Citation
Inheritance of nodulation and its association with genes controlling testa color in Arachis hypogaea L.

Material Information

Title:
Inheritance of nodulation and its association with genes controlling testa color in Arachis hypogaea L.
Alternate title:
Arachis hypogaea
Creator:
Dashiell, Kenton Eugene, 1954-
Publication Date:
Language:
English
Physical Description:
xii, 108 leaves : ; 28 cm.

Subjects

Subjects / Keywords:
Agronomy thesis Ph. D
Dissertations, Academic -- Agronomy -- UF
Nitrogen -- Fixation ( fast )
Peanuts -- Genetics ( fast )
Rhizobium ( fast )
Testa ( jstor )
Nodulation ( jstor )
Peanuts ( jstor )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1983.
Bibliography:
Includes bibliographical references (leaves 103-107).
Additional Physical Form:
Also available online.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Kenton Eugene Dashiell.

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09791664 ( OCLC )

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Full Text








INHERITANCE OF NODULATION AND ITS ASSOCIATION WITH GENES CONTROLLING TESTA COLOR IN Arachis hypogaea L.














BY

KENTON EUGENE DASHIELL



















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


UNIVERSITY OF FLORIDA


1983


































Dedicated to my parents, Robert and Rosemary Dashiell














ACKNOWLEDGMENTS


The author is sincerely grateful for the advice, guidance, and encouragement given to him by the chairman of the supervisory committee, Dr. D. W. Gorbet. Sincere appreciation is also extended to the cochairman of the supervisory committee, Dr. A. J. Norden, for the advice and guidance he provided when I was in Gainesville. Sincere thanks are also extended to other members of my committee, Drs. E. S. Hornier, D. H. Hubbell, and D. W. Dickson, whose doors were always open when I needed advice or assistance.

Special thanks are due to Wayne Branch, Charles Bryant, Mary

Chambliss, Harold Hewett, Willis Lipford, and Stanley Slay for their technical assistance in the field and laboratory.

I am also grateful to my parents, Robert and Rosemary Dashiell, and my sister, Karla Dashiell, for hand shelling thousands of peanuts for me during their Christmas vacations in 1980 and 1981, and for their encouragement throughout this study.

Thanks are- also given to Patricia French for typing this manuscript.













iii













TABLE OF CONTENTS

PAGE

ACKNOWLEDGMENTS......................................... iii

LIST OF TABLES.......................................... vi

ABSTRACT................................................ xi

CHAPTER 1 INTRODUCTION................................. 1

2 LITERATURE REVIEW............................ 2

Inoculating Peanuts with Rhizobium........... 2
Peanut-Rhizobium Interaction................. 3
Inheritance of Non-Nodulation and Ineffective
Nodulation................................... 5
Penetrance and Expressivity.................. 8
Paternal Inheritance......................... 10
Cytoplasmic Inheritance in Peanut............ 14
Inheritance of Testa Color in Peanut......... 16

3 INHERITANCE OF NON-NODULATION IN PEANUT...... 18

Introduction................................... 18
Materials and Methods........................ 19
Results and Discussion....................... 22

4 LINKAGE BETWEEN LOCI THAT CONTROL NODULATION
AND TESTA VARIEGATION IN PEANUT.............. 46

Introduction................................. 46
Materials and Methods........................ 47
Results and Discussion....................... 49

5 A GENE AFFECTING TESTA VARIEGATION COLOR AND
ITS ASSOCIATION WITH THE N2 LOCUS IN PEANUT.. 66

Introduction................................. 66
Materials and Methods........................ 67
Results and Discussion....................... 69

6 GENETIC RELATIONSHIP AND INHERITANCE OF NONNODULATION AND TESTA COLOR IN PEANUT LINES
FROM FLORIDA AND ICRISAT..................... 79

Introduction................................. 79
Materials and Methods........................ 80
Results and Discussion....................... 82


iv












PAGE

7 SUMMARY AND CONCLUSIONS........................ 101

LITERATURE CITED ......................................... 103

BIOGRAPHICAL SKETCH....................................... 108























































V













LIST OF TABLES

TABLE PAGE

3-1 A description of the peanut lines used as parents in crosses made to investigate the inheritance of
non-nodulation.................................... 20

3-2 The generations of peanuts grown each year with a description of the plot size, number of seed
planted per plot, rows per plot, seed spacing,
and age when dug.................................. 21

3-3 Description of nodulation ratings used to classify individual plants in all field plots.............. 23

3-4 The proposed genotypes for nodulation control of the peanut lines used as parents in crosses that were made to investigate the inheritance of nonnodulation........................................ 25

3-5 Nodulation ratings of F plants that were field
grown in 1979, 1981, anA 1982..................... 26

3-6 Calculation of percentage of non-nodulated F2
plants that produced an F3 population segregating
for nodulation.................................... 28

3-7 Calculation of percentage of F2 plants with few
nodules whose progeny did not segregate 1:1:2
(non-nod:few:normal).............................. 29

3-8 Method used to adjust the F nodulation data to
correct for incomplete peneirance when A, B, C, D, E, and F equal the number of plants rated 0, 1, 2,
3, 4, and 5, respectively......................... 30

3-9 F data with the adjusted frequency analyzed by
ci-square test for goodness-of-fit to the
proposed model.................................... 32

3-10 The number of F2 families derived from normally
nodulated F2 plants that segregate within each
of the proposed ratios with chi-square test for
goodness-of-fit to the proposed model............. 35

3-11 F BC data analyzed by chi-square test for goodness
ot-fit to the proposed model for inheritance of
non-nodulation in peanut.......................... 37


vi











TABLE PAGE

3-12 The number of F BC1 families derived from normally nodulated F BC plants that segregate within each
of the proposeA ratios with chi-square test for
goodness-of-fit to the proposed model............. 39

3-13 The number of F BC families derived from few and
1
non-nodulated F BC1 plants that segregated within
each of the proposed ratios....................... 41

4-1 A description of the four peanut lines used as
parents in crosses to determine if there is linkage
between loci that control nodulation and testa
variegation....................................... 48

4-2 The method used to adjust the F and F data to
2 3
correct for incomplete penetrance................. 50

4-3 Nodulation ratings of F1 plants that were field
grown in 1979 and 1981............................ 51

4-4 F variegated testa color data from three crosses
2
with chi-square tests for an expected 3:1 ratio... 52

4-5 F2 nodulation data with the adjusted frequency
2
analyzed by chi-square test for goodness-of-fit
to the expected ratio............................. 53

4-6 F2 segregation for variegated testa color and
nodulation with the adjusted frequency analyzed
by chi-square test for goodness-of-fit to the
expected ratio with no linkage or 50% crossing
over.............................................. 55

4-7 Calculated crossover percentages with number of
observations in each population and a listing of
the tables where the data is presented............ 56

4-8 Analysis of variance of the arcsine transformation
of percentage crossing over calculated on the three
parental combinations............................. 57

4-9 F2 segregation for variegated testa color and
nodulation with the adjusted frequency analyzed
by chi-square test for goodness-of-fit to the expected ratio with the appropriate crossover
percentage........................................ 59





vii











TABLE PAGE

4-10 F families (F plants) which segregated for variegated tesa color and nodulation analyzed by
chi-square test for goodness-of-fit to the expected
ratio with the appropriate crossover percentage.... 60

4-11 F1BC segregation for variegated testa color and nodulation with the observed frequency analyzed by chi-square test for goodness-of-fit to the expected
ratio with the appropriate crossover percentage.... 61

4-12 A comparison of testa variegation of F2 plants with segregation for nodulation in the F3 and chi-square tests for goodness-of-fit to the expected ratio of
parental, crossover, and two crossover types with
the appropriate crossover percentage............... 62

4-13 A comparison of testa variegation of F BC plants with segregation for nodulation in the F2 C and
chi-square tests for goodness-of-fit to the expected ratio of parental and crossover types with
the appropriate crossover percentage............... 64

5-1 A description of the peanut lines used as parents in crosses made to investigate the inheritance
of non-nodulation and testa color.................. 68

5-2 The method used to adjust the data in Table 5-6 to correct for incomplete penetrance.................. 70

5-3 The method used to adjust the data in Table 5-8 to correct for incomplete penetrance.................. 71

5-4 Segregation for red and pink testa color with chisquare test for goodness-of-fit to the expected
ratio.............................................. 72

5-5 F2 segregation of testa color and variegation analyzed by chi-square test for goodness-of-fit
to the expected ratio with independent segregation
of the two loci R2 and V........................... 74

5-6 F segregation of testa color and nodulation with t~e adjusted frequency analyzed by chi-square test
for goodness-of-fit to the expected ratio with
independent segregation of the three loci, NI, N2,
and R2 .......................... .................. 75

5-7 Segregation for white and tinted vareigation testa
color with chi-square test for goodness-of-fit to
the expected ratio................................. 76



viii










TABLE PAGE

5-8 F2 segregation of testa variegation color and
2
nodulation with the adjusted frequency analyzed by chi-square test for goodness-of-fit to the expected ratio with independent segregation of the two loci,
N2 and Wv.......................................... 77

6-1 A description of the peanut lines used as parents
in crosses made to investigate the inheritance of
nodulation and testa color......................... 81

6-2 Method used to adjust the F nodulation data to
correct for incomplete peneirance when A, B, C, D, E, and F equal the number of plants rated 0, 1, 2,
3, 4, and 5, respectively.......................... 83

6-3 Description of nodulation and testa color of F1
plants............................................. 84

6-4 F2 segregation for nodulation with the adjusted
2
frequency analyzed by chi-square test for goodness
of-fit to the proposed model....................... 85

6-5 F2 segregation for testa color with chi-square
2
test for goodness-of-fit to the expected ratio of
1 red:3 pink....................................... 89

6-6 F2 segregation for testa color with chi-square
2
test for goodness-of-fit to the expected ratio
of 1 pink:3 purple................................. 90

6-7 F2 segregation for testa color with chi-square test
for goodness-of-fit to the expected ratio of 1 red:
15 pink:48 purple.................................. 91

6-8 F2 segregation for variegated testa with chi-square
tests for goodness-of-fit to the expected ratio of
3 solid and trace-variegated:l variegated.......... 92

6-9 F segregation for testa color and variegation with
ci-square test for goodness-of-fit to the expected
ratio with independent segregation of the R2 and
V loci.................................... ........ 94

6-10 F2 segregation for testa color and variegation
analyzed by chi-square test for goodness-of-fit
to the expected ratio with independent segregation
of the four loci P, R2' R3, and V.................. 95



ix










TABLE PAGE

6-11 F segregation for testa variegation and nodulation
with the adjusted frequency analyzed by chi-square
test for goodness-of-fit to the expected ratio with
independent segregation of the V and N2 loci....... 96

6-12 F segregation for testa color and nodulation with
the adjusted frequency analyzed by chi-square test
for goodness-of-fit to the expected ratio with
independent segregation of the N2 and R2 loci...... 98

6-13 F segregation for testa color and nodulation with
the adjusted frequency analyzed by chi-square test
for goodness-of-fit to the expected ratio with
independent segregation of the NI, N2, and P loci.. 99

6-14 F segregation for testa color and nodulation with
the adjusted frequency analyzed by chi-square test
for goodness-of-fit to the expected ratio with
independent segregation of the P, R2, R3, and N2
loci .......................................... ... 100
































x













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


INHERITANCE OF NODULATION AND ITS ASSOCIATION WITH
GENES CONTROLLING TESTA COLOR IN Arachis hypogaeaL.


BY

KENTON EUGENE DASHIELL

April 1983

Chairman: Dr. D. W. Gorbet
Cochairman: Dr. A. J. Norden
Major Department: Agronomy

A study was conducted on peanut (Arachis hypogaea L.) to determine the inheritance of nodulation and its association with genes controlling testa color. A diallel cross was made using M4-2, a non-nodulating line, and three nodulating peanut lines, PI 262090, UF 487A, and 'Florunner.' Selected F1 plants were backcrossed to M4-2 or PI 262090. The F F 2, F3, FIBC1, and F2BC1 generations were field grown at the University of Florida Agricultural Research Center at Marianna, Florida. Nodulation classifications were determined by observing plants and rating roots on each plant from 0 (no nodules) through 5 (abundant nodules). Pod samples were taken and testa color was evaluated. These data were analyzed by chi-square test for goodness-of-fit to the proposed model.

The results indicate that inheritance of nodulation is controlled at two loci, N1 and N2. The non-nodulating genotype (M4-2) is n1 n 1n2n2 and all other genotypes have normal nodulation, except n1nlN2n2'



xi











which had few nodules when the parental male gamete was n1n2. The locus controlling testa variegation, V, was found to be linked to N with an average crossover rate of about 10%. Testa variegation color is controlled at one locus. The allele causing white variegation, Wv, is dominant to the allele causing tinted variegation, wv. The N2 and Wv loci segregated independently. The R2 locus, which controls red and pink testa color, segregated independently from the V locus. It was also determined that the N, N2, and R2 loci segregate independently.

In another study, two non-nodulating peanut lines, PI 445923 and PI 445924, were crossed with M4-2, PI 262090, UF 487A, and Florunner. Data were collected from the F and F2 generations for nodulation and testa color. The results do not fully support the model for inheritance of nodulation described in the first study. The allele causing purple testa color, P, appeared to be dominant to pink and red. There also appeared to be a duplicate locus of R2 which was designated R3. The following groups of loci were found to segregate independently (R2, V), (P, R2, R3, V), (V, N2), (R2, N2), (P, Nl, N2), and (P, R2, R3, N2).
















xii













CHAPTER 1
INTRODUCTION


Crops that fix their own N2 have an inherent advantage over crops which cannot. This advantage may become even more importan as the cost of N fertilizer, which is derived from fossil fuels, increases. Peanuts (Arachis hypogaea L.), when infected by an effective Rhizobium strain, form nodules which can fix amounts of N2 adequate to produce normal plant growth in soils that have reltively low levels of available N. If the peanut could fix N2 mor efficiently, higher yields might be possible and more residual N could remain in the soil for the succeeding crop.

One method of improving the N2-fixing ability of peanut woul be to develop cultivars that can fix N2 more efficiently. To mak efficient genetic gains in the N2-fixing ability, a knowledge of ,w this characteristic is inherited is required. One heritable trai which is a primary component of N2-fixing ability is nodulation. One objective of this study was to investigate the inheritance of nodulation in peanut.

The peanut lines that were used as parents in this study had different testa colors. Thus, another objective of this study wa! to determine if any of the genes controlling testa color were linl d to the gene or genes that control nodulation.







1












CHAPTER 2
LITERATURE REVIEW


Inoculating Peanuts with Rhizobium


There have been many studies to determine the effects of inoculating peanuts (Arachis hypogaea L.) with Rhizobium. Duggar (19) found that peanut yield was increased 30 to 40% when unshelled Spanish peanuts were inoculated and planted. Also, a significant positive correlation was found between the number of nodules per plant and yield of dry peanuts per plant.

Schiffmann (53) reported that peanut yield, 1000 pod weight, 1000 seed weight, and crude protein content of hay were all higher in plots that were inoculated with effective Rhizobium strains.

Van Der Merwe et al. (64) and Walker et al. (67) found no

significant increase of seed yield due to inoculation with Rhizobium. Iswaran and Sen (32) reported that a recommended rate of inoculant resulted in no yield increase but a 10x inoculation rate induced a yield response.

Ratner et al. (50) found that pod yield of inoculated plots was significantly higher than of uninoculated plots. Schiffmann and Alper (55) reported that best results were obtained when inoculant was placed in the seed furrow, and that as nodule number per plant increased the average nodule dry weight decreased. They (54) also evaluated the effect of placing the inoculant at a depth of 8, 12, or 15 cm. As the placement depth increased, the yield, 1000 seed weight, nodule number per plant, and nodule dry weight per plant


2






3



decreased. They also observed that when there were fewer nodules per plant, there was an increase in average weight per nodule.

According to Tonn and Weaver (63), the Virginia type cultivars 'Florunner' and 'Florigiant' had more N in their vegetative organs, accumulated N in their pods at a faster rate, developed more nodule mass, and had a higher C2H2 reduction rate than the two Spanish cultivars, 'Starr' and 'Tammut 74.' Duggar (20) found that Spanish peanuts averaged 11 nodules per plant at harvest and runner peanuts averaged 127 nodules per plant.

Nambiar et al. (38) randomly selected six lines from each of

the commercially grown botanical varieties and found that plants of A. hypogaea subsp. hypogaea var hypogaea had more nodules on the hypocotyl than the other two botanical varieties. They stated nodule formation on the hypocotyl may be a desirable trait because it has been observed that nodules on the hypocotyl remain active longer than those on the root.


Peanut-Rhizobium Interaction


The symbiotic relationship of peanut and Rhizobium has been investigated by many researchers. Whiting and Hansen (68) found that peanut is a member of the cowpea cross-inoculation group.

Allen and Allen (2) inoculated two peanut cultivars with 59

strains of Rhizobium that were isolated from 31 different leguminous species and found that all were infective. Gaur et al. (22) observed that Rhizobium obtained from 51 legume species representing 23 genera nodulated peanut plants. They (21) also reported that peanut








4



nodulated well in desert soil where no Arachis species had grown previously. This provided evidence that the peanut is less specific in its Rhizobium requirement than most legumes.

In peanut inoculation studies, Chandler (16) found that in contrast to Trifolium spp., no infection threads were formed. Root hairs were found only where there was an emerging lateral root and only root hairs with large basal cells were infected. Similar to Trifolium spp., root hairs were deformed when rhizobia were present but the rhizobia entered at the junction of root hairs and the epidermal and cortical cells. The bacteria moved intercellularly in the middle lamellae and entered the cortical, root hair, and large basal cells through the structurally altered cell wall. These invaded host cells divided repeatedly to form the nodule tissue, and when the host cells stopped dividing, bacteroids were formed.

Bhuvaneswari et al. (10) also reported peanut had nodules only where lateral roots emerged. No nodules were observed at the base of laterals where well-developed root hairs were present at the time of inoculation. Nodules developed at 25 to 50% of the sites where lateral roots emerged at the time of inoculation but basal root hairs emerged after inoculation. Nodules developed at 80 to 100% of the laterals which emerged after inoculation. Susceptibility of the peanut root to infection by Rhizobium may be more related to root hair development than to lateral root emergence (10).

Allen and Allen (2) observed spherical, plastid-like bodies in the bacteroidal area of infected cells. Staphorst and Strijdom (59) found the spherical structures located in mature nodules on peanut







5


roots were Rhizobium bacteroids. The nodules from peanut were the only ones that contained spheroplast-like cells and 13 other species contained "typical" bacteroid and rod-shaped cells. This characteristic of spheroplast-like cells in nodules seems to be a property of the genus Arachis because Staphorst and Strijdom (59) also observed the spheroplast-like cells in A. erecta, A. nambyquarae, A. villosulicarpa and an unidentified Arachis spp. Van Rensberg et al. (65), using electron microscopy, reported the spheroplast-like cells were protoplasts devoid of cell wall material.

Sen and Weaver (56) found that peanuts had three times more N in their plant tops per unit weight of nodules than cowpeas (Vigna unguiculata L. Walp) when inoculated with the same strain of Rhizobium.


Inheritance of Non-Nodulation and Ineffective Nodulation


There have been several reports on the inheritance of non-nodulation for species which normally nodulate. Nutman (42) found nonnodulation in red clover (Trifolium pratense L.) to be controlled by a recessive gene (r) and affected by a maternally transmitted component. Two additional factors proposed as influencing the inheritance of nodulation were dilution of a cytoplasmic factor and the presence of zygotic and post-zygotic lethals.

Williams and Lynch (69) crossed a non-nodulating soybean (Glycine max L. Merr.) with a nodulating line and then classified the roots of F1 F2, F3, and F BC1 plants as nodulated or non-nodulated. They determined that nodulation was controlled by one dominant gene with non-nodulating plants being homozygous recessive.







6


Gorbet and Burton (24) described a non-nodulating peanut line

which was originally identified in the F3 generation from the hybridization of UF 487A, a University of Florida breeding line, with PI 262090. They concluded that non-nodulation was not controlled by a single recessive gene.

Nigam et al. (39) determined the genetics of a non-nodulating peanut which was identified from the crosses of PI 259747, with two Virginia cultivars, 'NC 17' and 'NC Ac 2731.' They found that two independent genes controlled nodulation with the non-nodulating plants being homozygous recessive at both loci.

According to Holl (30), a mutant line of Pisum had one gene Sym2 controlling nodulation and another gene Sym3 controlling N2 fixation when nodules were present. The two genes segregated independently with nodulation and N2 fixation being dominant to non-nodulation and no N2 fixation.

Peterson and Barnes (48) found three alfalfa (Medicago sativa L.) clones in which ineffective nodulation was controlled by a single tetrasomically-inherited recessive gene symbolized as inl, in2, or in3. With a fourth clone they found that ineffective nodulation was controlled by two recessive genes symbolized as in and in5. Ineffective nodules were produced when both loci were nulliplex. The non-nodulating trait was controlled by two recessive genes, symbolized nn1 and nn2 with non-nodulating plants being nulliplex at both loci. Data for all F2 and backcross families with two of the clones showed consistent deficiencies of about 28% ineffective plants and expected ratios were calculated assuming a 28% deficiency







7



of the nulliplex genotype. A similar adjustment was made for another clone that showed consistent deficiencies of 32% ineffective plants.

Vest and Caldwell (66) found that the soybean cultivar 'Hill'

was ineffectively nodulated by Rhizobium japonicum (Kirchner) strain 61. This trait was controlled by a single gene and ineffective nodulation was dominant. Some plants had a few normal-appearing nodules and produced progeny that either segregated or were all ineffectively nodulated. Thus, these plants with a few normalappearing nodules were considered ineffectively nodulated.

Caldwell et al. (14) reported that the soybean cultivar 'Merrill' has an ineffective nodulation response to the R. japonicum serogroups 3-24-44 and 122. When Merrill was inoculated with strains of the serogroup 3-24-44, many small white (tumor-like) nodules but no normal nodules were formed. When Merrill was inoculated with strains of serogroup 122, a few normal-size nodules and a very few small white nodules developed. Caldwell (13) also reported that a single dominant gene Rj2 caused ineffective nodulation of soybeans by certain strains of serogroups 3-24-44 and 122 of R. japonicum.

Nutman (43) identified two red clover clones which were ineffectively nodulated by Rhizobium trifolii Dang. strain A. When he investigated the inheritance of this trait, he scored plants from 0 to 4 with 0 being completely ineffective and 4 being normally effective. When he analyzed the results of segregating generations, he considered half of the plants scored 1 as effective and half ineffective. With this adjustment, Nutman (44) concluded that the ineffective response







8



to strain A was controlled by a recessive allele at one locus but that it was also modified by other recessive characters.

Gibson (23) found that the 'Northern First Early' variety of

Trifolium subterraneum L. formed ineffective nodules with the normal effective NA30 strain of R. trifolii. When Northern First Early was crossed with other varieties of T. subterraneum, the F1. plants were intermediately effective. In the F2 generation plants were scored as ineffective, intermediate, and effective for nodulation. While the F2 data did not fit a 1:2:1 ratio, Gibson (23) concluded that a single locus with major effects and modifying genes probably controlled the plants' response to strain NA30.


Penetrance and Expressivity


Penetrance and expressivity have each been defined (1) as the frequency with which a gene produces a recognizable effect, and the degree or amount that a genetic character affects the phenotype, respectively. The penetrance and expressivity of a genetic character can be altered by genetic background.

Loesch (35) investigated five x-ray-induced morphological mutants from the peanut cultivar 'NC4.' He concluded that the variable expressivity of the mutant phenotypes observed in the F2 and F3 generations was caused by differences in the background genotype.

Gottschalk (25) transferred the bif-1 gene, which caused bifurcated main stems, into the genomes of other Pisum mutants. The gene efr, which caused early flowering, reduced the penetrance of bif-l. When efr and ion were combined there was no further reduction in the







9


penetrance of bif-l. A gene (sg-l) which caused a reduction in grain size increased the penetrance of bif-l.

There have been several reports in which adjustments were made

to the data or the expected values when investigating the inheritance of a trait with incomplete penetrance. These include four reports described previously in this chapter (44, 48, 66). Harris et al.

(27) working with corn (Zea mays L.), investigated the inheritance of second ear shoots that silk (SES). Lines that did not have 100% SES were designated AA, while lines that did have 100% were designated aa. To calculate the expected frequencies of SES in the F2 and F BC1 generations, the degree of penetrance of the parental genotypes were used. For example, from the cross AA x Aa, the segregation would be 1/2 AA:1/2 Aa. The expected frequency of SES would be 1/2 AA SES (from the parental data) + 1/2 Aa SES (from the F1 data). All goodness-of-fit and heterogeneity chi-square values were nonsignificant when the observed and expected values were compared. They concluded that the SES trait was controlled at one locus with the aa genotype having 100% SES.

Sorells et al. (58) investigated the inheritance of second ear formation in corn, a trait with incomplete penetrance. They used a technique for analyzing their data that was similar to that used by Harris et al. (27).

The spotted leaf trait in alfalfa was reported by Azizi and

Barnes (9) to be controlled by two tetrasomic genes, SA and SB, with random chromosome inheritance. The genotypes SA- - and sasasasa sbsbsbsb prevented leaf spotting, whereas the genotype sasasasa






10



SBSB- produced spotted leaves. If the simplex SB genotype sasasasa SBsbsbsb caused spotted leaves or normal leaves, then the Sl progeny would segregate 3 spotted:l normal or 1 spotted:3 normal, respectively. However, the SI progeny of a SB simplex genotype segregated 1 spotted: 2 normal. All expected ratios were adjusted so that 20% of the plants with simplex SB genotypes were expected to produce the spotted leaf trait. When this adjustment was made, the segregation observed from 13 different crosses supported the original genetic hypothesis.

Working with barley (Hordeum vulgare L.), Carroll et al. (15) investigated the inheritance of resistance to seed transmission of barley stripe mosaic virus (BSMV). 'Vantage,' the susceptible variety, had a relatively high rate of seed transmission of BSMV, ranging from 66.3 to 80.9%. Because of incomplete penetrance the classification of F2 plants as being resistant was not reliable. When F2 plants were progeny tested to determine the F2 genotypes, the F2 data then fit a 1:3 ratio for resistant and susceptible, respectively. Thus, resistance was being controlled by a recessive gene.


Paternal Inheritance


Working with corn, Lin (34) found that there was a 50% reduction of kernel size with the hypoploid-endosperm class when B translocations had a breakpoint near du in 10L. B translocations with a breakpoint further from du have only a 5% reduction of kernel size. Lin (34) found that with the TB-10 [19] translocation,kernels of the 29:2d endosperm class were normal in size like the normal endosperm (29:1d) class. Kernels of the 49:0d class had a 50% reduction in seed size,







11


like the hypoploid endosperm (29:0d) class. The two examples with tetraploid endosperms had different phenotypes. Thus, Lin (34) concluded that a paternal form of the chromosome region investigated is needed for normal endosperm development.

Crouse (18) reported that cells of a developing Sciara embryo

can differentiate between maternally and paternally derived homologous chromosomes and between the sex chromosome (X) and the autosomes. Evidence of this ability is found in the unusual cytogenetic behavior found in several species of Sciara. During the first spermatocyte division there is selective elimination of the paternal homologues. During the second division the maternally derived X chromosome does not divide; thus, the sperm nucleus contains two identical X's and three autosomes. Gamete formation by the female is through normal meiosis. The zygote then contains an extra X chromosome; however, during embryo development, one of the paternally derived chromosomes is eliminated from the somatic nucleus of the females and from the germ cells of both sexes. Both paternally derived X chromosomes are eliminated from the somatic nuclei of males. Crouse reported that "the dramatic chromosome unorthodoxies in Sciara are clearly unrelated to the genic make-up of the chromosomes: a chromosome which passes through the male germ line acquires an 'imprint' which will result in behavior exactly opposite to the 'imprint' conferred on the same chromosome by the female germ line." (18,P. 1442) In other words, the imprint a chromosome bears is unrelated to the genic constitution of the chromosome and is determined only by the sex of the germ line through which the chromosome has been inherited.






12



Simon and Peloquin (57) investigated the inheritance of the

origin of callus growth (anther or filament) during anther culture of Solanum hybrids. Stamens from five to twenty plants of each species or hybrid were cultured. Callus growth for each stamen was categorized as originating from the filament (F) or anther (A). A characteristic of each species and hybrid was that callus formation originated predominately from the F or the A. When a hybrid was made by crossing an A species (female) with an F species (male) the hybrid was F. When the reciprocal cross was made the hybrid was A. Simon and Peloquin (57) believed this type of inheritance could be caused by exclusive male transmission of a cytoplasmic factor. This was supported by the research of Nilsson-Tillgren and von WettsteinKnowles (40) who demonstrated that the male plastome was still present in uninucleate pollen. Also, Kutzelnigg and Stubbe (33) have shown that for some plastome mutants in Oenothera a cytoplasmic factor was transmitted only through the pollen. Further evidence to support this possibility was obtained when Tilney-Bassett (62) carefully analyzed normal and mutant plastids of Pelargonium zonale L. and their effects on fertilization and stages of embryo survival. They concluded that plastid transmission can be predominately paternal in this species. The second explanation for this type of inheritance was that paternal genes, possibly those near the locus controlling andric expression, were imprinted.

Simon and Peloquin (57) proposed that by making paired backcrosses one could determine if this unusual mode of inheritance was caused by male transmission of a cytoplasmic factor (paternal






13


inheritance) or by imprinting of paternal genes. From the crosses A x (A x F) and F x (F x A) paternal inheritance would produce all F or all A plants, respectively, while imprinting would produce equal numbers of A and F plants in both crosses. The crosses (A x F) x A and (F x A) x F would produce all A plants or all F plants, respectively, for both paternal inheritance or imprinting.

Mouli and Patil (37) investigated the inheritance of foliaceous stipule in the peanut. F plants had normal stipules when a normal line was the male parent, but when a normal line was used as the female parent, the F had foliaceous stipules. The segregation in the F2 was similar in reciprocal crosses. Three of the crosses segregated 12:4 and one segregated 1:1, normal:foliaceous, respectively, in the F2. To confirm the reciprocal differences found in the F1 generation the following crosses were made with the resulting FIBC 1 phenotypes:

Foliaceous F1 x normal 28 normal plants

Normal x foliaceous FI d 14 normal:8 foliaceous

Normal FI x normal 20 normal plants

Normal x normal F d 14 normal:6 foliaceous


The results in both the FI and F1BC1 generations provided strong evidence that the expression of the foliaceous stipule phenotype was dependent on the male gamete. A dihybrid model was proposed on the assumption that the foliaceous stipule was expressed only when the pollen contained both recessive genes. The normal parent that produced a 1:1 segregation in the F2 was homozygous recessive at one of







14



the loci, but the other three normal parents that produced 12 normal:

4 foliaceous segregation in the F2 were homozygous dominant at both loci. Results from the F3 generation also confirmed this proposed mode of inheritance.


Cytoplasmic Inheritance in Peanut


There have been a few studies that have indicated that different peanut cytoplasms may influence the inheritance of some characters.

Ashri (3) made reciprocal crosses between 'Virginia Beit Dagan No. 4' (V4) and six other peanut varieties which all have a bunch growth habit. In all crosses the Fl's were bunch when V4 was the female parent. When V4 was the male parent, the Fl's had a runner growth habit. The reciprocal crosses were evaluated for growth habit in the F2, F3, FIBC1, and F2BC1 generations and it was concluded that there were two plasmon types. One plasmon type (V4) was only found in V4 and the other plasmon ("others") was present in the other six varieties. To explain the inheritance of growth habit it was proposed that the two genes Hb1 and Hb2 interact differently with each plasmon. Ashri (4) further reported that when in the V4 plasmon the Hbl -Hb2genotype was runner-type while all other genotypes had bunch growth habit. In the "others" plasmon at least three dominant alleles were required to produce a runner, and plants with two or less dominant alleles produced plants with a bunch growth habit.

Resslar and Emery (51), using two of Ashri's (3, 4) cultivars, proposed that the reciprocal differences observed in the F and F2 generations were caused by dissipating maternal effects and not cytoplasmic inheritance.






15



In another study, Ashri (8) found that the HGl cultivar had a third plasmon (G) and a third locus (Hb5) which affect growth habit.

Coffelt and Hammons (17) determined the inheritance of pod

constriction in peanuts. No differences were detected in Fl's when reciprocal crosses were made between 'Argentine' (unconstricted) and 'Early Runner' (constricted). However, reciprocal differences were found in the F2. They proposed that three unlinked nuclear loci and one cytoplasmic factor controlled pod constriction in this cross. When any two of the four factors were homozygous recessive, the plant produced unconstricted pods.

Patil and Mouli (47) crossed a dwarf peanut which originated

as a spontaneous mutant from the peanut cultivar 'Kupergaon-3' with six other cultivars. Reciprocal differences were observed in the Fl's for plant height and secondary branching and they were assumed to be caused by the interaction of nuclear and cytoplasmic factors.

Parker et al. (45) used six peanut cultivars in a diallel cross. The F1 plants were evaluated for 17 seedling characters. Maternal effects were significant for leaf width at 18 days. Maternal and reciprocal effects were significant for number of leaves on cotyledonary branches at 15 days.

Isleib et al. (31) assessed the quantitative genetic aspects for N-fixing ability with a diverse group of peanut cultivars. The following characters were measured: nitrogenase activity, number of nodules, shoot dry weight, N content of the shoot, and dry weight per nodule. Reciprocal effects were observed for nodule number, nitrogenase activity, and total N. Interaction between nuclear and extranuclear






16



factors are generally believed to cause these effects. Maternal effects were significant for all the traits except nitrogenase activity. Maternal effects are generally thought tobe caused by heritable extranuclear factors, such as DNA in mitochondria and chloroplasts.


Inheritance of Testa Color in Peanut


There have been many reports on the genetics of testa color in peanut. A thorough review of this topic has been presented by Hammons (26).

Stokes and Hull (60) found red testa dominant to tan and controlled at one locus. Hayes(29) crossed 'Valencia' with 'Sine.' They had dark red and pale brown testa, respectively. He found that testa color was controlled at one locus with red being dominant.

Prasad and Srivastava (49) found that purple was dominant to rose and was controlled at two loci by duplicate genes. They also found that rose was dominant to light rose and was controlled by duplicate genes at two loci. In a cross between purple and light rose, the F1 was purple and the F2 data fit a 255 purple:l light rose ratio. They concluded there was a tetragenic difference between purple and light rose with purple being dominant.

Ashri (5, 7) provided evidence that two loci controlled red

testa color. At the R1 locus, the dominant allele R1 gives red color, but at the R2 locus the recessive r2 allele gives the red color.

Harvey (28) showed that red was dominant to pink and was controlled at one locus in the germplasm he was using. He also found






17



that purple was incompletely and monogenically dominant to pink and that the dominant gene for red testa affected the degree of purple pigmentation. Stokes and Hull (60) reported that the variegated testa of A. nambyguarae was incompletely dominant to the solid color of A. hypogaea testa.

Branch and Hammons (11, 12) found that inheritance of red on

white testa variegation in peanut fit a genetic model for incomplete dominance at one locus. The genotypes designated VV, Vv, and vv produced the phenotypes variegated, trace amount of variegation, and no variegation, respectively.

The inheritance of inner testa color in peanuts was reported to be controlled by at least four loci by Rodriguez and Norden (52). They found that a dominant allele (S) caused the inner testa color to be a neutral white.












CHAPTER 3
INHERITANCE OF NON-NODULATION IN PEANUT


Introduction


The peanut (Arachis hypogaea L.) is a legume which, when infected by effective Rhizobium strains, will form nodules on the root which are capable of N2 fixation. This characteristic is common to all legumes except those belonging to the subfamilies Caesalpinioideae and Mimosoidaea.

Reports in five species that normally nodulate indicate the

presence of a gene or genes which cause a plant to be non-nodulated. A single recessive gene caused non-nodulated plants in soybeans (Glycine max L. Merr.) (69) and peas (Pisum spp.) (30). Nutman (42) reported non-nodulation in red clover (Trifolium pratense L.) to be controlled by a recessive gene (r) and affected by a maternally transmitted component. He also proposed that dilution of the cytoplasmic factor and zygotic and post-zygotic lethals influenced the inheritance of nodulation. Non-nodulation in alfalfa (Medicago sativa L.) was reported to be caused by two tetrasomically inherited recessive genes (48).

Gorbet and Burton (24) described a non-nodulating peanut which

was originally identified in the F3 generation from the hybridization of UF 487A, a University of Florida breeding line, with PI 262090. Nigam et al. (39) also identified non-nodulating peanut plants from the cross of PI 259747 with 'NC 17' and 'NC Ac 2731.' They reported



18







19


that two independent duplicate genes control nodulation and that nonnodulated plants are homozygous recessive at both loci.

The objective of this study was to investigate the inheritance of nodulation in peanut using a non-nodulating peanut line, M4-2, selected from the non-nodulating germplasm described by Gorbet and Burton (24).


Materials and Methods


Four peanut genotypes (Arachis hypogaea subsp. hypogaea var hypogaea) were used as parents in this study and are described in Table 3-1. A diallel cross was made with the four parents and selected F1 plants were backcrossed to M4-2 or PI 262090. All crosses were made in the greenhouse using the method described by Norden and Rodriguez (41). All subsequent generations were field grown at the University of Florida Agricultural Research Center at Marianna, Florida, during the four growing seasons 1979-82 (Table 3-2). Recommended agronomic practices were utilized including inoculation of seed at planting with cowpea-type Rhizobium sp. manufactured by Nitragin.1

Leaf color ratings of individual plants were taken in the field by pulling a representative leaf from each plant and matching it to a color on "The Munsell Limit Color Cascade." These ratings were taken just prior to digging on individual F1 and FlBC1 plants in 1981 and 1982 and on F2 plants in 1980 and 1981. Individual plants



The listing of specific trade names does not constitute endorsement of these products by the Florida Agricultural Experiment Station in preference to others containing the same components.








20




Table 3-1. A description of the peanut lines used as parents in
crosses made to investigate the inheritance of nonnodulation.

Nodule
Parent classification Description or source
classification

M4-2 Non-nodulating A line selected from the cross UF 487A x PI 262090 PI 262090 Normal Plant harvested from farm near Robor6, Bolivia UF 487A Normal University of Florida breeding line

Florunner Normal Cultivar










Table 3-2. The generations of peanuts grown each year with a description of the plot
size, number of seed planted per plot, rows per plot, seed spacing, and
age when dug.


Generation Year Plots Seed planted Rows Within row Plant age per entry per plot per plot seed spacing at digging no. cm days F1 1979 1 2-8 1 57 120 F1 1981 1 2-8 1 57 127 F1 1982 1 2-8 1 57 120 F IBC1 1981 1 2-8 1 57 127 F1 BC1 1982 1 2-8 1 57 120
F2 1980 5 32 2 38 103-145t
F2 1981 5 32 2 38 106-126 F2 1982 3 60 2 19 93- 98 F2BC 1 1982 1 60 2 19 85- 98 F3 1981 1 60 2 19 101-146 F3 1982 1 60 2 19 75- 99 tlndicates the range of number of days from planting to digging.






22



were tagged so that foliage color could be compared with nodule characteristics for each plant. Plants were dug using a conventional peanut digger-inverter with the cutting blades set as deep in the soil as possible. Most roots were cut at about 20-25 cm below the soil surface. Nodulation of roots of individual plants was rated as described in Table 3-3 immediately after digging. Pods were hand-picked from individual plants that were to be progeny tested. Data were analyzed by chi-square tests for goodness-of-fit to the proposed model.


Results and Discussion


Leaf colors were yellow-green ordark-green and only a few plants had a color between these two extremes. Using the Munsell notation, the typical yellow-green plant was 5.0 GY 4.5/8.2, and the typical dark-green plant was 8.2 GY 3.2/6.1. Yellow-green plants had nodule ratings of 0, 1, or 2 and dark-green plants had nodule ratings of 3, 4, or 5. Plants that had an intermediate leaf color were rated 0, 1, or 2 for nodulation and had less plant competition near them, e.g. a plant at the end of a plot. These plants probably had darker green foliage than within plot plants with nodule ratings of 0, 1, or 2 because they could utilize a larger soil area to extract N. Some plants with nodule ratings of 3, 4, or 5 had an intermediate foliage color, which was apparently induced by stress conditions due to infection by Sclerotium rolfsiiSacc. or attack by lesser cornstalk borer (Elasmopalpus lignosellus Zeller).








23




Table 3-3. Description of nodulation ratings used to classify
individual plants in all field plots.


Nodule Nodule rating classification Description of phenotype rating classification

0 Non-nod No nodules

1 Few 1-10 largest nodules 2 Few 11-50 larget nodules

3 Normal >50 nodules but < than on a normal Florunner plant

4 Normal Similar to a normal Florunner plant

5 Normal More nodules than a normal Florunner plant

tNodules have about twice the diameter of nodules on a normal Florunner plant.







24


Although there were six different nodule ratings used in this study, there seemed to be only three distinct categories, non-nodulated (rated 0), few nodules (rated 1 or 2), and normally nodulated (rated 3, 4, or 5). Field observations strongly support this classification system. Most plants with few nodules also had larger nodules and a more yellow leaf color than a normally nodulated plant.

The genetic model proposed for the inheritance of nodulation in this study is similar to the model described by Nigam et al. (39), since it involves a pair of independent genes controlling nodulation with the non-nodulating genotype being homozygous recessive at both loci. For this reason, the gene symbols that Nigam et al. (39) proposed, N1 and N2, are used in this paper.

The genotypes proposed for the parents are given in Table 3-4. The proposed model has the non-nodulating genotype as nln 1n2n2 and all other genotypes have normal nodulation except n1nlN2n2, which has few nodules when the parental male gamete was n1n2. Evidence to support this is found in Table 3-5. Nearly all the FI plants had normal nodulation except those from PI 262090 x M4-2. Most of the F1 plants from the latter cross had few nodules; but, because this genotype does not have 100% penetrance, some of the plants were nonnodulated. Also, all the F1 plants that had M4-2 as the pollen source had a higher proportion of plants with a nodulation rating of 3 than the reciprocal crosses. This provides additional evidence that the n1n2 male gamete reduces nodulation. One plant was rated 0 from the cross of UF 487A x M4-2 and one plant rated 1 from the cross Florunner x M4-2. Since the female used in each of these crosses was






25





Table 3-4. The proposed genotypes for nodulation control of
the peanut lines used as parents in crosses that were made to investigate the inheritance of nonnodulation.



Parent Genotype M4-2 n1nln2n2 PI 262090 nlnlN2N 2 UF 487A NiNln2n2 Florunner N1N1N2N2






26






Table 3-5. Nodulation ratings of F1 plants that were field grown
in 1979, 1981, and 1982.


Cross Nodulation rating
9 0 1 2 3 4 Total no. of plants
UF 487A x M4-2 1 0 0 19 13 33 M4-2 x UF 487A 0 0 0 0 26 26 PI 262090 x M4-2 8 9 15 0 1 33 M4-2 x PI 262090 0 0 0 1 29 30 Florunner x M4-2 0 1 0 42 9 52 M4-2 x Florunner 0 0 0 4 27 31 UF 487A x PI 262090 0 0 0 0 17 17 PI 262090 x UF 487A 0 0 0 0 28 28 Florunner x PI 262090 0 0 0 0 25 25 PI 262090 x Florunner 0 0 0 5 13 18 Florunner x UF 487A 0 0 0 3 26 29 UF 487A x Florunner 0 0 0 0 20 20 to = no nodules, 1 and 2 = few nodules, 3 and 4 = normal nodulation.







27


normally nodulated, these could not have been from selfed seed. These were probably examples of plants that have genotypes which should produce a normally nodulated plant; but, because there was not 100% penetrance, plants with few or no nodules were produced. The plant rated 4 from the cross PI 262090 x M4-2 was probably from a selfed seed. Excluding these three exceptions, the F data support the proposed model.

To evaluate the data of segregating generations, it was necessary to adjust the data because of incomplete penetrance. There have been several reports in which adjustments were made to the data or the expected values when investigating the inheritance of a trait with incomplete penetrance (9, 27, 44, 48, 58, 66). Table 3-6 gives the calculated percentage of plants that were rated 0 in the F2 and produced an F3 segregating for nodulation, indicating that genetically the plants probably should have had a few nodules. Calculations on the percentage of plants in the F2 that were rated 1 or 2 whose progeny did not segregate 1:1:2 (non-nod:few:normal), thus indicating that they should have had normal nodulation, are shown in Table 3-7. In Tables 3-6 and 3-7, the data from Florunner x M4-2, UF 487A x PI 262090, and their reciprocals were combined because the genotype of their F1 plants and their expected F2 segregation ratios were the same. Also, relatively few F3 populations from F2 plants which rated 0, 1, or 2 were available from each cross.

Table 3-8 presents the method used to adjust the F2 data. All the values used to adjust the data were obtained from Tables 3-6 and 3-7. Also all adjustments are in one direction with a portion of





28






Table 3-6. Calculation of percentage of non-nodulated F2 plants
that produced an F3 population segregating for nodulation.


Cross F3 populations (F?)[0]1
9 d Segregating F plants [0]
Total for nodulation F2 plants [] no. % UF 487A x M4-2 127 0 0

PI 262090 x M4-2 170 51 30

Florunner x M4-2 &
71 22 31 UF 487A x PI 262090
tAlso includes reciprocal of cross shown. tF3 populations from an (F2) rated [0]. F plants rated [0] that gentically should have had few nodules.










Table 3-7. Calculation of percentage of F2 plants with few nodules whose progeny did not segregate
1:1:2 (non-nod:few:normal).

Cross F3 populations (F2)[11] F3 populations (F2)[2

9 Total Non-seg. 1:1:2 F2 plants [1] Total Non-seg. 1:1:2 F2 plants [2]#

no. --% no. -%
UF 487A x M4-2 3 3 100 4 4 100

PI 262090 x M4-2 49 0 0 26 0 0

Florunner x M4-2 & 13 8 14 8 43 UF 487A x PI 262090 13 1 8 14 8 43 tAlso includes reciprocal of cross.
tF3 populations from an (F2) rated [1]. F2 plants rates [1] that genetically should have been normally nodulated. IF3 populations from an (F2) rated [2]. #F 2 plants rated [2] that genetically should have been normally nodulated.







30






Table 3-8. Method used to adjust the F nodulation data to correct
for incomplete penetrance when A, B, C, D, E, and F
equal the number of plants rated 0, 1, 2, 3, 4, and 5,
respectively.


Cross Nodulation
9 d classification Adjusted frequency UF 487A x M4-2 Non-nod = A Normal = B + C + D + E + F PI 262090 x M4-2 Non-nod = A x 0.70 Few = (Ax 0.30) + B + C Normal = D + E + F Florunner x M4-2 & Non-nod = A x 0.69 PI 262090 x UF 487A
Few = (A x 0.31)+(B x 0.92)+(C x 0.57) Normal = (B x 0.08)+(C x 0.43)+ D + E + F tAlso includes reciprocal of cross shown.






31



the plants classified as non-nod being reclassified as few or few being reclassified as normal. For example, the observed F2 data (Table 3-9) for PI 262090 x M4-2 was 855 non-nod, 313 few, and 1149 normal. Table 3-6 shows that 30% of the F2 plants in this cross that were rated 0 genetically should have had few nodules, since their progeny segregated in the F3. To adjust the data, 855 was multiplied by 0.30, which is 257. The adjusted frequency was then obtained by subtracting 257 from 855 and adding 257 to 313. In this cross none of the plants classified as few needed to be reclassified as normal.

Table 3-9 presents the F2 data with the adjusted frequency analyzed by chi-square test for goodness-of-fit to the expected ratios of the proposed genetic model. The cross UF 487A x M4-2 and the reciprocal cross segregated 1:0:3. This indicates that the Nl allele is completely dominant to nl. The cross PI 262090 x M4-2 segregated 1:1:2 because half of the plants thatwereheterozygous at the N2 locus (n1nlN2n2) would have been formed as a result of the union of a n1N2 female gamete and a n1 n2 male gamete which would produce a plant with few nodules. The crosses Florunner x M4-2, UF 487A x PI 262090, and reciprocals should have produced all normal plants because both parents were homozygous dominant at the N1 or N2 locus and thus there was no segregation at that locus. The total summed and homogeneity chi-square values for all the F2 data had probabilities above the 5% level; thus, the F2 data support the proposed model.









Table 3-9. F2 data with the adjusted frequency analyzed by chi-square test for goodness-of-fit to the
proposed model.

Cross Nodule classification X2 test on adj. freq.
9 d F families Non-nod Few Normal Source df X P

no. -- -- no. of plants UF 487A x M4-2 12 ER 1 : 0 : 3 NIN1n2n2x n1n1n2n2t 0 426 : 51 : 1303 Total 12 8.99 A 426 : 0 : 1354 Summed 1 1.08 0.25-0.50 E 445 : 0 : 1335 Homog. 11 7.91 0.50-0.75

M14-2 x UF 487A 13 ER 1 : 0 : 3 n 1 nln2n2x NiN 1 1 n2n2 0 485 : 54 : 1386 Total 13 14.97 A 485 : 0 : 1440 Summed 1 0.03 0.75-0.90 E 481 : 0 : 1440 Homog. 12 14.94 0.10-0.25 PI 262090 x M4-2 18 ER 1 : 1 : 2 n1nlN2N2 x nln1n2n2 0 855 : 313 : 1149 Total 36 29.10 A 598 : 570 : 1149 Summed 2 0.88 0.50-0.75 E 579 : 579 : 1159 Homog. 34 28.22 0.50-0.75 M4-2 x PI 262090 13 ER 1 : 1 : 2 n1n1n2n2x n1n1 N2N2 0 668 : 220 : 857 Total 26 31.16 A 468 : 420 : 857 Summed 2 3.10 0.10-0.25 E 436 : 436 : 873 Homog. 24 28.06 0.25-0.50

Florunner x M4-2 15 ER 1 1 : 14 NIN1N2N2x n1n1n2n2 0 237 : 122 : 1960 Total 30 30.62 A 163 : 155 : 2001 Summed 2 3.42 0.10-0.25 E 145 : 145 : 2029 Homog. 28 27.21 0.50-0.75









Table 3-9.--continued.

Cross Nodule classification X2 test on adj. freq.
9 d F families Non-nod Few Normal Source df X P

--no.-- -- no. of plants-M4-2 x Florunner 12 ER 1 : 1 : 14 n1nln2n2x N1NIN2N2 0 198 : 83 : 1615 Total 24 11.51 A 137 : 118 : 1641 Summed 2 2.96 0.10-0.25 E 119 : 119 : 1659 Homog. 22 8.55 <0.995

UF 487A x PI 262090 8 ER 1 : 1 : 14 N1Nln2n2x n1nlN2N2 0 124 : 57 : 1037 Total 16 9.12 A 86 : 75 : 1057 Summed 2 1.25 0.50-0.75 E 76 : 76 : 1066 Homog. 14 7.88 0.75-0.90 PI 262090 x UF 487A 7 ER 1 : 1 : 14 n1nlN2N2x NINln2n2 0 83 : 51 : 892 Total 14 7.25 A 57 : 62 : 907 Summed 2 0.91 0.50-0.75 E 64 : 64 : 898 Homog. 12 6.34 0.75-0.90 Florunner x PI 252090 6 ER 0 : 0 : 1 NININ2N2x n1nlN2N2 0 1 : 3 : 910 PI 262090 x Florunner 5 ER 0 : 0 : 1 n1nlN2N2x NN11n2n2 0 0 : 1 : 743 Florunner x UF 487A 5 ER 0 : 0 : 1 NININ2N2x NIN1n2n2 0 0 : 0 : 803 UF 487A x Florunner 3 ER 0 : 0 : 1 NIN1n2n2x N1NIN2N2 0 0 : 1 : 458 tGenotypes of parents.
tER = Expected ratio, O = Observed frequency, A = Adjusted frequency, E = Expected frequency. Homog. = Homogeneity.







34



The number of F2 families derived from normally nodulated F2 plants that segregated within each of the proposed ratios are presented in Table 3-10. The expected values were determined by calculating the ratio of each expected F2 genotype that had normal nodulation and then determining the expected segregation ratio of the progeny for each genotype. For example, the F2 of M4-2 x PI 262090 would produce normally nodulated plants with the genotypes n 1nlN2N2 and n 1nlN2n2 in equal numbers. The n 1nlN2 N2 progeny would not segregate and the progeny of n1nlN2n2 would segregate 1:1:2. Thus, the expected values listed in Table 3-10 for the cross M4-2 x PI 262090 areone 0:0:1 and one 1:1:2. The number of families that were in each ratio classification was then tested for goodness-of-fit to the proposed model. All chi-square values had probabilities above the 5% level thus adding additional support to the proposed model.

The F BC1 data are presented in Table 3-11. These data were adjusted for four of the crosses in a manner similar to the method used for the F2, because of incomplete penetrance. Again, all the chi-square values had probabilities above the 5% level, thus supporting the proposed model. Table 3-12 presents the F BCI families from normally nodulated F1BC1 plants via the same form in which the F3 data were presented in Table 3-10. These data again support the proposed model with the exception of one population from the cross PI 262090 x (PI 262090 x M4-2) segregating 1:1:2. From this cross all families segregating 1:1:2 should have been progeny from a plant with few nodules. Because the female from the cross was









Table 3-10. The number of F families derived from normally nodulated F2 plants that segregate
within each of he proposed ratios with chi-square test for goodness-of-fit to the
proposed model.

Cross Ratios (non-nod:few:normal) X2 test
d 9 0:0:1 1:1:2 1:0:3 1:1:14 df X2 P no. of families
UF 487A x M4-2 ER 1 : 0 : 2 : 0 NiN1n2n2x n1n1n2n2t 0 26 : 0 : 61 : 0 1 0.47 0.25-0.50 M4-2 x UF 487A ER 1 : 0 : 2 : 0 nIn1n2n2x NIN1n2n2 0 47 : 0 : 84 : 0 1 0.37 0.50-0.75

PI 262090 x M4-2 ER 1 : 1 : 0 : 0 n1nlN2N2x nln1n2n2 0 71 : 60 : 0 : 0 1 0.92 0.25-0.50 M4-2 x PI 262090 ER 1 : 1 : 0 : 0 n1n1n2n2x n1nlN2N2 0 58 : 65 : 0 : 0 1 0.40 0.50-0.75 Florunner x M4-2 ER 7 : 1 : 2 : 4 NININ2N2x n1nln2n2 0 62 : 7 : 16 : 35 3 0.45 0.90-0.95 M4-2 x Florunner ER 7 : 1 : 2 : 4 n1n1n2n2x NININ2N2 0 33 : 3 : 7 : 14 3 1.51 0.50-0.75

UF 487A x PI 262090 ER 7 : 1 : 2 : 4 NiN1n2n2x n1nlN2N2 0 30 : 5 : 7 : 18 3 0.45 0.90-0.95

PI 262090 x UF 487A ER 7 : 1 : 2 : 4 n1nlN2N2x N1 Nn2n2 0 56 : 6 : 13 : 25 3 1.46 0.50-0.75 Florunner x PI 262090 ER 1 : 0 : 0 : 0 NIN1N2N2x nn NN N2 0 67 : 0 : 0 : 0 0









Table 3-10.--continued.


Cross Ratios (non-nod:few:normal) X2 test
9 6 0:0:1 1:1:2 1:0:3 1:1:14 df X2 P no. of families
PI 262090 x Florunner ER 1 : 0 : 0 : 0 n1nlN2N2x NININ2N2 0 27 : 0 : 0 : 0 0

Florunner x UF 487A ER 1 : 0 : 0 : 0 NIN1N2N2x NINln2n2 O 12 : 0 : 0 0 0

UF 487A x Florunner ER 1 : 0 : 0 : 0 N1Nln2n2x NIN1N2N2 0 10 : 0 : 0 : 0 0

tGenotypes of the parents.
tER = Expected ratio, O = Observed frequency.









Table 3-11. F 1BC data analyzed by chi-square test for goodness-of-fit to the proposed
model for inheritance of non-nodulation in peanut.

Entry Cross Nodulationt x2 test
9 x (9 x d) or
number (9 x d) x d Non-nod Few Normal df X2 P

-- no. of plants
1 M4-2 x (UF 487A x M4-2) & ER$ 1 : 0 : 1
M4-2 x (M4-2 x UF 487A) 0 33 : 0 : 38 1 0.35 0.50-0.75
nn 1 n2n2x (N1n1n2n2)t
2 (UF 487A x M4-2) x M4-2 & ER 1 : 0 : 1
(M4-2 x UF 487A) x M4-2 0 34 1 : 39
(N1nln2n2) x n1nln2n2 A 34 : 0 : 40 1 0.49 0.25-0.50

3 M4-2 x (PI 262090 x M4-2) & ER 1 : 0 : 1
M4-2 x (M4-2 x PI 262090) 0 12 : 0 : 16 1 0.57 0.25-0.50
n nn2n2x n 1nlN2n2

4 (PI 262090 x M4-2) x M4-2 & ER 1 : 1 : 0
(M4-2 x PI 262090) x M4-2 0 12 : 3 : 0
n1nlN2n2x n1nln2n2 A 8 : 7 : 0 1 0.22 0.50-0.75
5 M4-2 x (Florunner x M4-2) & ER 1 : 0 : 3
M4-2 x (M4-2 x Florunner) 0 13 : 0 : 40 1 0.01 0.90-0.95
n1 n1 n2n2x (N1nlN2n2)
6 (Florunner x M4-2) x M4-2 & ER 1 : 1 : 2
(M4-2 x Florunner) x M4-2 O 16 : 10 : 26
(Nln1N2n2) x n1n1n2n2 A 11 : 15 : 26 2 0.62 0.50-0.75









Table 3-11.--continued.


Entry Cross Nodulationt X2 test ntr 9 x (9 x d) or
number (9 x 6) x d Non-nod Few Normal df X2 P

no. of plants
7 PI 262090 x (M4-2 x PI 262090) & ER 0 : 1 : 1
PI 262090 x (PI 262090 x M4-2) 0 3 : 2 : 6 1 0.09 0.75-0.90
n1nlN2N2x (n1nlN2n2) A 0 : 5 : 6
8 (M4-2 x PI 262090) x PI 262090& ER 0 : 0 : 1
(PI 262090 x M4-2) x PI 262090 0 0 : 0 : 14 0
(n1nlN2n2) x n1nlN2N2

tGenotypes of the parents.
tER = Expected ratio, O = Observed frequency, A = Adjusted frequency.















00









Table 3-12. The number of F 1BC1 families derived from normally nodulated F1BC plants that 11 1
segregate within each of the proposed ratios with chi-square test for goodness-offit to the proposed model.

Entry Cross Ratios (non-nod:few:normal)t X2 test number 9 x (9 x ) or 0:0:1 1:1:2 1:0:3 1:1:14 df X P (9 x d) x d

no. of families
1 M4-2 x (UF 487A x M4-2) & ERt 0 : 0 : 1 : 0
M4-2 x (M4-2 x UF 487A) 0 0 : 0 : 9 : 0 0

2 (UF 487A x M4-2) x M4-2 & ER 0 : 0 : 1 : 0
(M4-2 x UF 487A) x M4-2 0 0 : 0 : 13 : 0 0

3 M4-2 x (PI 262090 x M4-2) ER 0 : 1 : 0 : 0
0 0 : 14 : 0 : 0 0

4 M4-2 x (Florunner x M4-2) & ER 0 : 1 : 1 : 1
M4-2 x (M4-2 x Florunner) 0 0 : 13 : 14 : 15 2 0.14 0.90-0.95

5 (Florunner x M4-2) x M4-2 & ER 0 : 0 : 1 : 1
(M4-2 x Florunner) x M4-2 0 0 : 0 : 11 : 12 1 0.04 0.75-0.90

6 PI 262090 x (PI 262090 x M4-2) ER 1 : 0 : 0 : 0 O 4: 1 : 0: 0 0
7 (PI 262090 x M4-2) x PI 262090 ER 1 : 1 : 0 : 0
0 6 : 4 : 0 : 0 1 0.40 0.50-0.75 tER = Expected ratio, O = Observed frequency.






40



PI 262090 and not an F1 plant, this unexpected segregation ratio could not have been the result of a selfed seed in the F BCl generation. The plant from which this family originated had a nodule rating of 3, but this was probably an error and the plant should have been rated 2.

The F BC1 families derived from few (1-2) and non-nodulated (0) FIBC1 plants are presented in Table 3-13. These data are in agreement with the proposed model. The seven families from non-nodulated F1 BC1 plants that segregated 1:1:2 should have had few nodules but because of incomplete penetrance of this character, the FlBC1 plants were non-nodulated. There were also three families that segregated 1:0:3 from F1BC1 plants that were non-nodulated. This was not expected; however, each of these three plants must have been created by an n1n2 pollen grain fertilizing an N1n2 egg. This is another example of the n 1n2 male gamete reducing nodulation. The same explanation would also apply to the one family which segregated 1:0:3 from an F BC1 plant with few nodules.

The genetic model that has been proposed in this study to describe the inheritance of nodulation in peanut is similar to the one described by Nigam et al. (39). The similarities are that both models assume that nodulation is controlled by two independent genes and that the genotype of a non-nodulating plant is n1n1n2n2. The difference between the two models is that the model described in this study contains a third phenotype classified as plants with few nodules. The model states that the genotype n1nlN2n2 produces plants with few nodules when the male gamete is n1n2. If the male gamete is









Table 3-13. The number of F BC families derived from few and non-nodulated F1BC1 plants that
segregated within each of the proposed ratios.

Cross Populations from a Populations from a FlBCl non-nodulated FIBCI with few nodules
9 x (9 x d) or11
9 x d) x d Ratios (non-nod:few:normal) Ratios (non-nod:few:normal) Entry ( x ) x d 0:0:1 1:1:2 1:0:3 0:0:1 1:1:2 1:0:3 no. of families
1 M4-2 x (UF 487A x M4-2) & ER 1 : 0 : 0 0 : 0 : 0
M4-2 x (M4-2 x UF 487A) 0 11 : 0 : 0 0 : 0 : 0

2 (UF 487A x M4-2) x M4-2 & ER 1 : 0 : 0 0 : 0 : 0
(M4-2 x UF 487A) x M4-2 O 17 : 0 : 2 0 : 0 : 0

3 M4-2 x (PI 262090 x M4-2) ER 1 : 0 : 0 0 : 0 : 0 0 11 : 0 : 0 0 : 0 : 0

4 (PI 262090 x M4-2) x M4-2 ER 1 : 0 : 0 0 : 1 : 0 0 8 : 4 : 0 0 : 3 : 0

5 M4-2 x (Florunner x M4-2) ER 1 : 0 : 0 0 : 0 : 0 M4-2 x (M4-2 x Florunner) 0 13 : 0 : 0 0 : 0 : 0

6 (Florunner x M4-2) x M4-2 ER 1 : 0 : 0 0 : 1 : 0 (M4-2 x Florunner) x M4-2 0 13 : 2 : 1 0 : 9 : 1

7 PI 262090 x (PI 262090 x ER 0 : 0 : 0 0 : 1 : 0 M4-2) 0 0 : 1 : 0 0 : 2 : 0 tER = Expected ratio, O = Observed frequency.








42




nlN2 then the plant will be normally nodulated. Evidence to support this model was provided in every generation but the strongest evidence is provided from the F1 and F BC1 data. The differences found in F1 plants of PI 262090 x M4-2 and M4-2 x PI 262090 support the model. These reciprocal differences could have been caused by the interaction of nuclear and cytoplasmic factors or by cytoplasmic factors alone. When cytoplasmic factors are involved, reciprocal differences would be expected also in the F2 and F3 generations; however, no reciprocal differences were observed in the F2 or F3 in this study (Tables 3-9 and 3-10). The F BC1 data (Table 3-11) also provide strong evidence to support the model. The only crosses that had plants with few nodules (with one exception that was explained earlier) were those that had the potential to produce n lN2 female gametes and n 1n2 male gametes. An example of this is found in the comparison of the F IBC1 results of Entries 3 and 4 in Table 3-11. The genotypes of the parents used in Entry 3 were n1nln2n2 x (nlnlN2n2 so when the male gamete was nlN2 the F IBC1 plant produced had normal nodules and a genotype of n1nlN2n2. The parental genotypes used in Entry 4 were (n1nlN2n2) x n1n1n2n2; thus when the female gamete was nlN2, the FIBC1 had few nodules and a genotype of (n1nlN2n2). The genotype of the plants with normal nodulation from Entry 3 and few nodules from Entry 4 should be the same and data in Table 3-12, Entry 3,and Table 3-13, Entry 4,support this because both segregated 1:1:2.

While the proposed model seems to be strongly supported by data from the Fl F2 F3, FBC, and F2 BC1 generations, the model assumes






43



that the phenotype of two plants will be different even though the genotypes are the same, and that cytoplasm has no effect. Mouli and Patil (37) reported a similar mode of inheritance for foliaceous stipule in peanut. They reported that normal x foliaceous produced foliaceous F1 plants but that foliaceous x normal produced normal F1 plants. This is similar to the reciprocal differences found in F1 plants from PI 262090 x M4-2 and M4-2 x PI 262090. They also reported that all normal plants were produced in the F1BC1 generation from the crosses (normal x foliaceous) x normal and (foliaceous x normal) x normal. This is similar to what was found in the F1BC1 plants from the crosses (PI 262090 x M4-2) x PI 262090 and (M4-2 x PI 262090) x PI 262090 in which all progeny had normal nodulation. Mouli and Patil (37) also reported that the F1BC1 plants segregated for foliaceous and normal stipules from the crosses normal x (normal x foliaceous) and normal x (foliaceous x normal). This is similar to what was found in the F BC1 results obtained from the crosses PI 262090 x (PI 262090 x M4-2) and PI 262090 x (M4-2 x PI 262090), in which the F1 BC1 generation segregated for plants with few nodules and plants with normal nodualtion. As in this study, Mouli and Patil (37) found no reciprocal differences in the F2 or F3. Since these similar findings have both been detected for different characters in the same species, it provides evidence that peanuts may have a mechanism of inheritance for some traits that is quite different from other species. Mouli and Patil concluded that the "modification of the segregation ratio was presumably due to the fact that both the recessive genes had to be present in the pollen carrying the functional factors." (37, P.29) The data in this study also support this type of inheritance.







44



One possible interpretation for this mode of inheritance was

discussed by Crouse (18) working with Sciara and Simon and Peloquin

(57) working with Solanum hybrids. They described the inheritance of traits that were controlled by chromosome imprinting. The imprint a chromosome bears is unrelated to the genic constitution of the chromosome and is determined only by the sex of the germ line through which the chromosome has been inherited. The mode of inheritance found at the N2 locus in this study could then be explained as follows. The imprint a peanut chromosome receives when transmitted through pollen activates the N2 locus, and the opposite imprint,which causes deactivation of the N2 locus,occurs when the chromosome is inherited through the egg. This imprint may not alter the N2 locus but the imprint may affect an element in the N2 gene control system which could be similar to the gene control system in corn (Zea mays L.) as described by McClintock (36).

The physiological mechanism which causes peanuts to be non-nodulated or have few nodules has not been reported and was not investigated in this study. However, a mutant peanut described by Ashri (6) has characteristics that are similar to peanuts that have few nodules. He observed diminutive plants that developed a normal side branch and called these plants mixed. He reported that when diminutive plants were sprayed with gibberellic acid they started to develop normally. This indicated that mixed plants may be caused by hormone levels which exceed a critical threshold level in certain developing bud primordia. It could be speculated that non-nodulated plants may be the result of plants that are deficient for a hormone. When a plant







45



has a few nodules the hormone exceeds a critical threshold level in the few locations where nodules are produced.

In summary, a genetic model has been proposed which describes the mode of inheritance of nodulation for the peanut lines used in this study. In the proposed model the non-nodulating genotype is n1n1n2n2 and all other genotypes have normal nodulation except nlnlN2n2 which has few nodules when the parental male gamete of the plant is n1n2. Further study is needed to determine what induces the alleles at the N2 locus to cause different phenotypes as a result of the n2 allele being inherited from the maternal or paternal parent.













CHAPTER 4
LINKAGE BETWEEN LOCI THAT CONTROL NODULATION AND TESTA VARIEGATION IN PEANUT


Introduction


There have been only three reports of linkage in peanut (Arachis hypogaea L.). Patel et al. (46) reported that growth habit and branching type did not segregate independently. They estimated the rate of crossing over between the genes for spreading and branching to be 30%. Patil, as reported by Hammons (26), found that the crossover rate between genes for growth habit and pod reticulation was 40.4% and the crossover rate between genes for stem hairiness and pod reticulation was 31.5%.

Non-nodulating peanuts were first identified by Gorbet and Burton

(24) in the F3 generation derived from the cross of UF 487A, a University of Florida breeding line, with PI 262090. Shortly thereafter, Nigam et al. (39) reported non-nodulating peanuts were identified in the F2 generation derived from the cross 'NC 17' x PI 259747. They stated nodulation was controlled by two independent genes with the non-nodulating plants being homozygous recessive at both loci.

Branch and Hammons (12) reported that the gene for testa variegation (V1) was incompletely dominant to solid'color, which confirmed an earlier report on the inheritance of testa variegation (11).

In preliminary studies on the non-nodulating peanut, it was

found that non-nodulated plants often had variegated testa. In this study, crosses were made in which there would be segregation for both



46







47



nodulation and testa variegation. The objective was to determine if the gene controlling testa variegation was linked to a gene(s) controlling nodulation.


Materials and Methods


Four peanut (Arachis hypogaea subsp. hypogaea var hypogaea) genotypes were used as parents (Table 4-1). The crosses, M4-2x 'Florunner,' M4-2 x UF 487A, PI 262090 x UF 487A, and their reciprocals were made in 1978 and 1980. F1 plants from M4-2 x Florunner and M4-2 x UF 487A were backcrossed to M4-2 in 1980 and 1981. Crosses were made in a greenhouse using the method described by Norden and Rodriguez (41). Subsequent generations were field grown at the University of Florida Agricultural Research Center, Marianna, Florida, during the four growing seasons of 1979-82. Recommended agronomic practices were utilized including inoculation of seed at planting with cowpea-type Rhizobium sp.

All F1, F2, and F1BC1 plants were tagged before digging and 30 plants were tagged in selected F3 plots immediately after digging. Plants were dug using a conventional peanut digger-inverter with the cutting blades set as deep (20-25 cm) in the soil as possible. Nodulation of roots of individual plants were rated as described in Chapter

3 immediately after digging. Pod samples were hand picked from all plants that were tagged. Testa were examined in the laboratory and were classified as solid, trace amount of variegation (trace-v), or variegated, as previously described (11, 12). These data were then analyzed by chi-square tests for goodness-of-fit to the proposed







48





Table 4-1. A description of the four peanut lines used as parents in
crosses to determine if there is linkage between loci that
control nodulation and testa variegation.



Parent Nodulation Testa color Genotype Description
or source

M4-2 Non-nodulating Variegated VVn1n 1n2n2 A line selected red-light red from the cross UF 487A x PI 262090

PI 262090 Normal Variegated VVn nlN2N2 Plant harvested red-white from farm near Robor6, Bolivia

UF 487A Normal Solid vvN iN 1n2n2 University of Flopink rida breeding line

Florunner Normal Solid vvNIN 1N2N2 Cultivar pink







49



model. When chi-square tests were used to analyze F2 or F3 data, the data were first adjusted to correct for incomplete penetrance. Reasons for adjusting the data were presented in Chapter 3 and the method used to adjust the data is presented in Table 4-2.


Results and Discussion


All F1 plants produced seed with trace-v testa and all were normally nodulated with two exceptions (Table 4-3). These results are consistent with the findings of other studies on inheritance of testa variegation (11, 12) and nodulation in peanuts (39, Chapter 3). The testa from F2 plants segregated into three phenotypic categories, solid, trace-v, and variegated. In some F2 plants which produced testa with trace-v, the variegated area on the seed was difficult to detect and could not be seen on all the seed. Because some plants that produced seed with trace-v were probably classified as solid, the two categories, trace-v and solid, were combined for analysis of the F2 data. Total, pooled, and homogeneity chi-square values fit a 3:1 ratio (Table 4-4), thus indicating that testa variegation is controlled at a single locus in these crosses. Based on the allele symbols used in previous studies (11, 12) on inheritance of testa variegation, the solid, trace-v, and variegated phenotypes have the genotypes vv, Vv, and VV, respectively. The F2 data for nodulation (Table 4-5) have been adjusted as described in Table 4-2 and the total, pooled, and homogeneity chi-square values were not significantly different (Table 4-5) when tested with the genetic model described in Chapter 3. The very low probability values obtained from the pooled






50





Table 4-2. The method used to adjust the F and F data to correct for
2 3
incomplete penetrance.


Data Nodule rating: 0 1 2 3 4 5

Testa classification: S Vt S V S V S V S V S V Number of plants: AB CD E F G H I J K L

Adjusted data with 1:0:3 nodulation ratio (non-nod:few:normal)

Nodule classification: Non-nod Normal Testa classification: S V S V Number of plants: M N M N

M= A N= B
0 =C + E + G + I +K P =D + F + H + J + L

Adjusted data with 1:1:14 nodulation ratio (non-nod:few:normal)
Nodule classification: Non-nod Few Normal Testa classification: S V S V S V Number of plants: Q R T U W X

Q = Ax 0.69 R = B x 0.69
T = (C x 0.92) + (E x 0.57) + (A x 0.31) U = (D x 0.92) + (F x 0.57) + (B x 0.31)
W = G + I + K + (Cx0.08) + (E x 0.43) X = H + J + L + (DxO.08) + (F x 0.43)

tS = Solid + trace-v testa and V = Variegated testa.






51





Table 4-3. Nodulation ratings of F plants that were field grown
in 1979 and 1981.


Cross Nodulation rating
9 d 0 1 2 3 4 no. of plants
UF 487A x M4-2 1 0 0 19 39 UF 487A x PI 262090 0 0 0 0 45 Florunner x M4-2 0 1 0 46 36 tAlso includes reciprocal of cross. $0 = no nodules, 1 and 2 = few nodules, and 3 and 4 = normal nodulation.










Table 4-4. F2 variegated testa color data from three crosses with chi-square tests for an



Crosst Testa colors X2 test
9 d Fl families S V Source df X2 P

-no. no. of plants UF 487A x M4-2 10 0 963 : 361 Total 10 6.88 E 993 : 331 Pooled 1 3.65 0.05-0.10 Homog. 9 3.23 0.95-0.98

UF 487A x PI 262090 4 0 239 : 93 Total 4 2.58 E 249 : 83 Pooled 1 1.61 0.10-0.25 Homog. 3 0.97 0.75-0.90

Florunner x M4-2 8 O 737 : 278 Total 8 8.74 E 761 : 254 Pooled 1 3.09 0.05-0.10 Homog. 7 5.65 0.50-0.75

tAlso includes reciprocal of cross shown. tS = Solid and trace-v, V = Variegated. 0 = Observed frequency, E = Expected frequency. Homog. = Homogeneity.










Table 4-5. F2 nodulation data with the adjusted frequency analyzed by chi-square test for
2
goodness-of-fit to the expected ratio.


Cross Nodulation classification X2 test on adj. freq.
9 d FI families Non-nod Few Normal Source df X2 P
no. no. of plants UF 487A x M4-2 10 ER$ 1 : 0 : 3 Total 10 7.34
0 351 : 4 : 969 Pooled 1 1.61 0.10-0.25 A 351 : 0 : 973 Homog. 9 5.73 0.75-0.90 E 331 : 0 : 993

UF 487A x PI 262090 4 ER 1 : 1 : 14 Total 4 5.05
0 33 : 15 : 284 Pooled 1 0.23 0.50-0.75 A 23 : 21 : 288 Homog. 3 5.22 0.10-0.25 E 21 : 21 : 290

Florunner x M4-2 8 ER 1 : 1 : 14 Total 8 9.99
0 98 : 35 : 882 Pooled 1 1.01 0.25-0.50 A 67 : 57 : 891 Homog. 7 8.98 0.25-0.50 E 64 : 64 : 888

tAlso includes reciprocal of cross shown. tER = Expected ratio, O = Observed frequency, A = Adjusted frequency, E = Expected frequency. Homog. = Homogeneity.






54



and the high probabilities for the homogeneity chi-square test indicate that testa variegation and nodulation did not segregate independently (Table 4-6). The F2 segregation for nodulation from the UF 487A x M4-2 cross was controlled at the N locus because both parents were n2n2. The evidence for linkage of testa variegation and nodulation in this cross indicates that the V and N loci are linked. Because all plants that were non-nodulated or had few nodules were n1n1, they are grouped into one classification for purposes of analysis and presentation.

The calculated crossover percentages of each population ranged from 6.2 to 20% (Table 4-7). However, within crosses involving each of the three parental combinations UF 487A and M4-2 (Entries 1-5), UF 487A and PI 262090 (Entries 6 and 7), and Florunner and M4-2 (Entries 8-14), the ranges were reduced to 6.2 to 8.0, 10.8 to 11.3, and 9.9 to 20%, respectively. The arcsine transformation of the calculated crossover percentage values were analyzed by grouping them into the three parental combinations and the results (Table 4-8) indicate that there is a significant difference caused by the parental combinations.

Means of the crossover percentage for each parental combination were weighted according to the number of observations in each population. The weighted means were 7.1, 11.2, and 12.2% for UP 487A and M4-2, UF 487A and PI 262090, and Florunner and M4-2, respectively. These values were then used as the best estimates of the crossover percentage for each parental combination when calculating the expected values to be used in chi-square test.









Table 4-6. F2 segregation for variegated testa color and nodulation with the adjusted frequency
analyzed by chi-square test for goodness-of-fit to the expected ratio with no
linkage or 50% crossing over.

Fl Non-nod and few Normal X2 test on adj. freq.
Crosst families S vit S V Source df X2 P
--no.-- no. of plants -UF 487A 10 ER 3 : 1 : 9 : 3 Total 40 1035.36
x 0 28 : 311 : 935 : 50 Pooled 3 1014.55 0.001
M4-2 A 39 : 312 : 924 : 49 Homog. J7 20.81 0.75-0.90 E 248 : 83 : 745 : 248

UF 487A 4 ER 6 : 2 : 42 : 14 Total 12 95.95
x 0 9 : 39 : 230 : 54 Pooled 3 84.20 0.001 PI 262090 A 8 : 36 : 231 : 57 Homog. 9 11.70 0.10-0.25 E 31 : 10 : 218 : 73

Florunner 8 ER 6 : 2 : 42 : 14 Total 24 233.73
x 0 28 : 105 : 710 : 172 Pooled 3 215.68 0.001 M4-2 A 24 : 101 : 714 : 176 Homog. 21 18.05 0.50-0.75 E 95 : 32 : 666 : 222

tResults of the reciprocal cross is also included. tS = Solid and trace-v, V = Variegated.
ER = Expected ratio, O = Observed frequency, A = Adjusted frequency, E = Expected frequency. Homog. = Homogeneity.





U









Table 4-7. Calculated crossover percentages with number of observations in each population
and a listing of the tables where the data is presented.

Experiment Number of Table
number Generation Crosst Crossover observations number
%
1 F2 [UF 487A x M4-2] 6.6 1324 4-9
2 F3 [UF 487A x M4-2] 7.5 1636 4-10 3 FIBC1 {M4-2 x [(UF 487A x M4-2)]} 8.0 137 4-11 4 F2 families [UF 487A x M4-2] 6.2 346 4-12 5 F 1BC1 families {M4-2 x [(UF 487A x M4-2)]} 7.7 52 4-13

6 F2 [UF 487A x PI 262090] 11.3 332 4-9 7 F2 families [UF 487A x PI 262090] 10.8 51 4-12 8 F2 [Florunner x M4-2] 12.4 1015 4-9
9 F3t [Florunner x M4-2] 12.3 372 4-10 10 F3 [Florunner x M4-2] 11.5 476 4-10 11 F1 BC1 M4-2 x [(Florunner x M4-2)] 14.3 49 4-11 12 FIBC1 [(Florunner x M4-2)] x M4-2 20.0 50 4-11 13 F2 families [Florunner x M4-2] 9.9 132 4-12 14 F IBC1 families (M4-2 x [Florunner x M4-2]} 12.1 99 4-13 tCross enclosed in []-G indicates that the results of the reciprocal cross is also included. Those populations which segregated 1:0:3 for none, few, and normally nodulated plants, respectively. u Those populations which segregated 1:1:14 for none, few, and normally nodulated plants, respectively.







57





Table 4-8. Analysis of variance of the arcsine transformation of
percentage crossing over calculated on the three parental
combinations.



Source df MS Total 13 Parental combinations 2 47.10** Error 11 4.05

**Indicates significant difference at 0.01 level.







58


When the adjusted frequencies for the F2 generation of each cross were analyzed for goodness-of-fit to the expected frequencies with linkage, no significant differences were found (Table 4-9). The results obtained from F3 plants which were in F2 families that were segregating for nodulation and testa variegation are presented in Table 4-10. The F2 families from the Florunner x M4-2 cross were separated into two groups. The first group segregated 1:0:3, and the second group segregated 1:1:14 (non-nod:few:normal). An insufficient amount of data was obtained from the F2 families of UF 487A x PI 262090; these data are not presented. There were no significant differences found when these F2 families were analyzed by chi-square test.

The observed frequencies in the F1BC1 generation were not significantly different from the expected frequencies calculated with the indicated crossover percentage (Table 4-11). In Table 4-12, F2 plants are classified as non-crossover, crossover, or two crossover types by comparing the F2 plant's testa variegation with the segregation for nodulation in the F3. For example, if an F2 population (F3 plants) segregated 1:1:2 (non-nod:few:normal), the F2 plant that the family was derived from had the genotype n1nlN2n2. If there was no crossover when this F2 plant was produced, then it would also have the genotype VV and thus have variegated testa. If one of the gametes that formed the F2 embryo had a crossover between the N and V loci, then the F2 plant would have the genotype Vv and thus be trace-v. When the numbers of non-crossover, crossover, and two crossover F2 plants were compared with the expected numbers assuming the appropriate crossover percentage, no significant difference was detected.









Table 4-9. F2 segregation for variegated testa color and nodulation with the adjusted frequency
2
analyzed by chi-square test for goodness-of-fit to the expected ratio with the appropriate crossover percentage.

F1 Non-nod and few Normal X2 test on adj. freq.
Cross families St V S V Source df X2 P

no. no. of plants
UF 487A 10 ER 0.55 : 3.45 : 11.45 : 0.55 Total 30 31.5
x 0 28.00 : 311.00 : 935.00 : 50.00 Pooled 3 4.2 0.10-0.25 M4-2 A 39.00 : 312.00 : 924.00 : 49.00 Homog. 27 27.3 0.25-0.50 E 45.00 : 286.00 : 948.00 : 45.00 (7.1% CO)#

UF 487A 4 ER 1.69 : 6.31 : 46.31 : 9.69 Total 12 12.94
x 0 9.00 : 39.00 : 230.00 : 54.00 Pooled 3 1.65 0.50-0.75 PI 262090 A 8.00 : 36.00 : 231.00 : 57.00 Homog. 9 11.29 0.25-0.50 E 9.00 : 33.00 : 240.00 : 50.00 (11.2% CO)

Florunner 8 ER 1.83 : 6.17 : 46.17 : 9.83 Total 24 18.99
x 0 28.00 : 105.00 : 710.00 : 172.00 Pooled 3 4.20 0.10-0.25 M4-2 A 24.00 : 101.00 : 714.00 : 176.00 Homog. 21 14.79 0.75-0.90 E 29.00 : 98.00 : 732.00 : 156.00 (12.2% CO)
tResults of the reciprocal cross is also included. tS = Solid and trace-v, V = Variegated. ER = Expected ratio, 0 = Observed frequency, A = Adjusted frequency, E = Expected frequency. Homog. = Homogeneity.
#CO = Crossover.

U,









Table 4-10. F2 families (F3 plants) which segregated for variegated testa color and nodulation
analyzed by chi-square test for goodness-of-fit to the expected ratio with the
appropriate crossover percentage.

F2 Non-nod and few Normal X2 test on adj. freq.
Crosst families ST V S V Source df X2 p

no. no. of plants
UF 487A 56 ER 0.55 : 3.45 : 11.45 : 0.55 Total 168 201.92
X 0 53.00 : 341.00 : 1174.00 : 68.00 Pooled 3 5.24 0.10-0.25 M4-2 A 48.00 : 339.00 : 1179.00 : 70.00 Homog.I165 196.68 0.05-0.10 E 56.00 : 353.00 : 1171.00 : 56.00 (7.1% CO)#

Florunner 13 ER 0.92 : 3.08 : 11.08 : 0.92 Total 39 53.41
x 0 18.00 : 66.00 : 260.00 : 28.00 Pooled 3 5.38 0.10-0.25 M4-2 A 15.00 : 65.00 : 263.00 : 29.00 Homog. 36 48.03 0.05-0.10 E 21.00 : 72.00 : 258.00 : 21.00 (12.2% CO)

Florunner 16 ER 1.83 : 6.17 : 46.17 : 9.83 Total 48 48.73
x 0 14.00 : 57.00 : 335.00 : 70.00 Pooled 3 0.81 0.75-0.90 M4-2 A 13.00 : 50.00 : 336.00 : 77.00 Homog. 45 47.02 0.25-0.50 E 14.00 : 46.00 : 343.00 : 73.00 (12.2% CO)
tResults of the reciprocal cross is also included. tS = Solid and trace-v, V = Variegated.
ER = Expected ratio, O = Observed frequency, A = Adjusted frequency, E = Expected frequency. Homog. = Homogeneity.
#CO = Crossover.









Table 4-11. F BC1 segregation for variegated testa color and nodulation with the observed
frequency analyzed by chi-square test for goodness-of-fit to the expected ratio
with the appropriate crossover percentage.

Crosst 2
9 (9 x d) or Non-nod and few Normal X test
(9 x d) x d St V S V df X2 P

{M4-2 ER 0.14 : 1.86 : 1.86 : 0.14
x 0 8.00 : 62.00 : 64.00 : 3.00 3 3.05 0.25-0.50 [(UF 487A x M4-2)]} E 4.65 : 63.85 : 63.85 : 4.65 (7.1% CO)

[(Florunner x M4-2)] ER 0.24 : 1.76 : 1.76 : 0.24
x 0 6.00 : 20.00 : 20.00 : 4.00 3 3.49 0.25-0.50
M4-2 E 3.10 : 21.90 : 21.90 : 3.10 (12.2% CO)

M4-2 ER 0.12 : 0.88 : 1.88 : 1.12
x 0 1.00 : 11.00 : 21.00 : 16.00 3 0.72 0.75-0.90 [(Florunner x M4-2)] E 1.50 : 10.80 : 23.00 : 13.70 (12.3% CO)
tCross enclosed in []I-{}indicates that the results of the reciprocal cross are also included. S = Solid and trace-v, V = Variegated. ER = Expected ratio, O = Observed frequency, E = Expected frequency. CO = Crossover.





ON'














Table 4-12. A comparison of testa variegation of F2 plants with segregation for nodulation in
the F3 and chi-square tests for goodness-of-fit to the expected ratio of parental,
crossover, and two crossover types with the appropriate crossover percentage.

F testa F3 segregation ratios (non-nod:few:normal) Crossover type X2 test
Crosst p enotypet 0:0:1 1:1:14 1:0:3 1:1:2 1:0:0 (NC) (CO) (TCO) df Xz P no. of families -no. of familiesUF 487A V 0 (TCO) 0 12 (CO) 0 112 (NC) 0# 305 41 1
x T 8 (CO) 0 127 (NC) 0 12 (CO) E 299 46 2 2 0.91 0.50-0.75 M4-2 S 66 (NC) 0 9 (CO) 0 1 (TCO) (7.1% CO)

UF 487A V 7 (U)1 1 (CO) 4 (CO) 11 (NC) 8 (NC) 0 41 9 1
x T 12 (U) 12 (NC) 10 (NC) 1 (CO) 2 (CO) E 40 10 1 2 0.35 0.75-0.90 PI 262090 S 25 (U) 1 (CO) 0 (CO) 0 (TCO) 1 (TCO) (11.2% CO)
Florunner V 15 (U) 7 (CO) 4 (CO) 29 (NC) 20 (NC) 0 108 22 2
x T 29 (U) 43 (NC) 16 (NC) 5 (CO) 3 (CO) E 102 28 2 2 1.77 0.25-0.50 M4-2 S 53 (U) 1 (CO) 2 (CO) 2 (TCO) 0 (TCO) (12.2% CO)

TResults of the reciprocal crosses are also included.
tV = Variegated, T = trace-v, S Solid.
(NC) = Non-crossover, (CO) Crossover, (TCO) Two crossover.
(U) = Cannot classify.
O0 Observed, E Expected.







63



Table 4-13 is similar to Table 4-12 except that the F BC1

plants are classified as non-crossover or crossover, instead of F2 plants. There is no two crossover classification for F 1BC1 plants because a crossover can be detected only if it occurs in gametogenesis of the F1 parent. No significant differences were detected when the number of non-crossover and crossover F 1BC1 plants was compared with the expected values.

These data support the hypothesis that the V and N loci are
--1
linked. However, the different crossover percentages observed in the three parental combinations were not expected. One factor that could cause some of the experiments to have a higher crossover rate is incomplete penetrance of normal nodulation. The method used to adjust the data (Table 4-2) assumes independent segregation of the V and N1 loci, and thus the adjusted values will tend to increase the crossover rate. These adjustments were made in experiments numbered 6, 8, and 10 (Table 4-7). In each of these three experiments the data adjustment has not caused a large increase in the calculated crossover percentage when compared with the crossover percentage found in other experiments of the same parental combination.

If it is assumed the difference in crossover rate observed in the three parental combinations is real and not caused by sampling error, then there are several factors that could affect recombination frequencies in different experiments. Factors known to influence recombination frequencies in Drosophila sp. are sex, maternal age, temperature, cytoplasm, nutrients, radiation, genotype, chromosomestructure, and the position of genes relative to the centromere (61).
















Table 4-13. A comparison of testa variegation of F1BC1 plants with segregation for nodulation
in the F2BC1 and chi-square tests for goodness-of-fit to the expected ratio of
parental and crossover types with the appropriate crossover percentage.


F BC testa F2BC1 segregation ratios (non-nod:few:normal) Crossover type X2 test Croset penotypet 1:1:14 1:0:3 1:1:2 1:0:0 (NC) (CO) df X2 P no. of families
M4-2 V 0 1 (CO) 0 25 (NC) 01 48 4
x T 0 23 (NC) 0 3 (CO) E 48 4 1 0.03 0.75-0.90 (UF 487A x M4-2) (7.1% CO)

M4-2 V 2 (CO) 3 (CO) 23 (NC) 22 (NC) 0 87 12
x T 20 (NC) 22 (NC) 4 (CO) 3 (CO) E 87 12 1 0.00 0.995 (Florunner x M4-2) (12.2% CO)

tReciprocal crosses are also included.
tV = Variegated, T = Trace-v.
(CO) Crossover, (NC) = Non-crossover.
10 = Observed, E Expected.








65


Additional studies would be required to prove or disprove that one or more of these factors caused the different crossover rates observed for the different parental combinations. However, the cytoplasm does not seem to be a factor, because no differences were found in the crossover rates of reciprocal crosses. Also, it seems improbable that maternal age would have an effect on crossover rates in peanut. The other factors mentioned may have an effect. For example, gametogenesis occurring at different times during the day in FI plants from different crosses could cause a temperature effect on crossover rates. Cytological studies of the parents and F1 plants could provide evidence of differences in chromosome structure in plants. For example, if UF 487A, Florunner, M4-2, and the F of M4-2 x Florunner all had similar karyotypes, and the F1 of M4-2 x UF 487A had a similar karyotype to the others except for one chromosome, this would provide evidence, but not proof, that chromosome structure caused the observed differences in crossover rates. It would also provide evidence that the N and V loci are on the chromosome which was different in the
1
F1 of M4-2 x UF 487A.

Evidence from the F2, F3, F1BC1, and F2BC1 generations have

shown that the N and V loci are linked. The recombination frequency
1
between N and V was 7.1, 11.2, and 12.2% for the three parental combinations, UF 487A and M4-2, UF 487A and PI 262090, and Florunner and M4-2, respectively. These data also provide additional support for the genetic model proposed for nodulation in Chapter 3.














CHAPTER 5
A GENE AFFECTING TESTA VARIEGATION COLOR AND
ITS ASSOCIATION WITH THE N2 LOCUS IN PEANUT


Introduction


The inheritance of testa color in peanut (Arachis hypogaea L.) has been the subject of many studies, and an extensive review was presented by Hammons (26). At the R locus the recessive r2 allele produces red testa color and the dominant R2 allele produces pink testa (5, 7). The variegated testa of A. nambygyarae L. was reported as incompletely dominant to solid color of A. hypogaea testa (60). The inheritance of red on white testa variegation in peanut was controlled at one locus and the allele for variegation (V) was incompletely dominant to the allele for solid testa color (v) (11). Recently, Branch and Hammons (12) found that the R and V loci seg2
regated independently with incomplete dominance gene action found at both loci.

Non-nodulating peanuts have been identified in progeny from certain crosses in Florida (24) and India (39). Nigam et al. (39) reported nodulation was controlled by two independent genes with the non-nodulating plants being homozygous recessive at both loci (n1n1n2n2). In Chapter 3, this inheritance was confirmed except that the n 1nlN2n2 genotype has few nodules when the male parental gamete was n1n2. In Chapter 4 it was reported that the V and N1 loci are linked.



66








67


In this study, crosses were made in which segregation was expected for both nodulation and color of the variegated area (white or light red) of the testa. The objective was to determine the inheritance of color of the variegated area of the testa and to determine if there was any linkage with the N2 gene that controls nodulation. In addition, crosses were made to determine whether the R2 locus was linked to the N1 or N2 loci. The relationship of the V and R loci was also investigated.
-2

Materials and Methods


Three peanut (Arachis hypogaea subsp. hypogaea var hypogaea)

lines were used as parents (Table 5-1). The crosses M4-2 x PI 262090, M4-2 x 'Florunner,' and their reciprocals were made and some of the F1 plants were backcrossed to M4-2. F1 plants of M4-2 x PI 262090 were also backcrossed to PI 262090. Crosses were made using the method described by Norden and Rodriguez (41). Subsequent generations were field grown at the University of Florida Agricultural Research Center, Marianna, Florida. Recommended agronomic practices were utilized including inoculation of seed at planting with cowpea-type Rhizobium sp.

All F1, F2, and F1BC1 plants were tagged before digging and 30 plants were tagged in randomly selected F3 plots immediately after digging. A conventional peanut digger-inverter was used to dig the plants,cutting the roots 20-25 cm below the soil surface. Nodulation of roots of individual plants were rated as described in Chapter 3 immediately after digging. Pod samples were hand picked from the










Table 5-1. A description of the peanut lines used as parents in crosses made
to investigate the inheritance of non-nodulation and testa color.


Parent Nodulation Testa color Genotype Description or source


M4-2 Non-nodulating Variegated VVn1n1n2n2 A line selected from the red-light cross UF 487A x PI 262090 red (tinted)

PI 262090 Normal Variegated VVn 1nlN2N2 Plant harvested from farm red-white near Robore, Bolivia Florunner Normal Solid vvN NIN2N2 Cultivar pink

















00







69



tagged plants and testa were classified as red or pink in the laboratory. When a plant had variegated testa, the lighter colored area of the testa (the variegated area) was classified as white or tinted. When comparedwiththe"Munsell Limit Color Cascade," using Munsell notation, typical pink, red, and tinted testa were 2.4 YR 8.2/4.4,

6.8 R 2.6/9.4, and 8.4 RP 7.3/9.2, respectively.

These data were analyzed by chi-square test for goodness-of-fit to the proposed model. When chi-square tests were used to analyze F2 data where there was segregation for nodulation, the data were first adjusted to correct for incomplete penetrance. Reasons for adjusting the data were reported in Chapter 3 and the method used to adjust the data is presented in Tables 5-2 and 5-3.


Results and Discussion


All 81 F1 plants derived from the cross Florunner x M4-2 had pink testa color with a trace amount of variegation. This indicates that pink was dominant to red and that variegation was incompletely dominant to solid testa color. The inheritance of testa variegation from this experiment was presented in Chapter 4 and will not be reported here. The data presented in Table 5-4 indicate that the R2 allele that produces pink testa color is dominant to the r2 allele which produces red testa and supports results reported by Ashri (5, 7), but others (29, 60) have reported that red is dominant.

The plants classified as having solid and trace amounts of

variegation were grouped together because some plants that produced trace variegated testa were probably classified as having solid (red










Table 5-2. The method used to adjust the data in Table 5-6 to correct for
incomplete penetrance.


Data
Nodule rating: 0 1 2 3 4 5 Testa color:t P R P R P R P R P R P R No. of plants: A B C D E F G H I J K L

Adjusted data
Nodule classification: Non-nod Few Normal Testa color: P R P R P R No. of plants: M N 0 Q S T

M = Ax 0.69 N = B x 0.69
O = (C x 0.92) + (E x 0.53) + (A x 0.31) Q = (D x 0.92) + (F x 0.53) + (A x 0.31)
S = G + I + K + (C x0.08) + (E x 0.47) T = H + J + L + (Dx0.08) + (F x 0.47) tP = pink, R = red.










Table 5-3. The method used to adjust the data in Table 5-8 to correct for
incomplete penetrance.


Data
Nodule rating: 0 1 2 3 4 5 Testa variegation:-t W T W T W T W T W T W T No. of plants: A B C D E F G H I J K L


Adjusted data Nodule classification: Non-nod Few Normal Testa variegation: W T W T W T No. of plants: M N 0 Q S T


M = Ax 0.70 N = B x 0.70
0 = (Ax0.30) + C + E Q = (B x 0. 30) + E + F
S=G+I+K T= H + J +L

tW = white, T = tint.









Table 5-4. Segregation for red and pink testa color with chi-square test for goodness-offit to the expected ratio.


Testa color X2 test
Cross Generation Families Red Pink Source df X2 P

no. no. of plants
Florunner F2 8 ERt 1 : 3 Total 8 4.32
x 0 262 : 753 Summed 1 0.36 0.50-0.75 M4-2 E 254 : 761 Homog. 7 3.96 0.75-0.90

Florunner F3t 57 ER 1 : 3 Total 57 66.44
x 0 496 : 1340 Summed 1 3.97 0.03-0.05 M4-2 E 459 : 1377 Homog. 56 62.47 0.10-0.25

M4-2 F IBC 1 ER 1 : 1 Total 1 0.04 0.75-0.90
x 0 52 : 50 (Florunner x M4-2) E 51 : 51

tER = Expected ratio, O = Observed frequency, E = Expected frequency. tThe F families that segregated for testa color. Homog. = Homogeneity.







73



or pink) testa color as reported in Chapter 4. These data (Table5-5) indicate that in the F2 generation the two loci, V and R2, segregate independently and thus support the results of Branch and Hammons (12).

In the F2 generation of Florunner x M4-2 the expected segregation ratio for nodulation is 1 non-nodulated:l few nodules:14 normally nodulated. Thus, 3:1:3:1:42:14 is the expected ratio for the segregation of nodulation and testa color (Table 5-6). Because of incomplete penetrance of nodulation, the data in Table 5-6 have been adjusted as described in Table 5-2. These data indicate that the loci N1, N2, and R2 segregate independently.

All of the 66 F1 plants derived from the cross of PI 262090 x

M4-2 had variegated red-white testa color, indicating that white variegation is dominant to tinted. Segregation ratios for the F2, F3, and F1BC1 generations were not significantly different from the expected ratios, assuming the trait is controlled at one locus with the allele causing white variegation(Wv)being dominant to the allele causing tinted variegation(wv)(Table 5-7). The inheritance of white and tinted variegation color is similar to the inheritance of inner seed-coat color in peanuts reported by Rodriguez and Norden (52). They stated that white inner seed-coat color was dominant. In the F2 generation derived from the cross PI 262090 x M4-2 the expected segregation ratio is 1 non-nodulated:l few nodules:2 normally nodulated; thus,3:1:3:1:6:2 is the expected ratio for the segregation of nodulation and testa variegation color (Table 5-8). These data were adjusted as described in Table 5-3 because of incomplete penetrance and indicate that the N and Wv loci segregate independently.










Table 5-5. F2 segregation of testa color and variegation analyzed by chi-square test for
goodness-of-fit to the expected ratio with independent segregation of the two
loci R and V.


F1 Pink Red X2 test
Cross families st V S V Source df X2 P

no. -- no. of plants-Florunner 8 ER$ 9 : 3 : 3 : 1 Total 24 27.82
x 0 554 : 199 : 184 78 Pooled 3 4.47 0.10-0.25 M4-2 E 571 : 190 : 190 64 Homog. 21 23.35 0.25-0.50

tS = Solid and trace-v, V = Variegated. tER = Expected ratio, O = Observed frequency, E = Expected frequency. Homog. = Homogeneity.









Table 5-6. F2 segregation of testa color and nodulation with the adjusted frequency analyzed
by chi-square test for goodness-of-fit to the expected ratio with independent
segregation of the three loci, NI, N2, and R2.


FI Non-nod Few Normal X2 test on adj. freq. Cross families Pink Red Pink Red Pink Red Source df X P
no. no. of plants
Florunner 8 ERt 3 : 1 : 3 : 1 : 42 : 14 Total 40 22.50
X 0 73 : 24 27 : 9 : 654 : 228 Pooled 5 1.20 0.90-0.95 M4-2 A 50 : 17 42 : 15 : 662 229 Homog.t 35 21.30 0.95-0.98 E 48 : 16 48 : 16 : 666 : 222

tER = Expected ratio, O = Observed frequency, A = Adjusted frequency, E = Expected frequency. tHomog. = Homogeneity.

















U,









Table 5-7. Segregation for white and tinted variegation testa color with chi-square test
for goodness-of-fit to the expected ratio.


Variegated testa color X2 test Crosst Generation Families Tinted White Source df X P

no. no. of plants
PI 262090 F2 11 ER 1 : 3 Total 11 10.36
x 0 336 : 1011 Summed 1 0.00 0.75-0.90 M4-2 E 337 : 1010 Homog. 10 10.35 0.25-0.50

PI 262090 F 40 ER 1 : 3 Total 40 50.62
x 0 271 : 851 Summed 1 0.43 0.50-0.75 M4-2 E 280 : 842 Homog. 39 50.19 0.10-0.25

M4-2 FlBC1 1 ER 1 : 1 Total 1 0.00 0.995
x 0 22 : 22 (PI 262090 x M4-2) E 22 : 22

PI 262090 FlBC 1 ER 0 : 1
x 0 0 : 22 (PI 262090 x M4-2) E 0 : 22

tResults of the reciprocal crosses are also included. tER = Expected ratio, 0 = Observed frequency, E = Expected frequency. Homog. = Homogeneity.










Table 5-8. F2 segregation of testa variegation color and nodulation with the adjusted frequency
analyzed by chi-square test for goodness-of-fit to the expected ratio with independent segregation of the two loci N2 and Wv.


Non-nod Few Normal X2 test on adj. freq. Cross Families White Tint White Tint White Tint Source df X2 P
no. no. of plants
PI 262090 11 ERt 3 : 1 : 3 : 1 : 6 : 2 Total 55 35.28
x 0 362 : 100 : 157 : 56 : 492 180 Pooled 5 4.24 0.50-0.75 M4-2 A 253 : 70 : 266 : 86 : 492 : 180 Homog.$ 50 31.04 0.97-0.99 E 253 : 84 : 253 : 84 : 505 168

tER = Expected ratio, O = Observed frequency, A = Adjusted frequency, E = Expected frequency. tHomog. = Homogeneity.







78



In summary, the results of this study support the reports by Ashri (5, 7) that the R2 allele, which produces pink testa, is dominant to the r2 allele, which produces red testa. They also support the report of Branch and Hammons (12) that the R and V 2
loci segregate independently. It was also determined that the N, N2, and R2 loci segregate independently. It was shown that testa variegation color is controlled at one locus and that the allele causing white variegation (Wv) is dominant to the allele controlling tinted variegation (wv). Finally, it was shown that the N2 and Wv loci segregate independently.













CHAPTER 6
GENETIC RELATIONSHIP AND INHERITANCE OF NON-NODULATION
AND TESTA COLOR IN PEANUT LINES FROM FLORIDA AND ICRISAT


Introduction


The peanut (Arachis hypogaea L.) is a legume which will form

root nodules that are capable of N2-fixation when infected by effective Rhizobium strains. Non-nodulating peanuts have been identified in Florida (24) and India (39). Nigam et al. (39) reported nodulation was controlled by two independent genes, with the non-nodulating plants being homozygous recessive at both loci (n1n1n2n2). Their report was confirmed in Chapter 3 except that the n1nlN2n2 genotype produced plants with only a few nodules when the parental male gamete of the plant was n1n2.

The inheritance of testa color in peanut has been the subject of many studies, and an extensive review was presented by Hammons (26). At the R2 locus the recessive r2 allele produces red testa color, and the dominant R2 allele produces pink testa (5, 7). Stokes and Hull

(60) reported that the variegated testa of A. nambyquarae L. was incompletely dominant to solid color of A. hypogaea testa. The inheritance of red on white testa variegation in peanut was controlled at one locus, and the allele for variegation (V) was incompletely dominant to the allele for solid testa color (v) (11). Recently, Branch and Hammons (12) found that the R and V loci segregated independently
2
with incomplete dominance gene action found at both loci.




79








80


This study was conducted to evaluate the inheritance of nodulation utilizing a non-nodulating peanut line developed from the nonnodulating germplasm described by Gorbet and Burton (24), and two non-nodulating lines developed at ICRISAT. In several of the crosses there was segregation for testa color; these data were also analyzed.


Materials and Methods


Six peanut genotypes were used as parents (Table 6-1). The lines PI 445923 and PI 445924 were crossed with UF 487A, PI 262090, 'Florunner,' and M4-2. Crosses were made using the method described by Norden and Rodriguez (41). About half of the F1 plants were field grown at the United States Department of Agriculture Research Station at Isabella, Puerto Rico and the remaining F1 plants were grown in a greenhouse at the University of Florida Agricultural Research Center, Marianna, Florida. The parents and F2 were grown in the field at Marianna in 1982 using recommended agronomic practices, including inoculation of seed at planting with cowpea-type Rhizobium sp.

All F2 plants from a sample of the FI families were tagged before digging. A conventional peanut digger-inverter was used to dig the plants;. roots were cut at 20-25 cm below the soil surface. Nodulation of roots of individual plants was rated as described in Chapter 3. Pod samples were handpicked and testa were classified as pink, red, light purple, or purple and also as solid, trace amount of variegation, or variegated. When compared with the "Munsell Limit Color Cascade," using Munsell notation, typical pink, red, light purple, and purple testa were 2.4 YR 8.2/4.4, 6.8 R 2.6/9.4, 2.6 RP 1.8/5.4, and 4.9 P 2.1/11.2, respectively.









Table 6-1. A description of the peanut lines used as parents in crosses made to investigate
the inheritance of nodulation and testa color.


Nodulation
Parent Phenotype Genotype Test color Description or source M4-2 Non-nodulating n1nln2n2 Variegated A line selected from the cross red-light red UF 487A x PI 262090 PI 262090 Normal n1n1 N2N2 Variegated Plant harvested from farm near red-white Robore, Bolivia

UF 487A Normal Ni N n2n2 Pink University of Florida breeding line

Florunner Normal NININ2N2 Pink Cultivar PI 445923 Non-nodulating n1n1n2n2 Pink ICRISATt PI 445924 Non-nodulating n1n1n2n2 Purple ICRISAT tInternational Crops Research Institute for the Semi-Arid Tropics.








H-.








82



The nodulation data were analyzed by chi-square test for goodness-of-fit to the proposed models for inheritance as described by Nigam et al. (39) and reported in Chapter 3. When the model described in Chapter 3 was used, the data were first adjusted to correct for incomplete penetrance as reported in Chapter 3 and presented in Table 6-2. When there was segregation for nodulation and testa color, the data were adjusted as explained in Table 6-2 with each testa color category adjusted independently.


Results and Discussion


Nodulation


The F data (Table 6-3) for nodulation cannot be fully explained by the genetic model reported by Nigam et al. (39) or in Chapter 3. The model described by Nigam et al. (39) would have predicted Entries 1, 2, and 3 to be non-nodulated and all others to be nodulated. The model described in Chapter 3 would have predicted Entries 1, 2, and 3 to be non-nodulated; Entries 10 and 12 to have few nodules; and all other entries to have normal nodulation. The only entries with normal nodulation were those that had a male parent with normal nodulation. All entries that had a non-nodulating line as the male parent were non-nodulated or had few nodules. This indicates that when the male gamete is n1n2 it tends to reduce the amount of nodulation, as was proposed in Chapter 3.

The F2 data (Table 6-4) have been adjusted as described in

Table 6-2. These adjustments were made assuming that the level of penetrance in these populations was the same as those discussed in







83





Table 6-2. Method used to adjust the F nodulation data to correct
for incomplete penetrance when A, B, C, D, E, and F
equal the number of plants rated 0, 1, 2, 3, 4, and 5,
respectively.


Nodule
Cross classification Adjusted frequency

UF 487A Non-nod = A
x
PI 445923 Normal = B + C + D + E + F
or
PI 445924


PI 262090 Non-nod A x 0.70
x
PI 445923 Few = (A x 0.30) + B + C
or
PI 445924 Normal = D + E + F


Florunner Non-nod = A x 0.69
x
PI 445923 Few = (A x0.31) + (B x0.92) + (C x 0.57)
or
PI 445924 Normal = (B x0.08) + (C x0.43) + D + E + F









Table 6-3. Description of nodulation and testa color of F plants.
1

Cross Nodulation classification Testa
Entry 9 d Non-nod Few Normal Color Variegation

-- no. of plants
1 PI 445923 x M4-2 6 0 0 pink trace 2 M4-2 x PI 445923 6 0 0 pink trace 3 PI 445924 x M4-2 6 0 0 light purple trace 4 UF 487A x PI 445923 0 5 0 pink solid 5 UF 487A x PI 445924 0 2 0 light purple solid 6 Florunner x PI 445923 0 1 0 pink solid 7 Florunner x PI 445924 0 2 0 light purple solid 8 PI 445924 x Florunner 0 0 1 light purple solid 9 PI 445923 x PI 262090 0 0 2 pink trace 10 PI 262090 x PI 445923 7 0 0 pink trace 11 PI 445924 x PI 262090 0 0 2 light purple trace 12 PI 262090 x PI 445924 1 0 0 light purple trace









Table 6-4. F2 segregation for nodulation with the adjusted frequency analyzed by chi-square test for
goodness-of-fit to the proposed model.

Fl Nodule classification X2 test on adj. freq. Entry Cross families Non-nod Few Normal Source df XZ p

no. -- no. of plants
1 UF 487A x PI 445923 5 ER$ 1 : 0 : 3 Total 5 3.20
Nl In2n2x nlnIn2n2t 0 98 : 29 : 286 Summed 1 0.36 0.50-0.75 A 98 : 0 : 315 Homog. 4 2.84 0.50-0.75 E 103 : 0 : 310

2 UF 487A x PI 445924 2 ER 1 : 0 : 3 Total 2 2.21 N1N1n2n2x n1n1n2n2 0 27 : 8 : 51 Summed 1 1.87 0.10-0.25 A 27 : 0 : 59 Homog. 1 0.34 0.50-0.75 E 21 : 0 : 65

3 PI 262090 x PI 445923 9 ER 1 1 : 2 Total 18 13.34
nnN1 2 N2x n1n1n2n2 0 268 : 94 : 345 Summed 2 0.90 0.50-0.75 A 188 : 174 : 345 Homog. 16 12.44 0.50-0.75 E 177 : 177 : 353

4 PI 262090 x PI 445924 3 ER 1 1 : 2 Total 6 1.70 n1n N2N2x nInln2n2 0 77 : 30 : 119 Summed 2 0.64 0.50-0.75 A 54 : 53 : 119 Homog. 4 1.06 0.75-0.90 E 56 : 57 : 113

5 Florunner x PI 445923 1 ER 1 : 1 : 14 Total 2 0.78 0.50-0.75 NININ2N2x nln1n2n2 0 9 : 5 : 61 A 6 : 6 : 63
E 5 : 5 : 65









Table 6-4.--continued.


F1 Nodule classification X2 test on adj. freq. Entry Cross families Non-nod Few Normal Source df XZ P

no. no. of plants
6 Florunner x PI 445924 3 ER 1 1 : 14 Total 6 10.43
NININ2N2x n1n1n2n2 O 20 : 14 : 107 Summed 2 7.46 0.01-0.03 A 14 : 14 : 113 Homog. 4 2.97 0.50-0.75 E 9 : 9 : 123

7 M4-2 x PI 445923 6 E 1 : 0 : 0
n1n1n2n2x nlnln2n2 0 372 : 0 : 0

8 M4-2 x PI 445924 12 E 1 : 0 : 0
n1n1n2n2x n1n1n2n2 0 897 : 0 : 0

tGenotype of the parents.
tER = Expected ratio, O = Observed frequency, A = Adjusted frequency, E = Expected frequency. Homog. = Homogeneity.









00







87




Chapter 3. A more accurate adjustment could be made if these F2 plants were progeny tested to calculate the degree of penetrance involved when these parents are used. This method would be preferred, because it has been reported that the level of penetrance of a trait can change when the genetic background is changed (25, 35). When a chi-square test for goodness-of-fit was conducted on the adjusted frequency, Florunner x PI 445924 was the only population that was significantly different from the expected frequency. This difference could have been caused by sampling error, a different level of penetrance than reported in Chapter 3, or by a different mode of inheritance than the one proposed. Further studies are needed to determine which of these explanations is best.

Based on the model described by Nigam et al. (39), the expected frequencies in the F2 and chi-square test would be the same as those presented in Table 6-4 for Entries 1, 2, 7, and 8. The expected ratio for Entries 3 and 4 would be 1 non-nodulated:3 nodulated, and the expected ratio for Entries 5 and 6 would be 1 non-nodulated:15 nodulated. The summed chi-square values, each having 1 df, for Entries 5, 6, 7, and 8 are 62.8, 9.92, 4.42, and 15.15, respectively. Each of these values has a probability of < 0.05. This indicates that the model reported in Chapter 3 describes the mode of inheritance of nodulation for this population better than the model reported by Nigam et al. (39).








88


Testa


The F1 and F2 data (Table 6-3, Entries 1, 2, and 10, and Table 6-5) provide evidence that pink testa color is dominant to red and supports the reports by Ashri (5, 7).

Harvey (28) found that purple was incompletely and monogenically dominant to pink and is controlled at the P locus. The F and F
1 2
results (Table 6-3, Entries 7 and 8, and Table 6-6) also indicate that purple is incompletely dominant to pink. However, the F2 data do not fit the 1:2:1 ratio for purple:light purple:pink that would be expected. This may have been because it is often difficult to distinguish between the purple and light purple phenotype.

The F1 data (Table 6-3, Entries 3, 11, and 12) indicate that

purple testa color is also dominant to red. The F2 data (Table 6-7) are not significantly different from the 1 red:15 pink:48 purple ratio. This ratio can be explained by the segregation of three independent loci,P, R2, and R3. The P and R2 loci have been described (5, 7, 28) but the R3 has not. The R locus has the same type of gene action as the R2 locus with the R allele producing pink testa and being dominant to the r3 allele, which produces red testa. Additional studies are needed to substantiate the presence of the R locus.

The F1 and F2 data (Table 6-3, Entries 1, 2, 3, 9, 10, and 12, and Table 6-8) indicate that testa variegation is incompletely dominant to solid color as previously reported (11, 12). For some F2 plants which produced testa with trace-variegated seed, the variegated area on the seed was difficult to detect and could not be seen on all the seed. Because some plants that produced seed with trace-variegated




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INHERITANCE OF NODULATION AND ITS ASSOCIATION WITH GENES CONTROLLING TESTA COLOR IN Arachls hypogaea L BY KENTON EUGENE DASHIELL A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1983

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Dedicated to my parents, Robert and Rosemary Dashiell

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ACKNOWLEDGMENTS The author is sincerely grateful for the advice, guidance, and encouragement given to him by the chairman of the supervisory committee. Dr. D. W. Gorbet. Sincere appreciation is also extended to the cochairman of the supervisory committee. Dr. A. J. Norden, for the advice and guidance he provided when I was in Gainesville. Sincere thanks are also extended to other members of my committee, Drs. E. S. Homer, D. H. Hubbell, and D. W. Dickson, whose doors were always open when I needed advice or assistance. Special thanks are due to Wayne Branch, Charles Bryant, Mary Chambliss, Harold Hewett, Willis Lipford, and Stanley Slay for their technical assistance in the field and laboratory. I am also grateful to my parents, Robert and Rosemary Dashiell, and my sister, Karla Dashiell, for hand shelling thousands of peanuts for me during their Christmas vacations in 1980 and 1981, and for their encouragement throughout this study. Thanks arealso given to Patricia French for typing this manuscript. iii

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TABLE OF CONTENTS PAGE ACKNOWLEDGMENTS Hi LIST OF TABLES vi ABSTRACT xi CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW 2 Inoculating Peanuts with Rhizobium 2 Peanut -Rhizobium Interaction 3 Inheritance of Non-Nodulation and Ineffective Nodulation 5 Penetrance and Expressivity 8 Paternal Inheritance 10 Cytoplasmic Inheritance in Peanut 14 Inheritance of Testa Color in Peanut 16 3 INHERITANCE OF NON-NODULATION IN PEANUT 18 Introduction 18 Materials and Methods 19 Results and Discussion 22 4 LINKAGE BETWEEN LOCI THAT CONTROL NODULATION AND TESTA VARIEGATION IN PEANUT 46 Introduction 46 Materials and Methods 47 Results and Discussion 49 5 A GENE AFFECTING TESTA VARIEGATION COLOR AND ITS ASSOCIATION WITH THE N^ LOCUS IN PEANUT.. 66 Introduction 55 Materials and Methods 67 Results and Discussion 69 6 GENETIC RELATIONSHIP AND INHERITANCE OF NONNODULATION AND TESTA COLOR IN PEANUT LINES FROM FLORIDA AND ICRISAT 79 Introduction 79 Materials and Methods 80 Results and Discussion 82 iv

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PAGE 7 SUMMARY AND CONCLUSIONS 101 LITERATURE CITED 103 BIOGRAPHICAL SKETCH 108 V

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LIST OF TABLES TABLE PAGE 3-1 A description of the peanut lines used as parents in crosses made to investigate the inheritance of non-nodulation 20 3-2 The generations of peanuts grown each year with a description of the plot size, number of seed planted per plot, rows per plot, seed spacing, and age when dug 21 3-3 Description of nodulation ratings used to classify individual plants in all field plots 23 3-4 The proposed genotypes for nodulation control of the peanut lines used as parents in crosses that were made to investigate the inheritance of nonnodulation 25 3-5 Nodulation ratings of plants that were field grown in 1979, 1981, and 1982 26 3-6 Calculation of percentage of non-nodulated F^ plants that produced an F„ population segregating for nodulation 28 3-7 Calculation of percentage of F2 plants with few nodules whose progeny did not segregate 1:1:2 (non-nod: few: normal) 29 3-8 Method used to adjust the F„ nodulation data to correct for incomplete penetrance when A, B, C, D, E, and F equal the number of plants rated 0, 1, 2, 3, 4, and 5, respectively 30 3-9 F data with the adjusted frequency analyzed by chi-square test for goodness-of-f it to the proposed model 32 3-10 The number of F^ families derived from normally nodulated F2 plants that segregate within each of the proposed ratios with chi-square test for goodness-of-f it to the proposed model 35 3-11 ^i^^j^ data analyzed by chi-square test for goodness of-fit to the proposed model for inheritance of non-nodulation in peanut 37 vl

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TABLE PAGE 3-12 The number of F^BC^ families derived from normally nodulated F-j^BC^ plants that segregate within each of the proposed ratios with chi-square test for goodness-of-f it to the proposed model 39 313 The number of Fj^BC^ families derived from few and non-nodulated P^^'^*^^ that segregated within each of the proposed ratios 41 41 A description of the four peanut lines used as parents in crosses to determine if there is linkage between loci that control nodulation and testa variegation 48 4-2 The method used to adjust the and F data to correct for incomplete penetrance 50 4-3 Nodulation ratings of F plants that were field grown in 1979 and 1981.7 51 4-4 F^ variegated testa color data from three crosses with chi-square tests for an expected 3:1 ratio... 52 4-5 F2 nodulation data with the adjusted frequency analyzed by chi-square test for goodness-of-f it to the expected ratio 53 4-6 F^ segregation for variegated testa color and nodulation with the adjusted frequency analyzed by chi-square test for goodness-of-f it to the expected ratio with no linkage or 50% crossing over 55 4-7 Calculated crossover percentages with number of observations in each population and a listing of the tables where the data is presented 56 4-8 Analysis of variance of the arcsine transformation of percentage crossing over calculated on the three parental combinations 57 4-9 F^ segregation for variegated testa color and nodulation with the adjusted frequency analyzed by chi-square test for goodness-of-f it to the expected ratio with the appropriate crossover percentage 59 vii

PAGE 8

TABLE PAGE 4-10 F2 families (F plants) which segregated for variegated testa color and nodulation analyzed by chi-square test for goodness-of-fit to the expected ratio with the appropriate crossover percentage.... 60 ^-11 ^i^^i segregation for variegated testa color and nodulation with the observed frequency analyzed by chi-square test for goodness-of-fit to the expected ratio with the appropriate crossover percentage. ... 61 4-12 A comparison of testa variegation of F„ plants with segregation for nodulation in the F and chi-square tests for goodness-of-fit to the expected ratio of parental, crossover, and two crossover types with the appropriate crossover percentage 62 413 A comparison of testa variegation of F BC plants with segregation for nodulation in the F BC and chi-square tests for goodness-of-fit to the expected ratio of parental and crossover tyjies with the appropriate crossover percentage 64 51 A description of the peanut lines used as parents in crosses made to investigate the inheritance of non-nodulation and testa color 68 5-2 The method used to adjust the data in Table 5-6 to correct for incomplete penetrance 70 5-3 The method used to adjust the data in Table 5-8 to correct for incomplete penetrance 71 5-4 Segregation for red and pink testa color with chisquare test for goodness-of-fit to the expected ratio 72 5-5 F^ segregation of testa color and variegation analyzed by chi-square test for goodness-of-fit to the expected ratio with independent segregation of the two loci and V 74 5-6 F segregation of testa color and nodulation with the adjusted frequency analyzed by chi-square test for goodness-of-fit to the expected ratio with independent segregation of the three loci, N N and _1 __2._ 73 5-7 Segregation for white and tinted vareigation testa color with chi-square test for goodness-of-fit to the expected ratio 75 viii

PAGE 9

TABLE PAGE 58 segregation of testa variegation color and nodulation with the adjusted frequency analyzed by chi-square test for goodness-of-f it to the expected ratio with independent segregation of the two loci, N2 and Wv 77 61 A description of the peanut lines used as parents in crosses made to investigate the inheritance of nodulation and testa color 81 6-2 Method used to adjust the F„ nodulation data to correct for incomplete penetrance when A, B, C, D, E, and F equal the number of plants rated 0, 1, 2, 3, 4, and 5, respectively 83 6-3 Description of nodulation and testa color of F plants T.... 84 6-4 F2 segregation for nodulation with the adjusted frequency analyzed by chi-square test for goodness of-fit to the proposed model 85 6-5 F^ segregation for testa color with chi-square test for goodness-of-fit to the expected ratio of 1 red: 3 pink 89 6-6 F^ segregation for testa color with chi-square test for goodness-of-fit to the expected ratio of 1 pink: 3 purple 90 6-7 F^ segregation for testa color with chi-square test for goodness-of-fit to the expected ratio of 1 red: 15 pink: 48 purple 91 6-8 F^ segregation for variegated testa with chi-square tests for goodness-of-fit to the expected ratio of 3 solid and trace-variegated :1 variegated 92 6-9 F segregation for testa color and variegation with chi-square test for goodness-of-fit to the expected ratio with independent segregation of the Rand V loci 94 6-10 F2 segregation for testa color and variegation analyzed by chi-square test for goodness-of-fit to the expected ratio with independent segregation of the four loci P, R^, R^, and V 95 ix

PAGE 10

TABLE PAGE 6-11 F2 segregation for testa variegation and nodulation with the adjusted frequency analyzed by chi-square test for goodness-of-f it to the expected ratio with independent segregation of the V and N2 loci 96 6-12 F„ segregation for testa color and nodulation with the adjusted frequency analyzed by chi-square test for goodness-of-fit to the expected ratio with independent segregation of the N2 and R2 loci 98 6-13 F segregation for testa color and nodulation with the adjusted frequency analyzed by chi-square test for goodness-of-fit to the expected ratio with independent segregation of the N^, and loci.. 99 6-14 Fsegregation for testa color and nodulation with the adjusted frequency analyzed by chi-square test for goodness-of-fit to the expected ratio with independent segregation of the P^, R R and N loci -A. .-rf -A-. .100 X

PAGE 11

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INHERITANCE OF NODULATION AND ITS ASSOCIATION WITH GENES CONTROLLING TESTA COLOR IN Arachis hypogaea L. BY KENTON EUGENE DASHIELL April 1983 Chairman: Dr. D. W. Gorbet Cochairman: Dr. A. J, Norden Major Department: Agronomy A study was conducted on peanut ( Arachis hypogaea L.) to determine the inheritance of nodulation and its association with genes controlling testa color. A diallel cross was made using M4-2, a non-nodulating line, and three nodulating peanut lines, PI 262090, UF 487A, and 'Florunner.' Selected F^ plants were backcrossed to M4-2 or PI 262090. The F^, F^, F^, Fj^BC^, and F^BC^ generations were field grown at the University of Florida Agricultural Research Center at Marianna, Florida. Nodulation classifications were determined by observing plants and rating roots on each plant from 0 (no nodules) through 5 (abundant nodules). Pod samples were taken and testa color was evaluated. These data were analyzed by chi-square test for goodness-of-flt to the proposed model. The results indicate that inheritance of nodulation is controlled at two loci, and N2. The non-nodulating genotype (M4-2) is n^n^n2n, and all other genotypes have normal nodulation, except n^n N n_. xi

PAGE 12

which had few nodules when the parental male gamete was n^n^. The locus controlling testa variegation, V, was found to be linked to with an average crossover rate of about 10%. Testa variegation color is controlled at one locus. The allele causing white variegation, Wv, is dominant to the allele causing tinted variegation, wv. The N2 and Wv loci segregated independently. The locus, which controls red and pink testa color, segregated independently from the V locus. It was also determined that the N^, and loci segregate independently. In another study, two non-nodulating peanut lines, PI 445923 and PI 445924, were crossed with M4-2, PI 262090, UF 487A, and Florunner. Data were collected from the and F^ generations for nodulation and testa color. The results do not fully support the model for inheritance of nodulation described in the first study. The allele causing purple testa color, P^, appeared to be dominant to pink and red. There also appeared to be a duplicate locus of R2 which was designated R^. The following groups of loci were found to segregate independently (R^, V), (P, R^, R^, V), (V, N^) (R2, N2) (P, N^, N2), and (P, R2, Ry xii

PAGE 13

CHAPTER 1 INTRODUCTION 1 Crops that fix their own N2 have an inherent advantage over crops which cannot. This advantage may become even more importan as the cost of N fertilizer, which is derived from fossil fuels, increases. Peanuts ( Arachis hypogaea L.), when infected by an effective Rhizobium strain, form nodules which can fix amounts of N2 adequate to produce normal plant growth in soils that have rel tively low levels of available N. If the peanut could fix N^ mort efficiently, higher yields might be possible and more residual N could remain in the soil for the succeeding crop. One method of improving the N2-fixing ability of peanut woul< be to develop cultivars that can fix N^ more efficiently. To mak. efficient genetic gains in the N^-fixing ability, a knowledge of ] ow this characteristic is inherited is required. One heritable trai which is a primary component of N2-fixing ability is nodulation. One objective of this study was to investigate the inheritance of nodulation in peanut. The peanut lines that were used as parents in this study had different testa colors. Thus, another objective of this study wa; to determine if any of the genes controlling testa color were linl ad to the gene or genes that control nodulation. 1

PAGE 14

CHAPTER 2 LITERATURE REVIEW Inoculating Peanuts with Rhizobiuin There have been many studies to determine the effects of inoculating peanuts ( Arachis hypogaea L.) with Rhizobium Duggar (19) found that peanut yield was increased 30 to 40% when unshelled Spanish peanuts were inoculated and planted. Also, a significant positive correlation was found between the number of nodules per plant and yield of dry peanuts per plant. Schiffraann (53) reported that peanut yield, 1000 pod weight, 1000 seed weight, and crude protein content of hay were all higher in plots that were inoculated with effective Rhizobium strains. Van Der Merwe et al. (64) and Walker et al. (67) found no significant increase of seed yield due to inoculation with Rhizobium Iswaran and Sen (32) reported that a recommended rate of inoculant resulted in no yield increase but a lOx inoculation rate induced a yield response. Ratner et al. (50) found that pod yield of inoculated plots was significantly higher than of uninoculated plots. Schiffmann and Alper (55) reported that best results were obtained when inoculant was placed in the seed furrow, and that as nodule number per plant increased the average nodule dry weight decreased. They (54) also evaluated the effect of placing the inoculant at a depth of 8, 12, or 15 cm. As the placement depth increased, the yield, 1000 seed weight, nodule number per plant, and nodule dry weight per plant 2

PAGE 15

3 decreased. They also observed that when there were fewer nodules per plant, there was an increase in average weight per nodule. According to Tonn and Weaver (63), the Virginia type ciiltivars 'Florunner' and 'Florigiant' had more N in their vegetative organs, accumulated N in their pods at a faster rate, developed more nodule mass, and had a higher C2H2 reduction rate than the two Spanish cultivars, 'Starr' and 'Tammut 74.' Duggar (20) found that Spanish peanuts averaged 11 nodules per plant at harvest and runner peanuts averaged 127 nodules per plant. Nambiar et al. (38) randomly selected six lines from each of the commercially grown botanical varieties and found that plants of hypogaea subsp. hypogaea var hypogaea had more nodules on the hypocotyl than the other two botanical varieties. They stated nodule formation on the hypocotyl may be a desirable trait because it has been observed that nodules on the hypocotyl remain active longer than those on the root. Peanut-Rhizobium Interaction The symbiotic relationship of peanut and Rhizobium has been investigated by many researchers. Whiting and Hansen (68) found that peanut is a member of the cowpea cross-inoculation group. Allen and Allen (2) inoculated two peanut cultivars with 59 strains of Rhizobium that were isolated from 31 different leguminous species and found that all were infective. Gaur et al. (22) observed that Rhizobium obtained from 51 legume species representing 23 genera nodulated peanut plants. They (21) also reported that peanut

PAGE 16

4 nodulated well in desert soil where no Arachis species had grown previously. This provided evidence that the peanut is less specific in its Rhizobium requirement than most legumes. In peanut inoculation studies. Chandler (16) found that in contrast to Trifolium spp., no infection threads were formed. Root hairs were found only where there was an emerging lateral root and only root hairs with large basal cells were infected. Similar to Trifolium spp., root hairs were deformed when rhizobia were present but the rhizobia entered at the junction of root hairs and the epidermal and cortical cells. The bacteria moved intercellularly in the middle lamellae and entered the cortical, root hair, and large basal cells through the structurally altered cell wall. These invaded host cells divided repeatedly to form the nodule tissue, and when the host cells stopped dividing, bacteroids were formed. Bhuvaneswari et al. (10) also reported peanut had nodules only where lateral roots emerged. No nodules were observed at the base of laterals where well-developed root hairs were present at the time of inoculation. Nodules developed at 25 to 50% of the sites where lateral roots emerged at the time of inoculation but basal root hairs emerged after inoculation. Nodules developed at 80 to 100% of the laterals which emerged after inoculation. Susceptibility of the peanut root to infection by Rhizobium may be more related to root hair development than to lateral root emergence (10) Allen and Allen (2) observed spherical, plastid-like bodies in the bacteroidal area of infected cells. Staphorst and Strijdom (59) found the spherical structures located in mature nodules on peanut

PAGE 17

5 roots were Rhizobium bacteroids. The nodules from peanut were the only ones that contained spheroplast-like cells and 13 other species contained "typical" bacteroid and rod-shaped cells. Thi& characteristic of spheroplast-like cells in nodules seems to be a property of the genus Arachis because Staphorst and Strijdom (59) also observed the spheroplast-like cells in erecta, nambyquarae A. villosulicarpa and an unidentified Arachis spp. Van Rensberg et al. (65), using electron microscopy, reported the spheroplast-like cells were protoplasts devoid of cell wall material. Sen and Weaver (56) found that peanuts had three times more N in their plant tops per unit weight of nodules than cowpeas ( Vigna unguiculata L. Walp) when inoculated with the same strain of Rhizobium Inheritance of Non-Nodulation and Ineffective Nodulation There have been several reports on the inheritance of non-nodulation for species which normally nodulate. Nutman (42) found nonnodulation in red clover ( Trifolium pratense L.) to be controlled by a recessive gene (r) and affected by a maternally transmitted component. Two additional factors proposed as influencing the inheritance of nodulation were dilution of a cytoplasmic factor and the presence of zygotic and pos't-zygotic lethals. Williams and Lynch (69) crossed a non-nodulating soybean (Glycine max L. Merr.) with a nodulating line and then classified the roots of ^1' ^2' ^3' ^^'^ ^l^^l P^^^*^s nodulated or non-nodulated. They determined that nodulation was controlled by one dominant gene with non-nodulating plants being homozygous recessive.

PAGE 18

6 Gorbet and Burton (24) described a non-nodulating peanut line which was originally identified in the generation from the hybridization of UF 487A, a University of Florida breeding line, with PI 262090. They concluded that non-nodulation was not controlled by a single recessive gene. Nigam et al. (39) determined the genetics of a non-nodulating peanut which was identified from the crosses of PI 259747, with two Virginia cultivars, 'NC 17' and 'NC Ac 2731.' They found that two independent genes controlled nodulation with the non-nodulating plants being homozygous recessive at both loci. According to Roll (30) a mutant line of Pisum had one gene S3rm2 controlling nodulation and another gene Sjrm^ controlling N2 fixation when nodules were present. The two genes segregated independently with nodulation and N2 fixation being dominant to non-nodulation and no N2 fixation. Peterson and Barnes (48) found three alfalfa ( Medicago sativa L.) clones in which ineffective nodulation was controlled by a single tetrasomically-inherited recessive gene symbolized as in^, in2, or in^. With a fourth clone they found that ineffective nodulation was controlled by two recessive genes symbolized as in^ and in^. Ineffective nodules were produced when both loci were nulliplex. The non-nodulating trait was controlled by two recessive genes, symbolized nn^ and nn2 with non-nodulating plants being nulliplex at both loci. Data for all F2 and backcross families with two of the clones showed consistent deficiencies of about 28% ineffective plants and expected ratios were calculated assuming a 28% deficiency

PAGE 19

7 of the nulliplex genotype. A similar adjustment was made for another clone that showed consistent deficiencies of 32% ineffective plants. Vest and Caldwell (66) found that the soybean cultivar 'Hill' was ineffectively nodulated by Rhizobium japonicum (Kirchner) strain 61. This trait was controlled by a single gene and ineffective nodulation was dominant. Some plants had a few normal-appearing nodules and produced progeny that either segregated or were all ineffectively nodulated. Thus, these plants with a few normalappearing nodules were considered ineffectively nodulated. Caldwell et al. (14) reported that the soybean cultivar 'Merrill' has an ineffective nodulation response to the japonicum serogroups 3-24-44 and 122. When Merrill was inoculated with strains of the serogroup 3-24-44, many small white (tumor-like) nodules but no normal nodules were formed. When Merrill was inoculated with strains of serogroup 122, a few normal-size nodules and a very few small white nodules developed. Caldwell (13) also reported that a single dominant gene Rj^ caused ineffective nodulation of soybeans by certain strains of serogroups 3-24-44 and 122 of R^ japonicum Nutman (43) identified two red clover clones which were ineffectively nodulated by Rhizobium trifolii Dang, strain A. When he investigated the inheritance of this trait, he scored plants from 0 to 4 with 0 being completely ineffective and 4 being normally effective. When he analyzed the results of segregating generations, he considered half of the plants scored 1 as effective and half ineffective. With this adjustment, Nutman (44) concluded that the ineffective response

PAGE 20

8 to strain A was controlled by a recessive allele at one locus but that it was also modified by other recessive characters. Gib son (23) found that the 'Northern First Early' variety of Trif olium subterraneum L. formed ineffective nodules with the normal effective NA30 strain of trifolii When Northern First Early was crossed with other varieties of subterraneum the F^, plants were intermediately effective. In the F^ generation plants were scored as ineffective, intermediate, and effective for nodulation. While the F^ data did not fit a 1:2:1 ratio, Gibson (23) concluded that a single locus with major effects and modifying genes probably controlled the plants' response to strain NA30. Penetrance and Expressivity Penetrance and expressivity have each been defined (1) as the frequency with which a gene produces a recognizable effect, and the degree or amount that a genetic character affects the phenotype, respectively. The penetrance and expressivity of a genetic character can be altered by genetic background. Loesch (35) investigated five x-ray-induced morphological mutants from the peanut cultivar 'NC4.' He concluded that the variable expressivity of the mutant phenotypes observed in the F^ and F^ generations was caused by differences in the background genotype. Gottschalk (25) transferred the bif-1 gene, which caused bifurcated main stems, into the genomes of other Pisum mutants. The gene efr, which caused early flowering, reduced the penetrance of bif-1 When efr and ion were combined there was no further reduction in the

PAGE 21

9 penetrance of blf-l A gene ( sg-1 ) which caused a reduction in grain size increased the penetrance of bif-1 There have been several reports in which adjustments were made to the data or the expected values when investigating the inheritance of a trait with incomplete penetrance. These include four reports described previously in this chapter (44, 48, 66). Harris et al. (27) working with com ( Zea mays L.), investigated the inheritance of second ear shoots that silk (SES) Lines that did not have 100% SES were designated AA, while lines that did have 100% were designated aa. To calculate the expected frequencies of SES in the 'F^ and F^BC^ generations, the degree of penetrance of the parental genotypes were used. For example, from the cross AA x Aa, the segregation would be 1/2 AA:l/2 Aa. The expected frequency of SES would be 1/2 AA SES (from the parental data) + 1/2 Aa SES (from the data). All goodness-of-fit and heterogeneity chi-square values were nonsignificant when the observed and expected values were compared. They concluded that the SES trait was controlled at one locus with the aa genotype having 100% SES. Sorells et al. (58) investigated the inheritance of second ear formation in com, a trait with incomplete penetrance. They used a technique for analyzing their data that was similar to that used by Harris et al. (27) The spotted leaf trait in alfalfa was reported by Azizi and Barnes (9) to be controlled by two tetrasomic genes, SA and S^, with random chromosome inheritance. The genotypes SA_ and sasasasa sbsbsbsb prevented leaf spotting, whereas the genotype sasasasa

PAGE 22

10 SBSBproduced spotted leaves. If the simplex genotype sasasasa SBsbsbsb caused spotted leaves or normal leaves, then the S^^ progeny would segregate 3 spotted: 1 normal or 1 spotted: 3 normal, respectively. However, the progeny of a SB simplex genotype segregated 1 spotted: 2 normal. All expected ratios were adjusted so that 20% of the plants with simplex SB^ genotypes were expected to produce the spotted leaf trait. When this adjustment was made, the segregation observed from 13 different crosses supported the original genetic hypothesis. Working with barley ( Hordeum vulgare L.), Carroll et al. (15) investigated the inheritance of resistance to seed transmission of barley stripe mosaic virus (BSMV) 'Vantage,' the susceptible variety, had a relatively high rate of seed transmission of BSMV, ranging from 66.3 to 80.9%. Because of incomplete penetrance the classification of plants as being resistant was not reliable. When F2 plants were progeny tested to determine the genotypes, the F^ data then fit a 1:3 ratio for resistant and susceptible, respectively. Thus, resistance was being controlled by a recessive gene. Paternal Inheritance Working with corn, Lin (34) found that there was a 50% reduction of kernel size with the hypoploid-endosperm class when B translocations had a breakpoint near du in lOL. B translocations with a breakpoint further from du have only a 5% reduction of kernel size. Lin (34) found that with the TB-10 [19] translocation, kernels of the 29:2
PAGE 23

11 like the hypoploid endosperm (29:0^^) class. The two examples with tetraploid endosperms had different phenotypes. Thus, Lin (34) concluded that a paternal form of the chromosome region investigated is needed for normal endosperm development. Grouse (18) reported that cells of a developing Sciara embryo can differentiate between maternally and paternally derived homologous chromosomes and between the sex chromosome (X) and the autosomes. Evidence of this ability is found in the unusual cytogenetic behavior found in several species of Sciara During the first spermatocyte division there is selective elimination of the paternal homologues. During the second division the maternally derived X chromosome does not divide; thus, the sperm nucleus contains two identical X's and three autosomes. Gamete formation by the female is through normal meiosis. The zygote then contains an extra X chromosome; however, during embryo development, one of the paternally derived chromosomes is eliminated from the somatic nucleus of the females and from the germ cells of both sexes. Both paternally derived X chromosomes are eliminated from the somatic nuclei of males. Grouse reported that "the dramatic chromosome unorthodoxies in Sciara are clearly unrelated to the genie make-up of the chromosomes: a chromosome which passes through the male germ line acquires an 'imprint' which will result in behavior exactly opposite to the 'imprint' conferred on the same chromosome by the female germ line." (18, P. 1442) In other words, the imprint a chromosome bears is unrelated to the genie constitution of the chromosome and is determined only by the sex of the germ line through which the chromosome has been inherited.

PAGE 24

12 Simon and Peloquin (57) investigated the inheritance of the origin of callus growth (anther or filament) during anther culture of Solanum hybrids. Stamens from five to twenty plants of ^ach species or hybrid were cultured. Callus growth for each stamen was categorized as originating from the filament (F) or anther (A) A characteristic of each species and hybrid was that callus formation originated predominately from the F or the A. When a hybrid was made by crossing an A species (female) with an F species (male) the hybrid was F. When the reciprocal cross was made the hybrid was A. Simon and Peloquin (57) believed this type of inheritance could be caused by exclusive male transmission of a cytoplasmic factor. This was supported by the research of Nilsson-Tillgren and von WettsteinKnowles (40) who demonstrated that the male plastome was still present in uninucleate pollen. Also, Kutzelnigg and Stubbe (33) have shown that for some plastome mutants in Oenothera a cytoplasmic factor was transmitted only through the pollen. Further evidence to support this possibility was obtained when Tilney-Bassett (62) carefully analyzed normal and mutant plastids of Pelargonium zonale L. and their effects on fertilization and stages of embryo survival. They concluded that plastid transmission can be predominately paternal in this species. The second explanation for this type of inheritance was that paternal genes, possibly those near the locus controlling andric expression, were imprinted. Simon and Peloquin (57) proposed that by making paired backcrosses one could determine if this unusual mode of inheritance was caused by male transmission of a cytoplasmic factor (paternal

PAGE 25

13 inheritance) or by imprinting of paternal genes. From the crosses A X (A X F) and F x (F x A) paternal inheritance would produce all F or all A plants, respectively, while imprinting would produce equal numbers of A and F plants in both crosses. The crosses (A X F) X A and (F x A) x F would produce all A plants or all F plants, respectively, for both paternal inheritance or imprinting. Mouli and Patil (37) investigated the inheritance of foliaceous stipule in the peanut. F^ plants had normal stipules when a normal line was the male parent, but when a normal line was used as the female parent, the F^ had foliaceous stipules. The segregation in the F2 was similar in reciprocal crosses. Three of the crosses segregated 12:4 and one segregated 1:1, normal : foliaceous respectively, in the F2. To confirm the reciprocal differences found in the F^ generation the following crosses were made with the resulting F^BC^ phenotypes: Foliaceous F^ x normal*^ 28 normal plants Normal x foliaceous F^cJ 14 normal: 8 foliaceous Normal F^ x normal*^ 20 normal plants Normal x normal F^cJ 14 normal: 6 foliaceous The results in both the F^ and F-j^BC^ generations provided strong evidence that the expression of the foliaceous stipule phenotype was dependent on the male gamete. A dihybrid model was proposed on the assumption that the foliaceous stipule was expressed only when the pollen contained both recessive genes. The normal parent that produced a 1:1 segregation in the F„ was homozygous recessive at one of

PAGE 26

14 the loci, but the other three normal parents that produced 12 normal: 4 foliaceous segregation in the F2 were homozygous dominant at both loci. Results from the generation also confirmed this proposed mode of inheritance. Cytoplasmic Inheritance in Peanut There have been a few studies that have indicated that different peanut cytoplasms may influence the inheritance of some characters. Ashri (3) made reciprocal crosses between 'Virginia Beit Dagan No. 4' (V4) and six other peanut varieties which all have a bunch growth habit. In all crosses the F-j^'s were bunch when V4 was the female parent. When V4 was the male parent, the F^'s had a runner growth habit. The reciprocal crosses were evaluated for growth habit in the F^, ^"'^ ^2^*^1 S^^^^^^tions and it was concluded that there were two plasmon types. One plasmon type (V4) was only found in V4 and the other plasmon ("others") was present in the other six varieties. To explain the inheritance of growth habit it was proposed that the two genes Hb^ and Hb^ interact differently with each plasmon. Ashri (4) further reported that when in the V4 plasmon the Hb^-Hb^genotype was runner-type while all other genotypes had bunch growth habit. In the "others" plasmon at least three dominant alleles were required to produce a runner, and plants with two or less dominant alleles produced plants with a bunch growth habit. Resslar and Emery (51), using two of Ashri's (3, 4) cultivars, proposed that the reciprocal differences observed in the F^ and F^ generations were caused by dissipating maternal effects and not cytoplasmic inheritance.

PAGE 27

15 In another study, Ashri (8) found that the HGl cultivar had a third plasmon (G) and a third locus (Hb^) which affect growth habit. Coffelt and Hammons (17) determined the inheritance of pod constriction in peanuts. No differences were detected in F-|^'s when reciprocal crosses were made between 'Argentine' (unconstricted) and 'Early Runner' (constricted). However, reciprocal differences were found in the They proposed that three unlinked nuclear loci and one cytoplasmic factor controlled pod constriction in this cross. When any two of the four factors were homozygous recessive, the plant produced unconstricted pods. Patil and Mouli (47) crossed a dwarf peanut which originated as a spontaneous mutant from the peanut cultivar 'Kupergaon-3' with six other cultivars. Reciprocal differences were observed in the F^'s for plant height and secondary branching and they were assumed to be caused by the interaction of nuclear and cytoplasmic factors. Parker et al. (45) used six peanut cultivars in a diallel cross. The plants were evaluated for 17 seedling characters. Maternal effects were significant for leaf width at 18 days. Maternal and reciprocal effects were significant for number of leaves on cotyledonary branches at 15 days. Isleib et al. (31) assessed the quantitative genetic aspects for N-fixing ability with a diverse group of peanut cultivars. The following characters were measured: nitrogenase activity, number of nodules, shoot dry weight, N content of the shoot, and dry weight per nodule. Reciprocal effects were observed for nodule number, nitrogenase activity, and total N. Interaction between nuclear and extranuclear

PAGE 28

16 factors are generally believed to cause these effects. Maternal effects were significant for all the traits except nitrogenase activity. Maternal effects are generally thought to be caused by heritable extranuclear factors, such as DNA in mitochondria and chloroplasts. Inheritance of Testa Color in Peanut There have been many reports on the genetics of testa color in peanut. A thorough review of this topic has been presented by Hammons (26) Stokes and Hull (60) found red testa dominant to tan and controlled at one locus. Hayes(29) crossed 'Valencia' with 'Sine.' They had dark red and pale brown testa, respectively. He found that testa color was controlled at one locus with red being dominant. Prasad and Srivastava (49) found that purple was dominant to rose and was controlled at two loci by duplicate genes. They also found that rose was dominant to light rose and was controlled by duplicate genes at two loci. In a cross between purple and light rose, the was purple and the data fit a 255 purple :1 light rose ratio. They concluded there was a tetragenic difference between purple and light rose with purple being dominant. Ashri (5, 7) provided evidence that two loci controlled red testa color. At the locus, the dominant allele gives red color, but at the R^ locus the recessive r^ allele gives the red color. Harvey (28) showed that red was dominant to pink and was controlled at one locus in the germplasm he was using. He also found

PAGE 29

17 that purple was incompletely and monogenically dominant to pink and that the dominant gene for red testa affected the degree of purple pigmentation. Stokes and Hull (60) reported that the variegated testa of nambyquarae was incompletely dominant to the solid color of hypogaea testa. Branch and Hammons (11, 12) found that inheritance of red on white testa variegation in peanut fit a genetic model for incomplete dominance at one locus. The genotypes designated VV, Vv, and w produced the phenotypes variegated, trace amount of variegation, and no variegation, respectively. The inheritance of inner testa color in peanuts was reported to be controlled by at least four loci by Rodriguez and Norden (52) They found that a dominant allele (S^) caused the inner testa color to be a neutral white.

PAGE 30

CHAPTER 3 INHERITANCE OF NON-NODULATION IN PEANUT Introduction The peanut (Arachis hypogaea L.) is a legume which, when infected by effective Rhizobium strains, will form nodules on the root which are capable of N^ fixation. This characteristic is common to all legumes except those belonging to the subfamilies Caesalpinioideae and Mimosoidaea. Reports in five species that normally nodulate indicate the presence of a gene or genes which cause a plant to be non-nodulated. A single recessive gene caused non-nodulated plants in soybeans ( Glycine max L. Merr.) (69) and peas (Pisumspp.) (30). Nutman (42) reported non-nodulation in red clover ( Trifolium pratense L.) to be controlled by a recessive gene (r) and affected by a maternally transmitted component. He also proposed that dilution of the cytoplasmic factor and zygotic and post-zygotic lethals influenced the inheritance of nodulation. Non-nodulation in alfalfa ( Medicago sativa L.) was reported to be caused by two tetrasomically inherited recessive genes (48) Corbet and Burton (24) described a non-nodulating peanut which was originally identified in the generation from the hybridization of UF 487A, a University of Florida breeding line, with PI 262090. Nigam et al. (39) also identified non-nodulating peanut plants from the cross of PI 259747 with 'NC 17' and 'NC Ac 2731.' They reported 18

PAGE 31

19 that two independent duplicate genes control nodulation and that nonnodulated plants are homozygous recessive at both loci. The objective of this study was to investigate the inheritance of nodulation in peanut using a non-nodulating peanut line, M4-2, selected from the non-nodulating germplasm described by Gorbet and Burton (24) Materials and Methods Four peanut genot3T)es (Arachis hypogaea subsp hypogaea var hypogaea ) were used as parents in this study and are described in Table 3-1. A diallel cross was made with the four parents and selected plants were backcrossed to M4-2 or PI 262090. All crosses were made in the greenhouse using the method described by Norden and Rodriguez (41) All subsequent generations were field grown at the University of Florida Agricultural Research Center at Marianna, Florida, during the four growing seasons 1979-82 (Table 3-2). Recommended agronomic practices were utilized including inoculation of seed at planting with cowpea-type Rhizobium sp. manufactured by Nitragin.-*Leaf color ratings of individual plants were taken in the field by pulling a representative leaf from each plant and matching it to a color on "The Munsell Limit Color Cascade." These ratings were taken just prior to digging on individual F^ and F-j^BC^ plants in 1981 and 1982 and on F^ plants in 1980 and 1981. Individual plants The listing of specific trade names does not constitute endorsement of these products by the Florida Agricultural Experiment Station in preference to others containing the same components.

PAGE 32

20 Table 3-1. A description of the peanut lines used as parents in crosses made to investigate the inheritance of nonnodulation. Parent Nodule classification Description or source M4-2 Non-nod ula t ing A line selected from the cross UF 487A X PI 262090 PI 262090 Normal Plant harvested from farm near Robore, Bolivia UF 487A Normal University of Florida breeding line Florunner Normal Cultivar

PAGE 33

21 4J O 0) •H 0) CO 00 -C 00 4J e>o u •H C C T3 CO o o iH n) CO o CO •H U "O OO o> OJ a •H Q) o •H t-l CO a o CO CO • c o. (U 4-) •H CO rH •a to a •H cu cu J5 U CO U 0) •H CU CO & o CO O rH 0) M CO & >^ •>> o U CU a o a. CO iH (U o p. u 00 cu CO 4J •w c 3 CO CO 0) & TJ OJ M-l (U O CO CO M-l C O O •H M 60 •U CU 3 CO ^ 73 u e 0) 3 C C C CU 0) 00 S 0) 0) N (U X -H 00 H CO CO CM I CO 0) CO H T3 (U 4-1 4-1 C O CO iH iH O, a u •o cu QJ O0) CO TO 4-1 o 4-1 c cu u (U (X V4 CO 0) c o CO u cu c o CO CO e o o c o CSI in VO CX3 CX3 00 o rH r-, m u-i lo in 00 00 0^ CO Cn rH rH CM (N CM CM CM CM 00 I CN 00 I CN 00 I CN 00 I CM 00 I CM CM CO CN o o o O in m CO C7\ CJN rH CM 00 00 00 CJ\ CM 00 o 00 C3^ 00 CJN CM 00 CM rH CN 00 00 00 0^ 0^ o> O pq CN CM CN CM CO CO pt, Pn [ii Pl< 00 a •H 00 00 •H TJ O 00 c •H s o u HH CO >, CO T3 lH O (4 0) 0) CD u tH T3 c

PAGE 34

22 were tagged so that foliage color could be compared with nodule characteristics for each plant. Plants were dug using a conventional peanut digger-inverter with the cutting blades set as deep in the soil as possible. Most roots were cut at about 20-25 cm below the soil surface. Nodulation of roots of individual plants was rated as described in Table 3-3 immediately after digging. Pods were hand-picked from individual plants that were to be progeny tested. Data were analyzed by chi-square tests for goodness-of-fit to the proposed model. Results and Discussion Leaf colors were yellow-green or dark-green and only a few plants had a color between these two extremes. Using the Munsell notation, the typical yellow-green plant was 5.0 GY 4.5/8.2, and the typical dark-green plant was 8.2 GY 3.2/6.1. Yellow-green plants had nodule ratings of 0, 1, or 2 and dark-green plants had nodule ratings of 3, 4, or 5. Plants that had an intermediate leaf color were rated 0, 1, or 2 for nodulation and had less plant competition near them, e.g. a plant at the end of a plot. These plants probably had darker green foliage than within plot plants with nodule ratings of 0, 1, or 2 because they could utilize a larger soil area to extract N. Some plants with nodule ratings of 3, 4, or 5 had an intermediate foliage color, which was apparently induced by stress conditions due to infection by Sclerotlum rolfsii Sacc. or attack by lesser cornstalk borer ( Elasmopalpus lignosellus Zeller)

PAGE 35

23 Table 3-3. Description of nodulation ratings used to classify individual plants in all field plots. Nodule Nodule „ ^ 1 ^Description of phenotype rating classification 0 1 2 3 Non-nod Few Few Normal Normal Normal No nodules I10 larget nodules II50 larget nodules > 50 nodules but < than on a normal Florunner plant Similar to a normal Florunner plant More nodules than a normal Florunner plant fNodules have about twice the diameter of nodules on a normal Florunner plant.

PAGE 36

24 Although there were six different nodule ratings used in this study, there seemed to be only three distinct categories, non-nodulated (rated 0) few nodules (rated 1 or 2) and normally nodulated (rated 3, 4, or 5). Field observations strongly support this classification system. Most plants with few nodules also had larger nodules and a more yellow leaf color than a normally nodulated plant. The genetic model proposed for the inheritance of nodulation in this study is similar to the model described by Nigam et al. (39) since it involves a pair of independent genes controlling nodulation with the non-nodulating genotype being homozygous recessive at both loci. For this reason, the gene symbols that Nigam et al. (39) proposed, and N^, are used in this paper. The genotypes proposed for the parents are given in Table 3-4. The proposed model has the non-nodulating genotype as n^n^n2n2 and all other genotypes have normal nodulation except n^n^N2n2, which has few nodules when the parental male gamete was ^j^2' Evidence to support this is found in Table 3-5. Nearly all the F^ plants had normal nodulation except those from PI 262090 x M4-2. Most of the F^ plants from the latter cross had few nodules; but, because this genotype does not have 100% penetrance, some of the plants were nonnodulated. Also, all the F^ plants that had M4-2 as the pollen source had a higher proportion of plants with a nodulation rating of 3 than the reciprocal crosses. This provides additional evidence that the n^n2 male gamete reduces nodulation. One plant was rated 0 from the cross of UF 487A x M4-2 and one plant rated 1 from the cross Florunner x M4-2. Since the female used in each of these crosses was

PAGE 37

Table 3-4. The proposed genotypes for nodulation control of the peanut lines used as parents in crosses that were made to investigate the inheritance of nonnodulation. Parent Genotype M4-2 n^n^n2n2 PI 262090 "l"l^2^2 UF 487A N^N^n^n^ Florunner N^N^N2N2

PAGE 38

26 Table 3-5. Nodulation ratings of F plants that were field grown in 1979, 1981, and 1982. Cross Nodulation ratingf 9 ^ 0 1 2 3 4 Total no. of plants UF 487A X M4-2 1 0 0 19 13 33 M4-2 X UF 487A 0 0 0 0 26 26 PI 262090 X M4-2 8 9 15 0 1 33 M4-2 X PI 262090 0 0 0 1 29 30 Florunner x M4-2 0 1 0 42 9 52 M4-2 x Florunner 0 0 0 4 27 31 UF 487A X PI 262090 0 0 0 0 17 17 PI 262090 X UF 487A 0 0 0 0 28 28 Florunner x PI 262090 0 0 0 0 25 25 PI 262090 x Florunner 0 0 0 5 13 18 Florunner x UF 487A 0 0 0 3 26 29 UF 487A X Florunner 0 0 0 0 20 20 to = no nodules, 1 and 2 = few nodules, 3 and 4 = normal nodulation.

PAGE 39

27 normally nodulated, these could not have been from selfed seed. These were probably examples of plants that have genotypes which should produce a normally nodulated plant; but, because there was not 100% penetrance, plants with few or no nodules were produced. The plant rated 4 from the cross PI 262090 x M4-2 was probably from a selfed seed. Excluding these three exceptions, the data support the proposed model. To evaluate the data of segregating generations, it was necessary to adjust the data because of incomplete penetrance. There have been several reports in which adjustments were made to the data or the expected values when investigating the inheritance of a trait with incomplete penetrance (9, 27, 44, 48, 58, 66). Table 3-6 gives the calculated percentage of plants that were rated 0 in the and produced an F^ segregating for nodulation, indicating that genetically the plants probably should have had a few nodules. Calculations on the percentage of plants in the F^ that were rated 1 or 2 whose progeny did not segregate 1:1:2 (non-nod : few: normal) thus indicating that they should have had normal nodulation, are shown in Table 3-7. In Tables 3-6 and 3-7, the data from Florunner x M4-2, UF 487A X PI 262090, and their reciprocals were combined because the genotype of their F^ plants and their expected F^ segregation ratios were the same. Also, relatively few F^ populations from F^ plants which rated 0, 1, or 2 were available from each cross. Table 3-8 presents the method used to adjust the F2 data. All the values used to adjust the data were obtained from Tables 3-6 and 3-7. Also all adjustments are in one direction with a portion of

PAGE 40

28 Table 3-6. Calculation of percentage of non-nodulated F2 plants that produced an population segregating for nodulatlon. Cross F3 populations (F?) [0] t Segregating Total for nodulation F2 plants [0]§ UF 487A X M4-2 no. 127 0 % 0 PI 262090 X M4-2 Florunner x M4-2 & UF 487A x PI 262090 170 71 51 22 30 31 fAlso includes reciprocal of cross shown. JF^ populations from an (F2) rated [0]. §F2 plants rated [0] that gentically should have had few nodules.

PAGE 41

.o 5 CM CO c CO a CM fa 1—1 CM CM • • 1—1 iH > CO to X. X. T) T3 rH rH 3 3 O O CO CO r— ^ rH >^ CM rH rH rH rH CO T3 CO to CO (U a *H CO T3 CO tu CO CU CO 0 4-1 C 4J (U o to o to T3 •H u •H U 3 4-1 4-1 rH to CD to CO a rH 4J rH 4-1 c 3 c 3 c •H a, CO ft to o rH 0 rH O ft ft CO CO CM CO CM <3 fa fa fa fa ++eOJ te= =s=

PAGE 42

30 Table 3-8. Method used to adjust the Fnodulation data to correct for incomplete penetrance when A, B, C, D, E, and F equal the number of plants rated 0, 1, 2, 3, 4, and 5, respectively. Crossf Nodulation classification Adjusted frequency UF 487A X M4-2 PI 262090 X M4-2 Florunner x M4-2 & PI 262090 X UF 487A Non-nod = A Normal =B+C+D+E+F Non-nod = A x 0.70 Few = (A X 0.30) + B + C Normal = D + E + F Non-nod = A x 0.69 Few = (A X 0.31)+(B x 0.92)+(C x 0.57) Normal = (B x 0.08)+(C x 0.43)+ D + E + F fAlso includes reciprocal of cross shown.

PAGE 43

31 the plants classified as non-nod being reclassified as few or few being reclassified as normal. For example, the observed data (Table 3-9) for PI 262090 x M4-2 was 855 non-nod, 313 few, and 1149 normal. Table 3-6 shows that 30% of the F2 plants in this cross that were rated 0 genetically should have had few nodules, since their progeny segregated in the F^. To adjust the data, 855 was multiplied by 0.30, which is 257. The adjusted frequency was then obtained by subtracting 257 from 855 and adding 257 to 313. In this cross none of the plants classified as few needed to be reclassified as normal. Table 3-9 presents the F^ data with the adjusted frequency analyzed by chi-square test for goodness-of-f it to the expected ratios of the proposed genetic model. The cross UF 487A x M4-2 and the reciprocal cross segregated 1:0:3. This indicates that the allele is completely dominant to n^. The cross PI 262090 X M4-2 segregated 1:1:2 because half of the plants that were heterozygous at the N2 locus (n^n^N2n2) would have been formed as a result of the union of a n^^N^ female gamete and a n^n2 male gamete which would produce a plant with few nodules. The crosses Florunner x M4-2, UF 487A x PI 262090, and reciprocals should have produced all normal plants because both parents were homozygous dominant at the ^ ^2 ^^^^ there was no segregation at that locus. The total summed and homogeneity chi-square values for all the F. data v 2 ./ had probabilities above the 5% level; thus, the F2 data support the proposed model.

PAGE 44

C vn ON iH m in CN m O O c O o o d O d o CO rH o CO CNl o vO • CM ON o ON c ON l~~f O NO CM * c < O CO r-H rn OO o J .H CN CM CM t-l n 1— vO CM O CN 00 r-l iH cn CO CNj CN cn CN CO) • -H .H a rH 0) bi) (U ci (0 o n) O CO o to o (0 o J-l i e 4-1 6 4J S E a O o o o O D o O a o o 3 o H CO Eh H H P3 H CO PS cn
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33 o in o csj ON *^ * o o o o o 1 1 1 1 1 o o in o in • rH o m V • <1) o o o o o rH to 00 rH i~l 00 csi CO X • • rH CO 0^ 1—1 r" — • O cd 1 — 1 f—t o CM CM vO
PAGE 46

34 The number of families derived from normally nodulated plants that segregated within each of the proposed ratios are presented in Table 3-10. The expected values were determined by calculating the ratio of each expected F2 genotype that had normal nodulation and then determining the expected segregation ratio of the progeny for each genotype. For example, the F2 of M4-2 x PI 262090 would produce normally nodulated plants with the genotypes ^]^^2^N2^2 n^n^N2n2 in equal numbers. The n^n^N2N2 progeny would not segregate and the progeny of n^n^N2n2 would segregate 1:1:2. Thus, the expected values listed in Table 3-10 for the cross M4-2 x PI 262090 are one 0:0:1 and one 1:1:2. The number of families that were in each ratio classification was then tested for goodness-of-f it to the proposed model. All chi-square values had probabilities above the 5% level thus adding additional support to the proposed model. The F-j^BC^ data are presented in Table 3-11. These data were adjusted for four of the crosses in a manner similar to the method used for the F2, because of incomplete penetrance. Again, all the chi-square values had probabilities above the 5% level, thus supporting the proposed model. Table 3-12 presents the F^^BC^ families from normally nodulated F^BC^ plants via the same form in which the F^ data were presented in Table 3-10. These data again support the proposed model with the exception of one population from the cross PI 262090 X (PI 262090 x M4-2) segregating 1:1:2. From this cross all families segregating 1:1:2 should have been progeny from a plant with few nodules. Because the female from the cross was

PAGE 47

35 (U 0) Cd 4J bO 0) O U u 00 0) 4J CD -H 4J I 0) M-l 42 O 4-> I CO CO CO 41 0) B C tJ iH O O. O OO < 3 >H a< iH CO CO I a -H 0 CJ c O -H >4-l CO •a o > •H CO 5i u 0) CMr^ CNCO OO ^00 t-( r-t OOO OO iHO iHin iHt^ iHCO iHm iHvO OO 00 in r-. CM CO CO PO CO 1 — vO 14-1 O (U o -a s: g M O S 0) CO B OJ 3 C CO C -H O 0) w o I CO XI CO H O CO CO o U ++ OS w o w o w o W O CM rH I c s c CM
PAGE 48

N X X O -aiH o O iH rH / — s l-l t u o c fO CO O CU o o 4 Z ft. z
PAGE 49

T3 0) CO o 4J CO o 0) u 4J CS X CN j: X 4J O 4J 14-1 a 4J •H 14-1 1 (4-1 td o G 1 CO O CD • +. a N 4J ^ -H .H >J tfl QJ 3 J= O •H ^•O (0 CO Xl 4-1 U CO X to o o X u u O X) 1-H X PQ 13 X rHO o e O* ^ — iH iH 1 ro M 0) 1 0) u u •i 3 3 H W c 3 Cd o 3 m d I o in in 00 o o en CO a! w o o in in Oi o m o I o in CM m • o i o Oi o o iH CTi O O iH O iH -aro CO C£5 w o <; o o CN Cd O O O O iH fo iH CN CX3 w o < ro o o o w o o m CM CM VO vO CM CM iH o in w o <: t ta /— s CM CN CM CN CM o 1 1 CN u 1 1 CM CN 1 ON sr — ^ 3 X 3 N — ^ rH CM CO <• m vO

PAGE 50

4J CO CN X &4 CN c o •H n) 3 a o z B u o z 1X4 X) O c I c o z O -D ^ — X o o CTi o I m O iH vo \D C3 I o o C2 CN in O ro o Pi o
PAGE 51

-ofPL, U 0) 4-1 CO cn CO (U u C 4-1 •a CO o CM •u o C 60 CO rH U a. o 14-) u -UI PQ CO iHO) T3 (1) 0) V4 CO t-l to 1—1 to 3 c iH cr o 3 CO 1 CO O -H C ^ 0) o O >4H >. 1-1 rH ^ T3 iH 4J o to Ti c 1 CN M c o CO o iH C O c •H iH e -u O CO CO o •H iH •a 4-1 T3 Q) CO O 0) CO > o O •H a u o 0) M (X CO 01 •H '(-I (U e o X) to o •*4 ^ e o iHtfl T3 O 0) > B3 m5 PS P5 CJ U O o o u o Ex] o o O C OJ 3 o" O 0) CM ON 1 O t4H — N — N CM CM 1 1 (1) '^r
PAGE 52

40 PI 262090 and not an plant, this unexpected segregation ratio could not have been the result of a selfed seed In the Fj^^C^ generation. The plant from which this family originated had a nodule rating of 3, but this was probably an error and the plant should have been rated 2. The F^BC^ families derived from few (1-2) and non-nodulated (0) F^BC^ plants are presented In Table 3-13. These data are In agreement with the proposed model. The seven families from non-nodulated F^BC^ plants that segregated 1:1:2 should have had few nodules but because of Incomplete penetrance of this character, the ^'-j^^C^ plants were non-nodulated. There were also three families that segregated 1:0:3 from F-|^BC^ plants that were non-nodulated. This was not expected; however, each of these three plants must have been created by an n-]^n2 pollen grain fertilizing an ^2_^2 This Is another example of the ^^^^ gamete reducing nodulatlon. The same explanation would also apply to the one family which segregated 1:0:3 from an F^^BC^ plant with few nodules. The genetic model that has been proposed In this study to describe the Inheritance of nodulatlon In peanut Is similar to the one described by Nlgam et al. (39). The similarities are that both models assume that nodulatlon Is controlled by two Independent genes and that the genotype of a non-nodulating plant is n^n^n2n2. The difference between the two models is that the model described in this study contains a third phenotype classified as plants with few nodules. The model states that the genotype n^n^N2n2 produces plants with few nodules when the male gamete is t^2_^2' male gamete is

PAGE 53

^ — s CO rH H to o u B PQ u rH ca .H o pti c 4-1 05 to Q) CO iH 0) 4J g 3 4H O "O CM T3 i-H c O rH P' 05 3 1 rH c (u c CJ O 4 H o CQ •H c iH 4J J3 — to rH iH 05 3 O rH (U D. iH 4J O 4-1 O CO r-l Pi o a O c 1 •i— c ^ — s CO o rH c CO O rH to u u rH c o to e a O 3 to u 0) o <4H 73 (U 4-1 1-1 4-l T) 4-1 T) 1 rH (U CO D c 05 rH o 0) o 3 r c > o. o. •H O o 3 M U Ph 0} 1 iH V4 J3 4-1 cfl c H w o o o o 0} 0) •H o c o o o o o o o o o o o o O CN o o o o o o o o o O o o o o rH lH rH rH rH rH rH CO rH rH rH rH Pi Pi Pi Cd O w o Cd o Cd O Cd o <^ CN /-^ CN 1 CN — S CN CN 1 < 1 0) CN
PAGE 54

42 n^N^ then the plant will be normally nodulated. Evidence to support this model was provided in every generation but the strongest evidence is provided from the and F^BC^ data. The differences found in plants of PI 262090 x M4-2 and M4-2 x PI 262090 support the model. These reciprocal differences could have been caused by the interaction of nuclear and cytoplasmic factors or by cytoplasmic factors alone. When cytoplasmic factors are involved, reciprocal differences would be expected also in the F^ and F^ generations; however, no reciprocal differences were observed in the F^ or F^ in this study (Tables 3-9 and 3-10), The F-^BC^ data (Table 3-11) also provide strong evidence to support the model. The only crosses that had plants with few nodules (with one exception that was explained earlier) were those that had the potential to produce t^j^2 f^^^l^ gametes and ^2_^2 ^^^^ gametes. An example of this is found in the comparison of the F^^BC^ results of Entries 3 and 4 in Table 3-11. The genotypes of the parents used in Entry 3 were n^n^n2n2 x (n^n^N2n2) so when the male gamete was ^-^^2 ^1^*^1 P-^^'^' produced had normal nodules and a genotype of n^n^N2n2. The parental genotypes used in Entry 4 were (n^n^N2n2) x n^n^n^n^; thus when the female gamete was n^N^, the F-j^^C^ had few nodules and a genotype of (n^n^N2n2) • The genotype of the plants with normal nodulation from Entry 3 and few nodules from Entry 4 should be the same and data in Table 3-12, Entry 3, and Table 3-13, Entry 4 support this because both segregated 1:1:2. While the proposed model seems to be strongly supported by data from the F^, F2, F^, F^BC^, and 7^SC^ generations, the model assumes

PAGE 55

43 that the phenotype of two plants will be different even though the genotypes are the same, and that cytoplasm has no effect. Mouli and Patil (37) reported a similar mode of inheritance for foliaceous stipule in peanut. They reported that normal x foliaceous produced foliaceous plants but that foliaceous x normal produced normal plants. This is similar to the reciprocal differences found in F^ plants from PI 262090 x M4-2 and M4-2 x PI 262090. They also reported that all normal plants were produced in the F-^BC^ generation from the crosses (normal x foliaceous) x normal and (foliaceous x normal) x normal. This is similar to what was found in the F^BC^ plants from the crosses (PI 262090 x M4-2) x PI 262090 and (M4-2 x PI 262090) x PI 262090 in which all progeny had normal nodulation. Mouli and Patil (37) also reported that the F^BC^ plants segregated for foliaceous and normal stipules from the crosses normal x (normal x foliaceous) and normal x (foliaceous x normal) This is similar to what was found in the F^BC^ results obtained from the crosses PI 262090 x (PI 262090 X M4-2) and PI 262090 x (M4-2 x PI 262090) in which the ^l^^l g^'^s'^^tion segregated for plants with few nodules and plants with normal nodualtion. As in this study, Mouli and Patil (37) found no reciprocal differences in the F^ or F^. Since these similar findings have both been detected for different characters in the same species, it provides evidence that peanuts may have a mechanism of inheritance for some traits that is quite different from other species. Mouli and Patil concluded that the "modification of the segregation ratio was presumably due to the fact that both the recessive genes had to be present in the pollen carrying the functional factors." (37, P. 29) The data in this study also support this type of inheritance.

PAGE 56

44 One possible interpretation for this mode of inheritance was discussed by Grouse (18) working with Sciara and Simon and Peloquin (57) working with Solanum hybrids. They described the inheritance of traits that were controlled by chromosome imprinting. The imprint a chromosome bears is unrelated to the genie constitution of the chromosome and is determined only by the sex of the germ line through which the chromosome has been inherited. The mode of inheritance found at the N2 locus in this study could then be explained as follows. The imprint a peanut chromosome receives when transmitted through pollen activates the N2 locus, and the opposite imprint, which causes deactivation of the N2 locus, occurs when the chromosome is inherited through the egg. This imprint may not alter the locus but the imprint may affect an element in the gene control system which could be similar to the gene control system in corn ( Zea mays L.) as described by McClintock (36). The physiological mechanism which causes peanuts to be non-nodulated or have few nodules has not been reported and was not investigated in this study. However, a mutant peanut described by Ashri (6) has characteristics that are similar to peanuts that have few nodules. He observed diminutive plants that developed a normal side branch and called these plants mixed. He reported that when diminutive plants were sprayed with gibberellic acid they started to develop normally. This indicated that mixed plants may be caused by hormone levels which exceed a critical threshold level in certain developing bud primordla. It could be speculated that non-nodulated plants may be the result of plants that are deficient for a hormone. When a plant

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45 has a few nodules the hormone exceeds a critical threshold level in the few locations where nodules are produced. In summary, a genetic model has been proposed which describes the mode of inheritance of nodulation for the peanut lines used in this study. In the proposed model the non-nodulating genotype is "l^l^z'^Z ^"'"^ other genotypes have normal nodulation except "l^l^z'^Z "^'^'^ nodules when the parental male gamete of the plant is n^n2. Further study is needed to determine what induces the alleles at the N2 locus to cause different phenotypes as a result of the n2 allele being inherited from the maternal or paternal parent.

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CHAPTER 4 LINKAGE BETWEEN LOCI THAT CONTROL NODULATION AND TESTA VARIEGATION IN PEANUT Introduction There have been only three reports of linkage in peanut ( Arachis hypogaea L.) Patel et al. (46) reported that growth habit and branching type did not segregate independently. They estimated the rate of crossing over between the genes for spreading and branching to be 30%. Patil, as reported by Haimnons (26), found that the crossover rate between genes for growth habit and pod reticulation was 40.4% and the crossover rate between genes for stem hairiness and pod reticulation was 31.5%. Non-nodulating peanuts were first identified by Gorbet and Burton (24) in the generation derived from the cross of UF 487A, a University of Florida breeding line, with PI 262090. Shortly thereafter, Nigam et al. (39) reported non-nodulating peanuts were identified in the F2 generation derived from the cross 'NC 17' x PI 259747. They stated nodulation was controlled by two independent genes with the non-nodulating plants being homozygous recessive at both loci. Branch and Hammons (12) reported that the gene for testa variegation (V^) was incompletely dominant to solid color, which confirmed an earlier report on the inheritance of testa variegation (11) In preliminary studies on the non-nodulating peanut, it was found that non-nodulated plants often had variegated testa. In this study, crosses were made in which there would be segregation for both 46

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47 nodulation and testa variegation. The objective was to determine if the gene controlling testa variegation was linked to a gene(s) controlling nodulation. Materials and Methods Four peanut ( Arachis hypogaea subsp. hypogaea var hypogaea ) genotypes were used as parents (Table 4-1). The crosses, M4-2 x Florunner,' M4-2 X UF 487A, PI 262090 x UF 487A, and their reciprocals were made in 1978 and 1980. F^ plants from M4-2 x Florunner and M4-2 x UF 487A were backcrossed to M4-2 in 1980 and 1981. Crosses were made in a greenhouse using the method described by Norden and Rodriguez (41) Subsequent generations were field grown at the University of Florida Agricultural Research Center, Marianna, Florida, during the four growing seasons of 1979-82. Recommended agronomic practices were utilized including inoculation of seed at planting with cowpea-type Rhizobium sp. All F^, F2, and F-j^BC^ plants were tagged before digging and 30 plants were tagged in selected F^ plots immediately after digging. Plants were dug using a conventional peanut digger-inverter with the cutting blades set as deep (20-25 cm) in the soil as possible. Nodulation of roots of individual plants were rated as described in Chapter 3 immediately after digging. Pod samples were hand picked from all plants that were tagged. Testa were examined in the laboratory and were classified as solid, trace amount of variegation (trace-v) or variegated, as previously described (11, 12). These data were then analyzed by chi-square tests for goodness-of-f it to the proposed

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48 Table 4-1. A description of the four peanut lines used as parents in crosses to determine if there is linkage between loci that control nodulation and testa variegation. Parent Nodulation Testa color Genotype Description or source M4-2 Non-nodulating Variegated Wnj^n^n2n2 A line selected red-light red from the cross UF 487A X PI 262090 PI 262090 Normal Variegated Wn^n^N2N2 Plant harvested red-white from farm near Robore, Bolivia UF 487A Normal Solid pink wN^N^n2n2 University of Florida breeding line Florunner Normal Solid pink wN^N^N2N2 Cultivar

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49 model. When chi-square tests were used to analyze or data, the data were first adjusted to correct for incomplete penetrance. Reasons for adjusting the data were presented in Chapter 3 and the method used to adjust the data is presented in Table 4-2. Results and Discussion All F^ plants produced seed with trace-v testa and all were normally nodulated with two exceptions (Table 4-3) These results are consistent with the findings of other studies on inheritance of testa variegation (11, 12) and nodulation in peanuts (39, Chapter 3). The testa from F^ plants segregated into three phenotypic categories, solid, trace-v, and variegated. In some F2 plants which produced testa with trace-v, the variegated area on the seed was difficult to detect and could not be seen on all the seed. Because some plants that produced seed with trace-v were probably classified as solid, the two categories, trace-v and solid, were combined for analysis of the F^ data. Total, pooled, and homogeneity chi-square values fit a 3:1 ratio (Table 4-4), thus indicating that testa variegation is controlled at a single locus in these crosses. Based on the allele symbols used in previous studies (11, 12) on inheritance of testa variegation, the solid, trace-v, and variegated phenotypes have the genotypes vv, Vv, and VV, respectively. The F2 data for nodulation (Table 4-5) have been adjusted as described in Table 4-2 and the total, pooled, and homogeneity chi-square values were not significantly different (Table 4-5) when tested with the genetic model described in Chapter 3. The very low probability values obtained from the pooled

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50 Table 4-2. The method used to adjust the incomplete penetrance. F2 and data to correct for Data Nodule rating: 0 1 2 3 4 5 Testa classification: s vt S V S V S V S V S V Number of plants: A B C D E F G H I J K L Adjusted data with 1:0:3 nodulation ratio (non-nod: few: normal) Nodule classification: Non-nod Normal Testa classification: S V S V Number of plants: M N M N M = A N = B 0 = C + E + G + I+ K P = D + F + H + J + L Adjusted data with 1:1:14 nodulation ratio (non-nod: few: normal) Nodule classification: Non-nod Few Normal Testa classification: S V S V S V Number of plants: Q R T U W X Q = A X 0.69 R = B X 0.69 T = (C X 0.92) + (E X 0.57) + (A x 0.31) U = (D X 0.92) + (F X 0.57) + (B x 0.31) W = G + I+ K+ (CxO.08) + (E X 0.43) X = H + J + L+ (DxO.08) + (F X 0.43) fS = Solid + trace-v testa and V = Variegated testa.

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51 Table 4-3. Nodulation ratings of F plants that were field grown in 1979 and 1981. Crossf Nodulation rating! ^ ^ 0 1 2 3 4 no. of plants UF 487A X M4-2 1 0 0 19 39 UF 487A X PI 262090 0 0 0 0 45 Florunner x M4-2 0 1 0 46 36 fAlso includes reciprocal of cross. |0 = no nodules, 1 and 2 = few nodules, and 3 and 4 = normal nodulation.

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CN X O O 00 r-f ON • • o o I I LTi in O 0^ o d 00 m 00 so cv) in o CM o^ o o I I o in d o 00 iH 1^ m vo ON ^ ro CO O rH ON i-H o ^ iH fO (U 00 CO W O O O O e o H pLi S3 T3 0) 6C O E o H PL, PS to 4-1 O O O o in d d I I in o o in d o < ON in o vo 00 fo in 00 iH T3 • (U 00 i o H 33 CO •w O O O ++ CO u > 4J .H 00 o C vO CO ON 00 m I-l CO fn m CM ON iH 9) ON vO H o ON (U CO J3 • O M O II B o O 32 COO C=

PAGE 65

(U u 13 c o u CO (U 4-1 CM X CM o •H 4J CO O •H •H CO CO to c o •H 3 T3 O T3 -i 3 O CO o T3 o c I c o z CO 1 CO CO o c CO O c o m O m m O o CM CM in in • • o o o o o o 1 o 1 m 1 o 1 o 1 m 1 in in CM CM O o o O o CD ro CO CM 00 vO o CN CM 0^ o CJN tH in in O IJ-I u u o (U CO >H Xi o o iH II to u o o Vi & o u •H •H •H U 4J (U 0) CO c a) 60 CO TJ o OJ (U B T3 J-i o 3 O iH (U O a II •H ci O II o

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54 and the high probabilities for the homogeneity chi-square test indicate that testa variegation and nodulation did not segregate independently (Table 4-6) The segregation for nodulation from the UF 487A X M4-2 cross was controlled at the N^^ locus because both parents were n2n2. The evidence for linkage of testa variegation and nodulation in this cross indicates that the V and loci are linked. Because all plants that were non-nodulated or had few nodules were ri-j^^-^j they are -\ grouped into one classification for purposes of analysis and presentation. The calculated crossover percentages of each population ranged from 6.2 to 20% (Table 4-7). However, within crosses involving each of the three parental combinations UF 487A and M4-2 (Entries 1-5) UF 487A and PI 262090 (Entries 6 and 7), and Florunner and M4-2 (Entries 8-14), the ranges were reduced to 6.2 to 8.0, 10.8 to 11.3, and 9.9 to 20%, respectively. The arcsine transformation of the calculated crossover percentage values were analyzed by grouping them into the three parental combinations and the results (Table 4-8) indicate that there is a significant difference caused by the parental combinations Means of the crossover percentage for each parental combination were weighted according to the number of observations in each population. The weighted means were 7.1, 11.2, and 12.2% for UF 487A and M4-2, UF 487A and PI 262090, and Florunner and M4-2, respectively. These values were then used as the best estimates of the crossover percentage for each parental combination when calculating the expected values to be used in chi-square test.

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55 a c 0) 3 cr 0) )-i o 0) 4J CO 3 •a J5 4J •H u c o to u -d a 4-l o I CO CO 0) c •T3 O O CO &0 o (30 c 0) CO CO u o CO o 3 cr o CO u u-i I a •H C J= 6>S o cj o •H in *J >> CO JQ ^ CO CO iH ^ CO c CMC -H CO r-( I J3 CO H X CO CO o M CJ c CO O O in CM i-H rH O O o o o 1 O 1 in o o O rH o O vO m O o ro m CO cyi CM iH CO m CO ro 00 (30 C3^ CO SO CJ> 00 CM CO ^ a in c r~0) rH 3 O o C o 1 0) o CD in 1s r-. vO c O •H c (U O 3 CO cr rH •T3 0) CM in rH CM CO (U o o CO 4J lU rH rH CO (0 •H M -a (U CO •H > CO U o CO 01 u > CO VO 00 m u Xi CM CN OS II o W O < W 00 u (U c c 3 o rH CM I s CO > a u > oI O O (U CO ^ ;j 4-1 (U J= T3 o O 4-1 •H -H 4-1 0) cs CO <4H O T) •H CO rH 4J O rH Cfl 3 CO II (U 0(5 Vi c (U CiO •o o 0) a 4-1 O O EC
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56 u 0) 0) ^ e n) 3 H C CO c 14-1 O O -H (U I o 0) > o CO CO o u o CO CO o u (J o to u c (1) o c (U M B (U •H ^ M s Qj a a c 1 I I I -aCM cn O CM 00 vo CM CM I I 00 00 fL4 CM I 00 C3 CM I Cvj I •4-l PQ i-l to pq iH CN rFl4 |i| |X4 CN CO T3 OJ T3 3 r-( O C CO c to o a CO rH to (1) 4-1 CO CO •H iH 3 CO T3 CO O o c to to o e O ^1 u o & c •H a T) (U c I-l CO OJ •> 4-1 OJ c o c o M 4-1 0) CO 4J O to 4-1 (U to 0) to OJ 00 CU CO J2 C CO H fl O T3 -H CD to O r-l -( 3 O P. c o (u a. CO (U CD CO o o U J2 O H I l-I CO H rH (U 3 > Ch CJ (J CO ex. CD O CO 01 ^ (U 1-1 H >J COS

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57 Table 4-8. Analysis of variance of the arcsine transformation of percentage crossing over calculated on the three parental combinations. Source df MS Total 13 Parental combinations 2 47.10** Error 11 4.05 **Indicates significant difference at 0.01 level.

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58 When the adjusted frequencies for the generation of each cross were analyzed for goodness-of-f it to the expected frequencies with linkage, no significant differences were found (Table 4-9). The results obtained from plants which were in F2 families that were segregating for nodulation and testa variegation are presented in Table 4-10. The F2 families from the Florunner x M4-2 cross were separated into two groups. The first group segregated 1:0:3, and the second group segregated 1:1:14 (non-nod: few: normal) An insufficient amount of data was obtained from the F^ families of UF 487A x PI 262090; these data are not presented. There were no significant differences found when these F2 families were analyzed by chi-square test. The observed frequencies in the F^^C^ generation were not significantly different from the expected frequencies calculated with the indicated crossover percentage (Table 4-11). In Table 4-12, F^ plants are classified as non-crossover, crossover, or two crossover types by comparing the F2 plant's testa variegation with the segregation for nodulation in the F^. For example, if an F2 population (F^ plants) segregated 1:1:2 (non-nod: few: normal) the F2 plant that the family was derived from had the genotype n^n^N2n2. If there was no crossover when this F2 plant was produced, then it would also have the genotype VV and thus have variegated testa. If one of the gametes that formed the F2 embryo had a crossover between the and V loci, then the F2 plant would have the genotype Vv and thus be trace-v. When the numbers of non-crossover, crossover, and two crossover F2 plants were compared with the expected numbers assuming the appropriate crossover percentage, no significant difference was detected.

PAGE 71

r-l O •a O 'U a 1-1 •o I 09 O I u m o n o a 2 § ta > u o •a M-i QJ U 4J 0) tfl CO GO GO oj m 0) 4J 4J •H C 1-4 0) Cd M M cr a o CO 4-1 I U H (U C J= > o o o 1-1 CO >v CO o u o CO ^ 60 Q) T3 )-i 0) GO N 0) Q) >> *-> M iH CO CO -H CO O. P-I a* >4-l •t3 CO X c o 4J CO cu 4-1 M a o> CM CM o o o rH O o o o o CO O o rH CM vO vO rH rH 00 CO CO CM o O CO CO CO in o o O o o o in o o o VO o o o o 00 m rH cn 00 <3V CM CO -vT O u rH OS w o <: w -"-^ o u CM w o <: w < 00 -J1=> -3<: 00 o CT\ O CM VO in o CM o\ o o I I o in rH d d cj\ o c^ o> CM r> • • • 00 <• CO rH CM CM (U GO O e o H pLi X CO 4J o O O U CO o o O 00 o o O • • • CM VO vO o in c rH rH rH CO 0) u rH 00 -JCJ^ 4J 14-1 CM CM CM CO CO •H GO TS 0) cu CO •H > CO o o CO 0) u > CO u 43 II O CM PS rH w o < w ^ 00 c c 3 M O CM X I o c. (U 3 cr o o X CO CD II O M • O GO i o o PC u

PAGE 72

0) u T3 to c o X to CM X 0) o u 3 o O z & 0) TJ c to o c I c o z CO 0) 1 to to CO o u c to o c in o m o iH CM rH o O o o 1 1 o 1 in 1 o m iH o rH o O o o o CN O 4-1 o a o o o o o o P3 H cu PC in o O o CN o o in o O o CJN o o o CO O vO o 00 CT> m CN CN m o o o 00 O o CO o o to (U o cr > bs II o CN rH to > II Cv) o rH o o >^ I-I tx > 1 H (U O CJ O -H (U CO 4J Vh V4 to 4J M ; 4J o W H W 3 II CO II Q) OS OS CO W 000 00 i o o

PAGE 73

o •H T3 JJ 0) Cfl > M CO (U O (U 4-1 a cu CO o •H I ^< -H td jz > a CO CO o u u u >, O J3 QJ M-l 4-) 'O td O N M •H >^ a td cd )-i 60 c a (U td & M td 60 (U U (U CO C J2 01 4J .H3 u cr x: cq II rH •H w o w V ^ #v O > 1 1 0) o o •H CN td 4J 1 < C •H 1-1 4-1 td u • s 0) XI O 0) X CO c Q) > CN o td 4-1 O 1 X rH U CO > )-l o c (U 0) x; 3 4-1 • cr XJ 4J td 60 4-1 Cd CO > II (1) > (U CO XI o

PAGE 74

u c C o 0) •H )-l OJ U to 00 (fl a CO iH 4J 3 u-i c T3 o cu O o c o u •H Qi U 4J o to u er c •T3 > o (U o •H •u CO o CO to 0) o 00 o. u a u 4J eu •H ex. 4-1 M-l to C 1 to M-l cu <-i O x; ex 1 CO 4J CO 0) 4J c •H 14-1 XI o o o CO c 00 (U o a •H u 4-1 o 4-1 to <4-l 60 M CO o to lU CO > 4J so to 01 u 4J u u CO to o CO o CO o o u <: 4-> o O o i iH u o c 9 M w U-t D -H O e 1 t4 B •H O c c*^ § ^ O n 0 1-4 •H 0 4J a u no c o iJ a eo f-t — V o tj o o z CJ u z ^ — o o o o ^ u o H U z O 00 CM m NO tH CM fH CM m > H > H tn > H M o b cr. 01 -< < o B CM B CO CM 00 NO 3 CM X 1 aX CM 1 •aO b. £ b. W 1-4 s 3 a. o 5 •H O (J O H h 1-1 I ^ (u o n o o O o li b 4J > OHO U (0 0) 4) O £ U !-> U U u u • 01 >> a H U 10 CO I I *M 00 C O 01 o H z CO V. jj n > 3 ^ CJ z ; OS > U O 0) E > C M n 0) u n

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63 Table 4-13 is similar to Table 4-12 except that the 'P-^^C^ plants are classified as non-crossover or crossover, instead of F 2 plants. There is no two crossover classification for F^^*^! P^^^ts because a crossover can be detected only if it occurs in gametogenesis of the parent. No significant differences were detected when the number of non-crossover and crossover F-j^BC^ plants was compared with the expected values. These data support the hypothesis that the V and loci are linked. However, the different crossover percentages observed in the three parental combinations were not expected. One factor that could cause some of the experiments to have a higher crossover rate is incomplete penetrance of normal nodulation. The method used to adjust the data (Table 4-2) assumes independent segregation of the V and loci, and thus the adjusted values will tend to increase the crossover rate. These adjustments were made in experiments numbered 6, 8, and 10 (Table 4-7) In each of these three experiments the data adjustment has not caused a large increase in the calculated crossover percentage when compared with the crossover percentage found in other experiments of the same parental combination. If it is assumed the difference in crossover rate observed in the three parental combinations is real and not caused by sampling error, then there are several factors that could affect recombination frequencies in different experiments. Factors known to influence recombination frequencies in Drosophlla sp. are sex, maternal age, temperature, cytoplasm, nutrients, radiation, genotype, chromosomestructure, and the position of genes relative to the centromere (61).

PAGE 76

*r*1 n1 \J f\ \J ri *H f~\ \J jj r* Rl vU M o T) 14-1 0) CO I } 1 ) r* r 1 r\ w ni j 1 M CTJ W 0) ^ x: ID 05 o o 4-1 CO JZ 4J U o •H •H )^ & 4-1 (J CO 1 <4-l o [ 1 c 1 to cS CO ^ [ iH CO p. (U C U o (-1 PQ o rH 00 u 14-1 o A0 >4-l ''^ c CO O 4J 4J •H CO •H 4J (U > rt CO (U (U •H & V4 nj >. 3 4-1 > cr CO u n) 1 u •H > CO o 0) a CD j-i CO X) o c u o to o c O u c CO P3 to •H CM u rH CO to 0) x: c o 4-1 o c CO <: •H I -aJ3 CQ o I o o 00 00 o u O W w CJ o o cj 9 O o o CJ o z o CJ o Z tJ O CJ CJ z O CJ CJ z u B I >• g I z -o 01 u u g u Q U 91 ki /-s u U H CJ o. CO Z X P ^ w 0) H I m u m u u O TJ > V4 O • O u 01 (Q CO Qi 00-0 > a a u u U -H (J CI O ki 0) a I ^ CL > o S a CJ

PAGE 77

65 Additional studies would be required to prove or disprove that one or more of these factors caused the different crossover rates observed for the different parental combinations. However, the cytoplasm does not seem to be a factor, because no differences were found in the crossover rates of reciprocal crosses. Also, it seems improbable that maternal age would have an effect on crossover rates in peanut. The other factors mentioned may have an effect. For example, gametogenesis occurring at different times during the day in plants from different crosses could cause a temperature effect on crossover rates. Cytological studies of the parents and F^ plants could provide evidence of differences in chromosome structure in plants. For example, if UF 487A, Florunner, M4-2, and the F^ of MA-2 x Florunner all had similar karyotypes, and the F^ of M4-2 x UF A87A had a similar karyot3npe to the others except for one chromosome, this would provide evidence, but not proof, that chromosome structure caused the observed differences in crossover rates. It would also provide evidence that the and V loci are on the chromosome which was different in the F^ of M4-2 X UF 487A. Evidence from the F2, F^, F^BC^, and ^2^^i generations have shown that the and V loci are linked. The recombination frequency between and V was 7.1, 11.2, and 12.2% for the three parental combinations, UF 487A and M4-2, UF 487A and PI 262090, and Florunner and M4-2, respectively. These data also provide additional support for the genetic model proposed for nodulation in Chapter 3.

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CHAPTER 5 A GENE AFFECTING TESTA VARIEGATION COLOR AND ITS ASSOCIATION WITH THE N2 LOCUS IN PEANUT Introduction The inheritance of testa color in peanut ( Arachis hypogaea L.) has been the subject of many studies, and an extensive review was presented by Hammons (26). At the R2 locus the recessive allele produces red testa color and the dominant R2 allele produces pink testa (5, 7). The variegated testa of A. nambyqyarae L. was reported as incompletely dominant to solid color of A^ hypogaea testa (60) The inheritance of red on white testa variegation in peanut was controlled at one locus and the allele for variegation (V) was incompletely dominant to the allele for solid testa color (v) (11) Recently, Branch and Hammons (12) found that the R2 and V loci segregated independently with incomplete dominance gene action found at both loci. Non-nodulating peanuts have been identified in progeny from certain crosses in Florida (24) and India (39). Nigam et al. (39) reported nodulation was controlled by two independent genes with the non-nodulating plants being homozygous recessive at both loci (n^n^n2n2) In Chapter 3, this inheritance was confirmed except that the n^n^N2n2 genotype has few nodules when the male parental gamete was ^^''^2' Chapter 4 it was reported that the V and N^ loci are linked. 66

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67 In this study, crosses were made in which segregation was expected for both nodulation and color of the variegated area (white or light red) of the testa. The objective was to determine the inheritance of color of the variegated area of the testa and to determine if there was any linkage with the N2 gene that controls nodulation. In addition, crosses were made to determine whether the R2 locus was linked to the or N2 loci. The relationship of the V and R2 loci was also investigated. Materials and Methods Three peanut ( Arachis hypogaea subsp. hypogaea var hypogaea ) lines were used as parents (Table 5-1). The crosses M4-2 x PI 262090, M4-2 X 'Florunner,' and their reciprocals were made and some of the plants were backcrossed to M4-2. plants of MA-2 x PI 262090 were also backcrossed to PI 262090. Crosses were made using the method described by Norden and Rodriguez (41) Subsequent generations were field grown at the University of Florida Agricultural Research Center, Marianna, Florida. Recommended agronomic practices were utilized including inoculation of seed at planting with cowpea-type Rhizobium sp. All F^, F2, and Fj^BCj^ plants were tagged before digging and 30 plants were tagged in randomly selected F^ plots immediately after digging. A conventional peanut digger-inverter was used to dig the plants, cutting the roots 20-25 cm below the soil surface. Nodulation of roots of individual plants were rated as described in Chapter 3 immediately after digging. Pod samples were hand picked from the

PAGE 80

o B 0^ l-l u .c O u J-l CM 14-1 ? VD o B CN a CO CO o o •H u M u > u Pm U-t •H o iH T3 X 13 O c dJ (U PQ o w < 4J •H a CO *s 4J Q) 00 0) & <-i •H o U CO FiCD u U o cS CO OJ Oj t> Q) c CO 4-1 •H O to c M 4J o td CO M 00 c •H 3 o a I c o z CNJ I OJ 0) 4-J 4-1 to (U ^ •H I CO 0) > V4 o z o CJ\ o VO CM rH C O -H O z V4 CU c § t-l o

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69 tagged plants and testa were classified as red or pink in the laboratory. When a plant had variegated testa, the lighter colored area of the testa (the variegated area) was classified as white or tinted. When compared with the "Munsell Limit Color Cascade," using Munsell notation, typical pink, red, and tinted testa were 2.4 YR 8.2/4.4, 6.8 R 2.6/9.4, and 8.4 RP 7.3/9.2, respectively. These data were analyzed by chi-square test for goodness-of-f it to the proposed model. When chi-square tests were used to analyze F2 data where there was segregation for nodulation, the data were first adjusted to correct for incomplete penetrance. Reasons for adjusting the data were reported in Chapter 3 and the method used to adjust the data is presented in Tables 5-2 and 5-3. Results and Discussion All 81 plants derived from the cross Florunner x M4-2 had pink testa color with a trace amount of variegation. This indicates that pink was dominant to red and that variegation was incompletely dominant to solid testa color. The inheritance of testa variegation from this experiment was presented in Chapter 4 and will not be reported here. The data presented in Table 5-4 indicate that the R2 allele that produces pink testa color is dominant to the r^^ allele which produces red testa and supports results reported by Ashri (5, 7), but others (29, 60) have reported that red is dominant. The plants classified as having solid and trace amounts of variegation were grouped together because some plants that produced trace variegated testa were probably classified as having solid (red

PAGE 82

70 u o U 9) U f-i o u o 4J vO I in rH •§ H a cd CO •o 0) .c 4J CO 3 •n • n (U rt o c o to •O Q) 0) c CO (U 3 Po E e o 0) o j= c CM I m 0) H Pi Pi Pi Pi PQ cd B u o z o •H 4J CO CJ Pi Pi Ph Pi Pi C/1 00 CO CO Ir CO c 4-1 4-) CO 4J •H U c CO CO U c 4-1 o CO •o CO O CO CO r-l iH iH iH M CO a ed o CO P0) (4-4 4J cu 14-1 tH CO o CO r-t CO o CO D 4J 3 3 4J iJ TD CO TH •T3 CO CO O a> o O m d Q z H z < z H z ^ — s rH m O o £> vO + + o CD O O M II X X + + Pi u Q <: PQ ^ — ^ II II II II II II z o cn H

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71 1^ B o PX4 CM H 13 o o c I c o e o o 4J >4-l CO to to to •H c 60 4J 4J CO •H (U C to CO •H to CO to 1-4 .H u to a o > CO H to 4-1 C CO O. IM O o o o o o X m II z + + o Pl4 + Ed + o o o x: PQ — II + + II CO + PS II H C •H 4J II H •H x: II

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lO o in in o ON o CsJ a\ • • o o o o o P-I 1 1 1 1 1 o in CO o m o r-l O o o o o CM vD vO . iH vD iH X T3 m 0) -o •o a iH a) 60 ,—1 tu 60 rH • u tfl B o to % o to 3 'USB 4J g o O o 3 o o o o c H M EC H CO PC H Q) 3 (U U to (4-1 u ^ 4J tn cn rH CO o iH O rH o C c <• in in rH •H cs cn CO 0) o Pm iH iH 4J O O (U tfl &. o X to w 0) t-H CM -nT iH rH CM rH H o 0^ in m m II • CM CM u w o rH •> o +>N O o fd O W M O u w o C to 0) 4-1 to 3 CO OJ C (U •H (U 4J iH o oo 1— 1 u •H c in B o tt) (U > T3 U Q) c Q) 4-1 o CD Cfl 1-t ,a 60 rH O Q) td CJ M ++ pq II 60 CN CO rH c o to (U o VI 4-1 o to ^ ^ 4J ^ CM cfl 1 u CO S 0) rH X V4 (U to u u 0) CX <4H to < 1 l-l 1 CM o o
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73 or pink) testa color as reported in Chapter 4. These data (Table 5-5) indicate that in the generation the two loci, V and segregate independently and thus support the results of Branch and Hammons (12). In the generation of Florunner x M4-2 the expected segregation ratio for nodulation is 1 non-nodulated: 1 few nodules: 14 normally nodulated. Thus, 3:1:3:1:42:14 is the expected ratio for the segregation of nodulation and testa color (Table 5-6) Because of incomplete penetrance of nodulation, the data in Table 5-6 have been adjusted as described in Table 5-2. These data indicate that the loci N^, N^, and R2 segregate independently. All of the 66 F^ plants derived from the cross of PI 262090 x M4-2 had variegated red-white testa color, indicating that white variegation is dominant to tinted. Segregation ratios for the F2, F^, and F^BC^ generations were not significantly different from the expected ratios, assuming the trait is controlled at one locus with the allele causing white variegation (Wv) being dominant to the allele causing tinted variegation (wv) (Table 5-7). The inheritance of white and tinted variegation color is similar to the inheritance of inner seed-coat color in peanuts reported by Rodriguez and Norden (52). They stated that white inner seed-coat color was dominant. In the F^ generation derived from the cross PI 262090 x M4-2 the expected segregation ratio is 1 non-nodulated: 1 few nodules: 2 normally nodulated; thus, 3:1:3:1:6:2 is the expected ratio for the segregation of nodulation and testa variegation color (Table 5-8). These data were adjusted as described in Table 5—3 because of incomplete penetrance and indicate that the N„ and Wv loci segregate independently.

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X CO 0) < CN CM fO r-H CN CM CO U O o o o e o H pL, PC 00 CO o C3^ C^ 0^ U CO (U > CO XI II o > II *> o > 1 (U o a •H •H CO 4J (U u CO c 4J l-l 60 -a T3 o c 0) e CO U o a 33 •a •H & II o w CO 60

PAGE 87

(U N 4J Hi a (U • e & a c 0) •o Ci fr ith (U 4J o CO •H 3 4J •r-i CO cd •a 0) (U X! u 4J ec J2 4J t< •H 15 • c OS o •H -a 4J o c CO 4J CO iH 3 4-1 •o •H O iw Z c 1 >4-< #> O 1— C 1 z CO CO CO t-i cu •w o c a .-1 o o o rH a o 00 (U CO 0) 4J M CO O x: M-l •u 4J dj CO O te 4-1 c 0 ^ o 00 0\ CN d iH CN CM CN (U CN CM CM 3 a" OJ fr CM CM VO m VO VO d) \o VO
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CO T3 Q) CO 0) e C o cfl u (U c 0) CO CO o u o o CJ^ to O O I I u-i in • • o o vO O to ro o 00 o o o iH O CO e09 (U 60 c Cfl O O c o ^ B O 3 O H M S5 m rH o o o fH vo ++ td O W O c CM O ON o CN VO CN CN X I S =^ O in to (N o O OS 1 1 o o O CO O rH o d o o in CO (50 rH O cfl e 4J 3 o O X H .H CM H in 00 00 rH O r- 00 CM CM Pi wow o o a\ o CM \D CN i 14H o d) w g II w [3 O o to c rH OJ Cfl 3 cr 0) CO M o CU u CO a O rH Cfl II o O o Q•W o CJ •H OJ 4J V1 to u (U J= •o cu 14H o o
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U 1 • c c cr OJ 3 a PL, W QJ CU T) c • •1-) T3 13 x: CO 0) 4J U -H c M CO ^ O X 3 •f-l o CO CC 4J OJ <4-l to •tJ (U M CM X (U | u 1 :s O CO iH CO TS 4-1 O (U C d CJ C Cfl •H H C O CM O O Z 0) •H 00 01 4J -H U rt (-1 O •H 00 O O Q) H-l tH •H M JJ O CO CO > > OJ -W 4J 4J CO 0) d o T-t CO )-l JJ c H (U CO 1 U 3 c cr o o 0) M-l CO 2 4J O 1 c •H •H O o a 4J •H CO 4J >^ 00 (0 j3 o; 00 M ^ OJ CO iH W •H to c H CMC 01 •H [ii to "O B to J-l 00 1 u-l OJ CO .H CO J5 o CO H U in CJ^ O O I I O • • o o CO ^ o 4J •H •H 4-1 OJ CO d ^ OJ 00 T3 o OJ s 4J o a tc OJ pII X a 00 II o B cd o W a:

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78 In summary, the results of this study support the reports by Ashri (5, 7) that the R2 allele, which produces pink testa, is dominant to the r^ allele, which produces red testa. They also support the report of Branch and Hammons (12) that the R2 and V loci segregate independently. It was also determined that the N^, N„ and R„ loci segregate independently. It was shown that testa variegation color is controlled at one locus and that the allele causing white variegation ( Wv ) is dominant to the allele controlling tinted variegation (wv) Finally, it was shown that the N2 and Wv loci segregate independently.

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CHAPTER 6 GENETIC RELATIONSHIP AND INHERITANCE OF NON-NODULATION AND TESTA COLOR IN PEANUT LINES FROM FLORIDA AND ICRISAT Introduction The peanut ( Arachis hypogaea L.) is a legume which will form root nodules that are capable of N2-fixation when infected by effective Rhizobium strains. Non-nodulating peanuts have been identified in Florida (24) and India (39). Nigam et al. (39) reported nodulation was controlled by two independent genes, with the non-nodulating plants being homozygous recessive at both loci (n^n^n2n2) Their report was confirmed in Chapter 3 except that the i^2"l^2"2 S^"type produced plants with only a few nodules when the parental male gamete of the plant was "2^2' The inheritance of testa color in peanut has been the subject of many studies, and an extensive review was presented by Hammons (26). At the R2 locus the recessive r^ allele produces red testa color, and the dominant R2 allele produces pink testa (5, 7). Stokes and Hull (60) reported that the variegated testa of A_^ nambyquarae L. was incompletely dominant to solid color of A^ hypogaea testa. The inheritance of red on white testa variegation in peanut was controlled at one locus, and the allele for variegation (V) was incompletely dominant to the allele for solid testa color (v) (11). Recently, Branch and Hammons (12) found that the R2 and V loci segregated independently with incomplete dominance gene action found at both loci. 79

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80 This study was conducted to evaluate the inheritance of nodulation utilizing a non-nodulating peanut line developed from the nonnodulating germplasm described by Gorbet and Burton (24) and two non-nodulating lines developed at ICRISAT. In several of the crosses there was segregation for testa color; these data were also analyzed. Materials and Methods Six peanut genotypes were used as parents (Table 6-1) The lines PI 445923 and PI 445924 were crossed with UF 487A, PI 262090, 'Florunner,' and M4-2. Crosses were made using the method described by Norden and Rodriguez (41) About half of the plants were field grown at the United States Department of Agriculture Research Station at Isabella, Puerto Rico and the remaining F^ plants were grown in a greenhouse at the University of Florida Agricultural Research Center, Marianna, Florida. The parents and F2 were grown in the field at Marianna in 1982 using recommended agronomic practices, including inoculation of seed at planting with cowpea-type Rhizobium sp. All F^ plants from a sample of the F^ families were tagged before digging. A conventional peanut digger-inverter was used to dig the plants; roots were cut at 20-25 cm below the soil surface. Nodulation of roots of individual plants was rated as described in Chapter 3. Pod samples were handpicked and testa were classified as pink, red, light purple, or purple and also as solid, trace amount of variegation, or variegated. When compared with the "Munsell Limit Color Cascade," using Munsell notation, typical pink, red, light purple and purple testa were 2.4 YR 8.2/4.4, 6.8 R 2.6/9.4, 2.6 RP 1.8/5.4, and 4.9 P 2.1/11.2, respectively.

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d o 3 T3 O Z CO u CkO CO Cfl c o 0) •H c e ee M u ACD 1 1 4-* n\ to a 13 S -a w u ^— O •H 3 M ON u u 14_) ^ o cn -i (1) CN (U •H o U CJ M su iv or d (U PL, OJ H o > O •H M pq 4J 4J CO Cfl •H +a. < x: CO Cfl H H •H (U u > <: M C 00 4-1 u (U •H CO cw O •H o > 0) U M M CO •H C rH Pi PS 0) o C -H 3 u CJ o 05 to rH U M M (U a o c (1) P4 c 01 u to (U ^4 •a o aj 4-1 u x: 4-1 4J O CO 60 CO -H O M -H 60 X Q) rH (U S JJ •H 1 •H 1 CO M T3 M c (U tC (U CO OJ •H H > M > U Ph 60 C 3 T) O c I d o z d CM I o z o o CM VO CM CO a o z 00 rH M O Z d 60 d •H 4J CO rH 3 •o o d I d o z & IH 3 P< CM CM CM CM CM CM d z d Z d c CM CM CM CM CM CM 4J d z d z d d o rH rH rH rH rH rH d d d Z • Z d d 0) rH rH rH rH rH rH C3 d d z z d d 60 d •H 4J CO rH 3 "13 O d I d o z u CO 4-J CM Cv) CO CT\ i m u-l e S3M
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82 The nodulation data were analyzed by chi-square test for goodness-of-fit to the proposed models for inheritance as described by Nigam et al. (39) and reported in Chapter 3. When the model described in Chapter 3 was used, the data were first adjusted to correct for incomplete penetrance as reported in Chapter 3 and presented in Table 6-2. When there was segregation for nodulation and testa color, the data were adjusted as explained in Table 6-2 with each testa color category adjusted independently. Results and Discussion Nodulation The data (Table 6-3) for nodulation cannot be fully explained by the genetic model reported by Nigam et al. (39) or in Chapter 3. The model described by Nigam et al. (39) would have predicted Entries 1, 2, and 3 to be non-nodulated and all others to be nodulated. The model described in Chapter 3 would have predicted Entries 1, 2, and 3 to be non-nodulated; Entries 10 and 12 to have few nodules; and all other entries to have normal nodulation. The only entries with normal nodulation were those that had a male parent with normal nodulation. All entries that had a non-nodulating line as the male parent were non-nodulated or had few nodules. This indicates that when the male gamete is n^n2 it tends to reduce the amount of nodulation, as was proposed in Chapter 3. The F2 data (Table 6-4) have been adjusted as described in Table 6-2. These adjustments were made assuming that the level of penetrance in these populations was the same as those discussed in

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83 Table 6-2. Method used to adjust the F„ nodulation data to correct for incomplete penetrance when A, B, C, D, E, and F equal the number of plants rated 0, 1, 2, 3, 4, and 5, respectively. Cross Nodule classification Adjusted frequency UP 487A X PI 445923 or PI 445924 Non-nod Normal B + C + D + E + F PI 262090 X PI 445923 or PI 445924 Non-nod Few Normal A X 0.70 (A X 0.30) + B + C D + E + F Florunner X PI 445923 or PI 445924 Non-nod Few Normal A X 0.69 (A x0.31) + (B xO.92) + (C x 0.57) (B xO.08) + (C xO.43) + D + E + F

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84 a O O u CO 4-l o a o •H o CO o I to H c o •H U (U (U 0) •a 73 T) •o (U (1) OJ 0) rt a o O •H •H •H •H •H O o CJ a 00 CO CO CO i-H iH I— i — 1 r-4 Co CO CO CO OJ t-i M M O O O O O U M u •H 4J CO CO CO CO CO 4J 4J u 4J u > (U (U (U (U 0) iH a p. p. IX a M 1-1 M M M 3 3 3 3 3 3 Oa a O. a a l-i *-> u u 4J o JS ^ x: J= ^ -C J= bO C c oo CjO c C C0 60 o •H •H •H •H •H •H •H •H •H •H •H OOi-t P. iH OrH rH a D. iH rH c c rH 0 CO •H e •U u CO o CJ z •H CO 14-1 4J •H C CO CO CO rH CO 15 a. tH cu O or c o • •H o 4J 13 c CO O rH c 3 1 T) c O o z z OOOOOOOrHCSOCslO OOOmCMrHCNJOOOOO vOvDvOOOOOOOr^O CO u o CO o <^ Csl CM OS eg CM ro 0^ c o CJ> o o\ CM CM in m § eg m CM m fa fa fa fa fa fa fa rH CM CO in 00 CJ^ o rH CM rH rH rH

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CO •p (U cN a 3 O 0^ • {M X -a" CO CO •H iH CO o B •H u JJ o CO z CO U 4J H c CO •H .H CO CO 0) CO <*-( iH D u 0) iH O 3 c X) 1 o c z o z o c in m d <3 I I o o u-i m o o in iH -3coo O 3 o a^ o o CM iH 00 00 PO o> cj> o W O Ci I I o o I— I in o o m m o d I I o o in m o d in rd I o m o CT\ d I in o o X) GO 0) 60 O CO O CO B 4J e u o o 3 o o H C/3 PC H CO CM VO T3 OJ. \0 CN -J60 O e o cn X O 00 o o iH CN CM CM OS w o < w CM t-~. ^ r~r> 0\ r-\ T-i 00 CO vo CO CM H t-l w o <; w iH o 1^ CO in m i-l 1^ vO in in in d I o in o vO rH >3 U rH CM St in

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0) u •a CD CM X 01 a u 3 O in C o •H 4-1 CO a (U 73 O c I c o z CO a) a CO CO CO o u u c in O • o o 1 rH 1 o m o o C3^ o CM rH CSl -3iH 01 ail CO o 4-1 B O 3 o H CO m rH o rH c CO P. o o c rH ~3<• CJN rH O CS rH w o <: w o c CM 0^ ir, <• c oCM c M rH C rH X c U X 0) CSl c 2 c CM 3 Z l-i rH o Z rH rH Z o o o o CM W O o o o o rH ex3 w o • II CM CM CO C c U O p-l CM C • CM c CM C >> a\ rH rH U O 4J in C m C CO -H •H ,a 4J ei O II o d 6 00 OJ Qt:; o +-++ coo

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87 Chapter 3. A more accurate adjustment could be made if these F2 plants were progeny tested to calculate the degree of penetrance involved when these parents are used. This method would be preferred, because it has been reported that the level of penetrance of a trait can change when the genetic background is changed (25, 35) When a chi-square test for goodness-of-fit was conducted on the adjusted frequency, Florunner x PI 445924 was the only population that was significantly different from the expected frequency. This difference could have been caused by sampling error, a different level of penetrance than reported in Chapter 3, or by a different mode of inheritance than the one proposed. Further studies are needed to determine which of these explanations is best. Based on the model described by Nigam et al. (39), the expected frequencies in the F2 and chi-square test would be the same as those presented in Table 6-4 for Entries 1, 2, 7, and 8. The expected ratio for Entries 3 and 4 would be 1 non-nodulated: 3 nodulated, and the expected ratio for Entries 5 and 6 would be 1 non-nodulated: 15 nodulated. The summed chi-square values, each having 1 df, for Entries 5, 6, 7, and 8 are 62.8, 9.92, 4.42, and 15.15, respectively. Each of these values has a probability of < 0.05. This indicates that the model reported in Chapter 3 describes the mode of inheritance of nodulation for this population better than the model reported by Nigam et al. (39)

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88 Testa The and F2 data (Table 6-3, Entries 1, 2, and 10, and Table 6-5) provide evidence that pink testa color is dominant to red and supports the reports by Ashri (5, 7). Harvey (28) found that purple was incompletely and monogenically dominant to pink and is controlled at the locus. The and F2 results (Table 6-3, Entries 7 and 8, and Table 6-6) also indicate that purple is incompletely dominant to pink. However, the F^ data do not fit the 1:2:1 ratio for purple: light purple: pink that would be expected. This may have been because it is often difficult to distinguish between the purple and light purple phenotype. The F^ data (Table 6-3, Entries 3, 11, and 12) indicate that purple testa color is also dominant to red. The F2 data (Table 6-7) are not significantly different from the 1 red: 15 pink: 48 purple ratio. This ratio can be explained by the segregation of three independent loci.P, and R^. The and R2 loci have been described (5, 7, 28) but the R^ has not. The R^ locus has the same type of gene action as the R2 locus with the R^ allele producing pink testa and being dominant to the r^ allele, which produces red testa. Additional studies are needed to substantiate the presence of the R^ locus. The F^ and F2 data (Table 6-3, Entries 1, 2, 3, 9, 10, and 12, and Table 6-8) indicate that testa variegation is incompletely dominant to solid color as previously reported (11, 12). For some F2 plants which produced testa with trace-variegated seed, the variegated area on the seed was difficult to detect and could not be seen on all the seed. Because some plants that produced seed with trace-variegated

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CM ^ 1 CO >4-l CO CD O )-i o c o in CJN CM o o I I in o o o in m O r>. o -ao -3
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0) PL, u o 1 1 u 1 4J M-t CO CNl O (U X 1 4J (0 M 0) X LM o o 00 u fo u (U (0 O 0) M 4J 3 o 0) C/3 M (d O" go 1 f* +0) (U iH J3 (X 4J • u CO •H (1) u 3 & rH l-M rH to >-i U O rH O 3 o a iH OO to MH O en O t to M (1) • 4-1 C H c o CO -H T-t c o 3 O H CO S3 (U rH o. u 00 •rl • rH O 00 T3 d C (U CO 3 cr ^ a a c (U OJ 4J CO 3 •H CO rH u 4J a 01 II CM CO CO Oi (U .o C5 in 4-1 rH o 60 3 O, U
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0 LP) 0 a) 0^ r* jj P-I 1 1 1 1 Q 0 M O CTi m CM 0 u • • >4-l 0 1 t *H 4J 1 ID pn CM u CN rH 1 — r-H vD 0) r\ \J j3 1 iH in iH 4J W Q) CO 4J C)0 fll (0 0 -< Q) 4J ^ 4J T5 vD CM CM T3 N 0) 60 X pL Q CjO COS 4J (U a) CO CJ 60 ( 00 )^ Q) u cfl 4H CJ lA CO H 14H CM& CO (14 (1) x; CO •3 4J CO a) CO > CO rH 0 4-1 a a; 1 0^ CM CM CO CO VO CO 0 a\ CTv (U XI CO CN in 4J 0 0) 0 \o
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"1 c^ LO t 1 lO CM P-i 1 •ri 1 1 1 1 ^ 1 1 1 1 1 1 1 1 ii t U 1 • CM lO 1 1 Cs] O O CM rH *W V • f\ \J 1 1 w (A w CO fit w csj 00 CO LO On nO fM *^ r*^ CM rH rH CM 00 *^ W rH O *H CO rH CO CM w • 4J (A w w Lj JJ 1 \ n [ U vu •ri ro i~H LO CO rH CM CO CM 0!) *H m (ft (11 w • M w rH CiO t! CO CO rr! 1 fll 1 Ui' •H •H r; 1 H O CO 4J 03 to ON vO CO 4J 4J 00 CO >f •J 1 LO 111 CO CM *H Cd (11 £ § 4J 6c Cd w W3 fll vU 4-1 i_i w 4-1 r-i Q CO ^rl Q) > \J tl dt •H CO 00 CO CO 00 lO LO cd ^ CO iH CM rH ^ o 1^ w o. M O (11 >Jh O tH M-l 4-) 11 II Cd Q W Q w Q w 00 4-{ u l-l 0) M-l -a c •o Cd 0) > 13 u 00 m o CO o
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93 seed were probably classified as solid, the two classifications were combined when the F2 data were analyzed. The F2 segregation of PI 262090 X PI 445923 were significantly different from the expected frequency. This was probably caused by some of the heterozygotes ( Vv ) being classified as variegated. Further studies would need to be conducted to determine if modifier genes or some other factor may be causing the deviation from the 3:1 ratio. The other populations in Table 6-8 were not significantly different from the expected 3:1 ratio. For testa color and variegation segregation in the F^, both the pooled and homogeneity chi-square test had probabilities below 0.025 when all four F^ families were tested (Table 6-9) When the family with the highest chi-square value was omitted, the probabilities were in the acceptable range. The family that was omitted had an excess number of plants with red variegated seed. This could have been caused by sampling error, but further study is needed to explain the abnormal segregation observed in this family. The results from the other three families indicate that the R2 and V loci segregate independently, supporing the results of Branch and Hammons (12) and Chapter 5. Table 6-10 provides evidence that the four loci R^* and V segregate independently. This also supports the data presented in Table 6-9. In Chapter 4, it was shown that the and V loci are linked. The data presented in Table 6-11 indicate that the N2 and V loci are independent. This would be expected because and N2 are also independent.

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o LO o o • • • • o o O o •H 1 1 1 1 ^-i in o 1 O O CN LO 4-1 • • • • O o o o o 1 CO CO 1— 1 in dl 4-1 O v£) 00 to ct CD • • • (U o CN o 4>J CN o bO CN CnI CO CT> ro vD u •H r-t o u 4-1 o rH 4-1 (U CO >l o iH iH 0) a!) 0) V4 .H o CO rH o 4J 13 3 U o a o S C o O o o o o o (U CO c/a H Oh PC H 35 CO CO 0) t-4 .-HiH ft* -H e CO CO o u u tHCjN 4^ o ro 00( mm Pi td OW CN C7^ ro CN O m moo u-i in (T> m iH u-i in wow CO . a c (U 0) 4-1 3 4J cr (U o 4-1 d "O (U (U 0) 4J XI o 0) CO a. CO X w CO CiO Qi o 0) U c •H CO >-i 3 3 CO cr a* > CO 0) • 1 II XI -H > XJ a 3 73 CJ Q) C 4-1 -H CO bO (U e CO 4-1 x) 0) rH (1) -++eoo COS •

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X eg X T3 P. U 3 ++ CO 4J C CO 4-1 o o O U-l o in 1 rH • • O o o o 1 1 1 1 Qj o m o rH 1 m o in o d o d 3 & n 00 o in in CO r-l O in in • • • • • • r-H CM 00 o iH rH o O in m o iH o u M QJ iH d) oO Q) oO 4J CO 1 — I O Cfl rH O H iH n) ve 13 M fie ate bse •H 60 O CO 0) CO •H II cfl rH CO o O > 1 >. (U 0) O 4J U o •H •H CO 4J QJ > u CO C 4-1 V4 0) C>0 CO T) x: c QJ i 4J CO Ct Ho CO T3 4-1 •H II CO rH CU o w 4-1 CO Crt CO II O (U CO II B rH Pi o H o w

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c n3 o Q) -H N JJ >^ tfl rH 60 to ^ to a C -u (U C s o XT "O (u c )-i 0) <4-l (U (U C 4-1 -H CO 3 x; •n 4J to S 0) O j: -h ^-| u to u •H > (U 4J U (U CI. 4J >i CO (U o 3 (U O 4-> c o C (0 4J •H C >4-l O I •H <4-i 4-1 O to I 60 CO O o to 60 4J CO U eu o o CJ o CO X c o CM M T3 to c CO to 3 60 tr Q) CO > M I 60 -H 0) (U JS £ CO O 4J I to H C301 U 14-1 CO CO CO CO (U •H CO CO o CJ c to ex. 14-1 O o c O lO • • o o I I in o CD O CVI 00 < vD i-H <• in lA o tO) -o tH on ro tx) fX) riH iH vo rH tH rH fO (30 sr O rH CO <3rH 1^ CM fO rH rH rH O c II >4H > •o en O in m St > M (U 0) 4J CO CO XI 60 O •n 0) II U O <: w CO o > 1 (U o 4J o •rt •H CO 4-1 (U u to a IH (U 60 TJ o c (U a to 4J o O HI •a (U O <• •H II o\ CN rH O o w CM m CO 60 vO II CM X II i O M M CO u 33 P4 +++ too o c Q} 3 cr , 60 O (u c •H Q) 3 CO cr > 0)

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97 The N2 and R2 loci segregated independently (Table 6-12) This supports the data reported in Chapter 5 which indicate that N^, N^, and R2 segregated independently. The data presented in Table 6-13 provide evidence that N^, N^, and segregated independently and Table 6-14 provides evidence that P^, R^* ^'<^^ ^2 regated independently. In summary, the inheritance of nodulation in peanut was investigated using a non-nodulating line derived from the non-nodulating germplasm identified by Corbet and Burton (24) two non-nodulating lines from ICRISAT, and three lines with normal nodulation. The results in the and F2 generations do not fully support the mode of inheritance proposed by Nigam et al. (39) or in Chapter 3. Further studies will need to be conducted to fully elucidate the mode of inheritance of nodulation. Evidence was also presented that there is another locus, R^, which controls testa color with pink being dominant to red. Also, the following groups of loci were found to segregate independently: (R2 V), (P, R2, R^, V), (V, N2) (R2, N2) (P, N^, N2), and (P, R2, R3, N2).

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bK O n *^ 4J QJ 00 Q) N % C CJ (U g X r-l C T3 o CNl Q) c3 3 4-l o C; CO 4-1 d H C CO CM O QJ *H 4-1 C 4J Cd CO OJ +too M c^j a) CO 2 w U 3 tX) CT* 4-4 CM r-( en 1 CM VD CO C3^ CO lO (U o u ^ u CO M H in o I o m o C3^ O I m o o O CN CN 13 (U rH O O PM ++ O 6 o PC in m vD o o o CSI o in CO CO m rH o o < w o <3\ O CN CM

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•H (U U 4-1 ^ O cr T3 C 0) 0) o u N •H >^ '*-• iH rt • (0 t>0 C 0) (0 M Cfl 00 >^ 01 C N U (0 o X c 9 C to cr 0) (U 14-1 QJ -o 4J H c N a X Q) T) o •u C to -H 3 3 o •nx: Cfl CO -H 13 Q) J2 O c 4J -H •H U Ph JS CO 1-1 4J M to •H 0) iH o & C 4J u O O 3 •H QJ to >< iH Q) CO a ^ 4-1 -o 4-l U O 1 (d m 4J CO c CO 0) •H 0) C PU o o • c +M O -H 1 (1) O 60 O c rH <+-( O o O^1 rH U C O 3 O l PU Ph •H 4J AJ -a to to c 60 0) to 60 Q) CMI CO 0) )-i 2 Q) to to •H 3 rH o •H c to Z 1 to U-l CO i-H 1 vO CO CO 0) o iH u to H in (U CN rH • • P. o o tH 1 1 3 o o a rH in • • • >> o o s: o 4-1 3 OJ m e 3 rH rH >-i CT* • • • o OJ 14H U rH DH o 4J m in o 0) rH rH 4J (U O x: OJ CO) 4J fr O • OJ rH CU 60 60 to f— ^ rj o 4-) o § 4J II O o O H p^ X T3 u tu & 3 O u -tf r00 0^ U 3 rH CM CS CM 60 0) 3 (U cr 0) j_i <4H CM CM VO 01 "3 -Jr 00 rH 0) o. 4J u CO 3 3 a •a 4J < rH ro <^ CN x: 60 II H rH <: -a S >^ ro rH o VO to CJ rH rH C 0) OJ rH 3 O. cr M OJ 3 u & IM rH VO CM CO •3 to > •a u 0) OJ •H CO CO CSl 00 vO iw X5 rH •H O to CO II to h+ Pi iH o O U o < W OJ O 4J u •H ^ OJ 4J 0) to c ro U 0) 4J 60 to T) O OJ a 4J 4J O O BC to OJ 4J a II fU CM CD X C 0^ 0) • w • c m 4J CO 60 3 CO II o M OJ CO ^ e O x: rH Pi o rH M H O W PC b PL( -i— ++e05

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cr Q) u CO 0) 4J X U-1 o ON o> • • • CO o o CD 1 1 CO o in iH ON O • o • o 0) r-( QCM in U o CM 3 • • • o • rH (1) X O U C < 00 VO Q) CM iH B 3 •T3 COO ij a* O 0) iH OJ n) iH o 4-1 o e O o o o JJ 13 H (U 1-1 .u (U U ^ CU uj a vO O O 0\ 00 00 00 Cd o o o in ro CO ro CM Cvj CNJ CM CM CO 00 iH 00 o 4-1
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CHAPTER 7 SUMMARY AND CONCLUSIONS The inheritance of nodulation and its association with genes controlling testa color were investigated on peanut ( Arachis hypogaea L.). A diallel cross was made using M4-2, a non-nodulating line, and three nodulating peanut lines, PI 262090, UF 487A, and 'Florunner.' Selected plants were backcrossed to M4-2 or PI 262090. The F^, F^, F^, F-j^BC^, and ^2^^i S^^^rations were field grown at the University of Florida Agricultural Research Center, Marianna, Florida. Nodulation classifications were determined by observing plants and rating roots on each plant from 0 (no nodules) through 5 (abundant nodules) Pod samples were taken and testa color was evaluated. These data were analyzed by chi-square test for goodness-of-f it to the proposed model. The results indicate that inheritance of nodulation is controlled at two loci, N^ and N2. The non-nodulating genotype (M4-2) is n^n^n2n2, and all other genotypes have normal nodulation, except n^n^N2n2 which had few nodules when the parental male gamete was n^n2. The locus controlling testa variegation, V, was found to be linked to N^ with an average crossover rate of about 10%. Testa variegation color is controlled at one locus. The allele causing white variegation, Wv is dominant to the allele causing tinted variegation, wv. The N2 and Wv loci segregated independently. The R2 locus, which controls red and pink testa color, segregated independently from the V locus. It was also determined that the N^, N2, and R2 loci segregate independently. 101

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102 In another study, two non-no dulating peanut lines, PI ^45923 and PI 44592A, were crossed with M4-2, PI 262090, UF 487A, and Florunner. Data were collected from the and F2 generations for nodulation and testa color. The results do not fully support the model for inheritance of nodulation described in the first study. The allele causing purple testa color, P^, appeared to be dominant to pink and red. There also appeared to be a duplicate locus of R2 which was designated R^. The following groups of loci were found to segregate independently: (R2, V), (P, R^, R^, V), (V, (R2, N2) (P, N^, N2), and (P, R2, R3, N2).

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LITERATURE CITED 1. Allard, R. W. 1960. Principles of plant breeding. John Wiley and Sons, Inc., New York. 2. Allen, 0. N., and E. K. Allen. 1940. Response of the peanut plant to inoculation with rhizobia, with special reference to morphological development of the nodules. Bot. Gaz. 102:121-142. 3. Ashri, A. 1964. Intergenic and genic-cytoplasmic interactions affecting growth habit in peanuts. Genetics. 50:363-372. 4. 1968. Genic-cytoplasmic interactions affecting growth habit in peanuts, A. hypogaea II. A revised model. Genetics. 60:807-810. 5. 1969. A second locus controlling red testa in peanuts, Arachis hypogaea Crop Sci. 9:515-517. 6. 1970. A dominant mutation with variable penetrance and expressivity induced by diethyl sulfate in peanuts, Arachis hypogaea L. Mutation Res. 9:473-480. 7. 1970. Further evidence for a second red testa gene in peanuts, A. hypogaea L. Oleagineux. 25:393-394. 8. 1976. Plasmon divergence in peanuts ( Arachis hypogaea ) ; A third plasmon and locus affecting growth habit. Theor. Appl. Genet. 48:17-21. 9. Azizi, M. R., and D. K. Barnes. 1977. Characterization and inheritance of a spotted leaf trait in alfalfa. Crop Sci. 17:126132. 10. Bhuvaneswari T. V., A. A. Bhagwat, and W. D. Bauer. 1981. Transient susceptibility of root cells in four common legumes to nodulation by rhizobia. Plant Physiol. 68:1144-1149. 11. Branch, W. D. and R. 0. Hammons. 1979. Inheritance of testa color variegation in peanut. Crop Sci. 19:786-788. 12. and 1980. Inheritance of a variegated testa color in peanuts. Crop Sci. 20:660-662. 13. Caldwell, B. E. 1966. Inheritance of a strain-specific ineffective nodulation in soybeans. Crop Sci. 6:427-428. 103

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104 14. K. Hinson, and H. W. Johnson. 1966. A strain-specific ineffective nodulation reaction in the soybean Glycine max L. Merrill. Crop Sci. 6:495-496. 15. Carroll, T. W., P. L. Gossel, and E. A. Hockett. 1979. Inheritance of resistance to seed transmission of barley stripe mosaic virus in barley. Phytopath. 69:431-433. 16. Chandler, M. R. 1978. Some observations on infection of Arachis hypogaea L. by Rhizobium J. Expl. Bot. 29:749-755. 17. Coffelt, T. A., and R. 0. Hammons. 1974. Inheritance of pod construction in peanuts. J. Hered. 65:94-96. 18. Crouse, H. V. 1960. The controlling element in sex chromosome behavior in Sciara Genetics. 45:1429-1443. 19. Duggar, J. F. 1935. The effects of inoculation and fertilization of Spanish peanuts on root nodule numbers. J. Am. Soc. Agron. 27:128-133. 20. 1935. Nodulation of peanut plants as affected by variety, shelling of seed, and disinfection of seed. J. Am. Soc. Agron. 27:286-288. 21. Gaur, Y. D., A. N. Sen, and N. S. Subba Rao. 1974. Problem regarding groundnut ( Arachis hypogaea L.) inoculation in tropics with special reference to India. Proc. Ind. Nat. Sci. Acad. Part B, Biol. Sci. 40:562-570. 22. and 1974. Promiscuity in groundnut Rhizobium association. Zbl. Bakt. Abt. II, Bd. 129:369-372. 23. Gibson, A. H. 1964. Genetic control of strain-specific ineffective nodulation in Trif olium subterraneum L. Aust. J. Agric. Res. 15:37-49. 24. Gorbet, D. W., and J. C. Burton. 1979. A non-nodulating peanut. Crop Sci. 19:727-728. 25. Gottschalk, W. 1978. The dependence of the penetrance of mutant genes on environment and genotypic background. Genetica. 49:21-29. 26. Hammons, R. 0. 1973. Genetics of Arachis hypogaea p. 135-173. I In Peanuts culture and uses. Am. Peanut Res. Educ. Asso., Stillwater, Okla. 27. Harris, R. E., R. H. Moll, and C. W. Stuber. 1976. Control and inheritance of prolificacy in maize. Crop Sci. 16:843-850.

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105 28. Harvey, J. E., Jr. 1967. Testa color inheritance in peanuts ( Arachis hypogaea L.). Ph.D. Thesis. Univ. Georgia. Univ. Microfilms, Ann Arbor, Mich. (Diss. Abstr. 19:1146). 29. Hayes, T. R. 1933. The classification of groundnut varieties. Trop. Agric. 10:318-325. 30. Holl, F. B. 1975. Host plant control of the inheritance of dinitrogen fixation in the Pisum-Rhizobium sjnnbiosis. Ephytica. 24:76 7-770. 31. Isleib, T. G., J. C. Wynne, G. H. Elkan, and T. J. Schneeweis. 1980. Quantitative genetic aspects of nitrogen fixation in peanuts ( Arachis hypogaea L.). Peanut Sci. 7:101-105. 32. Iswaran, V., and A. Sen. 1974. Some studies on the groundnut Rhizobium symbiosis. Zbl. Bakt. Abt. II, Bd. 129:477-480. 33. Kutzelnigg, K. and W. Stubbe. 1974. Investigations on plastome mutants in Oenothera 1. General considerations. Sub-cell. Biochem. 3:73-89. 34. Lin, Bor-yaw. 1978. A differential effect on kernal size of maternal and paternal forms of a chromosome region, p. 776. In D. B. Walden (ed.) Maize breeding and genetics. John Wiley and Sons, New York. 35. Loesch, P. J., Jr. 1964. Effect of mutated background genotype on mutant expression in Arachis hypogaea L. Crop Sci. 4: 73-78. 36. McClintock, B. 1967. Genetic systems regulating gene expression during development, p. 84-112. In M. Locke (ed.) Control mechanisms in developmental processes. Academic Press, New York. 37. Mouli, C, and S. H. Patil. 1975. Paternal inheritance of x-ray induced foliaceous stipule in the peanut. J. Hered. 66:28-30, 38. Nambiar, P. T. C, P. J. Dart, B. Srinivasa Rao, and V. Ramanatha Rao. 1982. Nodulation in the hypocotyl region of groundnut ( Arachis hypogaea ) Expl. Agric. 18:203-207. 39. Nigam, S. N., V. Arunachalam, R, W. Gibbons, A. Bandyopadhyay and P. T. C. Nambiar. 1980. Genetics of non-nodualtion in groundnut Arachis hypogaea L. Oleagineux. 35:453-455. 40. Nilsson-Tillgren, T. and P. von Wettstein-Knowles. 1970. When is the male plastome eliminated? Nature. 227:1265-1266. 41. Norden, A. J., and V. A. Rodriguez. 1971. Artificial hybridization of peanuts. Oleagineux. 26:159-162.

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106 42. Nutman, P. S. 1949. Nuclear and cytoplasmic Inheritance of resistance to infection by nodule bacteria in red clover. Hered. 3:263-291. 43. 1954. Symbiotic effectiveness in nodulated red clover. I. Variation in host and in bacteria. Hered. 8:35-46. 44. 1954. Symbiotic effectiveness in nodulated red clover. II. A major gene for ineffectiveness in the host. Hered. 8:47-60. 45. Parker, R. C, J. C. Wynne, and D. A. Emery. 1970. Combining ability estimates in Arachis hypogaea L. I. F^^ seedlings responses in a controlled environment. Crop Sci. 10:429-432. 46. Patel, J. S., C. M. John, and C. R. Seshadri. 1936. The inheritance of characters in the groundnut — Arachis hypogaea Proc. Ind. Acad. Sci. 3:214-233. 47. Patil, S. H., and C. Mouli. 1975. Genetics of a dwarf mutant in groundnut. Theor. Appl. Genet. 46:395-400. 48. Peterson, M. A., and D. K. Barnes. 1981. Inheritance of ineffective nodulation and non-nodulation traits in alfalfa. Crop Sci. 21:611-616. 49. Prasad, S., and D. P. Srivastava. 1967. Inheritance of testa colour in groundnut ( Arachis hypogaea L.). Sci. Cult. 33:489-490. 50. Ratner, E. I., R. Lobel, H. Feldhay, and A. Hartzook. 1979. Some characteristics of symbiotic nitrogen fixation, yield, protein and oil accumulation in irrigated peanuts (Arachis hypogaea L.). Plant Soil. 51:373-386. 51. Resslar, P. M. and D. A. Emery. 1978. Inheritance of growth habit in peanuts: Cytoplasmic or maternal modifications? J. Hered. 69:101-106. 52. Rodriguez, V. A., and A. J. Norden. 1970. Inheritance of inner seed-coat color in peanuts. J. Hered. 61:161-163. 53. Schiffmann, J. 1961. Field experiments on inoculation of peanuts in northern Negev soils. Israel J. Agric. Res. 11:151-158. 54. and Y. Alper. 1968. Effects of Rhizobiuminoculum placement on peanut inoculation. Expl. Agric. 4:203-208. 55. and 1968. Inoculation of peanuts by application of Rhizobium suspension into the planting furrows. Expl. Agric. 4:219-226.

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107 56. Sen, D. and R. W. Weaver. 1980. Nitrogen fixing activity of rhizobial strain 32 HI in peanut and cowpea nodules. Plant Sci. Letters. 18:315-318. 57. Simon, P. W., and S. J. Peloquin. 1977. The influence of paternal species on the origin of callus in anther culture of Solan um hybrids. Theor. Appl. Genet. 50:53-56. 58. Sorrells, M. E., J. H. Lonnquist, and R. E. Harris. 1979. Inheritance of prolificacy in maize. Crop Sci. 19:301-306. 59. Staphorst, J. L., and B. W. Strijdom. 1972. Some observations on the bacteroids in nodules of Arachis spp. and the isolation of Rhizobia from these nodules. Phytophylactica. 4:87-92. 60. Stokes, W. E., and F. H. Hull. 1930. Peanut breeding. J. Am. Soc. Agron. 22:1004-1019. 61. Strickberger, M. W. 1976. Genetics. Macmillan Publ. Co., New York. 62. Tilney-Bassett, R. A. E. 1969. Predominantly paternal inheritance of plastids in Pelargonium zonale Hered. 24:687 (Abstr.) 63. Tonn, W. H., Ill, and R. W. Weaver. 1981. Seasonal nitrogen fixation and dry matter accumulation by peanuts. Agron. J. 73:525-528. 64. Van Der Merwe, S. P., B. W. Strijdom, and C. J. Uys. 1974. Groundnut response to seed inoculation under extensive agricultural practices in South African soils. Phytophylactica. 6:295-302. 65. Van Rensburg, H. J., J. S. Hahn, and B. W. Strijdom. 1973. Morphological development of Rhizobium bacteroids in nodules of Arachis hypogaea L. Phytophylactica. 5:119-122. 66. Vest, G., and B. E. Caldwell. 1972. R.,— A gene conditioning ineffective nodulation in soybean. Crop Sci. 12:692-693. 67. Walker, M. E., N. A. Mlnton, and C. C. Dowler. 1976. Effects of herbicides, a nematicide and Rhizobium inoculant on yield, chemical composition and nodulation of Starr peanuts (Arachis hypogaea L.) Peanut Sci. 3:49-51. 68. Whiting, A. L., and R. Hansen. 1920. Cross inoculation studies with the nodule bacteria of lima beans, navy beans, cowpeas, and others of the cowpea group. Soil Sci. 10:291-300. 69. Williams, L. F., and D. L. Lynch. 1954. Inheritance of a nonnodulating character in the soybean. Agron. J. 46:28-29.

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BIOGRAPHICAL SKETCH Kenton Eugene Dashiell was born in Noblesville, Indiana, on October 25, 1954. His parents are Robert and Rosemary Dashiell. He graduated from Elkhart High School, Elkhart, Indiana, in June, 1972. He received the Bachelor of Science degree in May, 1976, from Purdue University and the Master of Science degree in agronomy in December, 1979, from Oklahoma State University. He completed the requirements for the Doctor of Philosophy degree in agronomy in April, 1983, at the University of Florida. He was a Peace Corps volunteer, agronomist, Diamond Estate, Antigua, West Indies, from June, 1976, through August, 1978. He also served as a research assistant in the Department of Agronomy, Okalahoma State University, from September, 1978, through December, 1979, and as a graduate research assistant in the Department of Agronomy, University of Florida Agricultural Research Center at Marianna, Florida, from January, 1980, through February, 1983. He is a member of the Crop Science Society of America, American Society of Agronomy, American Peanut Research and Education Society, Phi Kappa Phi, Sigma Xi, and Gamma Sigma Delta. 108

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. D. W. Gorbet, Chairman Associate Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. A. J^Norden, Cochairman^ Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. j2l E. S. Horner Professor of Agronomy

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Professor of Soil Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. D. W. Dickson Professor of Entomology and Nematology This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. April 1983 Dean for Graduate Studies and Research