci controlling albi nism in this population of pickerelweed.

PAGE 122

105 Materials and Methods The population utilized in this experime nt was developed using the strategy described in Appendix A of this dissertati on, with seeds germinat ed under ca. 5 cm of water in glass half-pint (250 mL) bottles. Da ta were recorded several times each week and germination conditions were maintained for a minimum of 8 wks. Identification of seedlings expressing albinism was possible 2 or 3 d after ra dicle emergence, as albino seedlings were pure white with no traces of gr een (Figure 6.1); affected seedlings were monitored for an additional 7 to 10 d after germ ination to ensure that the classification of albinism was accurate. Data from F1 and S1 populations were used to develop a working model to explain the type of gene action and number of loci controlling albinism in this population of pickerelweed. Development of this model allowed the assignment of genotypes to parents; the model was then verified by analyses of F2 populations. All data were analyzed using goodne ss-of-fit (chi-square or 2) tests with Yates correction for continuity. Results and Discussion Eight F1 families and five S1 families were examined in this experiment. No maternal effects were noted; ther efore, data presented for each F1 family are pooled within each cross/reci procal set. Three F1 families and three S1 families segregated for albinism, while five F1 families and two S1 families produced only green progeny (Table 6.1). Three of the five parents used in population developm ent (WS, WM and BL) played a role in the crea tion of the segregating F1 and S1 families. Numerous models were developed and tested in an effort to identify a system that explained the progeny ratios in the segregating and non-segregating F1 and S1 families. The simplest model that yielded the progeny types produced in all F1 and S1 families was one with three diallelic loci

PAGE 123

106 ( P A and B ) that interact in an epistatic manner. Two alleles ( P1 and P2) were identified for the epistatic locus P Gene action at the two hypostatic loci ( A and B ) is dominant; in addition, the hypostatic loci function as dupl icate factors. Production of a green (non-albino) seedling requires either two P1 alleles at the epistatic locus or one P1 allele at the epistatic locus and at least one dominant allele at either hypostatic locus. Parents that participated in the creation of segregating F1 and S1 families were assigned genotypes based on the model and on segreg ation of progeny. The parent WS was assigned the genotype P1P2aaBb while the parents WM and BL were assigned the genotypes P1P2AaBb and P1P2AaBB respectively. The genotypic identities of the two other parents (BS and BM) used in this experiment were unclear; however, these parents yielded no albino F1 or S1 progeny so it is likely that BS and BM are homozygous at all three loci ( P1P1AABB P1P1AAbb P1P1aaBB or P1P1aabb ). Punnett squares associated with the expected segregation ratios for F1, F2 and S1 families are shown in Figures 6.2 through 6.14. Goodness-of-fit values and their associated probabil ities are presented in Tabl es 6.2 through 6.9 and indicated that the model provided a good fit to the data and s upported the proposed genotype assignments. Seven of the eight F1 families under investigation were advanced to the F2 generation for further study. Analysis of th ese populations was less straightforward; as seen in Figures 6.2 through 6.9, most F1 populations were heterozygous and heterogeneous, so segregation ratios of F2 progeny differed among members of the same family. The Punnett squares in Figures 6.2 through 6.9 depict the frequency of each F1 genotype and the expected segregation ratio of F2 progeny created from

PAGE 124

107 self-pollination of each F1 genotype. Goodness-of-fit tests were utilized to assess segregation ratios for each F2 family; these data are pres ented in Tables 6.3 through 6.9 and are sorted by family. Summaries of the an alyses for each family are presented below. WMWS and WSWM. This F1 family was created by crossand reciprocal pollinations between the parents WM and WS; each F2 family was developed through self-pollination of an individual F1 plant. The Punnett square in Figure 6.2 provides a depiction of the expected segregation ratios of F1 and F2 progeny in this cross/reciprocal set. Parents WM and WS we re assigned the genotypes P1P2AaBb and P1P2aaBb respectively. The observed F1 progeny segregated in a manne r that was not different from the expected 22:10 (green:albino) ratio. It was not possible to evaluate the segregation of F2 progeny in this cross/reciprocal set, as all F2 seeds from this family were contaminated with fungi and failed to germinate. The F1 data from this cross/re ciprocal set provided evidence that supported the proposed model and genotypic assignments. WMBL and BLWM. This F1 family was created by crossand reciprocal pollinations between the parents WM and BL; each F2 family was developed through self-pollination of an individual F1 plant. The Punnett square in Figure 6.3 provides a depiction of the expected segregation ratios of F1 and F2 progeny in this cross/reciprocal set. Parents WM and BL we re assigned the genotypes P1P2AaBb and P1P2AaBB respectively. The observed F1 progeny segregated in a manne r that was not different from the expected 3:1 (green:albi no) ratio. Four segregation ra tios (46:18, 3:1, 10:6 and all green) were expected in the F2 generation. A total of 24 F2 families (derived from self-pollination of 24 F1 plants) were analyzed (Table 6.3). Seven F2 families did not segregate and only produced green seedli ngs. Seven families produced progeny that

PAGE 125

108 segregated in ratios that were not diff erent from 46:18 and 3:1, while ten families produced progeny that segregated in ratios that were not differ ent from 10:6, 46:18 and 3:1. These data provided evidence that supported the proposed model and genotypic assignments. WSBL and BLWS. This F1 family was created by crossand reciprocal pollinations between the parents WS and BL; each F2 family was developed through self-pollination of an individual F1 plant. The Punnett square in Figure 6.4 provides a depiction of the expected segregation ratios of F1 and F2 progeny in this cross/reciprocal set. Parents WS and BL we re assigned the genotypes P1P2aaBb and P1P2AaBB respectively. The observed F1 progeny segregated in a manne r that was not different from the expected 3:1 (green:albi no) ratio. Four segregation ra tios (10:6, 46:18, 3:1 and all green) were expected in the F2 generation. A total of 25 F2 families (derived from self-pollination of 25 F1 plants) were analyzed (Table 6.4). Three F2 families did not segregate and only produced green seedlings, while ten families produced progeny that segregated in ratios that were not differ ent from 46:18 and 3:1. Eleven families produced progeny that segregated in ratios that were not different from 10:6, 46:18 and 3:1, while one family produced progeny that segregated in a ratio that was not different from 10:6 and 46:18. More segregating families were produced than would be expected based on the proposed model and genotypic assignments; however, only 25 F2 families (from 25 F1 plants) were examined in this experiment so it is probable that sampling error was responsible for the production of more segr egating families than expected. With the exception of this discrepancy, these data pr ovided evidence that supported the proposed model and genotypic assignments.

PAGE 126

109 WSBM and BMWS. This F1 family was created by crossand reciprocal pollinations between the parents WS and BM; each F2 family was developed through self-pollination of an individual F1 plant. The Punnett square in Figure 6.5 provides a depiction of the expected segregation ratios of F1 and F2 progeny in this cross/reciprocal set. Parent WS was assigned the genotype P1P2aaBb The genotype of BM is unclear but BM is most likely homozygous at all thr ee loci, so all four possible homozygous genotypes ( P1P1AABB P1P1aaBB P1P1AAbb and P1P1aabb ) were tested in this experiment. All F1 progeny were green with no albino seedlings produced; based on these results, it is possible to eliminate P1P1aabb as a possible genotype for BM, as albino F1 progeny would have been produced had the genotype of BM been P1P1aabb Different sets of segregation ratios were expected in the F2 generation based on the proposed genotype of BM. If the genotype of BM were P1P1AABB then three segregation ratios (46:18, 3:1 and all green) woul d be expected in the F2 generation. If BM were assigned the genotype P1P1aaBB three segregation ratios (10: 6, 3:1 and all green) would be produced in the F2 generation, while if the genotype of BM were P1P1AAbb then three different segregation ratios (10:6, 46:18 a nd all green) should be recovered in the F2 generation. A total of 23 F2 families (derived from self-pollination of 23 F1 plants) were analyzed (Table 6.5). Eleven F2 families did not segregate and only produced green seedlings, while two F2 families produced progeny that segregated in a 10:6 ratio. Two families produced progeny that segregated in ratios that were not different from 46:18 and 3:1, while one family produced progeny th at segregated in a ratio that was not different from 10:6 and 46:18 and seven fam ilies produced progeny that segregated in ratios that were not different from 10:6, 46:18 and 3:1. Thes e data revealed that it was

PAGE 127

110 possible to eliminate P1P1AABB as a potential genotype for BM, since no F2 families generated in this cross/reci procal set would have been expected to produce progeny segregating in a 10:6 ratio if the genotype of BM were P1P1AABB Based on these data, it is probable that the genotype of BM is P1P1AAbb or P1P1aaBB The data from this cross/reciprocal set provided additional evidence in suppor t of the proposed model and genotype assignments. WMBS and BSWM. This F1 family was created by crossand reciprocal pollinations between the parents WM and BS; each F2 family was developed through self-pollination of an individual F1 plant. The Punnett square in Figure 6.6 provides a depiction of the expected segregation ratios of F1 and F2 progeny in this cross/reciprocal set. Parent WM was assigned the genotype P1P2AaBb The genotype of BS is unclear but BS is most likely homozygous at all thr ee loci, so all four possible homozygous genotypes ( P1P1AABB P1P1aaBB P1P1AAbb and P1P1aabb ) were tested in this experiment. All F1 progeny were green with no albino seedlings produced; based on these results, it was possible to eliminate P1P1aabb as a possible genotype for BS, as albino F1 progeny would have been produced had the genotype of BS been P1P1aabb Different sets of segregation ratios were expected in the F2 generation based on the proposed genotype of BS. If the genotype of BS were P1P2AABB then three segregation ratios (46:18, 3:1 and all green) woul d be expected in the F2 generation; however, if the genotype of BS was P1P1aaBB or P1P1AAbb then four segregati on ratios (10:6, 46:18, 3:1 and all green) should be recovered in the F2 generation. A total of 15 F2 families (derived from self -pollination of 15 F1 plants) were analyzed (Table 6.6). Six F2 families did not segregate and only produ ced green seedlings, while one F2 family produced

PAGE 128

111 progeny that segregated in a 3:1 ratio. Five families produced progeny that segregated in ratios that were not different from 46:18 and 3:1, while three families produced progeny that segregated in ratios that were not different from 10:6, 46:18 and 3:1. These data supported the proposed model and genotype assi gnment of WM, but did not provide any additional information regarding the assignm ent of a genotype to BS other than the elimination of P1P1aabb as a possible genotype. BSBM and BMBS. This F1 family was created by crossand reciprocal pollinations between the parents BS and BM; each F2 family was developed through self-pollination of an individual F1 plant. The Punnett square in Figure 6.7 provides a depiction of the expected segregation ratios of F1 and F2 progeny in this cross/reciprocal set. The genotypes of both parents used to create this family are unknown but both lines are most likely homozygous at all three loci; therefore, all combin ations of the four possible homozygous genotypes ( P1P1AABB P1P1aaBB P1P1AAbb and P1P1aabb ) were tested in this experiment. All F1 progeny were green with no albino seedlings produced; this was expected based on the pr oposed model. A total of 12 F2 families (derived from self-pollination of 12 F1 plants) were analyzed. None of the F2 families segregated for albinism and all families only produced green seedlings (data not shown). These data supported the proposed model but did not provi de any additional in formation regarding the assignment of genotypes to BS and BM. BSBL and BLBS. This F1 family was created by crossand reciprocal pollinations between the parents BS and BL; each F2 family was developed through self-pollination of an individual F1 plant. The Punnett square in Figu re 6.8 provides a depiction of the expected segregation ratios of F1 and F2 progeny in this cross/reci procal set. Parent BL

PAGE 129

112 was assigned the genotype P1P2AaBB The genotype of BS is unclear but BS is most likely homozygous at all three loci, so all four possible homozygous genotypes ( P1P1aaBB P1P1AAbb P1P1AABB and P1P1aabb ) were assessed in this experiment. All F1 progeny were green with no albino seedlings produced; this was expected based on the proposed model. Different sets of segreg ation ratios were expected in the F2 generation based on the proposed genotype of BS. If the genotype of BS were either P1P1AABB or P1P1aaBB then two segregation ratios (3:1 a nd all green) would be expected in the F2 generation; however, if the genotype of BS was P1P1AAbb then three segregation ratios (46:18, 3:1 and all green) should be recovered in the F2 generation. If the genotype of BS were P1P1aabb then three segregation ratios (10:6, 46:18 and all green) would occur in the F2 generation. A total of 12 F2 families (derived from self-pollination of 12 F1 plants) were analyzed (Table 6.7). Seven F2 families did not segregate and only produced green seedlings, while one F2 family produced progeny that segregated in a 3:1 ratio and four families produced progeny th at segregated in ra tios that were not different from 46:18 and 3:1. These data re vealed that it was possible to eliminate P1P1aabb as a potential genotype for BS, since no F2 families generated in this cross/reciprocal set produced progeny that se gregated in a 10:6 ratio. Based on these data, it is probable that the genotype of BS is P1P1AAbb P1P1aaBB or P1P1AABB These data supported the proposed model, the genotypic as signment of BL and the elimination of P1P1aabb as a possible genotype for BS, but did not provide any addi tional information regarding the assignment of a genotype to BS. BMBL and BLBM. This F1 family was created by crossand reciprocal pollinations between the parents BM and BL; each F2 family was developed through

PAGE 130

113 self-pollination of an individual F1 plant. The Punnett square in Figure 6.9 provides a depiction of the expected segregation ratios of F1 and F2 progeny in this cross/reciprocal set. Parent BL was assigned the genotype P1P2AaBB The genotype of BM is unclear but BM is most likely homozygous at all thr ee loci, so all four possible homozygous genotypes ( P1P1aaBB P1P1AAbb P1P1AABB and P1P1aabb ) were assessed in this experiment. All F1 progeny were green with no albino seedlings produced; this was expected based on the proposed model. Different sets of segregation ratios were expected in the F2 generation based on the proposed genotype of BM. If the genotype of BM were either P1P1AABB or P1P1aaBB then two segregation ratios (3:1 and all green) would be expected in the F2 generation; however, if the genotype of BM were P1P1AAbb then three segregation ratios (46:18, 3:1 a nd all green) should be seen in the F2 generation. If the genotype of BM were P1P1aabb then three different segr egation ratios (10:6, 46:18 and all green) would be recovered in the F2 generation. A total of 20 F2 families (derived from self-pollination of 20 F1 plants) were analyzed (Table 6.8). Nine F2 families did not segregate and only produced gr een seedlings, while one F2 family produced progeny that segregated in a 3:1 ratio and ten families pr oduced progeny that segreg ated in ratios that were not different from 46:18 and 3:1. These data revealed that it was possible to eliminate P1P1aabb as a potential genotype for BM, since no F2 families generated in this cross/reciprocal set produced progeny that se gregated in a 10:6 ratio. Based on these data, it is probable that the genotype of BM is P1P1AAbb P1P1aaBB or P1P1AABB These data supported the proposed model, the genotypic as signment of BL and the elimination of P1P1aabb as a possible genotype for BM, but did not provide any addi tional information regarding the assignment of a genotype to BM.

PAGE 131

114 WS This S1 family was created by self-pol lination of the parent WS. The Punnett square in Figure 6.10 provides a depictio n of the expected segregation ratio of S1 progeny derived from self-pollination of WS with the assigned genotype P1P2aaBb The observed S1 progeny segregated in a manner that was not different from the expected 10:6 (green:albino) ratio (Tab le 6.2). These data supporte d the proposed model and the assignment of the genotype P1P2aaBb to WS. WM This S1 family was created by self-pol lination of the parent WM. The Punnett square in Figure 6.11 provides a depictio n of the expected segregation ratio of S1 progeny derived from self-pollination of WM with the assigned genotype P1P2AaBb The observed S1 progeny segregated in a manner that was not different from the expected 46:18 (green:albino) ratio (Tab le 6.2). These data supported the proposed model and the assignment of the genotype P1P2AaBb to WM. BS This S1 family was created by self-pollin ation of the parent BS. The Punnett square in Figure 6.12 provides a depiction of the expected segr egation ratio of S1 progeny derived from self-pollination of BS with the assigned genotype P1P1AABB P1P1AAbb P1P1aaBB or P1P1aabb All observed S1 progeny were green and no albino S1 progeny were produced (Table 6.1). These data suppor ted the proposed model but did not provide any additional information regarding the assignment of a genotype to BS. BM This S1 family was created by self-pol lination of the parent BM. The Punnett square in Fig. 6.13 provides a depicti on of the expected segregation ratio of S1 progeny derived from self-pollination of BM with the assigned genotype P1P1AABB P1P1AAbb P1P1aaBB or P1P1aabb All observed S1 progeny were green and no albino

PAGE 132

115 S1 progeny were produced (Table 6.1). These data supported the proposed model but did not provide any additional in formation regarding the assignment of a genotype to BM. BL This S1 family was created by self-pol lination of the parents BL. The Punnett square in Figure 6.14 provides a depictio n of the expected segregation ratio of S1 progeny derived from self-pollinati on of BL with the assigned genotype P1P2AaBB The observed S1 progeny segregated in a manner that was not different from the expected 3:1 (green:albino) ratio (Table 6.2). Thes e data supported the proposed model and the assignment of the genotype P1P2AaBB to BL. Conclusions This experiment showed that albinism in this population of pickerelweed was controlled by three diallelic loci ( P A and B ) that interacted in an epistatic manner. Two alleles ( P1 and P2) were identified for the epistatic locus P Gene action at the two hypostatic loci ( A and B ) was dominant; in addition, the hypostatic loci functioned as duplicate factors. Production of a green (non-albino) seedling required either two P1 alleles at the epistatic locus, or one P1 allele at the epistatic locus and at least one dominant allele at either hypostatic locus. Based on these data, it was possible to assign genotypes to three of the five individuals used as parent al lines in this study. The genotypic identities of th e other two parents were unclear; however, these parents yielded no albino progeny when used in cross-pollina tions so it is likely that both were homozygous at all th ree loci, with two P1 alleles at the epistati c locus and two dominant alleles at one or both of the hypostatic loci ( P1P1AABB P1P1AAbb or P1P1aaBB ). This model requires the presence of at least one P1 allele at the epistatic P locus in order for chlorophyll production to occur. It is unusual to en counter a single locus that

PAGE 133

116 controls a trait as devastating as albinism, wh ich is lethal in this and other photosynthetic plant species. Fitness is reduced by 25% in individuals heterozygous at this epistatic locus, as they will produce a large number of albino progeny when self-pollinated or when mated with other individuals heterozygous at the same locus. This model employs two hypostatic diallelic lo ci that act as duplicate factors. The genetic control of albinism in peanut also had two hypostatic diallelic loci (Coffelt and Hammons 1971, 1973); gene action at the epistatic locu s in peanut differs from the gene action of the epistatic locus controlling albi nism in this population of pickerelweed, but the mechanism allowing the hypostatic loci to ac t as duplicate factors was similar in both systems. The presence of duplicate factors in these and other systems is not an unexpected occurrence, as biol ogically critical functions su ch as chlorophyll production typically have several loci that may play th e same role in creati ng an end product. This duplication allows a reasonable expectation that a single mutation will not result in a lethal condition.

PAGE 134

117 Table 6.1. Number of green and albino seedlings from F1 and S1 families of pickerelweed. Family Generation Seeds Green Albino# WSWM and WMWS F1 156 106 50 WMBL and BLWM F1 93 65 28 WSBL and BLWS F1 92 70 22 WSBM and BMWS F1 149 149 0 WMBS and BSWM F1 125 125 0 BSBM and BMBS F1 141 141 0 BSBL and BLBS F1 84 84 0 BMBL and BLBM F1 109 109 0 WS S1 66 44 22 WM S1 71 50 21 BS S1 10 10 0 BM S1 14 14 0 BL S1 104 77 27 Family: F1 or S1 family under investigation; F1 families were created through crossand reciprocal pollinations between two parents and F1 codes identify the parents (e.g., the F1 family WSWM was derived from cross-po llination between the parents WS and WM) Generation: Generation of th e family under investigation Seeds: Number of germinated seeds examined Green: Number of green seedlings # Albino: Number of albino seedlings

PAGE 135

118 Table 6.2. Goodness-of-fit tests for F1 and S1 families of pickerel weed segregating for albinism. Progeny were tested against a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. Family Expected1 Expected2 Observed 2# P WMWS 22:10 107.25:48.75 106:50 0.0168 0.8968 WMBL 24:8 (3:1) 69.75:23.25 65:28 1.0358 0.3088 WSBL 12:4 (3:1) 69.00:23.00 70:22 0.0145 0.9041 WS 10:6 41.25:24.75 44:22 0.3273 0.5672 WM 46:18 51.03:19.97 50:21 0.0197 0.8883 BL 3:1 78.00:26.00 77:27 0.0128 0.9098 Family: F1 or S1 family under investigation; F1 families were created through crossand reciprocal pollinations between two parents and F1 codes identify the parents (e.g., the F1 family WSWM was derived from cross-poll ination between the pa rents WS and WM); not shown: non-segregating families WSBM, WMBS, BSBM, BSBL, BMBL, BS and BM Expected1: Expected ratio of green progeny to albino progeny Expected2: Expected number of green progeny and albino progeny Observed: Number of green prog eny and albino progeny observed # 2: Chi-square value calculated from goodne ss-of-fit test; computed using Yates correction for continuity P: Probability associ ated with calculated 2 value

PAGE 136

119 Table 6.3. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism and derived from the initial cros s/reciprocal set WMBL (genotypes P1P2AaBb x P1P2AaBB ). Progeny were tested against a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. F2 F1 Parent Exp1 Exp2 Obs# 2 P F2 families WMBL 2:1 16:8 17:7 0.0001 0.9920 WM1 WMBL3 10:6 105:63 122:46 6.9143 0.0085 46:18 120.75:47.25 122:46 0.0166 0.8974 3:1 126:42 122:46 0.3889 0.5328 WM3 WMBL31 10:6 54.38:32.63 70:17 11.219 0.0008 46:18 62.53:24.47 70:17 2.9221 0.0873 3:1 65.25:21.75 70:17 1.1073 0.2926 WM4 WMBL48 10:6 39.38:23.63 46:17 2.5407 0.1109 46:18 45.28:17.72 46:17 0.0038 0.9508 3:1 47.25:15.75 46:17 0.0476 0.8272 WM6 BLWM8 10:6 70.63:42.38 89:24 12.064 0.0005 46:18 81.22:31.78 89:24 2.3209 0.1276 3:1 84.75:28.25 89:24 0.6637 0.4152 WL2 WMBL28 10:6 10.63:6.38 10:7 0.0042 0.9483 46:18 12.22:4.78 10:7 0.8596 0.3538 3:1 12.75:4.25 10:7 1.5882 0.2075 WL3 WMBL35 10:6 51.88:31.13 56:27 0.6755 0.4111 46:18 59.66:23.34 56:27 0.5937 0.4409 3:1 62.25:21.75 56:27 2.1245 0.1449 WL5 WMBL45 10:6 10.63:6.38 13:4 0.8824 0.3475 46:18 12.22:4.78 13:4 0.0230 0.8794 3:1 12.75:4.25 13:4 0.0000 0.9999 BM1 WMBL4 10:6 76.25:45.75 92:30 8.1333 0.0043 46:18 87.69:34.31 92:30 0.5894 0.4426 3:1 91.5:30.5 92:30 0.0000 0.9999 BM3 WMBL22 10:6 86.25:51.75 99:39 4.6396 0.0312 46:18 99.19:38.81 99:39 0.0000 0.9999 3:1 103.5:34.5 99:39 0.6184 0.4316 BM4 BLWM3 10:6 118.75:71.25 148:42 18.561 0.0000 46:18 136.56:53.44 148:42 3.1147 0.0775 3:1 142.5:47.5 148:42 0.7018 0.4021 BM5 BLWM6 10:6 53.13:31.88 57:28 0.5718 0.4495 46:18 61.09:23.91 57:28 0.7516 0.3859 3:1 63.75:21.25 57:28 2.4510 0.1174 BM6 BLWM9 10:6 36.88:22.13 46:13 5.3797 0.0203 46:18 42.41:16.59 46:13 0.8025 0.3703 3:1 44.25:14.75 46:13 0.1412 0.7070

PAGE 137

120 Table 6.3. Continued F2 F1 Parent Exp1 Exp2 Obs 2 P BL1 WMBL5 10:6 37.50:22.50 40:20 0.2844 0.5938 46:18 43.13:16.88 40:20 0.5681 0.4510 3:1 45:15 40:20 1.8000 0.1797 BL2 WMBL6 10:6 42.50:25.50 50:18 3.0745 0.0795 46:18 48.88:19.13 50:18 0.0284 0.8661 3:1 51:17 50:18 0.0196 0.8886 BL3 WMBL7 10:6 21.25:12.75 27:7 3.4588 0.0629 46:18 24.44:9.56 27:7 0.6189 0.4314 3:1 25.5:8.5 27:7 0.1569 0.6920 BL4 WMBL8 10:6 34.38:20.63 40:15 2.0376 0.1534 46:18 39.53:15.47 40:15 0.0000 0.9999 3:1 41.25:13.75 40:15 0.0546 0.8153 BL5 BLWM1 10:6 32.50:19.50 38:14 2.0513 0.1520 46:18 37.38:10.51 38:14 0.0015 0.9691 3:1 39:13 38:14 0.0256 0.8727 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families WM2, WM5, WL1, WL4, WL6, BM2 and BL6 F1 Parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents WM and BL Exp1: Expected ratio of green:albino seedlings ; bold type indicates th at the data are not significantly different from the ratio (p > 0.05) Exp2: Expected number of green:albino seedlings # Obs: Observed number of green:albino seedlings 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 138

121 Table 6.4. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism and derived from the initial cros s/reciprocal set WSBL (genotypes P1P2aaBb x P1P2AaBB ). Progeny were tested against a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. F2 F1 Parent Exp1 Exp2 Obs# 2 P F2 families WSBL 2:1 16.67:8.33 22:3 4.2036 0.0403 WS1 WSBL1 10:6 42.50:25.50 54:14 7.5922 0.0058 46:18 48.88:19.13 54:14 1.5561 0.2122 3:1 51:17 54:14 0.4902 0.4838 WS2 WSBL3 10:6 54.38:32.63 63:24 3.2375 0.0719 46:18 62.53:24.47 63:24 0.0000 0.9999 3:1 65.25:21.75 63:24 0.1877 0.6648 WS3 WSBL5 10:6 34.38:20.63 40:15 2.0376 0.1534 46:18 39.53:15.47 40:15 0.0000 0.9999 3:1 41.25:13.75 40:15 0.0546 0.8153 WS4 BLWS34 10:6 26.88:16.13 30:13 0.6837 0.4083 46:18 30.91:12.09 30:13 0.0190 0.8903 3:1 32.25:10.75 30:13 0.3798 0.5376 WS6 BLWS54 10:6 17.50:10.50 19:9 0.1524 0.6962 46:18 20.13:7.88 19:9 0.0690 0.7927 3:1 21:7 19:9 0.4286 0.5126 WL1 WSBL4 10:6 43.13:25.88 51:18 3.3633 0.0666 46:18 49.59:19.41 51:18 0.0589 0.8082 3:1 51.75:17.25 51:18 0.0048 0.9445 WL2 WSBL7 10:6 23.75:14.25 27:11 0.8491 0.3568 46:18 27.31:10.69 27:11 0.0000 0.9999 3:1 28.5:9.5 27:11 0.1404 0.7079 WL4 BLWS24 10:6 53.75:32.25 60:26 1.6403 0.2002 46:18 61.81:24.19 60:26 0.0991 0.7529 3:1 64.5:21.5 60:26 0.9923 0.3191 WL5 BLWS57 10:6 45.00:27.00 57:15 7.8370 0.0051 46:18 51.75:20.25 57:15 1.5502 0.2131 3:1 54:18 57:15 0.4630 0.4962 WL6 BLWS59 10:6 43.13:25.88 52:17 4.3372 0.0372 46:18 49.59:19.41 52:17 0.2605 0.6097 3:1 51.75:17.25 52:17 0.0000 0.9999 BS1 WSBL6 10:6 65.63:39.38 76:29 3.9625 0.0465 46:18 75.47:29.53 76:29 0.0001 0.9920 3:1 78.75:26.25 76:29 0.2571 0.6120 BS2 WSBL3 10:6 48.75:29.25 63:15 10.342 0.0013 46:18 56.06:21.94 63:15 2.6283 0.1049 3:1 58.5:19.5 63:15 1.0940 0.2955

PAGE 139

122 Table 6.4. Continued F2 F1 Parent Exp1 Exp2 Obs 2 P BS3 BLWS12 10:6 64.38:38.63 72:31 2.1029 0.1470 46:18 74.03:28.97 72:31 0.1126 0.7372 3:1 77.25:25.75 72:31 1.1683 0.2797 BS4 WSBL5 10:6 6.88:4.13 8:3 0.1515 0.6971 46:18 7.91:3.09 8:3 0.0000 0.9999 3:1 8.25:2.75 8:3 0.0000 0.9999 BS5 BLWS34 10:6 31.88:19.13 39:12 3.6719 0.0553 46:18 36.66:14.34 39:12 0.3297 0.5658 3:1 38.25:12.75 39:12 0.0065 0.9355 BS6 BLWS54 10:6 35.63:21.38 47:10 8.8526 0.0029 46:18 40.97:16.03 47:10 2.6552 0.1032 3:1 42.75:14.25 47:10 1.3158 0.2513 BS7 BLWS55 10:6 39.38:23.63 52:11 9.9566 0.0016 46:18 45.28:17.72 52:11 3.0367 0.0814 3:1 47.25:15.75 52:11 1.5291 0.2162 BL1 BLWS22 10:6 57.50:34.50 67:25 3.7565 0.0526 46:18 66.13:25.88 67:25 0.0076 0.9305 3:1 69.00:23.00 67:25 0.1304 0.7179 BL2 BLWS37 10:6 56.88:34.13 74:17 12.959 0.0003 46:18 65.41:25.59 74:17 3.5611 0.0591 3:1 68.25:22.75 74:17 1.6154 0.2037 BL3 BLWS39 10:6 63.75:38.25 80:22 10.377 0.0012 46:18 73.31:28.69 80:22 1.8568 0.1729 3:1 76.5:25.5 80:22 0.4706 0.4927 BL4 BLWS43 10:6 70.63:42.38 72:41 0.0289 0.8650 46:18 81.22:31.78 72:41 2.3919 0.1219 3:1 84.75:28.25 72:41 7.0826 0.0077 BL5 BLWS49 10:6 51.88:31.13 62:21 4.7622 0.0290 46:18 59.66:23.34 62:21 0.2026 0.6526 3:1 62.25:20.75 62:21 0.0000 0.9999 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families WS5, WL3, BL6 F1 Parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents WS and BL Exp1: Expected ratio of green:albino seedlings ; bold type indicates th at the data are not significantly different from the ratio (p > 0.05) Exp2: Expected number of green:albino seedlings # Obs: Observed number of green:albino seedlings

PAGE 140

123 Table 6.4. Continued 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 141

124 Table 6.5. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism and derived from the initial cros s/reciprocal set WSBM (genotypes P1P2aaBb x P1P1aaBB or P1P1AAbb or P1P1AABB ). Progeny were tested against a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. F2 F1 Parent Exp1 Exp2 Obs# 2 P F2 families WSBM 1:1 11.5:11.5 12:11 0.0000 0.9999 WS3 BMWS37 10:6 35.0:21.0 38:18 0.4762 0.4901 46:18 40.25:15.75 38:18 0.2705 0.6029 3:1 42:14 38:18 1.1667 0.2800 WS5 WSBM44 10:6 48.13:28.88 45:32 0.3818 0.5366 46:18 55.34:21.66 45:32 6.2253 0.0125 3:1 57.75:19.25 45:32 10.394 0.0012 WS6 WSBM51 10:6 56.25:33.75 65:25 3.2267 0.0724 46:18 64.69:25.31 65:25 0.0000 0.9999 3:1 67.5:22.5 65:25 0.2370 0.6263 WM1 WSBM13 10:6 6.25:3.75 6:4 0.0000 0.9999 46:18 7.19:2.81 6:4 0.2338 0.6287 3:1 7.5:2.5 6:4 0.5333 0.4652 WM2 WSBM26 10:6 37.50:22.50 41:19 0.6400 0.4237 46:18 43.13:16.88 41:19 0.2177 0.6407 3:1 45:15 41:19 1.0889 0.2967 WM3 BMWS85 10:6 21.25:12.75 22:12 0.0078 0.9296 46:18 24.44:9.56 22:12 0.5462 0.4598 3:1 25.5:8.5 22:12 1.4118 0.2347 WL2 BMWS41 10:6 9.38:5.63 11:4 0.3600 0.5485 46:18 10.78:4.22 11:4 0.0000 0.9999 3:1 11.25:3.75 11:4 0.0000 0.9999 BS5 WSBM21 10:6 13.13:7.88 10:11 1.4000 0.2367 46:18 15.09:5.91 10:11 4.9710 0.0257 3:1 15.75:5.25 10:11 7.0000 0.0081 BS6 WSBM41 10:6 63.75:38.25 75:27 4.8340 0.0279 46:18 73.31:28.69 75:27 0.0684 0.7936 3:1 76.5:25.5 75:27 0.0523 0.8191 BM2 WSBM45 10:6 90.63:54.38 96:49 0.6993 0.4030 46:18 27.31:10.69 96:49 2.0326 0.1539 3:1 108.75:36.25 96:49 5.5195 0.0188 BL1 BMWS33 10:6 23.75:14.25 26:12 0.3439 0.5575 46:18 27.31:10.69 26:12 0.0859 0.7694 3:1 28.5:9.5 26:12 0.5614 0.4536

PAGE 142

125 Table 6.5. Continued F2 F1 Parent Exp1 Exp2 Obs 2 P BL2 WSBM32 10:6 25:15 33:7 6.0000 0.0143 46:18 28.75:11.25 33:7 1.7391 0.1872 3:1 30:10 33:7 0.8333 0.3613 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families WS1, WS2, WS4, WS7, WL1, WL3, BS1, BS2, BS3, BS4, BM1 F1 Parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents WS and BM Exp1: Expected ratio of green:albino seedlings ; bold type indicates th at the data are not significantly different from the ratio (p > 0.05) Exp2: Expected number of green:albino seedlings # Obs: Observed number of green:albino seedlings 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 143

126 Table 6.6. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism and derived from the initial cros s/reciprocal set WMBS (genotypes P1P2AaBb x P1P1aaBB or P1P1AAbb or P1P1AABB ). Progeny were tested against a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. F2 F1 Parent Exp1 Exp2 Obs# 2 P F2 families WMBS 1:1 7.5:7.5 9:6 0.2667 0.6055 BS1 WMBS13 10:6 83.13:49.88 101:32 9.6847 0.0018 46:18 95.59:37.41 101:32 0.8953 0.3440 3:1 99.75:33.25 101:32 0.0226 0.8806 BS3 BSWM3 10:6 23.13:13.88 31:6 6.2721 0.0122 46:18 26.59:10.41 31:6 2.0401 0.1531 3:1 27.75:9.25 31:6 1.0901 0.2964 BS6 WMBS47 10:6 13.13:7.88 18:3 3.8889 0.0486 46:18 15.09:5.91 18:3 1.3639 0.2428 3:1 15.75:5.25 18:3 0.7778 0.3778 BS7 BSWM23 10:6 48.13:28.88 57:20 3.8866 0.0486 46:18 55.34:21.66 57:20 0.0859 0.7694 3:1 57.75:19.25 57:20 0.0043 0.9475 BM1 WMBS28 10:6 88.13:52.88 115:26 21.050 0.0000 46:18 101.34:39.66 115:26 6.0726 0.0137 3:1 105.75:35.25 115:26 2.8960 0.0888 BM3 BSWM16 10:6 31.88:19.13 38:13 2.6471 0.1037 46:18 36.66:14.34 38:13 0.0691 0.7926 3:1 38.25:12.75 38:13 0.0000 0.9999 BM4 BSWM19 10:6 88.75:53.25 107:35 9.4667 0.0020 46:18 102.06:39.94 107:35 0.6860 0.4075 3:1 106.5:35.5 107:35 0.0000 0.9999 BM6 WMBS24 10:6 27.50:16.50 32:12 1.5515 0.2129 46:18 31.63:12.38 32:12 0.0000 0.9999 3:1 33:11 32:12 0.0303 0.8618 BM8 WMBS82 10:6 28.75:17.25 34:12 2.0928 0.1479 46:18 33.06:12.94 34:12 0.0206 0.8858 3:1 34.5:11.5 34:12 0.0000 0.9999 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families BS2, BS4, BS5, BM2, BM5, BM7 F1 Parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents WM and BS Exp1: Expected ratio of green:albino seedlings ; bold type indicates th at the data are not significantly different from the ratio (p > 0.05)

PAGE 144

127 Table 6.6. Continued # Obs: Observed number of green:albino seedlings 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 145

128 Table 6.7. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism and derived from the initial cros s/reciprocal set BSBL (genotypes P1P1aaBB or P1P1AAbb or P1P1AABB x P1P2AaBB ). Progeny were tested against a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. F2 F1 Parent Exp1 Exp2 Obs# 2 P F2 families BSBL 1:1 6:6 5:7 0.0833 0.7728 UVS2 BLBS33 46:18 107.81:42.19 103:47 0.6133 0.4335 3:1 112.5:37.5 103:47 2.8800 0.0896 BS1 BLBS15 46:18 92:36 100:28 2.1739 0.1403 3:1 96:32 100:28 0.5104 0.4749 BS2 BLBS32 46:18 95.59:37.41 103:30 1.7740 0.1828 3:1 99.75:33.25 103:30 0.3033 0.5818 UVM2 BLBS19 46:18 110.69:43.31 121:33 3.0929 0.0786 3:1 115.5:38.5 121:33 0.8658 0.3521 BM2 BLBS11 46:18 103.5:40.5 115:29 4.1567 0.0414 3:1 108:36 115:29 1.5648 0.2109 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families VarBS, UVS3, BS3, UVM1, UVM3, BM1, BM3 F1 Parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents BS and BL Exp1: Expected ratio of green:albino seedlings ; bold type indicates th at the data are not significantly different from the ratio (p > 0.05) Exp2: Expected number of green:albino seedlings # Obs: Observed number of green:albino seedlings 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 146

129 Table 6.8. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism and derived from the initial cros s/reciprocal set BMBL (genotypes P1P1aaBB or P1P1AAbb or P1P1AABB x P1P2AaBB ). Progeny were tested against a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. F2 F1 Parent Exp1 Exp2 Obs# 2 P F2 families BMBL 1:1 10:10 11:9 0.0500 0.8230 WL1 BMBL34 46:18 29.47:11.53 26:15 1.0634 0.3024 3:1 30.75:10.25 26:15 2.3496 0.1253 WL4 BLBM13 46:18 38.81:15.19 37:17 0.1578 0.6911 3:1 40.50:13.50 37:17 0.8889 0.3457 BM1 BMBL43 46:18 122.91:48.09 117:54 0.8455 0.3578 3:1 128.25:42.75 117:54 3.6043 0.0576 BM2 BMBL56 46:18 56.78:22.22 58:21 0.0323 0.8573 3:1 59.25:19.75 58:21 0.0380 0.8455 BM5 BLBM4 46:18 82.66:32.34 87:28 0.6355 0.4253 3:1 86.25:28.75 87:28 0.0029 0.9570 BM6 BLBM18 46:18 63.97:25.03 75:14 6.1645 0.0130 3:1 66.75:22.25 75:14 3.5993 0.0578 BL2 BMBL49 46:18 64.69:25.31 64:26 0.0019 0.9652 3:1 67.5:22.5 64:26 0.5333 0.4652 BL4 BLBM1 46:18 37.338:14.63 39:13 0.1204 0.7286 3:1 39:13 39:13 0.0000 0.9999 BL5 BLBM5 46:18 22.28:8.72 24:7 0.2370 0.6263 3:1 23.25:7.75 24:7 0.0108 0.9174 BL6 BLBM7 46:18 22.28:8.72 22:9 0.0000 0.9999 3:1 23.25:7.75 22:9 0.0968 0.7557 BL8 BLBM11 46:18 44.56:17.44 49:13 1.2370 0.2660 3:1 46.5:15.5 49:13 0.3441 0.5574 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families WM1, WL2, WL3, BM3, BM4, BL1, BL3, BL7, BL9 F1 Parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents BM and BL Exp1: Expected ratio of green:albino seedlings ; bold type indicates th at the data are not significantly different from the ratio (p > 0.05) Exp2: Expected number of green:albino seedlings # Obs: Observed number of green:albino seedlings 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 147

130 Figure 6.1. Albino seedling of pickerelweed.

PAGE 148

131 WM x WS P1aB P1ab P2aB P2ab P1AB P1P1AaBB (F2 = NS) P1P1AaBb (F2 = NS) P1P2AaBB (F2 = 3:1) P1P2AaBb (F2 = 46:18) P1Ab P1P1AaBb (F2 = NS) P1P1Aabb (F2 = NS) P1P2AaBb (F2 = 46:18) P1P2Aabb (F2 = 10:6) P1aB P1P1aaBB (F2 = NS) P1P1aaBb (F2 = NS) P1P2aaBB (F2 = 3:1) P1P2aaBb (F2 = 10:6) P1ab P1P1aaBb (F2 = NS) P1P1aabb (F2 = NS) P1P2aaBb (F2 = 10:6) P1P2aabb P2AB P1P2AaBB (F2 = 3:1) P1P2AaBb (F2 = 46:18) P2P2AaBB P2P2AaBb P2Ab P1P2AaBb (F2 = 46:18) P1P2Aabb (F2 = 10:6) P2P2AaBb P2P2Aabb P2aB P1P2aaBB (F2 = 3:1) P1P2aaBb (F2 = 10:6) P2P2aaBB P2P2aaBb P2ab P1P2aaBb (F2 = 10:6) P1P2aabb P2P2aaBb P2P2aabb Figure 6.2. Punnett square of expe cted albinism genotypes of F1 populations of pickerelweed derived from the initial cross/reciprocal set WMWS (genotypes P1P2AaBb and P1P2aaBb ) and expected segregation of F2 progeny for albinism. No maternal effects were evid ent; therefore, only the Punnett square for the cross is shown. Expected segreg ation ratios based a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Gr een seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. Expected segregation ratio of F1 progeny is 22:10. Parental gametes are represented in the extreme upper a nd left cells, while genotypes of F1 progeny comprise the body of the Punnett square Expected segregation ratios for F2 generations derived from F1 progeny are given under the genotype of the F1 individual producing the F2 generation. Shaded cells within the Punnett square identify albino F1 progeny. NS = no segregation (all green).

PAGE 149

132 WM x BL P1AB P1aB P2AB P2aB P1AB P1P1AABB (F2 = NS) P1P1AaBB (F2 = NS) P1P2AABB (F2 = 3:1) P1P2AaBB (F2 = 3:1) P1Ab P1P1AABb (F2 = NS) P1P1AaBb (F2 = NS) P1P2AABb (F2 = 3:1) P1P2AaBb (F2 = 46:18) P1aB P1P1AaBB (F2 = NS) P1P1aaBB (F2 = NS) P1P2AaBB (F2 = 3:1) P1P2aaBB (F2 = 3:1) P1ab P1P1AaBb (F2 = NS) P1P1aaBb (F2 = NS) P1P2AaBb (F2 = 46:18) P1P2aaBb (F2 = 10:6) P2AB P1P2AABB (F2 = 3:1) P1P2AaBB (F2 = 3:1) P2P2AABB P2P2AaBB P2Ab P1P2AABb (F2 = 3:1) P1P2AaBb (F2 = 46:18) P2P2AABb P2P2AaBb P2aB P1P2AaBB (F2 = 3:1) P1P2aaBB (F2 = 3:1) P2P2AaBB P2P2aaBB P2ab P1P2AaBb (F2 = 46:18) P1P2aaBb (F2 = 10:6) P2P2AaBb P2P2aaBb Figure 6.3. Punnett square of expe cted albinism genotypes of F1 populations of pickerelweed derived from the initial cross/reciprocal set WMBL (genotypes P1P2AaBb and P1P2AaBB ) and expected segregation of F2 progeny for albinism. No maternal effects were evid ent; therefore, only the Punnett square for the cross is shown. Expected segreg ation ratios based a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Gr een seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. Expected segregation ratio of F1 progeny is 24:8 (3:1). Parental gametes are represented in the extreme upper a nd left cells, while genotypes of F1 progeny comprise the body of the Punnett square Expected segregation ratios for F2 generations derived from F1 progeny are given under the genotype of the F1 individual producing the F2 generation. Shaded cells within the Punnett square identify albino F1 progeny. NS = no segregation (all green).

PAGE 150

133 WS x BL P1AB P1aB P2AB P2aB P1aB P1P1AaBB (F2 = NS) P1P1aaBB (F2 = NS) P1P2AaBB (F2 = 3:1) P1P2aaBB (F2 = 3:1) P1ab P1P1AaBb (F2 = NS) P1P1aaBb (F2 = NS) P1P2AaBb (F2 = 46:18) P1P2aaBb (F2 = 10:6) P2aB P1P2AaBB (F2 = 3:1) P1P2aaBB (F2 = 3:1) P2P2AaBB P2P2aaBB P2ab P1P2AaBb (F2 = 46:18) P1P2aaBb (F2 = 10:6) P2P2AaBb P2P2aaBb Figure 6.4. Punnett square of expe cted albinism genotypes of F1 populations of pickerelweed derived from the initial cr oss/reciprocal set WSBL (genotypes P1P2aaBb and P1P2AaBB ) and expected segregation of F2 progeny for albinism. No maternal effects were evid ent; therefore, only the Punnett square for the cross is shown. Expected segreg ation ratios based a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Gr een seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. Expected segregation ratio of F1 progeny is 12:4 (3:1). Parental gametes are represented in the extreme upper a nd left cells, while genotypes of F1 progeny comprise the body of the Punnett square Expected segregation ratios for F2 generations derived from F1 progeny are given under the genotype of the F1 individual producing the F2 generation. Shaded cells within the Punnett square identify albino F1 progeny. NS = no segregation (all green).

PAGE 151

134 WS x BM P1aB or P1Ab or P1AB or P1ab P1aB P1P1aaBB (F2 = NS) P1P1AaBb (F2 = NS) P1P1AaBB (F2 = NS) P1P1aaBb (F2 = NS) P1ab P1P1aaBb (F2 = NS) P1P1Aabb (F2 = NS) P1P1AaBb (F2 = NS) P1P1aabb (F2 = NS) P2aB P1P2aaBB (F2 = 3:1) P1P2AaBb (F2 = 46:18) P1P2AaBB (F2 = 3:1) P1P2aaBb (F2 = 10:6) P2ab P1P2aaBb (F2 = 10:6) P1P2Aabb (F2 = 10:6) P1P2AaBb (F2 = 46:18) P1P2aabb Figure 6.5. Punnett square of expe cted albinism genotypes of F1 populations of pickerelweed derived from the initial cross/reciprocal set WSBM (genotypes P1P2aaBb and P1P1aaBB or P1P1AAbb or P1P1AABB or P1P1aabb ) and expected segregation of F2 progeny for albinism. No maternal effects were evident; therefore, only the Punnett square for the cross is shown. Expected segregation ratios based a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albi nism. Expected segregation ratios of F1 progeny differ and are dependent on the genotype of BM. Parental gametes are represented in the extrem e upper and left cells, while genotypes of F1 progeny comprise the body of the P unnett square. Expected segregation ratios for F2 generations derived from F1 progeny are given under the genotype of the F1 individual producing the F2 generation. Shaded cells within the Punnett square identify albino F1 progeny. NS = no segregation (all green).

PAGE 152

135 WM x BS P1aB or P1Ab or P1AB or P1ab P1AB P1P1AaBB (F2 = NS) P1P1AABb (F2 = NS) P1P1AABB (F2 = NS) P1P1AaBb (F2 = NS) P1Ab P1P1AaBb (F2 = NS) P1P1AAbb (F2 = NS) P1P1AABb (F2 = NS) P1P1Aabb (F2 = NS) P1aB P1P1aaBB (F2 = NS) P1P1AaBb (F2 = NS) P1P1AaBB (F2 = NS) P1P1aaBb (F2 = NS) P1ab P1P1aaBb (F2 = NS) P1P1Aabb (F2 = NS) P1P1AaBb (F2 = NS) P1P1aabb (F2 = NS) P2AB P1P2AaBB (F2 = 3:1) P1P2AABb (F2 = 3:1) P1P2AABB (F2 = 3:1) P1P2AaBb (F2 = 46:18) P2Ab P1P2AaBb (F2 = 46:18) P1P2AAbb (F2 = 3:1) P1P2AABb (F2 = 3:1) P1P2Aabb (F2 = 10:6) P2aB P1P2aaBB (F2 = 3:1) P1P2AaBb (F2 = 46:18) P1P2AaBB (F2 = 3:1) P1P2aaBb (F2 = 10:6) P2ab P1P2aaBb (F2 = 10:6) P1P2Aabb (F2 = 10:6) P1P2AaBb (F2 = 46:18) P1P2aabb Figure 6.6. Punnett square of expe cted albinism genotypes of F1 populations of pickerelweed derived from the initial cross/reciprocal set WMBS (genotypes P1P2AaBb and P1P1aaBB or P1P1AAbb or P1P1AABB or P1P1aabb ) and expected segregation of F2 progeny for albinism. No maternal effects were evident; therefore, only the Punnett square for the cross is shown. Expected segregation ratios based a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albi nism. Expected segregation ratios of F1 progeny differ and are dependent on the genotype of BS. Parental gametes are represented in the extrem e upper and left cells, while genotypes of F1 progeny comprise the body of the P unnett square. Expected segregation ratios for F2 generations derived from F1 progeny are given under the genotype of the F1 individual producing the F2 generation. Shaded cells within the Punnett square identify albino F1 progeny. NS = no segregation (all green).

PAGE 153

136 BS x BM P1aB or P1Ab or P1AB or P1ab P1aB P1P1aaBB (F2 = NS) P1P1AaBb (F2 = NS) P1P1AaBB (F2 = NS) P1P1aaBb (F2 = NS) or P1Ab P1P1AaBb (F2 = NS) P1P1AAbb (F2 = NS) P1P1AABb (F2 = NS) P1P1Aabb (F2 = NS) or P1AB P1P1AaBB (F2 = NS) P1P1AABb (F2 = NS) P1P1AABB (F2 = NS) P1P1AaBb (F2 = NS) or P1ab P1P1aaBb (F2 = NS) P1P1Aabb (F2 = NS) P1P1AaBb (F2 = NS) P1P1aabb (F2 = NS) Figure 6.7. Punnett square of expe cted albinism genotypes of F1 populations of pickerelweed derived from the initial cr oss/reciprocal set BSBM (genotypes P1P1aaBB or P1P1AAbb or P1P1AABB or P1P1aabb and P1P1aaBB or P1P1AAbb or P1P1AABB or P1P1aabb ) and expected segregation of F2 progeny for albinism. No maternal effects were evident; therefore, only the Punnett square for the cross is shown. Expected segregation ratios based a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. No segregation of F1 progeny is expected. Parental gametes are represented in the extreme upper a nd left cells, while genotypes of F1 progeny comprise the body of the Punnett square Expected segregation ratios for F2 generations derived from F1 progeny are given under the genotype of the F1 individual producing the F2 generation. Shaded cells within the Punnett square identify albino F1 progeny. NS = no segregation (all green).

PAGE 154

137 BS x BL P1AB P1aB P2AB P2aB P1aB P1P1AaBB (F2 = NS) P1P1aaBB (F2 = NS) P1P2AaBB (F2 = 3:1) P1P2aaBB (F2 = 3:1) or P1Ab P1P1AABb (F2 = NS) P1P1AaBb (F2 = NS) P1P2AABb (F2 = 3:1) P1P2AaBb (F2= 46:18) or P1AB P1P1AABB (F2 = NS) P1P1AaBB (F2 = NS) P1P2AABB (F2 = 3:1) P1P2AaBB (F2 = 3:1) or P1ab P1P1AaBb (F2 = NS) P1P1aaBb (F2 = NS) P1P2AaBb (F2 = 46:18) P1P2aaBb (F2 = 10:6) Figure 6.8. Punnett square of expe cted albinism genotypes of F1 populations of pickerelweed derived from the initial cr oss/reciprocal set BSBL (genotypes P1P1aaBB or P1P1AAbb or P1P1AABB or P1P1aabb and P1P2AaBB ) and expected segregation of F2 progeny for albinism. No maternal effects were evident; therefore, only the Punnett square for the cross is shown. Expected segregation ratios based a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. No segregation of F1 progeny is expected. Parental gamete s are represented in the extreme upper and left cells, while genotypes of F1 progeny comprise the body of the Punnett square. Expected segregation ratios for F2 generations derived from F1 progeny are given under the genotype of the F1 individual producing the F2 generation. Shaded ce lls within the Punnett square identify albino F1 progeny. NS = no segregation (all green).

PAGE 155

138 BM x BL P1AB P1aB P2AB P2aB P1aB P1P1AaBB (F2 = NS) P1P1aaBB (F2 = NS) P1P2AaBB (F2 = 3:1) P1P2aaBB (F2 = 3:1) or P1Ab P1P1AABb (F2 = NS) P1P1AaBb (F2 = NS) P1P2AABb (F2 = 3:1) P1P2AaBb (F2= 46:18) or P1AB P1P1AABB (F2 = NS) P1P1AaBB (F2 = NS) P1P2AABB (F2 = 3:1) P1P2AaBB (F2 = 3:1) or P1ab P1P1AaBb (F2 = NS) P1P1aaBb (F2 = NS) P1P2AaBb (F2 = 46:18) P1P2aaBb (F2 = 10:6) Figure 6.9. Punnett square of expe cted albinism genotypes of F1 populations of pickerelweed derived from the initial cross/reciprocal set BMBL (genotypes P1P1aaBB or P1P1AAbb or P1P1AABB or P1P1aabb and P1P2AaBB ) and expected segregation of F2 progeny for albinism. No maternal effects were evident; therefore, only the Punnett square for the cross is shown. Expected segregation ratios based a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. No segregation of F1 progeny is expected. Parental gamete s are represented in the extreme upper and left cells, while genotypes of F1 progeny comprise the body of the Punnett square. Expected segregation ratios for F2 generations derived from F1 progeny are given under the genotype of the F1 individual producing the F2 generation. Shaded ce lls within the Punnett square identify albino F1 progeny. NS = no segregation (all green).

PAGE 156

139 WS P1aB P1ab P2aB P2ab P1aB P1P1aaBB P1P1aaBb P1P2aaBB P1P2aaBb P1ab P1P1aaBb P1P1aabb P1P2aaBb P1P2aabb P2aB P1P2aaBB P1P2aaBb P2P2aaBB P2P2aaBb P2ab P1P2aaBb P1P2aabb P2P2aaBb P2P2aabb Figure 6.10. Punnett square of expe cted albinism genotypes of S1 progeny of pickerelweed from the parent WS (genotype P1P2aaBb ). Expected segregation ratios based a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. Expected segregation ratio of S1 progeny is 10:6. Parental gamete s are represented in the extreme upper and left cells, while genotypes of S1 progeny comprise the body of the Punnett square. Shaded cells within the Punnett square identify albino progeny.

PAGE 157

140 WM P 1AB P 1Ab P 1aB P 1ab P 2AB P 2Ab P 2aB P 2ab P 1AB P 1P1AABB P 1P1AABb P 1P1AaBB P 1P1AaBb P 1P2AABB P 1P2AABb P 1P2AaBB P 1P2AaBb P 1Ab P 1P1AABb P 1P1AAbb P 1P1AaBb P 1P1Aabb P 1P2AABb P 1P2AAbb P 1P2AaBb P 1P2Aabb P 1aB P 1P1AaBB P 1P1AaBb P 1P1aaBB P 1P1aaBb P 1P2AaBB P 1P2AaBb P 1P2aaBB P 1P2aaBb P 1ab P 1P1AaBb P 1P1Aabb P 1P1aaBb P 1P1aabb P 1P2AaBb P 1P2Aabb P 1P2aaBb P 1P2aabb P 2AB P 1P2AABB P 1P2AABb P 1P2AaBB P 1P2AaBb P 2P2AABB P 2P2AABb P 2P2AaBB P 2P2AaBb P 2Ab P 1P2AABb P 1P2AAbb P 1P2AaBb P 1P2Aabb P 2P2AABb P 2P2AAbb P 2P2AaBb P 2P2Aabb P 2aB P 1P2AaBB P 1P2AaBb P 1P2aaBB P 1P2aaBb P 2P2AaBB P 2P2AaBb P 2P2aaBB P 2P2aaBb P 2ab P 1P2AaBb P 1P2Aabb P 1P2aaBb P 1P2aabb P 2P2AaBb P 2P2Aabb P 2P2aaBb P 2P2aabb Figure 6.11. Punnett square of expe cted albinism genotypes of S1 progeny of pickerelweed from the parent WM (genotype P1P2AaBb ). Expected segregation ratios based a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. Expected segregation ratio of S1 progeny is 46:18. Parental gamete s are represented in the extreme upper and left cells, while genotypes of S1 progeny comprise the body of the Punnett square. Shaded cells within the Punnett square identify albino progeny.

PAGE 158

141 BS P1aB or P1Ab or P1AB or P1ab P1aB P1P1aaBB or P1Ab P1P1AAbb or P1AB P1P1AABB or P1ab P1P1aabb Figure 6.12. Punnett square of expe cted albinism genotypes of S1 progeny of pickerelweed from the parent BS (genotype P1P1aaBB P1P1AAbb P1P1AABB or P1P1aabb ). Expected segregation ra tios based a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. No segregation of S1 progeny is expected. Parental gametes are represented in the extreme upper a nd left cells, while genotypes of S1 progeny comprise the body of the Punnett square.

PAGE 159

142 BM P1aB or P1Ab or P1AB or P1ab P1aB P1P1aaBB or P1Ab P1P1AAbb or P1AB P1P1AABB or P1ab P1P1aabb Figure 6.13. Punnett square of expe cted albinism genotypes of S1 progeny of pickerelweed from the parent BM (genotype P1P1aaBB P1P1AAbb P1P1AABB or P1P1aabb ). Expected segregation ra tios based a model with three diallelic loci (epistatic locus P with alleles P1 and P2, hypostatic duplicate loci A and B with dominant gene action). Green seedlings have the genotype P1P1_ _ _, P1P2A _ or P1P2_ B_ ; all other genotypes result in albinism. No segregation of S1 progeny is expected. Parental gametes are represented in the extreme upper a nd left cells, while genotypes of S1 progeny comprise the body of the Punnett square.

PAGE 160

143 CHAPTER 7 INHERITANCE AND GENETIC C ONTROL OF FLOWER COLOR Introduction Pickerelweed ( Pontederia cordata L.) is an attractive di ploid (2n=2x=16) shoreline aquatic species that is frequently used in ornamental aquascapes. Wild-type plants of pickerelweed produce blue or vi olet flowers, but plants with white flowers are sometimes seen in nature (Figure 7.1) (Godfrey and Wooten 1979). While rare in nature, white-flowered specimens of pickerelweed are much easie r to locate in a retail environment; a simple search of the Worl d Wide Web reveals that white-flowered specimens of pickerelweed are readily availabl e from the many nurseries that sell aquatic plants, including The Water Garden 1, Arizona Aquatic Gardens 2 and The Water Garden Shop 3 Durbin et al. (2003) stated that variations in flower color are often transmitted in a simple Mendelian manner. Control of flower color by a single dial lelic locus has been described in several species, including st okes aster (Gaus et al. 2003), morning-glory (Zufall and Rausher 2003), crimson clover (M osjidis 2000), and gua yule (Estilai 1984). Genetic control of flower color is more complicated in other species; for example, 1 http://store.watergardenweb.com/whitpicrus.html Note: Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the product by the Florida Agricultural Experiment Station and does not imply its approval to the exclusion of other products that may be suitable. 2 http://www.azgardens.com/winter_hardy_bog_plants.php 3 http://www.thewatergardenshop.com/poncoralwhit.html

PAGE 161

144 Brewbaker (1962) reported that red flower co lor in white clover resulted from the presence of recessive alleles at two loci. Gr iesbach (1996) found that flower color in the common petunia was influenced by two loci th at were responsible for the production of anthocyanins and by two additional lo ci that controlled vacuole pH. Intraspecific differences in flower color are often associated with a change in the regulation of gene expression as opposed to mu tations in structural genes (Durbin et al. 2003). Zufall and Rausher (2003) found that th e recessive (pink) flower color in morning-glory resulted from an insertion in the gene encoding the anthocyanin biosynthetic gene responsible for expression of the dominant (purple) flower color; this insertion caused the production of a transcript that was shorte r than normal and generated a non-functional enzyme. There is no published information describing the inheritance and genetic control of flower color in pickerelweed. The objective of this experiment was to determine the type of gene action and number of loci contro lling flower color in this population of pickerelweed. Materials and Methods The population utilized in this experime nt was developed using the strategy described in Appendix A of this dissertation. Eight F1 families and five S1 families were examined in this experiment. Each F1 or S1 plant was grown to re productive maturity and evaluated for flower color. No maternal eff ects were noted; therefore, data presented for each F1 family were pooled within each cross/reciprocal set. Data from F1 and S1 populations were used to develop a working model to explain the type of gene action and number of loci controlling flower co lor in this population of pickerelweed. Development of this model allowed the assignment of genotypes to parents; the model

PAGE 162

145 was then verified by analyses of F2 populations. All data were analyzed using goodness-of-fit (chi-square or 2) tests with Yates correction for continuity. Results and Discussion Four F1 families and two S1 families segregated for flower color. One F1 family and two S1 families produced only white-fl owered progeny, while three F1 families and one S1 family had only blue-flowered progeny (Table 7.1). The F1 family that yielded only white-flowered progeny was developed usi ng both white-flowered parents (WS and WM); each S1 family from these parents comprised only white-flowered progeny as well. The S1 family from the parent BS had only blue fl owers; this parent also played a role in the development of all three F1 families with only blue-flowered progeny. The simplest model that yielded the proge ny types produced in all F1 and S1 families was a model with one diallelic locus ( W ) with completely dominant gene action so that individuals with heterozygous or homozygous dominant genotyp ic constitutions pr oduced blue flowers and homozygous recessive plants yielded white flowers. Genotypes were assigned to all five parents based on the proposed model and segregation of F1 progeny. Based on these data, the white-flowered parents WS and WM were homozygous recessive ( ww ), the blue-flowered parent BS was homozygous dominant ( WW ), and BM and BL were heterozygous ( Ww ). Goodness-of-fit values and their associated probabilities for F1 and S1 populations are presented in Table 7.2 and indicated that the model provided a good fit to the data and supported the proposed genotype assi gnments. Seven of the eight F1 families under investigation were advanced to the F2 generation for further study. Goodness-of-fit values and their associated probabilities for F2 families are presented in Tables 7.3 through 7.9 and are sorted by family. Summaries of the an alyses for each family are presented below.

PAGE 163

146 WMWS and WSWM. Parents WM and WS were assigned the genotypes ww and ww respectively. All F1 progeny produced white flowers (Table 7.1). F1 progeny were not advanced to the F2 generation as all F1 progeny were homozygous recessive ( ww ) and generation advancement would not have yielded additional information. The F1 data from this cross/reciprocal set provided evidence that supported the proposed model and genotypic assignments. WMBL and BLWM. Parents WM and BL were assigned the genotypes ww and Ww respectively. The observed F1 progeny segregated in a ma nner that was not different from the expected 1:1 (blue:white) ratio (Tab le 7.2). One of two segregation ratios was expected in the F2 generation based on the phenotype of the F1 plant used to create an F2 family. Blue-flowered F1 plants were expected to produce F2 progeny that segregated in a 3:1 (blue:white) ratio, while white-flowered F1 progeny were expected to produce all white-flowered F2 progeny. A total of 24 F2 families (derived from self-pollination of 12 white-flowered F1 plants and 12 blue-flowered F1 plants) were analyzed. All F2 families from white-flowered F1 plants produced only white-flowered progeny, while all F2 families from blue-flowered F1 plants produced progeny th at segregated in ratios that were not different from 3:1 (Table 7.3). These data provided evidence that supported the proposed model and genotypic assignments. WSBL and BLWS. Parents WS and BL were assigned the genotypes ww and Ww respectively. The observed F1 progeny segregated in a manne r that was not different from the expected 1:1 (blue:white ) ratio (Table 7.2). One of two segregation ratios was expected in the F2 generation based on the phenotype of the F1 plant used to create an F2 family. Blue-flowered F1 plants were expected to produce F2 progeny that segregated

PAGE 164

147 in a 3:1 (blue:white) ratio, while white-flowered F1 progeny were expected to produce all white-flowered F2 progeny. A total of 17 F2 families (derived from self-pollination of 8 white-flowered F1 plants and 9 blue-flowered F1 plants) were analyzed. All F2 families from white-flowered F1 plants produced only white-flow ered progeny. Seven of the nine F2 families from blue-flowered F1 plants produced progeny that segregated in ratios that were not different from 3:1, while two of the F2 families (BL3 and BL4) from blue-flowered F1 plants segregated in ra tios that differed from 3:1 (Table 7.4). As many as 1 in 33 populations of pickerelweed would be expected to show as much variation for flower color as BL3 and as many as 1 in 60 would be expected show as much variation for flower color as BL4; therefore, it is likely that sampling error was responsible for production of these families that did not segr egate as expected. W ith the exception of these discrepancies, these data provided evid ence that supported the proposed model and genotypic assignments. WSBM and BMWS. Parents WS and BM were assigned the genotypes ww and Ww respectively. The observed F1 progeny segregated in a ma nner that was not different from the expected 1:1 (blue:white) ratio (Tab le 7.2). One of two segregation ratios was expected in the F2 generation based on the phenotype of the F1 plant used to create an F2 family. Blue-flowered F1 plants were expected to produce F2 progeny that segregated in a 3:1 (blue:white) ratio, while white-flowered F1 progeny were expected to produce all white-flowered F2 progeny. A total of 23 F2 families (derived from self-pollination of 13 white-flowered F1 plants and 10 blue-flowered F1 plants) were analyzed. All F2 families from white-flowered F1 plants produced only white-flowered progeny. Nine of the ten F2 families from blue-flowered F1 plants produced progeny that segregated in

PAGE 165

148 ratios that were not diffe rent from 3:1, while one F2 family from a blue-flowered F1 plant segregated in a ratio that was different from 3:1 (Table 7.5). As many as 1 in 250 families would be expected to show as much variation for flower color as this family, so it is possible that sampling error was responsible for production of this family that did not segregate as expected. With the exception of this discrepancy, these data provided evidence that supported the proposed model and genotypic assignments. WMBS and BSWM. Parents WM and BS were assigned the genotypes ww and WW respectively. All F1 progeny produced blue flowers (Table 7.1) and all F1 plants were expected to produce F2 progeny that segregated in a 3: 1 (blue:white) ratio. A total of 15 F2 families (derived from self-pollination of 15 F1 plants) were analyzed; all F2 families produced progeny that segregated in ratios that were not different from 3:1 (Table 7.6). These data provided evidence that suppor ted the proposed model and genotypic assignments. BSBM and BMBS. Parents BS and BM were assigned the genotypes WW and Ww respectively. All F1 progeny produced blue flowers (Table 7.1) and F1 plants were expected to produce F2 progeny that either bore only blue flowers or segregated in a 3:1 (blue:white) ratio A total of 12 F2 families (derived from self-pollination of 12 F1 plants) were analyzed. Four F2 families produced only blue flowers, while seven F2 families produced progeny that segregated in ratios that were not different from 3:1 and one F2 family produced progeny that segregated in a ratio that was different from 3:1 (Table 7.7). Only 3 in 1000 popul ations of pickerelweed woul d be expected to show as much variation for flower color as this fam ily but it is possible that sampling error was responsible for the failure of this family to segregate as expected. With the exception of

PAGE 166

149 this discrepancy, these data provided ev idence that supported the proposed model and genotypic assignments. BSBL and BLBS. Parents BS and BL were assigned the genotypes WW and Ww respectively. All F1 progeny produced blue flowers (Table 7.1) and F1 plants were expected to produce F2 progeny that either bore only blue flowers or segregated in a 3:1 (blue:white) ratio A total of 12 F2 families (derived from self-pollination of 12 F1 plants) were analyzed. Eight F2 families produced only blue flowers and four F2 families produced progeny that segregated in ratios that were not different from 3:1 (Table 7.8). These data provided evidence that suppor ted the proposed model and genotypic assignments. BMBL and BLBM. Parents BM and BL were assigned the genotypes Ww and Ww respectively. The observed F1 progeny segregated in a manne r that was not different from the expected 3:1 (blue:white ) ratio (Table 7.2). One of three segregation ratios was expected in the F2 generation based on the phenotype and genotype of the F1 plant self-pollinated to create an F2 family. Blue-flowered F1 plants were expected to produce F2 progeny that either bore onl y blue flowers or that segr egated in a 3:1 (blue:white) ratio, while white-flowered F1 progeny were expected to produce all white-flowered F2 progeny. A total of 20 F2 families (derived from self-pollination of 5 white-flowered F1 plants and 15 blue-flowered F1 plants) were analyzed. All F2 families from white-flowered F1 plants produced only white-flowere d progeny and seven of the fifteen F2 families from blue-flowered F1 plants produced only blue-flowered progeny. Seven of the remaining eight F2 families segregated in ratios that were not different from 3:1, while one F2 family segregated in a ratio that was different from 3:1 (Table 7.9). The

PAGE 167

150 populations examined in this experiment were relatively small, so it is probable that sampling error was responsible for production of this family that did not segregate as expected. With the exception of this disc repancy, these data provided evidence that supported the proposed model and genotypic assignments. WS. The parent WS was assigned the genotype ww and all S1 progeny produced white flowers (Table 7.1); therefore, these S1 data provided eviden ce that supported the proposed model and assignment of the genotype ww to WS. WM. The parent WM was assigned the genotype ww and all S1 progeny produced white flowers (Table 7.1); therefore, these S1 data provided eviden ce that supported the proposed model and assignment of the genotype ww to WM. BS. The parent BS was assigned the genotype WW and all S1 progeny produced blue flowers (Table 7.1); therefore, these S1 data provided eviden ce that supported the proposed model and assignment of the genotype WW to BS. BM. The parent BM was assigned the genotype Ww and S1 progeny segregated in a ratio that was not different from 3:1 (Table 7.2); therefore, these S1 data provided evidence that supported the proposed m odel and assignment of the genotype Ww to BM. BL. The parent BL was assigned the genotype Ww and S1 progeny segregated in a ratio that was not different from 3:1 (Table 7.2); therefore, these S1 data provided evidence that supported the proposed m odel and assignment of the genotype Ww to BL. Conclusions The results of this experiment suggested that flower color in this population of pickerelweed was cont rolled by two alleles ( W and w ) at one locus ( W ); gene action was dominant and white flower color was recessive. This assessment was supported by the work of Durbin et al. (2003), which revealed that variations in fl ower color were often

PAGE 168

151 transmitted in a simple Mendelian manner. All eight F1 families and all five S1 families investigated in this experiment segregated as expected under this model; in addition, 119 of 124 F2 families also segregated as expected. Only five F2 families failed to conform to expected segregation patterns, but these discrepancies were most likely due to sampling error since the vast majority of the data supported the single di allelic locus model. It is possible that flower co lor in pickerelweed is infl uenced by an insertion or deletion in the gene responsible for the producti on of anthocyanins; th is type of system has been described in other species (Zufall and Rausher 2003). It is also possible that flower color in pickerelweed is actually co ntrolled by a more complicated system with multiple loci similar to that found in petuni a (Griesbach 1996); however, this experiment failed to reveal the presence of more than one locus controlling fl ower color in this population of pickerelweed.

PAGE 169

152 Table 7.1. Number of blue-flo wered and white-flowered F1 and S1 progeny of pickerelweed. Family Generation Plants Blue White# WSWM and WMWS F1 98 0 98 WMBL and BLWM F1 57 28 29 WSBL and BLWS F1 67 36 31 WSBM and BMWS F1 144 74 70 WMBS and BSWM F1 103 103 0 BSBM and BMBS F1 132 132 0 BSBL and BLBS F1 49 49 0 BMBL and BLBM F1 91 71 17 WS S1 21 0 21 WM S1 44 0 44 BS S1 9 9 0 BM S1 21 12 9 BL S 1 56 38 18 Family: F1 or S1 family under investigation; F1 families were created through crossand reciprocal pollinations between two parents and F1 codes identify the parents (e.g., the F1 family WSWM was derived from cross-po llination between the parents WS and WM) Generation: Generation of th e family under investigation Plants: Number of plants examined Blue: Number of plan ts with blue flowers # White: Number of plants with white flowers

PAGE 170

153 Table 7.2. Goodness-of-fit tests for F1 and S1 families of pickerel weed segregating for blue and white flower color. Progeny were tested against a model with two alleles ( W and w ) at one locus ( W ); gene action is dominant and white flower color is recessive. Family Parents Exp1 Exp2 Observed# 2 P WMBL ww x Ww 1:1 28.5:28.5 28:29 0.0000 0.9999 WSBL ww x Ww 1:1 33.5:33.5 36:31 0.2388 0.6250 WSBM ww x Ww 1:1 72:72 74:70 0.0625 0.8025 BMBL Ww x Ww 3:1 68.25:22.75 74:17 1.6154 0.2037 BM Ww 3:1 15.75:5.25 12:9 2.6825 0.1014 BL Ww 3:1 42:14 38:18 1.1667 0.2800 Family: F1 or S1 family under investigation; F1 families were created through crossand reciprocal pollinations between two parents and F1 codes identify the parents (e.g., the F1 family WSWM was derived from cross-poll ination between the pa rents WS and WM); not shown: non-segregating familie s WMWS, WMBS, BSBM, BSBL, WS WM BS Exp1: Expected ratio of blue-flo wered progeny to white-flowered progeny Exp2: Expected number of blue-flowe red progeny and white-flowered progeny Observed: Number of blue-flowered progeny and white-flowered progeny observed # 2: Chi-square value calculated from goodne ss-of-fit test; computed using Yates correction for continuity P: Probability associ ated with calculated 2 value

PAGE 171

154 Table 7.3. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived fr om the initial cross/reciprocal set WMBL (genotypes ww x Ww ). Progeny were tested against a model with two alleles ( W and w ) at one locus ( W ); gene action is dominant and white flower color is recessive. F 2 F 1 parent Exp1 Exp2 Obs# 2 P BM1 WMBL4 3:1 57:19 53:23 0.8596 0.3538 BM2 WMBL13 3:1 62.25:20.75 68:15 1.7711 0.1832 BM3 WMBL22 3:1 44.25:14.75 48:11 0.9548 0.3285 BM4 BLWM3 3:1 88.5:29.5 91:27 0.1808 0.6706 BM5 BLWM6 3:1 30:10 26:14 1.6333 0.2012 BM6 BLWM9 3:1 22.5:7.5 27:3 2.8444 0.0916 BL1 WMBL5 3:1 24:8 25:7 0.0417 0.8382 BL2 WMBL6 3:1 25.5:8.5 26:8 0.0000 0.9999 BL3 WMBL7 3:1 18.75:6.25 20:5 0.1200 0.7290 BL4 WMBL8 3:1 24:8 25:7 0.0417 0.8382 BL5 BLWM1 3:1 17.25:5.75 17:6 0.0000 0.9999 BL6 BLWM7 3:1 19.5:6.5 23:3 1.8462 0.1742 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families WM1, WM2, WM3, WM4, WM5, WM6, WL1, WL2, WL3, WL4, WL5, WL6 (white flowers only) F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents WM and BL Exp1: Expected ratio of blue-f lowered and white-flowered progeny Exp2: Expected number of blue-f lowered and white-flowered progeny # Obs: Observed number of blue-flowered and white-flowered progeny 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 172

155 Table 7.4. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived from the initial cross/reciprocal set WSBL (genotypes ww x Ww ). Progeny were tested against a model with two alleles ( W and w ) at one locus ( W ); gene action is dominant and white flower color is recessive. F 2 F 1 parent Exp1 Exp2 Obs# 2 P BS1 WSBL6 3:1 34.5:11.5 33:13 0.1159 0.7335 BS3 BLWS12 3:1 42:14 41:15 0.0238 0.8773 BS7 BLWS55 3:1 23.25:7.75 26:5 0.8710 0.3506 BL1 BLWS22 3:1 33:11 39:5 3.6667 0.0555 BL2 BLWS37 3:1 32.25:10.75 28:15 1.7442 0.1866 BL3 BLWS39 3:1 51.75:17.25 60:9 4.6425 0.0311 BL4 BLWS43 3:1 34.5:11.5 42:4 5.6812 0.0171 BL5 BLWS49 3:1 33:11 36:8 0.7576 0.3840 BL6 BLWS50 3:1 34.5:11.5 37:9 0.4638 0.4958 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families WS1, WS5, WL1, WL2, WL3, WL4, WL5, WL6 (white flowers only) F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents WS and BL Exp1: Expected ratio of blue-f lowered and white-flowered progeny Exp2: Expected number of blue-f lowered and white-flowered progeny # Obs: Observed number of blue-flowered and white-flowered progeny 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 173

156 Table 7.5. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived fr om the initial cross/reciprocal set WSBM (genotypes ww x Ww ). Progeny were tested ag ainst a model with two alleles ( W and w ) at one locus ( W ); gene action is dominant and white flower color is recessive. F 2 F 1 parent Exp1 Exp2 Obs# 2 P BS1 BMWS5 3:1 3:1 2:2 0.5000 0.4795 BS2 BMWS21 3:1 63.75:21.25 59:26 1.1333 0.2870 BS3 BMWS54 3:1 16.5:5.5 18:4 0.2424 0.6224 BS4 WSBM5 3:1 66:22 63:25 0.3788 0.5382 BS5 WSBM21 3:1 2.25:0.75 2:1 0.0000 0.9999 BS6 WSBM41 3:1 38.25:12.75 37:14 0.0588 0.8084 BM1 BMWS36 3:1 24.75:8.25 21:12 1.7071 0.1913 BM2 WSBM45 3:1 65.25:21.75 53:34 8.4636 0.0036 BL1 BMWS33 3:1 13.5:4.5 15:3 0.2963 0.5862 BL2 WSBM32 3:1 15:5 17:3 0.5806 0.4385 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families WS1, WS2, WS3, WS4, WS5, WS6, WS7, WM1, WM2, WM 3, WL1, WL2, WL3 (white flowers only) F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents WS and BM Exp1: Expected ratio of blue-f lowered and white-flowered progeny Exp2: Expected number of blue-f lowered and white-flowered progeny # Obs: Observed number of blue-flowered and white-flowered progeny 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 174

157 Table 7.6. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived fr om the initial cross/reciprocal set WMBS (genotypes ww x WW ). Progeny were tested against a model with two alleles ( W and w ) at one locus ( W ); gene action is dominant and white flower color is recessive. F2 F1 parent Exp1 Exp2 Obs# 2 P BS1 WMBS13 3:1 69.75:23.25 69:24 0.0036 0.9522 BS2 WMBS73 3:1 85.5:28.5 91:23 1.1696 0.2794 BS3 BSWM3 3:1 12.75:4.25 11:6 0.4902 0.4838 BS4 BSWM30 3:1 46.5:15.5 51:11 1.3763 0.2407 BS5 WMBS39 3:1 114.75:38.25 119:34 0.4902 0.4838 BS6 WMBS47 3:1 12:4 11:5 0.0833 0.7728 BS7 BSWM23 3:1 36.75:12.25 36:13 0.0068 0.9342 BM1 WMBS28 3:1 78.75:26.25 79:26 0.0000 0.9999 BM2 WMBS33 3:1 72:24 74:22 0.1250 0.7236 BM3 BSWM16 3:1 20.25:6.75 21:6 0.0123 0.9116 BM4 BSWM19 3:1 74.25:24.75 75:24 0.0034 0.9537 BM5 WMBS19 3:1 62.25:20.75 61:22 0.0361 0.8493 BM6 WMBS24 3:1 21:7 20:8 0.0476 0.8272 BM7 WMBS60 3:1 109.5:36.5 113:33 0.3288 0.5663 BM8 WMBS82 3:1 22.5:7.5 24:6 0.1778 0.6732 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents WM and BS Exp1: Expected ratio of blue-f lowered and white-flowered progeny Exp2: Expected number of blue-f lowered and white-flowered progeny # Obs: Observed number of blue-flowered and white-flowered progeny 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 175

158 Table 7.7. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived from the initial cross/reciprocal set BSBM (genotypes WW x Ww ). Progeny were tested against a model with two alleles ( W and w ) at one locus ( W ); gene action is dominant and white flower color is recessive. F2 F1 parent Exp1 Exp2 Obs# 2 P BS2 BMBS94 3:1 82.5:27.5 90:20 2.3758 0.1232 PBS1 BMBS88 3:1 42:14 32:24 8.5952 0.0033 PBS3 BMBS96 3:1 15:5 18:2 1.6667 0.1967 PBS4 BMBS104 3:1 30:10 31:9 0.0333 0.5838 BM3 BSBM2 3:1 26.25:8.75 25:10 0.0857 0.7697 PBM1 BMBS16 3:1 111.75:37.25 120:29 2.1499 0.1425 PBM2 BMBS71 3:1 81:27 81:27 0.0000 0.9999 PBM3 BSBM24 3:1 82.5:27.5 79:31 0.4364 0.5088 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families BS1, BS4, BM1, BM2 (blue flowers only) F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents BS and BM Exp1: Expected ratio of blue-f lowered and white-flowered progeny Exp2: Expected number of blue-f lowered and white-flowered progeny # Obs: Observed number of blue-flowered and white-flowered progeny 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 176

159 Table 7.8. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived from the initial cross/reciprocal set BSBL (genotypes WW x Ww ). Progeny were tested against a model with two alleles ( W and w ) at one locus ( W ); gene action is dominant and white flower color is recessive. F2 F1 parent Exp1 Exp2 Obs# 2 P UVM2 BLBS19 3:1 66.75:22.25 65:24 0.0936 0.7596 BM1 BLBS5 3:1 52.5:17.5 49:21 0.6857 0.4076 BM2 BLBS11 3:1 73.5:24.5 69:29 0.8707 0.3507 BM3 BSBL13 3:1 66:22 74:14 3.4091 0.0648 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families VarBS, UVS2, UVS3, BS1, BS2, BS3, UVM1, UVM3 (blue flowers only) F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents BS and BL Exp1: Expected ratio of blue-f lowered and white-flowered progeny Exp2: Expected number of blue-f lowered and white-flowered progeny # Obs: Observed number of blue-flowered and white-flowered progeny 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 177

160 Table 7.9. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue and white flower color and derived from the initial cross/reciprocal set BMBL (genotypes Ww x Ww ). Progeny were tested against a model with two alleles ( W and w ) at one locus ( W ); gene action is dominant and white flower color is recessive. F2 F1 parent Exp1 Exp2 Obs# 2 P BM1 BMBL43 3:1 38.25:12.75 37:14 0.0588 0.8084 BM2 BMBL56 3:1 28.5:9.5 19:19 11.368 0.0007 BM6 BLBM18 3:1 32.25:10.75 27:16 2.7984 0.0943 BL2 BMBL49 3:1 24:8 28:4 2.0417 0.1530 BL3 BMBL63 3:1 13.5:4.5 13:5 0.0000 0.9999 BL4 BLBM1 3:1 10.5:3.5 11:3 0.0000 0.9999 BL6 BLBM7 3:1 8.25:2.75 10:1 0.7576 0.3840 BL8 BLBM11 3:1 21.75:7.25 21:8 0.0115 0.9146 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families WM1, WL1, WL2, WL3, WL4 (white flowers only) and BM3, BM4, BM5, BL1, BL5, BL7, BL9 (blue flowers only) F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents BM and BL Exp1: Expected ratio of blue-f lowered and white-flowered progeny Exp2: Expected number of blue-f lowered and white-flowered progeny # Obs: Observed number of blue-flowered and white-flowered progeny 2: Chi-square value calculated from goodness-of-fit tes t; computed using Yates correction for continuity P: Probability associated with 2 value

PAGE 178

161 Figure 7.1. Flowers of pickerelweed. A) Wild -type blue flowers. B) White flowers. A B

PAGE 179

162 CHAPTER 8 INHERITANCE AND GENETIC C ONTROL OF FLORAL MORPH Introduction Pickerelweed is a diploid (2n=2x=16) erect, emergent, herbaceous aquatic perennial that reproduces by both sexual and vegetative means. Sexual reproduction in pickerelweed is influenced by tristyly, a t ype of heteromorphic incompatibility that promotes disassortative mating among floral morphs and encourages insect-mediated cross-pollination among different morphs (Crowe 1964; Darwin 1877; Ganders 1979; Vuillenmier 1967). Tristyly is thought to be the most comple x breeding system in plants; the system has an elaborate developmental basis and is rare, suggesting evolu tion of the trait is difficult (Kohn et al. 1996; Ri chards and Barrett 1987). Populati ons of species that utilize tristylous incompatibility systems have thre e distinct floral morphs, each with a unique set of characters. Floral mo rph differences include length of stigmatic papillae, style coloration and pollen exine sc ulpturing (Barrett 1988), but the most obvious visible difference among the morphs is style length. There are three positions within each flower, with each position occupied by either a single st yle or one of two sets of stamens. Floral morph designation is determined by style lengt h. Flowers with long styles are L-morphs, while those with styles in the mid or shor t positions are classified as M-morphs or S-morphs, respectively (Figure 8.1). Reciprocal positioning of anthers and stigmas occurs so that each plant produces flow ers with anthers borne at the same level as the stigma of the other morphs. This arrangement promotes insect-mediated cross-pollinations between

PAGE 180

163 anthers and stigmas of equivalent height, resulting in seed set. Darwin (1877) referred to this as legitimate pollination, while ill egitimate pollinations between anthers and stigmas at different levels result in little or no seed production. Inheritance of style length in tristylous systems was controlled by two diallelic loci in the species studied thus far [e.g., species of Lythrum (Anderson and Ascher 1995), Eichhornia (Barrett 1988) and Oxalis suksdorfi (Ordnuff 1964)]. The L-morph (long style) phenotype was produced by the completely recessive genotype ssmm while the M-morph (mid style) phenotype was due to a recessive condition at the S locus and the presence of at least one dominant allele at the M locus (genotype ssMM or ssMm ). The dominant S allele was present only in plants wi th S-morph (short style) flowers, which had the genotype SSMM SSMm SSmm SsMM SsMm or Ssmm The S locus was epistatic to the M locus and prevented expression of alleles at the M locus (Anderson and Ascher 1995; Barrett 1988; Charlesworth 1979). L-morp h plants were true-breeding for morph and self-pollination produced only L-morph progeny. Segregation of progeny resulting from self-pollination of S-morph and Mmorph plants would be dependent upon the genotype of the parent plant. There is no conclusive published informa tion describing the inheritance and genetic control of floral morph in pickerelweed. Ba rrett and Anderson (1985) assessed a small set of S1 progeny (20 seedlings) produced from cont rolled self-pollinations and found that two of the four populations under investigati on segregated for style length; however, the small population size precluded speculation ab out the inheritance of style length in pickerelweed. The objective of this experiment was to determine the type of gene action and number of loci controlling floral mo rph in this population of pickerelweed.

PAGE 181

164 Materials and Methods The populations utilized in this experi ment were developed using the strategy described in Appendix A of this dissertation. Each F1 and S1 plant was grown to reproductive maturity and evaluate d for floral morph. Data from F1 and S1 populations were used to develop a working model to expl ain the type of gene action and number of loci controlling floral morph in pickerelw eed. Development of this model allowed the assignment of genotypes to parents; the m odel was then verified by analyses of F2 populations. All data were analyzed using goodness-of-fit (chi-square or 2) tests with Yates correction for con tinuity when appropriate. Results and Discussion No maternal effects were noted in the eight F1 families examined in this experiment; therefore, data presented for each F1 family were pooled within each cross/reciprocal set. All eight F1 families segregated for floral morph, as did S1 progeny from self-pollination of S-morph and M-mor ph parents (Table 8.1). The only family that did not segregate was derived from self-polli nation of the L-morph parent BL. These data were compared to the two-locus diallelic m odel responsible for the control of floral morph in other tristylous species. Genotypes we re assigned to all five parents based on the proposed model and segregation of F1 progeny. Based on these data, the L-morph parent BL was homozygous recessive at both loci ( ssmm ), while the M-morph parents WM and BM were homozygous recessive at th e epistatic locus and heterozygous at the hypostatic locus ( ssMm ). The two S-morph parents examined in this experiment had different genotypes; the pare nt BS was heterozygous at the epistatic locus and homozygous dominant at the hypostatic locus ( SsMM ), while the parent WS was

PAGE 182

165 heterozygous at the epistatic locus and ho mozygous recessive at the hypostatic locus ( Ssmm ). Goodness-of-fit values and their associated probabilities for F1 and S1 populations are presented in Table 8.2; th ese values indicated that th e model provided a good fit to most of the data and supported the proposed genotype assignments. Seven of the eight F1 families under investigation were advanced to the F2 generation for further study. Goodness-of-fit values and their associated probabilities for F2 families are presented in Tables 8.3 through 8.9 and are sorted by fam ily. Summaries of the analyses for each family are presented below. WS. The parent WS was assigned the genotype Ssmm and S1 progeny segregated in a ratio that was not different from the expect ed 3:1 (short:long) (Tab le 8.2); therefore, these S1 data provided evidence th at supported the proposed model and assignment of the genotype Ssmm to WS. WM. The parent WM was assigned the genotype ssMm and S1 progeny segregated in a ratio that was not different from the e xpected 3:1 (mid:long) (T able 8.2); therefore, these S1 data provided evidence th at supported the proposed model and assignment of the genotype ssMm to WM. BS. The parent BS was assigned the genotype SsMM and S1 progeny segregated in a ratio that was not different from the exp ected 3:1 (short:mid) (Tab le 8.2); therefore, these S1 data provided evidence th at supported the proposed model and assignment of the genotype SsMM to BS. BM. The parent BM was assigned the genotype ssMm and S1 progeny segregated in a ratio that was not different from the e xpected 3:1 (mid:long) (T able 8.2); therefore,

PAGE 183

166 these S1 data provided evidence th at supported the proposed model and assignment of the genotype ssMm to BM. BL. The parent BL was assigned the genotype ssmm and all S1 progeny had L-morph flowers (Table 8.1); therefore, these S1 data provided evidence that supported the proposed model and assignment of the genotype ssmm to BL. WMWS and WSWM. Parents WM and WS were assigned the genotypes ssMm and Ssmm respectively. The observed F1 progeny segregated in a manner that was not different from the expected 2:1:1 (short:mid:l ong) ratio (Table 8.2). It was not possible to evaluate the segregation of F2 progeny from this cross/reciprocal set, as all F2 seeds from this family were contaminated with fungi and failed to germinate. The F1 data from this cross/reciprocal set provided evidence that supported the proposed model and genotypic assignments. WMBL and BLWM. Parents WM and BL were assigned the genotypes ssMm and ssmm respectively. The observed F1 progeny segregated in a manner that was not different from the expected 1:1 (mid:long) ratio (Table 8.2). One of two segregation ratios was expected in the F2 generation based on the phenotype of the F1 plant self-pollinated to create an F2 family. L-morph F1 plants were expected to produce all L-morph F2 progeny, while M-morph F1 plants were expected to produce F2 progeny that segregated in a 3:1 (mid:l ong) ratio. A total of 24 F2 families (derived from self-pollination of 12 L-morph F1 plants and 12 M-morph F1 plants) were analyzed (Table 8.3). All F2 families from L-morph F1 plants produced only L-morph progeny. Eleven of the twelve F2 families from M-morph F1 plants segregated in ratios that were not different from the expected 3:1 ratio, while the F2 family WM6 segregated in a

PAGE 184

167 manner that differed from the expected 3:1 ratio. Around 1 in 50 populations of pickerelweed with the proposed genotypes would be expected to exhibit as much variation for floral morph as WM6, so it is probable that sampling error was responsible for the recovery of this single family that did not segregate as expected. With the exception of the F2 progeny from WM6, these data pr ovided evidence th at supported the proposed model and genotypic assignments. WSBL and BLWS. Parents WS and BL were assigned the genotypes Ssmm and ssmm respectively. The observed F1 progeny segregated in a manner that was not different from the expected 1:1 (short:long) ratio (Table 8.2). One of two segregation ratios was expected in the F2 generation based on the phenotype of the F1 plant self-pollinated to create an F2 family. L-morph F1 plants were expected to produce all L-morph F2 progeny, while S-morph F1 plants were expected to produce F2 progeny that segregated in a 3:1 (short:long) ratio. A total of 16 F2 families (derived from self-pollination of 12 L-morph F1 plants and 4 S-morph F1 plants) were analyzed (Table 8.4). All F2 families from L-morph F1 plants produced only L-morph progeny. Three of the four F2 families from S-morph F1 plants produced progeny th at segregated in ratios that were not different from the expected 3:1 ratio, while the F2 family BS7 segregated in a manner that differed from the expected 3:1 ratio. Around 1 in 100 populations of pickerelweed with the proposed genotypes would be expected to exhibit as much variation for floral morph as BS7, so it is probable that sampling error was responsible for the recovery of this single family that did not segregate as expected. With the exception of the F2 progeny from BS7, these data pr ovided evidence that supported the proposed model and genotypic assignments.

PAGE 185

168 WSBM and BMWS. Parents WS and BM were assigned the genotypes Ssmm and ssMm respectively. The observed F1 progeny segregated in a manner that differed from the expected 2:1:1 (short:mid:long) ratio (Table 8.2). Around 1 in 250 populations of pickerelweed with the proposed genotypes w ould show as much variation for floral morph as the F1 progeny in this family, so the prob ability of recovering a progeny set that segregated in this manner was small (p =0.0035) but still poss ible. One of four segregation ratios was expected in the F2 generation based on the phenotype and genotype of the F1 plant self-pollinat ed to create an F2 family. L-morph F1 plants were expected to produce only L-morph F2 progeny, while M-morph F1 plants were expected to produce F2 progeny that segregated in a 3: 1 (mid:long) ratio and S-morph F1 plants were expected to produce F2 progeny that segregated eith er in a 12:3:1 (short:mid:long) ratio or a 3:1 (short:long) ratio. A total of 20 F2 families (derived from self-pollination of 5 L-morph F1 plants, 4 M-morph F1 plants and 11 S-morph F1 plants) were analyzed (Table 8.5). All F2 families from L-morph F1 plants only produced L-morph progeny. Three of the four F2 families from M-morph F1 plants produced progeny that segregated in ratios that were not diff erent from 3:1, while the F2 family from the M-morph F1 plant BM2 segregated in a manner that differed from the expected 3:1 rati o. The probability of recovering a population of pickerelweed with the proposed genotypes that showed as much variation for floral morph as BM2 is re mote, but still possible. Seven of the eleven F2 families from S-morph F1 plants segregated in ratios that were not different from 12:3:1; of the remaining four F2 families from S-morph plants, three segregated in ratios that were not different from 3:1. The F2 family from the S-morph plant WS5 produced only S-morph and L-morph progeny, but segregat ed in a manner that was different from

PAGE 186

169 the expected 3:1 ratio; however, around 1 in 20 populations of pickerelweed with the proposed genotypes would be expected to exhibi t as much variation for floral morph as WS5, so it is probable that samp ling error was responsible for th e recovery of this single family that did not segregate as e xpected. With the exception of the F2 progeny from BM2 and WS5, these data provided eviden ce that supported the proposed model and genotypic assignments. WMBS and BSWM. Parents WM and BS were assigned the genotypes ssMm and SsMM respectively. The observed F1 progeny segregated in a manner that was not different from the expected 1:1 (short:mid) ratio (Table 8.2). One of four segregation ratios was expected in the F2 generation based on the phenotype and genotype of the F1 plant self-pollinated to create an F2 family. M-morph F1 plants were expected to produce F2 progeny that either segreg ated in a 3:1 (mid:long) ratio or were all M-morph. S-morph F1 plants were expected to produce F2 progeny that segregated either in a 12:3:1 (short:mid:long) ratio or a 3:1 (s hort:long) ratio. A total of 15 F2 families (derived from self-pollination of 8 M-morph F1 plants and 7 S-morph F1 plants) were analyzed (Table 8.6). Four of the eight F2 families from M-morph F1 plants produced progeny that segregated in ratios that were not different from 3:1, while the remaining four F2 families from M-morph F1 plants produced only M-morph progeny. Four of the seven F2 families from S-morph F1 plants segregated in ra tios that were not differ ent from 12:3:1; of the remaining three F2 families from S-morph plants, two se gregated in ratios that were not different from 3:1. The F2 family from the S-morph plant BS5 produced only S-morph and L-morph progeny, but segregated in a mann er that was different from the expected 3:1 ratio; however, around 1 in 25 populati ons of pickerelweed with the proposed

PAGE 187

170 genotypes would be expected to exhibit as mu ch variation for floral morph as BS5, so it is probable that sampling error was responsible for the recovery of this single family that did not segregate as expected. With the exception of the F2 progeny from BS5, these data provided evidence that supported the propos ed model and genotypic assignments. BSBM and BMBS. Parents BS and BM were assigned the genotypes SsMM and ssMm respectively. The observed F1 progeny segregated in a manner that was not different from the expected 1:1 (short:mid) ratio (Table 8.2). One of four segregation ratios was expected in the F2 generation based on the phenotype and genotype of the F1 plant self-pollinated to create an F2 family. M-morph F1 plants were expected to produce F2 progeny that either segreg ated in a 3:1 (mid:long) ratio or were all M-morph. S-morph F1 plants were expected to produce F2 progeny that segregated either in a 12:3:1 (short:mid:long) ratio or a 3:1 (s hort:long) ratio. A total of 12 F2 families (derived from self-pollination of 6 M-morph F1 plants and 6 S-morph F1 plants) were analyzed (Table 8.7). Three of the six F2 families from M-morph F1 plants produced progeny that segregated in ratios that were not different from 3:1, while the remaining three F2 families from M-morph F1 plants produced only M-morph progeny. Three of the six F2 families from S-morph F1 plants segregated in ratios that were not different from 12:3:1; of the remaining three F2 families from S-morph plants, one segregated in a ratio that was not different from the expected 3:1. The F2 families from the S-morph plants BS1 and BS2 produced only S-morph and Lmorph progeny, but each segregated in a manner that differed from the expected 3:1 ratio. Around 1 in 125 populations of pickerelweed with the proposed genotypes w ould show as much variation for floral morph as BS1, so it is probable that sampling error was responsible for the recovery of

PAGE 188

171 this family that did not segregate as expect ed. The likelihood of recovering a progeny set that segregated in the same manner as BS2 wa s more remote, but still possible. With the exception of the F2 progeny from BS1 and BS2, these data provided evidence that supported the proposed model and genotypic assignments. BSBL and BLBS. Parents BS and BL were assigned the genotypes SsMM and ssmm respectively. The observed F1 progeny segregated in a manner that was not different from the expected 1:1 (short:mid ) ratio (Table 8.2). On e of two segregation ratios was expected in the F2 generation based on the phenotype of the F1 plant self-pollinated to create an F2 family. M-morph F1 plants were expected to produce F2 progeny that segregated in a 3: 1 (mid:long) ratio, while S-morph F1 plants were expected to produce F2 progeny that segregated in a 12: 3:1 (short:mid:long) ratio. A total of 12 F2 families (derived from self-pollination of 6 M-morph F1 plants and 6 S-morph F1 plants) were analyzed (Tab le 8.8). Five of the six F2 families from M-morph F1 plants produced progeny that segregated in ratios that were not different from the expected 3:1, while the F2 family from the M-morph F1 plant UVM2 segregated in a manner that differed from the expected 3:1 ratio. Around 1 in 100 populations of pickerelweed with the proposed genotypes would show as much va riation for floral morph as UVM2, so it is probable that sampling error was responsible for the recovery of this family that did not segregate as expected. Four of the six F2 families from S-morph F1 plants segregated in ratios that were not differe nt from 12:3:1, but the F2 families VarBS and UVS2 segregated in ratios that di ffered from the expected 12:3:1. Around 1 in 25 populations of pickerelweed with the proposed genotypes w ould show as much variation for floral morph as VarBS and as many as 1 in 30 would show as much variation for floral morph

PAGE 189

172 as UVS2, so it is probable that sampling error was responsible for the recovery of these families that did not segregate as expected. With the exception of the F2 progeny from UVM2, VarBS and UVS2, these data provide d evidence that supported the proposed model and genotypic assignments. BMBL and BLBM. Parents BM and BL were assigned the genotypes ssMm and ssmm respectively. The observed F1 progeny segregated in a manner that was not different from the expected 1:1 (mid:long) ratio (Table 8.2). One of two segregation ratios was expected in the F2 generation based on the phenotype of the F1 plant self-pollinated to create an F2 family. L-morph F1 plants were expected to produce only L-morph F2 progeny, while M-morph F1 plants were expected to produce F2 progeny that segregated in a 3:1 (mid:l ong) ratio. A total of 20 F2 families (derived from self-pollination of 13 L-morph F1 plants and 7 M-morph F1 plants) were analyzed (Table 8.9). All F2 families from L-morph F1 plants produced only L-morph progeny. Six of the seven F2 families from M-morph F1 plants segregated in ratios that were not different from the expected 3:1 ratio, while the F2 family BM5 segregated in a manner that differed from the expected 3:1 ratio. Around 1 in 250 populations of pickerelweed with the proposed genotypes would show as mu ch variation for floral morph as BM5, so it is possible that sampling error was responsible for the recovery of this family that did not segregate as expected. With the exception of the F2 progeny from BM5, these data provided evidence that supported the propos ed model and genotypic assignments. Conclusions The results of this experiment suggested that floral morph in this population of pickerelweed was controlled in a manner si milar to that described by Anderson and Ascher (1995), Barrett (1988) a nd Ordnuff (1964) for other tristy lous species. These data

PAGE 190

173 were provided the best fit by a model with tw o diallelic loci; gene action was dominant and expression was influenced by epistasis. The L-morph phenotype was produced by the completely recessive genotype ( ssmm ), while the M-morph ph enotype was due to a recessive condition at the S locus and at least one dominant allele at the M locus ( ssM _). The presence of a dominant allele at the S locus was required to produce the S-morph phenotype ( S _ _); the S locus was epistatic to the M locus so that the presence of the dominant S allele prevented expres sion of alleles at the M locus. This experiment examined the segregation patterns of five S1 families, eight F1 families and 119 F2 families of pickerelweed. All S1 families and seven of the eight F1 families segregated as expected. A total of 42 F2 families from L-morph F1 plants, 43 F2 families from M-morph F1 plants and 34 F2 families from S-morph F1 plants were studied in this experiment. All 42 F2 families from L-morph F1 plants segregated as expected (i.e., all F2 progeny from L-morph F1 plants were L-morph). Thirty-nine of the 43 F2 families from M-morph F1 plants segregated as expect ed, while the remaining four F2 families did not segregate as expected. It is interesting to note that all four F2 families from M-morph F1 plants that deviated significantly from the exp ected ratios produced an excess of L-morph F2 progeny and a deficit of M-morph F2 progeny. The reason for this excess of L-morph offspring is unknown; however it is possible that dominant alleles at the M locus were linked to an unidentified locu s that coded for a deleterious or lethal trait, which would have resulted in the pr oduction of non-viable progeny. Twenty-seven of the 34 F2 families from S-morph F1 plants segregated as expected, while the remaining seven F2 families did not segregate as expected. There was no evidence that suggested

PAGE 191

174 any one morph was consistently produced in excess as compared to the other morphs in F2 families derived from S-morph F1 plants. The vast majority of the data generated in this experiment supported the proposed model. All five S1 progeny sets, seven of eight F1 progeny sets and 108 of 119 F2 progeny sets segregated as expected. The failure of one F1 family and eleven F2 families to conform to expected segregation patterns wa s most likely due to sampling error, as all other data supported the proposed model. It is possible that inheritance of tristyly is influenced by epigenetic factors or by addi tional loci not included in the model; however, this experiment failed to reveal the existen ce of other factors that contributed to the genetic control of tristyly in this population of pickerelweed.

PAGE 192

175 Table 8.1. Number of S-morph, Mmorph and L-morph progeny in F1 and S1 families of pickerelweed. Family Generation Plants S M# L WSWM and WMWS F1 98 53 23 22 WMBL and BLWM F1 57 0 29 28 WSBL and BLWS F1 67 28 0 39 WSBM and BMWS F1 144 87 19 38 WMBS and BSWM F1 103 52 51 0 BSBM and BMBS F1 132 77 55 0 BSBL and BLBS F1 49 21 28 0 BMBL and BLBM F1 91 0 46 45 WS S1 21 16 0 5 WM S1 44 0 32 12 BS S1 9 7 2 0 BM S1 11 0 6 5 BL S1 56 0 0 56 Family: F1 or S1 family under investigation; F1 families were created through crossand reciprocal pollinations between two parents and F1 codes identify the parents (e.g., the F1 family WSWM was derived from cross-po llination between the parents WS and WM) Generation: Generation of th e family under investigation Plants: Number of plants examined S: Number of plants with S-morph flowers # M: Number of plants with M-morph flowers L: Number of plants with L-morph flowers

PAGE 193

176 Table 8.2. Goodness-of-fit tests for F1 and S1 families of pickerel weed segregating for short, mid and long floral morphs. Progeny were tested against a model with two diallelic loci ( S and M ) with epistasis; gene action at each locus is dominant and the S locus is epistatic to the M locus. S-morphs result from the genotypes S _ _, M-morphs from the genotypes ssM and L-morphs by the genotype ssmm Family Parents Expected Observed 2# P WMWS ssMm x Ssmm 2:1:1 53:23:22 0.6734 0.7141 WMBL ssMm x ssmm 0:1:1 0:29:28 0.0000 0.9999 WSBL Ssmm x ssmm 1:0:1 28:0:39 1.2821 0.2575 WSBM Ssmm x ssMm 2:1:1 87:19:38 11.264 0.0035 WMBS ssMm x SsMM 1:1:0 52:51:0 0.0000 0.9999 BSBM SsMM x ssMm 1:1:0 77:55:0 3.3409 0.0675 BSBL SsMM x ssmm 1:1:0 21:28:0 0.7347 0.3913 BMBL ssMm x ssmm 0:1:1 0:46:45 0.0000 0.9999 WS Ssmm 3:0:1 16:0:5 0.0000 0.9999 WM ssMm 0:3:1 0:32:12 0.0303 0.8618 BS SsMM 3:1:0 7:2:0 0.0000 0.9999 BM ssMm 0:3:1 0:6:5 1.1136 0.2913 Family: F1 or S1 family under investigation; F1 families were created through crossand reciprocal pollinations between two parents and F1 codes identify the parents (e.g., the F1 family WSWM was derived from cross-poll ination between the pa rents WS and WM); not shown: non-segregating family BL (L-morph progeny only) Parents: Genotypes of parents used to create family Expected: Expected ratio of S-mo rph to M-morph to L-morph progeny Observed: Number of S-morph, Mmorph and L-morph progeny observed # 2: Chi-square value calculated from goodness-of -fit test; values computed using Yates correction for continuity except for the families WMWS and WSBM P: Probability associ ated with calculated 2 value

PAGE 194

177 Table 8.3. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set WMBL (genotypes ssMm x ssmm ). Progeny were tested against a model with two diallelic loci ( S and M ) with epistasis; gene action at each locus is dominant and the S locus is epistatic to the M locus. S-morphs result from the genotypes S _ _, M-morphs from the genotypes ssM and L-morphs by the genotype ssmm F2 F1 parent Expected Observed 2# P WM1 WMBL3 0:3:1 0:45:15 0.0000 0.9999 WM2 WMBL25 0:3:1 0:11:5 0.0625 0.7728 WM3 WMBL31 0:3:1 0:44:11 0.4909 0.4835 WM4 WMBL48 0:3:1 0:32:9 0.0732 0.7867 WM5 BLWM5 0:3:1 0:25:11 0.3333 0.5637 WM6 BLWM8 0:3:1 0:44:27 5.7512 0.0164 BM1 WMBL4 0:3:1 0:53:23 0.8596 0.3538 BM2 WMBL13 0:3:1 0:64:19 0.1004 0.7513 BM3 WMBL22 0:3:1 0:49:10 1.6328 0.2013 BM4 BLWM3 0:3:1 0:96:22 2.2147 0.1367 BM5 BLWM6 0:3:1 0:31:9 0.0333 0.8552 BM6 BLWM9 0:3:1 0:23:7 0.0000 0.9999 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families WL1, WL2, WL3, WL4, WL5, WL6, BL1, BL2, BL3, BL 4, BL5, BL6 (L-morph progeny only) F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents WM and BL Expected: Expected ratio of S-mo rph to M-morph to L-morph progeny Observed: Observed number of Smorph, M-morph and L-morph progeny # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity P: Probability associated with 2 value

PAGE 195

178 Table 8.4. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set WSBL (genotypes Ssmm x ssmm ). Progeny were tested against a model with two diallelic loci ( S and M ) with epistasis; gene action at each locus is dominant and the S locus is epistatic to the M locus. S-morphs result from the genotypes S _ _, M-morphs from the genotypes ssM and L-morphs by the genotype ssmm F2 F1 parent Expected Observed 2# P WS1 WSBL1 3:0:1 29:0:12 0.2033 0.6520 BS1 WSBL6 3:0:1 40:0:6 2.8986 0.0886 BS3 BLWS12 3:0:1 38:0:18 1.1667 0.2800 BS7 BLWS55 3:0:1 30:0:1 6.7204 0.0095 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families WL1, WL2, WL3, WL4, WL5, WL6, BL1, BL2, BL3, BL 4, BL5, BL6 (L-morph progeny only) F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents WS and BL Expected: Expected ratio of S-mo rph to M-morph to L-morph progeny Observed: Observed number of Smorph, M-morph and L-morph progeny # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity P: Probability associated with 2 value

PAGE 196

179 Table 8.5. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set WSBM (genotypes Ssmm x ssMm ). Progeny were tested against a model with two diallelic loci ( S and M ) with epistasis; gene action at each locus is dominant and the S locus is epistatic to the M locus. S-morphs result from the genotypes S _ _, M-morphs from the genotypes ssM and L-morphs by the genotype ssmm F2 F1 parent Expected Observed 2# P WM1 WSBM13 0:3:1 0:2:1 0.0000 0.9999 WM2 WSBM26 0:3:1 0:19:10 0.9310 0.3346 BM1 BMWS36 0:3:1 0:21:12 1.7071 0.1913 BM2 WSBM45 0:3:1 0:48:39 17.199 0.0000 WS1 BMWS8 12:3:1 38:13:5 0.9583 0.6193 WS2 BMWS30 12:3:1 37:10:1 1.0347 0.6457 WS3 BMWS37 3:0:1 22:0:8 0.0000 0.9999 WS4 WSBM43 3:0:1 23:0:8 0.0000 0.9999 WS5 WSBM44 3:0:1 18:0:13 3.8817 0.0488 WS6 WSBM51 12:3:1 31:9:1 0.5264 0.7685 WS7 WSBM24 3:0:1 10:0:3 0.0000 0.9999 BS2 BMWS21 12:3:1 58:20:7 1.4941 0.3516 BS3 BMWS54 12:3:1 17:1:4 4.9545 0.0839 BS4 WSBM5 12:3:1 70:14:4 1.0303 0.5974 BS6 WSBM41 12:3:1 39:8:4 0.1503 0.9276 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families WL1, WL2, WL3, BL1, BL2 (L-morph progeny only) F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents WS and BM Expected: Expected ratio of S-mo rph to M-morph to L-morph progeny Observed: Observed number of Smorph, M-morph and L-morph progeny # 2: Chi-square value computed from goodness-of-f it test; values calculated using Yates correction for continuity except for families BS2 and BS4 P: Probability associated with 2 value

PAGE 197

180 Table 8.6. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set WMBS (genotypes ssMm x SsMM ). Progeny were tested against a model with two diallelic loci ( S and M ) with epistasis; gene action at each locus is dominant and the S locus is epistatic to the M locus. S-morphs result from the genotypes S _ _, M-morphs from the genotypes ssM and L-morphs by the genotype ssmm F2 F1 parent Expected Observed 2# P BM1 WMBS28 0:3:1 0:82:23 0.3841 0.5354 BM2 WMBS33 0:3:1 0:74:22 0.1250 0.7236 BM3 BSWM16 0:3:1 0:19:8 0.1111 0.7388 BM6 WMBS24 0:3:1 0:19:9 0.4286 0.5126 BS1 WMBS13 12:3:1 68:20:5 0.5332 0.7656 BS2 WMBS73 3:1:0 83:31:0 0.1871 0.6653 BS3 BSWM3 12:3:1 13:3:1 0.0000 0.9999 BS4 BSWM30 12:3:1 54:7:1 3.9731 0.1371 BS5 WMBS39 3:1:0 126:27:0 4.0283 0.0447 BS6 WMBS47 3:1:0 14:2:0 0.6429 0.3864 BS7 BSWM23 12:3:1 40:5:4 1.7483 0.4172 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families BM4, BM5, BM7, BM8 (M-morph progeny only) F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents WM and BS Expected: Expected ratio of S-mo rph to M-morph to L-morph progeny Observed: Observed number of Smorph, M-morph and L-morph progeny # 2: Chi-square value computed from goodness-of-f it test; values calculated using Yates correction for continuity except for the family BS1 P: Probability associated with 2 value

PAGE 198

181 Table 8.7. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set BSBM (genotypes SsMM x ssMm ). Progeny were tested against a model with two diallelic loci ( S and M ) with epistasis; gene action at each locus is dominant and the S locus is epistatic to the M locus. S-morphs result from the genotypes S _ _, M-morphs from the genotypes ssM and L-morphs by the genotype ssmm F2 F1 parent Expected Observed 2# P BM3 BSBM2 0:3:1 0:23:12 1.1524 0.2830 PBM1 BMBS16 0:3:1 0:117:32 0.8076 0.3688 PBM3 BSBM24 0:3:1 0:79:31 0.4364 0.5088 BS1 BMBS61 3:1:0 19:16:0 6.9429 0.0084 BS2 BMBS94 3:1:0 66:44:0 12.412 0.0004 BS4 BSBM28 3:1:0 54:20:0 0.0721 0.7883 PBS1 BMBS88 12:3:1 48:7:1 2.7202 0.2566 PBS3 BMBS96 12:3:1 15:4:1 0.0000 0.9999 PBS4 BMBS104 12:3:1 31:8:1 0.4083 0.8153 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families BM1, BM2, PBM2 (M-morph progeny only) F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents BS and BM Expected: Expected ratio of S-mo rph to M-morph to L-morph progeny Observed: Observed number of Smorph, M-morph and L-morph progeny # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity P: Probability associated with 2 value

PAGE 199

182 Table 8.8. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set BSBL (genotypes SsMM x ssmm ). Progeny were tested against a model with two diallelic loci ( S and M ) with epistasis; gene action at each locus is dominant and the S locus is epistatic to the M locus. S-morphs result from the genotypes S _ _, M-morphs from the genotypes ssM and L-morphs by the genotype ssmm F2 F1 parent Expected Observed 2# P UVM1 BLBS13 0:3:1 0:74:31 0.6881 0.3381 UVM2 BLBS19 0:3:1 0:56:33 6.2959 0.0121 UVM3 BSBL14 0:3:1 0:70:22 0.0145 0.9041 BM1 BLBS5 0:3:1 0:54:16 0.0762 0.7825 BM2 BLBS11 0:3:1 0:71:27 0.2177 0.6407 BM3 BSBL13 0:3:1 0:72:16 1.8333 0.1757 VarBS BSBL16 12:3:1 50:3:2 6.4061 0.0406 UVS2 BLBS33 12:3:1 53:4:1 6.8448 0.0326 UVS3 BSBL12 12:3:1 104:24:8 0.1569 0.9249 BS1 BLBS15 12:3:1 55:16:2 1.1724 0.5564 BS2 BLBS32 12:3:1 63:19:8 1.5704 0.4511 BS3 BSBL2 12:3:1 3:2:1 0.3889 0.8232 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents BS and BL Expected: Expected ratio of S-mo rph to M-morph to L-morph progeny Observed: Observed number of Smorph, M-morph and L-morph progeny # 2: Chi-square value computed from goodness-of-f it test; values calculated using Yates correction for continuity except for the families UVS3 and BS2 P: Probability associated with 2 value

PAGE 200

183 Table 8.9. Goodness-of-fit tests for F2 populations of pickerelweed segregating for floral morph and derived from the initial cr oss/reciprocal set BMBL (genotypes ssMm x ssmm ). Progeny were tested against a model with two diallelic loci ( S and M ) with epistasis; gene action at each locus is dominant and the S locus is epistatic to the M locus. S-morphs result from the genotypes S _ _, M-morphs from the genotypes ssM and L-morphs by the genotype ssmm F2 F1 parent Expected Observed 2# P WM1 BMBL28 0:3:1 0:42:20 1.3763 0.2407 BM1 BMBL43 0:3:1 0:37:14 0.0588 0.8084 BM2 BMBL56 0:3:1 0:23:15 3.5088 0.0610 BM3 BMBL11 0:3:1 0:94:31 0.0000 0.9999 BM4 BLBM3 0:3:1 0:55:12 1.4378 0.2304 BM5 BLBM4 0:3:1 0:33:24 8.0058 0.0046 BM6 BLBM18 0:3:1 0:33:10 0.0078 0.9298 F2: Code identifying F2 family; each F2 family was produced by self-pollination of the F1 plant listed in the F1 parent column; not shown: non-segregating families WL1, WL2, WL3, WL4, BL1, BL2, BL3, BL4, BL5, BL6, BL7, BL8, BL9 (L-morph progeny only) F1 parent: F1 plant self-pollinated to create F2 family; selected from the F1 family developed by crossand reciprocal pollinations between the parents BM and BL Expected: Expected ratio of S-mo rph to M-morph to L-morph progeny Observed: Observed number of Smorph, M-morph and L-morph progeny # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity P: Probability associated with 2 value

PAGE 201

184 Figure 8.1. The three floral morphs of pickerelweed. A) L-morph. B) M-morph. C) S-morph. A B C

PAGE 202

185 CHAPTER 9 LINKAGE RELATIONSHIP BETWEEN THE LOCI CONTROLLING FLOWER COLOR AND FLORAL MORPH Introduction Loci that are situated more than 50 ma p units (abbreviated m.u.; also called centiMorgans) apart on the same chromosome act in an independent manner since the distance between the loci is great enough that unrestricted recombination occurs between homologous chromosomes during meiosis. Th is free recombination results in the production of equal numbers of parental-type gametes and recombinant gametes. Genetic linkage occurs when the loci controlling multip le traits are less than 50 m.u. apart on the same chromosome. This close association be tween the loci reduces the frequency of recombination during meiosis; as a result, more parental-type gametes and fewer recombinant gametes are produced. The degr ee of linkage between loci ranges from complete linkage (loci located so close to each other that only parental-type gametes are produced) to complete independe nce (loci located 50 or more m.u. apart or on different chromosomes with the production of equal freq uencies of parental-type and recombinant gametes). Determination of the linkage relationship between loci controlli ng different traits requires that the type of gene action and number of loci cont rolling both traits be known. The distance between linked loci can be es timated by mating indivi duals with disparate genotypes and determining the frequency of progeny in the resulting segregating population that exhibit recombinant phenotypes. The percentage of recombinant offspring

PAGE 203

186 provides an estimate of the genetic distance and number of map units between the loci controlling the traits. There is no published information elucidat ing the linkage relationship between the loci controlling flower color and floral mor ph in pickerelweed. Research culminating in Chapter 7 of this dissertation showed that fl ower color in pickerelweed was controlled by a single diallelic locus with dominant gene act ion. Chapter 8 revealed that floral morph was conditioned by two diallelic loci; both loci exhibited dominant gene action and expression of floral morph was influenced by ep istasis. The objective of this experiment was to determine the linkage relationship a nd genetic distance or number of map units between the loci controlling flower colo r and floral morph in this population of pickerelweed. Materials and Methods The population utilized in this experime nt was developed using the strategy described in Appendix A of this dissertation. Each F1 plant was grown to reproductive maturity and evaluated for flower color and floral morph. Data from F1 populations were used to develop working models to explain the types of gene action and number of loci controlling flower color and floral morph in pickerelweed. Development of these models allowed the assignment of genotypes to pare nts; the models were then verified by analyses of F2 populations. Estimated genetic distance between the loci c ontrolling flower color and floral morph was computed based on segregation patterns of families that deviated from the ratios expected under th e assumption of independe nce of the loci of interest. A range of genetic distances was te sted to identify the distance estimate that provided the best fit to the F1 families that deviated from expected ratios, then expected segregation ratios for all F1 families were recalculated using the most likely distance

PAGE 204

187 estimate. Data for F2 families were also compared to expected values calculated with the most likely distance estimate. All data were analyzed usi ng goodness-of-fit (chi-square or 2) tests; Yates correction for continuity wa s employed when appropriate. In the interest of brevity, progeny will be referred to by their respective color and morph phenotypes (i.e., plants will blue L-morph flowers will be called blue long, plants with white M-morph flowers will be called white mid, etc.). Results and Discussion No maternal effects were noted in the seven F1 families examined in this experiment; therefore, data presented for each F1 family were pooled within each cross/reciprocal set. The assumption that th e loci controlling flow er color and floral morph act independently from one another was tested by comparing segregation of observed progeny to segregation ratios expected if the loci were 50 or more map units apart. Goodness-of-fit tests (T able 9.1) revealed that pr ogeny from two of the seven F1 families under investigation segregated in manners that were different from the ratios expected if the loci cont rolling flower color and flor al morph were independent. Evidence of linkage was only revealed in cross-pollinations that utilized BM as a parent. The F1 family BSBM was derived from the pa rents BS and BM but did not reveal linkage since BS was homozygous domi nant for flower color and all F1 progeny had blue flowers. Each of the other parents used in this experiment was he terozygous at only one locus; therefore, linkage did not affect segregation ratios of F1 progeny in families where BM played no parental role. Possible ge netic distances were computed based on segregation patterns produced by the two F1 families that deviated from the expected ratios. This was accomplished by identifying progeny with phenotypes that directly reflected recombinant genotypes.

PAGE 205

188 Linkage in the F1 family BMBL. The F1 family BMBL was derived from the parents BM and BL. The parent BM was heterozygous for flower color ( Ww ), homozygous recessive at the epistatic S locus ( ss ) and heterozygous at the hypostatic M locus ( Mm ) controlling floral morph. The pa rent BL was heterozygous at the W locus responsible for flower color ( Ww ) and homozygous recessive ( ssmm ) at both loci controlling floral morph. Progeny in the F1 family BMBL segregated as expected for flower color (75% blue flowers, 25% wh ite flowers) and for floral morph (50% M-morph, 50% L-morph) when these traits we re considered indivi dually. If the loci controlling the two traits were independent, the F1 progeny would have segregated in a distributive manner so that 50% of plants wi th blue flowers would have been M-morphs and 50% of plants with blue flowers would have been L-morphs; this same pattern of distribution would be expected to occur in white-flowered plan ts as well. Linkage between the loci controlling flower color and floral morph skewed this relationship. Free recombination does not occur when loci are linked, so parental-type and recombinant gametes were no longer produced in equal fr equencies and phenotypi c classes were no longer produced in a distributive fashion. The degree of linkage between the loci c ontrolling flower color and floral morph was estimated by examining recombinant progeny. In the case of BMBL, the possible genotypes and phenotypes of F1 progeny were determined by evaluating the gametes produced by each parent and combining thos e gametes to form progeny. The parent BL produced only two types of gametes Wms and wms and recombination would not be detected in gametes from this parent, sin ce a heterozygous condition existed only at the W locus; as a result, gametes produced from crossover events during meiosis in BL were

PAGE 206

189 genotypically identical to pare ntal-type gametes. Recombination was, however, detected in gametes from the parent BM, as BM wa s heterozygous at two of the three loci controlling flower color and floral morph. Th e two heterozygous loci of BM could have been linked either in coupling (domin ant alleles on the same chromosome WMs / wms ) or in repulsion (one dominant a llele on each chromosome i.e., Wms / wMs ). The linkage relationship or chromosomal arrangement of the dominant alleles of the linked loci determines the frequency of progeny types, as the genotypes of parental-type and recombinant gametes will differ based on the arrangement of the dominant alleles. If the W and M loci were linked in repulsion (i.e., Wms / wMs ), the most frequent gametes produced by BM would be Wms and wMs ; these gametes would fuse with gametes from BL to produce three types of F1 progeny: blue long ( Wms / Wms and Wms / wms ), blue mid ( wMs / Wms ) and white mid ( wMs / wms ). The parent BM would be expected to produce recombinant gametes ( WMs and wms ) less frequently; these gametes would fuse with gametes from BL to produce three types of F1 progeny: blue mid ( WMs / Wms and WMs / wms ), blue long ( wms / Wms ) and white long ( wms / wms ). This scenario with the W and M loci linked in repulsion is un likely, as the least frequent phenotype observed in BMBL was white mid (T able 9.1). If the loci controlling flower color and floral morph were linked in re pulsion, the white mid phenotype would have been produced at a high frequency, as it woul d have been derived from a parental-type gamete. If the W and M loci were linked in coupling (i.e., WMs / wms ), the most frequent gametes produced by BM would be WMs and wms ; these gametes would fuse with gametes from BL to produce three types of F1 progeny: blue mid ( WMs / Wms and

PAGE 207

190 WMs / wms ), blue long ( wms / Wms ) and white long ( wms / wms ). The parent BM would be expected to produce recombinant gametes ( Wms and wMs ) less frequently; these gametes would fuse with gametes from BL to produce three types of F1 progeny: blue long ( Wms / Wms and Wms / wms ), blue mid ( wMs / Wms ) and white mid ( wMs / wms ). Blue mid plants and blue long plants would be produced from both pa rental-type and recombinant gametes. White long plants would result only from parental-type gametes, while white mid plants would be derived only from r ecombinant gametes. The phenotypic class that was present in the lowest frequency in the F1 family BMBL was white mid (Table 9.1); it is therefore likely that the W and M loci were linked in c oupling in the parent BM. The frequency of progeny produced from r ecombinant gametes of BM provided an estimate of the genetic distance betw een the linked loci. A total of 91 F1 plants in the family BMBL were evaluated for flower color and floral morph; of these, one exhibited the recombinant white mid phenotype (Tab le 9.1). This phenotype represented one-quarter of all recombination events (recall the other three recombinant gametes produced offspring that were phenotypical ly identical to offspring produced by parental-type gametes); therefore, the map distance between the two loci should be approximately four times the observed fr equency of the phenotypically distinct recombinant F1 progeny. Based on these data, a r easonable estimate of the genetic distance between the W locus and the M locus would be 4(1/91) or 0.044 equivalent to 4.4 m.u.. Linkage in the F1 family WSBM. The second F1 family that segregated in a manner that was different from the ratios ex pected under the assumption of independence was coded WSBM and was derived from the parents WS and BM. The parent WS was

PAGE 208

191 homozygous recessive at the single lo cus responsible for flower color ( ww ), heterozygous at the epistatic S locus ( Ss ) and homozygous recessive at the hypostatic M locus ( mm ) controlling floral morph. The parent WS produced only two types of gametes wmS and wms and recombination was not detected sinc e a heterozygous condition existed only at the S locus; as a result, gametes produced fr om crossover events during meiosis were genotypically identical to pare ntal-type gametes. The parent BM was heterozygous for flower color ( Ww ), homozygous recessive at the epistatic S locus ( ss ) and heterozygous at the hypostatic M locus ( Mm ) controlling floral morph. Li nkage experiments are usually conducted with the understanding that progeny se gregated as expected when each trait was evaluated individually. Progeny that comprise the F1 family WSBM segregated as expected for flower color ( 50% blue flowers and 50% wh ite flowers); however, the family did not segregate as e xpected for floral morph and an excess of S-morph plants were produced (see Chapter 8 of this disserta tion). This difference made analysis of the family WSBM more complex; however, since the dominant epistatic S allele can only be transmitted by the parent WS in this cross-po llination, the ratios of parental-type and recombinant gametes produced by the parent BM could still be used to calculate an estimate of genetic distance with the information at hand. If the W and M loci were linked in repulsion (i.e., Wms / wMs ), the most frequent gametes produced by BM would be Wms and wMs ; these gametes would fuse with gametes from WS to produce four types of F1 progeny: blue short ( Wms / wmS ), blue long ( Wms / wms ), white short ( wMs / wmS ) and white mid ( wMs / wms ). The parent BM would be expected to produce recombinant gametes ( WMs and wms ) less frequently; these gametes would fuse with gametes from WS to produce four types of F1 progeny: blue short

PAGE 209

192 ( WMs / wmS ), blue mid ( WMs / wms ), white short ( wms / wmS ) and white long ( wms / wms ). This scenario with the W and M loci linked in repulsion is unlikely, as the least frequent phenotypes observed in WSBM were white mid a nd blue long (Table 9.1). If the loci controlling flower color and floral morph we re linked in repulsion, the white mid and blue long phenotypes would have been produ ced at a high frequency, since they would have been derived from parental-type gametes. If the W and M loci were linked in coupling (i.e., WMs / wms ), the most frequent gametes produced by BM would be WMs and wms ; these gametes would fuse with gametes from WS to produce four types of F1 progeny: blue short ( WMs / wmS ), blue mid ( WMs / wms ), white short ( wms / wmS ) and white long ( wms / wms ). The parent BM would be expected to produce recombinant gametes ( Wms and wMs ) less frequently; these gametes would fuse with gametes fr om WS to produce four types of F1 progeny: blue short ( Wms / wmS ), blue long ( Wms / wms ), white short ( wMs / wmS ) and white mid ( wMs / wms ). The blue short and white short phe notypes would be produced from both parental-type and recombinant gametes. Blue mid plants and white long plants would result only from parental-type gametes, wh ile the white mid and blue long phenotypes would be derived only from recombinant ga metes. This scenario corresponded well with the observed segregation of progeny in the fa mily BMBL (Table 9.1) and suggested that the W and M loci were indeed linked in coupling. The frequency of progeny produced from r ecombinant gametes of BM provided an estimate of the genetic distance betw een the linked loci. A total of 144 F1 plants in the family WSBM were evaluated for flower color and floral morph; of these, three exhibited the white mid phenotype and six produced the blue long phenotype (Table 9.1). These

PAGE 210

193 phenotypes represented half of all recombination events (recall the other two recombinant gametes produced offspring that were phenot ypically identical to offspring produced by parental-type gametes); therefore, the map distance between the linked loci should be approximately twice the observed frequency of the phenotypically distinct recombinant F1 progeny. A reasonable estimate of the genetic distance between the W locus and the M locus would be 2[(3+6)/144] or 0.125 equivalent to 12.5 m.u.. Goodness-of-fit tests were designed to test the two map distance estimates against observed progeny from both BMBL and WSBM. If the W and M loci were 12.5 m.u. apart as computed for the F1 family WSBM, the parent BM would be expected to produce parental-type gametes 87.5% of the time a nd recombinant gametes 12.5% of the time. These gametes would then fuse with gamete s from the parent WS to produce progeny with the phenotypic frequencies shown in Ta ble 9.2. The probability of recovering the array of segregating progeny seen in WSBM when 12.5 m.u. separate the W and M loci was 0.0139, so only about 1 in 70 populations would show as much variation as the F1 family WSBM. The poor fit of the model may seem questio nable, since the es timate of 12.5 m.u. was computed based on segregation of proge ny from this same family; however, recall that WSBM failed to segregate as expected for floral morph. Expected ratios were calculated based on the assumption that the tw o gamete types from WS were produced in equal frequencies, but an ex cess of S-morph progeny were produced. The reason for this excess of S-morph progeny was cryptic but not entirely unexpected, as other workers (e.g., Barrett et al. 1983; Morgan and Ba rrett 1988; Wolfe and Barrett 1989) have reported that an excess of S-morph progeny wa s often observed in na tural populations of

PAGE 211

194 pickerelweed. Adjusted expected ratios we re calculated based on the frequency of recovered S-morph progeny; this seemed reasonable since the only locus undergoing segregation in WS was the S locus. If ga mete production in WS were normal, each gamete type ( wmS and wms ) would be produced at a frequency of 0.50. Examination of the F1 family WSBM revealed that 60.4% of the progeny were S-morph and 39.6% of the progeny were M-morph and L-morph. Thes e data suggested that the gamete wmS was produced at a frequency of 0.604 and the gamete wms was produced at a frequency of 0.396; therefore, these gametic frequencies we re used to calculate adjusted expected ratios for the F1 family WSBM. The probability of recovering the array of segregating progeny seen in WSBM when 12.5 m.u. separate the W and M loci and when gametes were produced by WS in the frequencies of 0.604 ( wmS ) and 0.396 ( wms ) was 0.1271, so as many as 1 in 8 populations would be exp ected to show as mu ch variation as the F1 family WSBM (Table 9.2). This model provide d a better fit to the data and suggested that the estimate of 12.5 m.u. between the W and M loci was reasonable given the genotypic constitution of the population under investigation. The probability of recovering the array of segregating progeny seen in BMBL when 12.5 m.u. separate the W and M loci was 0.6531, so as many as 65 in 100 populations would be expected to sh ow as much variation as the F1 family BMBL (Table 9.3). These results s uggested that the model provid ed a good fit to the data and that the estimate of 12.5 m.u. between the W and M loci fell within th e range of genetic distances that would produ ce segregating progeny as seen in the family BMBL. If the W and M loci were 4.4 m.u. apart as computed for the F1 family BMBL, 95.6% of gametes from BM would be parental types and the remaining 4.4% of gametes

PAGE 212

195 would be recombinant types. These gametes would then fuse with gametes from the parent WS (for the family WSBM) or the pa rent BL (for the family BMBL) to produce progeny with the phenotypic frequencies shown in Table 9.4. The probability of recovering the array of segregating progeny seen in WSBM if the W and M loci were 4.4 m.u. apart was 0.0002, so only 2 in 10,000 populations would be expected to show as much variation as the F1 family WSBM. The fact that this model provided a poor fit to the data suggested that the genetic distance between the W and M loci was greater than the 4.4 m.u. tested w ith this model and that the estimate of 12.5 m.u. may have more accurately repr esented the relati onship between the W and M loci in the parent BM. The probability of recovering the array of segregating progeny seen in BMBL when 4.4 m.u. separate the W and M loci was 0.5287, so as many as 53 in 100 populations would be expected to sh ow as much variation as the F1 family BMBL (Table 9.4). This suggested that the model provided a good f it to the data and that the estimate of 4.4 m.u. between the W and M loci fell within the range of genetic distances that would produce segregating progeny as seen in the family BMBL. Estimates of map distance between the linked W and M loci. The goal of this experiment was to determine the genetic distance between the W and M loci; therefore, additional goodness-of-fit tests we re performed to identify the map distance that provided the best fit to both families exhibiting the e ffects of linkage. In the interest of brevity, only chi-square values and probabilities for these tests are presented in Table 9.5. The segregation of progeny from WSBM was not different from the ratios expected when the genetic distance between the W and M loci ranged from 10 m.u. to 20 m.u., with the best

PAGE 213

196 fit provided by a model where the linked loci were 16 m.u. apart. The segregation of progeny from BMBL was not di fferent from the ratios expected when the genetic distance between the W and M loci ranged from 4.4 m.u. to 22 m.u., with the best fit provided by a model where the linke d loci were 10 m.u. apart. Linkage in F2 families. Segregating F2 progeny were recovered from each F1 family and were subjected to anal yses like those described for the F1 families WSBM and BMBL. Some F2 families failed to produce progeny with one or two rare phenotypes. The analyses for these families were adjust ed to account for the missing phenotypic class (or classes) by dividing the expected freque ncy of each recovered phenotypic class by the sum of the expected frequencies of all observed phenotypic classes. This allowed comparison of the observed phenotypic freque ncies with frequenc ies that would be expected if the phenotypes that were actua lly recovered comprise d the entire population. The frequency of each expected phenotypic cl ass was computed using a genetic distance of 16 m.u. between the W and M loci, since this estimate prov ided a good fit to data from the F1 families WSBM and BMBL. Linkage in the F2 family WMBL. The genotypic constitutions of the parents of the F1 family WMBL were wMs / wms (for the parent WM) and Wms / wms (for the parent BL). Four types of F1 progeny were produced: blue mid ( Wms / wMs ), blue long ( Wms / wms ), white mid ( wms / wMs ) and white long ( wms / wms ). Linkage was not evident in the F1 generation as each parent was heterozygo us at only one locus; however, linkage was revealed in the F2 generation. Six F1 plants with the blue mid phenotype were self-pollinated and all produced F2 families that segregated for both flower color and floral morph (Table 9.6). All F2 families segregated as expected for each trait

PAGE 214

197 individually. All blue mid F1 plants were genotypically id entical and heterozygous at the W and M loci, with dominant alleles li nked in repulsion (i.e., genotype Wms / wMs ). Each F1 plant was expected to produce four types of gametes: parental-type gametes Wms and wMs at a frequency of 0.42 each and recombinant gametes WMs and wms at a frequency of 0.08 each. These gamete s would fuse to produce progeny with one of ten different genotypes. Five genotypes ( Wms / wMs Wms / WMs wMs / WMs WMs / WMs and WMs / wms ) were associated with the blue mid phenotype, which represented 50.64% of the F2 population. Two genotypes ( Wms / Wms and Wms / wms ) produced the blue long phenotype and two genotypes ( wMs / wMs and wMs / wms ) resulted in the white mid phenotype; each of these phenotypes accounted for 24.36% of the population. The white long phenotype was produced by progeny with the genotype wms / wms and composed 0.64% of the F2 population. Three of the six F2 families under investigation (BM2, BM5 and BM6) segregated as expected when compared to a model where 16 m.u. separate the B and M loci (Table 9.6). The family BM3 failed to produ ce any white long progeny; however, fewer than one offspring with this phenotype woul d be expected in a population the size of BM3 (n=59). The analysis for BM3 was adjusted to account for the missing phenotypic class by dividing the expected frequency of each recovered phenotypic class by the sum of the expected frequencies of all observed phenotypic classes. The F2 family BM3 segregated as expected when compared to th is adjusted model (Table 9.6). The families BM1 and BM4 each segregated in a manner that differed from the proposed model; however, in both families the difference was slig ht and a large proportion of the variation was attributable to segregants in the white long phenotypic class. Each family produced

PAGE 215

198 three progeny with the white long phenotype but fewer than one was expected in each family (0.4864 in the family BM1 and 0.7552 in the family BM4) (Table 9.6). This discrepancy seems small but had a profound eff ect on the chi-square value derived from the goodness-of-fit test; for example, if only two white long progeny instead of three had been produced in each family the segregation of observed progeny would not have been different from the model. Some of the F2 families from the F1 family WMBL did not segregate as expected, but this was most likely due to sampling e rror. The majority of the data from these F2 families supported the proposed m odel where 16 m.u. separate the W and M loci. Linkage in the F2 family WSBL. The genotypic constitutions of the parents of the F1 family WSBL were wmS / wms (for the parent WS) and Wms / wms (for the parent BL). Four types of F1 progeny were produced: blue short ( Wms / wmS ), blue long ( Wms / wms ), white short ( wms / wmS ) and white long ( wms / wms ). Linkage was not evident in the F1 generation as each parent was heterozygous at only one locus; in addition, linkage between the W and M loci was not revealed in the F2 generation, as the blue short F1 plants self-pollinated to create the F2 families were homozygous recessive at the M locus. Two F1 plants with the blue short pheno type were self-pollinated and both produced F2 families that segregated for flower co lor and floral morph (Table 9.7). Both F2 families segregated as expected for each trait individually. Both blue short F1 plants were genotypically identical and heterozygous at the W and S loci, with dominant alleles in repulsion (i.e., genotype Wms / wmS ). Each F1 plant was expected to produc e four types of gametes Wms wmS WmS and wms in equal frequencies. These gamete s would fuse to produce progeny with one

PAGE 216

199 of ten different genotypes. Five genotypes ( Wms / wmS Wms / WmS wmS / Wms WmS / WmS and WmS / wms ) were associated with the blue sh ort phenotype, which represented 56.25% of the F2 population. Two genotypes ( Wms / Wms and Wms / wms ) produced the blue long phenotype and two genotypes ( wmS / wmS and wmS / wms ) resulted in the white short phenotype; each of these phenotypes accounted for 18.75% of the population. The white long phenotype was produced by progeny with the genotype wms / wms and composed 6.25% of the F2 population. Both F2 families under investigation segregated as expected when compared to a model where 16 m.u. separate the W and M loci (Table 9.7). These data neither supported nor refuted the model where 16 m.u. separate the W and M loci; they did, however, provide evidence that the epistatic S locus responsible for floral morph was independent from the W locus. Linkage in the F2 family WSBM. The genotypic constitutions of the parents of the F1 family WSBM were wmS / wms (for the parent WS) and WMs / wms (for the parent BM). Six types of F1 progeny were produced: blue short ( WMs / wmS and Wms / wmS ), blue mid ( WMs / wms ), blue long ( Wms / wms ), white short ( wmS / wms and wMs / wmS ), white mid ( wMs / wms ) and white long ( wms / wms ). Linkage was evident in the F1 generation and was described in detail above; in additi on, linkage was also revealed in the F2 generation. Three F1 plants with the blue short phenotype and two F1 plants with the blue mid phenotype were self-pollinated and all produced F2 families that segregated for both flower color and floral morph (T able 9.8). Four of these five F2 families segregated as expected for each trait individually, while the family BM2 did not segregate as expected for either trait.

PAGE 217

200 The F1 blue short phenotype was pro duced by two different genotypes WMs / wmS and Wms / wmS Self-pollination of plants with the genotype WMs / wmS would have created populations with all three floral mor phs, while self-pollination of plants with the genotype Wms / wmS would be expected to generate pr ogeny with either the S-morph or L-morph phenotype. All three F2 families from blue short F1 plants had members with the M-morph phenotype; therefore, all blue short F1 plants in this experiment were genotypically identical and heterozygous at all three loci, with the dominant W and M alleles linked in coupling (i.e., genotype WMs / wmS ). Each F1 plant was expected to produce eight types of gametes: parental-type gametes WMs WMS wmS and wms at a frequency of 0.21 each and recombinant gametes Wms WmS wMS and wMs at a frequency of 0.04 each. These gametes would fuse to produce progeny with one of 36 differ ent genotypes. Nineteen genotypes ( WMs / wmS WMs / WmS WMs / WMS WMs / wMS wmS / WmS wmS / WMS wmS / Wms WmS / WmS WmS / wMs WmS / WMS WmS / wms WmS / Wms WmS / wMS wMs / WMS WMS / WMS WMS / wms WMS / Wms WMS / wMS and Wms / wMS ) were associated with the blue short phenotype, which represented 56.25% of the F2 population. The blue mid phenotype composed 16.91% of the population and was derived from five different genotypes ( WMs / WMs WMs / wMs WMs / wms WMs / Wms and wMs / Wms ). Two genotypes ( Wms / Wms and Wms / wms ) produced the blue long phenotype and two genotypes ( wMs / wMs and wMs / wms ) resulted in the white mid phenotype; each of these phenotypes accounted for 1.84% of the population. The whit e short phenotype was associated with seven genotypes ( wmS / wmS wmS / wMs wmS / wms wmS / wMS wMs / wMS wms / wMS and wMS / wMS ) and was 18.75% of the population, wh ile the white long phenotype was

PAGE 218

201 produced by progeny with the genotype wms / wms and composed 4.41% of the F2 population. The F2 family BS2 segregated as expected when compared to a model where 16 m.u. separate the W and M loci (Table 9.8). The family BS4 failed to produce any blue long progeny; however, fewer than two offspri ng with this phenotype would be expected in a population the size of BS4 (n=88). The analysis for BS4 was adjusted to account for the missing phenotypic class by dividing the expected frequency of each recovered phenotypic class by the sum of the expected frequencies of all observed phenotypic classes. The F2 family BS4 segregated as expected wh en compared to this adjusted model (Table 9.8). The family BS6 did not pr oduce any white mid progeny; however, fewer than one offspring with this phenotype woul d be expected in a population the size of BS6 (n=51). The analysis for BS6 was adjusted in the same manner as described for BS4; the family BS6 then segregated as expected when compared to this adjusted model (Table 9.8). Both F2 families from blue mid F1 plants were genotypically identical and heterozygous at the W and M loci, with the dominant W and M alleles linke d in coupling (i.e., genotype WMs / wms ). Each F1 plant was expected to pro duce four types of gametes: parental-type gametes WMs and wms at a frequency of 0.42 each and recombinant gametes Wms and wMs at a frequency of 0.08 each. These gametes would fuse to produce progeny with one of ten differe nt genotypes. Five genotypes ( WMs / WMs WMs / wms WMs / Wms WMs / wMs and Wms / wMs ) were associated with the blue mid phenotype, which represented 67.64% of the F2 population. Two genotypes ( wms / Wms and Wms / Wms ) produced the blue long phe notype and two genotypes ( wms / wMs and

PAGE 219

202 wMs / wMs ) resulted in the white mid phenotype ; each of these phe notypes accounted for 7.36% of the population. The white long phenot ype was produced by progeny with the genotype wms / wms and composed 17.64% of the F2 population. The F2 family BM1 segregated as expected when compared to a model where 16 m.u. separate the W and M loci (Table 9.8). The family BM2 failed segregate as expected; however, this family did not segregate as expected for either flower color or floral morph when these traits were evalua ted individually. Expected segregation ratios were calculated with the assu mption that the population under i nvestigation segregated as expected for both of the single traits; since this did not occur in the family BM2, it was unlikely that the family would conform to th e segregation ratios expected when alleles were equally represented. The families BS2 and BM1 segregated as expected when compared to a model where 16 m.u. separate the W and M loci. The families BS4 and BS6 failed to produce progeny in rare phenotypic classes; however, fewer than two offspring with the rare phenotypes were expected in each family and an alyses of adjusted frequencies for each family revealed that progeny segregated as expected when compared to the adjusted models. The family BM2 segregated in a manner that differed from the proposed model; however, this family failed to segregate as expected for both flow er color and floral morph individually so it was unlikely that the progeny would conform to a model that assumed equal representation of the alleles for each trait. Some of the F2 families from the F1 family WSBM did not segregate as exp ected, but this was most likely due to sampling error. The majority of the data from these F2 families supported the proposed model where 16 m.u. separate the W and M loci.

PAGE 220

203 Linkage in the F2 family WMBS. The genotypic constitutions of the parents of the F1 family WMBS were wMs / wms (for the parent WM) and WMS / WMs (for the parent BS). Two types of F1 progeny were produced: blue short ( WMS / wMs and WMS / wms ) and blue mid ( WMs / wMs and WMs / wms ). Linkage was not evident in the F1 generation as each parent was heterozygous at only one locus and all F1 progeny had blue flowers; however, linkage was revealed in the F2 generation. Seven F1 plants with the blue short phenotype and four F1 plants with the blue mid phenotype were self-pollinated; all produced F2 families that segregated for both flower color and floral morph (Table 9.9). All eleven F2 families segregated as expected for flower color and for floral morph individually. F1 plants with the blue short phenotype were produced by two different genotypes WMS / wMs and WMS / wms Self-pollination of plants with the genotype WMS / wMs would have created a population with progeny b earing either the S-morph or M-morph phenotype, while self-pollination of plants with the genotype WMS / wms would be expected to create populations with all three floral morphs. Three F2 families (BS2, BS5 and BS6) from blue short F1 plants were composed of progeny with either the S-morph or M-morph phenotype; therefore, the F1 parents of the F2 families BS2, BS5 and BS6 were genotypica lly identical and heterozygous at the W and S loci, with the dominant W and S alleles in coupling an d homozygous dominant at the M locus (i.e., genotype WMS / wMs ). Each of these F1 plants was expected to produce four types of gametes ( WMS WMs wMs and wMS ) in equal frequencies. These gametes would fuse to produce progeny with one of ten different genotypes. Five genotypes ( WMS / WMS WMS / wMs WMS / WMs WMS / wMS and WMs / wMS ) were associated with

PAGE 221

204 the blue short phenotype, whic h represented 56.25% of the F2 population. Two genotypes ( wMs / WMs and WMs / WMs ) produced the blue mid phenotype and two genotypes ( wMs / wMS and wMS / wMS ) resulted in the white shor t phenotype; each of these phenotypes accounted for 18.75% of the popul ation. The white mid phenotype was produced by progeny with the genotype wMs / wMs and composed 6.25% of the F2 population. All three F2 families derived from F1 plants with the genotype WMS / wMs segregated as expected when compared to a model where 16 m.u. separate the W and M loci (Table 9.9). These data neither s upported nor refuted the model where 16 m.u. separate the W and M loci; they did, however, provide additional evidence that the epistatic S locus responsible for floral morph was independent from the W locus. The remaining four F2 families from blue short F1 plants (BS1, BS3, BS4 and BS7) were composed of progeny that represented all three floral morphs; therefore, these families were derived from self-pollination of blue short F1 plants with the genotype WMS / wms The F1 parents of these F2 families were genotypically identical and heterozygous at all three loci, with the dominant W and M alleles linked in coupling (i.e., genotype WMS / wms ). Each F1 plant was expected to produce eight types of gametes: parental-type gametes WMs WMS wmS and wms at a frequency of 0.21 each and recombinant gametes Wms WmS wMS and wMs at a frequency of 0.04 each. These gametes would fuse to produce progeny with one of 36 differ ent genotypes. Nineteen genotypes ( WMs / wmS WMs / WmS WMs / WMS WMs / wMS wmS / WmS wmS / WMS wmS / Wms WmS / WmS WmS / wMs WmS / WMS WmS / wms WmS / Wms WmS / wMS wMs / WMS WMS / WMS

PAGE 222

205 WMS / wms WMS / Wms WMS / wMS and Wms / wMS ) were associated with the blue short phenotype, which represented 56.25% of the F2 population. The blue mid phenotype composed 16.91% of the population and was derived from five different genotypes ( WMs / WMs WMs / wMs WMs / wms WMs / Wms and wMs / Wms ). Two genotypes ( Wms / Wms and Wms / wms ) produced the blue long phenotype and two genotypes ( wMs / wMs and wMs / wms ) resulted in the white mid phenotype; each of these phenotypes accounted for 1.84% of the population. The whit e short phenotype was associated with seven genotypes ( wmS / wmS wmS / wMs wmS / wms wmS / wMS wMs / wMS wms / wMS and wMS / wMS ) and was 18.75% of the population, wh ile the white long phenotype was produced by progeny with the genotype wms / wms and composed 4.41% of the F2 population. The F2 family BS1 segregated as expected when compared to a model where 16 m.u. separate the W and M loci (Table 9.9). The families BS3, BS4 and BS7 failed to produce any blue long or white mid progeny; however, fewer than one offspring with each of these phenotypes would be expected in populations the size of these families (BS3 n=17, BS4 n=62, BS7 n=49). The analyses for BS3, BS4 and BS7 were adjusted to account for the missing phenotypic classes by di viding the expected frequency of each recovered phenotypic class by the sum of th e expected frequencies of all observed phenotypic classes. The F2 families BS3, BS4 and BS7 segregated as expected when compared to these adjusted models (Table 9.9). F1 plants with the blue mid phenotype we re produced by two different genotypes WMs / wMs and WMs / wms Self-pollination of plants with the genotype WMs / wMs would be expected to create popul ations composed entirely of progeny with the M-morph

PAGE 223

206 phenotype, while self-pollination of plants with the genotype WMs / wms would have resulted in populations with progeny b earing M-morphs and L-morphs. The four F2 families from blue mid F1 plants examined in this experiment were composed of progeny with either the mid or long phenotype; therefore, the F1 parents of the F2 families BM1, BM2, BM3 and BM6 were genotypically identical and he terozygous at the W and M loci, with the dominant W and M alleles linked in coupling (i.e., genotype WMs / wms ). Each F1 plant was expected to produce four types of gametes: parental-type gametes WMs and wms at a frequency of 0.42 each and recombinant gametes Wms and wMs at a frequency of 0.08 each. These gamete s would fuse to produce progeny with one of ten different genotypes. Five genotypes ( WMs / WMs WMs / wms WMs / Wms WMs / wMs and Wms / wMs ) were associated with the blue mi d phenotype, which represented 67.64% of the F2 population. Two genotypes ( wms / Wms and Wms / Wms ) produced the blue long phenotype and two genotypes ( wms / wMs and wMs / wMs ) resulted in the white mid phenotype; each of these phenotypes accounted for 7.36% of the population. The white long phenotype was produced by progeny with the genotype wms / wms and composed 17.64% of the F2 population. All four families from blue mid F1 plants segregated as expected when compared to a model where 16 m.u. separate the W and M loci (Table 9.9). These data provided additional support for the proposed model where 16 m.u. separate the W and M loci. The families BS1, BS2, BS5, BS6, BM1, BM2, BM3 and BM6 segregated as expected when compared to a model where 16 m.u. separate the W and M loci. The families BS3, BS4 and BS7 failed to produce progeny in rare phenotypic classes; however, fewer than two offspring with the rare phenotypes would be expected in each

PAGE 224

207 family and analyses of adjusted frequenc ies for each family revealed that progeny segregated as expected when compared to th e adjusted models. The majority of the data from these F2 families supported the proposed m odel where 16 m.u. separate the W and M loci. Linkage in the F2 family BSBM. The genotypic constitutions of the parents of the F1 family BSBM were WMS / WMs (for the parent BS) and WMs / wms (for the parent BM). Two types of F1 progeny were produced: blue short ( WMS / WMs WMS / wms WMS / Wms and WMS / wMs ) and blue mid ( WMs / WMs WMs / wms WMs / Wms and WMs / wMs ). Linkage was not evident in the F1 generation as all F1 progeny had blue flowers; however, linkage was revealed in the F2 generation. Four F1 plants with the blue s hort phenotype and three F1 plants with the blue mid phenotype were self-pollinated and all produced F2 families that segregated for both flower color and floral morph (T able 9.10). Five of the seven F2 families segregated as expected for flower color and fo r floral morph individually. The F2 family BS2 segregated as expected for flower color but did not segregate as expected for floral morph, while the F2 family PBS1 segregated as exp ected for floral morph but did not segregate as expected for flower color. F1 plants with the blue short phenotype were produced by four different genotypes WMS / WMs WMS / wms WMS / Wms and WMS / wMs Self-pollination of plants with the genotypes WMS / WMs and WMS / Wms would be expected to cr eate populations with only blue-flowered progeny and would therefore not be useful in this linkage study. Plants with the genotype WMS / wMs would produce progeny with both flower colors and S-morphs and M-morphs, while plants with the genotype WMS / wms would create a

PAGE 225

208 population with both flower colors a nd all three floral morphs. The F2 family BS2 was composed of progeny with either the Smorph or M-morph; therefore, the F1 parent of the F2 family BS2 was heterozygous at the W and S loci, with the dominant W and S alleles in coupling and homozygous dominant at the M locus (i.e., genotype WMS / wMs ). The F1 parent of the F2 family BS2 was expected to produce four types of gametes ( WMS WMs wMs and wMS ) in equal frequencies. These gametes would fuse to produce progeny with one of ten differe nt genotypes. Five genotypes ( WMS / WMS WMS / wMs WMS / WMs WMS / wMS and WMs / wMS ) were associated with the blue short phenotype, which represented 56.25% of the F2 population. Two genotypes ( wMs / WMs and WMs / WMs ) produced the blue mid phe notype and two genotypes ( wMs / wMS and wMS / wMS ) resulted in the white short phenotype ; each of these phenotypes accounted for 18.75% of the population. The wh ite mid phenotype was pro duced by progeny with the genotype wMs / wMs and composed 6.25% of the F2 population. The F2 family BS2 failed to segregate as expected when compared to a model where 16 m.u. separate the W and M loci (Table 9.10); howev er, this family did not segregate as expected when floral morph was considered individually and the M-morph was produced in excess when compared to the model where f( S ) = f( s ) = 0.50. The reason for this production of fewer gametes with the dominant allele S and an excess of gametes with the recessive allele s was unknown; however, it seemed appropriate to adjust the gametic frequencies in this family to more accurately reflect the ge notypic constitution of the population. The Hardy-Weinberg equation was used to de termine the frequency of the alleles in the population based on the segregation of observed progeny. The frequency of the

PAGE 226

209 recessive allele s was derived by examining the freque ncy of members of the phenotypic class produced by a homozygous recessive condi tion; in this case, the M-morph resulted from the genotype ss The frequency of the genotype ss was equal to 44/110; the square root of this ratio revealed that th e frequency of the recessive allele s was 0.6325 and therefore the frequency of the dominant allele S was 0.3675. The family BS2 was homo zygous dominant at the M locus, so gametes with the genotypes WM and wM were produced in equal frequenc ies. The frequencies of these gametes were multiplied by the frequencies of the S and s alleles to determine the expected frequency of each gamete type. Th is resulted in four types of gametes: WMS and wMS at a frequency of (0.50 )(0.3675) or 0.18375 each and WMs and wMs at a frequency of (0.50)(0.6325) or 0.31625 each. These gametes would fuse to produce progeny with one of ten differe nt genotypes. Five genotypes ( WMS / WMS WMS / wMs WMS / WMs WMS / wMS and WMs / wMS ) were associated with the blue short phenotype, which represented 44.996% of the F2 population. Two genotypes ( wMs / WMs and WMs / WMs ) produced the blue mid phenotype and accounted for 30.004% of the population, while two genotypes ( wMs / wMS and wMS / wMS ) resulted in the white short phenotype, which composed 14.998% of the population. The white mid phenotype was produced by progeny with the genotype wMs / wMs and composed 1.0001% of the F2 population. The F2 family BS2 segregated as expected when compared to a model where 16 m.u. separate the W and M loci and where f( S ) = 0.3675 and f( s ) = 0.6325 (Table 9.10). These data neither supported nor refu ted the model where 16 m.u. separate the W

PAGE 227

210 and M loci; they did, however, provide a dditional evidence that the epistatic S locus responsible for floral mor ph was independent from the W locus. The remaining three F2 families derived from blue short F1 plants were composed of progeny that represented all three floral morphs; therefore, these families were derived from self-pollination of genotypically identical blue short F1 plants that were heterozygous at all three loci, with the dominant W and M alleles linked in coupling (i.e., genotype WMS / wms ). Each F1 plant was expected to produce eight types of gametes: parental-type gametes WMs WMS wmS and wms at a frequency of 0.21 each and recombinant gametes Wms WmS wMS and wMs at a frequency of 0.04 each. These gametes would fuse to produce progeny with one of 36 differ ent genotypes. Nineteen genotypes ( WMs / wmS WMs / WmS WMs / WMS WMs / wMS wmS / WmS wmS / WMS wmS / Wms WmS / WmS WmS / wMs WmS / WMS WmS / wms WmS / Wms WmS / wMS wMs / WMS WMS / WMS WMS / wms WMS / Wms WMS / wMS and Wms / wMS ) were associated with the blue short phenotype, which represented 56.25% of the F2 population. The blue mid phenotype composed 16.91% of the population and was derived from five different genotypes ( WMs / WMs WMs / wMs WMs / wms WMs / Wms and wMs / Wms ). Two genotypes ( Wms / Wms and Wms / wms ) produced the blue long phenotype and two genotypes ( wMs / wMs and wMs / wms ) resulted in the white mid phenotype; each of these phenotypes accounted for 1.84% of the population. The whit e short phenotype was associated with seven genotypes ( wmS / wmS wmS / wMs wmS / wms wmS / wMS wMs / wMS wms / wMS and wMS / wMS ) and was 18.75% of the population, wh ile the white long phenotype was

PAGE 228

211 produced by progeny with the genotype wms / wms and composed 4.41% of the F2 population. Each of the three F2 families from completely heterozygous blue short F1 plants failed to produce rare progeny (Table 9.10). The family PBS1 did not produce any blue long progeny, but only one offspring with th e blue long phenotype was expected in a population the size of PBS1 (n=56), so this discrepancy was most likely due to sampling error. The families PBS3 and PBS4 both failed to produce white mid and white long progeny; however, fewer than one offspri ng with each of these phenotypes would be expected in families this size (PBS3 n=20, PBS4 n=40), so this difference may have been attributable to sampling error as well. Th e analyses for PBS1, PBS3 and PBS4 were adjusted to account for the missing phenotypic class by dividing the expected frequency of each recovered phenotypic class by the sum of the expected frequencies of all observed phenotypic classes. The F2 families PBS3 and PBS4 segregated as expected when compared to adjusted models where 16 m.u. separate the W and M loci (Table 9.10). The family PBS1 still did not segregate as expected; however, this fa mily did not segregate as expected when flower color was considered individua lly and the white-flowered phenotype was produced in excess when compared to the model where f( W ) = f( w ) = 0.50. The reason for this production of fewer gametes with the dominant allele W and an excess of gametes with the recessive allele w is unknown; however, as with the family BS2, it seemed appropriate to adjust the gametic frequencies in this family to more accurately reflect the genotypic constitution of the population. The frequency of the recessive allele w was derived by examining the frequency of me mbers of the phenotypic class produced by a

PAGE 229

212 homozygous recessive condition; in this cas e, the white-flowered phenotype resulted from the genotype ww The frequency of the genotype ww was equal to 24/56; the square root of this ratio revealed that th e frequency of the recessive allele w is 0.65465 and therefore the frequency of the dominant allele W is 0.34535. The M and S loci are presumed to be independent, so gametes with the genotypes MS Ms ms and mS would be produced in equal freque ncies. The frequencies of these gametes were multiplied by the frequencies of the W and w alleles to determine the expected frequency of each gamete type. Th is resulted in eight types of gametes: parental-type gametes WMS and WMs at a frequency of (0.34535)(0.50)(0.84) or 0.145047 each, parental-type gametes wms and wmS at a frequency of (0.65465)(0.50)(0.84) or 0.274953 each, recombinant gametes WmS and Wms at a frequency of (0.34535)(0.50)(0.16) or 0.027628 each and recombinant gametes wMs and wMS at a frequency of (0.65465)(0.50)(0.16) or 0.052372 each. These gametes would fuse to produce progeny with one of 36 different genotypes. Nineteen genotypes ( WMs / wmS WMs / WmS WMs / WMS WMs / wMS wmS / WmS wmS / WMS wmS / Wms WmS / WmS WmS / wMs WmS /WMS WmS / wms WmS / Wms WmS / wMS wMs / WMS WMS / WMS WMS / wms WMS / Wms WMS / wMS and Wms / wMS ) were associated with the blue short phenotype, which represented 42.853% of the F2 population. The blue mid phenotype composed 12.679% of the population and was derived from five different genotypes ( WMs / WMs WMs / wMs WMs / wms WMs / Wms and wMs / Wms ). Two genotypes ( Wms / Wms and Wms / wms ) produced the blue long phenotype and accounted for 1.595% of the population, while two genotypes ( wMs / wMs and wMs / wms ) resulted in the white mid phenotype and composed 3.154% of the population. The white short

PAGE 230

213 phenotype was associated with seven genotypes ( wmS / wmS wmS / wMs wmS / wms wmS / wMS wMs / wMS wms / wMS and wMS / wMS ) and was 31.943% of the population, while the white long phenotype was pr oduced by progeny with the genotype wms / wms and composed 7.599% of the F2 population. Recall that the family PBS1 failed to produ ce any blue long proge ny; therefore, the analysis was adjusted to account for the missing phenotypic class by dividing the expected frequency of each recovered phe notypic class by the sum of the expected frequencies of all observed phenotypic classes. The F2 family PBS1 segregated as expected when compared to this adjusted model where f( W ) = 0.34535 and f( w ) = 0.65465 and where 16 m.u. separate the W and M loci (Table 9.10). F1 plants with the blue mid phenotype were produced by four different genotypes WMs / WMs WMs / wms WMs / Wms and WMs / wMs Self-pollination of plants with the genotypes WMs / WMs and WMs / Wms would be expected to cr eate populations with only blue-flowered progeny and would therefore not be useful in this linkage study. Self-pollination of plants with the genotype WMs / wMs would be expected to produce progeny with both flower colors but only M-morphs, while self -pollination of plants with the genotype WMs / wms would be expected to create popul ations with both flower colors and M-morphs and L-morphs. All three F2 families from blue mid F1 plants were composed of M-morph and L-morph progeny; therefore, the F1 parents of the F2 families BM3, PBM1 and PBM3 were genotypically identical and heterozygous at the W and M loci, with the dominant W and M alleles linked in coupling (i.e., genotype WMs / wms ). These F1 plants were expected to produce parental-type gametes WMs and wms at a frequency of 0.42 each and recombinant gametes Wms and wMs at a frequency of 0.08

PAGE 231

214 each. These gametes would fuse to produce prog eny with one of ten different genotypes. Five genotypes ( WMs / WMs WMs / wms WMs / Wms WMs / wMs and Wms / wMs ) were associated with the blue mid phenotype which represented 67.64% of the F2 population. Two genotypes ( wms / Wms and Wms / Wms ) produced the blue long phenotype and two genotypes ( wms / wMs and wMs / wMs ) resulted in the white mid phenotype; each of these phenotypes accounted for 7.36% of the popul ation. The white long phenotype was produced by progeny with the genotype wms / wms and composed 17.64% of the F2 population. All three families from blue mid F1 plants segregated as expected when compared to a model where 16 m.u. separate the W and M loci (Table 9.10). The families PBS3, PBS4, BM3, PBM1 and PBM3 segregated as expected when compared to a model where 16 m.u. separate the W and M loci. The family BS2 did not segregate as expected; however, this family failed to segregate as expected for floral morph and did segregate as e xpected when compared to an adjusted model where 16 m.u. separate the W and M loci and where f( S ) = 0.3675 and f( s ) = 0.6325. The family PBS1 failed to segregate as expected; however, this family failed to segregate as expected for flower color and did segregate as expected when compared to a model where 16 m.u. separate the W and M loci and where f( W ) = 0.34535 and f( w ) = 0.65465. The majority of the data from these families supported the proposed model where 16 m.u. separate the W and M loci and provided additional evidence that the epistatic S locus is independent from the W locus controlling flower color. Linkage in the F2 family BSBL. The genotypic constitutions of the parents of the F1 family BSBL were WMS / WMs (for the parent BS) and Wms / wms (for the parent BL).

PAGE 232

215 Two types of F1 progeny were produced: blue short ( WMS / Wms and WMS / wms ) and blue mid ( WMs / Wms and WMs / wms ). Linkage was not evident in the F1 generation, as all F1 progeny had blue flowers; however linkage was revealed in the F2 generation. Four F1 plants with the blue mid phenotype were self-pollinated and all produced F2 families that segregated for both flower co lor and floral morph (Table 9.11). Three of the four F2 families segregated as expected for each trait individually. The F2 family UVM2 segregated as expected for flower colo r, but did not segreg ate as expected for floral morph. F1 plants with the blue mid phenotype we re produced by two different genotypes WMs / Wms and WMs / wms Self-pollination of plants with the genotype WMS / Wms would create populations with only bl ue-flowered progeny and would therefore not be useful in this linkage study; however, self-polli nation of plants with the genotype WMs / wms would create populations with both flower colors and both M-morphs and L-morphs. The F1 parents of the F2 families UVM1, BM1, BM2 and BM3 were genotypically identical and heterozygous at the W and M loci, with the dominant W and M alleles linked in coupling (i.e., genotype WMs / wms ). These F1 plants were expected to produce parental-type gametes WMs and wms at a frequency of 0.42 each and recombinant gametes Wms and wMs at a frequency of 0.08 each. These gametes would fuse to produce prog eny with one of ten different genotypes. Five genotypes ( WMs / WMs WMs / wms WMs / Wms WMs / wMs and Wms / wMs ) were associated with the blue mid phenotype which represented 67.64% of the F2 population. Two genotypes ( wms / Wms and Wms / Wms ) produced the blue long phenotype and two genotypes ( wms / wMs and wMs / wMs ) resulted in the white mid phenotype; each of these

PAGE 233

216 phenotypes accounted for 7.36% of the popul ation. The white long phenotype was produced by progeny with the genotype wms / wms and composed 17.64% of the F2 population. The F2 families BM1 and BM2 segregated as expected when compared to a model where 16 m.u. separate the W and M loci, but the F2 families UVM2 and BM3 did not segregate as expected when compared to th e same model (Table 9.11). The reason for the poor fit of the family BM3 to the model was unknown, as the family segregated as expected for both flower color and floral morph individually; however, the family UVM2 did not segregate as expected when floral morph was considered individually and the L-morph was produced in excess when compared to the model where f( M ) = f( m ) = 0.50. The reason for this production of fewer gametes with the dominant allele M and an excess of gametes with the recessive allele m was unknown; however, it again seemed appropriate to adjust the gametic frequencies in this family to more accurately reflect the genotypic constitution of the population. The frequency of the recessive allele m was derived by examining the frequency of me mbers of the phenotypic class produced by a homozygous recessive condition; in this case, the L-morph phenotype resulted from the genotype mm The frequency of the genotype mm was equal to 33/89; the square root of this ratio revealed that the fr equency of the recessive allele m was 0.609 and therefore the frequency of the dominant allele M was 0.391. The expected frequencies of parental and recombinant gametes were multiplied by the frequencies of the W and w alleles to determine the expected frequency of each gamete type. This resulted in four types of gametes: parental-type gamete WMs at a frequency of (0.391)(.84) or 0.32844, parental-type gamete wms at a frequency of

PAGE 234

217 (0.609)(0.84) or 0.51156, recombinant gamete Wms at a frequency of (0.609)(0.16) or 0.09744 and recombinant gamete wMs at a frequency of (0.391)(0.16) or 0.06256. These gametes would fuse to produce progeny with one of ten different genotypes. Five genotypes ( WMs / WMs WMs / wms WMs / Wms WMs / wMs and Wms / wMs ) were associated with the blue mid phenot ype, which represented 56.1199% of the F2 population. Two genotypes ( wms / Wms and Wms / Wms ) produced the blue long phenotype and composed 10.9187% of the population and two genotypes ( wms / wMs and wMs / wMs ) resulted in the white mid phenot ype and accounted for 6.7920% of the population. The white long phenotype was pr oduced by progeny with the genotype wms / wms and composed 26.1796% of the F2 population. The F2 family UVM2 still deviated from th e segregation ratio expected when compared to this adjusted model where f( M ) = 0.391 and f( m ) = 0.609 and where 16 m.u. separate the W and M loci (Table 9.11). As many as 1 in 25 populations the size of UVM2 (n=89) would show as much variation as the F2 family UVM2, so this discrepancy may have been attr ibutable to sampling error. The families BM1 and BM2 segregated as expected when compared to a model where 16 m.u. separate the W and M loci. The family UVM2 did not segregate as expected; however, this family did not segregat e as expected for floral morph and a better fit was accomplished by comparing segregants to an adjusted model where 16 m.u. separate the W and M loci and where f( M ) = 0.391 and f( m ) = 0.609. The family BM3 failed to segregate as expected and the r eason for this was unknown; however, this may have been due more to sampling error than to flaws in the model, as the majority of the

PAGE 235

218 data from these families supported the pr oposed model where 16 m.u. separate the W and M loci. Linkage in the F2 family BMBL. The genotypic constitutions of the parents of the F1 family BMBL were WMs / wms (for the parent BM) and Wms / wms (for the parent BL). Four types of F1 progeny were produced: blue mid ( WMs / Wms WMs / wms and wMs / Wms ), blue long ( wms / Wms and Wms / Wms ), white mid ( wMs / wms ) and white long ( wms / wms ). Linkage was evident in the F1 generation and was described ad nauseum earlier in this chapter; linkage was evident in the F2 generation as well. Three F1 plants with the blue mid phenotype were self-pollinated and all produced F2 families that segregated for both flower color and floral morph (Table 9.12). Two of the three F2 families segregated as expected for each trait individually. The F2 family BM2 segregated as expected for floral mo rph, but did not segregate as expected for flower color. F1 plants with the blue mid phenotype we re produced by three different genotypes WMs / Wms WMs / wms and wMs / Wms Self-pollination of pl ants with the genotype WMs / Wms would have created popul ations with only blue-flowered progeny and would therefore not be useful in this linkage study; however, self-pollinati on of plants with the genotype WMs / wms or Wms / wMs would have created popula tions with both flower colors and both M-morphs and L-morphs. The F1 parents of the F2 families BM1, BM2 and BM6 were genotypically iden tical and heterozygous at the W and M loci, but the dominant W and M alleles may have been linke d in coupling (i.e., genotype WMs / wms ) or in repulsion (i.e., genotype wMs / Wms ).

PAGE 236

219 Gametic frequencies were determined by the linkage relationship present in the F1 parent. If dominant alleles were linked in coupling (i.e., WMs / wms ), then parental-type gametes WMs and wms would be produced at a frequency of 0.42 each and recombinant gametes Wms and wMs would be produced at a fre quency of 0.08 each. These gametes would fuse to produce progeny with one of ten different genotypes. Five genotypes ( WMs / WMs WMs / wms WMs / Wms WMs / wMs and Wms / wMs ) were associated with the blue mid phenotype, which represented 67.64% of the F2 population. Two genotypes ( wms / Wms and Wms / Wms ) produced the blue long phenotype and two genotypes ( wms / wMs and wMs / wMs ) resulted in the white mid phenotype; each of these phenotypes accounted for 7.36% of the population. The white long phenotype was produced by progeny with the genotype wms / wms and composed 17.64% of the F2 population. If dominant alleles were linked in repulsion (i.e., Wms / wMs ), then parental-type gametes Wms and wMs would be produced at a freque ncy of 0.42 each and recombinant gametes WMs and wms would be produced at a fre quency of 0.08 each. These gametes would fuse to produce progeny with one of ten different genotypes. Five genotypes ( WMs / WMs WMs / wms WMs / Wms WMs / wMs and Wms / wMs ) were associated with the blue mid phenotype, which represented 50.64% of the F2 population. Two genotypes ( wms / Wms and Wms / Wms ) produced the blue long phenotype and two genotypes ( wms / wMs and wMs / wMs ) resulted in the white mid phenotype; each of these phenotypes accounted for 24.36% of the population. The white long phenotype was produced by progeny with the genotype wms / wms and composed 0.64% of the F2 population. The F2 family BM1 did not segregate as e xpected when compared to the model with dominant alleles linked in coupl ing and where 16 m.u. separate the W and M loci,

PAGE 237

220 but the family segregated as expected wh en compared to a model where the dominant alleles were linked in repulsion and where 16 m.u. separate the W and M loci (Table 9.12). These results supported the model where the W and M loci are 16 m.u. apart and revealed that the genotype of the F1 parent of the F2 family BM1 was Wms / wMs The F2 family BM2 did not segregate as expe cted when compared to models for both linkage arrangements, but was provide d a much better fit by the model where dominant alleles were linked in coupling (Tab le 9.12). This family did not segregate as expected when flower color was consid ered individually and the white-flowered phenotype was produced in excess when compared to the model where f( W ) = f( w ) = 0.50. The reason for this production of fewer gametes with the dominant allele W and an excess of gametes with the recessive allele w was unknown; however, it again seemed appropriate to adjust the gametic frequencies in this family to more accurately reflect the genotypic constituti on of the population. The white-flowered phenotype resulted from the genotype ww and occurred at a frequency of 19/38 in the family BM2; the square root of this ratio revealed that the frequency of the recessive allele w was 0.707 and the frequency of the dominant allele W was 0.293. The expected frequencies of parental and recombinant gametes were multiplied by the frequencies of the W and w alleles to determine the expected frequency of each gamete type. The W and M alleles were most likely linked in coupling in the F1 parent of the F2 family BM2, as that model provided a much better fit to the data than the model where dominant alleles were linked in repulsion; therefore, gametic frequencies were computed based on the former model. Four di fferent types of game tes would be produced by BM2: parental-type gamete WMs at a frequency of (0.293)(.84) or 0.24612,

PAGE 238

221 parental-type gamete wms at a frequency of (0.707)(0.84) or 0.59388, recombinant gamete Wms at a frequency of (0.293)(0.16) or 0.04688 and recombinant gamete wMs at a frequency of (0.707)(0.16) or 0.11312. These gametes would fuse to produce progeny with one of ten different genotypes. Five genotypes ( WMs / WMs WMs / wms WMs / Wms WMs / wMs and Wms / wMs ) were associated with th e blue mid phenotype, which represented 44.227% of the F2 population. Two genotypes ( wms / Wms and Wms / Wms ) produced the blue long phenotype and co mposed 5.788% of the population and two genotypes ( wms / wMs and wMs / wMs ) resulted in the white mid phenotype and accounted for 14.716% of the population. The white long phenotype was produced by progeny with the genotype wms / wms and composed 35.269% of the F2 population. The F2 family BM2 segregated as expected when compared to this adjusted model where f( W ) = 0.293 and f( w ) = 0.707 and where the W and M loci were 16 m.u. apart and linked in coupling (Table 9.12). These results supported th e model where the W and M loci are 16 m.u. apart and reveal ed that the genotype of the F1 parent of the F2 family BM2 was WMs / wms The F2 family BM6 did not segregate as expe cted when compared to models for both linkage arrangements, but was provide d a much better fit by the model where dominant alleles are linked in coupling (Table 9.12). This family segregated as expected for both flower color and floral morph indivi dually, so the reason for this failure to segregate as expected when compared to either linkage model was unknown. As many as 1 in 50 populations the size of BM6 (n=43) a nd with dominant allele s linked in coupling would show as much variation as BM6; ther efore, it is likely that sampling error was responsible for the discrepancy. These results provided some support for the model where

PAGE 239

222 the W and M loci were 16 m.u. apart and revealed that the most likely genotype of the F1 parent of the F2 family BM6 was WMs / wms The family BM1 segregated as expect ed when compared to a model where dominant alleles were linked in repulsion and the W and M loci were separated by 16 m.u.. The family BM2 did not segregate as expected; however, this family did not segregate as expected for flower color and di d segregate as expected when compared to a model where dominant alleles were linked in coupling, the W and M loci were separated by 16 m.u. and f( W )=0.293 and f( w )=0.707. The family BM6 failed to segregate as expected and the reason for this was unknown; however, this may have been due to sampling error, as the population size of BM6 was small. Conclusions This study provided evidence that the W locus controlling flower color and the hypostatic M locus controlling floral morph in this population of pickerelweed were located 16 m.u. apart on the same chromosome. Evaluation of seven F1 families revealed that all segregated as expected wh en compared to a model where the W and M loci were separated by 16 m.u.. A to tal of thirty-eight F2 families were examined; twenty-two families were derived from F1 plants with the blue mid phenotype and the remaining sixteen families were produced from F1 plants with the blue s hort phenotype. A majority (21/38) of the F2 families studied segregated as expected when compared to the model where the W and M loci were 16 m.u. apart and thir teen of the remaining seventeen F2 families segregated as expected when the model was adjusted to account for rare or missing phenotypic classes or for poor fit of either trait individua lly. The model provided a poor fit to progeny from only four F2 families; two of these families segregated in a

PAGE 240

223 manner that was close to that expected under the model and two deviated from the model with no apparent cause. The vast majority of these data supported the model where the W and M loci were 16 m.u. apart on the same chromosome; in add ition, this study provided evidence that the epistatic S locus was independent from the W locus controlling flow er color and from the hypostatic M locus that contributed to the control of floral morph. It is not unusual for loci that control different traits related to the same tissue to be located on the same chromosome. Halvankar and Patil (1994) noted linkage among floral traits of soybean; it is possible that a sim ilar arrangement occurs in pickerelweed. This experiment revealed that the W locus controlling flower color and the hypostatic M locus contributing to floral morph were close t ogether on the same chromosome, while the epistatic S locus responsible for the control of floral morph was independent from both the W and M loci. If the S locus were situated on the same chromosome as the W and M loci but was more than 50 m.u. from either locus, it would be possible to determine the position of the S locus by identifying a trait linked to both the S locus and to the W or M locus; however, that analysis was beyond the scope of this study.

PAGE 241

224 Table 9.1. Segregation of proge ny and goodness-of-fit tests for F1 families of pickerelweed segregating for flower co lor and floral morph. Expected values were computed assuming independence of the loci controlling flower color and floral morph. F1 families are coded to identify the parents used to create the F1 family (e.g., the F1 family WSBM was create d by cross-pollination of the parents WS and BM). Not shown: F1 families that did not reveal linkage (WMBL, WSBL, WMBS, BMBS and BSBL). F1 family WSBM (n=144) Phenotype Exp. frequency Exp. number Obs 2# Blue short 0.250 36 52 7.11111 Blue mid 0.125 18 16 0.22222 Blue long 0.125 18 6 8.00000 White short 0.250 36 35 0.02778 White mid 0.125 18 3 12.5000 White long 0.125 36 32 0.44444 P = 0.0000 Grand chi-square = 28.3056 F1 family BMBL (n=91) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.375 34.125 45 3.46566 Blue long 0.375 34.125 29 0.76969 White mid 0.125 11.375 1 9.46291 White long 0.125 11.375 16 1.88049 P = 0.0013 Grand chi-square = 15.5788 Phenotype: Phenotype of F1 progeny Exp. frequency: Expected ratio of progeny in each phenotypic class Exp. number: Expected number of progeny in each phenotypic class Obs.: Observed number of progeny in each phenotypic class # 2: Chi-square value comput ed from goodness-of-fit test Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 242

225 Table 9.2. Goodness-of-fit test for the F1 family WSBM segregating for flower color and floral morph. Expected values were co mputed using a genetic distance of 12.5 m.u. between the W locus and the M locus with dominant alleles linked in coupling in the parent BM. Unadjust ed expected values assume gametes from WS are produced in e qual frequencies so that f( wmS ) = f( wms ) = 0.5. Adjusted values calculated with f( wmS ) = 0.604 and f( wms ) = 0.396. Unadjusted Phenotype Exp. frequency Exp. number Obs. 2# Blue short 0.25000* 36.0 52 6.6736 Blue mid 0.21875 31.5 16 7.1429 Blue long 0.03125 4.50 6 0.2222 White short 0.25000* 36.0 35 0.0069 White mid 0.03125 4.50 3 0.2222 White long 0.21875 31.5 32 0.0000 P = 0.0139 Grand chi-square=14.2678 Adjusted Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.30200* 43.488 52 1.4761 Blue mid 0.17325 24.948 16 2.8607 Blue long 0.02475 3.5640 6 1.0517 White short 0.30200* 43.488 35 1.4673 White mid 0.02475 3.5640 3 0.0011 White long 0.17325 24.948 32 1.7207 P = 0.1271 Grand chi-square = 8.5776 Phenotype: Phenotype of F1 progeny Exp. frequency: Expected ratio of proge ny in each phenotypic class; frequencies followed by are a combination of parental and recombinant genotypes Exp. number: Expected number of progeny in each phenotypic class Obs.: Observed number of progeny in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 243

226 Table 9.3. Goodness-of-fit test for the F1 family BMBL segregating for flower color and floral morph. Expected values were co mputed using a genetic distance of 12.5 m.u. between the W locus and the M locus with dominant alleles linked in coupling in the parent BM. Phenotype Exp. frequency Exp. number Obs. 2# Blue mid 0.46875* 42.656 45 0.0797 Blue long 0.28125* 25.594 29 0.3300 White mid 0.03125 2.844 1 0.6351 White long 0.21875 19.906 16 0.5828 P = 0.6531 Grand chi-square = 1.6276 Phenotype: Phenotype of F1 progeny Exp. frequency: Expected ratio of proge ny in each phenotypic class; frequencies followed by are a combination of parental and recombinant genotypes Exp. number: Expected number of progeny in each phenotypic class Obs.: Observed number of progeny in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 244

227 Table 9.4. Goodness-of-fit tests for the F1 families WSBM and BMBL segregating for flower color and floral morph. Expected values were computed with a genetic distance of 4.4 m.u. between the W locus and the M locus with dominant alleles linked in c oupling in the parent BM. Exp ected values for WSBM were computed using adjusted gametic frequencies for the parent WS. F1 family WSBM Phenotype Exp. frequency Exp. number Obs. 2# Blue short 0.3020* 43.4880 52 1.4761 Blue mid 0.1893 27.2592 16 4.2467 Blue long 0.0087 1.2528 6 14.399 White short 0.3020* 43.4880 35 1.4673 White mid 0.0087 1.2528 3 1.2416 White long 0.1893 27.2592 32 0.6598 P = 0.0002 Grand chi-square=23.4901 F1 family = BMBL Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.489* 44.499 45 0.0000 Blue long 0.261* 23.751 29 0.9496 White mid 0.011 1.001 1 0.0000 White long 0.239 21.749 16 1.2668 P = 0.5287 Grand chi-square = 2.2164 Phenotype: Phenotype of F1 progeny Exp. frequency: Expected ratio of proge ny in each phenotypic class; frequencies followed by are a combination of parental and recombinant genotypes Exp. number: Expected number of progeny in each phenotypic class Obs.: Observed number of progeny in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 245

228 Table 9.5. Summary of goodness-of-fit tests for genetic distances ranging from 4.4 m.u. to 22 m.u. for the linked W and M loci in the F1 families WSBM and BMBL. F1 family = WSBM Distance 2 P Independent 37.2900 0.0000 4.4 23.4901 0.0002 direct estimate of distance from BMBL 5 20.2546 0.0011 6 16.4840 0.0055 8 12.1756 0.0324 10 10.0032 0.0751 12 8.7847 0.1179 12.5 8.5776 0.1271 direct estimate of distance from WSBM 14 8.1851 0.1463 16 8.0809 0.1518 18 9.8626 0.0792 20 10.3549 0.0657 22 11.0766 0.0498 F1 family = BMBL Distance 2 P Independent 15.5788 0.0013 4.4 2.21640 0.5287 direct estimate of distance from BMBL 5 2.10045 0.5518 6 1.91627 0.5899 8 1.63790 0.6508 10 1.55586 0.6694 12 1.60100 0.6591 12.5 1.62760 0.6531 direct estimate of distance from WSBM 14 1.73810 0.6284 16 1.94980 0.5828 18 2.22677 0.5266 20 2.56380 0.4638 22 2.95799 0.3981 Distance: Number of map units between the W and M loci; independent = W and M loci are greater than 50 m.u. apart 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity except for tests for independence in both families and for genetic distance estimates of 18, 20 and 22 m.u. in the family WSBM P: Probability associated with 2

PAGE 246

229 Table 9.6. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family WMBL segregating for flower co lor and floral morph. Each F2 family was derived from self-pollina tion of an individual F1 plant developed by cross-pollination of the parents WM and BL. Expected values were computed assuming 16 m.u. between the W and M loci. F2 family = BM1 (n=76) Phenotype Exp. frequency Exp. number Obs. 2# Blue mid 0.5064 38.4864 33 0.6461 Blue long 0.2436 18.5136 20 0.0526 White mid 0.2436 18.5136 20 0.0526 White long 0.0064 0.4864 3 8.3359 P = 0.0281 Grand chi-square =9.08707 F2 family = BM2 (n=83) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.5064 42.0312 51 1.7064 Blue long 0.2436 20.2188 17 0.0000 White mid 0.2436 20.2188 13 2.2327 White long 0.0064 0.5312 2 1.7669 P = 0.1268 Grand chi-square = 5.7060 F2 family = BM3 (n=59) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.50966 30.0699 38 2.0913 Blue long 0.24517 14.4650 10 1.3782 White mid 0.24517 14.4650 11 0.8300 White long (adjusted) (adjusted) 0 (n/a) P = 0.1165 Grand chi-square = 4.2996 F2 family = BM4 (n=118) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.5064 59.7552 72 2.3084 Blue long 0.2436 28.7448 19 2.9733 White mid 0.2436 28.7448 24 0.6268 White long 0.0064 0.7552 3 4.0312 P = 0.0190 Grand chi-square = 9.9397 F2 family = BM5 (n=40) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.5064 20.256 18 0.1522 Blue long 0.2436 9.744 8 0.1588 White mid 0.2436 9.744 13 0.7795 White long 0.0064 0.256 1 0.2326 P = 0.7236 Grand chi-square = 1.3231

PAGE 247

230 Table 9.6. Continued F2 family = BM6 (n=30) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.5064 15.192 21 1.8546 Blue long 0.2436 7.308 6 0.0893 White mid 0.2436 7.308 2 3.1632 White long 0.0064 0.192 1 0.4941 P = 0.1327 Grand chi-square = 5.6012 Phenotype: Phenotype of F2 progeny Exp. frequency: Expected ratio of progeny in each phenotypic class Exp. number: Expected number of progeny in each phenotypic class Obs.: Observed number of progeny in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity fo r all families except BM3 Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 248

231 Table 9.7. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family WSBL segregating for flower co lor and floral morph. Each F2 family was derived from self-pollina tion of an individual F1 plant developed by cross-pollination of the parents WS and BL. Expected values were computed assuming 16 m.u. between the W and M loci. F2 family = BS1 (n=46) Phenotype Exp. frequency Exp. number Obs. 2# Blue short 0.5625 25.875 29 0.2663 Blue long 0.1875 8.625 4 1.9728 White short 0.1875 8.625 11 0.4076 White long 0.0625 2.875 2 0.0489 P = 0.4409 Grand chi-square = 2.6957 F2 family = BS3 (n=56) Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.5625 31.5 28 0.2857 Blue long 0.1875 10.5 13 0.3810 White short 0.1875 10.5 10 0.0000 White long 0.0625 3.5 5 0.2857 P = 0.8127 Grand chi-square = 0.9524 Phenotype: Phenotype of F2 progeny Exp. frequency: Expected ratio of progeny in each phenotypic class Exp. number: Expected number of progeny in each phenotypic class Obs.: Observed number of progeny in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 249

232 Table 9.8. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family WSBM segregating for flower co lor and floral morph. Each F2 family was derived from self-pollina tion of an individual F1 plant developed by cross-pollination of the parents WS and BM. Expected values were computed assuming 16 m.u. between the W and M loci. F2 family = BS2 (n=85) Phenotype Exp. frequency Exp. number Obs. 2# Blue short 0.5625 47.8125 38 1.8138 Blue mid 0.1691 14.3735 18 0.6801 Blue long 0.0184 1.5640 3 0.5602 White short 0.1875 15.9375 20 0.7963 White mid 0.0184 1.5640 2 0.0000 White long 0.0441 3.7485 4 0.0000 P = 0.5711 Grand chi-square = 3.8504 F2 family = BS4 (n=88) Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.5730 50.424 50 0.0000 Blue mid 0.1723 15.162 13 0.1823 Blue long (adjusted) (adjusted) 0 n/a White short 0.1910 16.808 20 0.4312 White mid 0.0187 1.646 1 0.0129 White long 0.0449 3.951 4 0.0000 P = 0.9600 Grand chi-square = 0.6263 F2 family = BS6 (n=51) Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.5730 29.2230 26 0.2536 Blue mid 0.1723 8.7873 8 0.0094 Blue long 0.0187 0.9537 3 2.5071 White short 0.1910 9.7410 13 0.7815 White mid (adjusted) (adjusted) 0 n/a White long 0.0449 2.2899 1 0.2725 P = 0.4303 Grand chi-square = 3.8241 F2 family = BM1 (n=33) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 22.3212 17 1.0413 Blue long 0.0736 2.4288 4 0.4724 White mid 0.0736 2.4288 4 0.4724 White long 0.1764 5.8212 8 0.4842 P = 0.4806 Grand chi-square = 2.4704

PAGE 250

233 Table 9.8. Continued F2 family = BM2 (n=87) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 58.8468 34 10.491 Blue long 0.0736 6.4032 19 24.781 White mid 0.0736 6.4032 14 9.0129 White long 0.1764 15.3468 20 1.4109 P = 0.0000 Grand chi-square = 45.696 Phenotype: Phenotype of F2 progeny Exp. frequency: Expected ratio of progeny in each phenotypic class Exp. number: Expected number of progeny in each phenotypic class Obs.: Observed number of progeny in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity fo r all families except BM2 Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 251

234 Table 9.9. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family WMBS segregating for flower co lor and floral morph. Each F2 family was derived from self-pollina tion of an individual F1 plant developed by cross-pollination of the parents WM and BS. Expected values were computed assuming 16 m.u. between the W and M loci. F2 family = BS1 (n=93) Phenotype Exp. frequency Exp. number Obs. 2# Blue short 0.5625 52.3125 50 0.0629 Blue mid 0.1691 15.7263 17 0.0381 Blue long 0.0184 1.7112 2 0.0000 White short 0.1875 17.4375 18 0.0002 White mid 0.0184 1.7112 3 0.3636 White long 0.0441 4.1013 3 0.0882 P = 0.9900 Grand chi-square = 0.5529 F2 family = BS2 (n=114) Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.5625 64.125 63 0.0197 Blue mid 0.1875 21.375 28 2.0534 White short 0.1875 21.375 20 0.0885 White mid 0.0625 7.125 3 2.3882 P = 0.2078 Grand chi-square = 4.5497 F2 family = BS3 (n=17) Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.58399 9.92783 8 0.2054 Blue mid 0.17556 2.98452 3 0.0000 Blue long (adjusted) (adjusted) 0 n/a White short 0.19466 3.30922 5 0.4285 White mid (adjusted) (adjusted) 0 n/a White long 0.04578 0.77826 1 0.0000 P = 0.8886 Grand chi-square = 0.6338 F2 family = BS4 (n=62) Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.58399 36.20738 44 1.4688 Blue mid 0.17556 10.88472 7 1.0525 Blue long (adjusted) (adjusted) 0 n/a White short 0.19466 12.06892 10 0.2040 White mid (adjusted) (adjusted) 0 n/a White long 0.04578 2.83836 1 0.6311 P = 0.3398 Grand chi-square = 3.3564

PAGE 252

235 Table 9.9. Continued F2 family = BS5 (n=153) Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.5625 86.0625 98 1.6558 Blue mid 0.1875 28.6875 21 2.0601 White short 0.1875 28.6875 28 0.0165 White mid 0.0625 9.5625 6 1.3272 P = 0.1674 Grand chi-square = 5.0596 F2 family = BS6 (n=16) Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.5625 9 10 0.0278 Blue mid 0.1875 3 1 0.7500 White short 0.1875 3 4 0.0833 White mid 0.0625 1 1 0.0000 P = 0.8348 Grand chi-square = 0.8611 F2 family = BS7 (n=49) Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.58399 28.61551 31 0.1241 Blue mid 0.17556 8.60244 5 1.1189 Blue long (adjusted) (adjusted) 0 n/a White short 0.19466 9.53834 9 0.0002 White mid (adjusted) (adjusted) 0 n/a White long 0.04578 2.24322 4 0.7041 P = 0.5834 Grand chi-square = 1.9473 F2 family = BM1 (n=105) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 71.022 71 0.0000 Blue long 0.0736 7.728 8 0.0096 White mid 0.0736 7.728 11 1.3854 White long 0.1764 18.522 15 0.6697 P = 0.5590 Grand chi-square = 2.0647 F2 family = BM2 (n=96) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 64.9344 65 0.0001 Blue long 0.0736 7.0656 9 0.5296 White mid 0.0736 7.0656 9 0.5296 White long 0.1764 16.9344 13 0.9141 P = 0.5779 Grand chi-square = 1.9733

PAGE 253

236 Table 9.9. Continued F2 family = BM3 (n=27) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 18.2628 17 0.0319 Blue long 0.0736 1.9872 4 1.1517 White mid 0.0736 1.9872 2 0.0000 White long 0.1764 4.7628 4 0.0145 P = 0.7534 Grand chi-square = 1.1980 F2 family = BM6 (n=28) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 18.9392 17 0.1094 Blue long 0.0736 2.0608 3 0.0936 White mid 0.0736 2.0608 2 0.0000 White long 0.1764 4.9392 6 0.0637 P = 0.9661 Grand chi-square = 0.2666 Phenotype: Phenotype of F2 progeny Exp. frequency: Expected ratio of progeny in each phenotypic class Exp. number: Expected number of progeny in each phenotypic class Obs.: Observed number of progeny in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity for all fa milies except BS2, BS5, BM1 and BM2 Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 254

237 Table 9.10. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family BSBM segregating for flower co lor and floral morph. Each F2 family was derived from self-pollina tion of an individual F1 plant developed by cross-pollination of the parents BS and BM. Exp ected values were computed assuming 16 m.u. between the W and M loci. F2 family = BS2 (n=110) Phenotype Exp. frequency Exp. number Obs. 2# Blue short 0.5625 61.875 53 1.2730 Blue mid 0.1875 20.625 37 13.001 White short 0.1875 20.625 13 2.8189 White mid 0.0625 6.875 7 0.0023 P = 0.0006 Grand chi-square = 17.095 F2 family = BS2 adjusted so that f( S ) = 0.3675 and f( s ) = 0.6325 Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.44996 49.4956 53 0.2481 Blue mid 0.30004 33.0044 37 0.4837 White short 0.14998 16.4978 13 0.7416 White mid 0.10001 11.0011 7 1.4552 P = 0.4027 Grand chi-square = 2.9286 F2 family = PBS1 (n=56) Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.5730 32.0880 27 0.6560 Blue mid 0.1723 9.6488 5 1.7839 Blue long (adjusted) (adjusted) 0 n/a White short 0.1910 10.6960 21 8.9864 White mid 0.0187 1.0472 2 0.1958 White long 0.0449 2.5144 1 0.4093 P = 0.0171 Grand chi-square = 12.031 F2 family = PBS1 adjusted so that f( W ) = 0.34535 and f( w ) = 0.65465 Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.43550 24.387 27 0.1831 Blue mid 0.12885 7.215 5 0.4077 Blue long (adjusted) (adjusted) 0 n/a White short 0.32461 18.178 21 0.2966 White mid 0.03205 1.795 2 0.0000 White long 0.07722 4.324 1 1.8444 P = 0.6036 Grand chi-square = 2.7317

PAGE 255

238 Table 9.10. Continued F2 family = PBS3 (n=20) Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.6000 12.000 13 0.0208 Blue mid 0.1804 3.608 4 0.0000 Blue long 0.0196 0.392 1 0.0298 White short 0.2000 4.000 2 0.5625 White mid (adjusted) (adjusted) 0 n/a White long (adjusted) (adjusted) 0 n/a P = 0.8934 Grand chi-square = 0.6131 F2 family = PBS4 (n=40) Phenotype Exp. frequency Exp. number Obs. 2 Blue short 0.6000 24.000 23 0.0104 Blue mid 0.1804 7.216 8 0.0112 Blue long 0.0196 0.784 1 0.0000 White short 0.2000 8.000 8 0.0000 White mid (adjusted) (adjusted) 0 n/a White long (adjusted) (adjusted) 0 n/a P = 0.9991 Grand chi-square = 0.0216 F2 family = BM3 (n=35) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 23.674 21 0.1996 Blue long 0.0736 2.576 4 0.3314 White mid 0.0736 2.576 2 0.0022 White long 0.1764 6.174 8 0.2848 P = 0.8451 Grand chi-square = 0.8181 F2 family = PBM1 (n=149) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 100.7836 104 0.1027 Blue long 0.0736 10.9664 16 2.3104 White mid 0.0736 10.9664 13 0.3771 White long 0.1764 26.2836 16 4.0235 P = 0.0780 Grand chi-square = 6.8137

PAGE 256

239 Table 9.10. Continued F2 family = PBM3 (n=110) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 74.404 69 0.3925 Blue long 0.0736 8.096 10 0.4478 White mid 0.0736 8.096 10 0.4478 White long 0.1764 19.404 21 0.1313 P = 0.7010 Grand chi-square = 1.4193 Phenotype: Phenotype of F2 progeny Exp. frequency: Expected ratio of progeny in each phenotypic class Exp. number: Expected number of progeny in each phenotypic class Obs.: Observed number of progeny in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity for all fa milies except BS2, PBM1 and PBM3 Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 257

240 Table 9.11. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family BSBL segregating for flower co lor and floral morph. Each F2 family was derived from self-pollina tion of an individual F1 plant developed by cross-pollination of the parents BS and BL. Exp ected values were computed assuming 16 m.u. between the W and M loci. F2 family = UVM2 (n=89) Phenotype Exp. frequency Exp. number Obs. 2# Blue mid 0.6764 60.1996 48 2.4723 Blue long 0.0736 6.5504 17 16.670 White mid 0.0736 6.5504 8 0.3208 White long 0.1764 15.6996 16 0.0058 P = 0.0002 Grand chi-square = 19.469 F2 family = UVM2 adjusted so that f( M ) = 0.391 and f( m ) = 0.609 Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.561199 49.9467 48 0.0759 Blue long 0.109187 9.7177 17 5.4573 White mid 0.067920 6.0449 8 0.6323 White long 0.261796 23.2998 16 2.2870 P = 0.0375 Grand chi-square = 8.4525 F2 family = BM1 (n=70) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 47.348 44 0.2367 Blue long 0.0736 5.152 5 0.0045 White mid 0.0736 5.152 10 4.5619 White long 0.1764 12.348 11 0.1472 P = 0.1754 Grand chi-square = 4.9503 F2 family = BM2 (n=98) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 66.2872 61 0.4217 Blue long 0.0736 7.2128 8 0.0859 White mid 0.0736 7.2128 10 1.0770 White long 0.1764 17.2872 19 0.1697 P = 0.6249 Grand chi-square = 1.7544 F2 family = BM3 (n=88) Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 59.5232 62 0.1031 Blue long 0.0736 6.4768 12 4.7100 White mid 0.0736 6.4768 10 1.9165 White long 0.1764 15.5232 4 8.5539 P = 0.0015 Grand chi-square = 15.284

PAGE 258

241 Table 9.11. Continued Phenotype: Phenotype of F2 progeny Exp. frequency: Expected ratio of progeny in each phenotypic class Exp. number: Expected number of progeny in each phenotypic class Obs.: Observed number of progeny in each phenotypic class # 2: Chi-square value comput ed from goodness-of-fit test Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 259

242 Table 9.12. Goodness-of-fit tests for F2 families of pickerelweed from the F1 family BMBL segregating for flower co lor and floral morph. Each F2 family was derived from self-pollina tion of an individual F1 plant developed by cross-pollination of the parents BM and BL. Expected values were computed assuming 16 m.u. between the W and M loci. F2 family = BM1 (n=51) coupling Phenotype Exp. frequency Exp. number Obs. 2# Blue mid 0.6764 34.4964 24 2.8968 Blue long 0.0736 3.7536 13 20.380 White mid 0.0736 3.7536 13 20.380 White long 0.1764 8.9964 1 6.2465 P = 0.0000 Grand chi-square = 49.904 F2 family = BM1 (n=51) repulsion Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.5064 25.8264 24 0.0681 Blue long 0.2436 12.4236 13 0.0005 White mid 0.2436 12.4236 13 0.0005 White long 0.0064 0.3264 1 0.0923 P = 0.9835 Grand chi-square = 0.1614 F2 family = BM2 (n=38) coupling Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 25.7032 16 3.2953 Blue long 0.0736 2.7968 3 0.0000 White mid 0.0736 2.7968 7 4.9034 White long 0.1764 6.7032 12 3.4326 P = 0.0087 Grand chi-square = 11.631 F2 family = BM2 coupling and adjusted so that f( W ) = 0.293 and f( w ) = 0.707 Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.44227 16.8063 16 0.0056 Blue long 0.05788 2.1994 3 0.0411 White mid 0.14716 5.5919 7 0.1475 White long 0.35269 13.4024 12 0.0608 P = 0.9682 Grand chi-square = 0.2549 F2 family = BM2 (n=38) repulsion Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.5064 19.2432 16 0.3911 Blue long 0.2436 9.2568 3 3.5802 White mid 0.2436 9.2568 7 0.3334 White long 0.0064 0.2432 12 521.03 P = 0.0000 Grand chi-square = 525.34

PAGE 260

243 Table 9.12. Continued F2 family = BM6 (n=43) coupling Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.6764 29.0852 24 0.7228 Blue long 0.0736 3.1648 3 0.0000 White mid 0.0736 3.1648 9 8.9941 White long 0.1764 7.5852 7 0.0010 P = 0.0211 Grand chi-square = 9.7179 F2 family = BM6 (n=43) repulsion Phenotype Exp. frequency Exp. number Obs. 2 Blue mid 0.5064 21.7752 24 0.1366 Blue long 0.2436 10.4748 3 4.6443 White mid 0.2436 10.4748 9 0.0907 White long 0.0064 0.2752 7 140.80 P = 0.0000 Grand chi-square = 145.67 Phenotype: Phenotype of F2 progeny Exp. frequency: Expected ratio of progeny in each phenotypic class Exp. number: Expected number of progeny in each phenotypic class Obs.: Observed number of progeny in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test; calculat ed using Yates correction for continuity Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 261

244 CHAPTER 10 INHERITANCE AND GE NETIC CONTROL OF SCAPE PUBESCENCE Introduction Pubescence refers to soft, short, downy ha irs that occur on vari ous surfaces. This pubescence may provide plants with protec tion against insect feeding damage by reducing access to succulent tissue or may increase the ambient temperature around the pubescent structure by creating an insulating effect (Maes et al. 2001; Miller 1986). The inheritance and genetic control of pubescence is often simple w ith a dominant allele at a single locus conditioning the pube scent phenotype; this scenar io has been described in red clover ( Trifolium pratense ) (Broda 1979), lentil ( Lens culinaris ) (Hoque et al. 2002; Sarker et al. 1999), Triticum dicoccoides (Lange and Jochemsen 1987) and soybean ( Glycine max ) (Halvankar and Patil 1994). Pubescen ce is less often expressed as the result of a recessive condition at a single diallelic locus (D eren 1987) or as the product of a system with epistatic interaction be tween two diallelic loci (Gorsic 1994). An assessment of morphologi cal characters in an e xperimental population of pickerelweed revealed that some plants had pubescent inflorescence scapes, while the scapes of other plants lacked pubescence a nd were glabrous. The objective of this experiment was to determine the mode of i nheritance and number of loci controlling the expression of scape pubescence in this population of pickerelweed. Materials and Methods The parents used in this experiment were selected based on phenotype and cross-compatibility from a collection of pl ants maintained for breeding and genetics

PAGE 262

245 studies at the University of Florida in Ga inesville. The parent WM was collected in southeastern Florida and had white M-mo rph flowers borne on a glabrous scape (Figure 10.1), while the parent RI was coll ected in Rhode Island and had blue L-morph flowers produced on a pubescen t scape (Figure 10.2). Crossand reciprocal pollinations were performed between the parental lines a nd utilized compatible pollen (i.e., l-pollen from WM was used to pollinate RI and m-po llen from RI was used to pollinate WM). Barrett and Glover (1985) st ated that emasculation was not necessary to prevent self-pollination, so anthers borne on long f ilaments were removed only from WM to increase access to the stigma. Fine forceps we re used to remove dehisced anthers from the pollen parent; an anther was then brushed gently across the stigma of the seed parent to effect pollination. Magnifying headgear was worn during all pollinations to allow visual confirmation of successf ul transfer and adhesion of pollen grains to the stigma. Flowers from a given inflorescen ce were used as either pol len donors or seed parents to eliminate contamination from self-produced pollen. Pollinations commenced with the opening of the first flowers of an inflorescence and continued until all flowers on the inflores cence had been pollinated (ca. 7 to 12 d). A total of eighty-one flowers were pollinated us ing WM as the seed parent and RI as the pollen donor; eighty-one flowers were utilized in the reciproc al as well. All pollinations were performed between 9 am and 3 pm daily and pollination data were recorded on jewelry tags placed on each inflorescence. Each completed inflorescence was enclosed in a small mesh bag and secured with a plastic-c overed twist-tie until fruits were ripe. Fruits were considered ripe when the bearing infr uctescence shattered (usu ally 23 to 30 d after completion of pollinations).

PAGE 263

246 Fruits were collected in their mesh bag and air-dried for ca. 7 d, then de-husked using a rubber-covered rub board. Cleaned seeds were stored at 4 C in labeled coin envelopes until all pollinations were completed. A total of fifty-six hybrid seeds were produced in this experiment: twenty-two seeds from RI x WM (coded RIWM) and thirty-four seeds from WM x RI (coded WMR I). Seeds were germinated under ca. 5 cm of water in glass half-pint (250 mL) bottles, with additional water added as needed to maintain a constant depth. Germination vessels were placed on a bench in the greenhouse with temperature at 27 C (day) and 16 C (night) and daylength artificially extended to 16 h. Germinated seeds were allowed to remain under water for 2 or 3 d after germination, then were transplanted in to 612 cell packs fill ed with Metro-Mix 5001. Twenty-two F1 seedlings were produced in this manner and each was labeled with a tag indicating parental iden tity (i.e., RIWM or WMRI) and germination rank (i.e., seedling number from a particular pollination event). Seedlings were kept in cell packs and irrigated with an automatic mist system (irrigation events every 2 h from 6 am until 8 pm; duration of each event 3 min) for 3 to 4 wks until the seedlings were ca. 30 cm tall. Seedlings were then transplanted into 1L nursery containers filled with Metro-Mix 500 and amended with 10 g of Osmocote Plus 15-9-12 per container. Plants were sub-irrigated and kept in a pollinator -free glasshouse; supplemental lighting was employed to artificially extend daylength to 16 h and air temperature was maintained at 27 C (day) and 16 C (night). 1 Note: Mention of a trademark or a proprietary produc t does not constitute a guarantee or warranty of the product by the Florida Agricultural Experiment Station and does not imply its approval to the exclusion of other products that may be suitable.

PAGE 264

247 These F1 plants were grown to reproductive maturity and screened for scape pubescence. Due to limited resources, only two F1 plants (BM1 and BM2) were selected and self-pollinated to generate F2 families. Self-pollination of 183 flowers on BM1 produced 147 seeds, while self-pollinati on of 195 flowers on BM2 produced 130 seeds. F2 seeds were collected, cleaned and stored following the protocols described above for F1 seeds. Seeds were germinated under water as before but germination vessels were placed on a bench in a greenhouse with no climate control and a daytime temperature range of ca. 30 C to 40 C. Sixty-three F2 seeds from BM1 germinated, as did forty-one seeds from BM2. Fifty-four F2 seedlings from BM1 and thirty-two F2 seedlings from BM2 were transplanted into 612 cell packs as before, but were stepped up to slightly smaller (12 cm) nursery containers filled with Metro-Mix 500 and amended with 10 g of Osmocote Plus 15-9-12 per container. Pl ants were sub-irrigated and grown to reproductive maturity in a sc reen house with no climate controls or supplemental lighting. Data from the F1 family were used to develop a working model to explain the type of gene action and number of loci cont rolling scape pubescence in pickerelweed. Development of this model allowed the assignment of genotypes to parents; the model was then verified by analyses of F2 families. All data were analyzed using goodness-of-fit (chi-square or 2) tests with Yates correc tion for continuity. Each F2 family was small (BM1 n = 54 and BM2 n = 32); therefore, ch i-square analysis was performed on pooled data from both families as well as on data from each family indi vidually. Heterogeneity chi-square analysis was conducted using uncorre cted chi-square values to confirm that

PAGE 265

248 pooling across the families was appropriate (i.e., that F2 families were from populations with the same segregation ratios). Results and Discussion All F1 plants evaluated in this experiment had pubescen t scapes, which indicated that the trait was dominant. These results also suggested that the parent RI was homozygous dominant at the locus or loci co ntrolling the trait and that the parent WM was homozygous recessive at the same locus or loci. Self-pollination of both BM1 and BM2 produced F2 populations that segregated for scape pubescence. Progeny from both F2 families were compared to a model where the trait was controlled by one diallelic locus with dominant gene action (Table 10.1) and to models where the trait was controlled by two diallelic loci (Tables 10.2 through 10.4). The model tested in Table 10.1 assumed th at the genotypes of RI and WM were GG and gg respectively. This would have cr eated a homogenous and heterozygous F1 population composed of me mbers with the genotype Gg and expressing the pubescent phenotype. Self-pollination of these F1 plants would be expected to produce F2 progeny that segregated in a 3:1 (pubescent:glabr ous) ratio, since the genotypic ratio of the population would be 0.25 GG :0.50 Gg :0.25 gg and the occurrence of dominance would cause the phenotypes of the heterozygous cl ass and the homozygous dominant class to be indistinguishable from one another. This mode l provided a poor fit to the data and less than 3 in 100 populations would show as much variation for this trait as BM1 or BM2 (Table 10.1). Heterogeneity chi-square anal ysis (not shown) re vealed that pooling progeny from BM1 and BM2 was appropriate; the model provided a poor fit to these pooled data as well (Table 10.1). These resu lts indicated it was unlikely that scape pubescence in this population of pickerelweed was controlled by a single diallelic locus.

PAGE 266

249 Only two phenotypes (pubescent and glabrous) were observed in this experiment; if pubescence were controlled by two loci, then segregation of F2 progeny must have been influenced by epistasis, which modified the 9:3:3:1 ratio expected when two loci control a trait. The epistatic models tested in this experiment assumed that the genotypes of RI and WM were GGHH and gghh respectively. Cross-pollinat ion between RI and WM would have created a hom ogenous and heterozygous F1 population composed of members with the genotype GgHh and expressing the pubescent scape phenotype. Self-pollination of F1 plants would have resulted in F2 progeny that segregated in different ways based on the type of epistatic interaction between the two loci controlling scape pubescence in pickerelweed. Self-pollination of heterozygous plan ts would be expected to produce F2 progeny that segregated in a 9:7 (pubescent:glabrous ) ratio if scape pubes cence were controlled by two diallelic loci influenced of duplicate recessive epistasis. This type of epistasis causes an alteration in the classical 9:3:3:1 ratio exp ected when two diallelic loci control a trait since duplicate recessive epista sis requires a dominant allele at each locus to effect expression of the trait of interest. Duplicat e recessive epistasis may occur when the two loci work in tandem to produce an end product; for example, a dominant allele at the first locus may be needed to produce a precursor substance, while a dominant allele at the second locus is required to c onvert the precursor to the end product. The result of this type of epistasis is the production of only two phenotypic classes in stead of four since three of the four classes ( G_hh = ggH_ = gghh ) are phenotypically identical. The class with a dominant allele at each locus ( G_H_ ) would express the pubescent phenotype, while the three classes with a homozygous rece ssive condition at one locus (or both loci)

PAGE 267

250 would exhibit the recessive glabrous phenotype. The parent RI was homozygous dominant at both loci and expressed the pubescent phenotype; the parent WM was homozygous recessive at both loci and e xhibited the glabrous phenotype, while all F1 progeny were heterozygous at both loci and expressed the pubescent phenotype. These observed phenotypes for parental and F1 generations conformed to those expected if scape pubescence in pickerelweed were cont rolled by two loci influenced by duplicate recessive epistasis. The validity of the model was tested by comparing the observed segregation of F2 progeny with the 9:7 (pubescent:gl abrous) ratio expe cted under this model (Table 10.2). This model provided a poor fit to the data and less than 1 in 10,000 populations would be expected to show as much variation fo r this trait as BM1 or BM2 (Table 10.2). Heterogeneity chi-square anal ysis (not shown) re vealed that pooling progeny from BM1 and BM2 was appropriate; the model provided a poor fit to these pooled data as well (Table 10.2). These resu lts indicated it was unlikely that scape pubescence in this population of pickerelweed was contro lled by two diallelic loci influenced by duplicate recessive epistasis. Self-pollination of heterozygous plan ts would be expected to produce F2 progeny that segregated in a 13:3 ( pubescent:glabrous) rati o if scape pubescence were controlled by two diallelic loci influen ced by dominant-and-recessive epistasis. This type of epistasis causes an alteration in the classical 9:3:3:1 ratio expected when two diallelic loci control a trait since the same phenotype is pr oduced by individuals with a dominant allele at one locus and/or a homozygous rece ssive condition at the second locus. Dominant-and-recessive epistasis may occur if a product such as a pigment is produced by the dominant allele at the first locus (in this case, the G locus); the same product is

PAGE 268

251 made by a homozygous recessive condition at the second locus (in this case, the H locus), while synthesis of the product does not occur if a dominant allele exists at the second locus (the H locus). The end result of this type of epistasis is the production of only two phenotypic classes instead of four si nce three of the four classes ( G_H_ = G_hh = gghh ) are phenotypically identical. The three classes with at least one dominant allele at the G locus and/or a recessi ve condition at the H locus would express the pubescent scape phenotype, while the class with a recessive condition at the G locus and at least one dominant allele at the H locus ( ggH_ ) would produce the glabrous scape phenotype. As with the previous epistatic model, th e observed phenotypes for parental and F1 generations conformed to t hose expected if scape pubes cence in pickerelweed were controlled by two loci influen ced by dominant-and-recessive ep istasis. The validity of the model was tested by comparing the observed segregation of F2 progeny with the 13:3 (pubescent:glabrous) ratio expected under this model (T able 10.3). This model provided a good fit to the data; as many as 21 in 100 popula tions would show as much variation for this trait as BM1 and as many as 11 in 100 popul ations would show as much variation as BM2 (Table 10.3). Heterogeneity chi-square an alysis (not shown) revealed that pooling progeny from BM1 and BM2 was appropriate; the model provided a poor fit to these pooled data and less than 4 in 100 populations would show as much variation for scape pubescence as the pooled families BM1 and BM2 (Table 10.3). These results indicated it was unlikely that scape pubescence in this population of pickerelw eed was controlled by two diallelic loci influenced by dominant-and-recessive epistasis. Self-pollination of heterozygous plan ts would be expected to produce F2 progeny that segregated in a 15:1 ( pubescent:glabrous) rati o if scape pubescence were controlled

PAGE 269

252 by two duplicate gene loci. This type of genetic control causes an alteration in the classical 9:3:3:1 ratio expected when two di allelic loci control a trait since systems controlled by duplicate gene loci require only a single dominant allele at either locus to effect expression of the trait of interest. Duplicate gene lo ci may occur when a dominant allele at either locus produces the same product; the end result of this type of epistasis is the production of only two phenot ypic classes instead of four since three of the four classes ( G_H_ = G_hh = ggH_ ) are phenotypically identical. The three classes with at least one dominant allele would expre ss the pubescent scape phenotype, while the completely homozygous recessive class ( gghh ) would exhibit glabrous scape phenotype. As with the previous models, the obs erved phenotypes for parental and F1 generations conformed to those expected if scape pubescen ce in pickerelweed were controlled by two duplicate gene loci. The validity of the m odel was tested by comparing the observed segregation of F2 progeny with the 15:1 (pubescent:g labrous) ratio expected under this model (Table 10.4). This model provided a very good fit to the data ; as many as 23 in 100 populations would show as much variation for this trait as BM 1 and virtually all populations would show as much variati on as BM2 (Table 10.4). Heterogeneity chi-square analysis (not s hown) revealed that pooling progeny from BM1 and BM2 was appropriate; the model provided a good fit to these pooled data and as many as 34 in 100 populations would show as much variation for scape pubescence as the pooled families BM1 and BM2 (Table 10.4). These results s uggested that scape pubescence in this population of pickerelweed may have been controlled by two duplicate gene loci. Conclusions These data indicated that scape pubescence in pickerelweed was not controlled by a single diallelic locus or by two diallelic loci influenced by duplicate recessive epistasis,

PAGE 270

253 as these models provided poor fits to the data observed for the F2 families BM1 and BM2. The model with dominant-a nd-recessive epistasis provided an adequate fit to the observed segregation of F2 progeny of both families when examined individually, but analysis of pooled data from both families re vealed the model provided a poor fit to the larger dataset. The model where two duplicate gene loci conditioned scape pubescence provided the best fit to th e observed segregation of F2 progeny of both families when examined individually and when pooled. Thes e results suggested that scape pubescence in this population of pickerelweed wa s controlled by two dupl icate gene loci ( G and H ) with dominant gene action. Pube scence resulted from the presence of a dominant allele at either locus ( G _ or _ H _), while a fully recessive genotype ( gghh ) resulted in a glabrous scape. It is worth noting that the parent RI (c ollected in Rhode Island) expressed the pubescent scape phenotype, while the parent WM (collected in southern Florida) produced the glabrous scape phenotype. It make s sense that a plant with a northern, more temperate provenance would have a pubescent inflorescence scape, as pubescence can create an insulating effect th at results in increased floral temperature (Maes et al. 2001; Miller 1986). This increase in temperature would allow flowers to open and anthers to dehisce earlier in the day and would result in a longer window of opportunity for insects to facilitate pollination. Ecotype s of pickerelweed collected from northern climes exhibit other adaptations to temperate environments (i.e., dormancy see Appendix B of this dissertation). It would be in teresting to determine the re lationships among traits that allow plants to survive a nd reproduce in northern environments; however, this was beyond the scope of this experiment.

PAGE 271

254 Table 10.1. Segregation and goodness-of-fit tests for F2 families of pickerelweed segregating for scape pubescence. Goodne ss-of-fit tests were computed based on a model where scape pubescence is cont rolled by one diallelic locus with dominant gene action with the glabro us phenotype produced as a result of a recessive condition. F2 family = BM1 (n = 54) Phenotype Expected frequency Expected number Observed 2# Pubescent 0.75 40.5 48 1.20988 Glabrous 0.25 13.5 6 3.62963 P = 0.0278 Grand chi-square = 4.83951 F2 family BM2 (n = 32) Phenotype Expected frequency Expected number Observed 2 Pubescent 0.75 24 30 1.26042 Glabrous 0.25 8 2 3.78125 P = 0.0247 Grand chi-square = 5.04167 Pooled F2 families BM2 (n = 86) Phenotype Expected frequency Expected number Observed 2 Pubescent 0.75 64.5 78 2.62016 Glabrous 0.25 21.5 8 7.86047 P = 0.0012 Grand chi-square = 10.4806 Phenotype: Phenotype of F2 progeny Expected frequency: Expected ra tio of progeny in each phenotypic class Expected number: Expected number of progeny in each phenotypic class Observed: Number of progeny obs erved in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test using Yates correction for continuity Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 272

255 Table 10.2. Segregation and goodness-of-fit tests for F2 families of pickerelweed segregating for scape pubescence. Goodne ss-of-fit tests were computed based on a model where scape pubescence is cont rolled by two diallelic loci under the influence of duplicate recessive ep istasis. The pubescent phenotype would be expressed only by individuals with a dominant allele at each locus. F2 family = BM1 (n = 54) Phenotype Expected frequency Expected number Observed 2# Pubescent 0.5625 30.375 48 9.65484 Glabrous 0.4375 23.625 6 12.4134 P = 0.0000 Grand chi-square = 22.0682 F2 family BM2 (n = 32) Phenotype Expected frequency Expected number Observed 2 Pubescent 0.5625 18 30 7.34722 Glabrous 0.4375 14 2 9.44643 P = 0.0000 Grand chi-square = 16.7937 Pooled F2 families (n = 86) Phenotype Expected frequency Expected number Observed 2 Pubescent 0.5625 48.375 78 17.5352 Glabrous 0.4375 37.625 8 22.5453 P = 0.0000 Grand chi-square = 40.0805 Phenotype: Phenotype of F2 progeny Expected frequency: Expected ra tio of progeny in each phenotypic class Expected number: Expected number of progeny in each phenotypic class Observed: Number of progeny obs erved in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test using Yates correction for continuity Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 273

256 Table 10.3. Segregation and goodness-of-fit tests for F2 families of pickerelweed segregating for scape pubescence. Goodne ss-of-fit tests were computed based on a model where scape pubescence is cont rolled by two diallelic loci under the influence of dominant-and-recessiv e epistasis. The pubescent phenotype would be expressed only by i ndividuals with the genotypes G_H_ G_hh or gghh F2 family = BM1 (n = 54) Phenotype Expected frequency Expected number Observed 2# Pubescent 0.8125 43.875 48 0.29950 Glabrous 0.1875 10.125 6 1.29784 P = 0.2062 Grand chi-square = 1.59734 F2 family BM2 (n = 32) Phenotype Expected frequency Expected number Observed 2 Pubescent 0.8125 26 30 0.47115 Glabrous 0.1875 6 2 2.04167 P = 0.1129 Grand chi-square = 2.51282 F2 families pooled (n=86) Phenotype Expected frequency Expected number Observed 2 Pubescent 0.8125 69.875 78 0.83207 Glabrous 0.1875 16.125 8 3.60562 P = 0.0351 Grand chi-square = 4.43769 Phenotype: Phenotype of F2 progeny Expected frequency: Expected ra tio of progeny in each phenotypic class Expected number: Expected number of progeny in each phenotypic class Observed: Number of progeny obs erved in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test using Yates correction for continuity Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 274

257 Table 10.4. Segregation and goodness-of-fit tests for F2 families of pickerelweed segregating for scape pubescence. Goodne ss-of-fit tests were computed based on a model where scape pubescence is c ontrolled by two duplicate gene loci. The pubescent phenotype would be expre ssed by individuals with a dominant allele at either locus. F2 family = BM1 (n = 54) Phenotype Expected frequency Expected number Observed 2# Pubescent 0.9375 50.625 48 0.08920 Glabrous 0.0625 3.375 6 1.33796 P = 0.2322 Grand chi-square = 1.42716 F2 family BM2 (n = 32) Phenotype Expected frequency Expected number Observed 2 Pubescent 0.9375 30 30 0.00000 Glabrous 0.0625 2 2 0.00000 P = 0.9999 Grand chi-square = 0.00000 F2 families pooled (n = 86) Phenotype Expected frequency Expected number Observed 2 Pubescent 0.9375 80.625 78 0.05601 Glabrous 0.0625 5.375 8 0.84012 P = 0.3438 Grand chi-square = 0.89612 Phenotype: Phenotype of F2 progeny Expected frequency: Expected ra tio of progeny in each phenotypic class Expected number: Expected number of progeny in each phenotypic class Observed: Number of progeny obs erved in each phenotypic class # 2: Chi-square value computed from goodnessof-fit test using Yates correction for continuity Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 275

258 Figure 10.1. Glabrous infloresce nce scape of pickerelweed.

PAGE 276

259 Figure 10.2. Pubescent inflorescen ce scape of pickerelweed.

PAGE 277

260 CHAPTER 11 INHERITANCE AND GENETIC C ONTROL OF A SECOND LOCUS INFLUENCING FLOWER COLOR Introduction Flower color in pickerelweed was describe d in Chapter 7 of this dissertation as being controlled by a single di allelic locus with blue flow er color dominant to white flower color. A series of cr osses involving one plant (WL) revealed the existence of a second locus that played a role in the expres sion of flower color in this population of pickerelweed. The objective of this experiment was to determine the inheritance of this second locus and to assess its effect on the genetic control of fl ower color in this population of pickerelweed. Materials and Methods The parents used in this experiment were selected from a collection of plants maintained for breeding and genetics studies at the University of Florida in Gainesville and were grown as described in Appendix A. The parent WL had white L-morph flowers and the parent WM had white M-morph flowers. The parent WL was also cross-pollinated with the parents BS, BM and WS, but the main focus of this study involved cross-pollinations between WL and WM Each parent was self-pollinated with pollen produced by the anther levels desc ribed by Ordnuff (19 66) as being most productive (i.e., WM was pollinated using se lf-produced l-pollen and WL utilized self-produced m-pollen). Stylar surgery (see Ch apter 4 of this dissert ation) was employed in self-pollinations of WL, as normal self-pollination of this parent failed to produce any

PAGE 278

261 S1 seeds. Crossand reciprocal pollinations were performe d between the parental lines and utilized compatible pollen (i.e., l-polle n from WM was used to pollinate WL and m-pollen from WL was used to pollinate WM ). Barrett and Glover (1985) stated that emasculation was not necessary to prevent self-pollination, so anthers borne on long filaments were removed only from WM to increase access to the stigma. Fine forceps were used to remove dehi sced anthers from th e pollen parent; an anther was then brushed gently across the stig ma of the seed parent to effect pollination. Magnifying headgear was worn during all pollinations to allow visual confirmation of successful transfer and adhesion of pollen gr ains to the stigma. Flowers from a given inflorescence were used as either poll en donors or seed parents to eliminate contamination from self-produced pollen. Pol linations commenced with the opening of the first flowers of an inflorescence and con tinued until all flowers in the inflorescence had been pollinated (ca. 7 to 12 d). All pol linations were performed between 9 am and 3 pm daily and pollination data were recorded on jewelry tags placed on each inflorescence. Each completed inflorescen ce was enclosed in a small mesh bag and secured with a plastic-covered twist-tie until fru its were ripe. Fruits were considered ripe when the bearing infructescence shattered (usually 23 to 30 d after completion of pollinations). Fruits were collected and cleaned as described in Appendix A; seeds were germinated and seedlings were grown out us ing the methods outlined in Appendix A as well. Eight F1 plants were selected for generation advancement; each was self-pollinated using pollen from anthers described by Ordnu ff (1966) as being most productive (i.e., l-pollen was used to pollinated M-morph pl ants and m-pollen was used to pollinate

PAGE 279

262 L-morph plants). This resulte d in the creation of eight F2 families. Three of the eight F1 plants (BL1, BL2 and BL3) selected for generation advancement were L-morphs; as with the parent WL, normal self-pollinati on of these plants failed to produce any S1 seeds, so stylar surgery was employed to facilitate seed set. Data from F1 and S1 populations were used to determine the type of gene action controlling flower color in pickerelweed and to assign likely genotype s to the parents WL and WM. Working models to explain the t ype of gene action and number of loci controlling flower color in these families were developed based on segregation of progeny from F2 populations. Models were then te sted by comparing the number of progeny with blue or white flowers observed in F2 populations with the number expected under the proposed models. All data were an alyzed using goodness-of -fit (chi-square or 2) tests with Yates correction for continuity. Results and Discussion All S1 progeny from WM and WL bore white flowers; this revealed that each parent was homozygous for flower color, as S1 progeny did not segregate for the trait. All F1 plants derived from cross-pollinations be tween WL and WM bore blue flowers. The parent WL was also hybridized with the parents BS, BM and WS; all F1 progeny from these cross-pollinations had blue flowers as well, but the populations that resulted from these cross-pollinations were very small and were not advanced to the F2 generation (data not shown). These results suggested that a second locus influenced fl ower color in pickerelweed and that the parents WL and WM were most likely homozygous dominant and homozygous recessive, respectively, at one locus and homozygous recessive and homozygous dominant, respectiv ely, at the second locus. Self-pollination of eight F1

PAGE 280

263 plants yielded eight F2 populations that segregated fo r flower color (Table 11.1). Only two phenotypes (blue and white) were observed in this experiment; therefore, segregation of F2 progeny appeared to be influenced by epis tasis, which modified the 9:3:3:1 ratio expected when two loci control a trait. Epistasis can influence the expression of so me traits controlled by two loci so that two phenotypic classes are produced instead the expected four classes. The models tested in this experiment assumed that the genotypes of WL and WM were WWcc and wwCC respectively. This would result in each pare nt breeding true for flower color, as each parent was homozygous at bot h loci. Cross-pollination betw een WL and WM would be expected to create a hom ogenous and heterozygous F1 population composed of members with the genotype WwCc and expressing the blue-flowered phenotype. Self-pollination of F1 plants would be expe cted to result in F2 progeny that segregated in different ways based on interaction between the two loci co ntrolling flower color in this population of pickerelweed. Self-pollination of heterozygous plan ts would be expected to produce F2 progeny that segregated in a 15:1 (blue:white) ratio if flower color were controlled by two diallelic loci influenced by duplicate dominant epistasis. This type of epistasis would cause an alteration in the classical 9:3: 3:1 ratio expected when two di allelic loci control a trait since duplicate dominant epistasis requires only a single dominant allele at either locus to effect expression of the trait of interest. Duplicate dominant epistasis may occur when a dominant allele at either locu s produces the same product; the end result of this type of epistasis is the production of only two phenotypi c classes instead of four since three of the four classes ( W_C_ = W_cc = wwC_ ) are phenotypically identical. The three classes

PAGE 281

264 with at least one dominant al lele would be expected to express the blue-flowered phenotype, while the completely homozygous recessive class ( wwcc ) would be expected to exhibit the white-flowered phenotype. Each parent in th is experiment was homozygous dominant at one of the two loci but each expressed the recessive white-flowered phenotype; based on this model, each parent would be expected to express the blue-flowered phenotype. It was therefore unlikel y that flower color in this experiment was influenced by duplicate dominant epistasis. Self-pollination of heterozygous plan ts would be expected to produce F2 progeny that segregated in a 13:3 (blue:white) ratio if flower color were controlled by two diallelic loci influenced by dominant-and -recessive epistasis. This ty pe of epistasis would cause an alteration in the classical 9:3:3:1 ratio exp ected when two diallelic loci control a trait, since the same phenotype is produced by individua ls with a dominant allele at one locus and/or a homozygous recessive condition at the second locus. Dominant-and-recessive epistasis may occur if a product such as a pi gment is produced by the dominant allele at the first locus (in this case, the W locus); the same product is made by a homozygous recessive condition at the second locus (in this case, the C locus), while synthesis of the product does not occur if a dominant alle le exists at the second locus (the C locus). The end result of this type of epistasis is the production of only two phenotypic classes instead of four since thre e of the four classes ( W_C_ = W_cc = wwcc ) are phenotypically identical. The three classes with at least one dominant allele at the W locus and/or a recessive condition at the C locus would be expected to express the blue-flowered phenotype while the class with a recessive condition at the W locus and at least one dominant allele at the C locus ( wwC_ ) would be expected to exhibit the white-flowered

PAGE 282

265 phenotype. The parents in this expe riment were assigned the genotypes wwCC (parent WM) and WWcc (parent WL), so each parent was homozygous dominant at one locus and homozygous recessive at the other locus. If flower color in this experiment were influenced by dominant-and-recessive epis tasis, the parent WM with the genotype wwCC would have been expected to express the blue-flowered phenotype, but both parents bore white flowers. It was therefore unlikely th at flower color in this experiment was influenced by dominant-and-recessive epistasis. Self-pollination of heterozygous plan ts would be expected to produce F2 progeny that segregated in a 9:7 (blue:white) ratio if flower color in this experiment were controlled by two diallelic loci influenced by duplicate recessive epistasis. This type of epistasis causes an alteration in the classical 9:3:3:1 ratio expected when two diallelic loci control a trait, since duplicate recessive epistasis requires a do minant allele at each locus to effect expression of the trait of interest Duplicate recessive epistasis may occur when the two loci work in tandem to produce an end product; for example, a dominant allele the first locus may be needed to produce a pi gment precursor, while a dominant allele at the second locus is required to convert the precu rsor to the end product. The result of this type of epistasis is the production of only two phenotypic classes in stead of four since three of the four classes ( W_cc = wwC_ = wwcc ) are phenotypically identical. The class with a dominant allele at each locus ( W_C_ ) would be expected to express the blue-flowered phenotype, while the three cla sses with a homozygous recessive condition at one locus (or both loci) w ould be expected to exhib it the white-flowered phenotype. Each parent and S1 offspring in this experiment wa s homozygous dominant at one locus and homozygous recessive the other locus; in addition, all F1 progeny were heterozygous

PAGE 283

266 at both loci. If flower color in this population of pickerelweed were controlled by two loci acting under the influence of duplicate rece ssive epistasis, each parent and each S1 plant would have expressed the white-flowere d phenotype, since the parent or S1 genotype would have been either wwCC (parent WM and S1 progeny from WM) or WWcc (parent WL and S1 progeny from WL). All F1 progeny would have produced the blue-flowered phenotype, as each F1 plant would have had the genotype WwCc The phenotypes of the parents, S1 progeny and F1 progeny conformed to those expected under a model where two loci control flower color, with expression influenced by duplicate recessive epistasis. The validity of the model was tested by comparing the observed segregation of F2 progeny with the 9:7 (blue:white) ratio e xpected under this model (Table 11.2). The model where flower color was controlled by two diallelic loci influenced by duplicate recessive epistasis provided a good fit to seven of the eight F2 families examined in this experiment. The family BM1 failed to segreg ate as expected under this model; however, as many as 1 in 25 populations of pickerel weed the size of BM1 (n=50) would be expected to show as much vari ation for flower color as the F2 family BM1, so this discrepancy may have been attr ibutable to sampling error. Conclusions These data suggested that a second locus ( C ) influenced the expression of flower color in this population of pickerelweed. E xpression of blue flower color required the presence of a dominant allele at each of th e two loci in this system (i.e., genotype W C _), while an individual with a homozygous r ecessive condition at either locus (i.e., W cc wwC or wwcc ) would be expected to bear white fl owers. The model tested in Table 11.2 provided a good fit to the phenot ypes observed in parental, S1, F1 and F2 generations;

PAGE 284

267 therefore, these data suggested that flower color in this population of pickerelweed was controlled by two diallelic loci influenced by duplicate recessive epistasis. It was not unusual to find that flower co lor in this population of pickerelweed was influenced by another lo cus in addition to the W locus described in Chapter 7 of this dissertation. The parents utilized in Chapte r 7 (BS, BM and WS) may each have been homozygous dominant at the C locus, as all F1 progeny derived from cross-pollinations between these parents and WL had blue flower s. Flower color is often controlled by a single locus (Estilai 1984; Gaus et al. 2003; Mosjidis 2000; Zufall and Rausher 2003); however, systems where multiple loci influence flower color have been described as well (Brewbaker 1962; Griesbach 1996). There are several explanations as to why WL had a homozygous recessive condition at the C locus; this may have been the result of two forward mutations ( C c ), or WL may have been derived from a populatio n with a different genetic base than the other parents examined in this experiment. The parent WL was obtained from a commercial source for this pr oject and the provenance of the plant was unknown; based on this information and on the rare occurren ce of mutations, it is possible that the homozygous recessive condition at the C locus of WL was a function of the genic composition of the population from which the pl ant was selected, as opposed to the result of mutation.

PAGE 285

268 Table 11.1. Flower color of progeny in S1, F1 and F2 families of pickerelweed derived from the parents WL and WM. Family Gen Flrs Seeds Germ# Plants B W WL S1 130 33 6 6 0 6 WM S1 176 116 71 44 0 44 WM x WL F1 107 97 71 71 71 0 WL x WM F1 329 82 47 47 47 0 BM1 (WMWL51) F2 173 158 57 50 36 14 BM2 (WMWL53) F2 146 119 24 13 7 6 BM3 (WMWL15) F2 216 128 18 14 11 3 BM4 (WMWL31) F2 192 132 29 22 13 9 BM5 (WMWL19) F2 154 106 15 11 4 7 BL1 (WMWL24) F2 282 128 56 28 17 11 BL2 (WMWL65) F2 568 268 34 28 20 8 BL3 (WMWL25) F2 131 60 25 22 15 7 Family: S1, F1 or F2 family under investigation; each S1 family was developed by self-pollination of one of the parents (WM or WL); F1 families were created by cross-pollinations between the parents WM and WL; each F2 family was generated by self-pollination of an individual F1 plant (in parentheses) Gen: Generation of family under investigation Flrs: Number of flowers pollinated; stylar su rgery was utilized in th e self-pollination of all L-morph plants (WL, BL1, BL2, BL3) Seeds: Number of seeds produced # Germ: Number of seeds germinated; all seeds placed under germination conditions except for the family BL2, where 100 seeds placed under germination conditions Plants: Number of F1, S1 or F2 plants examined B: Number of blue-flowered progeny W: Number of white-flowered progeny

PAGE 286

269 Table 11.2. Segregation for flower co lor and goodness-of-fit tests for F2 families of pickerelweed. Each F2 family was derived from self-pollination of an individual F1 plant created by cross-pollinations between the parents WM and WL. Goodness-of-fit tests were based on a model where flower color was controlled by two diallelic loci with dup licate recessive epista sis. Blue flower color results from the presence of a dominant allele at each locus ( W C _). F2 family = BM1 (n = 50) Phenotype Expected frequency Expected number Observed 2# Blue 0.5625 28.125 36 1.93389 White 0.4375 21.875 14 2.48643 P = 0.0355 Grand chi-square = 4.42032 F2 family = BM2 (n = 13) Phenotype Expected frequency Expected number Observed 2 Blue 0.5625 7.3125 7 0.00000 White 0.4375 5.6875 6 0.00000 P = 0.9999 Grand chi-square = 0.00000 F2 family = BM3 (n = 14) Phenotype Expected frequency Expected number Observed 2 Blue 0.5625 7.875 11 0.87500 White 0.4375 6.125 3 1.12500 P = 0.1572 Grand chi-square = 2.00000 F2 family = BM4 (n = 22) Phenotype Expected frequency Expected number Observed 2 Blue 0.5625 12.375 13 0.00126 White 0.4375 9.625 9 0.00162 P = 0.9571 Grand chi-square = 0.00289 F2 family = BM5 (n = 11) Phenotype Expected frequency Expected number Observed 2 Blue 0.5625 6.1875 4 0.46023 White 0.4375 4.8125 7 0.59173 P = 0.1836 Grand chi-square = 1.05195 F2 family = BL1 (n = 28) Phenotype Expected frequency Expected number Observed 2 Blue 0.5625 15.75 17 0.03571 White 0.4375 12.25 11 0.04592 P = 0.7751 Grand chi-square = 0.08163

PAGE 287

270 Table 11.2. Continued F2 family = BL2 (n = 28) Phenotype Expected frequency Expected number Observed 2 Blue 0.5625 15.75 20 0.89286 White 0.4375 12.25 8 1.14796 P = 0.1531 Grand chi-square = 2.04082 F2 family = BL3 (n = 22) Phenotype Expected frequency Expected number Observed 2 Blue 0.5625 12.375 15 0.36490 White 0.4375 9.625 7 0.46916 P = 0.3611 Grand chi-square = 0.83405 Phenotype: Flower color of F2 progeny Expected frequency: Expected ra tio of progeny in each phenotypic class Expected number: Expected number of progeny in each phenotypic class Observed: Number of progeny obs erved in each phenotypic class # 2: Chi-square value comput ed from goodness-of-fit test Grand chi-square: Sum of chi-square values computed for each phenotypic class P: Probability associated with grand chi-square value

PAGE 288

271 CHAPTER 12 SUMMARY AND CONCLUSIONS The experiments discussed in this disser tation yielded valuable information about seed germination, pollination techniques, pollen grain diameter, in vitro pollen germination and genetic control of mor phological characters in pickerelweed ( Pontederia cordata L.). Parts of this work were simila r to reports published by other workers; however, the majority of the topics contained within this document had not been explored prior to this study. Morphometric data analyzed in Chapter 3 confirmed the results of Barrett and Glover (1985) and Price a nd Barrett (1982, 1984) and showed that the diameters of pollen grains produced by anthers borne by th e three filament lengt hs of pickerelweed were significantly different from one another. Diameters of grains of s-pollen averaged 20.46 0.34 m, while mean diameters of m-pollen and l-pollen measured 35.04 0.49 m and 44.97 0.34 m, respectively. No overlap in grain diameter occurred among the three classes of pollen. In vitro pollen germination did not produce the same results as those reported in vivo by Anderson and Barrett (1986). The rela tionships among pollen tube lengths produced in vitro by the three pollen grain diameter classes differed from those described by Anderson and Barrett (1986), who reporte d significant differences among the pollen tubes produced by all three pollen grain diameter classes. The experiment outlined in Chapter 3 of this dissertation showed that there was no signifi cant difference in lengths of pollen tubes produced by l-pollen and those produced by m-pollen 240 min after

PAGE 289

272 germination. Pollen tubes produced in vitro by l-pollen and m-pollen averaged 486.43 m and 431.14 m in length, respectively, 240 min after germination, while pollen tubes from s-pollen reach ed an average length of 265.57 m. The reason for these conflicting results was unknown but it is possible that factors such as stylar interaction (i.e., the presence of inhibitory or stimulato ry substances) with the germinating pollen grain influenced in vivo germination. A significant positive re gression between pollen grain diameter and in vitro pol len tube length was identified; these results were similar to those described by Anderson and Barrett (1986) for in vivo pollen germination and suggested that pollen diameter had a positiv e impact on the growth of pollen tubes produced during in vitro germination. Self-incompatibility and the resultant poor seed set in some floral morphs of pickerelweed was overcome with the use of the novel pollination techniques developed, tested and described in Chapter 4 of this dissertation. Physical c onstraints (i.e., style length in L-morphs and stigma access in S-mo rphs) played an important role in the prevention of self-pollination in pickerelweed; results presented in Chapter 4 showed that the use of stylar surgery in L-morph plants and corolla removal in S-morph plants greatly increased seed set after self-pollination. Seed set after control self -pollination of a group of L-morph plants averaged 1.97%; the use of st ylar surgery on this same group of plants increased mean seed set af ter self-pollination to 29.93% Seed set after control self-pollination of a group of S-morph pl ants averaged 19.76%; the use of corolla removal on this same group of plants increase d mean seed set afte r self-pollination to 45.53%. This information will be helpful for pl ant breeders and geneticists interested in studying this and other tristylous species as these techniques can be utilized to improve

PAGE 290

273 seed set after self-pollination and facilitate the development of S1 populations and inbred lines. The study described in Chapter 5 tested the e ffect of various seed storage intervals, conditions and germination environments on the seeds of pickerelweed in an effort to determine optimum seed storage and germ ination conditions. Results from this experiment showed that best germination of seeds of pickerelweed occurred when de-husked seeds were germinated under water. These results confirmed previous reports that highest germination rates occurred when seeds were not buried but were germinated under water; this experiment also provide d new evidence that de-husked seeds of pickerelweed germinated at a higher rate than intact fruits. Stratification was not necessary to induce germination of seeds of pickerelweed a nd germination percentage of seeds stored for 6 mo was less than germinati on rates of fresh seeds or seeds stored for 3 mo, which suggested that seed ag e had a negative impact on viability. The genetic control and inheri tance of albinism in this population of pickerelweed was described in Chapter 6. Albinism is a lethal trait and seedlings exhibiting this condition did not survive for more than 3 wks after germination. Data collected from the families examined in Chapter 6 showed that al binism in this population of pickerelweed was controlled by three diallelic loci ( P A and B ) that interacted in an epistatic manner. Two alleles ( P1 and P2) were identified for the epistatic locus P Gene action at the two hypostatic loci ( A and B ) was dominant and the hypostatic loci functioned as duplicate factors. Production of a green (non-a lbino) seedling required either two P1 alleles at the epistatic locus, or one P1 allele at the epistatic locus a nd at least one dominant allele at

PAGE 291

274 either hypostatic locus. Albinism occu rred in seedlings with the genotypes P2P2_ _ or P1P2aabb Results presented in Chapter 7 demonstrated that flower color in this population of pickerelweed was controlled by a single diallelic locus ( W ) with completely dominant gene action. Individuals with homozygous dominant ( WW ) or heterozygous ( Ww ) genotypes produced blue flowers, while whit e flowers were borne by plants with the homozygous recessive genotype ( ww ). The inheritance and genetic control of floral mor ph in this population of pickerelweed was examined in Chapter 8 and the data suggested that floral morph was determined by two dialleic loci ( S and M ) influenced by epistasis. This system was the same as that described in other tristylous species; gene action at each locus was dominant and the S locus was epistatic to the M locus. Individuals with the genotypes S _ were S-morphs and had short styles, while those with the genotypes ssM were M-morphs and bore mid styles. Long styles were produced by L-morph plants and resulted from a completely recessive condition at both loci ( ssmm ). Data analyzed in Chapter 9 of this di ssertation revealed that linkage existed between the W locus responsible for flower color and the M locus of the system controlling floral morph in this population of pickerelweed. These loci were close together on the same chromosome and segreg ation of progeny suggested that 16 m.u. was a viable estimate of the genetic distance between the W and M loci. This experiment also showed that the epistatic S locus controlling floral mor ph was independent from both the W locus and the M locus.

PAGE 292

275 Analyses in Chapter 10 suggested that scape pubescence in this population of pickerelweed was controlled by duplicate ge ne loci with two diallelic loci ( G and H ). Individuals with a dominant al lele at either locus would be expected to produce the pubescent phenotype, while a completely r ecessive condition would result in the production of an individual with a glabrous scape. A second locus that influenced the expressi on of flower color in this population of pickerelweed was described in Chapter 11. Thes e data showed that flower color in this population of pickerelweed was influenced by the locus C in addition to the W locus described in Chapter 7 of this dissertation. Expression of blue flow er color required the presence of a dominant allele at each of the tw o loci in this system, while an individual with a homozygous recessive c ondition at either locus ( ww _ or _ cc ) would be expected bear white flowers. This work is by no means comprehensive but adds to the body of scientific knowledge available for pickerelweed. This is the first publication of the inheritance of morphological traits in pickerel weed and one of a handful of studies that has attempted to overcome self-incompatibility through stylar surgery. Pickerelweed is a naturally out-crossed species and a great d eal of natural vari ation exists, even in the small gene pool examined during the course of this proj ect. I hope other investigators will use this dissertation as a resource to he lp them exploit the variation in pickerelweed to create new lines with desirable characteristics and to conduct genetic research on this and other tristylous species.

PAGE 293

276 APPENDIX A POPULATION DEVELOPMENT FOR INHERITANCE STUDIES Introduction Pickerelweed ( Pontederia cordata L.) is an attractive shoreline aquatic species that is frequently used in ornamental aquas capes, as the showy purplish-blue or white inflorescences of this native herbaceous pe rennial make pickerelweed a prime candidate for inclusion in water gardens. There is a gr eat deal of morphologi cal variation in the species and it may be possible to exploit this variation to de velop cultivars of pickerelweed with more desirable ornamental characteristics. Breeding programs are most productive when the mode of inheri tance and type of gene action are known, as experiments can be planned with more precision and efficiency. There is little published inform ation describing the inheritance or genetic control of any trait in pickerelweed. Ba rrett and Anderson (1985) examined a small number of progeny in an attempt to confirm th e genetic control of tristyly in the species, but the population under investigation was so sm all that definitive conclusions were not possible. Studies designed to investigat e the type of gene action a nd mode of inheritance of a given trait often examine several generations of the organism of interest, including progeny derived from self-pollination. The objective of this project was to develop a multigenerational population of pickerelweed to determine the type of gene action and number of loci controlling a vari ety of traits in pickerelweed.

PAGE 294

277 Materials and Methods Plants used in these experiments were se lected from a diverse collection of plants being maintained for breeding and genetics st udies at the University of Florida in Gainesville. All plants were grown in 1-L nursery containe rs filled with Metro-Mix 5001, a commercially available growing substr ate that contains 40 to 50% composted pine bark, 20 to 35% horticultural grade ve rmiculite and 12 to 22% Canadian sphagnum peat moss by volume with a nutrient charge and pH adjustment (Scotts-Sierra, Marysville, OH). Nutrition was supplied by the incorporation of 10 g of Osmocote Plus 15-9-12 (Scotts-Sierra, Marysville OH) per container. Plants were sub-irrigated and kept in a pollinator-free glasshouse with air temperature maintained at 27 C (day) and 16 C (night). During preliminary experiments, we observed that some genotypes were more floriferous when grown under long days; ther efore, supplemental lighting was employed to artificially extend dayle ngth to 16 h in this study. Plants used as parental li nes were selected based on phenotype and flowering time. Six parents were chosen to represent all combin ations of flower colo r (white or blue) and floral morph (short, mid or long), then all possible compatible crossand reciprocal pollinations were performed between the pare ntal lines. Selfpollinations were also performed on each of the six parents and uti lized pollen from anther levels described by Ordnuff (1966) as being most productive (i .e., L-morphs were pollinated with m/L pollen, M-morphs with l/M pollen and Smorphs with m/S pollen). Pollen was transferred in a similar manner in cross-pollinations and self-pollinations. 1 Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the product by the Florida Agricultural Experiment Station and does not imply its approval to the exclusion of other products that may be suitable.

PAGE 295

278 Barrett and Glover (1985) st ated that emasculation was not necessary to prevent self-pollination, so anthers were removed onl y when their presence restricted access to the stigma (i.e., anthers borne on the long f ilaments of M-morphs, anthers borne on the long and mid filaments of S-morphs). Fine forc eps were used to remove dehisced anthers from the pollen parent; an anther was then br ushed gently across the stigma of the seed parent to effect pollination. Magnifying hea dgear was worn during all pollinations and allowed visual confirmation of successful transfer and adhe sion of pollen grains to the stigma. Forceps were flame-sterilized between pollination of different plants to prevent contamination with foreign pollen. Pollinations commenced with the opening of the first flowers of an inflorescence and continued until all flowers in the in florescence had been pollinated (ca. 7 to 12 d). All pollinations we re performed between 9 am and 3 pm daily and all flowers in each inflorescence were pollinated using the same method. Daily pollination data were recorded on jewelry tags placed on each inflorescence. Each completed inflorescence was enclosed in a small mesh bag and secured with a plastic-covered twist-tie until fruits were ri pe. Fruits were considered ripe when the bearing infructescence shattered (usually 23 to 30 d after completion of pollinations). Fruits were collected in their mesh bag (F igure A.1) and air-dried for ca. 7 d, then de-husked using a rubber-covered rub boar d. Cleaned seeds were stored at 4 C in labeled coin envelopes until all pollinations were completed. Seeds were germinated under ca. 5 cm of water in glass half-pint (250 mL) bottles, with additional water added as needed to maintain a constant depth. Germin ation vessels were placed on a bench in the greenhouse (Figure A.2) under the temperatur e and light conditions described above. Germinated non-albino seeds were allowed to remain under water for 2 or 3 d after

PAGE 296

279 germination, then were transplanted in to 612 cell packs fill ed with Metro-Mix 500 (Figure A.3). F1 seedlings produced in this manner were labeled with a tag indicating parental identity and germination rank (i.e., seedling number from a particular pollination event). Seedlings were kept in cell packs a nd irrigated with an automatic mist system (irrigation events every 2 h from 6 am until 8 pm; duration of each event 3 min) for 3 to 4 wks until the seedlings were ca. 30 cm tall. Seedlings were then transplanted into 1-L nursery containers filled with Metro-Mix 500 and amended with 10 g of Osmocote Plus 15-9-12 per container. Plants were sub-irriga ted and kept in a pollinator-free glasshouse; supplemental lighting was used to artificiall y extend daylength and air temperature was maintained at 27 C (day) and 16 C (night). These F1 plants were grown to reproductive maturity and screened for flower color and floral morph. Representative sa mples were selected from each F1 family and self-pollinated to generate F2 families. Each F2 family was created by self-pollination of an individual F1 plant. Each flower of picker elweed has only one functional ovule (Richards and Barrett 1987); th erefore, seed set was repres entative of polli nation success. F1 plants that produced less than 10% seed se t after self-pollination using the pollination methods described above were classified as self-incompatible and subjected to either stylar surgery (L-morph plants ) or corolla removal (S-mor ph plants) as described in Chapter 4 of this dissertation. F2 seeds were collected, cleaned and stored following the same protocols as described above for F1 seeds. Seeds were germinated under water as before, but germination vessels were placed on a bench in a greenhouse with no climate control and a daytime temp erature range of ca. 30 C to 40 C. The resultant F2 seedlings were transplanted into 612 cell packs as before, but were grown in the greenhouse with

PAGE 297

280 no climate control (Figure A.4). Seedlings we re stepped up to slightly smaller (12 cm) nursery containers filled with Metro-Mix 500 and amended with 10 g of Osmocote Plus 15-9-12 per container. Plants were sub-irriga ted and grown to reproductive maturity in a screen house with no climate controls or supplemental ligh ting (Figure A.5). Descriptions of the Families Six parents were chosen for inclusion in these inheritance studies, but the plant selected to represent the white-flowered Lmorph phenotype had poor fertility in most pollination events. Populations that were deve loped using this parental line were too small to provide meaningful data; theref ore, populations developed using this white-flowered L-morph parent are not include d in the following descriptions. Each of the other five plants selected for use as pa rental lines was assigne d a code indicative of the lines phenotype. WS was a white-f lowered S-morph line, while WM was a white-flowered M-morph line. BS, BM and BL all had blue flowers; BS was an S-morph line, while BM and BL were M-mor ph and L-morph lines, respectively. Data for S1, F1 and F2 families are presented in Tables A.1 through A.8. Families were assigned codes based on pedigree. F1 families were labeled with the identity of the parents used to create the family; for example, the F1 family WMBL was derived from cross-pollinations between th e parents WM and BL. Each F2 family was coded with the phenotype and selection rank of the F1 plant self-pollinated to create the F2 family; for example, the F2 family BM2 was derived from self-pollination of the second blue mid F1 plant selected for generation advancement in a particular family. There was considerable overlap of coding among F2 families; for example, six of the seven F1 families produced blue mid progeny and each had members selected for generation advancement, so six F2 families were assigned the code BM2. Color-coded labels were

PAGE 298

281 used with F2 populations to identify the F1 family from which the F2 families were derived; for example, all F2 families produced from self-pollination of F1 plants from the parents WM and BL were labeled with an or ange planter tag. Most families derived from cross-pollinations between the parent al lines were advanced to the F2 generation. Self-pollinations were performed on F1 plants from the cross/ reciprocal set WSWM and WMWS; however, the resultant F2 seeds were colonized by an unknown fungal pathogen and failed to germinate. Results and Discussion This project developed a multigenerat ional population to be used in the determination of the type of gene action and number of loci control ling various traits in this population of pickerelweed. The inheritanc e studies that utilized this population were described in detail in Chapters 6, 7, 8 and 9 of this dissertation.

PAGE 299

282 Table A.1. F1 progeny of pickerelweed derived from the parents WS and WM, and S1 progeny derived from self-pollinations of the parental lines WS, WM, BS, BM and BL. PE Gen Code Flrs Seeds# Germ PGen Prog WS x WM P x P WSWM 239 102 88/102 F1 59 WM x WS P x P WMWS 83 75 68/75 F1 39 WS P WS 332 119 66/119 S1 21 WM P WM 176 116 71/116 S1 44 BS P BS 299 89 10/89 S1 9 BM P BM 358 104 14/38 S1 11 BL P BL 582 204 104/133 S1 56 PE: Pollination event; identity of plants used in cross-pollinations or self-pollinations Gen: Generation of plants used in pollination event; P = parental Code: Code used to identify progeny produced from pollination event Flrs: Number of flowers pollinated # Seeds: Number of seeds produced Germ: Number of seeds germinated / numbe r of seeds placed in germination vessels PGen: Generation of plants produced from pollination event Prog: Number of progeny ev aluated for floral traits

PAGE 300

283 Table A.2. F1 progeny of pickerelweed derived from the parents WS and BM, and F2 progeny derived from self-pollination of F1 progeny. Each F2 family was developed through self-pollination of an individual F1 plant. PE Gen Code Flrs Seeds# Germ PGen Prog WS x BM P x P WSBM 158 61 54/61 F1 50 BM x WS P x P BMWS 112 98 95/98 F1 94 BMWS8 F1 WS1 796 165 72/165 F2 56 BMWS30 F1 WS2 464 165 51/165 F2 48 BMWS37 F1 WS3 278 122 56/122 F2 30 WSBM43 F1 WS4 392 109 41/109 F2 31 WSBM44 F1 WS5 336 145 77/145 F2 31 WSBM51 F1 WS6 601 190 90/190 F2 41 WSBM24 F1 WS7 894 128 21/128 F2 13 WSBM13 F1 WM1 244 173 10/100 F2 3 WSBM26 F1 WM2 184 148 60/100 F2 29 BMWS85 F1 WM3 230 171 34/100 F2 11 BMWS60 F1 WL1 564 206 48/100 F2 5 BMWS41 F1 WL2 335 135 15/135 F2 10 WSBM14 F1 WL3 1155 120 10/120 F2 6 BMWS5 F1 BS1 714 157 11/157 F2 4 BMWS21 F1 BS2 585 207 141/207 F2 85 BMWS54 F1 BS3 529 167 30/167 F2 22 WSBM5 F1 BS4 450 173 119/173 F2 88 WSBM21 F1 BS5 492 148 21/148 F2 3 WSBM41 F1 BS6 700 262 102/262 F2 51 BMWS36 F1 BM1 256 119 65/119 F2 33 WSBM45 F1 BM2 313 206 145/206 F2 87 BMWS33 F1 BL1 1002 120 38/100 F2 18 WSBM32 F1 BL2 929 103 40/103 F2 20 PE: Pollination event; identity of plants used in cross-pollinations or self-pollinations Gen: Generation of plants used in pollination event; P = parental, F1 = derived from cross-pollinations betwee n the parents WS and BM Code: Code used to identify progeny produced from pollination event Flrs: Number of flowers pollinated # Seeds: Number of seeds produced Germ: Number of seeds germinated / numbe r of seeds placed in germination vessels PGen: Generation of plants produced from pollination event Prog: Number of progeny ev aluated for floral traits

PAGE 301

284 Table A.3. F1 progeny of pickerelweed derived from the parents WS and BL, and F2 progeny derived from self-pollination of F1 progeny. Each F2 family was developed through self-pollination of an individual F1 plant. PE Gen Code Flrs Seeds# Germ PGen Prog WS x BL P x P WSBL 83 12 8/12 F1 7 BL x WS P x P BLWS 171 138 84/138 F1 60 WSBL1 F1 WS1 746 157 68/100 F2 41 WSBL3 F1 WS2 609 216 87/100 F2 n/a WSBL5 F1 WS3 644 251 55/100 F2 n/a BLWS34 F1 WS4 535 122 43/100 F2 n/a BLWS40 F1 WS5 555 201 45/100 F2 29 BLWS54 F1 WS6 397 240 28/100 F2 n/a WSBL4 F1 WL1 602 155 69/100 F2 6 WSBL7 F1 WL2 874 134 38/100 F2 7 BLWS10 F1 WL3 697 162 53/100 F2 9 BLWS24 F1 WL4 698 188 86/100 F2 6 BLWS57 F1 WL5 586 165 72/100 F2 6 BLWS59 F1 WL6 1132 108 69/100 F2 9 WSBL6 F1 BS1 814 187 105/187 F2 46 WSBL3 F1 BS2 280 196 78/100 F2 n/a BLWS12 F1 BS3 771 192 103/192 F2 56 WSBL5 F1 BS4 526 256 11/100 F2 n/a BLWS34 F1 BS5 285 114 51/100 F2 n/a BLWS54 F1 BS6 414 231 57/100 F2 n/a BLWS55 F1 BS7 462 179 63/179 F2 31 BLWS22 F1 BL1 249 165 92/165 F2 44 BLWS37 F1 BL2 784 182 91/182 F2 43 BLWS39 F1 BL3 1485 201 102/201 F2 69 BLWS43 F1 BL4 427 114 113/114 F2 46 BLWS49 F1 BL5 897 137 83/137 F2 44 BLWS50 F1 BL6 1214 189 93/189 F2 46 PE: Pollination event; identity of plants used in cross-pollinations or self-pollinations Gen: Generation of plants used in pollination event; P = parental, F1 = derived from cross-pollinations betwee n the parents WS and BL Code: Code used to identify progeny produced from pollination event Flrs: Number of flowers pollinated # Seeds: Number of seeds produced Germ: Number of seeds germinated / numbe r of seeds placed in germination vessels PGen: Generation of plants produced from pollination event Prog: Number of progeny evaluated for fl oral traits; n/a = progeny screened for albinism only

PAGE 302

285 Table A.4. F1 progeny of pickerelweed derived from the parents WM and BS, and F2 progeny derived from self-pollination of F1 progeny. Each F2 family was developed through self-pollination of an individual F1 plant. PE Gen Code Flrs Seeds# Germ PGen Prog WM x BS P x P WMBS 113 101 90/101 F1 73 BS x WM P x P BSWM 115 65 35/65 F1 30 WMBS13 F1 BS1 372 203 133/203 F2 93 WMBS73 F1 BS2 372 184 145/184 F2 114 BSWM3 F1 BS3 346 82 37/82 F2 17 BSWM30 F1 BS4 364 169 78/169 F2 62 WMBS39 F1 BS5 358 254 164/254 F2 153 WMBS47 F1 BS6 306 170 21/170 F2 16 BSWM23 F1 BS7 634 175 77/175 F2 49 WMBS28 F1 BM1 283 213 141/213 F2 105 WMBS33 F1 BM2 267 185 120/185 F2 96 BSWM16 F1 BM3 227 184 51/184 F2 27 BSWM19 F1 BM4 278 233 142/233 F2 99 WMBS19 F1 BM5 331 182 95/182 F2 83 WMBS24 F1 BM6 272 228 44/228 F2 28 WMBS60 F1 BM7 329 268 193/268 F2 146 WMBS82 F1 BM8 229 122 46/122 F2 30 PE: Pollination event; identity of plants used in cross-pollinations or self-pollinations Gen: Generation of plants used in pollination event; P = parental, F1 = derived from cross-pollinations betwee n the parents WM and BS Code: Code used to identify progeny produced from pollination event Flrs: Number of flowers pollinated # Seeds: Number of seeds produced Germ: Number of seeds germinated / numbe r of seeds placed in germination vessels PGen: Generation of plants produced from pollination event Prog: Number of progeny ev aluated for floral traits

PAGE 303

286 Table A.5. F1 progeny of pickerelweed derived from the parents WM and BL, and F2 progeny derived from self-pollination of F1 progeny. Each F2 family was developed through self-pollination of an individual F1 plant. PE Gen Code Flrs Seeds# Germ PGen Prog WM x BL P x P WMBL 104 82 75/82 F1 47 BL x WM P x P BLWM 31 19 18/19 F1 10 WMBL3 F1 WM1 272 209 168/209 F2 60 WMBL25 F1 WM2 149 125 26/125 F2 16 WMBL31 F1 WM3 322 262 87/100 F2 55 WMBL48 F1 WM4 246 211 63/211 F2 41 BLWM5 F1 WM5 171 152 54/152 F2 36 BLWM8 F1 WM6 397 331 113/331 F2 71 WMBL9 F1 WL1 488 220 86/100 F2 5 WMBL28 F1 WL2 351 168 17/100 F2 4 WMBL35 F1 WL3 334 154 17/100 F2 5 WMBL44 F1 WL4 278 133 26/100 F2 10 WMBL45 F1 WL5 455 175 17/100 F2 6 BLWM2 F1 WL6 293 105 84/100 F2 6 WMBL4 F1 BM1 349 299 122/299 F2 76 WMBL13 F1 BM2 228 206 94/100 F2 83 WMBL22 F1 BM3 361 316 138/316 F2 59 BLWM3 F1 BM4 328 271 190/271 F2 118 BLWM6 F1 BM5 246 233 85/233 F2 40 BLWM9 F1 BM6 171 160 59/160 F2 30 WMBL5 F1 BL1 454 253 60/100 F2 32 WMBL6 F1 BL2 532 234 70/100 F2 34 WMBL7 F1 BL3 880 228 34/228 F2 25 WMBL8 F1 BL4 538 162 55/100 F2 32 BLWM1 F1 BL5 932 195 52/195 F2 23 BLWM7 F1 BL6 468 142 75/100 F2 26 PE: Pollination event; identity of plants used in cross-pollinations or self-pollinations Gen: Generation of plants used in pollination event; P = parental, F1 = derived from cross-pollinations betwee n the parents WM and BL Code: Code used to identify progeny produced from pollination event Flrs: Number of flowers pollinated # Seeds: Number of seeds produced Germ: Number of seeds germinated / numbe r of seeds placed in germination vessels PGen: Generation of plants produced from pollination event Prog: Number of progeny ev aluated for floral traits

PAGE 304

287 Table A.6. F1 progeny of pickerelweed derived from the parents BS and BM, and F2 progeny derived from self-pollination of F1 progeny. Each F2 family was developed through self-pollination of an individual F1 plant. PE Gen Code Flrs Seeds# Germ PGen Prog BS x BM P x P BSBM 76 39 31/39 F1 29 BM x BS P x P BMBS 147 133 110/133 F1 103 BMBS61 F1 BS1 865 118 59/118 F2 35 BMBS94 F1 BS2 480 227 187/227 F2 110 BSBM28 F1 BS4 658 136 91/136 F2 74 BMBS88 F1 PBS1 687 149 83/149 F2 56 BMBS96 F1 PBS3 569 108 34/108 F2 20 BMBS104 F1 PBS4 589 99 66/99 F2 40 BMBS103 F1 BM1 247 126 62/126 F2 59 BMBS105 F1 BM2 322 120 45/120 F2 31 BSBM2 F1 BM3 290 160 55/160 F2 35 BMBS16 F1 PBM1 609 335 182/335 F2 149 BMBS71 F1 PBM2 352 163 126/163 F2 108 BSBM24 F1 PBM3 384 260 150/260 F2 110 PE: Pollination event; identity of plants used in cross-pollinations or self-pollinations Gen: Generation of plants used in pollination event; P = parental, F1 = derived from cross-pollinations between the parents BS and BM Code: Code used to identify progeny produced from pollination event Flrs: Number of flowers pollinated # Seeds: Number of seeds produced Germ: Number of seeds germinated / numbe r of seeds placed in germination vessels PGen: Generation of plants produced from pollination event Prog: Number of progeny ev aluated for floral traits

PAGE 305

288 Table A.7. F1 progeny of pickerelweed derived from the parents BS and BL, and F2 progeny derived from self-pollination of F1 progeny. Each F2 family was developed through self-pollination of an individual F1 plant. PE Gen Code Flrs Seeds# Germ PGen Prog BS x BL P x P BSBL 71 31 22/31 F1 16 BL x BS P x P BLBS 77 64 62/64 F1 33 BSBL16 F1 VarBS 436 134 81/134 F2 55 BLBS33 F1 USV2 497 171 150/171 F2 58 BSBL12 F1 UVS3 749 292 200/292 F2 136 BLBS15 F1 BS1 385 220 128/220 F2 73 BLBS32 F1 BS2 384 185 133/185 F2 90 BSBL2 F1 BS3 393 111 15/111 F2 6 BLBS13 F1 UVM1 169 167 160/167 F2 105 BLBS19 F1 UVM2 265 240 154/240 F2 89 BSBL14 F1 UVM3 300 217 110/217 F2 92 BLBS5 F1 BM1 210 197 116/197 F2 70 BLBS11 F1 BM2 286 203 144/203 F2 98 BSBL13 F1 BM3 300 249 235/249 F2 88 PE: Pollination event; identity of plants used in cross-pollinations or self-pollinations Gen: Generation of plants used in pollination event; P = parental, F1 = derived from cross-pollinations betwee n the parents BS and BL Code: Code used to identify progeny produced from pollination event Flrs: Number of flowers pollinated # Seeds: Number of seeds produced Germ: Number of seeds germinated / numbe r of seeds placed in germination vessels PGen: Generation of plants produced from pollination event Prog: Number of progeny ev aluated for floral traits

PAGE 306

289 Table A.8. F1 progeny of pickerelweed derived from the parents BM and BL, and F2 progeny derived from self-pollination of F1 progeny. Each F2 family was developed through self-pollination of an individual F1 plant. PE Gen Code Flrs Seeds# Germ PGen Prog BM x BL P x P BMBL 100 94 91/94 F1 74 BL x BM P x P BLBM 30 19 18/19 F1 17 BMBL28 F1 WM1 435 255 79/100 F2 62 BMBL34 F1 WL1 891 117 41/100 F2 9 BMBL65 F1 WL2 336 108 61/100 F2 6 BLBM8 F1 WL3 353 167 70/100 F2 11 BLBM13 F1 WL4 884 114 54/100 F2 6 BMBL43 F1 BM1 331 225 171/225 F2 51 BMBL56 F1 BM2 262 127 79/127 F2 38 BMBL11 F1 BM3 415 222 142/222 F2 125 BLBM3 F1 BM4 341 199 97/199 F2 67 BLBM4 F1 BM5 244 171 115/171 F2 57 BLBM18 F1 BM6 343 159 89/159 F2 43 BMBL46 F1 BL1 801 88 32/88 F2 22 BMBL49 F1 BL2 461 163 90/100 F2 32 BMBL63 F1 BL3 468 109 44/100 F2 18 BLBM1 F1 BL4 565 76 52/76 F2 14 BLBM5 F1 BL5 878 125 31/100 F2 6 BLBM7 F1 BL6 954 104 31/100 F2 11 BLBM2 F1 BL7 565 122 37/122 F2 23 BLBM11 F1 BL8 632 146 62/146 F2 29 BMBL58 F1 BL9 751 118 36/118 F2 22 PE: Pollination event; identity of plants used in cross-pollinations or self-pollinations Gen: Generation of plants used in pollination event; P = parental, F1 = derived from cross-pollinations between the parents BM and BL Code: Code used to identify progeny produced from pollination event Flrs: Number of flowers pollinated # Seeds: Number of seeds produced Germ: Number of seeds germinated / numbe r of seeds placed in germination vessels PGen: Generation of plants produced from pollination event Prog: Number of progeny ev aluated for floral traits

PAGE 307

290 Figure A.1. Fruits of pickerel weed. A) Fresh fruits collect ed in mesh bag. B) Fruits drying in greenhouse. A B

PAGE 308

291 Figure A.2. Seeds of pickerelweed germina ting in jars of water in the greenhouse.

PAGE 309

292 Figure A.3. Newly transplanted F1 seedling of pickerel weed in 612 cell pack.

PAGE 310

293 Figure A.4. F2 seedlings of pickerelweed in the greenhouse.

PAGE 311

294 Figure A.5. F2 plants in the greenhouse.

PAGE 312

295 APPENDIX B OBSERVATIONS Introduction There were many observations made during th e course of this project that did not belong in a chapter but were certainly worth mentioning. Some notes related to plant care, culture and maintenance in a gr eenhouse setting, while others focused on differences in phenotypes and response to envi ronmental and chemical stimuli. It seemed appropriate to discuss these observations under the broad categories described below. Plant Care and Maintenance in the Greenhouse Pickerelweed is very vigorous and quickl y produces large amounts of biomass; as a result, the species can be challenging to main tain in a greenhouse setting, as plants in neighboring pots rapidly become intertwined and grow into each others containers (Figure B.1). I found the best way to keep pl ants restrained was through the use of long wooden stakes and long plasticcoated wire twist-ties. Over grown plants were divided and a rooted rhizome with 5 to 7 leaves wa s selected and placed on a 7 to 10 cm deep layer of potting mix in the center of a 1-L nursery container. Additional potting mix was added to cover the rhizome, then ca. 10 g of controlled-release fert ilizer was placed on the top of the potting mix. The container was filled to the top with potting mix and the wooden stake was inserted behind the newly transplanted division. The petioles were grouped with the stake using the long twist-ti e; these were bound as loosely as possible and the ends of the twist-ties were folded over one another (not tw isted) to keep the assembly intact. The staked plant was then pl aced in a 20 cm deep container filled with

PAGE 313

296 water to allow saturation of the potting mix through capillary action. Plants divided in this manner were low-maintenance for 7 to 10 d after division; at that time, it was necessary to adjust the twist-tie to prevent girdling of the growing plant and to release trapped emergent petioles. Most plants could be maintained in the same container for up to 3 mo, but needed to be divided and repotted when rhizomes began to creep over the edge of the pot and into the container of neighboring plants. Greenhouse Pests (a.k.a. Everybody Loves Pickerelweed) Greenhouse pests loved the succulent lush growth of pickerelweed. Pickerelweed was susceptible to all the usual greenhouse pe sts including caterpillars and lepidopterans of all kinds (Figure B.2), spider mites (Figure B.3), slugs (Figure B.4), aphids (Figure B.4), scale, mealybugs and thrips. Sl ugs, caterpillars and a phids were able to remain hidden by positioning themselves betw een the clasping petioles, while spider mites preferred to encase the leaves and infl orescences of the plants. Applications of insecticides were effective against these menaces but adequate coverage was often difficult to achieve due to the growth habit of pickerelweed. Pesticides were applied as a tank mix of the following chemicals: Conserve SC1 (spinosad 11.6% a.i.) (Dow AgroSc ience): 2 to 4 mL per gallon for thrips and lepidopteran larvae Enstar II (S-Kinoprene 65.1% a.i.) (Sandoz, Des Plaines, IL): 4 to 6 mL per gallon for aphids and mealybugs Mavrik AquaFlow [( RS,2R)-fluvalinate 22.3% a.i.] (Sandoz, Des Plaines, IL): 4 to 6 mL per gallon for aphids, thrips, mites and caterpillars 1 Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the product by the Florida Agricultural Experiment Station and does not imply its approval to the exclusion of other products that may be suitable.

PAGE 314

297 Pyrenone (pyrethrins 6% a.i. + piperonyl butoxide 60% a.i.) (Roussel Uclaf Corp., Montvale NJ): 4 to 6 mL per gallon for aphid, thrips, mites and caterpillars Kicker (pyrethrins 6% a.i. + piperonyl butoxide 60% a.i.) (AgrEvo, Montvale NJ): 4 to 6 mL per gallon for aphid, thrips, mites and caterpillars No phytotoxicity was noted with any of these chemicals; however, seed set appeared to be depressed when chemicals to control thrips we re applied. Biological control agents were also used to redu ce insect populations; these included Bt ( Bacillus thurengensis var. israelensis ) to control lepidopteran larvae and ladybeetles to control aphids. A single caterpillar coul d effectively wipe out one hundr ed or more seedlings in a matter of hours, so seedlings were protected from lepidopteran pests with custom-made screen enclosures designed to fit snugly over the custom-made fl ood trays (Figure B.5). No serious fungal or bacterial problems were noted during the course of this experiment. Response to Colchicine Morphological evidence suggested that it may have been possible to induce polyploidy in pickerelweed through the use of colchicine. A group of 50 seedlings derived from open-pollinated s eeds were treated with a 0.5% solution of colchicine. Two drops of this solution were appl ied to the apex of seedlings that were 3 cm tall twice per day for 3 d. A majority of the seedlings (ca. 80%) were killed by this treatment; however, four of the surviving plants exhibited signs of polyploidy (e. g., foliar and floral gigantism). One of these plants (coded M oonglow) had exceptionally large flowers and may have utility as a novel ornamental lin e (Figure B.6). Ploidy level in Moonglow has not been verified at this date; however, pollen grains of Moonglow were significantly larger than pollen produced by the same anth er levels of wild-type plants (data not shown) and flowers were consistently much larger than flowers of wild-type plants (Figure B.7).

PAGE 315

298 Variegation Foliar variegation was noted in a small num ber of plants (< 2%) during the course of this experiment (Figure B.8). Variega tion had not previously been reported in pickerelweed and plants expressing the trait may have commercial value as ornamental plants. Expressivity of the trait was va riable; some plants produced many highly variegated leaves, while others had only a few lightly varieg ated leaves. The inheritance of variegation was not investig ated, as the frequency of the trait was too low to conduct any meaningful analysis; therefore, pene trance of the trait wa s unknown. Expression of variegation was stable in plants propagated using vegetative means a nd did not appear to be affected by environmental conditions. Va riegation was manifested as distinct light-green sectors or as a speckled or s cattered pattern; however, the former phenotype was noted more frequently than the latter. Most F1 plants that produced variegated leaves had white flowers and were derived from cross-pollinations between the parents WS and WM, while F2 plants expressing varieg ation had either white or blue flowers and were recovered from a wide range of F2 families. A group of ca. 15 variegated plants with a high degree of variegation was selected for further study and comprised both flower colors. These plants were compared to one another; then two lines were selected to represent the most attractive white-flowered and blue-flowered vari egated plants. These selections were propagated using vegetative means and may have great potential for use as ornamental cultivars for the water garden or aquascape. Leaf Shape There was a great deal of variation in the leaves of pickerelweed, but foliage could be grouped into a range of types based on the shapes of the base and apex of the leaf (Figure B.9); in addition, the ratio of the blade width to length was also useful. Thirty-one

PAGE 316

299 distinct lines of pickerelweed derived from various sources were characterized for leaf base and apex forms; blade widths and lengths of five mature leaves from each plant were recorded as well. Many of the leaf sh apes differed only slight ly from one another; however, plants with lanceolate leaves were strikingly different from plants with other leaf types in shape and in blade width to le ngth ratio. Brief descriptions and width to length ratios of the leaf types of pickerelweed are listed belo w, along with the identity of members of the population bearing the leaf type. Sagittate: Deep, pointed l obes; acute apex; width to length ratio of 0.531 to 0.571; borne by NYKBS, NYKBM and PINKL Sub-sagittate: Shallow, point ed lobes; acute apex; widt h to length ratio of 0.526 to 0.584; produced by KANAP and NCCI Meta-cordate: Deep, rounded lobes; acute apex; width to length ratio of 0.575 to 0.684; borne by NCCII, WEDWL, MANBL and ECOPA Acute-cordate: Shallow, rounded lobes; acute apex; width to length ratio of 0.514 to 0.657; produced by STEIN, HOFWL, WALBS, WHTOL, USDA and RINAR Cordate: Deep, rounded lobe s; rounded apex; width to length ratio of 0.545 to 0.622; borne by WEDWL, WALWL, FLAUD, PPWL and NYKBH Cord-ovate: Shallow, rounded lobes; r ounded apex; width to length ratio of 0.483 to 0.579; SFLWM, LILBS and CRNPT Lanceolate: Rounded base; acute apex; width to length ratio of 0.255 to 0.404; LANCE, VB, MANWS, MINNIE, SFL BM, LANCEMAN, SFLBS and SFLBL Provenance and Dormancy Some of the plants used in this projec t produced new vegetative growth throughout the year, while others entere d a state of dormancy during wi nter (October through March in Florida) (Figure B.10). A ll plants were grown in th e same climate-controlled greenhouse, so air temperature did not appear to play a role in th e induction of dormancy. The only discernible difference between plants that entered dormancy and plants that

PAGE 317

300 continued to grow was provenance. Plants collected in southern Florida (e.g., SFLBS, SFLBM, SFLBL, SFLWM) produced new vegeta tive growth year-r ound, while plants derived from northern climes like New York (NYKBM, NYKBS) or Rhode Island (RINAR, RIBL) ceased normal growth. When days become short (12 h or less), dormant types ceased normal growth and instead produc ed short, stubby overwintering structures. The utilization of supplemental lighting to si mulate long days (ca. 16 h daylength) caused dormant plants to resume normal growth; it is therefore likely that daylength was a critical factor in the induc tion of dormancy. Plants from s outhern regions did not enter dormancy under short days, but ceased flower ing when daylength was 12 h or less. When daylength was extended thr ough the use of supplemental lig hting, reproductive growth and flowering was induced in pl ants from southern regions. These observations suggested that provenance played an important role in the life cycle of pick erelweed and that the use of locally adapted ecotypes is critical to ensure the success of wetland mitigation projects.

PAGE 318

301 Figure B.1. Growth of pickerelweed in th e greenhouse. A) Plants before division. B) Same plants after division. A B

PAGE 319

302 Figure B.2. Lepidopteran pests of pickerelweed and feeding damage. A, B) Lepidopteran larvae. C) Feeding damage. A B C

PAGE 320

303 Figure B.3. Spider mites on leaves and inflor escence of pickerelweed. A) Mites on leaf. B) Mites encasing inflorescence. C) Plant infested with mites. A B C

PAGE 321

304 Figure B.4. Slug and aphids on pick erelweed. A) Slug. B) Aphids. A B

PAGE 322

305 Figure B.5. Screen enclosures designed to exclude lepidopteran pests from seedlings.

PAGE 323

306 Figure B.6. Flowers of Moonglow and wild-t ype plant. A) Moonglow. B) Wild-type. A B

PAGE 324

307 Flower area (in mm2) 0 100 200 300 400 500 600 Moonglow SFLBL SFLBS SFLBM WS2 BL2 543.732 259.792 254.486 248.787246.404 242.666 Figure B.7. Mean area in mm2 of flowers of pickerelweed. Area was computed as height x width with flower height measur ed from the top of the banner tepal to the bottom of the lip tepal and width m easured from the distal ends of the lower tepals. Bars represent the mean of 20 flowers for each plant and error bars indicate one standard error from the mean.

PAGE 325

308 Figure B.8. Variegated leaves of pickerelweed.

PAGE 326

309 Figure B.9. Leaf shapes of pickerelweed. A) Sagittate. B) Sub-sagittate. C) Meta-cordate. D) Acute cordate. E) Cordate F) Cord-ovate. G) Lanceolate. A BC D E F G

PAGE 327

310 Figure B.10. Growth habit of dormant and nondor mant plants during winter in southern Florida. A) Close-up of dormant-type plant. B) Dormant plant next to nondormant plant. A B

PAGE 328

311 LITERATURE CITED Agharkar, S.P. and I. Banerji. 1930. Studies in the pollination and seed formation of water-hyacinth ( Eichhornia speciosa Kunth). Agric. J. India 25:286-296. Anderson, J.M. and S.C.H. Barrett. 1986. Pollen tube growth in tristylous Pontederia cordata (Pontederiaceae). Can. J. Bot. 64:2602-2607. Anderson, N.O. and P.D. Ascher. 1995. St yle morph frequencies in Minnesota populations of Lythrum (Lythraceae). II. Tristylous L. salicaria L. Sex. Plant Reprod. 8:105-112. Arroyo, M.T.K. 1974. Chiasma frequency evid ence on the evolution of autogamy in Limnanthes floccusa (Limnanthaceae). Evolution 27:679-688. Bailey, L.H. 1949. Manual of cultivated plants. Macmillan, New York, NY. Baker, H.G. 1955. Self-compatibility and esta blishment after long-distance dispersal. Evolution 9:347-348. Baker, H.G. 1967. Support for Bakers Law as a rule. Evolution 21:853-856. Barbieri, R. and F. deA. Esteves. 1991. The chemical composition of some aquatic macrophyte species and implications for th e metabolism of a tropical lacustrine ecosystem Lobo Reserve, So Paulo, Brazil. Hydrobiologia 213:133-140. Barrett, S.C.H. 1977. The breeding system of Pontederia rotundifolia L., a tristylous species. New Phytol. 78:209-220. Barrett, S.C.H. 1978. Floral biology of Eichhornia azurea (Swartz) Kunth (Pontederiaceae). Aquat. Bot. 5:217-228. Barrett, S.C.H. 1985. Floral trimorphism a nd monomorphism in c ontinental and island populations of Eichhornia paniculata (Spreng.) Solms. (Pontederiaceae). Biol. J. Linn. Soc. 25:41-60. Barrett, S.C.H. 1988. Evolution of breeding systems in Eichhornia (Pontederiaceae): a review. Ann. Missouri Bot. Garden 75:741-760. Barrett, S.C.H. 1993. The evolutionary biology of tristyly. In: Futuyama, D. and J. Antonovics (eds.). Oxford Surveys Evol. Biol. 9:283-326. Oxford University Press, Oxford, UK.

PAGE 329

312 Barrett, S.C.H. and J.M. Anderson. 1985. Va riation of expression of trimorphic incompatibility in Pontederia cordata L. (Pontederiaceae). Theoret.Appl. Genet. 70:355-362. Barrett, S.C.H. and D.E. Glover. 1985. On the Darwinian hypothesis of the adaptive significance of tristyl y. Evolution 39:766-774. Barrett, S.C.H., S.D. Price and J.S. Shor e. 1983. Male fertilit y and anisoplethic population structure in tristylous Pontederia cordata L. (Pontederiaceae). Evolution 37:745-759. Bateman, A.J. 1956. Cryptic self-inc ompatibility in the wildflower: Cheiranthus cheiri L. Heredity 10:257-261. Becerra, J.X. and D.G. Lloyd. 1992. Competitiondependent abscission of self-pollinated flowers of Phormium tenax (Agavaceae): a second action of self-incompatibility at the whole-flower leve l? Evolution 46:458-469. Berjak, P.J., M. Ferrat and N.W. Pammen ter. 1990. The basis of recalcitrant seed behavior, pp.89-108. In: R.B. Taylorson (ed.) Recent advances in the development and germination of seeds. Plenum Press, NY. Bell, C.R. and B.J. Taylor. 1982. Florida wild flowers and roadside plants. Laurel Hill Press, Chapel Hill, NC. Bowman, R.N. 1987. Cryptic self-incompa tibility and the breeding system of Clarkia unguicalata (Onagraceae). Am. J. Bot. 74:471-476. Brewbaker, J.L. 1962. Cyanidin-red white clover. Heredity 53:163-167. Brewbaker, J.L. 1967. The distribution and phylogenetic significan ce of binucleate and trinucleate pollen grains in the an giosperms. Am. J. Bot. 54:1069-1083. Broda, Z. 1979. Inheritance of some morphologica l characters in multifoliolate mutant of diploid red clover ( Trifolium pratense ). Genet. Polonica 20(1):75-88. Charlesworth, D. 1979. The evolution and br eakdown of tristyly. Evolution 33:486-498. Coffelt, T.A. and R.O. Hammons. 1971. Inheri tance of an albino s eedling character in Arachis hypogaea L. Crop Sci. 11:753-755. Coffelt, T.A. and O. Hammons. 1973. Influe nce of sizing peanut seed on two phenotypic ratios. Heredity 64(1):39-42. Crowe, L.K. 1964. The evolution of outbreeding in plants. I. The angiosperms. Heredity 19:435-457.

PAGE 330

313 Cruzan, M.B. and S.C.H. Barrett. 1993. Contri bution of cryptic incompatibility to the mating system of Eichhornia paniculata (Pontederiaceae). Evolution 47:925-934. Darwin, C. 1877. The different forms of flow ers on plants of the same species. John Murray, London. Davies, A.J.S. 1957. Successful crossing in the genus Lathyrus through stylar amputation. Nature (London) 180:612. Deren, C.W. 1987. Inheritance of photoperiodinduced flowering and a glabrous-stem marker gene in Aeschynomene americana Diss. Abst. Intl., B Sciences and Engineering 48(4):924B. Dulberger, R. 1981. Dimorphic exine sculpt uring in three distylous species of Linum (Linaceae). Plant System. Evol. 139:113-119. Durbin, M.L, K.E. Lundy, P.L. Morrell, C.L. Torres-Martinez, and M.T. Clegg. 2003. Genes that determine flower color: the role of regulatory changes in the evolution of phenotypic adaptations. Mol. Phylog. Evol. 29:507-518. East, E.M. 1940. The distributi on of self-sterility in the flowering plants. Proc. Am. Philos. Soc. 82:449-518. Eckert, C.G. and S.C.H. Barrett. 1994. Tristyl y, self-compatibility and floral variation in Decodon verticillatus (Lythraceae). Biol. J. Linn. Soc. 53:1-30. Eckert, C.G., D. Manicacci and S.C.H. Ba rrett. 1996. Frequency-de pendent selection on morph ratios in tristylous Lythrum salicaria (Lythraceae). Heredity 77:581-588. Estilai, A. 1984. Inheritance of flower color in guayule. Crop Sci. 24:760-762. Faegri, K. and L. van der Pilj. 1979. The principles of pollination ecology. Oxford: Pergamon Press. Francois, J. 1964. Observations sur lhtrostylie chez Eichhornia crassipes (Mart.) Solms. Acad. Roy. Soc. dOutr e-Mer. Bull. Sances 1964:501-519. Galinato, M.I. and A.G. van der Valk. 1986. S eed germination traits of annuals and emergents recruited during drawdowns in the Delta Marsh, Manitoba, Canada. Aquat. Bot. 26:89-102. Ganders, F.R. 1979. The biology of hete rostyly. N. Z. J. Bot. 17:607-635. Garbisch, E.W. and S. McIninch. 1992. Seed information for wetland plant species of the Northeast United States. Rest. Mgt. Notes 10:85-86. Gaus, J., D. Werner, L. Gettys and R. Gr iesbach. 2003. Genetics and biochemistry of flower color in stokes aster. Acta Hort. 624:449-453.

PAGE 331

314 Gettys, L.A. S.J. Peters and D.L. Sutt on. 2001. Culture and production of pickerelweed using three different substrates. Proc Florida State Hort. Soc. 114:252-254. Glover, D.E. and S.C.H. Barrett. 1983. Trimorphic incompatibility in Mexican populations of Pontederia sagittata Presl. (Pontederiaceae). New Phytol. 95:439-455. Glover, D.E. and S.C.H. Barrett. 1986. S tigmatic pollen loads in populations of Pontederia cordata from the southern U.S. Am. J. Bot. 73:1607-1612. Godfrey, R.K. and J.W. Wooten. 1979. Aquatic and wetland plants of southeastern United States: monocotyledons. The Univer sity of Georgia Press, Athens, GA. Gorsic, J. 1994. Inheritance of eleven new variants of Collinsia heterophylla J. Hered. 85(4):314-318. Griesbach, R.J. 1996. The inher itance of flower color in Petunia hybrida Vilm. J. Hered. 87:241-245. Grime, J.P., G. Mason, A.V. Curtis, J. Rodman, S.R. Band, M.A.G. Mowforth, A.M. Neal and S. Shaw. 1981. A comparative study of germination characteristics in a local flora. J. Ecol. 69:1017-1059. Halsted, B. 1889. Pickerel weed pollen. Bull. Torrey Bot. Club 14:255-257. Halvankar, G.B. and V.P. Patil. 1994. Inherita nce and linkage studies in soybean. Indian J. Genet. Plant Breed. 54(3):216-224. Harder, L.D. and S.C.H. Barrett. 199 3. Pollen removal from tristylous Pontederia cordata : effects of anther position and po llinator specialization. Ecology 74:10591072. Hazen, T. 1918. The trimorphism and insect visitors of Pontederia Memoirs Torrey Bot. Club 17:459-484. Heisey, R.M. and A.W.H. Damma n. 1982. Biomass and production of Pontederia cordata and Potamogeton epihydrus in three Connecticut rivers. Am. J. Bot. 69:855-864. Hermann, B.P., T.K. Mal, R.J. Willia ms and N.R. Dollahon. 1999. Quantitative evaluation of stigma polymorphism in a tristylous weed, Lythrum salicaria (Lythraceae). Am. J. Bot. 86:1121-1129. Hopkins, W.G. 1995. Introduction to plant physiology. John Wiley & Sons, Inc., New York, NY.

PAGE 332

315 Hoque, M.E., S.K. Mishra, Y. Kumar, R. Kumar, S.M.S. Tumar and B. Sharma. 2002. Inheritance and linkage of leaf col our and plant pubescence in lentil ( Lens culinaris Medik.). Indian J. Genet. Plant Breed. 62(2):140-142. Johnson, A.M. 1924. The mid-styled form of Piaropus paniculatus Bull. Torrey Bot. Club 51:25-28. Kane, M.E. and N.L. Philman. 1992. Effect of culture vessel type on in vitro multiplication of Pontederia cordata L. Proc. Florida State Hort. Soc. 105:213-215. Kane, M.E. and N.L. Philman. 1997. In vitro propagation and selection of superior wetland plants for habitat restoration. Combined Proc. Intl. Plant Prop. Soc. 47:556-560. Kohn, J.R., S.W. Graham, B. Morton, J. J. Doyle and S.C.H. Barrett. 1996. Reconstruction of the evolution of reproduc tive characters in Pontederiaceae using phylogenetic evidence from chloroplast DNA restriction-site va riation. Evolution 50:1454-1469. Laberge, W.E. 1956. A revision of the bees of the genus Melissodes in North and Central America. II. Hymenoptera, Apidae. Univ. Kansas Sci. Bull. 38:533-578. Lange, W. and G. Jochemsen. 1987. Inhe ritance of hairy leaf sheath in Triticum dicoccoides Cereal Res. Co mmun. 15(2-3):139-142. Larson, R. 1995. Swamp song: a natural history of Floridas swamps. University Press of Florida, Gainesville, FL. Leck, M.A. 1996. Germination of macrophytes from a Delaware River tidal freshwater wetland. Bull. Torrey Bot. Club 123:48-67. Leck, M.A. and R.L. Simpson. 1993. Seeds and seedlings of the Hamilton marshes, a Delaware River tidal freshwater wetland. Proc Acad. of Natural Sci. (Philadelphia) 144:267-281. Leggett, W.H. 1875a. Pontederia cordata L. Bull. Torrey Bot. Club 6:62-63. Leggett, W.H. 1875b. Pontederia cordata L. Bull. Torrey Bot. Club 6:170-171. Lewis, D. and A.N. Rao. 1971. Evolution of dimorphism and population polymorphism in Pemphis acidula Fors. Proc. R. Soc. London, Ser. B (Biol. Sci.) 178:79-94. Linneaus, C. 1753. Species plantarum, vol.1. L. Salvii, Stockholm. (Facsimile edition: Stearn, W.T. 1957. Ray Society, London). Lowden, R.M. 1973. Revision of the genus Pontederia L. Rhodora 75:426-487.

PAGE 333

316 Maes, B., R.M. Trethowan, M.P. Reynolds M. van Ginkel and B. Skovmand. 2001. The influence of glume pubescence on spikelet temperature of wheat under freezing conditions. Aust. J. Plant Physiol. 28(2):141-148. Mal, T.K., J. Lovett-Doust and L. Lovett -Doust. 1999. Maternal and paternal success among flower morphs in tristylous Lythrum salicaria Aquat. Bot. 63:229-239. Martin, A.C. 1946. The comparative inner morphology of seeds. Am. Midl. Nat. 36:513-560. Miller, G.A. 1986. Pubescence, floral temp erature and fecundity in species of Puya Bromeliaceae in the Ecuadorian Andes. Oecologia 70(1):155-160. Moore, D.M. and H. Lewis. 1965. The evolution of self-pollination in Clarkia xantiana Evolution 19:104-114. Morgan, M.T. and S.C.H. Barrett. 1988. Hist orical factors and anisoplethic population structure in tristylous Pontederia cordata : a reassessment. Evolution 42:496-504. Mosjidis, J.A. 2000. Inheritance of bright pi nk flower color with ornamental value in crimson clover. HortScience 35:1175. Muenscher, W.C. 1936. Storage and germination of seeds of aquatic plants. Bulletin of Cornell Univ. Agric. Exp. Stn. 652:1-17. Mulcahy, D.L. 1964. The reproductive biology of Oxalis priceae Am. J. Bot. 51:1045-1050. ONeill, P. 1994. Genetic incompatibility and offspring quality in the tristylous plant Lythrum salicaria (Lythraceae). Am. J. Bot. 81:76-84. Ordnuff, R. 1964. The breeding system of Oxalis suksdorfi Am. J. Bot. 51:307-314. Ordnuff, R. 1966. The breeding system of Pontederia cordata L. Bull. Torrey Bot. Club 93:407-416. Ordnuff, R. 1972. The breakdown of trimorphic incompatibility in Oxalis section Corniculatae. Evolution 26:52-65. Ortiz, R. and D.R. Vuylsteke. 1994. Inheritanc e of albinism in banana and plantain ( Musa spp.) and its significance in br eeding. HortScience 29(8)903-905. Price, S.D. and S.C.H. Barrett. 1982. Tristyly in Pontederia cordata L. (Pontederiaceae). Can. J. Bot. 60:897-905. Price, S.D. and S.C.H. Barrett. 1984. The func tion and adaptive significance of tristyly in Pontederia cordata L. (Pontederiaceae). Biol. J. Linn. Soc. 21:315-329.

PAGE 334

317 Richards, J.H. and S.C.H. Barrett. 1987. Development of tristyly in Pontederia cordata (Pontederiaceae). I. Mature floral struct ure and patterns of relative growth of reproductive organs. Am. J. Bot. 74:1831-1841. Roberts, E.H. and M.W. King. 1980. The characte ristics of recalcitrant seeds. In: H.F. Chin and E.H. Roberts (eds.), Recalcitrant crop seeds. Tropical Press, SDN, BHD, Kuala Lumpur, Malaysia. Roggen, H.P. Jr. and A.J. van Dij k. 1972. Breaking incompatibility in Brassica oleracea L. by steel brush pollination. Euphytica 21:424-425. Salisbury, E. 1970. The pioneer vegetation of e xposed muds and its biological features. Philos. Trans. R. Soc. London 259:207-255. Salkind, N.J. 2004. Statistics for people who (think they) hate statistics. 2nd ed. Sage Publications Inc., Thousand Oaks, CA. Sarker, A., W. Erskine, B. Sharma and M.C. Tyagi. 1999. Inheritance and linkage relationships of days to flower and morphological loci in lentil ( Lens culinaris Medikus subsp. culinaris). J. Hered. 90(2):270-275. Schultz, A.G. 1942. Las Pontederiaceas de la Argentina. Darwiniana 6:45-82. Schwartz, O. 1930. Pontederiaceae. In: A. E ngler, Die natrlichen pflanzenfamilien 15a:1-707. Shipley, B. and M. Parent. 1991. Germination responses of 64 wetland species in relation to seed size, minimum time to reproducti on and seedling relative growth rate. Functional Ecol. 5:111-118. Shull, G.H. 1915. Albinism in maize. Bot. Gaz. 60(4)324-325. Simpson, G.M. 1966. A study of germination in th e seed of wild rice (Zizania aquatica). Can. J. Bot. 44:1-9. Snedecor, G.W. 1946. Statistical methods. 4th ed. The Iowa State College Press, Ames, IA. Solms-Laubach, H. 1883a. Pontederiaceae. In : A. and C. DeCandolle, Monographiae Phanerogamarum 4:501-535. Solms-Laubach, H. 1883b. Ueber das vorkommen cleistogamer Blthen in der familie der Pontederiaceae. Bot. Zeit. 18:302-303. Speichert, G. and S. Speichert. 2001. Ort hos all about water ga rdening. Ortho Books Meredith Corp., Des Moines, IA.

PAGE 335

318 Stebbins, G.L. 1957. Self fertilization and popula tion variability in the higher plants. Am. Nat. 41:337-354. Steel, R.G.D., J.H. Torrie and D.A. Dickey. 1997. Principles and procedures of statistics: a biometrical approach. 3rd ed. WCB McGraw-Hill, New York, NY. Taylor, W.K. 1992. The guide to Florida wi ldflowers. Taylor Publishing Company, Dallas, TX. Tobe, J.D., K.C. Burks, R.W. Cantrell, M. A. Garland, M.E. Sweeley, D.W. Hall, P. Wallace, G. Anglin, G. Nelson, J.R. Cooper, D. Bickner, K. Gilbert, N. Aymond, K. Greenwood and N. Raymond. 1998. Florid a wetland plants: an identification manual. Florida Department of Enviro nmental Protection, Tallahassee, FL. Turner, R.L. 1996. Use of stems of emerge nt plants for oviposition by the Florida applesnail, Pomacea paludosa and implications for marsh management. Florida Sci. 59:34-49. Turner, R.J. Jr., and E. Wasson. 1998. Botani ca. Mynah for Random House Australia Pty. Ltd., NSW Australia. Vuillenmier, B.S. 1967. The origin and evolutio nary development of heterostyly in the angiosperms. Evolution 21:210-226. Weller, S.G. 1976. Breeding system polymorphism in a heterostylous species. Evolution 30:442-454. Whigham, D.F. and R.L. Simpson. 1982. Ge rmination and dormancy studies of Pontederia cordata L. Bull. Torrey Bot. Club 109:524-528. Williges, K.A. and T.T. Harris. 1995. Seed bank dynamics in the Lake Okeechobee marsh ecosystem. Arch. Hydrobiol. Sp ecial Issues Adv. Limn. 45:79-94. Wolfe, L.M. and S.C.H. Barrett. 1989. Patte rns of pollen removal and deposition in tristylous Pontederia cordata L. (Pontederiaceae). Biol. J. Linn. Soc. 36:317-329. Wunderlin, R.P. and B.F. Hansen. 2000. Atlas of Florida Vascular Plants (http://www.plantatlas.usf.edu ). Institute for Systematic Botany, University of South Florida, Tampa, FL. Wyatt, R. 1983. Pollinator-plant interactions and the evolution of breeding systems. In: Real, L. (ed.). Pollination Biology. Academic Press, Inc., New York, NY. Zar, J.H. 1996. Biostatistical analysis. 3rd ed. Prentice Hall, Upper Saddle River, NJ. Zomlefer, W.B. 1994. Guide to flowering pl ant families. The University of North Carolina Press, Chapel Hill, NC.

PAGE 336

319 Zufall, R.A. and M.D. Rausher. 2003. The ge netic basis of a flow er color polymorphism in the common morning glory ( Ipomoea purpurea ). J. Hered. 94:442-448.

PAGE 337

320 BIOGRAPHICAL SKETCH Lyn Anne Gettys was born and raised in West Palm Beach, Florida. She studied Environmental Horticulture full-time at the University of Floridas Fort Lauderdale Research and Education Center while worki ng full-time at a printing company in West Palm Beach. Lyn was awarded a Bachelor of Science degree with Highest Honors in 1996 and earned a Master of Science degree in Horticultural Scienc e from North Carolina State University in 2000. Lyn is the moth er of Mykel Haymond and the daughter of Nancy and Joe Gettys.


Permanent Link: http://ufdc.ufl.edu/UFE0009585/00001

Material Information

Title: Inheritance of Morphological Characters of Pickerelweed (Pontederia cordata L.)
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0009585:00001

Permanent Link: http://ufdc.ufl.edu/UFE0009585/00001

Material Information

Title: Inheritance of Morphological Characters of Pickerelweed (Pontederia cordata L.)
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0009585:00001


This item has the following downloads:


Full Text












INHERITANCE OF MORPHOLOGICAL CHARACTERS OF PICKERELWEED
(Pontederia cordata L.)















By

LYN ANNE GETTYS


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005
























Copyright 2005

by

Lyn Anne Gettys















ACKNOWLEDGMENTS

I would like to thank Dr. David Wofford for his guidance, encouragement and

support throughout my course of study. Dr. Wofford provided me with laboratory and

greenhouse space, technical and financial support, copious amounts of coffee and

valuable insight regarding how to survive life in academia. I would also like to thank

Dr. David Sutton for his advice and support throughout my program. Dr. Sutton supplied

plant material, computer resources, career advice, financial support and a long-coveted

copy of Gray's Manual of Botany.

Special thanks are in order for my advisory committee. Dr. Paul Pfahler was an

invaluable resource and provided me with laboratory equipment and supplies, technical

advice and more lunches at the Swamp than I can count. Dr. Michael Kane contributed

samples of his extensive collection of diverse genotypes of pickerelweed to my program.

Dr. Paul Lyrene generously allowed me to take up residence in his greenhouse when my

plants threatened to overtake all of Gainesville.

My program could not have been a success without the help of Dr. Van Waddill,

who provided significant financial support to my project. I appreciate the generosity of

Dr. Kim Moore and Dr. Tim Broschat, who allowed me to use their large screenhouses

during my tenure at the Fort Lauderdale Research and Education Center. Thanks also go

to my friends and advocates at the FLREC: Nancy Gaynor, Joanne Korvick, Luci Fisher

and Susan Thor for technical and greenhouse assistance; Bill and Sarah Kern for

identifying greenhouse critters; Bill Latham for providing me with access to the









chemistry lab; and Bridge Desoran and his crew for building anything I asked them to in

a completely unreasonable timeframe. I extend my sincere appreciation to Mr. Eric

Ostmark and Mr. Doug Manning for providing technical support to my program in

Gainesville. Thanks are also in order to Dr. Eastonce Gwata and Ms. Gabriela Luciani for

helping out when I was in a bind and to the faculty and graduate students of the

Agronomy Department for providing moral support throughout my tenure at the

University of Florida. I truly appreciate the assistance provided by Kim, Paula, Sandy and

Nancy (in Gainesville) and Cherie, Sarah, Veronica and Sue (in Fort Lauderdale), who

helped navigate the labyrinth of paperwork to ensure that I got paid on time.

I am eternally grateful to my family for their unflagging support of this and all of

my endeavors. My parents, Mykel and Jody have been and continue to be my most

enthusiastic cheerleaders; without their support I would probably be running a print shop

somewhere. Thanks also go to Mr. Paul J. Best II, who provided unwavering friendship,

moral support and a nice swampy home for my culled plants and to Dr. Ed Duke for

being a fantastic mentor, advisor, inspiration and friend.

Finally, I would like to thank the University of Florida Alumni Association for

providing me with a 4-year Outstanding Alumni Fellowship and the Crop Science

Society of America for awarding me a Gerald 0. Mott Scholarship.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iii

LIST OF TABLES .. ................... ............ ......... .............. viii

LIST OF FIGURES ............................... ........... ............................ xiii

A B S T R A C T ..................................................................................................................... x v i

CHAPTER

1 INTRODUCTION AND OVERVIEW ..................................................1...

2 A REVIEW OF THE LITERA TURE ........................................................ ............... 3

E con om ic Im p ortan ce .............................................................................. ............... 3
Classification, O rigin and D istribution.................................................... ...............4...
M orphology .............. ....................................................... ...............6 6..... ....... . 6
C u ltu re ........................................................................................................ .......... 8
P ropagation and D orm ancy ..................................................................... ...............8...
G general H eterom orphic Incom patibility................................................ ............... 12
G en eral T risty ly .............. .... ................................ ... ............................................... 12
M orph Inheritance in Tristylous Species............................................... ................ 14
Population Structure of Tristylous Species............................................ ................ 15
B reakdow n of T ristyly ....................................................................... ............... 15
C ryptic Self-incom patibility .................................................................. ............... 17
Prevalence of Tristyly in Species of the Pontederiaceae......................................19
Self-incompatibility in Species of the Pontederiaceae .........................................20
Morph Inheritance in Species of the Pontederiaceae.............................................20
M orph Inheritance in Pickerelw eed....................................................... ................ 21
Pollen Diameter Trimorphism and Production in Pickerelweed ..............................21
Floral Structure and Reproductive Organ Arrangement in Pickerelweed...............22
Pollen Physiology and Male Fitness in Pickerelweed...........................................25
Self, Intramorph and Intermorph Compatibility in Pickerelweed ..............................26
P ollen G row th in v iv o ................................................... .. ........................... .........2 7
Population Structure of Pickerelweed ...................................................28
Im pact of P ollinator B behavior ............................................................... ................ 28
Stigm atic Pollen Loads in Pickerelw eed ............................................... ................ 31
Greenhouse Production vs. Natural Populations of Pickerelweed ..........................32


v









3 POLLEN GRAIN DIAMETER, IN VITRO POLLEN GERMINATION AND
REGRESSION BETWEEN GRAIN DIAMETER AND IN VITRO
G E R M IN A T IO N ..................................................................... ...................................4 2

In tro d u ctio n ................................................................................................................ 4 2
M materials and M ethods .................... ................................................................ 46
R results and D discussion ............. .. ............... .............................................. 50
C o n c lu sio n s............................................................................................................... .. 5 2

4 DEVELOPMENT OF NOVEL POLLINATION TECHNIQUES TO REDUCE
SELF-INCOMPATIBILITY RESULTING FROM HERKOGAMY.....................59

In tro d u ctio n ............................................................................................................... .. 5 9
M materials and M ethods .. ..................................................................... ................ 62
R results and D discussion ................ .............. ............................................ 66
C o n c lu sio n s............................................................................................................... .. 6 9

5 OPTIMUM SEED STORAGE AND GERMINATION CONDITIONS................... 79

In tro d u ctio n ................................................................................................................. 7 9
M materials and M ethods .. ..................................................................... ................ 81
R results and D discussion ................ .............. ............................................ 85
C o n c lu sio n s............................................................................................................... .. 9 0

6 INHERITANCE AND GENETIC CONTROL OF ALBINISM .......................... 104

In tro d u ctio n ............................................................................................................... 1 0 4
M materials and M ethods ................... .............................................................. 105
R results and D discussion ................ .............. ............................................ 105
C o n c lu sio n s.............................................................................................................. 1 1 5

7 INHERITANCE AND GENETIC CONTROL OF FLOWER COLOR............... 143

In tro d u ctio n ............................................................................................................... 14 3
M materials and M ethods ................... .............................................................. 144
R results and D discussion ................ .............. ............................................ 145
C o n c lu sio n s.............................................................................................................. 1 5 0

8 INHERITANCE AND GENETIC CONTROL OF FLORAL MORPH............... 162

In tro d u ctio n ............................................................................................................... 1 6 2
M materials and M ethods ................... .............................................................. 164
R results and D discussion ............... ...................................... 164
C o n c lu sio n s.............................................................................................................. 17 2









9 LINKAGE RELATIONSHIP BETWEEN THE LOCI CONTROLLING
FLOWER COLOR AND FLORAL MORPH ............................... ..................... 185

In tro d u ctio n ............................................................................................................... 1 8 5
M materials and M ethods ................... .............................................................. 186
R results and D discussion ............... ...................................... 187
C o n c lu sio n s.............................................................................................................. 2 2 2

10 INHERITANCE AND GENETIC CONTROL OF SCAPE PUBESCENCE..........244

In tro d u ctio n ............................................................................................................... 2 4 4
M materials and M ethods ................... ............................................................... 244
R results and D discussion ................... ............................................................... 248
C o n c lu sio n s.............................................................................................................. 2 5 2

11 INHERITANCE AND GENETIC CONTROL OF A SECOND LOCUS
INFLUENCING FLOWER COLOR .......... ..........................260

In tro d u ctio n .............................................................................................................. 2 6 0
M materials and M ethods ................... ............................................................... 260
R results and D discussion ................... ............................................................... 262
C o n c lu sio n s.............................................................................................................. 2 6 6

12 SUMMARY AND CONCLUSIONS............... ......................271

APPENDIX

A POPULATION DEVELOPMENT FOR INHERITANCE STUDIES...................276

In tro d u ctio n ............................................................................................................... 2 7 6
M materials and M ethods ................... ............................................................... 277
D descriptions of the Fam ilies .......................................................... 280
R results and D discussion ............... ...................................... 281

B O B SE R V A T IO N S .................................................. ............................................ 295

In tro d u ctio n ........................ ....... ....... ...................................................................... 2 9 5
Plant Care and Maintenance in the Greenhouse..................................................295
Greenhouse Pests (a.k.a. Everybody Loves Pickerelweed...)................................296
R response to C olchicine. ................................................................. ................ 297
V arieg atio n ............................................................................................................... 2 9 8
L e af S h ap e ................................................................................................................ 2 9 8
Provenance and D orm ancy ................. ........................................................... 299

L IT E R A T U R E C IT E D .................................................. ............................................3 11

BIOGRAPH ICAL SKETCH .................. .............................................................. 320















LIST OF TABLES


Table page

3.1. Analysis of variance of pollen grain diameter in microns of s-pollen, m-pollen
and 1-pollen of pickerelw eed ..................................... ...................... ................ 54

3.2. Analysis of variance of pollen tube length in microns produced in vitro by
s-pollen, m -pollen and 1-pollen of pickerelweed................................. ................ 55

4.1. Seed set after self-pollination of L-morph plants of pickerelweed subjected to
control and stylar surgery pollination treatments................................ ................ 72

4.2. Analysis of variance of arcsine transformed percent seed set in control and stylar
surgery pollinations of L-morph plants of pickerelweed. ...................................73

4.3. Seed set after self-pollination of S-morph plants of pickerelweed subjected to
control and corolla removal pollination treatments............................. ................ 74

4.4. Analysis of variance of arcsine transformed percent seed set in control and
corolla removal pollinations of S-morph plants of pickerelweed .........................76

5.1. Analysis of variance of arcsine-transformed data for germination of fresh fruits
and seeds of pickerelw eed ....................................... ........................ ................ 92

5.2. Analysis of variance of arcsine-transformed data for germination of fruits and
seeds of pickerelw eed stored for 3 m o ............................................... ................ 93

5.3. Analysis of variance of arcsine-transformed data for germination of fruits and
seeds of pickerelw eed stored for 6 m o ............................................... ................ 95

5.4. Analysis of variance of arcsine-transformed data for germination of seeds of
pickerelw eed germ inated under w ater................................................. ................ 97

6.1. Number of green and albino seedlings from Fi and S families of pickerelweed.. 117

6.2. Goodness-of-fit tests for Fi and S, families of pickerelweed segregating for
a lb in ism ............................................................................................................ . 1 1 8

6.3. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism
and derived from the initial cross/reciprocal set WMBL genotypess P1P .4,Bb x
P 1P .4,I B B ).............................................................................................................. 1 19









6.4. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism
and derived from the initial cross/reciprocal set WSBL genotypess PiP2aaBb x
P 1P .4, B B ) ................................................... ..................................................... 12 1

6.5. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism
and derived from the initial cross/reciprocal set WSBM genotypess PiP2aaBb x
P1PiaaBB or PiP1AAbb or PiPIAABB).......... ........................................ 124

6.6. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism
and derived from the initial cross/reciprocal set WMBS genotypess PiP _ABb x
P1PiaaBB or PiP1AAbb or PiPiAABB).......... ........................................ 126

6.7. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism
and derived from the initial cross/reciprocal set BSBL genotypess PiPiaaBB or
P1P1AAbb or PJPJAABB x P1PA .IB)............... ........................ 128

6.8. Goodness-of-fit tests for F2 families of pickerelweed segregating for albinism
and derived from the initial cross/reciprocal set BMBL genotypess P1PiaaBB or
P1P1AAbb or PiPiAABB x PP2AaBB)............... .........................129

7.1. Number of blue-flowered and white-flowered Fi and Si progeny of
p ick erelw ee d ........................................................................................................... 15 2

7.2. Goodness-of-fit tests for Fi and Si families of pickerelweed segregating for blue
and w hite fl ow er color........................................ ......................... ............... 153

7.3. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue
and white flower color and derived from the initial cross/reciprocal set WMBL
genotypess w w x W w ) ........................................................................ ...............1...... 54

7.4. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue
and white flower color and derived from the initial cross/reciprocal set WSBL
genotypess w w x W w ) ........................................................................ ................ 155

7.5. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue
and white flower color and derived from the initial cross/reciprocal set WSBM
genotypess w w x W w ) ........................................................................ ................ 156

7.6. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue
and white flower color and derived from the initial cross/reciprocal set WMBS
genotypess w w x W W ) ................... ............................................................. 157

7.7. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue
and white flower color and derived from the initial cross/reciprocal set BSBM
genotypess W W x W w ). ................. ............................................................. 158









7.8. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue
and white flower color and derived from the initial cross/reciprocal set BSBL
genotypess W W x W w ). ................. ............................................................. 159

7.9. Goodness-of-fit tests for F2 populations of pickerelweed segregating for blue
and white flower color and derived from the initial cross/reciprocal set BMBL
genotypess W w x W w ) ................... ............................................................. 160

8.1. Number of S-morph, M-morph and L-morph progeny in Fi and Si families of
p ick erelw ee d ........................................................................................................... 17 5

8.2. Goodness-of-fit tests for Fi and Si families of pickerelweed segregating for
short, mid and long floral morphs. ............. ...... ......................176

8.3. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral
morph and derived from the initial cross/reciprocal set WMBL genotypess ssMm
x ssm m ).............................................................................................................. ... 1 7 7

8.4. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral
morph and derived from the initial cross/reciprocal set WSBL genotypess Ssmm
x ssm m ).............................................................................................................. ... 1 7 8

8.5. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral
morph and derived from the initial cross/reciprocal set WSBM genotypess Ssmm
x ssM m ). ................................................................................................................ 1 7 9

8.6. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral
morph and derived from the initial cross/reciprocal set WMBS genotypess ssMm
x S s M ) ................................................................................................................ 1 8 0

8.7. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral
morph and derived from the initial cross/reciprocal set BSBM genotypess SsMIM
x ssM m ). ................................................................................................................ 1 8 1

8.8. Goodness-of-fit tests for F2 families of pickerelweed segregating for floral
morph and derived from the initial cross/reciprocal set BSBL genotypess SsMM
x ssm m ).............................................................................................................. ... 1 8 2

8.9. Goodness-of-fit tests for F2 populations of pickerelweed segregating for floral
morph and derived from the initial cross/reciprocal set BMBL genotypess ssMm
x ssm m ).............................................................................................................. ... 1 8 3

9.1. Segregation of progeny and goodness-of-fit tests for Fi families of pickerelweed
segregating for flower color and floral morph. ...................................224

9.2. Goodness-of-fit test for the Fi family WSBM segregating for flower color and
fl o ra l m o rp h ............................................................................................................ 2 2 5









9.3. Goodness-of-fit test for the Fi family BMBL segregating for flower color and
flo ra l m o rp h ............................................................................................................ 2 2 6

9.4. Goodness-of-fit tests for the Fi families WSBM and BMBL segregating for
flower color and floral m orph. ...... ............ ............ ...................... 227

9.5. Summary of goodness-of-fit tests for genetic distances ranging from 4.4 m.u. to
22 m.u. for the linked W and Mloci in the Fi families WSBM and BMBL ..........228

9.6. Goodness-of-fit tests for F2 families of pickerelweed from the Fi family WMBL
segregating for flower color and floral morph. ..................................229

9.7. Goodness-of-fit tests for F2 families of pickerelweed from the Fi family WSBL
segregating for flower color and floral morph. ..................................231

9.8. Goodness-of-fit tests for F2 families of pickerelweed from the Fi family WSBM
segregating for flower color and floral morph. ..................................232

9.9. Goodness-of-fit tests for F2 families of pickerelweed from the Fi family WMBS
segregating for flower color and floral morph. ..................................234

9.10. Goodness-of-fit tests for F2 families of pickerelweed from the Fi family BSBM
segregating for flower color and floral morph. ..................................237

9.11. Goodness-of-fit tests for F2 families of pickerelweed from the Fi family BSBL
segregating for flower color and floral morph. ..................................240

9.12. Goodness-of-fit tests for F2 families of pickerelweed from the Fi family BMBL
segregating for flower color and floral morph. ..................................242

10.1. Segregation and goodness-of-fit tests for F2 families of pickerelweed
segregating for scape pubescence. ...... ......... ........ ...................... 254

10.2. Segregation and goodness-of-fit tests for F2 families of pickerelweed
segregating for scape pubescence. ...... ......... ........ ...................... 255

10.3. Segregation and goodness-of-fit tests for F2 families of pickerelweed
segregating for scape pubescence. ...... ......... ........ ...................... 256

10.4. Segregation and goodness-of-fit tests for F2 families of pickerelweed
segregating for scape pubescence. ...... ......... ........ ...................... 257

11.1. Flower color of progeny in S1, Fi and F2 families of pickerelweed derived from
the parents W L and W M ................. ........................................................... 268

11.2. Segregation for flower color and goodness-of-fit tests for F2 families of
p ick erelw eed ........................................................................................................... 2 6 9









A. 1. Fi progeny of pickerelweed derived from the parents WS and WM, and
S1 progeny derived from self-pollinations of the parental lines WS, WM, BS,
BM and BL ............................................................................ ...... ......... ............... 282

A.2. Fi progeny of pickerelweed derived from the parents WS and BM, and
F2 progeny derived from self-pollination of Fi progeny ................................283

A.3. Fi progeny of pickerelweed derived from the parents WS and BL, and
F2 progeny derived from self-pollination of Fi progeny ................................284

A.4. Fi progeny of pickerelweed derived from the parents WM and BS, and
F2 progeny derived from self-pollination of Fi progeny ................................285

A. 5. Fi progeny of pickerelweed derived from the parents WM and BL, and
F2 progeny derived from self-pollination of Fi progeny ................................286

A.6. Fi progeny of pickerelweed derived from the parents BS and BM, and
F2 progeny derived from self-pollination of Fi progeny ................................287

A.7. Fi progeny of pickerelweed derived from the parents BS and BL, and
F2 progeny derived from self-pollination ofF1 progeny .............. .................... 288

A. 8. Fi progeny of pickerelweed derived from the parents BM and BL, and
F2 progeny derived from self-pollination of Fi progeny ................................289















LIST OF FIGURES


Figure page

2.1. Pre-packaged water garden kit with bare-root pickerelweed plants, damp
sphagnum moss, soilless planting substrate, fertilizer tablets and planting basket..33

2.2. Pickerelweed growing along the margin of Robert's Pond in Bainbridge, New
Y o rk ..................................................................................................... . ...... .. 3 4

2.3. Inflorescence of white-flowered pickerelweed. ............. ..................................... 35

2.4. Yellow nectar guides eyespotsts") on banner tepal of pickerelweed flower ..........36

2.5. Fresh fruit, seed and dried fruit of pickerelweed. ............................... ................ 37

2.6. Flow ers of 'Singapore Pink' pickerelw eed ......................................... ................ 38

2.7. L-m orph flow ers of pickerelw eed ....................................................... ................ 39

2.8. M -m orph flow ers of pickerelw eed...................................................... ................ 40

2.9. S-m orph flow ers of pickerelw eed ....................................................... ................ 41

3.1. Grain diameter in microns of s-pollen, m-pollen and 1-pollen of pickerelweed ......56

3.2. Mean length of pollen tubes in microns produced in vitro by s-pollen, m-pollen
and 1-pollen of pickerelweed 30, 60, 120 and 240 min after germination ...............57

3.3. Regression between pollen grain diameter and in vitro tube length 30, 60, 120
and 240 m in after germ nation ......................................................... 58

4.1. Stylar surgery of an L-morph flower of pickerelweed........................................77

4.2. Pollination of an S-morph flower of pickerelweed after corolla removal .............78

5.1. Fruits and seeds of pickerelweed germinated under water in half-pint (250 mL)
b o title s ................................................................... ................................................ ... 9 8

5.2. Fruits and seeds of pickerelweed germinated on or 0.5 cm below the soil surface
under m ist irrigation ......................................................................... 99

5.3. Percent germination of fresh fruits and seeds of pickerelweed............................100









5.4. Percent germination of fruits and seeds of pickerelweed stored for 3 months ......101

5.5. Percent germination of fruits and seeds of pickerelweed stored for 6 months ...... 102

5.6. Percent germination of seeds of pickerelweed germinated under water............. 103

6.1. Albino seedling of pickerelweed ....... ....... ...................... 130

6.2. Punnett square of expected albinism genotypes ofF1 populations of
pickerelweed derived from the initial cross/reciprocal set WMWS genotypess
P1P .4lBb and PiP2aaBb) and expected segregation of F2 progeny for albinism .131

6.3. Punnett square of expected albinism genotypes ofF1 populations of
pickerelweed derived from the initial cross/reciprocal set WMBL genotypess
P1P .4,ABb and PIP .4IBB) and expected segregation ofF2 progeny for albinism. 132

6.4. Punnett square of expected albinism genotypes of Fi populations of
pickerelweed derived from the initial cross/reciprocal set WSBL genotypess
PiP2aaBb and PP .4,ABB) and expected segregation ofF2 progeny for albinism .133

6.5. Punnett square of expected albinism genotypes of Fi populations of
pickerelweed derived from the initial cross/reciprocal set WSBM genotypess
P1P2aaBb and P1PlaaBB or P1P1AAbb or PIPJAABB or PiPiaabb) and expected
segregation ofF2 progeny for albinism ....... .......... ........................................ 134

6.6. Punnett square of expected albinism genotypes of Fi populations of
pickerelweed derived from the initial cross/reciprocal set WMBS genotypess
P1P_.4uBb and P1PlaaBB or P1P1AAbb or PIP1AABB or P1Plaabb) and expected
segregation of F2 progeny for albinism ...... ........... ........................................ 135

6.7. Punnett square of expected albinism genotypes of Fi populations of
pickerelweed derived from the initial cross/reciprocal set BSBM genotypess
P1PiaaBB or P1P1AAbb or PIPJAABB or P1Plaabb and P1PlaaBB or P1P1AAbb
or PIPIAABB or PiPiaabb) and expected segregation of F2 progeny for albinisml36

6.8. Punnett square of expected albinism genotypes ofF1 populations of
pickerelweed derived from the initial cross/reciprocal set BSBL genotypess
P1PiaaBB or P1P1AAbb or PIPIAABB or P1Pjaabb and P1P .4ABB) and
expected segregation of F2 progeny for albinism ...... ................ ................... 137

6.9. Punnett square of expected albinism genotypes of Fi populations of
pickerelweed derived from the initial cross/reciprocal set BMBL genotypess
P1PiaaBB or P1P1AAbb or PIPIAABB or P1Pjaabb and P1P .4,BB) and
expected segregation of F2 progeny for albinism ...... ................ ................... 138

6.10. Punnett square of expected albinism genotypes of Si progeny of pickerelweed
from the parent W S (genotype PIP2aaBb) ....... ... ...................................... 139









6.11. Punnett square of expected albinism genotypes of S progeny of pickerelweed
from the parent W M (genotype P P_ .uBb) ...................................... ............... 140

6.12. Punnett square of expected albinism genotypes of S progeny of pickerelweed
from the parent BS (genotype P1PlaaBB, PIPiAAbb, PJPJAABB or PiPiaabb) ...141

6.13. Punnett square of expected albinism genotypes of S, progeny of pickerelweed
from the parent BM (genotype P1PlaaBB, P1P1AAbb, P1PJAABB or PiPiaabb).. 142

7.1. Flow ers of pickerelw eed .................. .......................................................... 161

8.1. The three floral m orphs of pickerelweed ....... .......... ...................................... 184

10.1. Glabrous inflorescence scape of pickerelweed. ...................................258

10.2. Pubescent inflorescence scape of pickerelweed...... .................... ................... 259

A 1. Fruits of pickerelw eed ........................................ ......................... ................ 290

A.2. Seeds of pickerelweed germinating in jars of water in the greenhouse ...............291

A.3. Newly transplanted Fi seedling of pickerelweed in 612 cell pack.......................292

A.4. F2 seedlings of pickerelweed in the greenhouse...... ................... ................... 293

A .5. F2 plants in the greenhouse.................................... ...................... ................ 294

B. 1. Growth of pickerelweed in the greenhouse....... ... ....................................... 301

B.2. Lepidopteran pests of pickerelweed and feeding damage.................................302

B.3. Spider mites on leaves and inflorescence of pickerelweed...............................303

B.4. Slug and aphids on pickerelweed ....... ......... ........ ...................... 304

B.5. Screen enclosures designed to exclude lepidopteran pests from seedlings..........305

B.6. Flowers of M oonglow and wild-type plant....... ... ....................................... 306

B.7. M ean area in mm2 of flowers of pickerelweed ....... .................. ................... 307

B .8. V ariegated leaves of pickerelw eed .......................... .................... .....................308

B .9. Leaf shapes of pickerelw eed .......................................................... 309

B. 10 Growth habit of dormant and nondormant plants during winter in southern
F lo rid a ................................................................................................................ .. 3 1 0















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

INHERITANCE OF MORPHOLOGICAL CHARACTERS OF PICKERELWEED
(Pontederia cordata L.)

By

Lyn Anne Gettys

August 2005

Chair: David S. Wofford
Cochair: David L. Sutton
Major Department: Agronomy

Pickerelweed (Pontederia cordata L.) is a diploid (2n=2x=16) perennial aquaphyte

used in wetland mitigation and restoration and in ornamental aquascapes. There have

been no reports of the inheritance of characters of pickerelweed so the primary goal of

this work was to provide information regarding the genetic control of albinism, flower

color, floral morph and scape pubescence in pickerelweed.

Pickerelweed and other heterostylous species often exhibit some degree of

self-incompatibility due to structural differences among the heteromorphic flowers. One

factor thought to contribute to self-incompatibility in pickerelweed is differential pollen

tube growth among the three types of pollen produced by this species. A study of in vitro

pollen germination showed that pollen tubes from 1-pollen and m-pollen were longer than

those from s-pollen 240 min after germination. Pollen diameter measurements revealed

that 1-pollen was larger than m-pollen or s-pollen and that m-pollen was larger than









s-pollen. A significant positive regression between pollen grain diameter and pollen tube

length 240 min after germination was identified as well.

Only the M-morph of pickerelweed is self-fertile; therefore, novel pollination

techniques (corolla removal and stylar surgery) were developed to circumvent

self-incompatibility in S-morph and L-morph flowers, respectively. The effect of seed

storage intervals, conditions and germination environments were studied; this experiment

revealed that best germination occurred when seeds stored for less than 6 months were

germinated under water.

Albinism in pickerelweed was conditioned by three diallelic loci with expression

influenced by epistasis. The P locus was epistatic, while the A and B loci were hypostatic

and functioned as duplicate factors with dominant gene action at each locus. Flower color

was usually controlled by a single diallelic locus (W), with blue flowers dominant and

white flowers recessive, but a second locus influencing flower color was identified as

well. Floral morph was conditioned by two diallelic loci (S and M), with dominant gene

action; expression was influenced by epistasis. Scape pubescence was controlled by

duplicate gene loci, with pubescence dominant to a glabrous condition. The W locus

controlling flower color and the Mlocus involved in floral morph were linked by 16 m.u.














CHAPTER 1
INTRODUCTION AND OVERVIEW

Pickerelweed (Pontederia cordata L.) is a diploid (2n=2x=16) perennial native

aquatic species that is frequently used in wetland mitigation and restoration and in

ornamental aquascapes. There have been no reports describing the inheritance of

morphological characters in pickerelweed, so the primary goal of this work was to

provide information regarding the genetic control of albinism, flower color, floral morph

and scape pubescence in pickerelweed.

A review of the current literature is presented in Chapter 2. This describes the work

that has been reported to date regarding the taxonomy, culture, propagation and

reproductive biology of pickerelweed. Chapter 2 also includes a review of heterostyly

and provides a body of knowledge to draw from throughout this dissertation.

Heterostylous species often exhibit at least some degree of self-incompatibility due

to anatomical, structural or physiological differences among the heteromorphic flowers;

these differences encourage insect-mediated cross-pollinations between disparate

members of the same species and also act as barriers against self-fertilization. One of the

factors thought to contribute to self-incompatibility in pickerelweed is the different

lengths of pollen tubes produced in vivo by the three types of pollen borne by this

species. Chapter 3 examines the in vitro germination of pollen in an effort to determine

whether differences in pollen tube growth are evident in an in vitro system as well.

An objective of this work was to design a breeding program and develop a

population of pickerelweed that would yield the information needed to conduct









inheritance studies. Most plant breeding and genetics experiments benefit greatly when

self-produced progeny can be analyzed; however, only one of the three floral morphs of

pickerelweed is self-fertile. Novel pollination techniques were developed to increase seed

production after self-pollination; these techniques are described in Chapter 4 and can be

used by future investigators to bypass or overcome self-incompatibility in pickerelweed.

Population development requires the production not only of seeds, but also of seedlings;

therefore, Chapter 5 examines the effect of various seed storage intervals, conditions and

germination environments on the seeds of pickerelweed to determine optimum storage

and germination conditions.

Chapters 6 through 11 explore the inheritance of multiple morphological traits in

pickerelweed. Albinism is a lethal trait and seedlings exhibiting this condition do not

survive for more than 3 weeks after germination; the genetic control and inheritance of

this deleterious trait is described in Chapter 6. The inheritance and genetic systems

controlling flower color and floral morph are examined in Chapters 7 and 8, while the

linkage relationship between these two traits is elucidated in Chapter 9. The genetic

control of scape pubescence is described in Chapter 10 and the inheritance of non-allelic

or complementary flower color is discussed in Chapter 11.

This work is offered as a contribution to the body of scientific knowledge available

for pickerelweed. It is by no means comprehensive, but I hope that other investigators

will find this dissertation to be a useful resource for further studies.














CHAPTER 2
A REVIEW OF THE LITERATURE

Economic Importance

Pickerelweed (Pontederia cordata L.) is an attractive shoreline aquatic species that

is frequently used in wetland mitigation and restoration and in ornamental aquascapes.

The showy purplish-blue or white inflorescences of this herbaceous perennial make

pickerelweed a prime candidate for inclusion in water gardens and its status as a native

plant provides many opportunities for use in projects where ecosystem fidelity is critical.

Pickerelweed is classified as an obligate wetland species (Garbisch and McIninch 1992),

but the species tolerates a wide range of moisture levels and may be useful in less

saturated areas as well.

Many nurseries that produce aquatic plants offer pickerelweed for use in

ornamental water gardens and aquascapes. Pickerelweed may even be purchased at many

of the "big box" retail stores (e.g., Wal-Mart, Lowe's); in fact, Wal-Mart offers

pickerelweed with either purplish-blue or white flowers for sale in a pre-packaged kit

with two plants, fertilizer, a planting container and growing substrate for a very

reasonable price (Figure 2.1). Plants are bare-root specimens packed in damp sphagnum

and recover quickly after planting.

When used in wetland mitigation or restoration, pickerelweed provides a refuge and

habitat for many types of fauna. The flowers attract butterflies, skippers and

hummingbirds (Larson 1995; Speichert and Speichert 2001). Florida apple snails

(Pomaceapaludosa) frequently lay their eggs on the sturdy emergent stems (Turner









1996), while dragonflies and damselflies use the upright stems as perches to shed their

final larval stage before reaching adulthood (Speichert and Speichert 2001). The fruit of

pickerelweed is an important food source for ducks and small animals (Tobe et al. 1998);

Speichert and Speichert (2001) and Taylor (1992) also state that humans may safely eat

fruits, leaves and stems.

Classification, Origin and Distribution

Pickerelweed was described by Linnaeus (1753) and named in honor of Italian

botanist Giulio Pontedera (1688-1757), an Italian professor of botany at the University of

Padua (Bailey 1949; Turner and Wasson 1998). The type specimen used by Linnaeus for

the original description of the species was found in Virginia and was most likely supplied

to him by Gronovius (Lowden 1973). The genus Pontederia is namesake for the

monocotyledonous family Pontederiaceae, which encompasses ca. nine genera and thirty

species (Zomlefer 1994). The Pontederiaceae is of New World origin and includes the

invasive genus Eichhornia, whose member species E. crassipes (waterhyacinth) is

infamous for its ability to dominate aquatic habitats and to render waterways impassible

through prodigious production of floating vegetative mats. Other genera in the family

include Heteranthera, Monochoria, Eurystemon, Hydrothrix, Scholleropsis, Reussia and

Zosterella.

Species in the genus Pontederia include P. cordata L., P. rotundifolia L.,

P. subovata (Seub.) Lowd., P. parviflora Alex. and P. sagittata Presl.; however, only

P. cordata is commonly found in the eastern United States (Lowden 1973; Zomlefer

1994). There also exists a form commonly referred to as "lance-leaf pickerelweed" that is

classified as either P. cordata var. lanceolata or as P. lanceolata (Muhl.) Torrey. The

primary characteristics used to distinguish between P. cordata and lance-leaf









pickerelweed are lance-shaped leaves and persistent pubescence in the mature floral tube.

There is a great amount of natural variation in these characters and many taxonomists

lack confidence in the classification of this type as a separate species (Godfrey and

Wooten 1979).

The taxonomic hierarchy for pickerelweed is Kingdom Plantae, Subkingdom

Tracheobionta, Superdivision Spermatophyta, Division Magnoliophyta, Class Liliopsida,

Subclass Liliidae, Order Liliales, Family Pontederiaceae, Genus Pontederia L., Species

Pontederia cordata L. (USDA 20021). Multiple synonyms exist for P. cordata and

include species of Narukila and Unisema; these genera no longer exist and all members

previously placed in these genera have been reclassified as forms of P. cordata (USDA

2002; Wunderlin and Hansen 2000).

Pickerelweed is hardy in USDA Zones 4 through 11 and has a North American

geographic range that extends from Prince Edward Island to the Florida Keys (Godfrey

and Wooten 1979). The species is also found in Central America, Brazil, the West Indies

and Argentina (Godfrey and Wooten 1979; Lowden 1973). The center of diversity for the

family is thought to be lowland South America (Kohn et al. 1996). Emergent vegetation

dies back in colder regions during the winter, but rhizomes maintained below the frozen

epilimnion will overwinter and produce new shoots in spring. In Ontario and eastern

Connecticut, new shoots begin to emerge from rhizomes in April and many

well-developed leaves have formed by June. Flowering commences in June and peaks in

July, with maximum fruit dispersal occurring in August. Shoots begin to die back by

autumn and most aboveground biomass is dead by December (Heisey and Damman

1 Retrieved 12 July 2002 from the Integrated Taxonomic Information System online database
(Ihtp \ %\ \ .it is.usda.gov)









1982; Price and Barrett 1984). Plants in more hospitable climes like southern Florida

remain green throughout the winter and will flower for up to 10 months of the year.

Morphology

Pickerelweed is a diploid (2n=2x=16), erect, emergent, herbaceous aquatic

perennial and produces vegetative growth up to 1.5 m tall. The species is found in

marshes, swamps, streams, ditches and the shallow water along the margins of lakes and

ponds (Figure 2.2) (Bell and Taylor 1982; Godfrey and Wooten 1979; Tobe et al. 1998).

Pickerelweed is most common along shorelines and in flooded areas that are fairly still

and shallow (less than 30 cm in depth); seasonal fluctuations in water levels do not

adversely impact growth of pickerelweed (Heisey and Damman 1982). Pickerelweed

reproduces using both sexual and vegetative strategies. Single-seeded fruits are produced

in large amounts and allow for dispersion of the species, while creeping rhizomes rooted

in the substrate encourage the formation of large, extensive, clonal colonies.

Leaves are glabrous, entire, basal, erect and borne individually on long petioles.

Blade size and shape is highly variable and ranges from cordate to lanceolate. The

racemose inflorescence is borne at the distal end of a stem and is subtended by a single

leaf-like bract. Each inflorescence measures from 5 to 20 cm in length and bears up to

250 individual flowers [although Ornduff (1966) noted more than 450 individual flowers

on a single inflorescence] (Figure 2.3). Anthesis begins in the morning and flowers

remain open for up to 12 h; an average of 20 individual flowers are open on any given

day on a single inflorescence. The perianth is composed of six petaloid tepals arranged in

two whorls of three; tepals range in color from violet-blue to lilac to rarely white, with

yellow nectar guides ("eye spots") marking the median upper tepal of the floral envelope

(Figure 2.4). Each flower is zygomorphic, basally connate and perfect, bearing one style









and two sets of three stamens. Concentrated nectar with up to 55% sucrose equivalents is

produced during anthesis. Fruits of pickerelweed are buoyant and surrounded by light

aeriferous tissue and may float for up to 15 d (Schultz 1942). The fruit has been described

as a nutlet (Richards and Barrett 1987) or utricle (Bailey 1949); the difference between

the two classifications lies in the degree of attachment of the ovary wall to the seed. The

wall of the fruit is formed from the floral tube and is ridged with a dentate crest

(Figure 2.5). The seed contained within the fruit is filled with starchy endosperm and

contains a linear embryo that traverses the entire length of the seed (Martin 1946).

Garbisch and Mclninch (1992) stated that 1 kg contained ca. 11,000 moist seeds (seeds

were stored in water but all excess water was removed before weights were recorded).

Pickerelweed produces flowers from mid-June to mid-August in the northern extremes of

the species' range, while flowering is almost continuous in southern Florida (Bell and

Taylor 1982; Godfrey and Wooten 1979; Price and Barrett 1984; Tobe et al. 1998; Wolfe

and Barrett 1989; Zomlefer 1994).

Roots of pickerelweed are white, purplish or fuchsia when young and may turn

rusty red due to accumulation of oxidized iron. Roots darken as they age and become

brown or black as they decay and die (Heisey and Damman 1982).

In addition to the species, another type of pickerelweed is available from large

aquatic plant nurseries. This form is referred to as 'Singapore Pink'; the original plant

was reportedly found in Thailand and is propagated through clonal tissue culture or

micropropagation techniques. Some sources list 'Singapore Pink' as a cultivar of

P. cordata, while other sources list the clone as Pontederia sp.. Several characters

suggest that 'Singapore Pink' may genetically distinct from the species. Flowers are pink









(as opposed to the wild-type blue-violet found in natural habitats) and lack the

anthocyanins produced at the throat of both blue-violet and white flowers (Figure 2.6).

Anecdotal reports suggest that 'Singapore Pink' is sterile and much less tolerant of

temperature extremes (high or low) than the species; in addition, this form is often

shorter, more compact and less vigorous than the species and produces uniformly hastate

leaves.

Culture

Vegetative growth of pickerelweed is affected by substrate moisture level and

nutritional status. Heisey and Damman (1982) stated that greatest biomass accumulation

occurred at sites high in nutrients; maximum biomass was achieved 100 to 150 d after

significant growth was first noted in spring. Gettys et al. (2001) found that plants being

cultured in nursery containers grew most vigorously when the rooting substrate provided

high water-holding capacity. Barbieri and Esteves (1991) found that 1.50, 2.49 and

0.15% of total plant dry mass was attributable to N, K and P, respectively. Barbieri and

Esteves (1991) also stated that Ca contributed 1.10% of total plant dry mass, while Mg,

Na and C (ash) were responsible for 0.27, 0.12 and 10.7% of total plant dry mass,

respectively. Growth of pickerelweed is also influenced by light availability during plant

growth. Heisey and Damman (1982) showed that net photosynthetic efficiency during the

growing period was 1.5% based on peak biomass values and 1.3% when seasonal net

production was considered. Plants grown under full sun are usually more compact than

plants produced under shade.

Propagation and Dormancy

The environmental conditions that mimic a drawdown (i.e., the reduction in water

depth that occurs during the dry season) often induce germination in wetland plants.









These conditions include cold stratification in the light with an alternation of

temperatures (i.e., 200C / 300C) (Shipley and Parent 1991). Cold stratification is a

common requirement to ensure the perpetuation and survival of many seed-propagated

temperate species. Seeds that germinate soon after being shed by the parent plant in late

summer or early fall produce plantlets that will most likely be killed by winter conditions.

Chilling requirements ensure that seeds remain dormant and will not germinate until

freezing temperatures have passed.

Galinato and van der Valk (1986) and Leck and Simpson (1993) noted that

stratification was necessary for germination of seeds of most aquatic species. Shipley and

Parent (1991) studied germination of stratified seeds (9 mo in moist sand at 4C) of 64

wetland species; seeds from 10 of the species experienced poor germination (less than

10%), while seeds from the remaining 54 species had adequate germination (60 to 80%

on average).

Muenscher (1936), Speichert and Speichert (2001) and Whigham and Simpson

(1982) stated that seeds of pickerelweed required a cold, moist period of stratification

before germination. Whigham and Simpson (1982) showed that less than 5% of freshly

collected unstratified seeds of pickerelweed germinated 16 wks after being placed in Petri

plates lined with moistened filter paper and that 8 wks of moist stratification at 4C was

adequate to initiate germination. Whigham and Simpson (1982) found that best

germination of stratified seeds of pickerelweed occurred when a minimum constant

temperature of 20C to 30C was maintained or when a regime of alternating

temperatures (>10C / >20C with 12 h thermoperiods) was used. Leck (1996) stated that

freshly collected seeds of pickerelweed from Delaware or New Jersey stored in jars of









water at 5C for 7.5 mo germinated only when moved to an alternating temperature

regime of 25C / 15C with 12 h thermoperiods.

Salisbury (1970) and Grime et al. (1981) found that most mudflat and wetland

species germinated better or faster in light than in dark; Galinato and van der Valk (1986)

also noted better germination was realized in the presence of light than in dark, but

further stated that dark germination was improved by stratification. Whigham and

Simpson (1982) stated that presence or absence of light did not affect germination of

seeds of pickerelweed.

Several authors (Berjak et al. 1990; Leck 1996; Roberts and King 1980; Simpson

1966) noted that seeds of aquatic species tend to be recalcitrant (i.e., desiccation

sensitive); in fact, as little as 2 weeks of dry conditions can negatively impact

germination in sensitive species (e.g., Zizania aquatic) (Simpson 1966). Muenscher

(1936) found that germination of seeds of 40 aquatic species (seeds stored dried for 2 to

7 mo at 1C to 3C) was only 8%; germination of seeds of 45 aquatic species (seeds

stored at room temperature) was only 13%. Grime et al. (1981) found that seeds from 37

of 45 wetland species were capable of germination after being stored for 1 yr at 5C.

Whigham and Simpson (1982) suggested that seeds of pickerelweed lost viability within

1 yr of being shed. Garbisch and McIninch (1992) found that seeds of pickerelweed

collected in Maryland remained viable for more than 3 yrs and had no dormancy

requirement; however, seeds were stored in water at 1. 1C to 4.4C and should be

considered stratified.

Williges and Harris (1995) conducted greenhouse experiments under natural

conditions and stated that germination was significantly higher in inundated treatments









than in non-flooded treatments. All material used by Williges and Harris (1995) was

collected in the area around Lake Okeechobee as part of a seed-bank density sampling

experiment and was refrigerated for an unspecified length of time before germination

experiments commenced. Galinato and van der Valk (1986) found that seed burial

reduced germination percentage, while Leck (1996) stated that seeds would not germinate

in Petri plates. Barrett et al. (1983) reported that seeds germinated poorly in water at 30C

to 40C; only 76 seedlings were produced from 15 inflorescences, which theoretically

could have produced up to 3,000 seeds.

Rhizomes and roots account for a large percentage of total plant biomass at all

times. The ratio of new living below-ground biomass to new above-ground biomass

ranges from 0 in spring to 1.71 in autumn as new rhizomes and roots develop. Energy is

stored in rhizomes as compounds that can be metabolized to support new growth or as

structural compounds that remain in the rhizome. Most rhizomes produced during the

course of one growing season overwinter, then produce new shoots and rhizomes when

growth commences in spring. Structural compounds remain in the original rhizome,

which usually dies by autumn (Heisey and Damman 1982).

Rhizomes narrow when growth slows in autumn and become wider when growth

resumes in spring. These differing growth habits form a constriction and the rhizome is

easily fragmented at the junction. Energy fixed during the previous year is stored in the

rhizome and subsidizes new growth when active growth begins in spring. Heisey and

Damman (1982) estimated that as much as 30% of the biomass present in live

overwintered rhizomes and roots at the beginning of spring will be used to produce new

tissue when active growth commences. Rhizomes and rootstocks of pickerelweed do not









require stratification to overcome dormancy, as plants begin to grow as soon as soil

temperatures exceed freezing. Whigham and Simpson (1982) stated that rhizomes of

dormant plants maintained in cold storage (2C to 4C) for 8 to 16 wks produced new

growth within 15 d after being removed from cold storage and being placed in a

greenhouse with a temperature of 20C to 30C. Large-scale production of pickerelweed

is frequently accomplished using micropropagation and tissue-culture techniques (Kane

and Philman 1992, 1997).

General Heteromorphic Incompatibility

Heteromorphic incompatibility is found in 24 angiosperm families and refers to a

reproductive system where differences among floral morphs, or heterostyly, determine

incompatibility types. Heterostylous species are considered simultaneous hermaphrodites,

but unequal fitness of pollen and ovule contributions is common in many heteromorphic

species, especially those that exhibit distyly (Wyatt 1983). Heterostyly promotes

disassortative mating among floral morphs and encourages insect-mediated

cross-pollination between different morphs (Crowe 1964; Darwin 1877; Ganders 1979;

Vuillenmier 1967). Species with floral heteromorphisms are typically associated with a

self-incompatibility system that operates under sporophytic control to regulate mating

patterns in populations (Barrett 1977; Ganders 1979; Ordnuff 1966). Heteromorphic

sporophytic incompatibility is not known in any monocotyledonous plant family other

than the Pontederiaceae (Kohn et al. 1996).

General Tristyly

Tristyly is likely the most complex breeding system in plants; the system has an

elaborate developmental basis and is rare, suggesting that evolution of the trait is difficult

(Kohn et al. 1996; Richards and Barrett 1987). Tristyly is a type of heteromorphic









incompatibility and is only found in four angiosperm families: Amaryllidaceae,

Lythraceae, Oxalidaceae and Pontederiaceae. Some tristylous species are self-compatible,

while others have degrees of self-incompatibility (Barrett 1988, 1993; Barrett and

Anderson 1985; Darwin 1877; Eckert and Barrett 1994; O'Neill 1994). Populations of

species that employ tristylous incompatibility systems have three distinct floral morphs,

each with a unique set of characters. Floral morph differences include length of stigmatic

papillae, style coloration and pollen exine sculpturing (Barrett 1988), but the most

obvious visible difference among the morphs is style length. There are three positions

within each flower, with each position occupied by either a single style or one of two sets

of stamens. Floral morph designation is determined by style length; flowers with long

styles are L-morphs (Figure 2.7), while those with mid styles and short styles are

classified as M-morphs (Figure 2.8) and S-morphs (Figure 2.9), respectively. Reciprocal

positioning of anthers and stigmas occurs so that each plant produces flowers with

anthers borne at the same level as the stigmas of the other morphs. This arrangement

promotes insect-mediated cross-pollination between anthers and stigmas of equivalent

height, resulting in seed set. Darwin (1877) referred to this as "legitimate pollination",

while "illegitimate pollination" between anthers and stigmas at different levels results in

little or no seed production. Style color in some species may be an indicator of floral

morph. For example, long styles of Eichhorniapaniculata and E. crassipes are purple,

while mid or short styles are lilac/lavender or white, respectively (Barrett 1985, 1988).

Stigma and pollen polymorphisms can lead to correlations between the relative size or

spacing of stigmatic papillae and the diameter of pollen grains; these structural









polymorphisms may function as a "lock and key" mechanism to encourage and facilitate

legitimate pollination (Dulberger 1981).

Self-pollinations with pollen from anthers positioned closer to the stigma may

result in increased seed production, as the structural differences typically found in

heteromorphic flowers (i.e., stigmatic papillae density, pollen heteromorphism) may be

less pronounced in reproductive structures with reduced herkogamy. Pollen produced by

anthers borne on mid-length filaments is classified as m-pollen, while pollen produced by

anthers borne on long or short filaments is classified as 1-pollen or s-pollen, respectively.

Morph Inheritance in Tristylous Species

Inheritance of style length in tristylous systems was controlled by two diallelic loci

in the species studied thus far [e.g., species of Lythrum (Anderson and Ascher 1995),

Eichhornia (Barrett 1988) and Oxalis suksdorfi (Ordnuff 1964)]. The L-morph (long

style) phenotype was produced by the completely recessive genotype ssmm, while the

M-morph (mid style) phenotype was due to a recessive condition at the S locus and the

presence of at least one dominant allele at the M locus (genotype ssMM or ssMm). The

dominant S allele was present only in plants with S-morph (short style) flowers, which

have the genotype SSMM, SSMm, SSmm, SsMMI, SsMm or Ssmm. The sporophytic

incompatibility system present in these species drastically reduced successful

self-pollination, so homozygosity of the S and M alleles was unlikely. The S locus was

epistatic to the M locus and prevented expression of alleles at the M locus (Anderson and

Ascher 1995; Barrett 1988; Charlesworth 1979). L-morph plants were true-breeding for

floral morph so self-pollination produced only L-morph progeny. Segregation ratios of

progeny resulting from the self-pollination of S-morph and M-morph plants would be

dependent on the genotype of the parent plant. Populations of a tristylous species that









include plants bearing S-morph flowers could only produce this phenotype in one of three

ways: the founder population had at least one S-morph plant, pollen from an S-morph

plant in a nearby population was transported by pollinators, or a chance mutation arose to

change an s to an S (Anderson and Ascher 1995).

Population Structure of Tristylous Species

If all three morphs are of equal fitness and if the genes controlling tristyly are

independent from one another, the only possible condition in large tristylous populations

is isoplethic equilibrium (1:1:1) (Barrett et al. 1983). Members of the species Eichhornia

and Pontederia are exceptions and exhibit anisoplethic population structures. The gene

pool of a population may be influenced by tristyly, as there is unequal representation of

the alleles. The S-morph is most often present in low frequencies or may be lost

altogether from populations; this may be attributable to the low frequency of the S allele,

as the S allele is present only in the S-morph. Successful development of an isoplethic

population would require the presence of all three floral morphs and the production of

pollen and nectar rewards necessary to attract bees and other specialized long-tongued

pollinators (Barrett 1988; Charlesworth 1979). Ordnuff (1964, 1966) suggested that

populations of tristylous species may have anisoplethic structures with unequal

representation of floral morphs due to low numbers of founders in colonizing

populations, weak recruitment in later generations and intensive clonal propagation as a

means to increase population size.

Breakdown of Tristyly

Self-pollinating populations of normally cross-pollinated species usually occur on

the fringe of a species' geographic range or at marginal sites within the species' range.

Self-compatible individuals often have a selective advantage in low-density situations









and may be favored in pioneer habitats or under conditions associated with the

development of population bottlenecks. Self-compatibility is an important strategy if

specialized pollinators typically required by the species are not present to facilitate

cross-pollination (Arroyo 1974; Baker 1955, 1967; Barrett 1985; Moore and Lewis 1965;

Stebbins 1957).

The breakdown of tristyly and resulting development of predominantly

self-pollinating populations with semi-homostylous forms has been documented in three

species of Eichhornia (E. paniculata, E. azurea and E. crassipes). The S allele is lost,

resulting in the disappearance of the S-morph, while the loss of the m allele causes

production of the L-morph to cease. The loss of these alleles and morphs may be due to

the lower frequency of the S allele, or may be due to a reduction or loss of long-tongued

bees and other specialized pollinators that facilitate cross-pollination (Barrett 1988).

Members of the Lythraceae and Oxalidaceae may form semi-homostylous populations in

addition to stable distylous populations (Barrett 1988; Charlesworth 1979; Lewis and Rao

1971; Mulcahy 1964; Ordnuff 1972; Weller 1976). Newly formed populations serviced

by unspecialized generalist pollinators provide a selective advantage that favors

semi-homostylous variants, as autonomous self-pollination and self-compatibility results

in reproductive assurance (Barrett 1988).

Pollen diameter overlap increases in monomorphous and dimorphous populations

of species of Eichhornia. Pollen borne by same-level anthers may differ in diameter

based on the presenting floral morph. In E. azurea, 1-pollen produced by anthers borne on

long filaments of M-morph flowers may be significantly larger than 1-pollen produced by

anthers borne on long filaments of S-morph flowers (Barrett 1978). This condition also









occurs in E. paniculata, but considerable pollen diameter overlap is evident in this

species. This is most likely due to relaxed selection pressure and random accumulation of

small mutations that influence pollen diameter (Barrett 1988).

Other morphological differences have been noted in semi-homostylous populations

of E. azurea. The size and prominence of yellow nectar guides and the degree of perianth

limb extension is greatly reduced and all flowers in an inflorescence open and senesce

within 1 or 2 days. In addition, the axes of inflorescences in semi-homostylous

populations are shorter and produce more condensed inflorescences that are enclosed in a

sheath. These modifications suggest a reduced need to attract pollinators, as pollen

transfer from an individual with a different genotype is no longer necessary (Barrett

1978).

Members of monomorphous populations of E. paniculata in Jamaica uniformly

bear mid-length styles and up to three sets of anthers (most commonly one or two sets)

adjacent to the stigmas; in addition, plants are self-compatible and autogamous (Barrett

1985). Brazilian monomorphous populations of E. paniculata have small flowers borne

in reduced numbers on shorter plants than Brazilian tristylous populations (Barrett 1985).

A host of floral abnormalities are associated with developmental instability of floral

morph expression. In E. paniculata, these abnormalities include the formation of five

tepals instead of the typical six, collapsed perianth limbs, twisted or asymmetric perianth

parts, male sterility in pollen produced by anthers borne on long filaments, uniform pale

flowers and weakly developed nectar guides (Barrett 1985).

Cryptic Self-incompatibility

Cryptic self-incompatibility (also called weak self-incompatibility) describes the

production of primarily out-crossed progeny by a self-compatible species after pollination









with a mix of self-produced and externally-produced pollen. Pollen competition results in

fewer self- and intramorph matings and an excess of intermorph matings when mixtures

of all three pollen types (e.g., self, intramorph and intermorph) are deposited in amounts

in excess of the number of receptive ovules available. Legitimate pollen grew faster and

outcompeted illegitimate pollen in all three floral morphs of E. paniculata (Cruzan and

Barrett 1993). This permits a flexible mating strategy that leads to the genesis of

out-crossed progeny during pollinator abundance and the production of self-produced

progeny during pollinator scarcity (Bateman 1956; Becerra and Lloyd 1992; Bowman

1987; Cruzan and Barrett 1993). The system is referred to as "cryptic" since it is difficult

to detect without the use of genetic markers.

Case Study Lythrum salicaria L.

Lythrum salicaria L. (purple loosestrife) uses heteromorphic incompatibility in the

form of tristyly to reduce the likelihood of self-produced progeny. Mal et al. (1999) found

that legitimate pollination events resulted in greater fruit and seed set than illegitimate

intermorph, intramorph and self-pollinations. In addition, seeds produced from legitimate

pollinations had increased germination rates when compared to seeds produced from any

illegitimate pollination (81.4 and 72.7%, respectively). Mal et al. (1999) also noted that

the floral morphs differed in maternal fitness and in level of incompatibility. L-morph

flowers of purple loosestrife produced more seeds after legitimate pollination than did

S-morph flowers; incompatibility (as measured by seed set) was strongest in S-morph

flowers and weakest in M-morph flowers. Mal et al. (1999) described differences in

siring ability of pollen borne by different anther whorls within the same flower.









Self-pollinations of L-morph and M-morph flowers with 1-pollen resulted in significantly

more seed set than self-pollinations of these morphs with s-pollen (Mal et al. 1999).

Hermann et al. (1999) found that the mean diameter of L-morph stigmas of

L. salicaria was significantly larger than the mean diameters of stigmas of M-morph and

S-morph flowers; in addition, the stigmatic papillae on L-morph stigmas were

significantly larger and less dense than papillae on M-morph and S-morph stigmas. There

was no difference in the number of papillae on stigmas of all three morphs.

Studies by Eckert et al. (1996) provided the first evidence of frequency-dependent

selection on morph ratios in natural populations ofL. salicaria and found that morph

evenness and the frequency of rare morphs increased significantly over a 5-year period.

Eckert et al. (1996) suggested that their study was successful because the species'

self-incompatibility system resulted in disassortative matings among morphs, rare morphs

were present in moderate (0.05 to 0.15) frequencies, high recruitment occurred and

populations were large and resistant to the short-term effects of genetic drift.

Prevalence of Tristyly in Species of the Pontederiaceae

Solms-Laubach (1883a) characterized the floral morphology of the Pontederiaceae

and reported that Pontederia (with the exception of P. parviflora), Reussia and

E. paniculata (Spreng.) Solms were trimorphic, while E. crassipes (Mart.) Solms was

dimorphic (L-morph and M-morph only). Solms-Laubach (1883a, b) also stated that

E. natans (P. Beauv.) Solms and Heteranthera were monomorphic. The species

E. paradoxa (Mart.) Solms was described by Solms-Laubach (1883a) as possibly

trimorphic and by Schwartz (1930) as homomorphic. The heteromorphic composition of

E. azurea (Sw.) Kuntz has also been debated; East (1940) and Solms-Laubach (1883a)









referred to the species as trimorphic, while others (Johnson 1924; Miller 1883; Schultz

1942) stated that E. azurea was dimorphic.

Self-incompatibility in Species of the Pontederiaceae

Workers have described moderate self-compatibility in the M-morphs of several

species of Eichhornia similar to that found in Pontederia; however, species of

Eichhornia lack the pollen trimorphism evident in Pontederia. Agharkar and Banerji

(1930) and Francois (1964) found that the M-morph of E. crassipes was moderately

self-compatible; Agharkar and Banerji (1930) also noted that pollen from the upper set of

anthers was more productive in self-pollinations than pollen from the lower set of

anthers. Johnson (1924) witnessed the same weak self-incompatibility in the M-morph in

E. paniculata. Barrett (1985) found illegitimate pollinations of the S-morph of

E. paniculata to be much less productive than illegitimate pollinations in the other

morphs, as flowers with the S-morph produced little or no seeds when undisturbed;

Barrett (1985) suggested that herkogamy might have been sufficient to prevent autogamy

in the S-morph of the species.

The M-morphs of both P. rotundifolia and P. sagittata were moderately

self-compatible when pollinated with 1-pollen, while S-morphs and L-morphs exhibited

much stronger self-incompatibility (Barrett 1977, 1988; Barrett and Anderson 1985;

Glover and Barrett 1983; Ordnuff 1966).

Morph Inheritance in Species of the Pontederiaceae

Francois (1964) analyzed progeny resulting from illegitimate pollinations of

dimorphic E. crassipes and theorized that a single diallelic locus was responsible for

inheritance of style length in the species, with the M-morph dominant and the L-morph

recessive. The loss of the S-morph (and therefore the S allele) from the population would









result in a homozygous recessive state at the S-locus; therefore, the genotypes in the

population under investigation were most likely ssmm (L-morph) and ssMm (M-morph)

rather than mm (L-morph) and Mm (M-morph). If this is the case, the inheritance of style

length in E. crassipes is probably similar to the system proposed for other tristylous

species (e.g., Lythrum and Oxalis); segregation for all three morphs would occur if the

S-morph and S allele were present.

Morph Inheritance in Pickerelweed

Barrett and Anderson (1985) assessed a small set of S progeny (20 seedlings)

produced from controlled self-pollinations. Two of the 4 observed populations segregated

for style length and self-pollination of S plants yielded self-compatibility rates similar to

those of their parents. Barrett and Anderson (1985) thought these data suggested that

self-incompatibility was associated with style length; however, the small population size

precludes serious speculation about the inheritance of style length in pickerelweed.

Pollen Diameter Trimorphism and Production in Pickerelweed

Pollen production in pickerelweed varies based on the position of the anthers

bearing the pollen. Anthers borne by long filaments produce small amounts of large

pollen grains; anthers borne by short filaments produce large quantities of small pollen

grains and anthers held by mid-length filaments produce an intermediate amount of

medium pollen grains (Barrett 1985, 1988; Price and Barrett 1982, 1984). Two distinct

classes of anthers have been identified. Large anthers measure 1.02 + 0.06 mm and are

borne by long filaments of M-morph and S-morph flowers and mid-length filaments of

L-morph flowers. Small anthers measure 0.85 0.05 mm and are borne by short

filaments of M-morph and L-morph flowers and mid-length filaments of S-morph

flowers (Price and Barrett 1982). Barrett et al. (1983) and Price and Barrett (1982) found









that anthers borne by mid-length filaments of the S-morph produce nearly twice as much

m-pollen as anthers borne by mid-length filaments of the L-morph.

Several workers have described differences in the diameters of pollen grains

produced by the three sets of anthers. Three distinct pollen diameter classes are evident,

with the largest pollen produced by anthers borne on long filaments and the smallest

pollen produced by anthers borne on short filaments (Barrett and Glover 1985; Halsted

1889; Hazen 1918; Leggett 1875a,b; Ordnuff 1966; Price and Barrett 1982, 1984). There

is no overlap in pollen diameter so pollen origin may be identified without ambiguity

(Price and Barrett 1982, 1984). Diameter classes are preserved regardless of whether

pollen is fresh or acetolyzed (Barrett and Glover 1985; Price and Barrett 1984).

Grains of 1-pollen (produced by anthers borne on long filaments) are largest and

measure 65.65 3.22 |jm in diameter when fresh and 43.82 2.43 |jm in diameter when

acetolyzed. Pollen grains from anthers borne on short filaments are smallest; freshly

collected grains of s-pollen are 34.52 2.57 |tm in diameter, while acetolyzed grains are

25.59 1.43 |jm in diameter. Anthers borne on mid-length filaments generate m-pollen

that is intermediate in size, with fresh pollen grains measuring 53.95 3.60 |tm in

diameter and acetolyzed grains measuring 36.93 1.78 |tm in diameter (Barrett and

Glover 1985). Harder and Barrett (1993) found no difference in pollen release times

among the three morphs.

Floral Structure and Reproductive Organ Arrangement in Pickerelweed

Flowers of pickerelweed are trimerous with two series of three tepals, two series of

three stamens and a tricarpellate ovary with a single ovule (Richards and Barrett 1987).

The tepals are joined to form a perianth tube into which stamens are inserted. Individual









flowers are 12 to 16 mm long, zygomorphic and marked with a bright yellow nectar

guide eyespotot") on the large upper (banner) tepal. There is a close relationship between

stigma and anther height in organs with reciprocal positions (Price and Barrett 1982).

Richards and Barrett (1987) theorized that the rate of development of reproductive

organs could be affected by the diallelic system that is thought to control inheritance of

floral morph. Richards and Barrett (1987) suggested, for example, that the presence of an

M allele could alter reproductive growth rates to increase filament length and decrease

style length.

Differences in anther heights are attributable to the position of insertion of the

filament on the floral tube and to differences in filament length. Filaments bearing long

anthers grow quickly, while filaments bearing mid anthers elongate more slowly. Length

differences between filaments bearing long and short anthers may be due primarily to

growth rate. Filaments of short anthers experience rapid early growth but elongation is

sharply reduced when floral bud length exceeds 3 mm; in addition, filaments of short

anthers are inserted farther down in the floral tube than filaments bearing mid or long

anthers (Richards and Barrett 1987).

The filaments bearing the long anthers of both M-morph and S-morph flowers are

inserted at the same point in the floral tube. The filaments bearing the short anthers of

both L-morph and M-morph flowers are also inserted at the same point but further down

the floral tube than filaments bearing the long anthers. Mid anthers of S-morph flowers

constitute the lower anther set of the morph and filaments are inserted on the adaxial side

of the perianth tube, while mid anthers of L-morph flowers are the upper anther set of the









morph and filaments are inserted on the abaxial side of the perianth tube (Barrett et al.

1983; Price and Barrett 1982; Richards and Barrett 1987).

Stamens of the same level within a flower are produced in different ways. Short

anthers of L-morph and M-morph flowers and mid anthers of S-morph flowers are

classified as upper shorter stamen level; within this level the central stamen is longest and

is inserted on one of the inner tepal series, while the outer two stamens are shorter and are

inserted on the outer tepal series. The long anthers of M-morph and S-morph and the mid

anthers of L-morph flowers are classified as lower longer stamen levels; within this level

the central stamen is shortest and is inserted on one of the outer tepal series, while the

outer two stamens are longer and are inserted on the inner tepal series. As a result, each

stamen level within a flower has members that represent both inner and outer tepal series

(Richards and Barrett 1987).

Stigmatic height variation is attributable to style length, as ovary length is similar

in all three morphs. Styles of L-morphs, M-morphs and S-morphs measure 12.6 0.7,

7.6 0.3 and 2.7 + 0.1 mm, respectively (Richards and Barrett 1987). Price and Barrett

(1982) recorded similar measurements.

Price and Barrett (1982) found that styles of L-morph, M-morph and S-morph

flowers of pickerelweed were purple, lilac and pink, respectively. Price and Barrett

(1982) also noted differences in the density of stigmatic papillae among the morphs.

Stigmas of L-morphs had low-density papillae and thus large interstitial spaces, while

stigmas of S-morphs had very dense papillae and small interstitial spaces. Price and

Barrett (1982) speculated that these differences corresponded to the pollen diameter









trimorphisms noted in pickerelweed and may present a physical barrier to exclude

incompatible pollen.

There are no significant differences among the morphs in regard to flower and

inflorescence production, fecundity or ability to produce seed, fruit weight or germination

(Barrett and Anderson 1985; Price and Barrett 1982).

Pollen Physiology and Male Fitness in Pickerelweed

The pollen of pickerelweed is binucleate (Brewbaker 1967). Ordnuff (1966)

suggested that pollen grains produced by anthers occupying corresponding positions in

different morphs (e.g., 1-pollen from M-morphs and 1-pollen from S-morphs) were

physiologically equivalent when utilized in legitimate pollination events. However, later

work by Barrett et al. (1983) and Price and Barrett (1982) found that anthers borne by

mid-length filaments of the S-morph produced nearly twice as many grains of m-pollen

as anthers borne by mid-length filaments of the L-morph. This difference in pollen

production is attributable to the larger size and therefore volume of mid anthers produced

in the S-morph as compared to the mid anthers of the L-morph.

Barrett et al. (1983) proposed a "differential male fertility hypothesis", which stated

that this differential pollen production rendered m/S pollen (m-pollen from a S-morph)

more fit than m/L pollen (m-pollen from a L-morph). Simulation models derived by

Barrett et al. (1983) suggested that the anisoplethic population structures found in many

populations of pickerelweed could be due to the two-fold difference in m-pollen

production between the S-morphs and L-morphs. Barrett et al. (1983) examined a small

number of flowering progeny (76 seedlings) collected from fifteen open-pollinated

M-morph plants in a mixed S-morph-dominated population of pickerelweed and assessed

segregation of S-morphs versus non-S-morphs; these ratios were identical to expected









values calculated using their hypothesis, but the small sample size precluded the level of

confidence needed to ensure that results were not skewed. Field studies of natural

populations by Barrett et al. (1983) provided limited support for their hypothesis.

Self, Intramorph and Intermorph Compatibility in Pickerelweed

The different floral morphs of pickerelweed exhibit varying levels of

self-incompatibility, but all morphs produce more seeds after legitimate pollination than

after illegitimate pollination (Barrett and Anderson 1985; Barrett and Glover 1985;

Ordnuff 1966).

Ordnuff (1966) found that "own-form" (e.g., M x 1/M or M x s/M) and

"other-form" (e.g., M x 1/S or M x s/L) illegitimate pollinations were less successful than

legitimate pollinations (e.g., M x m/S or M x m/L). Ordnuff (1966) also noted that

illegitimate pollinations of L-morphs and S-morphs were most productive when

self-produced m-pollen was used, while illegitimate pollinations of M-morphs had

greatest seed yield when self-produced 1-pollen was utilized. Ordnuff (1966) stated that

differential self-incompatibility response among morphs may have been largely

attributable to carpellary (sporophytic) factors and cited the following example: 1-pollen

from M-morph plants was highly productive when used in self-pollination (M x l/M) but

poorly productive when applied to S-morph plants (S x l/M).

Self-incompatibility is strongest in S-morphs. "Own-form" illegitimate pollinations

resulted in an average of 2.8% (S x 1/S) and 12.7% (S x m/S) seed set, while "other-form"

illegitimate pollinations yielded 3.1% (S x l/M) and 2.7% (S x m/L) seed set. Legitimate

pollinations of S-morphs (S x s/M and S x s/L) produced an average of 61.3% seed set

(Ordnuff 1966).









Self-incompatibility is slightly weaker in L-morphs. "Own-form" illegitimate

pollinations L-morphs resulted in an average of 7.1% (L x s/L) and 18.7% (L x m/L) seed

set, while "other-form" illegitimate pollinations yielded 11.7% (L x m/S) and 7.0%

(L x s/M) seed set. Legitimate pollinations of L-morphs (L x 1/M and L x 1/S) produced

an average of 71.1% seed set (Ordnuff 1966).

Self-incompatibility is very weak in M-morphs. "Own-form" illegitimate

pollinations of M-morphs resulted in an average of 21.3% (M x s/M) and 53.9%

(M x l/M) seed set, while "other-form" illegitimate pollinations yielded 39.8% (M x l/S)

and 27.6% (M x s/L) seed set. Legitimate pollinations (M x m/S and M x m/L) of

M-morphs produced an average of 82.7% seed set (Ordnuff 1966).

Pollen Growth in vivo

The growth rate of compatible and incompatible pollen grain tubes is of

importance, as flowers of pickerelweed have an anthesis period of only 6 to 8 h.

Anderson and Barrett (1986) found that both compatible and incompatible grains

germinated readily on stigmas, which suggested that incompatibility in the species was

not due to strong stigmatic inhibition. Incompatible pollen tubes that reached the base of

the style enlarged, curled or lost direction, but frequently were able to enter the ovary.

Compatible pollen grew more quickly in vivo than incompatible pollen.

Pollinations by Anderson and Barrett (1986) showed that compatible pollen grains

reached the base of the style more often than incompatible pollen grains; exceptions were

noted in pollinations of the S-morph, where all grains of all three pollen types

successfully reached the ovary. All compatible pollinations resulted in pollen tubes

reaching the base of the ovary within 2 h after pollination, while incompatible

pollinations took 4 h or longer to reach the base of the ovary. Pollinations of M-morphs









and L-morphs with s-pollen rarely resulted in pollen tubes reaching the ovary and growth

of the pollen tubes from s-pollen ceased after 8 h (Anderson and Barrett 1986).

Anderson and Barrett (1986) found a correlation between pollen grain diameter and

pollen tube growth. Pollen tubes from s-pollen, m-pollen and 1-pollen reached 4 to 7 mm,

7 to 9 mm and 14 mm in length, respectively. These results suggested that storage

reserves played a role in compatibility of some combinations, but Anderson and Barrett

(1986) stated that some form of ovarian inhibition was most likely responsible for many

incompatibility reactions as well. Reduced seed set in illegitimate pollinations in spite of

ovule penetration suggests the presence of an ovarian inhibitory system to retard seed

production after self-pollination (Anderson and Barrett 1986).

Population Structure of Pickerelweed

All three morphs are usually present in natural populations of pickerelweed (Barrett

et al. 1983; Price and Barrett 1982, 1984), but S-morphs are often over-represented while

L-morphs are frequently under-represented (Barrett et al. 1983; Morgan and Barrett 1988;

Wolfe and Barrett 1989). Price and Barrett (1982) speculated that the abundance of

S-morphs may be due to the increased production of m-pollen by S-morphs as compared

to production of m-pollen by L-morphs. Morgan and Barrett (1988) stated that population

structure was strongly influenced by the genotypic constitutions of the founders and that

historical factors played an important role in determining population structure.

Impact of Pollinator Behavior

Price and Barrett (1984) studied four natural populations of pickerelweed and

found that total stigmatic pollen loads decreased in conjunction with a decrease in

inflorescence density and pollinator activity. Barrett and Glover (1985) found that

stigmatic pollen loads were typically 13.6% 1-pollen, 22% m-pollen and 64.4% s-pollen.









The proportion of legitimate pollen present in total stigmatic pollen loads on L-morphs

and M-morphs remained constant throughout the season, but the legitimate pollen

component increased as the season progressed.

Legitimate pollination was inhibited and illegitimate geitonogamous pollination

was favored by non-random foraging behavior of local pollinators and by non-random

distribution of morphs attributable to the clonal nature of the species. Price and Barrett

(1984) found that 75% of flights by Bombus spp. were among the five nearest neighbors;

in addition, the probability that the three nearest neighbors possessed the same morph

was greater than 70%. Legitimate pollination was found in spite of these constraints,

which suggested substantial pollen carryover by pollinators. Legitimate pollination was

highest in L-morph flowers and lowest in S-morph flowers, while legitimate pollination

of M-morph flowers was intermediate. Price and Barrett (1984) suggested that this might

have been due to partitioning of pollen on the bodies of pollinators. Northern populations

of pickerelweed are serviced by non-specialized bumblebee pollinators (e.g., Bombus

spp.) with broad foraging preferences that are not highly co-adapted for the floral

arrangement found in P. cordata, while long-tongued solitary bees (Melissodes apicata)

visit populations in southern regions. The zygomorphic flowers of pickerelweed present

pollinators with a limited number of orientations for floral entry (Faegri and van der Pilj

1979). Laberge (1956) suggested that species of Melissodes were specialized pollinators

of Pontederia spp. and had hairs on their proboscis to allow collection of pollen

concealed in the short anthers. The abdomens of the pollinators came into intimate

contact with 1-pollen and the stigmas of L-morphs, while the face and head (proboscis









base) contacted m-pollen and the stigmas of M-morphs. The proboscis tip was aligned

with s-pollen and the stigmas of S-morphs (Laberge 1956).

Wolfe and Barrett (1989) stated that pollinators (Bombus spp., Apis mellifera and

Melissodes apicata) typically visited less than 10 flowers on a single inflorescence and

removed 45% of total pollen production during single visits to previously unvisited

flowers. Each anther level retained less than 40% of its original pollen complement

within 90 min after dehiscence. The largest number of total pollen grains was removed

from anthers borne by short filaments, while lesser amounts were removed from anthers

borne by mid and long filaments. Rapid pollen depletion occurred, with 69% of 1-pollen,

50% of m-pollen and 38% of s-pollen removed in a single visit (Wolfe and Barrett 1989).

Harder and Barrett (1993) found slightly different results, with pollinators removing 39%

of 1-pollen, 24% of m-pollen and 28% of s-pollen during a first visit. Pollen distribution

on the bodies of pollinators was non-random; 1-pollen and m-pollen was found at greatest

concentrations where it was initially deposited (i.e., the abdomen and head), while most

s-pollen was displaced to the exterior of the pollinator, probably due to grooming

activity. M-morph flowers captured the greatest total pollen load (mean = 68 pollen

grains per stigma) and S-morph flowers received the smallest total pollen load

(mean = 18 pollen grains per stigma). The largest proportion of compatible pollen grains

was found on stigmas of L-morphs (59% of total stigmatic load); stigmas of M-morphs

and S-morphs had much smaller proportions of compatible pollen grains (22% and 25%

of total stigmatic load, respectively). Many pollinator visits failed to deposit compatible

pollen; 40% of visits to S-morphs, 21% of visits to M-morphs and 26% of visits to

L-morphs did not result in deposition of compatible pollen on stigmatic surfaces (Wolfe









and Barrett 1989). Pollinators showed no preference for any of the three morphs (Price

and Barrett 1982).

Stigmatic Pollen Loads in Pickerelweed

Stigmas of M-morphs received highest pollen loads and intact flowers captured

more pollen grains than flowers that had been emasculated. Emasculation caused some

contamination with self-produced pollen, but the mean number of self-produced pollen

grains on unpollinated stigmas borne by emasculated flowers was only 4.14% of the total

stigmatic pollen load found on intact flowers (Barrett and Glover 1985). The difference in

the number of legitimate pollen grains captured by intact vs. emasculated flowers was

insignificant, which provided evidence that stamens did not obstruct the transfer and

receipt of legitimate out-crossed pollen (Barrett and Glover 1985). Barrett and Glover

(1985) also found that even large amounts of illegitimate pollen did not cause stigmatic

clogging and did not interfere with growth of legitimate pollen tubes. Pollinator visitation

was unaffected by emasculation (Barrett and Glover 1985).

Stigmas of S-morphs and L-morphs received only small amounts of illegitimate

pollen, with 13% (S-morph) and 14.4% (L-morph) of stigmatic pollen attributable to

self-produced or geitonogamous pollen. The amount of illegitimate pollen on stigmas of

M-morphs was considerably higher, as self-produced or geitonogamous pollen accounted

for 64.3% of total stigmatic pollen load (Barrett and Glover 1985). L-morphs received the

largest amount of legitimate pollen, while pollen deposition on stigmatic surfaces of

M-morphs and S-morphs was characteristic of random pollination (Glover and Barrett

1986). Flowers of pickerelweed have only one functional ovule and typical pollen loads

have copious amounts of compatible pollen grains; as a result, seed set in the species in

rarely pollen-limited (Barrett and Glover 1985; Glover and Barrett 1986).









Greenhouse Production vs. Natural Populations of Pickerelweed

Ordnuff (1966) noted that seed production in a greenhouse environment was

reduced (especially in L-morphs and S-morphs) when compared to natural populations.

Legitimate pollinations of S-morphs in greenhouse populations and field populations

produced an average of 61.3 and 88.9% seed set, respectively. Legitimate pollinations of

L-morphs in greenhouse populations and field populations produced an average of

71.7 and 94.2% seed set, respectively. Legitimate pollinations of M-morphs in

greenhouse populations and field populations produced an average of 82.7% and 89.0%

seed set, respectively (Ordnuff 1966). Ordnuff (1966) speculated that differential seed

production among the morphs in the greenhouse was eliminated under field conditions,

where all three morphs were equally capable of seed production.


































Figure 2.1. Pre-packaged water garden kit with bare-root pickerelweed plants, damp
sphagnum moss, soilless planting substrate, fertilizer tablets and planting
basket. A kit with blue-flowered plants is shown, but a kit with white-
flowered plants is also available.


















P"kTLYPW J~
Jit I.')'


~.' < L- I
A n .4J) At*1~


IA -t, I


Figure 2.2. Pickerelweed growing along the margin of Robert's Pond in Bainbridge, New
York.


S'^'
.i.l.:?A I ;.
















k 1M


Figure 2.3. Inflorescence of white-flowered pickerelweed.






















Or ......


Figure 2.4. Yellow nectar guides eyespotsts") on banner tepal of pickerelweed flower.





















Figure 2.5. Fresh fruit, seed and dried fruit of pickerelweed.



























Figure 2.6. Flowers of 'Singapore Pink' pickerelweed. Note pale floral throat due to lack
of anthocyanins.


I




































Figure 2.7. L-morph flowers of pickerelweed. Note the single style in the long position,
three anthers in the mid position and the barely-visible three anthers in the
short position.




































Figure 2.8. M-morph flowers of pickerelweed. Note the three anthers visible in the long
position and the single style in the mid position; anthers in the short position
are barely visible in the lowest flower of this inflorescence.




































Figure 2.9. S-morph flowers of pickerelweed. Note the three anthers in the long position
and the three anthers in the mid position; the single style in the short position
is obscured by the throat of the flower and is not visible.














CHAPTER 3
POLLEN GRAIN DIAMETER, IN VITRO POLLEN GERMINATION AND
REGRESSION BETWEEN GRAIN DIAMETER AND IN VITRO GERMINATION

Introduction

Pickerelweed (Pontederia cordata L.) is a naturally outcrossed tristylous species

that relies on heteromorphic incompatibility to reduce or prevent self-pollination. Some

tristylous species are self-compatible, while others have degrees of self-incompatibility

(Barrett 1988, 1993; Barrett and Anderson 1985; Darwin 1877; Eckert and Barrett 1994;

O'Neill 1994). Three distinct floral morphs are produced by tristylous species, but each

plant always produces flowers of the same morph. Floral morphs may differ from one

another in characters including length or density of stigmatic papillae, style coloration

and pollen exine sculpturing (Barrett 1988), but the most obvious visible difference

among the floral morphs is style length. Stigmatic height variation is attributable to style

length, as ovary length is similar in all three floral morphs (Richards and Barrett 1987).

There are three positions within each flower, with each position occupied by either

a single style or one of two sets of stamens. Floral morph designation is determined by

style length; flowers with long styles are L-morphs, while those with mid styles and short

styles are classified as M-morphs and S-morphs, respectively. Reciprocal positioning of

anthers and stigmas occurs so that each plant produces flowers with anthers borne at the

same level as the stigmas of the other morphs. This arrangement promotes

insect-mediated cross-pollination between anthers and stigmas of equivalent height,

resulting in seed set. Darwin (1877) referred to this as "legitimate pollination", while









"illegitimate pollinations" between anthers and stigmas at different levels result in little

or no seed production.

Several workers have described differences in the diameter of pollen grains

produced by the three sets of anthers in pickerelweed. Three distinct pollen diameter

classes are evident; anthers in the long position (borne by long filaments) produce the

largest pollen and anthers in the short position (borne by short filaments) produce the

smallest pollen, while anthers in the mid position (borne by mid-length filaments)

produce pollen that is intermediate in diameter (Barrett and Glover 1985; Halsted 1889;

Hazen 1918; Leggett 1875a,b; Ordnuff 1966; Price and Barrett 1982, 1984). Pollen

produced by anthers borne on mid-length filaments is classified as m-pollen, while pollen

produced by anthers borne on long or short filaments is classified as 1-pollen or s-pollen,

respectively. Pollen is further identified as s/M or s/L (s-pollen originating from

M-morph plants or L-morph plants, respectively), m/S or m/L (m-pollen derived from

S-morph plants or L-morph plants, respectively) and 1/S or 1/M (1-pollen from produced

by S-morph plants or M-morph plants, respectively).

There is no overlap in pollen diameter so pollen origin (i.e., anther level) may be

identified without ambiguity (Price and Barrett 1982, 1984). Diameter classes are

preserved regardless of whether pollen is fresh or acetolyzed (Barrett and Glover 1985;

Price and Barrett 1984). Barrett and Glover (1985) reported that fresh grains of 1-pollen

measured 65.65 3.22 |tm in diameter, while fresh grains of s-pollen and m-pollen were

34.52 2.57 |tm and 53.95 3.60 |tm in diameter, respectively.

There are no differences among the floral morphs in regard to flower and

inflorescence production, fecundity or ability to produce seed after cross-pollination, fruit









weight or seed germination (Barrett and Anderson 1985; Price and Barrett 1982). The

different floral morphs of pickerelweed exhibit varying levels of self-incompatibility but

all morphs produce more seeds after legitimate pollination than after illegitimate

pollination (Barrett and Anderson 1985; Barrett and Glover 1985; Ordnuff 1966).

Ordnuff (1966) found that illegitimate pollinations (e.g., L x m or s, M x 1 or s,

S x 1 or m) were less successful than legitimate pollinations (e.g., L x 1, M x m, S x s).

Ordnuff (1966) also noted that illegitimate pollinations of L-morphs and S-morphs were

most productive when self-produced m-pollen was used, while illegitimate pollinations of

M-morphs had greatest seed yield when self-produced 1-pollen was utilized.

Self-incompatibility was strongest in S-morphs. Illegitimate pollinations resulted in an

average of 2.7 to 12.7% seed set, while legitimate pollinations produced an average of

61.3% seed set. Self-incompatibility was slightly weaker in L-morphs. Illegitimate

pollinations produced from 7.0 to 18.7% seed set, while legitimate pollinations averaged

71.1% seed set. Self-incompatibility was very weak in M-morphs. Illegitimate

pollinations resulted in an average of 21.3 to 53.9% seed set, while legitimate pollinations

produced an average of 82.7% seed set (Ordnuff 1966).

Anderson and Barrett (1986) found that both compatible and incompatible pollen

grains germinated readily on stigmas, which suggested that incompatibility in

pickerelweed was not sporophytic (i.e., due to strong stigmatic inhibition). Incompatible

pollen tubes that reached the base of the style enlarged, curled or lost direction, but

frequently were able to enter the ovary. Anderson and Barrett (1986) showed that

compatible pollen grains grew more quickly and reached the base of the style more often

than incompatible pollen grains; exceptions were noted in pollinations of the S-morph,









where all grains successfully reached the ovary. Compatible pollination of all floral

morphs resulted in pollen tubes that reached the base of the ovary within 2 h after

pollination, while incompatible pollinations took 4 h or longer to reach the base of the

ovary. Pollinations of M-morphs and L-morphs with s-pollen rarely resulted in pollen

tubes reaching the ovary and growth of the pollen tubes ceased after 8 h (Anderson and

Barrett 1986).

Anderson and Barrett (1986) noted a correlation between pollen grain diameter and

in vivo pollen tube growth. Pollen tubes from s-pollen, m-pollen and 1-pollen reached 4 to

7 mm, 7 to 9 mm and 14 mm, respectively. Richards and Barrett (1987) found that

stigmatic heights of S-morphs, M-morphs and L-morphs measured 2.7 + 0.1 mm,

7.6 0.3 mm and 12.6 0.7 mm, respectively, with similar measurements recorded by

Price and Barrett (1982). These results suggested that pollen storage reserves played a

role in compatibility of some combinations, but Anderson and Barrett (1986) stated

reduced seed set in illegitimate pollinations in spite of ovule penetration suggested the

presence of an ovarian inhibitory system that may have retarded seed production after

self-pollination.

The objectives of this experiment were threefold. The first objective was to

compare pollen grain diameter of same-level pollen produced by different floral morphs

(s/M vs. s/L, m/S vs. m/L and 1/S vs. l/M) and to identify differences in grain diameter

among pollen produced by the three different anther levels (s-pollen vs. m-pollen vs.

1-pollen). The second objective was to compare in vitro pollen tube growth of same-level

pollen produced by different floral morphs (s/M vs. s/L, m/S vs. m/L and 1/S vs. l/M) and

to determine whether the differences in pollen tube growth in vivo described by









Anderson and Barrett (1986) also exist in an in vitro system. The final objective of this

experiment was to define the relationship between pollen grain diameter and in vitro

pollen tube length 240 min after germination.

Materials and Methods

Pollen grains from both anther levels of twelve plants were examined in this

experiment; these comprised four each of L-morph plants (BL2, HWL, PBL and WL1),

M-morph plants (BM2, PBM, PWM and WM1) and S-morph plants (BS1, BS2, BS5 and

PWS). Plants were grown in a greenhouse in 1-L nursery containers filled with

Metro-Mix 500 1, a commercially available growing substrate that contains 40 to 50%

composted pine bark, 20 to 35% horticultural grade vermiculite and 12 to 22% Canadian

sphagnum peat moss by volume with a nutrient charge and pH adjustment (Scotts-Sierra,

Marysville OH). Nutrition was supplied by the incorporation of 10 g of Osmocote Plus

15-9-12 (Scotts-Sierra, Marysville OH) per container. Plants were sub-irrigated and kept

in a pollinator-free glasshouse with air temperature maintained at 270C (day) and 16C

(night). During earlier experiments, we observed that some genotypes were more

floriferous when grown under long days; therefore, supplemental lighting was employed

to artificially extend daylength to 16 h for the duration of this study.

Grains of s-pollen from 8 plants were studied; 4 of these plants (BM2, PBM, PWM

and WM1) were M-morphs and the remaining 4 plants (BL2, HWL, PBL and WL1) were

L-morphs. Grains of m-pollen from 8 plants were studied; 4 of these plants (BL2, HWL,

PBL and WL1) were L-morphs and the remaining 4 plants (BS1, BS2, BS5 and PWS)



1 Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the product
by the Florida Agricultural Experiment Station and does not imply its approval to the exclusion of other
products that may be suitable.









were S-morphs. Grains of 1-pollen from 8 plants were studied; 4 of these plants (BS1,

BS2, BS5 and PWS) were S-morphs and the remaining 4 plants (BM2, PBM, POWM and

WM1) were M-morphs.

Pollen grain diameter. Dehisced anthers were removed from open flowers with

fine forceps and placed in ca. 2 mL of pollen killing and fixing solution in 6-well culture

plates (BD FalconTM Multiwell Cell Culture Plates #353046, BD Biosciences, Bedford

MA; well volume 15 mL, well surface area 9.6 cm2). The pollen killing and fixing

solution consisted of 5 parts formaldehyde, 3 parts glacial acetic acid, 20 parts glycerin

and 72 parts deionized water and allowed hydration of the pollen grains but prevented

germination. Three anthers bearing the same type of pollen from an individual plant were

placed in each well. Each plate contained 6 wells, so an individual plate contained pollen

from both anther levels of 3 different plants. Each plate assembly included a fitted cover,

which was labeled with the source of the pollen in each well (donor identity and anther

level).

Grain diameter was measured for 50 pollen grains from each plant/anther level

combination. Grains were magnified and visualized using a Bausch and Lomb

microprojector (Leica Microsystems, Wetzlar, Germany). Diameters of magnified pollen

grains were recorded in millimeters then converted to actual size in microns with a

multiplier appropriate for the magnification used to visualize the sample. Means of

converted values were calculated for each plant/anther level combination and these

values were subjected to standard analysis of variance procedures. The model was

constructed to identify differences among same-level pollen produced by plants with

different floral morphs (s/M pollen vs. s/L pollen, m/S pollen vs. m/L pollen, 1/S pollen









vs. 1/M pollen). The model also tested for differences among diameters of pollen grains

produced by the three anther levels (s-pollen vs. m-pollen vs. 1-pollen). Morph was nested

within anther level, as each anther level was present in only two of the three morphs

(s-pollen from M-morphs and L-morphs, m-pollen from S-morphs and L-morphs,

1-pollen from S-morphs and M-morphs). Means were separated using t-tests to detect

least significant differences.

In vitro pollen germination. This experiment utilized an agarose germination

medium consisting of 10% sucrose, 0.6% agar, 0.02% Ca3NO4 and 0.01% boric acid

dissolved in deionized water. This mixture was boiled for 5 min then allowed to cool

slightly before being transferred to the 6-well culture plates. Prepared culture plates were

cooled to room temperature then stored at 4C for up to 72 h before pollen collection.

Pollen was collected from dehisced anthers at ca. 10:00 am. Fine forceps were used

to remove anthers from open flowers and pollen was transferred to the surface of the

germination medium by gently dragging the anthers across the surface of the medium.

Each well was dusted with pollen from three anthers collected from the same level of an

individual plant. Each plate contained 6 wells, so each plate contained pollen from both

anther levels of three different plants. Each plate assembly included a fitted cover, which

was labeled with the source of the pollen in each well (donor identity and anther level)

and with the collection time. Plates were placed in a germination chamber maintained at

ca. 30C and treated with killing and fixing solution (components described in the

previous section) at specified time intervals. Pollen grains from each anther level of each

plant were killed at one of four time intervals: 30, 60, 120 and 240 min. All pollen









samples in a single plate were killed at the same time interval by adding ca. 2 mL of

killing and fixing solution to each well.

Pollen tube length was measured for 200 germinated pollen grains of each

plant/anther level/interval combination. Pollen tubes were magnified and visualized using

the Bausch and Lomb microprojector described above and tube length data were obtained

by utilizing digital calipers to measure pollen tubes from the point of emergence from the

pollen grain to the distal end of the pollen tube. These data for magnified pollen tubes

were recorded in millimeters then converted to actual size in microns using a multiplier

appropriate for the magnification used to visualize the sample. Means of converted values

were calculated for each plant/anther level/interval combination and these values were

subjected to standard analysis of variance procedures. The model was constructed to

identify differences in lengths of pollen tubes from same-level pollen produced by plants

with different floral morphs (s/M pollen vs. s/L pollen, m/S pollen vs. m/L pollen,

1/S pollen vs. 1/M pollen) at all four intervals. The model also tested for differences

among lengths of pollen tubes from pollen produced by the three anther levels (s-pollen

vs. m-pollen vs. 1-pollen) at all four intervals. Morph was nested within anther level, as

each anther level was present in only two of the three morphs (s-pollen from M-morphs

and L-morphs, m-pollen from S-morphs and L-morphs, 1-pollen from S-morphs and

M-morphs). Means were separated using t-tests to detect least significant differences.

Relationship between pollen grain diameter and tube length. The relationship

between pollen grain diameter and tube length 240 min after germination was examined

by computing a regression coefficient between the two variables. The Pearson

product-moment correlation formula was used for this analysis.









Results and Discussion

Pollen grain diameter. There was no difference in same-level pollen grain

diameter produced by the different floral morphs (Table 3.1), so further discussion of

grain diameter will refer to pollen only by the anther level producing the pollen (i.e.,

s-pollen instead of s/M and s/L, m-pollen instead of m/S and m/L, 1-pollen instead of 1/S

and l/M). Significant differences were evident among grain diameters of s-pollen,

m-pollen and 1-pollen (Table 3.1). Grains of 1-pollen averaged 44.97 0.30 tm in

diameter, while grains of m-pollen and s-pollen were 35.04 0.49 tm and 20.46 +

0.34 [m in diameter, respectively (Figure 3.1).

In vitro pollen germination. There was no difference in the lengths of pollen

tubes generated by same-level pollen produced by the different floral morphs 30, 60, 120

or 240 min after germination (Table 3.2), so data for all pollen produced by the same

anther level were pooled prior to comparisons among the three anther levels. Since floral

morph did not have a significant impact on pollen tube length, further discussion of grain

size will refer to pollen only by the anther level producing the pollen (i.e., s-pollen,

m-pollen and 1-pollen). Significant differences in pollen tube lengths were evident during

in vitro germination of s-pollen, m-pollen and 1-pollen (Table 3.2). Pollen tubes from

1-pollen and m-pollen were longer than tubes from s-pollen at all time intervals under

investigation (Figure 3.2).

Tubes from 1-pollen and m-pollen were longer than tubes from s-pollen 30 min

after germination, but there was no difference between tubes from 1-pollen and tubes

from m-pollen during the same time interval. Pollen tubes from s-pollen averaged

80.49 tm in length 30 min after germination, while pollen tubes from m-pollen and









1-pollen reached average lengths of 106.26 tm and 118.16 am, respectively, 30 min after

germination (least significant difference 15.41 am) (Figure 3.2).

Pollen tubes from 1-pollen and m-pollen were longer than tubes from s-pollen

60 min after germination; in addition, pollen tubes from 1-pollen were longer than pollen

tubes from m-pollen during the same time interval. Pollen tubes from s-pollen reached an

average length of 137.27 am 60 min after germination; pollen tubes from m-pollen grew

to 175.18 am in length and pollen tubes from 1-pollen reached an average length of

195.07 am 60 min after germination (least significant difference 17.34 am) (Figure 3.2).

Pollen tubes from 1-pollen and m-pollen were longer than pollen tubes from

s-pollen 120 min after germination, but there was no difference between pollen tubes

from 1-pollen and pollen tubes from m-pollen during the same time interval. Pollen tubes

from s-pollen averaged 187.77 am in length 120 min after germination, while pollen

tubes from m-pollen and 1-pollen reached average lengths of 306.34 am and 313.74 am,

respectively, 120 min after germination (least significant difference 63.62 am)

(Figure 3.2).

Pollen tubes from 1-pollen and m-pollen were longer than pollen tubes from

s-pollen 240 min after germination, but there was no difference between pollen tubes

from 1-pollen and pollen tubes from m-pollen during the same time interval. Pollen tubes

from s-pollen reached an average length of 265.57 am 240 min after germination; pollen

tubes from m-pollen grew to 431.14 am in length and pollen tubes from 1-pollen reached

an average length of 486.43 am 240 min after germination (least significant difference

64.27 am) (Figure 3.2).









Relationship between pollen grain diameter and pollen tube length. There was

a highly significant regression between pollen grain diameter and pollen tube length

240 min after germination (Figure 3.3). The result of this regression was an increase of

ca. 9.13 pm in in vitro pollen tube length for each micron increase in pollen grain

diameter 240 min after germination, with similar trends noted at the three other intervals

as well. These results suggested that pollen grain diameter had a significant positive

impact on pollen tube growth in an in vitro system.

Conclusions

This experiment confirmed the results of Barrett and Glover (1985) and Price and

Barrett (1982, 1984) and showed that diameters of pollen grains produced by the three

anther levels of pickerelweed were significantly different from one another with no

overlap in grain diameter among the classes. Measurements of pollen in this experiment

differed from those reported by Barrett and Glover (1985) and Price and Barrett

(1982, 1984); however, this was most likely a function of the disparate methods used to

collect data as opposed to a true difference in grain diameter.

In vitro pollen germination did not produce the same results as those reported in

vivo by Anderson and Barrett (1986). Pollen tubes germinated in vitro did not generate

the impressive growth reported in vivo, but this is not unexpected since pollen

germination in vitro is often less vigorous than pollen germination in vivo. The

relationships among pollen tubes from the three pollen grain diameter classes differed

from those described by Anderson and Barrett (1986) as well; these workers reported

significant differences among all three classes but this experiment showed no significant

difference between pollen tubes produced by 1-pollen and those produced by m-pollen.

The reason for these conflicting results is unknown but it is possible that factors such as









stylar interaction (e.g., the presence of inhibitory or stimulatory substances) with the

germinating pollen grain influence in vivo germination.

This experiment also detected a highly significant regression between pollen grain

diameter and in vitro pollen tube length; these results were similar to those described by

Anderson and Barrett (1986) for in vivo pollen germination and suggested that pollen

diameter had a positive impact on the growth of pollen tubes produced as a result of in

vitro germination. These results provided an explanation for Ordnuff's (1966) findings

that self-pollinations of M-morphs and L-morphs were most fruitful when pollen from

upper-level anthers was used; as grain diameter conditions pollen tube length, larger

pollen is more likely to produce a pollen tube long enough to travel down the length of

the style and reach the ovary. These results also supported Anderson and Barrett's (1986)

hypothesis that storage reserves played a role in compatibility of some combinations;

however, Anderson and Barrett (1986) also pointed out that reduced seed set in

illegitimate pollinations in spite of ovule penetration suggested the presence of an ovarian

inhibitory system (i.e., somatoplastic incompatibility) that may have retarded seed

production after self-pollination.






54


Table 3.1. Analysis of variance of pollen grain diameter in microns of s-pollen, m-pollen
and 1-pollen of pickerelweed. Data analyzed were the means of 50 pollen
grains from both anther levels of 12 plants.
Source DFt MSJ F-value Pr > F
Anther 2 1215.68 7902.82 <.0001
Morph(anther) 3 0.25 1.63 0.2185
Error 18 0.15
Total 23
t DF: Degrees of freedom
% MS: Mean square
Anther: Anther level (s-pollen, m-pollen, 1-pollen)
Morph(anther): Anther level nested within morph (s/M vs. s/L, m/S vs. m/L, 1/S vs.
l/M)









Table 3.2. Analysis of variance of pollen tube length in microns produced in vitro by
s-pollen, m-pollen and 1-pollen of pickerelweed. Data analyzed were the
means of 200 pollen tubes from each pollen diameter class of 12 plants at
4 intervals.
Source DFt MSJ F-value Pr > F
Pollen 2 103457.94 43.06 <.0001
Morph(pollen) 3 2070.26 0.86 0.4650
Time# 3 359125.88 149.49 <.0001
Pollen*timett 6 16675.24 6.94 <.0001
Morph(pollen)*time 9 2352.19 0.98 0.4646
Error 72 2587.88
Total 95
t DF: Degrees of freedom
$ MS: Mean square
Pollen: Pollen diameter class (s-pollen, m-pollen, 1-pollen)
T Morph(pollen): Morph nested within pollen diameter class (s/M vs. s/L, m/S vs. m/L,
1/S vs. l/M)
# Time: In vitro germination interval (30 min, 60 min, 120 min, 240 min)
ft Pollen*time: Interaction between pollen grain diameter and in vitro germination
interval
%% Morph(pollen)*Time: Interaction between morph nested within pollen grain diameter
and in vitro germination interval



























10 --


II s-pollen
II m-pollen
- I-pollen


44.97a


35.04b


20.46c


Figure 3.1. Grain diameter in microns of s-pollen, m-pollen and 1-pollen of pickerelweed.
Bars represent the mean diameter of 400 grains per pollen class. Means were
separated using a t-test to detect least significant differences. Grain diameters
coded with different letters are significantly different at p=0.05.







57



600 -
------ Short

500 --*-- Mid
S-* Long

S400-


*300-


200 -


100 .. ...


0--"
30 minutes 60 minutes 120 minutes 240 minutes

Figure 3.2. Mean length of pollen tubes in microns produced in vitro by s-pollen,
m-pollen and 1-pollen of pickerelweed 30, 60, 120 and 240 min after
germination. Symbols represent the mean length of 2,000 pollen tubes for
each anther level/interval combination.











2
30 min:y =1.398x + 51.246, r = 0.519, r = 0.7204
60 min: y = 2.228x + 97.430, r2 = 0.6002, rxy = 0.7749
120 min: y = 5.574x + 93.555, r2 = 0.406, rxy = 0.6372
240 min: y = 9.134x + 92.167, r2 = 0.660, rxy = 0.8124


500 -



400 -



300 -



200 -



100 -



0 -


Pollen grain diameter in microns


Figure 3.3. Regression between pollen grain diameter and in vitro tube length 30, 60, 120
and 240 min after germination. Regressions computed using 24 XY pairs with
the mean diameter of 50 pollen grains (X) and the mean length of 200 pollen
tubes (Y) for each plant/anther level/interval combination.


R60


- --- 200














CHAPTER 4
DEVELOPMENT OF NOVEL POLLINATION TECHNIQUES TO REDUCE
SELF-INCOMPATIBILITY RESULTING FROM HERKOGAMY

Introduction

Genetic studies designed to investigate the type of gene action and mode of

inheritance of a given trait often examine several generations of the organism of interest,

including progeny derived from self-pollination. Pickerelweed (Pontederia cordata L.) is

a naturally out-crossed tristylous species that relies on herkogamy (spatial separation of

reproductive organs) and heteromorphic incompatibility to reduce or prevent

self-pollination. Some tristylous species are self-compatible, while others have degrees of

self-incompatibility (Barrett 1988, 1993; Barrett and Anderson 1985; Darwin 1877;

Eckert and Barrett 1994; O'Neill 1994).

Three distinct floral morphs are produced by tristylous species, but each plant

always produces flowers of the same morph. Floral morphs may differ from one another

in characters including length or density of stigmatic papillae, style coloration and pollen

exine sculpturing (Barrett 1988), but the most obvious visible difference among the floral

morphs is style length. Stigmatic height variation is attributable to style length, as ovary

length is similar in all three morphs (Richards and Barrett 1987). There are three

positions within each flower, with each position occupied by either a single style or one

of two sets of stamens. Floral morph designation is determined by style length; flowers

with long styles are L-morphs, while those with mid styles and short styles are classified

as M-morphs and S-morphs, respectively. Reciprocal positioning of anthers and stigmas









occurs so that each plant produces flowers with anthers borne at the same level as the

styles of the other morphs. This arrangement promotes insect-mediated cross-pollination

between anthers and stigmas of equivalent height, resulting in seed set. Darwin (1877)

referred to this as "legitimate pollination", while "illegitimate pollination" between

anthers and stigmas at different levels results in little or no seed production.

Several workers have described differences in the diameter of pollen grains

produced among the three lengths of filaments in pickerelweed. Three distinct pollen

diameter classes are evident, with the largest diameter pollen produced by anthers borne

on long filaments and the smallest diameter pollen produced by anthers borne on short

filaments (Barrett and Glover 1985; Halsted 1889; Hazen 1918; Leggett 1875a,b; Ordnuff

1966; Price and Barrett 1982, 1984). Pollen grains produced by anthers with mid-length

filaments are classified as m-pollen, while pollen grains produced by anthers borne on

long or short filaments are classified as 1-pollen or s-pollen, respectively. There is no

overlap in pollen diameter so pollen origin (i.e., anther level or filament length) may be

identified without ambiguity (Price and Barrett 1982, 1984). Diameter classes are

preserved regardless of whether pollen is fresh or acetolyzed (Barrett and Glover 1985;

Price and Barrett 1984). Fresh grains of 1-pollen measured 65.65 3.22 |tm in diameter,

while the diameters of fresh grains of s-pollen and m-pollen averaged 34.52 2.57 |tm

and 53.95 3.60 |tm, respectively (Barrett and Glover 1985).

There are no significant differences among the morphs in regard to flower and

inflorescence production, fecundity or ability to produce seed after cross-pollination, fruit

weight or seed germination (Barrett and Anderson 1985; Price and Barrett 1982). The

different floral morphs of pickerelweed exhibit varying levels of self-incompatibility but









all morphs produce more seeds after legitimate pollination than after illegitimate

pollination (Barrett and Anderson 1985; Barrett and Glover 1985; Ordnuff 1966).

Ordnuff (1966) found that illegitimate pollinations (e.g., L x m or s, M x 1 or s,

S x 1 or m) were less successful than legitimate pollinations (e.g., L x 1, M x m, S x s).

Ordnuff (1966) also noted that illegitimate pollinations of L-morphs and S-morphs were

most productive when self-produced m-pollen was used, while illegitimate pollinations of

M-morphs had greatest seed yields when self-produced 1-pollen was utilized.

Self-incompatibility was strongest in S-morphs. Illegitimate pollinations resulted in an

average of 2.7 to 12.7% seed set, while legitimate pollinations produced an average of

61.3% seed set. Self-incompatibility was slightly weaker in L-morphs. Illegitimate

pollinations produced from 7.0 to 18.7% seed set, while legitimate pollinations averaged

71.1% seed set. Self-incompatibility was very weak in M-morphs. Illegitimate

pollinations resulted in an average of 21.3 to 53.9% seed set, while legitimate pollinations

produced an average of 82.7% seed set (Ordnuff 1966).

Anderson and Barrett (1986) found that both compatible and incompatible pollen

grains germinated readily on stigmas in vivo, which suggested that incompatibility in

pickerelweed was not sporophytic (i.e., due to strong stigmatic inhibition). Incompatible

pollen tubes that reached the base of the style enlarged, curled or lost direction, but

frequently were able to enter the ovary. Anderson and Barrett (1986) showed that

compatible pollen grains grew more quickly and reached the base of the style more often

than incompatible pollen grains; exceptions were noted in pollinations of S-morphs,

where all grains successfully reached the ovary. Compatible pollination of all morphs

resulted in pollen tubes that reached the base of the ovary within 120 min after









pollination, while incompatible pollinations took 240 min or longer to reach the base of

the ovary. Pollinations of M-morphs and L-morphs with s-pollen rarely resulted in pollen

tubes reaching the ovary and growth of the pollen tubes ceased after 480 min (Anderson

and Barrett 1986).

Anderson and Barrett (1986) hypothesized that a correlation existed between pollen

grain diameter and in vivo pollen tube growth. Pollen tubes from s-pollen, m-pollen and

1-pollen reached 4 to 7 mm, 7 to 9 mm and 14 mm in length, respectively. Richards and

Barrett (1987) found that stigmatic heights of S-morphs, M-morphs and L-morphs

measured 2.7 0.1, 7.6 0.3 and 12.6 0.7 mm, respectively, with similar

measurements recorded by Price and Barrett (1982). These results suggested that pollen

storage reserves may have played a role in compatibility of some combinations; however,

Anderson and Barrett (1986) also stated that reduced seed set after illegitimate

pollinations in spite of ovule penetration suggested the presence of somatoplastic

incompatibility or an ovarian inhibitory system that may have retarded seed production

after self-pollination.

The objectives of this experiment were twofold. The first objective was to assess

the level of self-incompatibility among members of a greenhouse population of

pickerelweed and to assign a designation of self-compatible or self-incompatible to each

member of the population. The second objective was to develop methods to overcome or

avoid self-incompatibility mechanisms and to improve seed set after self-pollination in

members of the population classified as self-incompatible.

Materials and Methods

Plants used in this experiment were from experimental Fi greenhouse populations

of pickerelweed created and maintained at the University of Florida in Gainesville. Plants









were grown in 1-L nursery containers filled with Metro-Mix 500 a commercially

available growing substrate that contains 40 to 50% composted pine bark, 20 to 35%

horticultural grade vermiculite and 12 to 22% Canadian sphagnum peat moss by volume

with a nutrient charge and pH adjustment (Scotts-Sierra, Marysville OH). Nutrition was

supplied by the incorporation of 10 g of Osmocote Plus 15-9-12 (Scotts-Sierra,

Marysville OH) per container. Plants were sub-irrigated and kept in a pollinator-free

glasshouse with air temperature maintained at 270C (day) and 160C (night). During

earlier experiments, we observed that some genotypes were more floriferous when grown

under long days; therefore, supplemental lighting was employed to artificially extend

daylength to 16 h for the duration of this study.

All plants were pollinated using self-produced pollen from anthers borne on

filament lengths described by Ordnuff (1966) as being most productive (i.e., L-morphs

were pollinated with m/L pollen, M-morphs with 1/M pollen and S-morphs with

m/S pollen). Anthers were removed from flowers with fine forceps and pollen transfer

was accomplished by brushing the stigma with an anther (L- and M-morph flowers) or by

depositing a whole anther deep in the throat of the flower (S-morph flowers). Magnifying

headgear was worn during all pollinations to allow visual confirmation of successful

transfer and adhesion of pollen grains to the stigma. Forceps were flame-sterilized

between pollinations of different plants to prevent contamination with foreign pollen.

Pollinations commenced with the opening of the first flowers of an inflorescence and

continued until all flowers on the inflorescence had been pollinated (ca. 7 to 12 d). All


1 Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the product
by the Florida Agricultural Experiment Station and does not imply its approval to the exclusion of other
products that may be suitable.









pollinations were performed between 10 am and 2 pm daily and all flowers in each

inflorescence were pollinated using the same method. Daily pollination data were

recorded on jewelry tags placed on each inflorescence. Each completed inflorescence was

enclosed in a small mesh bag and secured with a plastic-covered twist-tie until fruits were

ripe. Fruits were considered ripe when the bearing infructescence shattered (usually 23 to

30 d after completion of pollinations).

Fruits were collected in their mesh bag and air-dried for ca. 7 d, then fruits were

de-husked using a rubber-covered rub board. The use of the rub board allowed removal

of the outer husk of the fruit without scarification of the enclosed seed. Percent seed set

was calculated by dividing the total number of seeds by the total number of flowers

pollinated on each inflorescence. Genotypes that produced less than 10% seed set using

the methods described above were classified as self-incompatible and were subjected to

one of two novel pollination techniques.

L-morph genotypes deemed self-incompatible were treated with stylar surgery. A

thumbnail was placed under the style of the flower; the style was then shortened with a

surgical blade held between the thumb and forefinger of the other hand. The style was cut

so that the tip of the style was at ca. the same level as the mid anthers, then m-pollen

from the same flower was immediately transferred to the cut tip of the style (Figure 4.1).

All flowers on each inflorescence were pollinated using stylar surgery and completed

inflorescences were treated as described above.

The floral envelope was completely removed from self-incompatible S-morph

genotypes to increase access to the stigma. Removal of the floral envelope was

accomplished by firmly grasping the center of the open flower with forceps and gently









pulling to remove the corolla. This protocol effectively exposed the stigma in most cases;

however, some plants required further manipulation to remove tepal tissue obstructing

access to the stigma. Removal of the floral envelope resulted in emasculation of the

flower, so m-pollen was taken from the removed portion of the flower and transferred to

the exposed stigma (Figure 4.2). All flowers on each inflorescence were pollinated in this

manner and completed inflorescences were treated as described above. The seeds

produced during the course of this experiment were used for inheritance studies

(presented in later chapters of this dissertation). Multiple inflorescences were pollinated

on most plants to ensure production of sufficient quantities of seeds.

Most statistical tests require that the samples under investigation meet three basic

conditions in order for the analyses to be valid. Data for each group to be studied must be

drawn at random from a normally distributed population and sampled populations must

have homogeneity of variances; in addition, any factor or treatment effect must be

additive (or linear) in nature (Zar 1996). The use of percentage or proportion data such as

those utilized in this experiment violates the requirement of population normality, as

percentages form a binomial distribution; however, the deviation from normality

presented by these data can be corrected by transformation of the percentage data

(Snedecor 1946; Steel et al. 1997; Zar 1996).

Several types of transformation may be employed; however, the most appropriate

for percentage or proportion data is arcsine transformation (also referred to as angular or

inverse sine transformation). Arcsine transformation is very useful for the modification of

percentage data where all data points fall between 1.0 (100%) and 0.0 (0%), as the

mathematical manipulation of the original binomially distributed dataset results in the









creation of a transformed dataset that is normally distributed. The equation for arcsine

transformation is

p'= arcsin Jp

where p'represents the transformed percentage and p symbolizes the original percentage

data (Snedecor 1946; Steel et al. 1997; Zar 1996). While this equation is useful in most

circumstances, it is less accurate when data fall at the extreme ends of the range (between

0 to 30% and 70 to 100%). Zar (1996) suggested that more accurate results could be

obtained if actual proportions were used in the modified equation


p= arcsin + arcsin x-


where x/n symbolizes the actual proportion data. Proportion data were recorded for this

experiment, so statistical analyses were conducted on data transformed using the latter,

more accurate equation. These transformed data were then subjected to standard analysis

of variance procedures, with means of control and treatment pollinations separated using

t-tests to detect least significant differences.

Results and Discussion

M-morph plants. A total of 14,425 flowers were self-pollinated on 53 M-morph

plants. Seed set ranged from 37.3 to 98.8%, with 10,979 seeds produced. All M-morph

genotypes studied in this experiment were classified as self-compatible, so further data

regarding this group of plants will not be presented. This high level of seed production

after self-pollination corresponded well with previous reports (i.e., Barrett and Anderson

1985; Barrett and Glover 1985; Ordnuff 1966) that self-incompatibility was weakest in

M-morph flowers of pickerelweed.









L-morph plants. There were 51 L-morph plants self-pollinated in this experiment;

28 of these plants were classified as self-compatible. Self-pollination of 13,412 flowers in

the self-compatible group produced a total of 4,429 seeds. Seed set ranged from

15.3 to 66.3%. Stylar surgery was not performed on these self-compatible plants and

further data regarding this group will not be presented.

The 23 remaining L-morph plants were classified as self-incompatible. Control

(normal) self-pollination of 9,237 flowers in this group resulted in the production of only

262 seeds, with seed set ranging from 0 to 9.8%. Stylar surgery and subsequent

self-pollination of 11,049 flowers in the self-incompatible group resulted in the

production of 3,248 seeds, with seed set ranging from 14.7 to 61.6% (Table 4.1).

Data from L-morph plants subjected to control and stylar surgery pollinations were

transformed using the arcsine transformation procedure described above. Analysis of

variance revealed that mean seed set in self-incompatible L-morph plants subjected to

stylar surgery differed from mean seed set in the same plants after control pollinations

(Table 4.2). Mean seed set of all self-incompatible L-morph plants after stylar surgery

was 29.93% (arcsine transformed mean 33.17), while mean seed set of the same plants

after control pollinations was 1.97% (arcsine transformed mean 8.06). T-tests showed

that mean seed set in self-incompatible L-morph plants subjected to stylar surgery was

significantly greater than mean seed set in the same plants after control pollinations (least

significant difference between arcsine transformed means 3.57). These results suggested

that stylar surgery effectively improved seed set in L-morph plants that had been

classified as self-incompatible when pollinated using control techniques.









S-morph plants. There were 72 S-morph plants self-pollinated in this study. A

total of 32 plants were pollinated using only one of the methods (i.e., either control or

corolla removal). A group of 12 S-morph plants flowered early in the experiment and had

already produced sufficient quantities of seeds before the corolla removal technique was

fully developed; as a result, these plants received only the control treatment. A total of

1,741 seeds were produced from control pollination of 5,730 flowers and seed set ranged

from 22.6 to 41.2%. Corolla removal was a reasonably simple manipulation and early

results showed that seed set was greatly increased following removal of the corolla;

therefore, all S-morph plants pollinated during the last few months of this experiment

were subjected to this protocol. A group of 20 plants were pollinated using only the

corolla removal treatment. A total of 3,380 seeds were produced after corolla removal

pollination of 7,627 flowers and seed set ranged from 13.6 to 78.1%. Since the plants

described above were subjected to only one of the two pollination techniques, further data

obtained from these groups will not be presented.

The 40 remaining S-morph plants were pollinated using both control and corolla

removal techniques. Control self-pollination of 11,144 flowers in this group resulted in

the production of 2,199 seeds, with seed set ranging from 4.6 to 42.9%. Corolla removal

and subsequent self-pollination of 11,644 flowers of the same plants produced 5,012

seeds, with seed set ranging from 18.0 to 86.0% (Table 4.3).

Data from S-morph plants subjected to control and corolla removal pollinations

were transformed using the arcsine transformation procedure described above. Analysis

of variance revealed that mean seed set in S-morph plants subjected to corolla removal

differed from mean seed set in the same plants after control pollinations (Table 4.4).









Mean seed set of all S-morph plants after corolla removal was 45.53% (arcsine

transformed mean 42.44), while mean seed set of the same plants after control

pollinations was 19.76% (arcsine transformed mean 26.39). T-tests showed that mean

seed set in S-morph plants subjected to corolla removal was significantly greater than

mean seed set in the same plants after control pollinations (least significant difference

between arcsine transformed means 3.90). These results suggested that corolla removal

effectively improved seed set in S-morph plants when compared to seed set after control

techniques.

Conclusions

Self-incompatibility and the resultant poor seed set of some floral morphs of

pickerelweed may be overcome with the use of the novel pollination techniques

developed, tested and described in this experiment. Some workers (e.g., Anderson and

Barrett 1986) have stated that poor seed set after self-pollination may be due to the

presence of somatoplastic incompatibility or an ovarian inhibitory system that retards

seed production after self-pollination. This study suggested that physical constraints

(e.g., style length in L-morphs and stigma access in S-morphs) played an important role

in the prevention of self-pollination in pickerelweed and could be bypassed to effect

adequate production of seeds after self-pollination.

It is likely that the pollen tube growth limitations described by Anderson and

Barrett (1986) are at least partly responsible for self-incompatibility and poor seed

production after normal self-pollination of the L-morph of pickerelweed. The largest

diameter pollen grain produced by the L-morph is m-pollen; Anderson and Barrett (1986)

found that m-pollen formed a pollen tube that was 7 to 9 mm in length, but Price and

Barrett (1982) and Richards and Barrett (1987) stated that styles of L-morphs measured









12.6 0.7 mm in length. The stylar surgery technique employed in this experiment

artificially shortened the style and lowered the pollen reception surface, which reduced

the travel distance required for a pollen tube to reach the ovule and effect fertilization.

Roggen and van Dijk (1972) used a steel brush to simultaneously mutilate and

pollinate the stigma of Brassica oleracea L. in order to increase seed set, but this species

is self-incompatible due to sporophytic factors (i.e., the stigma is the site of inhibition), so

the goal of their experiment was to eliminate the stigmatic barriers responsible for

incompatibility. Stylar surgery is more similar to the stump pollination technique

employed by Davies (1957) to facilitate seed set in interspecific crosses between

members of the genus Lathyrus. The two species investigated by Davies, L. odoratus and

L. hirsutus, produced styles of different lengths; the length of the style of L. odoratus was

10 mm, while the length of the style of L. hirsutus was 4 mm. The interspecific

cross-pollination ofL. hirsutus x L. odoratus resulted in fertilization, but the reciprocal

event normally failed to produce seeds. Davies (1957) removed the stigma and part of the

style of L. odoratus and applied pollen from L. hirsutus to the cut end of the style; this

resulted in fertilization as the pollen had to travel a shorter distance to reach the ovary.

It is likely that reduced access to the stigma is at least partly responsible for

self-incompatibility and poor seed production after normal self-pollination of the

S-morph of pickerelweed. Price and Barrett (1982) and Richards and Barrett (1987)

stated that styles of S-morph flowers measured only 2.7 + 0.1 mm in length; also, gross

visual observation revealed that the reproductive structure was ensconced deep within the

throat of the flower. Barrett and Anderson (1985) used forceps to split the floral perianth

of S-morph flowers to increase access to the stigma, but stated that it was still difficult to









conduct pollinations using S-morph flowers as seed parents. We found that removal of

the corolla to allow increased access to the stigma was a reasonably simple manipulation

if done properly and the increased seed production resulting from utilization of the

technique was well worth the small effort required.

This information will be helpful for plant breeders and geneticists interested in

studying this and other tristylous species. Genetic studies designed to investigate the

inheritance and genetic control of a given trait often examine several generations of the

organism of interest, including progeny derived from self-pollination; therefore,

geneticists can use this information to improve seed set after self-pollination. Plant

breeders may employ these techniques to develop inbred lines of tristylous species,

barring the presence of severe inbreeding depression in the species of interest.









Table 4.1. Seed set after self-pollination of L-morph plants of pickerelweed subjected to
control and stylar surgery pollination treatments.
Plant Controlj CPCT Treatment TPCT# Diffftt
L1 0/150 0.00 146/482 30.29 30.29
L2 1/272 0.37 117/479 24.43 24.06
L3 3/280 1.07 111/604 18.38 17.31
L4 32/522 6.13 85/369 23.04 16.91
L5 6/500 1.20 98/457 21.44 20.24
L6 1/313 0.32 124/565 21.95 21.63
L7 0/47 0.00 76/518 14.67 14.67
L8 19/437 4.35 69/364 18.96 14.61
L9 3/476 0.63 354/875 40.46 39.83
L10 26/372 6.99 156/412 37.86 30.87
L11 30/958 3.13 176/527 33.40 30.27
L12 0/385 0.00 137/512 26.76 26.76
L13 0/501 0.00 189/713 26.51 26.51
L14 8/408 1.96 129/488 26.43 24.47
L15 17/230 7.39 149/242 61.57 54.18
L16 22/421 5.23 148/446 33.18 27.95
L17 2/238 0.84 131/409 32.03 31.19
L18 2/489 0.41 101/452 22.35 21.94
L19 1/537 0.19 119/465 25.59 25.40
L20 50/512 9.77 201/471 42.68 32.91
L21 19/319 5.96 111/226 49.12 43.16
L22 1/360 0.28 148/439 33.71 33.43
L23 19/510 3.73 173/534 32.40 28.67
t Plant: Identification code of plant subjected to control and stylar surgery pollinations
Control: Number of seeds produced using control method / number of flowers
pollinated
CPCT: Percent seed set after control pollination
TS/P: Number of seeds produced using stylar surgery / number of flowers pollinated
# TPCT: Percent seed set after stylar surgery
ft Diff: Difference in percent seed set; TPCT CPCT






73


Table 4.2. Analysis of variance of arcsine transformed percent seed set in control and
stylar surgery pollinations of L-morph plants of pickerelweed.
Source DFt MSJ F-value Pr > F
Treatment 1 7246.32 201.37 <.0001
Error 44 35.99
Total 45
t DF: Degrees of freedom
$ MS: Mean square
Treatment: Pollination type (control, stylar surgery)









Table 4.3. Seed set after self-pollination of S-morph plants of pickerelweed subjected to
control and corolla removal pollination treatments.
Plant CS/PP CPCT TS/P TPCT# Difftt
S1 24/111 21.62 110/325 33.85 12.23
S2 40/264 15.15 252/485 51.96 36.81
S3 10/53 18.87 161/444 36.26 17.39
S4 18/138 13.04 81/451 17.96 4.92
S5 36/220 16.36 113/467 24.20 7.84
S6 75/428 17.52 61/230 26.52 9.00
S7 59/384 15.36 116/250 46.40 31.04
S8 35/130 26.92 148/262 56.49 29.57
S9 106/286 37.06 54/118 45.76 8.70
S10 77/315 24.44 102/147 69.39 44.95
Sl1 19/96 19.79 117/332 35.24 15.45
S12 70/186 37.63 161/228 70.61 32.98
S13 67/185 36.22 47/100 47.00 10.78
S14 157/366 42.90 99/160 61.88 18.98
S15 61/418 14.59 131/353 37.11 22.52
S16 30/333 9.01 92/192 47.92 38.91
S17 78/374 20.86 173/270 64.07 43.21
S18 19/183 10.38 197/426 46.24 35.86
S19 71/558 12.72 86/188 45.74 33.02
S20 100/572 17.48 65/224 29.02 11.54
S21 66/169 39.05 56/109 51.38 12.33
S22 117/625 18.72 64/211 30.33 11.61
S23 48/189 25.40 334/473 70.61 45.21
S24 36/372 9.68 92/512 18.97 9.29
S25 63/329 19.15 127/272 46.69 27.54
S26 11/99 11.11 134/247 54.25 43.14
S27 35/325 10.77 65/287 22.65 11.88
S28 13/124 10.48 160/326 49.08 38.60
S29 43/308 13.96 114/396 28.79 14.83
S30 34/168 20.24 173/417 41.49 21.25
S31 5/85 5.88 143/417 34.29 28.41
S32 19/149 12.75 243/551 44.10 31.35
S33 83/408 20.34 117/263 44.49 24.15
S34 17/371 4.58 131/451 29.05 24.47
S35 84/385 21.82 92/155 59.35 37.53
S36 74/412 17.96 66/133 49.62 31.66
S37 96/322 29.81 87/149 58.39 28.58
S38 80/356 22.47 96/200 48.00 25.53
S39 42/117 35.90 260/316 82.28 46.38
S40 81/201 40.30 92/107 85.98 45.68






75


Table 4.3. Continued

f Plant: Identification code of plant subjected to control and corolla removal pollinations
$ Control: Number of seeds produced using control method / number of flowers
pollinated
CPCT: Percent seed set after control pollination
TS/P: Number of seeds produced using corolla removal / number of flowers pollinated
# TPCT: Percent seed set after corolla removal
ft Diff: Difference in percent seed set; TPCT CPCT






76


Table 4.4. Analysis of variance of arcsine transformed percent seed set in control and
corolla removal pollinations of S-morph plants of pickerelweed.
Source DFt MSJ F-value Pr > F
Treatment 1 5149.33 67.06 <.0001
Error 78 76.79
Total 79
t DF: Degrees of freedom
$ MS: Mean square
Treatment: Pollination type (control, corolla removal)






77





























Figure 4.1. Stylar surgery of an L-morph flower of pickerelweed. Note pollen on cut
surface of surgically shortened style.






















































Figure 4.2. Pollination of an S-morph flower of pickerelweed after corolla removal. A)
Exposed stigma. B) Pollen on the exposed stigma.














CHAPTER 5
OPTIMUM SEED STORAGE AND GERMINATION CONDITIONS

Introduction

Pickerelweed (Pontederia cordata L.) is an attractive shoreline aquatic species that

is frequently used in wetland mitigation and restoration and in ornamental aquascapes.

Pickerelweed reproduces utilizing both sexual and vegetative strategies, but dispersion of

the species is accomplished primarily through the production of copious amounts of

single-seeded fruits. The fruit has been described as a nutlet (Richards and Barrett 1987)

or utricle (Bailey 1949); the difference between the two classifications lies in the degree

of attachment of the ovary wall to the seed. The wall of the fruit is formed from the floral

tube and is ridged with a dentate crest. Fruits of pickerelweed are buoyant, surrounded by

light aeriferous tissue and may float for up to 15 d (Barrett 1978; Schultz 1942). Garbisch

and McIninch (1992) stated that 1 kg contained ca. 11,000 moist seeds; seeds were stored

in water but all excess water was removed before weights were recorded. The seed

contained within the fruit is filled with starchy endosperm and contains a linear embryo

that traverses the entire length of the seed (Martin 1946).

Several authors (Berjak et al. 1990; Leck 1996; Roberts and King 1980; Simpson

1966) noted that seeds of aquatic species were recalcitrant (i.e., desiccation sensitive); in

fact, as little as 2 wks of dry conditions negatively impacted germination in sensitive

species (e.g., Zizania aquatic) (Simpson 1966). Muenscher (1936) found that

germination occurred in only 8% of seeds of 40 aquatic species stored dried for 2 to 7 mo

at 1 to 30C and 13% of seeds of 45 species stored at room temperature, but Grime et al.









(1981) found that seeds from 37 of 45 wetland species were capable of germinating after

being stored for 1 yr at 5C.

Whigham and Simpson (1982) stated that presence or absence of light did not

affect germination of seeds of pickerelweed, but Salisbury (1970) and Grime et al. (1981)

found that most mudflat and wetland species germinated better or faster in light than in

dark. Galinato and van der Valk (1986) also noted better germination was realized in the

presence of light than in dark, but further stated that dark germination was improved by

stratification.

Muenscher (1936), Speichert and Speichert (2001) and Whigham and Simpson

(1982) stated that seeds of pickerelweed required a cold, moist period of stratification

prior to germination. Whigham and Simpson (1982) showed that less than 5% of freshly

collected unstratified seeds germinated 16 wks after being placed in Petri plates lined

with moistened filter paper and that 8 wks of moist stratification at 4C was adequate to

initiate germination; however, Leck (1996) stated that seeds would not germinate in Petri

plates. Whigham and Simpson (1982) found that best germination of stratified seeds

occurred when a minimum constant temperature of 20 to 30C was maintained or when a

regime of alternating temperatures (>10C / >20C; 12 h thermoperiods) was utilized.

Leck (1996) stated that freshly collected seeds from Delaware or New Jersey stored in

jars of water at 5C for 7.5 mo germinated only when moved to an alternating

temperature regime of 25C / 15C (12 h thermoperiods).

Garbisch and McIninch (1992) found that seeds of pickerelweed collected in

Maryland remained viable for more than 3 yrs and had no dormancy requirement;

however, seeds were stored in water at 1. 1C to 4.4C and should be considered stratified.









Whigham and Simpson (1982) suggested that seeds of pickerelweed lost viability within

1 yr of being shed. Williges and Harris (1995) conducted greenhouse germination

experiments and stated that germination of pickerelweed was significantly higher in

inundated treatments than in non-flooded treatments. All material utilized by Williges

and Harris (1995) was collected in the area around Lake Okeechobee as part of a

seed-bank density sampling experiment and was refrigerated for an unspecified length of

time before germination experiments commenced. Galinato and van der Valk (1986)

found that seed burial reduced germination percentage. Barrett et al. (1983) found seeds

germinated poorly in water at 30C to 40C; only 76 seedlings were produced from

15 inflorescences, which theoretically could have produced up to 3,000 seeds.

The fulfillment of conditions required for germination determines the success of

long-distance dispersal of pickerelweed but also plays a critical role in inheritance

experiments. Genetic studies require the evaluation of sexually derived progeny to

determine the mode of transmission of the trait of interest from parents to offspring, so

the ability to efficiently produce seed-borne progeny is of great importance. Several

workers have studied storage and germination of seeds of pickerelweed; however, as

shown above, the literature is conflicting and does not conclusively define optimum

storage and germination conditions for the species. The objective of this experiment was

to determine the storage and germination conditions that induced optimum germination in

seeds of this population of pickerelweed.

Materials and Methods

Open-pollinated fruits were collected in December 2003 from a heterozygous and

heterogenous population of plants being grown outdoors at the University of Florida Fort

Lauderdale Research and Education Center and were air-dried for ca. 7 d. Dried fruits









were counted into lots of 100 fruits; each lot was placed in a small zip-lock bag and

sealed. Some fruits were used immediately after the 7 d drying period to assess the effect

of germination conditions in fresh fruits and seeds, while others were stored at either RT

(room temperature ca. 25 C) or at 4C for 3 or 6 mo before being moved to a

germination environment.

Two pre-germination treatments were studied in this experiment. Before placement

in a germination environment, some dried fruits were cleaned to remove the "husk"

surrounding the seed; this was accomplished using a rubber-covered rub board.

De-husked fruits were classified as seeds, while intact fruits were classified as fruits.

Three germination environments were examined: under water, on the soil surface

and 0.5 cm below the soil surface. Fruits and seeds to be germinated under water were

placed in glass half-pint (250 mL) bottles and covered with ca. 5 cm of water

(Figure 5.1); additional water was added as needed throughout the course of the

experiment to maintain a constant depth. Fruits and seeds to be germinated on or below

the soil surface were placed in propagation flats filled with Metro-Mix 5001

(Scotts-Sierra, Marysville, OH) and maintained under a mist irrigation system (duration

5 sec, interval 10 min, 24 h / d) (Figure 5.2).

This experiment studied three storage periods (fresh, 3 mo and 6 mo), two storage

temperature regimes (RT and 4C), two pre-germination treatments (fruits and seeds) and

three germination environments (under water, on the soil surface and 0.5 cm below the

soil surface). Fresh fruits and seeds were planted immediately after the 7 d drying period;



1 Note: Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the
product by the Florida Agricultural Experiment Station and does not imply its approval to the exclusion of
other products that may be suitable.









therefore, no storage temperature regime was applied to fresh fruits and seeds. Each

storage period/storage temperature regime/pre-germination treatment/germination

environment combination was replicated four times, with 100 fruits or seeds per replicate.

The first part of this experiment utilized fresh fruits and seeds and was started on

12 January 2004; fruits being stored for 3 or 6 mo were moved to the appropriate storage

temperature regime on this date as well. The second part of this experiment commenced

on 14 April 2004 and utilized fruits and seeds that had been stored for 3 mo, while the

final part of this experiment began on 12 July 2004 and used fruits and seeds that had

been stored for 6 mo. All treatments were monitored on a weekly basis for 12 wks after

being moved to the appropriate germination environments. Radicle emergence was

considered evidence of germination and germinated fruits and seeds were removed from

the experiment after data were recorded to eliminate redundant data collection.

Most statistical tests require that the samples under investigation meet three basic

conditions in order for the analyses to be valid. Data for each group to be studied must be

drawn at random from a normally distributed population, and sampled populations must

have homogeneity of variances. In addition, any factor or treatment effect must be

additive (or linear) in nature (Zar 1996). The use of percentage or proportion data such as

those utilized in this experiment violates the requirement of population normality, as

percentages form a binomial distribution; however, the deviation from normality

presented by these data can be corrected by transformation of the percentage data

(Snedecor 1946; Steel et al. 1997; Zar 1996). While several types of transformation may

be employed, the most appropriate for percentage or proportion data is arcsine

transformation (also referred to as angular or inverse sine transformation). Arcsine