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PCR-Based Polymorphisms in Bermudagrass (Cynodon spp.)


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PCR-BASED POLYMORPHISMS IN BERMUDAGRASS ( Cynodon spp.) By NEIL RAY WILLIAMS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Neil Ray Williams

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I dedicate this thesis and the research it represents to Suzanne C. Williams who has supported my efforts and made possible all th at you see here through her sacrifice and loving support.

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ACKNOWLEDGMENTS I appreciate the contributions of my supervisory committee (Drs. Christine Chase, Brian Scully, Laurie Trenholm and Terry Kamps). I thank Victor Ortega and the Chase Lab; and Ginger Clark in the ICBR for their invaluable input and help. I also acknowledge the financial support of the Horticultural Sciences Department at the University of Florida; the G. C. Horn Fellowship, awarded by the Florida Turfgrass Association; the Davidson Travel Grant; and the Dade County Scholarship. I especially appreciate my family (Suzanne, Genavieve, Tyler Ian, and soon Collin Micheal Williams as well as mothers, fathers, brothers and sisters). I acknowledge the hand of Jesus Christ in this work. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.........................................................................................................................x CHAPTER 1 REVIEW OF LITERATURE.......................................................................................1 Problem.........................................................................................................................1 Introduction...................................................................................................................1 Breeding................................................................................................................4 Molecular Markers................................................................................................6 Isozymes.........................................................................................................7 Random Fragment Length Polymorphism (RFLP)........................................7 Random Amplified Polymorphic DNA (RAPD)...........................................9 DNA-Amplification Fingerprinting (DAF)..................................................10 Amplification Fragment Length Polymorphism (AFLP).............................11 Simple Sequence Repeat (SSR)...................................................................12 2 CROSS-TAXA PRIMERS APPLIED TO BERMUDAGRASS...............................22 Introduction.................................................................................................................22 Materials and Methods...............................................................................................24 Plant Material......................................................................................................24 DNA Preparation.................................................................................................25 Cross Taxa Primers..............................................................................................25 PCR......................................................................................................................25 PCR Product Sequence Analysis.........................................................................26 Results.........................................................................................................................27 Discussion...................................................................................................................29 3 SPECIES-SPECIFIC PRIMERS................................................................................49 Introduction.................................................................................................................49 Materials and Methods...............................................................................................51 v

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Plant Material......................................................................................................51 DNA Preparation.................................................................................................52 Enriched Library Construction............................................................................52 Primer Development............................................................................................54 Analysis...............................................................................................................55 Results.........................................................................................................................55 Discussion...................................................................................................................58 4 CONCLUSIONS........................................................................................................82 APPENDIX A INDIVIDUAL GENETIC SIMILARITY MATRICES FOR CROSS-TAXA PRIMER SETS ZMADH2N AND LPSSRHO1A10.................................................83 B NUCLEOTIDE SEQUENCE FOR FUTURE PRIMER DEVELOPMENT.............85 C INDIVIDUAL GENETIC SIMILARITY MATRICES FOR BER 1, BER9, AND BER11.........................................................................................................................91 LIST OF REFERENCES...................................................................................................94 BIOGRAPHICAL SKETCH.............................................................................................99 vi

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LIST OF TABLES Table page 2.1 Origins and uses of bermudagrass cultivars evaluated for simple sequence repeats.......................................................................................................................33 2.2 Origins of cross-taxa primer pairs used in PCR reactions on bermudagrass templates...................................................................................................................34 2.3 Basic step-down PCR programs used to test primer amplification..........................36 2.4 Sorghum bicolor cross-taxa amplicon sizes estimated in base pairs........................37 2.5 Lolium perenne cross-taxa amplicon sizes estimated in base pairs.........................38 2.6 Zea mays cross-taxa amplicon sizes estimated in base pairs...................................39 2.7 Genetic similarity matrix including combined data from primer sets LPSSRHO1A10 and ZMADH2N............................................................................40 3.1 Origins and uses of bermudagrass cultivars evaluated for simple sequence repeats.......................................................................................................................62 3.2 Cynodon spp.-specific primers developed from the sequencing of fragments in the enriched DNA library.........................................................................................63 3.3 Basic step-down PCR programs used to test primer amplification..........................64 3.4 BER1 PCR product length on 23 bermudagrass DNA templates from gel #20027 (Figure3.4)...................................................................................................65 3.5 BER9 PCR product length on 23 bermudagrass DNA templates from gel #20735 (Figure 3.5)..................................................................................................66 3.6 BER11 PCR product length on 23 bermudagrass DNA templates from gel #20763 (Figure 3.6)..................................................................................................67 3.7 Genetic similarity matrix including combined data from all BER primer sets........68 A.1 ZMADH2N genetic similarity matrix......................................................................83 A.2 LPSSRHO1A10 genetic similarity matrix...............................................................84 vii

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LIST OF FIGURES Figure page 1.1 Anatomical features of a grass stem and leaf section used in grass taxonomy and separation of one species from another....................................................................18 1.2 Two closely related turf varieties that appear similar..............................................19 1.3 Diagram of the PCR process by which small amounts of DNA can quickly be copied by DNA polymerase to produce large numbers of a single fragment..........20 1.4 Diagram of ideal SSR marker system showing fragments are visualized and how they can show allele inheritance...............................................................................21 2.1 SB4-22 primer pair amplification of 23 bermudagrass DNA templates..................41 2.2 SB6-36 primer pair amplification of 23 bermudagrass DNA templates..................42 2.3 LPSSRHO1A10 primer pair amplification of 23 bermudagrass DNA templates....43 2.4 LPSSRHO1AO7 primer pair amplification of 23 bermudagrass DNA templates...44 2.5 CLUSTAL W (1.81) alignment of LPSSRHO1AO7 amplification product sequences..................................................................................................................45 2.6 MZEPPDKA2 primer pair amplification of 23 bermudagrass DNA templates.......46 2.7 ZMADH2N primer pair amplification of 23 bermudagrass DNA templates...........47 2.8 Sequence of ZMADH2N amplification products from Figure 2.6 aligned by CLUSTAL W (1.81)................................................................................................48 3.1 Dot-blot hybridization of dilution series of enriched libraries to verify enrichment for (CA) n repeats...................................................................................69 3.2 Colony transfer and hybridization of initial bacteria transformation and hybridization to fluorescent probe to show transformation success rate.................70 3.3 Nucleotide sequences of the clones from which species-specific primers were developed.................................................................................................................71 viii

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3.4 BER1 amplification products of replicated PCR reactions using 23 bermudagrass DNA templates, fractionated on agarose gels, showing the reproduction of fragment length patterns...........................................................................................73 3.5 BER9 amplification products of replicated PCR reactions using 23 bermudagrass DNA templates, fractionated on agarose gels, showing the reproduction of fragment length patterns...........................................................................................74 3.6 BER11 amplification products of replicated PCR reactions using 23 bermudagrass DNA templates, fractionated on agarose gels, showing the reproduction of fragment length patterns...........................................................................................75 3.7 Products of BER9 primer pair amplification on bermudagrass DNA fractionated on polyacrylamide gels showing the amplicon pattern for the bermudagrass template shown at the top each lane.........................................................................76 3.8 Gel #16088-2, showing amplification of BER 1 on a different group of DNA templates...................................................................................................................77 3.9 Phylogenetic analysis of BER1 fragment length data from gel #20027 using PAUP..............................................................................................................78 3.10 Phylogenetic analysis of BER9 fragment length data from gel #20735 using PAUP........................................................................................................................79 3.11 Phylogenetic analysis of BER11 fragment length data from gel #20763 using PAUP........................................................................................................................80 3.12 Phylogenetic analysis of the collective fragment length data from Tables 3.5-3.7 using PAUP..............................................................................................................81 B.1 Nucleotide sequences of the clones from which future species-specific primers may be developed.....................................................................................................85 ix

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PCR-BASED POLYMORPHISM IN BERMUDAGRASS (Cynodon spp.) By Neil Ray Williams December 2003 Chair: Brian Scully Cochair: Chris Chase Major Department: Horticultural Sciences Hybrid bermudagrass is an important turf-type grass in Florida and other southern states. Cultivars with similar appearance, but differences in economic value-added traits, make genetic markers desirable. For this reason, a microsatellite DNA marker system that will provide immediate cultivar identification was studied. Two methods of primer development were used to identify microsatellite DNA polymorphisms. In the first, thirty-five primer pairs developed for other grasses in the Poaceae family resulted in only two patterns that reproduced reliably. In the second, a genomic library enriched for (AC) n repeats was developed and sequences were used for primer development. This method of microsatellite amplification provided three primer pairs that reliably amplified useful polymorphisms on bermudagrass DNA templates. x

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CHAPTER 1 REVIEW OF LITERATURE Problem Commercial, clonally propagated varieties of improved bermudagrass used for turf are not easily distinguished by morphological and physiological characteristics. Varieties exhibit only subtle morphological differences that are subject to environmental influences. While similar in appearance, varieties may differ significantly in cost because of reactions to drought, high foot traffic, fertilizers, and herbicides. Thus, distinguishing among varieties becomes important for enforcement of uniform commercial code (which states that people will receive what they have paid for). The purpose of this study was to develop a consistent, easily analyzed microsatellite marker system for identification of improved and hybrid bermudagrass varieties. This was attempted in two phases 1) the trial and use of published, cross-taxa microsatellite primers and 2) the development of new, species-specific primers. Introduction The genus Cynodon is a member of the family Poaceae; group Chlorideae. Cynodon contains nine species, all of which are characterized as sod forming, perennial, warm-season grasses with a broad economic influence in tropical to transitional regions (Beard 1973; Taliaferro 1995). The turf-type species of interest to this study are Cynodon dactylon (L.) Pers (C. dactylon); Cynodon transvaalensis (Burtt-Davy) (C. transvaalensis); and their hybrids 1

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2 Cynodon dactylon, known in the U.S. as common bermudagrass, is a tetraploid with a chromosome number of 2n=4x=36. It is widely distributed between 45 o N and 45 o S latitudes with some cold-hardy varieties reaching farther north (Taliaferro 1995). It is generally characterized by a folded vernation, no auricle, and a fringe of hairs on the ligule, using the diagram in Figure 1.1 to reference these features. It is rhizomatous, stoloniferous, and produces seed excessively on 4-5 digitate spikes raised above the leaf canopy. Specifically, leaf character is highly variable, with color ranging from light to dark green; and texture ranging from medium to coarse. Leaves turn light brown to white during dormancy; and the species tolerates neither shade nor low temperatures well. It does tolerate drought, heat, and high foot traffic; and forms an aggressive sod on a wide range of soil types. The strong, creeping growth habit makes bermudagrass a weed in ornamental beds (and with turfgrasses of finer textures). Because of its versatility and specifically selected enhancements (such as durability, drought tolerance, and uniformity of leaf texture), C. dactylon is fit for use as a turfgrass; or for utility, soil stabilization, or low-maintenance playing fields and golf course roughs (Beard 1973). The African Bermudagrass (C. transvaalensis), is a diploid species with a chromosome number of 2n=2x=18 (Beard 1973). It is confined to the South African Transvaal, its center of origin (Taliaferro 1995). General characteristics resemble C. dactylon, but leaf color is light green to yellow with fine, dense leaf texture. It spreads by rhizomes and stolons, although less aggressively than C. dactylon; and seed production (though poor), is also above the canopy. In lower temperatures, the yellow-green leaves and stolons turn reddish-purple (Beard 1973). Because of uniform morphology maintained within the varieties and its ability to breed interspecifically with C. dactylon,

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3 it has become important in the U.S. turfgrass industry, where it is maintained specifically for breeding purposes. In Africa it is used as a turfgrass and for forage (Beard 1973; Taliaferro 1995). The hybrid of C. dactylon X C. transvaalensis is a sterile triploid with 2n=3x=27 chromosomes. Hybrids are used in the southern US and in subtropical climates throughout the world that do not drop below -12 o C during dormancy. Cold is the main limiting factor to bermudagrass success (Beard 1973; Taliaferro 1995). Morphologically similar to C. dactylon (auricles, ligules, etc.), hybrids can be selected with a combination of desirable turf traits from the two species (Taliaferro 1995). It spreads by rhizomes and stolons more quickly than C. transvaalensis, but has shorter internodes and denser growth than C. dactylon. Mowing heights vary by cultivar and use, ranging from below to 1". In Florida it is preferred by golfers because it provides a smooth playing surface; and by golf course superintendents because it keeps a medium to dark green color during dormancy, eliminating the need to overseed. It is also improved to meet the needs of the golf course industrys demanding requirements for salt tolerance and lower fertilization rates (Busey and Dudeck 1999). It is intolerant to shade, and root systems are shortened by low mowing, making it susceptible to drought stress when not properly maintained. These triploid hybrid varieties are propagated vegetatively from root and shoot cuttings (Taliaferro 1995). Hybrid bermudagrass is used extensively on golf courses and athletic fields, but because of its medium to higher maintenance requirements, bermudagrass is not recommended for home turf (Beard 1973). Hybrid and improved bermudagrass is an economically important grass in the state of Florida. According to the 2000 economic report, 59 million rounds of golf were

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4 played on the 1,334 golf courses statewide. Golf courses cover 207,582 acres of the state and were responsible for $4.4 billion in revenue (Haydu and Hodges 2002). Forty-one of these golf courses were new, supporting the $307.2 million sod industry (Haydu et al. 2002) that covers 80,347 acres of state and private land (Haydu et al. 2002). Bermudagrass accounts for over half of the golf course grasses or 136,000 acres (Hodges and Haydu 2002) and covers 6% of the sod industry production acreage, or 4,556 acres total. Turf bermudagrass garnered $16.3 million at $0.114ft -2 which accounted for 19% of the sod industry revenue that year (Haydu et al. 2002). Breeding Breeding and selection of bermudagrass has been almost continuous over the past century. Because of the genetic variability of C. dactylon (and its wide geographic adaptability), seeded varieties (both named and unnamed) are common. Cynodon transvaalensis selection has been centered in South Africa, from which much of the first germplasm for hybrid bermudagrass was obtained through cooperative breeding efforts between Africa and the U.S. (Busey and Dudeck 1999; Taliaferro 1995). Busey and Dudeck (1999 pg. 97) describe both seeded and vegetatively propagated bermudagrass as a complex of interbreeding species undergoing rapid evolution through natural and human intervention. Developments in seeded varieties have yielded grasses that are more cold tolerant, have lower fertilization requirements, are finer textured, and provide more uniform plant quality than the wild relatives. Cynodon dactylon is currently the main source of seeded varieties in the U.S. (Taliaferro 1995). Numex, Sahara, and Mirage are a few of the limited number of seeded bermudagrass varieties raised in the Southern U.S. Seeded varieties are more vigorous and more heterogeneous than hybrids, making encroachment and contamination by unimproved varieties less of a

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5 problem (Busey and Dudeck 1999). Other C. dactylon varieties are released for vegetative propagation although they do produce some seed. Examples of these are Floratex (Trenholm et al. 2000), GN-1 (Greg Norman Turf), and Tiflawn (Hein 1953). Hybrid bermudagrass is the result of the interspecific cross of C. dactylon X C. transvaalensis, which provides uniformity of leaf texture, color, foot-traffic resistance, and winter color that are of great value to athletic fields and golf courses. Hybrids used for golf courses are blocked into two major selection groups 1) greens grasses and 2) fairway grasses. Much of the original turf-type Cynodon spp. material was imported from South Africa in the 1940s and 1950s (Taliaferro 1995). The crossing of vigor and variable green colors with fine texture and uniform performance has produced grasses that are fine and short enough for closely mown greens. Other varieties grow vigorously with coarser-textured leaves and are better adapted to well-traveled fairways and football, soccer, and baseball fields (Beard 1973). Tifgreen (Hein 1961) was the first such hybrid developed by the USDA-ARS in Tifton, Georgia; followed by Tiffine (Busey and Dudeck 1999) in 1953; and Tifway 419 (Burton 1966b) in 1960. Interspecific crosses resulted in Midiron (Anderson et al. 1988) from Kansas State University, and MS Choice (Krans et al. 1995) from the Mississippi Agriculture and Forestry Experiment Station in 1995. Tifgreen and Tifway are interspecific hybrids that were subjected to induced mutation by gamma irradiation, and resulted in Tifgreen II (Busey and Dudeck 1999) and Tifway II (Burton 1985) respectively. Tifway II was then irradiated to produce TifEagle (Zhang et al. 1999). Midiron was also irradiated and Tifsport (Haring 1998) was selected. Natural mutation, contamination, or somatic variations have given rise to Tifdwarf (Burton 1966a) and MS Supreme (Krans et al. 1995) from Tifgreen. Each of

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6 these mutations have been compared to existing varieties and selected based on foot traffic resistance; shade tolerance; fertilizer, water, and mowing requirements; and winter hardiness and leaf color and texture. Taliaferro (1995) discussed the decreasing diversity of germplasm that may lead to disease problems if not addressed. A lack of readily visible morphological variability presents another problem within categories of grasses (Figure 1.2). While the varieties appear similar, their reaction to drought, wear, fertilizers, and herbicides can be sufficiently different (warranting significant differences in cost). As a limited genetic base of parent material is used to produce new cultivars, homopheneity is unavoidable. For this reason, technology using a genetic marker system was sought and the various systems were compared and analyzed for usefulness in this task. This will help enforce uniform commercial code, which protects the buyer from receiving something other than what he or she paid for. Molecular Markers There are many types of marker systems available to the researcher in the molecular world, but we will discuss two categories: those not based on the polymerase chain reaction (PCR); and PCR-based systems. Molecular markers not based on PCR include isozymes (which show protein polymorphisms) and restriction fragment length polymorphisms (RFLPs), which are DNA based. PCR-dependent markers include random amplified polymorphic DNA (RAPD), DNA amplification fingerprinting (DAF), amplified fragment length polymorphism (AFLP), and simple sequence repeats (SSR) also known as microsatellites. The purpose of these systems is to detect polymorphism (mutation) within the genome of an organism. All rely on electrophoresis for separation of polymorphic proteins or DNA fragments.

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7 Isozymes Isozyme markers use the positive and negative charges in the amino acids as the driving force for separation. Because protein molecules of the same size differ in charge because of one or more amino acid differences, plants with differing polymorphisms may be distinguished (Hartl 2000). Sample preparation is as simple as lysing cells to release the proteins from the cytoplasm. These cellular contents are then fractionated by a gel matrix in the presence of an electrical current and stained for enzyme activity. Vermeulen et al. (1991) ran starch gel electrophoresis on crude protein of 21 bermudagrass cultivars that were not closely related and successfully distinguished all of them. Other studies were able to distinguish 7 forage type bermudagrass varieties (Dabo et al. 1990), indicating that isoenzymes could be used to identify different cultivars. Concern was raised because of the plant growth/environment influence associated with this technique. There is no apparent connection between morphological characteristics or botanic species and isozyme banding patterns in bermudagrass (Vermeulan et al. 1991). There are fewer protein polymorphisms than DNA polymorphisms because replacement of an encoded amino acid is required for polymorphisms to be seen. Furthermore, isozyme analysis is restricted to proteins that can be identified by an activity stain. Protein polymorphisms are also more difficult to interpret because a change in amino acid sequence may reflect a number of DNA mutation events (Hartl 2000). Random Fragment Length Polymorphism (RFLP) RFLPs are DNA markers based on the premise that some DNA molecules in a population contain a particular restriction site, whereas others lack it because of mutation (Hartl 2000). This polymorphism is shown by placing highly purified nuclear DNA in

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8 contact with an enzyme recognizing a sequence of 4-8 nucleotides. Fractionating the fragments on a gel and doing a southern blot (Hartl 2000) allows hybridization of a radio labeled or fluorescent probe. The probe will reveal a pattern of DNA fragments based on the number of restriction sites present or absent along its sequence or flanking sequence. Xu and Sleper (1991) applied RFLP analysis on Festuca spp., using 174 cDNA, species-specific probes to differentiate between cultivars. With this information, a linkage map was developed that provides an indirect selection tool for agronomic traits. From the development of these probes, a cross-taxa experiment involving Lolium perenne (L.) (L. perenne) was conducted with 2/3 of Festuca spp. (L.) probes cross-hybridizing to L. perenne DNA. Of the probes that successfully hybridized, 69% were polymorphic on 5 genotypes (Xu et al. 1992). This study shows the successful application of cross-taxa RFLP work in gene tracking through interspecific and intergenic hybridization and breeder selection (Xu and Sleper 1993). RFLPs require sufficient highly purified genomic DNA from each of a large number of individuals. They also require a library of probes, and radio-labeling for the most sensitive detection. For these reasons many researchers have turned to PCR based methods (Reiter 1994). PCR utilizes oligonucleotide primers as priming sites for polymerase from Thermus aquaticus to begin the in vitro amplification of DNA (Figure 1.3). Using nucleotides, magnesium, and repeated heating and cooling cycles to copy a small segment of the genomic DNA, one molecule of template DNA can be amplified into microgram quantities within hours. PCR itself is technically simple, because reagents are easily mixed and heating is done by a programmed machine. Primer information can be

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9 transferred from one lab to another without the need to transfer live cultures or libraries. The transfer of sequence information and the ease of oligonucleotide construction increase the speed of information transfer (Reiter 1994). Random Amplified Polymorphic DNA (RAPD) RAPD is a sequence-arbitrary PCR method used in genetic analysis, which was developed by two groups simultaneously (Welsh and McClelland; 1990Williams et al. 1990). RAPDs use short (10 mer) oligonucleotide primers in the PCR reaction allowing multiple amplicons when a primer pair binds in opposite orientation in close proximity. When analyzed by gel electrophoresis, a fairly simple fragment pattern is detectable after ethidium bromide staining. The fragment length patterns of individuals are compared and differences or similarities noted (Reiter 1994). RAPD markers have been used for a variety of studies in turf and other grasses. Huff et al. (1993) showed RAPD variation within and among natural populations of Buchloe dactyloides (Nutt.) Engelm, an out crossing species, by means of statistical evaluation to eliminate within population polymorphism. Research has been done following migration in Triticum dicoccoides (L.) (Fahima et al. 1999) and also in Poa annua (L.) (Sweeney and Dannegerger 1995) using RAPDs to disprove isolation theories and to follow gene flow. Golembiewski et al. (1997) used RAPDs to test Agrostis spp. (L.) bulk seed samples and were able to identify most with assurance. This information is useful for researchers, breeders, and seed producers who are interested in more precise monitoring and control of germplasm sources in open-pollinated varieties. Sweeney and Dannegerger (2000) tried to modernize RFLP by combining it with RAPD on L. perenne, first amplifying RAPD products and then performing RFLP analysis on these products.

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10 While theoretically it was possible to obtain more information, only one reliable probe was discovered. Of particular interest to the present study, Busey et al. (2000) attempted the use of RAPDs to distinguish off-types within the variety Tifdwarf. Analysis was sensitive enough to distinguish between Tifdwarf and a contaminant of different morphology, proving that it was contaminated and not merely mutated. Tifdwarf and Tifgreen proved to be too closely related for separation using RAPD analysis. As with other arbitrarily primed PCR systems, RAPD markers have low reproducibility because multiple amplicons are competing for available enzyme and substrate, and because low stringency thermal cycling allows mismatch annealing between primer and substrate. RAPD polymorphism is seen when priming sites are present or absent and products are scored by a plus/minus system revealing mainly dominant markers (Reiter 1994). DNA-Amplification Fingerprinting (DAF) Caetano-Anolles et al. (1997) developed a second arbitrarily primed, PCR-based marker method termed DNA amplification fingerprinting (DAF). DAF uses a single, short oligonucleotide primer of 5-8 nucleotides long, at high concentration in combination with either low (40-50 o ) or high stringency annealing temperature, high resolution fragment separation on polyacrylamide gels, and sensitive fragment detection with silver staining. DAF was used in a genetic stability evaluation (Caetano-Annoles et al. 1995) to show that Tifway 419 off-types are planting contaminations, and not mutations, based on the genetic stability among repetitions of Tifway 419 plantings. Tifway and Tifway II, however, were indistinguishable using this technique. In a later study (Caetano-Annoles

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11 1998) Tifdwarf and Tifgreen off-types were shown to be somatic mutations, generally having similar, but distinct genotypes compared to the true types. Anderson et al. (2001) also used DAF to determine homogeneity of a named grass and proved that U-3 bermudagrass populations have undergone genetic drift while Tifway 419 populations remain more genetically similar. DAF produces a banding pattern that is highly complex, containing many more fragment lengths than RAPD. This allows an increased chance of observing polymorphism, but is still unable to show heterozygosis. Because it is arbitrarily primed, it is also susceptible to low reproducibility. Amplification Fragment Length Polymorphism (AFLP) Amplified fragment length polymorphism (AFLP) is a PCR-based, sequence independent method wherein genomic DNA is first digested with one or more restriction enzymes, and an adapter of known sequence is ligated to the fragments. PCR primers have sequence specificity for the adaptor with one or more arbitrary, selective bases at their 3' ends. The arbitrary base limits the number of amplification products, and polymorphisms are detected as differences in the restriction sites and in the sequences flanking those sites. Adapter specific primers allow for increased stringency making AFLP more robust than the arbitrarily primed methods (Reiter 1994). Bert et al. (1999) developed a high density linkage map of L. perenne using AFLP. This map can be used in genetic studies and marker-assisted breeding, two important uses of molecular markers. The cultivars were grouped as previously expected from RFLP and RAPD work, but showed a wider distribution of markers for linkage to quantitative and qualitative traits. Manifesto et al. (2001) did a similar study in Triticum aestivum (L.), studying the private and public breeders stocks in Argentina. The genetic diversity

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12 was lower than expected, indicating great similarities in selection of germplasm and the use of released lines in breeding programs. Zhang et al. (1999) at Tifton, GA studied 27 cultivars of Cynodon spp. using AFLP markers. Testing 18 primer sets with three replications of successful markers, they were able to separate all cultivars with a high level of confidence. They showed a small dissimilarity coefficient of 0.08 between Tifgreen and its mutant Tifdwarf, but were able to successfully distinguish them. AFLP produced some unexpected results, separating Tifway, its mutant Tifway II, and a subsequent mutant, TifEagle, into different groups on the dendrogram. In contrast, DAF was unsuccessful in separating them (Caetano-Annoles 1998). Also of interest, two seeded varieties had dissimilarity coefficients of 0.05, which was the lowest in the subset. The use of radio-labeling was originally required to visualize AFLP bands, but has now been replaced by fluorescent markers and capillary electrophoresis. AFLPs reveal primarily dominant markers because of moderately complex banding patterns. Fluorescent labels, computer analysis, and capillary electrophoresis units present a considerable expense and require a high level of technical expertise (Reiter 1994). Simple Sequence Repeat (SSR) SSR markers belong to a class of molecular markers whose primers are sequence dependent. Known also as microsatellites, SSRs are ubiquitous DNA motifs repeated tandemly (Figure 1.4). Factors influencing the success of a particular motif include: frequency throughout genome, structural constraints of the DNA helix, and the size of the fragment to be amplified. Di-, tri-, or tetra nucleotide motifs are repeated perfectly at least three times, with the shorter motifs being more likely to mutate in most organisms (Cho et al. 2000). These additions or deletions cause the number of repeats to vary

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13 greatly between alleles of a given locus. The variability in the number of repeat units is the basis of observed polymorphism (Reiter 1994). SSRs have become popular because they show easily distinguished co-dominance, are more easily reproduced than sequence independent markers, assay robustness, high rate of polymorphism, and distribution throughout eukaryotic genomes. Primer sequence is easily transferred between labs and primer synthesis is readily available. Currently, they are used for marker-assisted breeding, genetic analysis, and to obtain relationship, evolution, and migration information for an organism (Gethi et al. 2002). Primers are designed using the sequence of the more highly conserved region flanking a region of the repeat motif. Mutation in the primer site and insertion and deletion events of the repeat motif, are the sources of polymorphism, thus the choice for repeat motif composition is important. Variables for motif success consist of mutation rate, overcrowding, structural functions, and size. The ideal size for an amplification product is between 100-300 bp in length, allowing for the most rapid fractionation (Reiter 1994). A partial list of molecular marker studies in monocots using SSRs include: L. perenne (Kubik et al. 1999; Jones et al. 2001), Oryza sativa (L.) (O. sativa) (Chen et al. 1997; Temnykh et al. 2000; Cho et al. 2000), Zea mays (L.) (Z. mays) (Chin et al. 1996; Gethi et al. 2002; Matsuoke et al. 2002; Senior et al. 1998), Paspalum vaginatum (Swartz) (P. vaginatum) (Liu et al. 1995), Saccharum officinarum (L.) (Cordiero et al. 2000), Hordeum vulgare (L.) (Sanchez et al. 1996), and Secale cereale (L.) (Saal and Wricke 1999). Comparisons between SSR and RAPD markers (Sanchez et al. 1996), SSRs and RFLPs, (Chen et al. 1997), and SSRs and isozymes (Senior et al. 1998) found SSRs to be either comparable or superior in their results for cultivar differentiation. The

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14 advantage of simple co-dominance makes this genetic tool ideal for following parentage or trait inheritance. The drawback inherent with sequence-dependent primer development is that genetic material must be sequenced. Sequence information of O. sativa and Z. mays is readily available in large databases. From sequences of both microsatellites and the regions flanking them, studies have been developed to identify microsatellite type, abundance, and length. There is also sufficient length of sequence information on either side of the repeat motif to provide ample choice for primer selection. Database mining is the least expensive method of primer design but requires large quantities of genome information to be available. Cho et al. (2000) indicated that sequence information available on GenBank produces fewer polymorphisms than sequence information obtained from a genomic library. For organisms that are not highly studied, it is theoretically feasible to use primer sets developed in the study of closely related species. First used in the study of animals (Moore et al. 1991), and later in plants (Kubik et al. 1999), cross-taxa SSR technology has been applied successfully between closely related species, generally where one species is better characterized genetically, or is more economically valuable. Such is the case with Festuca spp. and L. perenne, wherein RFLP probes (Xu et al. 1992) and SSR primer pairs have been successfully applied to follow the inheritance of traits through inter-genic crosses. In the SSR study of Kubik et al. (1999), thirteen primers developed from L. perenne genomic DNA were used to amplify DNA templates from species of varying genetic relatedness. Primer sets developed for L. perenne successfully amplified products from genomes of Festuca spp. and Lolium multiflorum (L.), closely related

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15 grasses, and produced polymorphic information. This primer development technique relies on evolutionary conservation of the primer region, and is more expensive than database searching because of the uncertainty of amplification and the possibility of lower rates of polymorphism with increasing genetic distance from the source species (Kubik et al. 1999). Kubik et al. (1999) tested the use of three primers sets developed for SSR amplification of P. vaginatum on L. perenne template DNA and was unsuccessful. In a study using primer pairs characterized on L. perenne, SSR primers (Jones et al. 2001) were applied to Phalaris spp. (L.) and Avena sativa (L.), members of the tribe Aveneae, with 38% and 12% efficiency of cross-taxa amplification, respectively. Similar results were obtained when primers developed for Z. mays were used on Sorghum bicolor (L.) (Moench) (S. bicolor). One hundred polymorphic primer sets were developed from Z. mays information on GenBank. Of these 12 primer sets amplified products on S. bicolor DNA templates, but only 2-3% of the 100 primer pairs were reliable and polymorphic (Brown et al. 1996). Kamps and Okagaki (Maximizing Taxonomical Transfer in Molecular Marker Development and Cross-taxa SSRs for Tripsacum. Used by permission.) developed a universally applied stringency modification to reveal cross-taxa SSR markers using a set of four touchdown PCR programs. Starting at 65-55 o C with progressively lower stringency, 59 cross-taxa primers from Z. mays and 32 from S. bicolor were applied to Tripsacum dactyloides (L.) (T. dactyloides). A five degree drop in stringency for PCR protocols revealed 14% more polymorphism from Z. mays primers and 19% more from S. bicolor primers. Zea mays and T. dactyloides are both in tribe Maydeae, while S. bicolor

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16 is in tribe Andropogoneae. All three are in super tribe Andropogonodae (Devos and Gale 1997). When attempting cross-taxa amplification, PCR products must conform to the following requirements: only amplification products found within a specified length of the parental polymorphic or monomorphic fragment sizes and containing a region of repeat motif should be considered. This is because of the possibility of mispriming under the reduced stringency programs (Jones et al. 2001). Development, sequencing, and primer selection from genomic libraries is the most expensive and time consuming method of SSR primer development, but has been shown to yield more informative markers than GenBank and is more reliable than the use of cross-taxa primer sets in showing polymorphism. The high cost of primer development arises from the low number of primer sets that can be developed from the sequence available and the low percentage of those that show polymorphism. This is true even in highly enriched libraries. Jones et al. (2001), in their study of L. perenne, sequenced 1,853 clones using multiplex enrichment and identified 859 that contained viable SSRs. Only 366 of these had flanking sequence long enough to develop 16-24 bp primers. From these 100 primer pairs were found to be polymorphic. Thus, only 5% of sequenced clones produce polymorphic information. While set-up is costly and time consuming, once primers have been identified, the actual running of the SSR reactions is relatively easy. PCR products can be fractionated on an inexpensive, high-concentration agarose gel and analyzed with as little equipment as an ultraviolet box, ethidium bromide staining and a camera. Comparison of PCR

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17 fragments to a marker ladder of known molecular lengths is done so that accurate sizing of the products may be determined by fractionation on a crude (agarose) gel system. SSR markers were developed for this study of bermudagrass cultivar identification. Two sources for primer sets were used. First, SSR primer pairs developed in the study of other members in the Poaceae family will be applied to bermudagrass DNA templates to determine if this less expensive method may be used. Second, an enriched genomic library will be developed and sequencing of that library done to develop bermudagrass-specific SSR primer sets. In this initial study it is expected that the grasses will be distinguished as groups, but further research will be needed to individually identify each cultivar. Statistical and phylogenetic analysis of the PCR product length will be done and the gel fractionation pictures compared to determine the success of these initial primer sets. The ultimate goal is to uniquely identify each named cultivar and the new breeding lines that are to be released so that a genetic method of cultivar identification may affirm phenotypic assessment. Future applications of this research may include genetic analysis, marker-assisted breeding, relationship, evolution, and migration information for Cynodon spp.

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18 Figure 1.1 Anatomical features of a grass stem and leaf section used in grass taxonomy and separation of one species from another (from http://www.weeds.montana. edu/crop/jgg.htm; Visited April 30, 2003).

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19 Figure 1.2 Two closely related turf varieties that appear similar. Despite their appearance, they have slightly different growth habits, which may only be seen after two or more years of growth and maintenance. Left: Champion bermudagrass. Right: Floradwarf.

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20 Figure 1.3 Diagram of the PCR process by which small amounts of DNA can quickly be copied by DNA polymerase to produce large numbers of a single fragment (from http://members.aol.com/BearFlag45/Biology1A/LectureNotes/LNPics /Recomb/pcr.gif; Visited April 30, 2003).

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21 Figure 1.4 Diagram of ideal SSR marker system showing fragments are visualized and how they can show allele inheritance. Notice that there is one solid band surrounded by lighter extraneous bands (from http://www.nal.usda.gov/ pgdic/Probe/v2n1/chart.gif; Visited April 30, 2003).

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CHAPTER 2 CROSS-TAXA PRIMERS APPLIED TO BERMUDAGRASS Hybrid bermudagrass is an important turf-type grass in Florida and other southern states. Cultivars with similar appearance, but differences in economic value-added traits, make genetic markers desirable. A microsatellite DNA marker system to provide immediate cultivar identification was studied using thirty-five cross-taxa primer pairs developed for SSR study on other grasses in the Poaceae family. Cross-taxa primer application resulted in only two patterns that reproduced reliably applied to all 23 bermudagrass DNA templates. This method of PCR amplification was successfully able to group grasses into major grass types, but did not show microsatellites in the amplification product. Introduction Commercial, clonally propagated varieties of improved bermudagrass used for turf are not easily distinguished by morphological and physiological characteristics because their subtle differences are subject to environmental influences. Because of the limited genetic base of parent material used to produce new cultivars, physical similarity is unavoidable. While the varieties appear similar, their reaction to drought, foot traffic, fertilizers, and herbicides can be significantly different. In order to distinguish between commercially important varieties, technology using a genetic marker system was sought. Simple sequence repeat (SSR) markers, known also as microsatellites, are one to eight nucleotide DNA motifs repeated three times or more. Polymorphisms in SSRs are because of the difference in the number of times the motif occurs at a given locus (Reiter 22

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23 1994). Once characterized and tested SSR primer pair sequences are easily transferable between labs, are easily synthesized, and allow higher stringency than random amplified DNA (RAPD) or DNA amplification fingerprinting (DAF) during PCR. Furthermore, the analysis is technically simpler than amplified fragment length polymorphism (AFLP) and SSRs set up for PCR with minimal DNA manipulation. AFLPs require the additional steps of restriction digestion and adapter ligation and must be fractionated on more labor-intensive DNA sequencing gels. Cross-taxa molecular markers have been successfully used among closely related plant species but tend to break down as the distance from the species of origin increases. One previous study in cross-taxa application used restriction fragment length polymorphism (RFLP) molecular markers developed for Festuca spp. (L.) (Xu and Sleper 1992). In this study probes from Festuca spp. were applied to Lolium perenne (L.) (L. perenne), a closely related monocot, and interspecific crosses were tracked for inheritance of important traits. SSRs have also been applied in cross-taxa studies of closely related species including the use of 16 L. perenne primer sets on Festuca spp. (Kubik et al. 1999). Another study applied 59 cross-taxa primer sets from Zea mays (L.) (Z. mays) and 32 from Sorghum bicolor (L.) (Moench) (S. bicolor), to Tripsacum dactyloides (L.) (T. dactyloides) using lower stringency of annealing temperature to increase PCR amplifications (Kamps and Okagaki, in prep). In a previous AFLP study researchers were successful in distinguishing 27 named bermudagrass cultivars and breeding lines (Zhang et al. 1999). In this study we attempted to apply SSRs to named bermudagrass cultivars to confirm the unusual placement of Tifeagle, which is an irradiated mutant of Tifway II (Zhang et al. 1999)

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24 with the greens grasses. Primer development being the largest cost in SSR primer research (Kubik et al. 1999), cross-taxa primers published for other grasses were used as an inexpensive source of primer sets. The hypotheses of this study are that when cross-taxa primers from other members of the Poaceae family are applied to bermudagrass DNA templates amplification products will occur, homologous regions will be amplified, SSRs will occur within these products and the use of step-down PCR will increase the events of cross-taxa polymorphism. In the event that polymorphism is observed amplification products must be within 100 base pairs (bp) of the size of the positive control, contain a confirmed SSR, and be clearly and reliably reproduced. To test these hypotheses, we used 36 cross-taxa primers developed for Saccharum officinarum (L.) (S. officinarum), S. bicolor, Z. mays, L. perenne, and Oryza sativa (L.) (O. sativa) all of which are in different subfamilies of Poaceae (Devos and Gale 1997). The genus Cynodon is the only member of the subfamily Chloridoideae represented in this study. Materials and Methods Plant Material The names or identification numbers of the 23 genotypes tested in this study are listed in Table 2.1. Named cultivars were obtained as breeders or foundation seed from the same sources used by the AFLP study of Zhang et al. (1999). Grass samples were maintained in black, one and two gallon plastic containers, using a soil less mixture of three parts sand to one part peat and one part perlite, in a greenhouse on the University of Florida campus.

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25 DNA Preparation Using the method of Dellaporta et al. (1983) DNA was extracted from two grams of frozen leaf tissue. DNA concentrations were read by a SmartSpec 3000 (BIO-RAD, Hercules, CA). Genomic DNA samples were diluted to 1ug/ul and were maintained at -20 o C. The following positive control DNAs were graciously provided for this research: L. perenne by Dr. C.M. Styles, O. sativa, by Dr. W.Y. Song, S. bicolor by Dr. D. Pring, and S. officinarum, and Z. mays by Dr. C.D. Chase. Cross Taxa Primers Thirty six primer pairs were taken from published studies within the family Poaceae (Table 2.2). These cross-taxa primers were first tested on six plant introductions (PI) (Table 2.1) of Cynodon spp. with four step-down PCR programs (Table 2.3) of varied stringency starting as high as 65 o C and going as low as 45 o C. The annealing temperature in the first ten cycles was lowered by one degree per cycle, followed by thirty cycles at the lowest annealing temperature in order to increase cross-taxa amplification. Primers amplifying DNA fragments of 60-700 bp were subsequently used to test 23 different bermudagrass templates, including utility, greens, fairway, and ball field types as well as breeding accessions (BA) and PIs (Table 2.1). Sizes of PCR products were estimated based on 100 bp and 25 bp marker ladders (Invitrogen, Carlsbad, CA) run on each gel. PCR amplification of the entire set of genomic samples was done at least twice and reproducibility was determined based on the comparison of the results. PCR All PCR amplifications were done in a PTC-100 Peltier-effect thermal cycler (MJ Research, Inc.; Waltham, MA). PCR was performed using 1.25 units Hot Start Polymerase (Promega), 1 X supplied buffer (50 mM KCl, 10 mM Tris HCl (pH 8.0),

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26 1.63 mM (volume/volume) Triton X 100), 0.2 mM of each dNTP, 1.5 mM MgCl 2 1.0 uM of each primer (forward and reverse), and 1.0 g of template DNA, in a total volume of 50 L. The PCR products were fractionated on 3% Agarose 1000 (Invitrogen; Carlsbad, CA) gels in 1X TBE buffer (1.2% Trizma base, 0.6% Boric acid, 0.07% EDTA) at 110 V for four hours and stained with a 0.1 g/ mL ethidium bromide solution for 30 min. Gels were visualized over a UV light box and images captured on the AlphaImager 2200 system (Alpha Innotech Corp. San Leandro, CA). PCR Product Sequence Analysis PCR products for cross-taxa alignment were either sequenced directly, or cloned and reamplified prior to sequencing. In the latter case, PCR products were ligated into P-Gem T Easy Vector (Promega) and transformed into JM-109 (Promega) competent cells according to manufacturers recommendations. Colonies were grown on LB media with 100 g/ mL ampicillin, 80 g/ mL x-gal, and 0.5 mM IPTG then selected based on blue-white color according to instructions for JM-109 competent cells. Cloned inserts were amplified by PCR using M13 forward (5-GTTTTCCCAGTCACGAC) and reverse (5-CAGGAAACAGCTATGAC) primers. Inserts were then sequenced by the ICBR DNA Sequencing Core Laboratory at the University of Florida, through use of ABI 373a Stretch/377 DNA sequencers ( http://www.biotech.ufl.edu/DNASequencing/ services.html ). DNA sequences were aligned with CLUSTAL W (1.81) Multiple Sequence Alignments (Thompson et al. 1994) on the San Diego Workbench (http://workbench.sdsc.edu/ Visited April 30, 2003). Statistical analysis of fragment patterns was done using the genetic similarity formula GS=m/(m+n) where GS=genetic

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27 similarity between individuals, m=matches, and n=fragments not matching (Senior et al. 1998). Results After successful amplification of product on their species of origin, thirty six primer pairs (Table 2.2) were used to screen six bermudagrass PIs (Table 2.1). Eighteen primer pairs (50%) showed no cross-taxa amplification, twelve (33%) amplified fragments without polymorphism or with multiple amplification products that were too numerous to discern easily. Both of these observations are designated as not informative. Six (17%) primer sets initially produced informative polymorphic amplification products (Table 2.2) and two of these showed reproducible fragment length patterns. All primer sets were screened using four touchdown PCR programs (Table 2.3), KAM4NW most often producing amplification products. From the five species that contributed primers the S. officinarum (Cordiero et al. 2000) and O. sativa (Chen et al. 1997; Temnykh et al. 2000) primer sets either did not amplify from bermudagrass templates at all, or amplified products that were judged not informative on either the subset of six bermudagrass PIs or the full set of 23 genotypes (Table 2.2). Five of the seven S. bicolor primer pairs amplified products on bermudagrass DNA templates (Table 2.2). One amplified only monomorphic products, two amplified products that were difficult to discern, and two primer sets were initially informative (Table 2.4). Primer pair SB4-22 (Figure 2.1) failed to show amplification product after the initial trial even though it occasionally revealed polymorphisms and amplification products of a size similar to the positive control. The amplification products of SB6-36 on bermudagrass templates were also occasionally polymorphic, but more often appeared

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28 monomorphic (Figure 2.2) with lengths of 500-600 bp, three fold larger than the size of the positive control amplicon. Three of the seven L. perenne primer pairs amplified PCR products on bermudagrass DNA templates (Table 2.2). Only two primer sets revealed polymorphism when tested on all templates. Of these two, LPSSRHO1A10 amplified multiple polymorphic products as well as some faint, inconsistent amplicons (Figure 2.3). The fragments that were common between gels (Table 2.5) placed grasses into the groups indicated by Zhang et al. (1999) and were used to calculate genetic similarity. Primer pair LPSSRHO1A07 did not predictably amplify products in the named cultivars, but the amplification products from the PIs, while faint, were usually distinguishable (Figure 2.4). Sequencing showed that the LPSSRHO1AO7 positive control product was 251 bp and contained a (GA) 10 SSR. Fragments of 108 bp and 452 bp in length from BA 475 (Figure 2.4) were also sequenced and contained no SSR (Figure 2.5). When aligned, sequences showed a 21 bp region of conserved sequence in the three amplicons near the 3 end. The LPSSRHO1A07 control template sequence was submitted to BLASTN (Altschul et al. 1990) and subjected to a redundancy search against all plant sequences in GenBank using default settings, and the closest match was on chromosome 12 in O. sativa. A total of eight Z. mays primer pairs were tested on the bermudagrass templates. Three pairs did not yield detectable amplification products and another three sets amplified only monomorphic products on the 23 bermudagrass templates (Table 2.2). MZEPPDKA2 (Figure 2.6) amplified inconsistently, but amplified products of the same approximate size for a specific cultivar (Table 2.6). Primer pair ZMADH2N amplified

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29 consistently, showing two or more polymorphic fragments, and placed the grasses into groups predicted by Zhang et al. (1999). Fragment length patterns showed that the breeding lines were individually distinguishable one from another. Greens grasses and fairway grasses formed separate groups, but grasses within each group were similar (Figure 2.7, Table 2.6). Sequencing of the ZMADH2N primer pair (Figure 2.8) positive control product revealed a 128 bp fragment containing an SSR of (AG) 8 Fragments of 174 bp and 339 bp from PI 289-916 DNA template (Figure 2.7) were also sequenced, but contained no SSR. Alignment showed that Random base pairs matched between fragments, but no extended sequence was conserved among all three. The sequence of the positive control for Z. mays was submitted to BLAST (Altschul et al. 1990) against all plant sequences in GenBank and, as expected, showed homology to the gene for alcohol dehydrogenase II. Genetic similarity analysis of LPSSRHO1A10 (Table 2.5) and ZMADH2N (Table 2.7) fragment length patterns showed high similarities within the greens grasses and three of the fairway grasses (Table 2.7). By this statistical analysis Midiron and MS Choice were considered dissimilar from each other and from the other fairway grasses. The greens grasses were distinct from the other groups and individuals when matrices were combined. Similarity matrices for individual primer sets are in Appendix A. Discussion We assessed cross-taxa primers developed for Poaceae species that are not closely related to bermudagrass, but were hoped to provide an inexpensive method of primer development. For this study, we designate successful cross-taxa amplicons as being within 100 bp of the length of the positive control, containing SSRs, and repeatable through PCR amplification. In the study of Kamps and Okagaki (In prep), primer pairs

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30 from Z. mays and S. bicolor successfully amplified polymorphic regions from T. dactyloides DNA. The occurrence of amplification events was increased by using annealing temperatures below that of the species-specific primers. All three of these plants are in super tribe Andropogonodae, with Z. mays and T. dactyloides in the same tribe of Maydeae, while S. bicolor is from tribe Andropogoneae (Devos and Gale 1997). In correlating work done by Jones et al. (2001) using primer pairs derived from L. perenne, polymorphic amplifications were found between L. perenne, Lolium multiflorum (L.), and the Festuca spp. (Schreb), the same genus and same tribe (Poeae) respectively. When L. perenne primers were applied to Poa spp. (L.), Phalaris spp. (L.), and Avena sativa (L.), the last being a member of a different tribe, lower levels of cross-amplification were detected. In general, most of the primer pairs used in this study amplified products that were 300 bp larger than the positive control and failed to reproduce successfully through repeated trials. Many of the primer sets designated as informative separated the grasses into expected groups of phenotypic similarity such as fairways and greens grasses when they successfully amplified. Amplification products that were many times larger than the positive control amplicon may be PCR artifacts where only one primer sat down, but even in some of those cases the breeding lines were distinguishable one from another. In the case of MZEPPDKA2, amplification products were the same size when trials were repeated, but no sequencing was done to confirm presence or absence of an SSR or the cause of erratic amplification patterns. It seems possible that further manipulation of PCR conditions may uncover the means of reliably amplifying polymorphic product.

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31 It is encouraging that the amplification pattern of the two primer pairs that amplified similar products twice on all DNA templates, ZMADH2N and LPSSRHO1A10, grouped the grasses as expected from the AFLP study done by Zhang et al. (1999). Knowing that the Z. mays primer pair ZMADH2N, which successfully amplified products from S. bicolor (Brown et al. 1996), also amplifies a product from bermudagrass templates encourages the possibility of success with certain primers from organisms that are distantly related. The lack of SSR is disappointing, but understandable given the genetic distance. Primer set LPSSRHO1A10 (Figure 2.3) also produced a repeatable pattern through subsequent amplifications on bermudagrass DNA, but fragments of multiple lengths suggested that mispriming is a concern with this primer pair. It was interesting to note that bermudagrass amplicons from the ZMADH2N and LPSSRHO1AO7 primer pairs showed no SSRs and poor homology with the SSR containing positive controls when aligned by CLUSTALW (Thompson et al. 1994) (Figures 2.5, 2.8). Lack of SSR in cross-taxa amplification indicates that the lower stringencies in the PCR reactions allow cross-taxa SSR primers to function as little more than long RAPD primers. The genetic similarity analysis shows that while the grasses are grouped together, there is still similarity between the fairway grasses and the breeding lines. This system is a beginning, but eventually we would like to be able to find much lower similarities and much larger differences between cultivars and groups. Based on the findings of this and other studies (Jones et al. 2001; Brown et al. 1996), the use of cross-taxa primers from distantly related species for inexpensive, simple detection of polymorphic SSRs is not supported. Lower stringency PCR programs

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32 increased the number of cross-taxa primer pair amplification events from bermudagrass DNA, but the number and quality of polymorphic products were unacceptably low to encourage further investigation for quick, confident identification of cultivars. Perhaps, as more closely related species are studied, and their genomic sequences become available, cross-taxa amplification success will increase. Primer pairs developed for Cynodon spp. will also be useful to other members of the Chlorideae tribe. These indications suggest that the development of a species-specific set of primers used for the detection of SSR polymorphism would be advantageous for hybrid bermudagrass and its parents for marker assisted breeding, cultivar identification, and genetic relatedness studies.

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Table 2.1 Origins and uses of bermudagrass cultivars evaluated for simple sequence repeats 33 Type Identification Ploidy Taxon Source Reference Fairway MS Choice 27 (3n) txd J. Kranz & W. Philley, MSU Krans et al. 1995 Midiron 27(3n) txd J. Fry, Kansas State University Anderson et al. 1988 Tifsport 27(3n) txd W. Hanna, USDA-ARS. Tifton, GA Zhang et al. 1999 TifwayII 27(3n) txd W. Hanna, USDA-ARS. Tifton, GA Burton 1985 Tifway419 27(3n) txd W. Hanna, USDA-ARS. Tifton, GA Burton 1966b Greens Tifgreen 27 (3n) txd W. Hanna, USDA-ARS. Tifton, GA Hein 1961, Tifdwarf 27(3n) txd W. Hanna, USDA-ARS. Tifton, GA Burton 1966a Tifeagle 27(3n) txd W. Hanna, USDA-ARS. Tifton, GA Zhang et al. 1999 MS Supreme 27 (3n) txd J. Kranz & W. Philley, MSU, MI Krans et al. 1999 Champion 27(3n) txd Mike Brown, Coastal Turf, TX www.champion_dwarf.com Floradwarf 27(3n) txd Al Dudeck, Dept. Env. Hort. UF, FL Dudeck 1991 Turf/ GN-1 ND ND Jimmy Doublle, GNT, Florida www.shark .com/gnturf/introduction Ball field Floratex 36 (4n) d Al Dudeck, Dept. Env. Hort. UF, FL Dudeck 1991 Tiflawn 36(4n) d W. Hanna, USDA-ARS. Tifton, GA Hein 1953 Tifton 10 54 (6n) d W. Hanna, USDA-ARS. Tifton, GA Hanna et al. 1990 Breeding BA 481 ND ND B. Scully, EREC-IFAS-UF, FL EREC, Breeding line Accessions BA 157 ND ND B. Scully, EREC-IFAS-UF, FL EREC, possible Wintergreen BA 475 ND ND B. Scully, EREC-IFAS-UF, FL EREC, Breeding line Plant *PI 290-868 36 (4n) d PI collection, Athens, GA Royal Cape South Africa Introductions *PI 290-895 36 (4n) d PI collection, Athens, GA Reitz South Africa *PI 289-916 27 (3n) txd PI collection, Athens, GA Magennis South Africa *PI 290-900 27 (3n) txd PI collection, Athens, GA Damascus' South Africa *PI 290-894 18 (2n) t PI collection, Athens, GA 'Sekapplos Fine' South Africa C. dactylon is designated d, C. transvaalensis is designated t, and their hybrid is designated txd. MSU: Mississippi State University, UF: University of Florida, GNT: Gregg Norman Turf, MS: Mississippi, EREC: Everglades Research and Education Center, ND: not determined. Five templates used as subset plus PI 290-905 (2n), which was later dropped, for a total of six designates by (*).

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34 Table 2.2 Origins of cross-taxa primer pairs used in PCR reactions on bermudagrass templates Primers from L. perenne Amplification Results Program (Table 2.3) LPSSRH01A02 + not informative LPSSRH01A07 + informative KAM4NW LPSSRH01A10 + informative KAM3NW LPSSRH01E10 LPSSRH01H06 LPSSRH02C11 LPSSRK01A11 Primers from S. bicolor Sb4-32 + not informative Sb5-236 + not informative Sb5-85 + not informative Sb6-36 + informative KAM3NW Sb6-84 Sb4-22 + informative KAM4NW Sb4-15 Primers from S. officinarum SMC222CG SMC226CG + not informative SMC248CG SMC319CG SMC477CG SMC863CG + not informative SMC1039CG Primers from O. sativa RM110 + not informative RM280 + not informative RM349 + not informative RM23 RM44 RM227 RM256

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35 Table 2.2. Continued Primers from Z. mays Amplification Results Program MZE-PPDKA2 + informative KAM4NW ZMADH2N + informative KAM4NW CMBMC1893 ZMBNLG1633 + not informative p-umc2042 + not informative p-bnlg1252 p-dupssr1 p-umc1308 + not informative Cross-taxa primers applied to bermudagrass DNA. Informative fragment length patterns were bright, polymorphic in nature, and clear. Not informative patterns were monomorphic under more stringent conditions, but under less stringent conditions the fragments amplified are too numerous to discern easily. Primer sources are L. perenne (Jones et al. 2001); S. bicolor (Brown et al. 1996); S. officinarum (Cordiero et al. 2000); O. sativa (Temnykh et al. 2000; Chen et al. 1997); Z. mays (Brown et al. 1996); and Maize Genome Database mapping to chromosome 2 ( http://www.agron.missouri.edu/ ).

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36 Table 2.3 Basic step-down PCR programs used to test primer amplification Step Minutes Temp (degrees C) 1 5 95 2 1 95 3 1 annealing temp 4 1.5 72 5 Repeat steps 2-4 ten times, lowering annealing temp 1 degree in each cycle. 6 1 95 7 1 lowest annealing temp 8 1.5 72 9 Repeat steps 6-8 thirty times using lowest annealing temp. 10 5 72 11 cool down 10 The four step-down annealing temperatures (65-55 o 60-50 o 55-45 o and 50-40 o ) used for this project were originally developed by Kamps and Okagaki (In prep). These programs were identified in this project as KAM1NW: 65-55 o KAM2NW: 60-50 o KAM3NW 55-45 o and KAM4NW: 50-40 o respectively.

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37 Table 2.4 Sorghum bicolor cross-taxa amplicon sizes estimated in base pairs Accession DNA Amplification Products SB4-22 primers SB6-36 primers Gel#19570-2 Gel#19872 Gel#19621 MS Choice 550 650 600 Midiron 300 650 Tifsport 300 550 650 600 Tifway II 550 Tifway 419 550 650 Tifgreen 300 550 650+ 600 Tifdwarf 300 550 650+ 600 Tifeagle 550 600 MS Supreme 300 550 600 Champion 550 Floradwarf 300 550 600 GN-1 300 600 550 650 600 Floratex 300 550 650 600 Tiflawn 550 650 600 Tifton 10 300 550 650 600 BA 481 300 550 650 600 BA 157 300 550 650 BA 475 300 550 650 600 PI 290-905 550 600 PI 289-916 550 650 600 PI 290-900 300 500 PI 290-868 300 400 550 650 600 PI 290-895 300 400 550 650+ 600 S. bicolor 300 200 This table describes the major products amplified from the entire set of genotypes to determine reproducibility. Estimated length of PCR products is given in nucleotide base pairs based on 100 bp and 25 bp marker ladders (Invitrogen) run on each gel. Based on sequencing lengths vs. visual determination, an error of ~50 bp was determined for fragments because of resolution of agarose gels and human error. Positive control PCR product was the only clear amplification in gel # 19924 therefore no results are reported.

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38 Table 2.5 Lolium perenne cross-taxa amplicon sizes estimated in base pairs Accession DNA Amplification Products LPSSRHO1A1O primers LPSSRHO1AO7 primers Gel #19570-1 Gel #19950 Gel #19621 Gel #19898-1 MS Choice 250 300 250 300 Midiron 300 600 300 600 Tifsport 150 300 150 300 600 Tifway II 150 300 600 Tifway 419 150 300 600 Tifgreen 225 600 600 600 Tifdwarf 225 600 Tifeagle 225 600 MS Supreme 225 600 600 600 600 Champion 600 600 600 Floradwarf 225 600 600 600 GN-1 225 300 700 Floratex 300 300 Tiflawn 250 300 250 110 500 500 Tifton 10 200 250 300 200 250 300 110 BA 481 200 300 200 250 300 110 500 500 BA 157 200 300 200 250 300 110 500 500 BA 475 300 250 300 110 500 500 PI 290-905 150 300 150 200 300 600 600 PI 289-916 150 300 150 300 600 600 PI 290-900 125 300 300 410 PI 290-868 200 300 200 400 600 400 600 PI 290-895 250 300 250 600 410 600 L. perenne 150 200 150 200 200 200 Length of PCR products is given in nucleotide base pairs, and was scored by estimation based on 100 bp and 25 bp marker ladders (Invitrogen) run on each gel. Based on sequencing lengths vs. visual determination, an error of ~50 bp was determined for fragments because of resolution of agarose gels and human error. This table describes the major products amplified on the entire set of genotypes to determine reproducibility.

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39 Table 2.6 Zea mays cross-taxa amplicon sizes estimated in base pairs Accession DNA Amplification Products MZEPP-DKA2 primers ZMADH2N primers Gel #19364-2 Gel #20001 Gel #19975 Gel #19364 MS Choice 500 650 275 400 275 400 Midiron 175 175 650 275 400 600 275 400 Tifsport 650 650 275 600 275 600 Tifway II 650 275 600 Tifway 419 650 275 600 Tifgreen 550 650 200 400 200 400 Tifdwarf 550 650 200 400 200 400 Tifeagle 550 650 200 400 400 MS Supreme 550 650 200 400 200 400 Champion 550 650 200 400 400 Floradwarf 550 650 200 400 200 400 GN-1 400 400 Floratex 650 275 400 275 400 Tiflawn 650 275 275 Tifton 10 275 275 275 400 275 400 BA 481 650 275 400 275 400 BA 157 650 400 400 BA 475 650 275 400 275 400 PI 290-905 650 600 600 PI 289-916 650 200 400 200 400 PI 290-900 650 PI 290-868 650 275 400 275 400 PI 290-895 650 275 600 275 600 Z. mays 125 125 150 150 Length of PCR products is given in nucleotide base pairs, and was scored by estimation based on 100 bp and 25 bp marker ladders (Invitrogen) run on each gel. Based on sequencing lengths vs. visual determination, an error of ~50 bp was determined for fragments because of resolution of agarose gels and human error. This table describes the major products amplified on the entire set of genotypes to determine reproducibility.

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Table 2.7 Genetic similarity matrix including combined data from primer sets LPSSRHO1A10 and ZMADH2N MS ChoiceMidironTifsport Tifway II Tifway 419TifgreenTifdwarfTifeagleMS SupremeChampionFloradwarfGN-1FloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895POSITIVEMS Choice1Midiron0.331Tifsport0.110.111Tifway II0.110.1111Tifway4190.110.11111Tifgreen00.110001Tifdwarf00.1100011Tifeagle00.17000111MS Supreme00.110001111Champion00.250000.50.50.50.51Floradwarf00.1700011110.51GN-10.130.130000.130.130.130.1300.131Floratex0.50.50.170.170.170000000.171Tiflawn0.50.170.170.170.1700000000.171Tifton 100.660.250.080.080.080000000.10.250.331BA 4810.330.330.110.110.1100000000.330.170.661BA 1570.170.170000000000.250.1700.330.51BA 4750.50.50.110.110.110000000.170.50.250.330.50.251PI 290-905000.330.330.3300000000000001PI 289-9160.170.170000000000.130.1700.130.170.170.2501PI 290-900000000000000000000001PI 290-8680.330.330.110.110.110000000.130.330.170.6610.50.500.1101PI 290-8950.330.110.330.330.3300000000.330.50.220.1100.170.17000.111POSITIVE000000000000000000000001 40 Genetic similarity was determined by the formula GS=m/ (m+n) where GS=genetic similarity, m=matches, and n=fragments not matching.

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41 100 bp ladderMSChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMSSupremeChampionFloradwarfGN-125 bp ladderFloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895PositiveNegative100 bp ladder 600 bp600 bp FairwayGreens Lawn Breeding Lines 19570-219924 100 bp 100 bp Figure 2.1 SB4-22 primer pair amplification of 23 bermudagrass DNA templates. The positive control PCR reaction was performed on S. bicolor DNA template. The negative control consists of the entire PCR reaction mix, including primers, but without template DNA.

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42 600 bp600 bp 1962119872100 bp ladderMSChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMSSupremeChampionFloradwarfGN-125 bp ladderFloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895PositiveNegative100 bp ladder FairwayGreens Lawn Breeding Lines 100 bp 200 bp Figure 2.2 SB6-36 primer pair amplification of 23 bermudagrass DNA templates. The positive control PCR reaction was performed on S. bicolor DNA template. The negative control consists of the entire PCR reaction mix, including primers, but without template DNA.

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43 600 bp 19570-1100 bp ladderMSChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMSSupremeChampionFloradwarfGN-125 bp ladderFloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895PositiveNegative100 bp ladder FairwayGreens Lawn Breeding Lines 100 bp 600 bp 19950100 bp Figure 2.3 LPSSRHO1A10 primer pair amplification of 23 bermudagrass DNA templates. The positive control PCR reaction was performed on L. perenne DNA template. The negative control consists of the entire PCR reaction mix, including primers, but without template DNA.

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44 600 bp 19621 Sequenced products 100 bp ladderMSChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMSSupremeChampionFloradwarfGN-125 bp ladderFloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895PositiveNegative100 bp ladder FairwayGreens Lawn Breeding Lines 100 bp 600 bp 19898-1 1 00 bp Figure 2.4 LPSSRHO1AO7 primer pair amplification of 23 bermudagrass DNA templates. The positive control PCR reaction was performed on L. perenne DNA template. The negative control consists of the entire PCR reaction mix, including primers, but without template DNA.

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45 LPSSRHO1AO7_F -TGGAGGGCTCGTGGAGAAGT--------------------------------------UPPER -TGGAGGGCTCGTGGAGAAGTCCAGTAAAGAAAACTCACCATCAAAAATGGGGTGGGCTC LOWER -TGGAGGGCTCGTGGAGAAGT--------------------------------------CONTROL TTGGAGGGCTCGTGGAGAAGT--------------------------------------^^^^^^^^^^^^^^^^^^^^^ UPPER CCTCAAGGGGCGCCGCCAGTCACAATGAGTTGCCTAAGCTGGAACTATCATGGACTTGGC LOWER -----------------------------------------------------------CONTROL -----------------------------------------------------------UPPER AATGCCGCGACAGTTAAAGAACTTCGCGATCTTGTGAAGCGTTTTGCCCCATCTGTGTTG LOWER -----------------------------------------------------------CONTROL -----------------------------------------------------------UPPER TGCGTTCAAGAAACTTAGATTCGTAACTCGCGGGTGGAAAGTTTGAAAAATATACTTGGC LOWER -----------------------------------------------------------CONTROL ---------------------------------------------------ACATCGACC UPPER TTTGACC-AAGCGTTTGTTGTTAGTAGTCAACGCCGTAGTGGAGGCTTAGGAATCTTCTA LOWER -----------------------------------------------------------CONTROL CGCAGCCGAAGACTTCGGTGTT-----TCGGGAGGC GAGAGAGAGAGAGAGAGAGA TGCA UPPER GAATAATAATATAAGAATGACTATTTTACCGTACTCGCAATATCATATTGACATGATTTT LOWER ----------------------------------------------AAAGACTGGGTG-CONTROL GGTGGTGAATGCGACGCATGCGCTTTGGACGTGGCACCGGAACGACGACGACGAGCCT-*** UPPER CAACGAGGGGCAAGCTGATCCGTGGAGGTTGACATGCGTGTACGGGGAGGCGTAGACAAG LOWER -GATCAAGGTAAATGTTG-ATGGCGCATTTGTGGCACAAACAGATCAAGCAGGT-GCAAG CONTROL --GTGGTGGCCGATCAAGTGTGGATCACCAGCCTAGCATCCAAGCCGGCTTGTA-ACAAG ** * * * **** UPPER TGAGTGGCTCGTCCTAATCACTAGTGCGGCCGCCTGCAGGTCGACCATATGGGAGCGGTT LOWER TGAGTGGCTCGTCCTACTTCTC---------------------------------CGGTT CONTROL TGAGTGGCTCGTCCTAATCACTAGTGCGGCCGCCTGCAGGTCGACCATATGGGAGCGGTT LPSSRHO1AO7_R -------------------------------------------------------CGGTT **************** ^^^^^ UPPER CCCACGCCTTGC LOWER CCCACGCCTTGC CONTROL CCCACGCCTTGC LPSSRHO1AO7_R CCCACGCCTTGC ^^^^^^^^^^^^ Figure 2.5 CLUSTAL W (1.81) alignment of LPSSRHO1AO7 amplification product sequences. The forward primer is not seen in the product designated Lower because this product was sequenced directly with the LPSSRHO1AO7 forward primer. The other products were first cloned into the pGemT plasmid vector (Promega) and sequenced using the M13 forward primer. Sequence was then trimmed to include only those sequences within the LPSSRHO1AO7 forward and reverse primers. Bases that are conserved between fragments are marked by (*). The positive control SSR is underlined and the primer sequences are designated by (^).

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46 600 bp600 bp20001 19364-2100 bp ladderMSChoiceMidiroTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMSSupremeChampionFloradwarfGN-125 bp ladderFloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895PositiveNegative100 bp ladder FairwayGreens Lawn Breeding Lines 100 bp 100 bp n Figure 2.6 MZEPPDKA2 primer pair amplification of 23 bermudagrass DNA templates. The positive control PCR reaction was performed on Z. mays DNA template. The negative control consists of the entire PCR reaction mix, including primers, but without template DNA.

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47 600 bp Sequenced products19975100 bp ladderMSChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMSSupremeChampionFloradwarfGN-125 bp ladderFloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895PositiveNegative100 bp ladder FairwayGreens Lawn Breeding Lines 100 bp 19364600 bp 100 bp Figure 2.7 ZMADH2N primer pair amplification of 23 bermudagrass DNA templates. The positive control PCR reaction was performed on Z. mays DNA template. The negative control consists of the entire PCR reaction mix, including primers, but without template DNA.

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48 Upper AATTGGACCGTACTAGTACTCTAGCCATTTTTCTTTGGTATGCGTTAATGTTCAAATGTT Lower -----------------------------------------------------------Control -----------------------------------------------------------Upper TACTTCCAATATCGTTTTAAATAGTGAGGAAGAGGAGGGCAGGAGGCCTATCATGGTTTT Lower -----------------------------------------------------------Control -----------------------------------------------------------Upper CTTCAGCTGCACACAACCCCTTGTCTCAATCTCAGTGGCCAGTGGGAGATGATGAGCATC Lower -----------------------------------GCACGCCAAGGATCTGAGGAAACTC Control -----------------------------------------------------------Upper GACCCGAACGGACCGATCGTAAGACTGAATCCAAAAGGATGGCGACAACCTCTCCCACAA Lower GAATAACATATATCAAAACAGAATAACAGATTACTGATACAGAATAGCTCTACTAATTTC Control ---------------------GATTCCATTCTCGTGTTCTTGGAGTGGTCCATCGAT--C * Upper TAATTACGCCGCGGATCGGCGGTTACTAATCTCTTGATGTTCAGTAATATATAAAGCAAT Lower AAATTTTTTCATCAATTAGTGATGAGTAAAGTTAGC--TAACAAAAGCAATGAAAACCAC Control GAGC---TCCGTGAA AGAGAGAGAGAGAGAG CAAGCAATGGCGACAGCAGGGAAGGTGAT * * * * ** Upper GCGCAAATTGCGACAGCGCCGTGCGTCTTCTACCTCCAA Lower ---TATA-----AATGTTTCGTGCGTCTTCTACCTCCAA Control ---CAAGTGC--AGAGGTGCGTGCGTCTTCTACCTCCAA ZMADH2NR -------------------CGTGCGTCTTCTACCTCCA* * ^^^^^^^^^^^^^^^^^^^ Figure 2.8 Sequence of ZMADH2N amplification products from Figure 2.6 aligned by CLUSTAL W (1.81). Amplification products on bermudagrass and the product amplified on the positive control template are shown, as well as the alignment of the reverse primer. These fragments were directly sequenced using the forward primer in a single pass from PCR product. The positive control SSR is underlined. Bases that are conserved between fragments are marked by (*). Reverse primer is designated by (^) and forward primer sequence is (5 TGCGAAG AAAGCAGTAGCAAA).

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CHAPTER 3 SPECIES-SPECIFIC PRIMERS Hybrid bermudagrass is an important turf-type grass in Florida and other southern states. Cultivars with similar appearance, but differences in economic value-added traits, make genetic markers desirable. For this reason, a microsatellite DNA marker system that will provide immediate cultivar identification was studied. A genomic library was enriched for (AC) n repeats for primer development. This method of microsatellite primer development provided three primer pairs that reliably amplified polymorphisms on bermudagrass DNA templates. Unique genetic fingerprinting in many cultivars was also shown using a simple genetic similarity matrix and phylogenetic analysis. Introduction Cynodon dactylon (L.) Pers. (C. dactylon), known in the U.S. as common bermudagrass, is a tetraploid with chromosome number of 2n=4x=36 (Taliaferro 1995). Generally C. dactylon is described as having a folded vernation, no auricle, and a fringe of hairs on the ligule. It is rhizomatous, stoloniferous, and produces seed excessively on 4-5 digitate spikes raised above the leaf canopy. Cynodon transvaalensis (Burtt-Davy) (C. transvaalensis), known as African bermudagrass, is a diploid with a chromosome number of 2n=2x=18 (Beard 1973). Cynodon transvaalensis is used in the U.S. turfgrass industry because of its uniform morphology maintained within varieties and its ability to cross interspecifically with C. dactylon (Taliaferro 1995; Beard 1973). The hybrid of C. dactylon X C. transvaalensis, is a sterile triploid with 2n=3x=27 chromosomes (Taliaferro 1995; Beard 1973). Morphologically similar to C. dactylon 49

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50 (auricles, ligules, etc.), the hybrid possesses a desirable combination of turf traits from the two species (Taliaferro 1995). In Florida, bermudagrass accounts for over half of the golf course grasses or 136,000 acres (Haydu et al. 2002a) and 6% of the sod industry production acreage, or 4,556 acres. Turf bermudagrass garnered $16.3 million at $0.114 ft -2 which accounted for 19% of the sod industry revenue in 2000 (Haydu et al. 2002b). Commercial, clonally propagated varieties of improved bermudagrass used for turf are not easily distinguished by morphological and physiological characteristics because of subtle differences subject to environmental influences. Although the varieties appear similar, their reaction to drought, foot traffic, fertilizer application rates, and herbicides can be significantly different. A genetic marker system was sought in order to more quickly distinguish between economically valuable cultivars. Simple sequence repeat (SSR) markers, also known as microsatellites, are one, two or three nucleotide DNA motifs repeated three times or more (Edwards et al. 1996). The variation in the number of times the motif occurs between alleles of a given locus is the basis of observed polymorphism. SSRs have become popular because they show easily distinguished co-dominance, are more easily reproduced than sequence independent markers, assay robustness, high rate of polymorphism, and distribution throughout eukaryotic genomes. Primer sequence is easily transferred between labs and primer synthesis is readily available. Currently, they are used for marker-assisted breeding, genetic analysis, and to obtain relationship, evolution, and migration information for an organism (Gethi et al. 2002). One major drawback to SSR markers is that large amounts of genomic sequence is needed to produce primers that flank a high copy number of the repeat (Reiter 1994). According to previous studies, the rate of mutation observed at a

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51 given locus is dependant on the type of motif, thus di-nucleotide repeats mutate more frequently than trior tetra-nucleotide repeats (Cho et al. 2000). SSR primers are 16-20 base pair (bp) long, which allows higher stringency during the polymerase chain reaction (PCR) than the 10 bp random amplified polymorphic DNA (RAPD) primers or the 5-8 bp DNA amplification fingerprinting (DAF) primers. Furthermore, SSR analysis is technically simpler than amplified fragment length polymorphism (AFLP) analysis. SSRs require minimal DNA manipulation while AFLPs require restriction digestion and adapter ligation. Size fractionation of SSRs is done on inexpensive agarose gels, which are easy to set up and use (Reiter 1994). AFLP markers were successful in distinguishing 27 named bermudagrass cultivars and breeding lines (Zhang et al. 1999). Studies in Zea mays (Gethi et al. 2002) have shown that 44 primer pairs detect small genetic variations within inbred lines, which are presumably genetically identical. In this study we attempted to apply species-specific SSRs to many of the same cultivars to confirm the unusual placement of Tifeagle, which is an irradiated mutant of Tifway II (Zhang et al. 1999). We will seek to determine if it is possible to differentiate between closely related species of bermudagrass using primers developed from bermudagrass genomic sequence enriched for (CA/GT) n repeats, the third most common SSR in plant genomes (Morgante and Olivieri 1993). Materials and Methods Plant Material The 23 genotypes tested in this study are listed in Table 3.1 along with their origin, ploidy, and use. Named cultivars were obtained as breeders or foundation seed from the same sources used by the AFLP study of Zhang et al. (1999). GN-1, which is generally classified as a fairway grass, is more cytogenetically similar to the lawn grasses and was

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52 therefore grouped with them. Grass samples were maintained in black, one and two gallon size plastic containers, using a soil-less mixture of three parts sand to one part peat and one part perlite, in a greenhouse on the University of Florida campus. Dr. B. Scully graciously provided additional stocks of Tifsport, Tifway 419, Tifway II, Tifdwarf, Tifeagle, Champion, MS Supreme, and Floradwarf, from his breeding program at the Everglades Research and Education Center, University of Florida. DNA Preparation DNA was extracted from two grams of frozen leaf tissue by the method of Dellaporta et al. (1983). Nucleotide concentration was read on a SmartSpec 3000 (BIO-RAD. Hercules, CA). DNAs were diluted to 1.0 g/ul and stocks maintained at -20 o C. Enriched Library Construction An enriched microsatellite library (Kandpal et al. 1994) was developed for Tifway 419 and plant introduction (PI) 290-984 DNA for (CA) n repeats. The Tifway 419 library was used for all primer development in this project. Library enrichment was accomplished by digesting 5.0 g of genomic DNA with Sau 3A I enzyme overnight and fragments between 0.4-1.5 kilobase pairs in length were isolated by passing restricted DNA through Chroma Spin columns (Clontech Laboratories, Palo Alto, CA). Sau 3A I (Forward Sau-L-A: 5' GCGGTACCCGGGAAGCTTGG; Reverse Sau-L-A: 5' GATCCCAAGCTTCCCGGGTACCGC) linkers were ligated to the fragments and the ligation mixture was washed through Chroma Spin columns (Clontech Laboratories) to remove excess linker. Fragments were amplified by PCR using primers annealing to the Sau 3A-I linkers with random base pair overhangs into the genomic sequence and 15.0 L of the product was denatured and hybridized to a biotinylated probe [5-(CA) 15 TATAAGATA-Biotin]. This hybridization solution was washed using the VECTREX

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53 Avidin D matrix (Vector Laboratories, Burlingame, CA), which binds to the biotinylated probe. In the wash solution (0.5 M phosphate), only DNA fragments hybridized to the probe remain attached to the matrix, but when eluted with distilled water, the DNA fragments were released from the probe and removed. Both the impoverished fraction and the enriched fraction were saved and tested for (CA) n enrichment by dot blotting a 10% dilution series (from 100% down to 0.1%) of both fractions on 3MM paper and hybridizing with a fluorescent probe. Film was exposed to the paper for 2 hrs (Figure 3.1). Fragments of enriched library were again amplified by PCR using the Sau 3A I primers, ligated into a vector and transformed into Escherichia coli (One Shot, Invitrogen; Carlsbad, CA) using the TOPO TA Cloning vector (Invitrogen). Transformed bacterial colonies were raised on blue/white inducing media (LB media with 80 g/ mL x-gal, and 0.5 mM IPTG) and were also tested for repeat presence by colony lift on 3MM paper and hybridization of a fluorescent probe (Figure 3.2). Those colonies fluorescing and showing white color were chosen. After the initial test transformation, subsequent ligations used P-Gem T Easy Vector (Promega Corp.; Madison, WI) transformed into JM-109 (Promega) competent cells. Transformed colonies were grown on LB media with 100 g/ mL ampicillin, 80 g/ mL x-gal, and 0.5 mM IPTG and selection was based on blue/white color according to manufacturers instructions. Cloned inserts were amplified by PCR using M13 forward (5 GTTTTCCCAGTCACGAC) and reverse (5 CAGGAAACAGCTATGAC) primers. Inserts were then sequenced by the University of Florida ICBR DNA Sequencing Core using ABI 373a Stretch/377 DNA sequencers (http://www.biotech.ufl.edu/DNASequencing/services.html).

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54 Primer Development The genomic insert sequences that contained a microsatellite of at least five repeats, and sufficient flanking sequence to develop primer pairs (Figure 3.3), were chosen and primer pairs were developed using Primer 3 (Rozen and Skaletsky 2000) on the San Diego Workbench ( http://workbench.sdsc.edu/ ). The criteria used for primer selection was a 58 o C annealing temperature and a primer length of 16-20 bp (Table 3.2). Species-specific primers were first tested for amplification on plant introduction (PI) 290-905, PI 290-900, PI 290-895, and Tifway 419 (Table 3.1). Primer amplification was tested using four step-down PCR programs (Table 3.3) of varied stringencies, which consisted of ten cycles with one degree lower annealing temperature each cycle. These were followed by 30 cycles at the lowest annealing temperature. The primer sets that amplified DNA fragments were then used to test 23 different bermudagrass genotypes, including fairway, greens, turf/ ball field types, and breeding material (Table 3.1). All PCR was amplified in a PTC-100 Peltier-effect thermal cycler (MJ Research, Inc.; Waltham, MA). TaqBead Hot Start Polymerase (Promega) was used in all reactions with final concentrations of reactants being: 1.0 X Promega reaction buffer (50 mM KCl, 10 mM Tris HCl, 1.63 mM (volume/volume) Triton X 100), 0.2 mM of each dNTP, 1.5 mM MgCl 2 1.0 uM of each primer (forward and reverse), 1.0 g of template DNA, and 1.25 units of Taq polymerase in a total volume of 50 L. The PCR products were fractionated on 3% Agarose 1000 (Invitrogen) gels in 1X TBE buffer (1.2% Trizma base, 0.6% Boric acid, 0.07% EDTA) at 110 V for four hours and stained with a 0.1 g/ mL ethidium bromide solution for 30 min. To obtain a more precise estimate of fragment size, polymorphic PCR products were fractionated on 6% polyacrylamide gels and then stained with a 0.1 g/ mL ethidium bromide solution for 10 min. Gels were visualized

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55 over a UV light box and the images were captured on the AlphaImager 2200 system (Alpha Innotech Corp. San Leandro, CA). PCR product length was determined by estimation based on a 100 bp and 25 bp marker ladder (Invitrogen) run on each gel. The amplification products of the entire set of template DNAs from at least two separate PCR reactions were fractionated on separate gels to confirm reproducibility of the results. Analysis Fragment lengths for phylogenetic tree construction and genetic similarity were determined by computer (Tables 3.4-3.6), using Kodak Digital Science 1D version 2.0.2 (Eastman Kodak Co. New Haven, CO) and Excel was used to present the results as 0 (absence) or 1 (presence) of a fragment within a range of 10 bp. The phylogenetic trees were constructed using PAUP Version: 4.0 beta 10 (Swofford 2000), run on Power Macintosh (PPC). Genetic similarities were calculated using the formula GS=m/(m+n) where GS=genetic similarity between individuals, m=matches, and n=fragments not matching (Senior et al. 1998). Results An SSR enriched library was developed using Tifway 419 as the source of genomic DNA. DNA fragments were enriched for (CA) n repeats and a dilution series hybridized to a fluorescent probe (Figure 3.1) to attempt quantification. Following amplification and cloning of the library, bacterial colonies were screened for the presence of the repeat motif (Figure 3.2). Out of 250 colonies on the test plate, 178 contained the (CA) n repeat. After ligation and transformation were optimized, 50% of the colonies chosen by blue/white color selection contained genomic inserts of 600 bp, based on PCR amplification of the insert.

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56 From 96 inserts of 600 bp, 56 yielded sequences longer than 100 bp because of problems with sequencing. Of these, 21 genomic fragments carried SSRs with (CA) n and n > 5. Eleven fragments contained sufficient flanking sequence for primer development (Figure 3.3; Table 3.2). There were other imperfect SSR motifs represented within several sequences. Predicted amplicon sizes ranged from 97 to 612 bp. For future study, 106 additional clones were sequenced yielding 74 that contained inserts, and 27 that contained confirmed SSRs and enough flanking sequence for primer development. These sequences are reported in Appendix B and are presently being exploited. All 11 primer pairs developed from cloned sequences (Table 3.2) were tested for amplifications on a subset of DNA templates (Table 3.1) using PCR programs KAM1NW, KAM2NW, and KAM3NW (Table 3.3). PCR products were scored for clarity and brightness of fragments on the agarose gels. Only three of the 11 primer pairs amplified a product over the three PCR protocols. The program producing the clearest amplicons was then used to amplify from the entire 23 templates (Figures 3.4-3.7). Replicated trials of each primer set amplification were fractionated on separate agarose gels and higher resolution polyacrylamide gels. BER1 and BER11 amplified products that were smaller than expected, but products amplified using BER9 were within 75 bp of the expected size (Table 3.2). Tables 3.4-3.6 list estimated amplicon sizes using primers BER1 (Figure 3.4), BER9 (Figure 3.5), and BER11 (Figure 3.6) to amplify from the 23 bermudagrass templates. The five fairway grasses (Table 3.1) were separated into smaller groups consisting of Midiron and MS Choice, which were unique from each other and from the other fairway grasses. Tifway 419, Tifway II, Tifsport consistently showed similar

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57 amplicons. Amplicons from greens grasses (Table 3.1) showed little variation. Tifeagle may have a longer BER9 amplification product visible on the polyacrylamide gel (Figure 3.7), but the pattern of fragment length remained undistinguishable on the agarose gels (Figure 3.5). The four lawn grasses were differentiated into three groups by amplification of product using the BER1 primer pair (Figures 3.4). BER9 (Figures 3.5) showed no product for Tiflawn or Tifton 10, but GN-1 and Floratex appeared similar. BER11 appeared able to distinguish all four lawn grasses by agarose fractionation (Figure 3.6). The breeding lines, which consist of the breeding accessions (BAs) and PIs, were separated into a group containing BA 481 and BA 157, while the PIs were individually distinguished by unique PCR products. To verify the grouping of Tifeagle with the greens grasses and Tifsport with Tifway 419 and Tifway II, DNA was isolated from another sample of these grasses, and BER1 was used to amplify products from all named varieties (Figure 3.10). The same results were obtained. BER1 amplicons fractionated on gel #20027 (Figure 3.4) were scored (Table 3.4) and used to produce a phylogenetic tree (Figure 3.9). Grasses from different categories were grouped together by this method. PI 290-900, Floratex, Tifway 419, Tifway II Tifsport, and MS Choice were all placed in one group. Greens grasses were grouped together, as were BA 475, BA 157, BA 481, Tifton 10, and Tiflawn. Two groups of interest were formed, GN-1 with PI 290-895 and Midiron with PI 289-916. BER9 amplicons fractionated on gel #20735 (Figure 3.5) were scored (Table 3.5), and showed two groups of six (Figure 3.10). The first group consisted of Tifton 10, PI 290-868, BA 475, BA 157, BA 481, and Tiflawn while the greens grasses formed the

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58 second. MS Choice and Midiron were widely separated, with Midiron being closer to the three other fairway grasses. PI 289-916 and PI 290-895 were grouped together. BER 11 amplicons from gel #20763 (Figure 3.6) were scored (Table 3.6) and grouped the greens grasses together, with Midiron closer to them than to a group of the three fairway grasses. Both groups were distinguished from MS Choice. BA 475 was separated from BA 481 and BA 157 by these amplicons. A combined phylogenetic analysis (Figure 3.13) showed that the six greens grasses were indistinguishable, as were the two groups of the fairway grasses and the BAs. Each of the other grasses had unique amplicons from one or another primer pair. A genetic similarity matrix (Table 3.7) was produced from the results shown in Tables 3.4 to 3.6. By this means also greens grasses were clearly similar, as were the three fairway grasses and BA 481 and BA 157. The largest similarity outside of these groups was 33% between MS choice and PI 290-868. Individual matrices for each primer set are in Appendix C. Discussion Enrichment for (CA) n repeats in the bermudagrass genome was successful. Although problems occurred with sequencing we obtained three polymorphic primers from 96 sequences, similar to numbers from other enriched libraries (Kubik et al. 1999; Cordiero et al. 2000). PCR amplification using primers developed from the enriched library usually showed single or double amplification products with no apparent correspondence to the ploidy level. From the amplicon patterns produced by each primer pair, phylogenetic and statistical analyses showed distinct groups, and when combined, showed great dissimilarity and uniquely identified many of the lawn grasses, breeding lines, and fairway grasses.

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59 Nine out of the eleven primer pairs developed failed to produce amplification products. It is possible that some of the regions were inaccessible to normal PCR reactions and would be available if the DNA were first restriction digested as it was in the building of the enriched library. It is also possible that there were problems in the editing of the sequence so the primers themselves did not function. Perhaps variations in the PCR program and PCR reaction mixture would allow amplification to occur. PCR products produced by BER1 and BER11 from Tifway 419 bermudagrass DNA were smaller than expected (Table 3.3). In this case it is possible that stem-loop formations occurred or primers amplified different alleles of the site that was sequenced. Sequencing of products may explain these size discrepancies. The generally low level of microsatellite polymorphism within the groups of fairway grasses and greens grasses is in agreement with previous studies (Busey et al. 2000; Taliaferro 1995; Caetano-Annoles 1998). Taliaferro (1995) proposed that the restricted background in turfgrass breeding programs may become a serious problem in agreement with our results revealing identical grouping of BA 157 and BA 481. Midiron, MS Choice, the lawn grasses, and the PIs are hybrids from different breeding programs and were different for the SSR markers. The relationships between mutants and irradiated parents came into question by the use of SSR and AFLP markers. The documented histories state that the greens grasses are mutants of Tifgreen, and that Tifeagle is a mutant of Tifway II (Taliaferro 1995; Zhang et al. 1999). This explains the difficulty in distinguishing the greens grasses by molecular markers. However there are questions about the origin of Tifeagle. Both in this work and in the work of Zhang et al. (1999) Tifeagle is more similar to the greens

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60 grasses than it is to its irradiated parent. In a similar case, Tifway 419 and Tifway II are related (Burton 1985) but have shown similarity to Tifsport, which is a mutant of irradiated Midiron (Zhang et al. 1999). Both genetic similarity and phylogenetic trees produced from the gel pictures fail to group these grasses with their irradiated parents. When DNA samples from other individuals of these cultivars were tested our observations were sustained (Figure 3.10). Because Tifeagle came from the irradiation of a fairway grass it would be expected to appear similar to Tifway II. The same is true of Tifsport. We observed that Tifeagle was not similar to Tifway II, but was similar to the greens grasses, which would require at least five independent changes. Tifsport also showed no similarity to Midiron by genetic similarity (Table 3.7). More probably, mislabeling or contamination of seed material occurred at some point in the irradiation and selection process. Although closely related cultivars were not distinguished, this study represents the first attempt to use microsatellites for the identification of bermudagrass cultivars. The (CA) n repeat is the third most common repeat in plant genomes (Morgante and Olivieri 1993), and was chosen to avoid overcrowding during the library enrichment steps. Sequencing and primer development of fragments from the Tifway 419 library (Appendix B) are currently being exploited to continue this work. When this library is exhausted the PI 290-984 library could be exploited or new libraries could attempt enrichment for (AG) n which is more common in plants. Sequencing of fragments enriched for (CA) n revealed that microsatellites contain numerous motifs (Figure 3.3). As more primer pairs are characterized, and more than one individual from a cultivar is used, unweighted pair group method with arithmetic mean (UPGMA), Neis genetic

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61 distance, and other statistical analyses may be conducted. A larger number of SSR loci would separate groups that are unresolved in this study.

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Table 3.1 Origins and uses of bermudagrass cultivars evaluated for simple sequence repeats Type Identification Ploidy Taxon Source Reference Fairway MS Choice 27 (3n) txd J. Kranz & W. Philley, MSU Krans et al. 1995 Midiron 27(3n) txd J. Fry, Kansas State University Anderson et al. 1988 Tifsport 27(3n) txd W. Hanna, USDA-ARS. Tifton, GA Zhang et al. 1999 TifwayII 27(3n) txd W. Hanna, USDA-ARS. Tifton, GA Burton 1985 *Tifway 419 27 (3n) txd W. Hanna, USDA-ARS. Tifton, GA Burton 1966b Greens Tifgreen 27 (3n) txd W. Hanna, USDA-ARS. Tifton, GA Hein 1961, Tifdwarf 27(3n) txd W. Hanna, USDA-ARS. Tifton, GA Burton 1966a Tifeagle 27(3n) txd W. Hanna, USDA-ARS. Tifton, GA Zhang et al. 1999 MS Supreme 27 (3n) txd J. Kranz & W. Philley, MSU, MI Krans et al. 1999 Champion 27(3n) txd Mike Brown, Coastal Turf, TX www.champion_dwarf.com Floradwarf 27(3n) txd Al Dudeck, Dept. Env. Hort. UF, FL Dudeck ,1991 Turf/ GN-1 ND ND Jimmy Doublle, GNT, Florida www.shark.com/gnturf/introduction Ball field Floratex 36 (4n) d Al Dudeck, Dept. Env. Hort. UF, FL Dudeck 1991 Tiflawn 36(4n) d W. Hanna, USDA-ARS. Tifton, GA Hein 1953 Tifton 10 54 (6n) d W. Hanna, USDA-ARS. Tifton, GA Hanna et al. 1990 Breeding BA 481 ND ND B. Scully, EREC-IFAS-UF, FL EREC, Breeding line Accessions BA 157 ND ND B. Scully, EREC-IFAS-UF, FL EREC, possible 'Wintergreen' BA475 ND ND B. Scully, EREC-IFAS-UF, FL EREC, Breeding line Plant *PI 290-868 36 (4n) d PI collection, Athens, GA 'Royal Cape' South Africa Introductions PI 290-895 36 (4n) d PI collection, Athens, GA 'Reitz' South Africa *PI 289-916 27 (3n) txd PI collection, Athens, GA 'Magennis' South Africa PI 290-900 27(3n) txd PI collection, Athens, GA 'Damascus' South Africa *PI 290-894 18 (2n) t PI collection, Athens, GA 'Sekapplos Fine' South Africa 62 Cynodon dactylon is designated d, C. transvaalensis is designated t, and their hybrid is designated txd. MSU: Mississippi State University, UF: University of Florida, GNT: Gregg Norman Turf, MS: Mississippi, EREC: Everglades Research and Education Center, ND: not determined. The four templates used as a subset are designated by (*).

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63 Table 3.2 Cynodon spp.-specific primers developed from the sequencing of fragments in the enriched DNA library Primer Sequence Results Program Product Size Repeat and Length BER1 F 5-GCTATACACAATGTGCAGCGT Polymorphic Kam2nw 256 bp (CA) 19 R 5-GATGATTTCGAGCACAGCAG BER2 F 5-ACCAATAGAGGGGGTGTAGC No amplification 171 bp (GT) 16 R 5-CACCCATATTTGTATTGGGATT BER3 F 5-AAAGCGAGCCCCCACTA No amplification 97 bp (GT) 12 R 5-CGAAGAGTTGACCTCCGAA BER4 F 5-CCCCATGTGGTTTTCTGTATC No amplification 279 bp (GT) 6 R 5-TACGGACCCCGACTCAT BER5 F 5-TCTCGTGTCAATAAGGCACA No amplification 612 bp (CA) 10 R 5-ATCATGTAGCAGCTCCCAAC BER6 F 5-GAGCCTTCACAAGAAGTGGA No amplification 231 bp (GT) 30 R 5-ATGACACATGGAGAGGGAAG BER7 F 5-CGACCTTTGGCCACTATAAA No amplification 246 bp (CA) 19 R 5-ATGACACATGGAGAGGGAAG (GT) 31 BER8 F 5-CTAAAGGCGCCATCTCGTAG No amplification 127 bp (GT) 11 R 5-GTTCAAACCCAGCTCTCTGC BER9 F 5-TCTTAATCCATGATGCCGAT Polymorphic Kam3nw 293 bp (CA) 22 R 5-GGAACCTAACACCGTGAGTG BER10 F 5-CATCAGACAACAGGGTGACA No amplification 270 bp (GT) 24 R 5-ACATTCATTTGCATTGCCTT BER11 F 5-TTCCTGAAGTCGATGGGTAA Polymorphic Kam3nw 456 bp (CA) 10 R 5-TGACTTGGAGACATGAGCAA Sequence of the eleven primer pairs developed from Tifway 419 genomic DNA (Figure 3.3) for use in this project. Primers that successfully amplified are underlined. Also indicated are the results of amplification on a subset of 4 genotypes (Table 3.1), the PCR programs (Table 3.3) that were used to successfully amplify products on bermudagrass templates, the expected product length, and the length of the largest (CA/GT) n repeat. Product size indicates the length predicted by Primer 3 (Rozen and Skaletsky 2000).

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64 Table 3.3 Basic step-down PCR programs used to test primer amplification Step Minutes Temp (degrees C) 1 5 95 2 1 95 3 1 annealing temp 4 1.5 72 5 Repeat steps 2-4 ten times, lowering annealing temp 1 degree in each cycle. 6 1 95 7 1 lowest annealing temp 8 1.5 72 9 Repeat steps 6-8 thirty times using lowest annealing temp. 10 5 72 11 cool down 10 The four step-down annealing temperatures (65-55 o 60-50 o 55-45 o and 50-40 o ) used for this project were originally developed by Kamps and Okagaki (In prep). They were identified in this project as KAM1NW: 65-55 o KAM2NW: 60-50 o KAM3NW 55-45 o and KAM4NW: 50-40 o respectively.

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65 Table 3.4 BER1 PCR product length on 23 bermudagrass DNA templates from gel #20027 (Figure3.4) Grass ID Product size MS Choice 82 105 Midiron 83 130 Tifsport 82 100 Tifway II 81 101 Tifway 419 81 101 Tifgreen 83 116 132 Tifdwarf 82 118 132 Tifeagle 83 118 135 MS Supreme 82 116 134 Champion 82 116 137 Floradwarf 83 118 139 GN-1 83 124 Floratex 83 108 Tiflawn 85 Tifton 10 85 BA 481 87 BA 157 86 BA 475 86 PI 290-905 105 141 PI 289-916 88 130 PI 290-900 86 103 PI 290-868 89 100 PI 290-895 88 127 PCR product length (in bp) was determined by the computer program Kodak Digital Science 1D version 2.0.2 (Eastman Kodak Co.) based on 100 bp and 25 bp marker ladders (Invitrogen) run on each gel. This information was used to produce phylogenetic tree information (Figure 3.9) and compute genetic similarity (Table 3.7).

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66 Table 3.5 BER9 PCR product length on 23 bermudagrass DNA templates from gel #20735 (Figure 3.5) Grass ID Product size MS Choice 330 Midiron 345 Tifsport 298 345 Tifway II 298 345 Tifway 419 298 345 Tifgreen 307 Tifdwarf 307 Tifeagle 307 MS Supreme 307 Champion 307 Floradwarf 307 GN-1 316 Floratex 303 330 Tiflawn Tifton 10 BA 481 BA 157 BA 475 PI 290-905 286 321 PI 289-916 316 PI 290-900 290 PI 290-868 PI 290-895 316 350 PCR product length (in bp) was determined by the computer program Kodak Digital Science 1D version 2.0.2 (Eastman Kodak Co.) based on 100 bp and 25 bp marker ladders (Invitrogen) run on each gel. This information was used to produce phylogenetic tree information (Figure 3.10) and compute genetic similarity (Table 3.7).

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67 Table 3.6 BER11 PCR product length on 23 bermudagrass DNA templates from gel #20763 (Figure 3.6) Grass ID Product size MS Choice 209 236 Midiron 224 Tifsport 248 Tifway II 251 Tifway 419 251 Tifgreen 209 254 Tifdwarf 209 251 Tifeagle 209 251 MS Supreme 209 254 Champion 206 251 Floradwarf 209 251 GN-1 230 251 Floratex 206 260 Tiflawn 199 Tifton 10 222 248 BA 481 236 BA 157 236 BA 475 214 PI 290-905 260 PI 289-916 245 PI 290-900 214 257 PI 290-868 107 230 PI 290-895 107 273 PCR product length (in bp) was determined by the computer program Kodak Digital Science 1D version 2.0.2 (Eastman Kodak Co.) based on 100 bp and 25 bp marker ladders (Invitrogen) run on each gel. This information was used to produce phylogenetic tree information (Figure 3.11) and compute genetic similarity (Table 3.7).

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Table 3.7 Genetic similarity matrix including combined data from all BER primer sets MS ChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMS SupremeChampionFloradwarfGN-1FloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-89 5 MS Choice1Midiron01Tifsport001Tifway II0011Tifway41900111Tifgreen000001Tifdwarf0000011Tifeagle00000111MS Supreme000001111Champion0000011111Floradwarf00000111111GN-1000000000001Floratex0000000000001Tiflawn00000000000001Tifton 100.250.250000000000001BA 4810.2500000000000.170001BA 157000000000000.1700011BA 475000000000000000001PI 290-9050000000000000000001PI 289-916000000000000000.2500001PI 290-900000000000000000000.25001PI 290-8680.3300000000000.110000.250.2500001PI 290-89500000000000000000000001 68 Genetic similarity was determined by the formula GS=m/(m+n) where GS=genetic similarity, m=matches, and n=fragments not matching.

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69 Enriched genomic fractionImpoverished genomic fraction 100%10%1%0.1% Figure 3.1 Dot-blot hybridization of dilution series of enriched libraries to verify enrichment for (CA) n repeats. Two rows of dark spots in the enriched genomic fraction indicate genomic libraries from Tifway 419 and PI 290-894. The dark spots show that the fluorescent probe has hybridized to the microsatellite in the sample. Two spots corresponding to the two impoverished fractions were placed directly above the enriched fraction. Going from left to right a 10% dilution rate of enriched DNA was tested to show that sufficient concentration of DNA library had occurred. Inaccuracies in pipetting account for the darker spots in lower concentrations.

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70 Figure 3.2 Colony transfer and hybridization of initial bacteria transformation and hybridization to fluorescent probe to show transformation success rate. Bacterial colonies were checked for the presence of repeat of interest in correspondence with blue/white color selection. Most of the colonies that fluoresced after hybridization were also white, indicating the presence of insert. Only those colonies that were both white and had fluoresced were chosen for amplification and sequencing.

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71 Plate 14441 C1 BER1 GCGGGGAGCCTGGGAAGCTTGTGGATCGCACGAAAC GCTATACACAATGTGCAGCGT GCAGTGACGTGTC AACGTGACACACACACACACACACACACCACACACACACACACACACGCACACACAAGCTTATTTTTGGT CGGAATGTGAAATGCAAATATATACTTTCATTAAGAATACACACACAAATTAGCTTATTTGTACAATGTC TACAAAGAAAAGTCACTAGATAGGGACTGACAAAGTTCAAGAATAATACCTTGTGTGCCATTTC CTGCTG TGCTCGAAATC ATCTGAGGAACATCAGGTAAAATAGATATATCCAGCGAAACATCATTCAATATATTATC CATCACATAAAAATTATCGGGCAACTCCTGTGACTCTGAGGATATAACATTGTACGTATTGAGCACACTA TTGGTATA Plate 14441 D6 BER2 GCGTTAGCCGGGAAGCTTGGGATCTACATCTGACG ACCAATAGAGGGGGTGTAGC TCCTCG AGTTGATGG TGAGTAGCTACCCACGGTTGTAGCTAGTTATCTCCGTATCTCTTATATGTTGCGTGCTGATTCTCTCTCT CTCTCTCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTAGTAATCCC AATACAAATATGGGTG Plate 14441 E7 BER3 CCATACAAGAGG AAAGCGAGCCCCCACTA TATCATCAATGAGAAAG TGACTTGTGGAAAAGAAGGGGTAG AGAGGGTGTGTGTGTGTGTGTGTGTGTGTGCAGGAAATTATA TTCGGAGGTCAACTCTTCG GT Plate 14441 F7 BER4 AAATGATG CCCCATGTGGTTTTCTGTATC TTATGCTGGTTTGTGTATACTCCTACACATGAATCAGGAGT ACTTACAAGTTTGCTTGGATAAGTTNTGTTACCAAGAAAGAACCGGACGACGTTTCTACAGTATCACAAA AGAAAAAACAAAGAGGAATGCCATGTCCGTGTGTGTGTGTTGAAGAACCACGGACGTACTCCTGCTCGTA GCAGAGGCAGAGCACGCAGCAGCAGGCGTTCGCAGCGGAACAGCAGCGACGTGATGAGTC TACTCAGCCC CAGGCAT GGGGTCCGTACCCTCCGACGCCG Plate 14441 F10 BER5 AGCTATTATCAAATGTCATTTCTCTTATTCA ACGATAAATTATCCATTTCAGCTCGCTCTCGTGTCAATA AGGCACA CACCCACTCCCATTCACTGCCCCTCTCTCTCTCTCTCTCTCTCTCTTTCTCTCTCTCTCTCTC TCTCTCTCTCTCTCTACACACACACACACACACACTCAAAAAGAAGCAATCATTCCCTCTTTCACATAGT AATTTCCAATTCCTAGTGGAATTTAGTCTAGATTGAGCCTTGCTCATGTCTCCAAGTCATAGGAGTATTG GTATGGCACTTCTATTCCCTTTATGTATGACTTTGCTCTATTTCAATAATATATTTCTTTCATATAGTGT GCTTATGATATATGTTCCTTGTTTGGTTATATATAATGCTCATGTCATACGACTAAAGGCATGCATACTT TCATAATAGTATAGGCTTTCTACCTTGCTGTTATGATTGGCATATACCAGGACTATAATTGTTATAATAC TACTTTATACTTAGCTCTAGTTGTTATGCCTCAGCATAGGTTTAATATCCAATTAGGTTTAGGGAAGTTC TAGAGCTTGATCCCAAGCTTCCCGGGTACCGCAATCACTAGTGCGGCCGCCTGCAGGTCGACCATATGGG AGAGCTCCCAACGCGTTG GTTGGGAGCTGCTACATGAT GCATAGCTTGAGTATTCTATAGTGTCACCTAA ATAGCTTGGCGT Plate 14441 F5 BER6 GCGTTTTGCCTGGGAAGCTTGTGTGATCGATGTGAGTCAGAGATTTGTGCCTGTGAGCTGGTGCTTGCAA TTTCACAAGTTCAAAGAAAGTGCTT GAGCCTTCACAAGAAGTGGAT AACAGCCCACGTCCCTCTCTCTCT CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCATCACTCTCTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGAGAGAGAGAGAGAGAGAGAGGGGGGAGCATCCGGGC GACATCTAGTGCAGGCCCTCAACCGG CTTCCCTCTCCATGTGTCAT ATCTCTAGCTAGCTAGTACTGTCT ATATAAGCGCACCAATGGCCCCATCACGGCTTCAGTTCACCGTGGTTGCATTTGTTCAACTCTGGTTAGT CTTCATTTCCATCTATTTATGTATAGAAGTTGTATTTGTTCAGCTCTGGTTAGACTTCCTTCGCGTAGAA GTCTATTGTTGAAAACATAAATCGTGTATGCATTTCGTTGATACGGGCTCTACCATCCGAGTCATTAACT ATGAAGACCTTGTTTCTCCAACCCAGGGCGTGAATCAGTTCTGATTAATTTGATATTTTTGATAAGTTCT TAAGCCCAAGTGGGCGATCCCAAGCTTCCCGGGTACCGCAAGGGCGAATTCCAGCACACTGGCGGCCGTT ACTAGTGGATCCGAGCTCGGTACCAAGCTTGGCGTAA Plate 14441 C12 BER7 GCGGTACCTGGGAAGCTTGGGATCGCTGACGTGGTTGCACAGGATTCTTCTGACCTGTCAGCCACCCAGA AGAAGCATAGGCTGAGGGGGCCCAAGCCTAGGTTGGCCAACCTAGGGTTGAGGCGGTTACTCTACTGCCT CAACGTGGCACTCCGTGGTGGCTTCCTGAAGGCGGTTGCATGGCGATTACTTCGTGGTTACTCCTTCTTA AC CGACCTTTGGCCACTATAAAT AGAGGGTCACATTCTTCACTCCAACACACACACACACACACACACAC ACACACACACACACACACAAACACTCATATATAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGTGTGTGTGAGAGAGAGAGAGAGAGAGAGGGGGGAGCATCCGGGCGACATCTA GTGCAGGCCCTCAACCGG CTTCCCTCTCCATGTGTCAT ATCTCTAGCTAGCTAGTACTGTCTATATAAGC GCACCAATGGCCCCATCACGGCTTCAGTTCACCGTGGTTGCATTTGTTCAACTCTGGTTAGTCTTCATTT CCATCTATTTATGTATAGAAGTTGTATTTGTTCAGCTCTGGTTAGACTTCCTTCGCGTAGAAGTCTATTG TTGAAAACATAAATCGTGTATGCATTTCGTTGATACGGGCTCTACCATCCGAGTCATTAACTATGAAGAC CTTGTTTCTCCAACCCAGGGCGTGAATCAGTTCTGATTAATTTGATATTTTTGATAAGTTCTTAAGCCCA AGTGGGCGATCCCAAGCTTCCCGGGTACCGCAAGGGCGAATTCCAGCACACTGGCGGCCGTTACTAGTGG ATCCGAGCTCGGTACCAAGCTTGG Figure 3.3 Nucleotide sequences of the clones from which species-specific primers were developed. Underlined sequences show the location of SSR primers, and bolded sequences indicate all of the potential microsatellites.

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72 Plate 14441 C5 lBER8 GCGTTAGCCGGGAAGCTTGGGATGCACTTGTCGGATCAGATGGCGGTGGCGGCCTAACGTCATGACCTTC CTAAAGGCGCCATCTCGTAG CAGCTCCCAACCACTACGGCGTCCATGTCGTTTTTGTGTGTGTGTGTGTG TGTGTGTCGCGCAGTGAGCTGGAGGCTCGATTTGGAA GCAGAGAGCTGGGTTTGAAC TGTGGAGTATTGC ACTTTCGGTAGGGATTGGTTTCGGTTTGAAATTCGAATGAAACGCATGGTTTCGTCGCTGCAAAAGGCAA GGAAATTTGGGGAGGCGAAAACAGTCCACAAGCAAGCAAAAGAGGGAAGTGCTATATCGGGCACATGCTA CTCTGTTTTACCATAATGCTAGCATTTACTTTTTAAATACGCAGGAAAAATTACTTTATTTGGTTCTTCC AATAGTGTAAATACAACTTTATAAAAAAAAGGTCTAGTATAGTCTTTTCAATATCTTTCTTTTAGCACCA GATTAATGCATAAATATCTTGATTTTTACCTCTTGTTAATTGACGTGTTGATAATTAGATGGGTGTTGAT TTTTTTTTTTCTTTTTTGAGAACCATAGTACAAACTAATACCATAACACTATGAGTACATACAAGCACAA TCTGGGTCGTTCTATCTGGAAATTTACAAACTTATCACATATGCCTCGTTATTGATGGAGCCATCACTAA TACGATCGTAACATTCATGTTGAATTTAGGGCTCCAAAATAGGATCCCAAGCTTCCCGGGTACCGCAAGG GCGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGGCGT Plate 14441 C2 BER9 GCTTTTAGCCGGGAAGCTTGGGATCTCCT TCTTAATCCATGATGCCGAT ATGACTCAATAAATATAATTC TTCCATATCCTTTTAGTTTGTCTTGGTCTATCACTAGTTGGTGCAATATTCTTGCACTTACAGTTCAAAC TCAATGTTTCCTCCACCTCTACTATATCTAGTAGTTTGGATACTTGGTAAGACAAGAAATATATTTTTTG TTACTTCATACACCCATGCACACACACAGCACACACACACACACACACACACACACACACACACAATACA ACAAACAGACAAACTTAAGAAT CACTCACGGTGTTAGGTTCC GTGATATTATGCAGTGCGCGAGAAAATC TAACCGACCATTTGGGAAGACCACACGTGACTGATTGATTCTCACCTCAATATGACAAACTTTACCAAGG GGGAAGTAAGCATGGCCGTATTATCTTTAAGGTGCTGTTTGGCAATGTGTCAATCCAAAAACTCAGGAGG TATTAGTTGCAGGTATGCATGAAAATAGTGAGGTCACAAAATGCAAATAAAGAAACAAAGTGCAGCAAAT AGCTGAATCATGATGAACTGTTATTTACGAGGGTCAACAATGGTTGCATGTGGTGGTTTCTACTAAATAA AAAATAGAAAGATTTTCCCGATTAGCTTTCCTGCTAGGGAGTTGGTGTGCAGACTGGAAAGTCTCCACAT GCGCCAGATCCCAAGCTTCCCGGGTACCGCAAGGGCGAATTCCAGCACACTGGCGGCCGTTACTAGTGGA TCCGAGCTCGGTACCA Plate 14441 B7 BER10 CTTAAACTCATTTGCAACAGGTGCGGTTGCGTGGCTATATCTAGCATTCCTTTTGCAACACTTCAGTTTT GTGTTGCGATAGCATATAGTCTATTGCGGCAATCGATACTAACTATAACAACATTTTGTATCAATTGCGA TATAGACTATTATTGCAAT CATCAGACAACAGGGT GACA ATAACATCGCCCTAAGTACAACGCACATATC ATCGTTGCAATATAGTCTACTTATTGCAATTAAGTGTGTGTTTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGATTGCGCTCTCGCGTGTGCTCCAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA GAGAGAGAGAGAGAGAGTTGGCATGTATAGCGGGTAGCGGCAGCTCTGGCAAAACCCAA AAGGCAATGCA AATGAATGT GTGGATTTGAATTCTCCTGCCGTGCAGCAATGATGCTAGGCAACAGCAGCAGAGAAAACAG GCCATGTATTACTGTTTCTGCACTACAGGATGTAAGCTCGCTGCGGCTTTGCTTGTAACGCTGAGTATTT TTGCTTGTACTTAGCTGTTCTGGAGCATTAGTAAAATCGAATGATCCCAAGCTTCCCGGGTACCGCAATC ACTAGTGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTAT TCTATAGTGTCACCTAAATAGCTGG Plate 14441 A2 BER11 GCGGTAGGGGGATGCGAGGTATGAAAGTGTGGGCTGCATAGGGTGTCGCGTTAGAAGCACCATGGCTAGC AGGTGCAGCATGGTAGTGACCGGGAAGAAAACACATCGACCGACCTTGTCCTGAGGCGGTTCAGCCTGAG CTTCCATGTGTCACTCCCTTGTCCAT TTCCTGAAGTC GATGGGTAA CGGTTTCATGGCGTTTGCTCCTAG ATCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC AAACCGACCTTTGGACACTATAAAAGGATGGTAGATCTCTCTCTCTCTCTCACACACACACACACACACACTCAAAGAGAAGCAAACATTCTCTCTCTCACATAG TAATTTCCAATTCCTAGTGGAATTTAGTCTAGATTGAGCCTTGCTCATGTCTCCAAGTCATAGGAGTATT GGTATGGCACTTCTATTCCCTTTATGTATGACTTTGCTCTATTTCAATAATATATTTCTTTCATATAGTG TGCTTATGATATATGTTCCTTGTTTGGTTATATATAATGCTCATGTCATACGACTAAAGGCATGCATACT TTCATAATAGTATAGGCTTTCTACCTTGCTGTTATGATTGGC TGACTTGGAGACATGAGCAA ATATACCA GGACTATAATTGTTATAATACTACTTTATACTTAGCTCTAGTTGTTATGCCTCAGCATAGGTTTAATATC CAATTAGGTTTAGGGAAGTTCTAGAGCTTGATCCCAAGCTTCCCGGGTACCGCAATCACTAGTGCGGCCG CCTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCAC CTAAATAGCTTGGCGT Figure 3.3 Continued

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73 100 bp100 bp20027 20791100 bp ladderMSChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMSSupremeChampionFloradwarfGN-1MarkerFloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895PositiveNegative100 bp ladder FairwayGreens Lawn Breeding Lines 600 bp 600 bp Figure 3.4 BER1 amplification products of replicated PCR reactions using 23 bermudagrass DNA templates, fractionated on agarose gels, showing the reproduction of fragment length patterns. Names of genotypes are at the top of each lane, 100 bp and 25 bp marker ladder (Invitrogen) was run on each gel with bands of significant length indicated. The middle marker lane is 25 bp in the top gel and 100 bp in the lower gel. The positive control PCR reaction in gel #20027 was run on Tifway 419 DNA template. Tifway 419 DNA was used as the positive control in the top gel, but was dropped because it is one of the 23 DNA templates. The negative controls consist of the entire PCR reaction mix, including primers, but without template DNA.

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74 2073521017100 bp ladderMSChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMSSupremeChampionFloradwarfGN-1MarkerFloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895NegativeNegative100 bp ladder FairwayGreens Lawn Breeding Lines 600 bp 600 bp 100 bp Figure 3.5 BER9 amplification products of replicated PCR reactions using 23 bermudagrass DNA templates, fractionated on agarose gels, showing the reproduction of fragment length patterns. Names of genotypes are at the top of each lane as well as 100 bp and 25 bp marker ladder (Invitrogen), which was run on each gel with bands of significant length indicated. The middle marker lane is 100 bp in the top gel and 25 bp in the lower gel. The negative controls consist of the entire PCR reaction mix, including primers, but without template DNA.

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75 100 bp600 bp20763 21043100 bp 100 bp ladderMSChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMSSupremeChampionFloradwarfGN-1MarkerFloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895PositiveNegative100 bp ladder FairwayGreens Lawn Breeding Lines 600 bp Figure 3.6 BER11 amplification products of replicated PCR reactions using 23 bermudagrass DNA templates, fractionated on agarose gels, showing the reproduction of fragment length patterns. Names of genotypes are at the top of each lane, 100 bp and 25 bp marker ladder (Invitrogen) was run on each gel with a band of significant length indicated. The middle marker lane is 100 bp in the top gel and 25 bp in the lower gel. The negative controls consist of the entire PCR reaction mix, including primers, but without template DNA.

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76 600 bp21050 2104925 bp ladderMSChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMSSupremeChampionFloradwarf25 bp ladder GN-1FloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-895 FairwayGreens Lawn Breeding Lines 125 bp Figure 3.7 Products of BER9 primer pair amplification on bermudagrass DNA fractionated on polyacrylamide gels showing the amplicon pattern for the bermudagrass template shown at the top each lane. A 25 bp marker ladder (Invitrogen) marks the approximate length of DNA fragments. This confirms reproduction of DNA fragments of a predictable length for this primer set.

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77 100 bp ladderPI 290-905 PI 290-894 PI 289-916PI 290-900TifdwarfMSSupreme ChampionTifeagleFloradwarfTifgreenTifsportMarkerTifway 419Tifway IIGN-1BA 481BA 157BA 475PI 290-868PI 290-895FloratexNegativeNegative100 bp ladder 2n 3n 3n or 4n 4n100 bp16088-2600 bp Figure 3.8 Gel #16088-2, showing amplification of BER 1 on a different group of DNA templates. Tifsport is grouped with the fairway grasses and Tifeagle is grouped with the greens grasses as in the amplification of the other samples of these grasses. Tifgreen is grouped with the fairway grasses, but this proved to be an error in the sample of that cultivar. Breeders seed was obtained to insure correct identification and grouped Tifgreen appropriately with the greens grasses.

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78 Tifgreen Tifdwarf Tifeagle MS Supreme Champion Floradwarf Midiron PI 289-916 GN-1 P I290-895 PI 290-868 BA 475 BA 157 BA 481 Tifton 10 Tiflawn PI 290-905 PI 290-900 Floratex Tifway 419 Tifway II Tifsport MS Choice Figure 3.9 Phylogenetic analysis of BER1 fragment length data (Table 3.5) from gel #20027 (Figure 3.4) using PAUP (Swofford 2000).

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79 Tifsport Tifway II Tifway 419 Midiron GN-1 PI 289-916 PI 290-895 Tifton 10 PI 290-868 BA 475 BA 157 BA 481 Tiflawn Tifgreen Tifdwarf Tifeagle MS Supreme Champion Floradwarf PI 290-905 PI 290-900 Floratex MS Choice Figure 3.10 Phylogenetic analysis of BER9 fragment length data (Table 3.6) from gel #20735 (Figure 3.5) using PAUP (Swofford 2000).

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80 Champion Floradwarf MS Supreme Tifeagle Tifdwarf Tifgreen Tifsport Tifway II Tifway 419 GN-1 BA 475 PI 290-900 Floratex Tifton 10 PI 289-916 PI 290-868 PI 290-895 Midiron Tiflawn PI 290-905 BA 481 BA 157 MS Choice Figure 3.11 Phylogenetic analysis of BER11 fragment length data (Table 3.7) from gel #20763 (Figure 3.6) using PAUP (Swofford 2000).

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81 Tifgreen Tifdwarf Tifeagle MS Supreme Champion Floradwarf Midiron Tifton 10 PI 290-868 PI 289-916 GN-1 PI 290-895 BA 481 BA 157 Tiflawn BA 475 Tifsport Tifway II Tifway 419 PI 290-900 PI 290-905 Floratex MS Choice Figure 3.12 Phylogenetic analysis of the collective fragment length data from Tables 3.5-3.7 (Figures 3.3-3.5) using PAUP (Swofford 2000).

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CHAPTER 4 CONCLUSIONS In summary, primers amplifying three SSR containing products with repeats of >5 were isolated from the genomic library of C. dactylon X C.transvaalensis, var. Tifway 419, one of the 23 varieties tested in this study. Species-specific primers yielded reliability of reproduction from one PCR reaction to another and consistently amplified products of an acceptable length. On the whole, cross-taxa primer pairs would require more optimization in order to exhibit reliable amplification. As other markers are found it is likely that even tightly linked groups may be distinguishable. Two varieties, Tifeagle and Tifsport, were not similar to their irradiated parents. This brings into question the source of their origin and should be the topic of further research into the relationship of commercial varieties of bermudagrass. 82

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APPENDIX A INDIVIDUAL GENETIC SIMILARITY MATRICES FOR CROSS-TAXA PRIMER SETS ZMADH2N AND LPSSRHO1A10 Table A.1 ZMADH2N genetic similarity matrix. ZMADH2N MS ChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMS SupremeChampionFloradwarfGN-1FloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895POSITIVEMS Choice1Midiron11Tifsport0.50.51Tifway II0001Tifway41900001Tifgreen0.330.330001Tifdwarf0.330.3300011Tifeagle0.50.50000.50.51MS Supreme0.330.33000110.51Champion0.50.50000.50.510.51Floradwarf0.330.33000110.510.51GN-10.50.50000.50.510.510.51Floratex110.5000.330.330.50.330.50.330.51Tiflawn0.50.510000000000.51Tifton 10110.5000.330.330.50.330.50.330.510.51BA 4810.50.50000.50.510.510.510.500.51BA 157110.5000.330.330.50.330.50.330.510.510.51BA 475110.5000.330.330.50.330.50.330.510.510.511PI 290-9050000000000000000001PI 289-9160.330.33000110.510.510.50.3300.330.50.330.3301PI 290-900000000000000000000001PI 290-868110.5000.330.330.50.330.50.330.510.510.51100.3301PI 290-89500000000000000000000001POSITIVE000000000000000000000001 83

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84 Table A.2 LPSSRHO1A10 genetic similarity matrix. LPSSRHO1A10 MS ChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMS SupremeChampionFloradwarfGN-1FloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895POSITIVEMS Choice1Midiron0.331Tifsport0.330.331Tifway II0001Tifway41900001Tifgreen00.50001Tifdwarf0000001Tifeagle00000001MS Supreme00.50001001Champion00.500010011Floradwarf00000000001GN-1000000000001Floratex0.50.50.50000000001Tiflawn0.500.3300000000001Tifton 100.250.250.250000000000.330.331BA 4810.330.330.330000000000.500.661BA 1570.330.330.330000000000.500.6611BA 4750.50.50.5000000000100.330.50.51PI 290-9050.330.3310000000000.500.250.330.330.51PI 289-9160.330.3310000000000.500.250.330.330.511PI 290-900000000000000000000001PI 290-868000000000000000.330.50.500001PI 290-8950.500000000000010.3300000001POSITIVE000.33000000000000.250.330.3300.330.330001

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APPENDIX B NUCLEOTIDE SEQUENCE FOR FUTURE PRIMER DEVELOPMENT Plate 20972 A2 Gatctagagttctagacaaagatagtgctactaga acacacacacacacacacacacacacacacacacacacacacacaca cacacacac agagcggaccaccctagccagggacccaggagggaaatcaaatctttaaccttttgtccgaagcaagctaagggaaccacatgaacagtgacacagtgtcatgcatgcatgcctgcgttgcggcctcgacaaagggaagggcagctataataatggcgccaaaggcacgcagcaatgcagctcaatgcatcgtattatgcgttgcgtgcgcgcctggaaagagagcagagatgcttgggtcgccacggtacggcacggcaatggctgctgcatgtagacggagacctggcccggccgggcggccggcagggggaggcccagcctgggccctgaagcgtaggccgtgccaggcc Plate 20972 A4 Ccgggaagcttgggatctgctttgcgtttttggttcacaagacatcacaaagtaaaaaatcatgcatccaaagtctcaacatcgtgttacattctccgccccagcctcatcaaagtaagaggagattgaatttaactaacattaggcagcaacaatgcagacatttgttttttttttt tgtgtgtgtgtgtgtgtgtgtgtgtg tcaatttggtgtagaaaacgtgaaagacgcgatgctgctagggattcttgggactgctgtgatgttttaattttgacataccacaaatacatggacctgccagtagtttgtcagcagtgttgtattaaagatacaagcggttttagccttgttaagagttacttcattcgtaagtatagtttaagttgttaaatctcttgtacaaactcttttaataacatttttctcttggtcattcatgtcaactttctttctcttgctcattaat Plate 20972 A5 Gatccgttgtttaccccaagagtatcttgtcccatccgagaaatcatgatagagttagtagtattcactggaacattaacccccctttaattggcaaaaaaaggtgtattctttcttagtgcttaaggagtagctaggcatgcaggtgatagaggtgatatgcagcaacacatcttgtctgctctgtctcttccctctatagaagcttttgttgacttgtgtggaatatggtatgcctgagattcaagctcactatgatacctgatgagtcaggctcctcaagttgcttgtgcctgcttctttacttgtcatttgctc tgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtggtgtgt gtgtg acataatctttatgtaactgcaacactacagaacccaccaattattggaacatgcatgaaagatttcgtgtaattattggcaagagatcccaagcttcccgggtgcaatcactagtgcggccgcctgcaggtcgaccatatgggagagctcccaacgcgttggatgcatagcttgagtattctatagtgtcacctaaatagcttggcgtaatcatggtcatagctgtttcctg Plate 20972 A12 Gatctttcgggtcgaaactactgtgaactagagtgttatagtcaacccccttaagggatgatgccctcagtagcgcacc acacac acacacacacacacacacacacacacacacacacac agagagagagagagagagagagagagagagagggagagagagagagcaagctgatacccagaatcttctctcagtacaagaccgtgttgcgggtctatccacacgcatacgtagacatgcaatgaaagttaatgctctgaaaccactgtaacacctaccatccaaactacgtttttcttctagccataccatatatgggagggtatgcctattcttccatagcacatcttttatattttcgttctcgttttatgtaaggggttaattcgaaggtgcaatgattggaacaacgatgattttatgcttacccttacccactaaacttctgttatgggattaatcacatctctacttcactttattggccttctgagccaaaactactactactacgggttttcaatgggttgggatgttacaccttattagtgctcaagtattgtgagtatgtccaatatggtgtaaaacatatgaaaacacactgaaaatatacgcaagtagaatccagta Figure B.1 Nucleotide sequences of the clones from which future species-specific primers may be developed. Motif greater than five repeats underlined. 85

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86 Plate 20972 B2 Acggccagtgaattgtaatacgactcactatagggcgaattgggcccgacgtcgcatgctcccggccgccatggccgcgggattgcggtgcccgggaagcttggctctcaagattgtaaaagactgtcccaggcaaagaacctaacagggttgatgaccgcgagggagtgactctcacggtctcatcgcatctctataatgatatatcccctccaccccatgcacgcgcgcgc acacacacacacacaca cacacacacac ctgctggccataagacccaacaaaattcacactatcacatataatcattaattatgttaaattccctgcaagtcggtataattttaatctacaaagcagagttcaaagttcaaacacaaacaaaaagagagaatgttcaaaatggaaaaactattcatttagggacaaaaggaatgcatagatttctacacgtggtattattttccctttgccatatggcaaatgggaatgaaaagcgaataactaagtgaagcaactaatttttttgctattttgcagactgaattctctccccatcccatccatttcccggattcctaaagagagtaagaacaaaaattagcacagggaagt Plate 20972 B4 ggatcaataggcagaaaagaaagagcctagaacttgtgcattattgtactagcacaagataaaagta acacacacacacacac ataaaagtcagttctcttatcatcgagagaacaatagaagtcagaaggaagagaggagagaaatacttagttcgtcatgatgcaaggagctgcatctcaggcccaaggtatatttattggtataatacatgagagaatgtcgcacatattctatccggaaaatatacgacaagctctatatcacagagtgtaatatattgttatataaggttaaatcagtcttatgctaacat Plate 20972 B5 gatcgtctagggatgcttaaacagttagatgttcatgtcgttatgtctcaagacacatatctgaaaaacaaagcgacgagcccctacttttgatgcaaatgctgcatatcgctcgagcgagatga acacacacacacacacacacac cctcgagcaagtgcattacatcacggtgatgttcctttgtctccgtccccggccgaccgtgcatgcctctgcatgccaccaaagcctttctacctttaatcagtgcactacgaaaaggagaggccggcattaccgaaaatttgtccaaggattttcgaccgatggtgatgacagaggagagcaaaggctggcttttgcttccacagggaaatgtttcagagcaggcagccaggctctctcaacttttgcatggtaatgccaacggttagacaaggtgttggcctgttggcactacatgactagctatttggttaagtaccacaactgagattgatgagtacggtttattgttggtaatgagctgaaacaataactgtagcgctagcacgctcagaagaatatatatactaaagaaatatgtgcatggtgtcagtagatgtcacgaaagttcgcagcggaacatatttgtagaaa Plate 20972 B6 ccgggaagcttggatctaagaaaacaaataaatgagctagaaacagttaatttttttattacatcaacagtgttcaattgactacgtttttttcattgaagaacaaagatggcaccttttcaacatacgagtgcaagagaactactcctttattggaggtgagtttttg tgtgtgtgtg tgtgtgtgtgtgtgtg tactgcaaaaagtaggcttatttgttaccttatttgctacgttattgttgcattggtgaatatgcccctaactaggattttttttgttcctttttct Plate 20972 B7 gatcccctgtcgactgccgggcgcgcccgggtggtagagcacccggggcccgggtcatgtaaccccctcctccttggtctataaaaggaggaggcgagcaccgtgtaaaaggacagagaaagaaatatactcagacaagcaggacgtagggtgttacgcatcaagcgacccgaacctgggtaaaacacgcgtgttcctacatccgttcgcctgaccacccgagaccgggatccgagctccccataacctttcccaagggcggccttgcagacccccatttggaggtgctc acacacacacacacac cgacgacaacactcaaagaaaacgtttcatgagaagcatgtctgacaaatgtcacgggcccaacggcagaaggcaaaactgctgcatcaccgctgacacatcagggctgtgacgagggctaaccctgcacttaacgcagtgcatctccgtgtgtctgccagagcgcccaggagcctagttaatactactgtattatgaagtgtactagtaatagtaatagcatggcgatactacatatgagctttgaaattcctcactgccaaggtttttttttttgaggcgcttctcactgccagtgggtcatcagcgtccgactttgagaggaggaggcttgaagcggcggaagggcctaagagctgtcggtccg Figure B.1 Continued

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87 Plate 20972 B12 atacgactcactatagggcgaattgggcccgacgtcgcatgctcccggccgccatggccgcgggattattaataatattggtgcaaatggaaagcctgattcaaaaacatgtttagaagttttaagaaattcttcaagaaacggtataagtatttgagtggtgaccaatatgtacactaacattatacaataacttaacaatgaaaacttaaagtgttaatcctgtagttatttctcttctctctagtgtgaacagatatgcaaccctgtttcaaaaacacgtttagaagtttccaatttattgtgaaaaattggcataagtatttgagtatactaacttaatagaatgactagctactaatgacaatttaaagtgttcatctttggggtggaaaccaatactataacaagggcacagaaccaatagtgt tgtgcgtgtgt gtgtgtgtg tctggggggctcaagcccccgctccattagtgcatctgctgctcccttcacttgaacactatttacgccatatgcaaacttgtcaatgtttaatctgctctgattcaaatacctcaaaggtatacgtattaaatgatgctacctcaaatatcactttttgtttctgccaccccaagaatcatatagtccttttttctctatcattttgtatattttatttacttgcgttggctaccttataaataataagatatatggatgtggtcatgtaatagtttatcttaccctagtttaagatatataatcgattcctttaccttggtt Plate 20972 C2 gatccagcgtcacccgaagctgcgacgaccagatctggctaccccctctctggcggcgcggcggcacatcccctcctctcaggcggtgcagcatgttcaacggtgtggcaggacccatagtggacccaaacacatttttttttgtttattaatcagactagtagcagcgggtgatattaccacctgtcgctacaaacatctttaaagcggcgggtaaccgctactgctgctacaactcgcgatttgtagtttcacaagcatagcggcggtgtattgctgtagtgtgcaatacaatattttttttctacttctaggagttgttactttcacatatatgtatctcacaaagtagagtgtatctcttcatacttgaggtttctaaaaaaacatctacatatatttgacttttaaaagtagtatatgaaaatacttttataagaaatgtgatatatcacatgatgcaaaaatatatgagttttgctcaaagtttttcgttgtttatcactttatatatataaatgttgctatcttatttttaaatcactatacacgc acacacacacacacacacacacacacacacacacacac tcacctttctataagtgtggcgtgagagatttattagctcaaactatagaacataggagtatatagtatatacctacatagatgtttatgccaacaggttaaataatt Plate 20972 C4 gatcagaattattcgcaaattccagcgccatgggttgtaaagtgcttgcttcttctcacccgggcctccgctatcggatattgcgacatcggcagcgcatggcgaggactccccctacttcaccggttggaaagcctacgacgagaacccctacgactccgtcaccaaccccgctggtgtcatccagatgggccttgcagagaaccaggtt tgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgt gtgtgtgtgtgtgtgtg tttgaccttgcacagcactgcaaaatgcacatcatcgtcatgacgaagctgattgattagcgatggccgtgcaggtgtcctttgatcccaagcttc Plate 20972 C6 aatacgactcactatagggcgaattgggcccgacgtcgcatgctcccggccgccatggccgcgggattttatctcaatgtttgtttc tgtgtgtgtgtggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg gaagatgttagacataacccctttgtctgcagcttatttctcctttattgttggcgcacttggagggtacattttcctattattaagtcgataaagaagaatgtagaggatgccgcttgtttctcacttgagattaagacaaaggggataaaaagggttggccgtatgatggccttcgtgcatggtatctttccatatttttggtatccaccatccatgtcacgaagaatgatatgaggtcagtctttgctgtcctgtctatgtccttatagcctgcaggttatgtgaagtttgaattatgttagtgcagtatgtatgctatcgtaactcgtaagtcatgacagttgtgttaactggtcttgcatttatatttatcccgaccaattctctgattgaccgattctcctgaccatttgtattccacttttatttaaagaatattgtttgctctctaatgctatgtattccacttttattctcccgattctcttgcattaattaccaggctctaatttgtggtctttgtgtcagggtatggttg Plate 20972 C9 gatcgcccccaaacttgacgcagactagtttcgtgcaatccctagcggcaagaccaagctcgatggtggggcaagacagttttcgtcctatctcgttggagtccatgacaatgccgaccgccttgtaaaaaggaactcccatgttttagaaaaacaaatggtgtgtgtgtg tgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgttgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcgtgtgtgtgt gtgtgtgtgtgt gatgattntctactttggtttagtgcgaagtaccatcgaagccgctttcttggaatgaacatgctcatattccttcaa Figure B.1 Continued

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88 Plate 20972 C9 ataacttcagaaccacataatattgttattatatctatataagttgaataatgttatcttctatatttgcacttccctcacagggattataggcatacaatccgtacaaaaccagaagtttttttctttgtgaatcatcccaaacctgaagttgtgcacttgagtcatgccaacctactgcg Plate 20972 C10 gatccttcaccactgcacaatagagaaaggaaaaagggtagaggaaatgtgaagttcacatgctattacccctgggtgcactcctatcatccgtgttagccagatattttccatatgtcgtaatagtgaaatgtatggacatataactatcaaatgaagacatacatagtaaaagagtatagctggttaggttttttgtgatgtgacctacccacttcagtttttattattctacagctctgaatgagtgctagcatttacaagtgattattttttcattggcaggggaatatattacccattaaatctcctttaggtgtt tgtgtgtgtgtgtgtgtgtgtgtgtgtg gtggtggtggtggtacttgtgcgtatgaccatgtctatactcttcttctgaatgaaatatcaagggatgcggaaaagaactatacaacatgcgtttactttaagacatgaccaaatatttcctacgacattcctttgtggtggctaccgagagatgtagatatagagttagcttc Plate 20972 C12 ggatcaataggcagaaaagaaagagcctagaacttgtgcattattgtactagcacaagataaaagta acacacacacacacac ataaaagtcagttctcttatcatcgagagaacaatagaagtcagaaggaagagaggagagaaatacttagttcgtcatgatgcaaggagctgcatctcaggcccaaggtatatttattggtataatacatgagagaatgtcgcacatattctatccggaaaatatacgacaagctctattcacagagtgtaatatattgttatataaggttaaatcagtcttatgctaacat Plate 20972 D1 gatctggtatttgcaggcttgcaactatggtggagataaagcaagcagaaatgaagttagaagggtgttaccagtcaacatagcaactgtaattagctttagtttctttacggtcttgtagcgattatgtaagagcagccacactctttcggtcgtttgaacatcaaatcagatttatagaagcaccgttgcctcgattttatgcttgcccacagtcataacaccgaaacttgaacaaaaaaactgaccccacaaagaataaaacgag acacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacac agagagagagagttattttcaagcatcgggtgaatgttgagcaccgatgctctgtttttgagttgaagcacctgtggtttcaacggaaagcatcgatgcttgaacaaaaaagtagagaaaaagttggttaagacacggatgactgtgggtgctctaaaatgttgccgaccgtatagtggaaagcatgtttttgcatatctgatatcccttctagcgtgcatattaatgttgcatccatgatttctgatttttttttaaatgatttctaaagttggttaagtcaatggaatgatgcaaactgactgtgattgatgccatattcatgtagta Plate 20972 D2 gatcctgtgggaattatcttaattgccttgacgagcaagcagcccaaaggttacttccccaacaaagccttcatccgaaacacagaaaatcacagttcagattactgtgttggctagcaaagacaggtgcctaaagatgatgagaaaagtaaacacagaattaa acacaca cacacacacacacacacacacacacacacacacacacacacacacacac aaaacagaataagatgtcttataaagggtacaaaagaacttaccagccagtcttttgttgaaaatgcaccgaatcagccacatatgagatatgactggcaaacaatcactctgctcaatcggcctcctctgtcagtcatcaagatgttctcaaagcctggccatagagttttccgcctttcttttggcatattcatgaactatgctagttgtgctctccctgtggcgcaagtagaactcagcaagcaatattctattttcttcaggtaaga Plate 20972 D5 atacgactcactatagggcgaattgggcccgacgtcgcatgctcccggccgccatggccgcgggattcagcagtaacgtggctactagagacgattaaatgggacatgatgactgttggcatccgggtatgccatacctttttttttaaaaaaaataatgggtaggagtatgccatacgctgaccgcattgaaccgaatttgcgtggtcaagcaaaaaataatgggtagtagtagcaggttggtggtggttactttttttttctcgaaaacgcgggacagttaacaggacaagaacccgcaacggcaccaacaatacaaaaaacaccacgacacacacacacacagcacaaacacatacgaacaacgatgaaacgcacgcacaaatacacg acacacacac aggacaggacaccgtgaggt taagggttgcttcaagatggcgccttcaagaaggtcatgacacttagacgccaccgccatccgatccggcgagccggatccca Nucleotide sequences of the clones from which future species-specific primers may be developed.

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89 Plate 20972 D9 gatcaattgatacccgcctgactcccacctgactctagctgctcaagcaggaacgtacatcacatgcccctcaagaagcttttgttttgataatcaaatggttcattcaaattataagacaatgagatttcatattaccgtcagctggaaaggtggtgggtacaaaataattttaaaagaaaatcatctattacaattcgggtgtactcttatttgcaatggtagggcgaaaacaaaatcctcatatgggtgcatgatacaattaattgggtaatttgaaagaaacaaa tgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg agagagagagagagagagagagagagagagagagagagatgcttctgcctcctgtcgggactgcatgtgagtccagcttcatagtctgtcggctgcatatatataattctctcggtttccaccggcagaaaacacgacaggacgcttgaagttgctacaattctaacgacgcctacgtgaagttaattccctaattattggtagaagcatactatattttagaatttaaattagggtagaattgttttaaatatttttataatgacagaggtggtta Plate 20972 D10 ggatcaaacgttgtcactgaactttaaacacgagtgagggggaaattaagcactcaggagtccttgtgtgacggctcaaatcgaaaccaataaagatgatgacgaattgaggatagaaccaacaactgcaattgaag tgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgt gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg ttttgcgccttgtgcttggtgaacatgcaatgattcacaccaactgcctcatttcttctgatactctaggatagtcgattgcgatcccaagcttc Plate 20972 D12 ccgggaagcttgggatcaagcaaaatagtcccaaaactgtaaaccacactctctgggcttactctacctgcagaaacatatagcagttgatgaagaacattagtatcgtgtgtgcgcgcgcgcgcgtgcgtgggtgtgtgtgtgtgtgcc tgtgtgtgtgtgtgtgtgtgtg tgtgtgtgtgtgtgtgtgtgtgtgtgt ctgtgtgtgtgttacacttgtctgtcttaaggnactctggaggtgtgaatgctaagtttgtactgtaactctttccgtcacggctgttcttcatcagaccgaaacaagatagtctagggttaccatnctacaattcaccatggaaaaaaggattagtgattctgtaagtgtatgcaaggtctgagaagaaatcaaggnatacacttgaaggctcaccanatcgaagacgactctatatgcatgcaggtcatgataaagcgctctctctttg Plate 20972 F1 atcgtatcttttctagattgtatctttttatatgttttctgttgtaaactccttaacttggcatgtactccaatccatagtcttggtcttctcgtacttcaagtcgtgcacgaccatgccgggggctgttcccggtcatataaatgaaatgacatcaggttcattcattttggcccgacaaataaaaatcaatctagcacgacatcttgcacgcacaggtccagccgtccaggttgtgtttctagagttcatcagaccctgctgacccaaaggggtgcaccttaattacatagaggtagcatgggcacgatgaca acacacacacacacacacacacacacacacacacac acacacac acaaaatgatgattcactttagtagaaacgaactggcggcagcaattactctggcagt Plate 20972 F11 Aacgacggccagtgaattgtaatacgactcactatagggcgaattgggcccgacgtcgcatgctcccggccgccatggccgcgggattagaacccgtaacggccccgacaaaacatacaaacccacgacacacacatggacacaaacaaacaacacacacacacgacgc acacacacacacac gaacccacatacacatacaccacgaggacactgcgagattaggggttgctcccagatgacgcctttaggaaggacatgacgtttagacgccatcgccatct Plate 20972 F12 tacgactcactatagggcgaattgggcccgacgtcgcatgctcccggccgccatggccgcgggattattt tgtgtgtgtgtgtgtg tgtgtgtgtgtgtgtgtgtgtgtgtgt tgtggggtggaaggttacttcttggagctaagaaatacatcgacaaccactttgagccatggtcctatctttgcagattcaagttttgttaattgggagatgaggttaagagttcaagcgccagaccgcttcaatcccatcgttgagaagaaacacatcaaatttcttatcgagggcatccatgacctatggtgtgccatgataactttatgaagactttgatttgcctctgcaaggaaataaatcaagagtgaaatgcatggtgaacatgcattcctttgtgaagatttgggttgaaagatactctactctttatgaatcccccaaaagagatgtaggaactgtatttatgg Figure B.1 Continued

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90 Plate 20972 G4 gatcgagagcattagtgaggatggcgaagcaacattactaacgctaatatgtggacgtacaaaaggagctcatgcatgtgtcatcgtgcgataggcttggccccaggtgaacgatttcaacgttgatgacgacttgggtgatttcacag tgtgtgtgtgtgtgtg tttcatccctgacattggggtgacatggactagtaatatcgtacagagaggagcaccatgtgtaaatatacaaaatgtaaaataaggtgcgtgagtatgaggggtacagactaattgcaattataacctaataatcttttgtattgctgaacttaatttcttcttaaataaaacagcggtgctcctgcctatttctcgaaaacaaagagggtgtgtcatgttggatagtcatgtgatggataagtcacaattatacgtataacacatcatcaaaatacatggtaatgaccgtgttgatactaagaagcgagcttcatggccacaatttgcagtttcatgaaattaaggatcccaagctt Plate 20972 H5 gatccatggacatgcacaaggagcattccgctgtccatggtgtcaagggacggcatgaggtaggagcgctcattgaagccagtgcatactaactcacctgtaatcattgctacttacctgaagcctggccagcacttgttgagttccacagtgaggacgtcgctggcatatgtcgcgacacagaagtggcctcgcacggagaggcccccgccctgaatgagtggcaggtcctcgtcctcctcttcatgtacagccgcagttcatgaactctagtgggacccaatgatcgcccccaaacttgacgcagactagtttcgtgcaatccctagcggcaagaccaagctcgatggtggggcaagacagttttcatcctatctcgtccgagcccatgacaatgccgaccgccttgtaaaaaggaactcccatgtttaggaaaaacaaatgg tgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcgtgtgtgtgtgtgtgtgtgtgtg atgattttctacttcggtttagtgcgaagtaccatctaagctgaataatgtggaatgaacatgctcatattccttcaaataacttcagaaccacataatatttttattttatctttataagttgaataatgttatcttctttatttgcacttccctcaca Figure B.1 Continued

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APPENDIX C INDIVIDUAL GENETIC SIMILARITY MATRICES FOR BER 1, BER9, AND BER11 Table C.1 BER1 genetic similarity matrix. BER1MS ChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMS SupremeChampionFloradwarfGN-1FloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895MS Choice1Midiron0.331Tifsport10.331Tifway II10.3311Tifway41910.33111Tifgreen0.250.660.250.250.251Tifdwarf0.250.660.250.250.2511Tifeagle0.250.660.250.250.25111MS Supreme0.250.660.250.250.251111Champion0.250.660.250.250.2511111Floradwarf0.250.660.250.250.25111111GN-110.331110.250.250.250.250.250.251Floratex0.50.50.50.50.50.250.250.250.250.250.250.331Tiflawn0.50.50.50.50.50.330.330.330.330.330.330.50.51Tifton 100.50.50.50.50.50.330.330.330.330.330.330.50.511BA 4810.50.50.50.50.50.330.330.330.330.330.330.50.5111BA 1570.50.50.50.50.50.330.330.330.330.330.330.50.51111BA 4750.50.50.50.50.50.330.330.330.330.330.330.50.511111PI 290-9050.500.50.50.500000000.5000001PI 289-9160.510.50.50.50.660.660.660.660.660.660.330.50.50.50.50.50.501PI 290-90010.331110.250.250.250.250.250.250.3310.50.50.50.50.50.330.331PI 290-86810.331110.250.250.250.250.250.250.3310.50.50.50.50.50.330.3311PI 290-8950.330.330.330.330.330.250.250.250.250.250.2510.330.50.50.50.50.500.330.330.331 91

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92 Table C.2 BER9 genetic similarity matrix. BER9MS ChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMS SupremeChampionFloradwarfGN-1FloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895MS Choice1Midiron01Tifsport00.51Tifway II00.511Tifway41900.5111Tifgreen000001Tifdwarf0000011Tifeagle00000111MS Supreme000001111Champion0000011111Floradwarf00000111111GN-1000000000001Floratex0000000000001Tiflawn00000000000001Tifton 10000000000000001BA 4810000000000000001BA 15700000000000000001BA 475000000000000000001PI 290-9050000000000000000001PI 289-916000000000000.500000001PI 290-900000000000000000000001PI 290-8680000000000000000000001PI 290-895000000000000.50000000100 1

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93 Table C.3 BER11 genetic similarity matrix. BER11MS ChoiceMidironTifsportTifway IITifway 419TifgreenTifdwarfTifeagleMS SupremeChampionFloradwarfGN-1FloratexTiflawnTifton 10BA 481BA 157BA 475PI 290-905PI 289-916PI 290-900PI 290-868PI 290-895MS Choice1Midiron01Tifsport001Tifway II0011Tifway41900111Tifgreen0.3300.50.50.51Tifdwarf0.3300.50.50.511Tifeagle0.3300.50.50.5111MS Supreme0.3300.50.50.51111Champion0.3300.50.50.511111Floradwarf000.50.50.5111111GN-10.3300.50.50.50.330.330.330.330.330.331Floratex000000.330.330.330.330.330.3301Tiflawn00000000000001Tifton 100.50.50000000000001BA 4810.500000000000.330001BA 157000000000000.3300011BA 475000000000000000001PI 290-9050000000000000.5000001PI 289-916000000000000000.500001PI 290-900000.50.50.50.330.330.330.330.330.330.33000000.5001PI 290-8680.3300000000000.330000.50.500001PI 290-89500000000000000000000001

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96 Hartl DL. 2000. A Primer of Population Genetics, Third Edition (20-45). Sinaur Assoc. Inc., Sunderland, MA. Haydu JJ, Hodges AW. (2002) Economic Impacts of the Florida Golf Course Industry in 2000. University of Florida Economic Information Report #02-4. Haydu JJ, Saterthwaite LN, Cisar JL. (2002) An Agronomic and Economic Profile of Floridas Sod Industry in 2000. University of Florida Economic Information Report #02-6. Hein MA. (1953) Registration of Varieties and Strains of Bermudagrass, II. (Cynodon Dactylon (L.) Pers.) Agronomy J 45:572-573. Hein MA (1961) Registration of Varieties and Strains of Bermudagrass, III. (Cynodon Dactylon (L.) Pers.) Agronomy J 45:572-573. Hodges AW, Haydu JJ. (2002) Economic Impacts of the Florida Environmental Horticulture Industry, 2000. University of Florida Economic Information Report #02-3. Huff DR, Peakall R, Smouse PE. (1993) RAPD variation withing and among natural populations of outcrossing buffalograss[Buchloe dactyloides (Nutt.) Engelm]. Theor Appl Genet. 86:927-934. Jones ES, Dupal MP, Kolliker R, Drayton MC, Forster JW. (2001) Development and characterization of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.). Theor Appl Genet. 102:405-415. Kandpal RP, Dandpal G, Weissman SM. (1994) Construction of libraries enriched for sequence repeats and jumping clones, and hybridization selection for region-specific markers. Proc Natl Acad Sci.91:88-92 Krans JV, Philley HW, Goatley JM, Tomaso-Peterson M, and Maddox VL, 1995. Registration of MS-Choice bermudagrass. Crop Sci. 35:1506. Kubik C, Meyer WA, Gaut BS. (1999) Assessing the abundance and polymorphism of simple sequence repeats in perennial ryegrass. Crop Sci. 39:1136-1141. Liu ZW, Jarret RL, Kresovich S, Duncan RR. (1995) Characterization and analysis of simple sequence repeat (SSR) loci in seashore paspalum (Paspalum vaginatum Swartz). Theor Appl Genet. 91:47-52. Manifesto MM, Schlatter AR, Hopp HE, Suarez EY, Dubcovsky J. (2001) Quantitative evolution of genetic diversity in wheat germplasm using molecular markers. Crop Sci. 41:682-690. Matsuoka Y, Vigouroux Y, Goodman M, Sanchez J. (2002) A single domestication for maize shown by multilocus microsatellite genotyping. Proc Nat Acad of Sci. 99:6080-6084.

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97 Moore SS, Sargeant LL, King TJ, Mattick JS, Georges M, Hetzel DJS. (1991) The conservation of dinucleotide microsatellites among mammalian genomes allows the use of hetrologous PCR primer pairs in closely related species. Genomics 10:654-660 Morgante M, Olivieri AM. (1993) PCR-amplified microsatellites as markers in plant genetics. The Plant Journal. 3(1):175-182. Powell TB, Burton GW, Young JR. (1974) Mutations induced in vegetatively propagated turf bermudagrasses by gamma radiation. Crop Sci. 14:327-330. Reiter R. (1994) PCR-based marker systems. In Phillips RL, Vasil IK (Eds), DNA-based Markers in Plants. Dordrecht ; Boston : Kluwer Academic. Rozen S, Skaletsky HJ. (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ. pp 365-386. Saal B, Wricke G. (1999) Development of simple sequence repeat markers in rye (Secale cereale L.). Genome 42:964-972. Sanchez de la Hoz MP, Davila JA, Loarce Y, Ferrer E. (1996) Simple sequence repeat primers used in polymerase chain reaction amplifications to study genetic diversity in barley. Genome 39:112-117. Senior ML, Murphy JP, Goodman MM Stuber CW. (1998) Utility of SSRs for determining genetic similarities and relationships in maize using an agarose gel system. Crop Sci. 38:1088-1099 Sweeney P, Danneberger K. (2000) Inheritance of restriction amplification fragment length polymorphisms in perennial ryegrass. Crop Sci. 40:1126-1129. Sweeney PM, Dannegerger TK. (1995) RAPD characterization of Poa annua L. populations in golf course greens and fairways. Crop Sci. 35:1676-1680. Swofford DL. (2000) PAUP*: Phylogenetic analysis using parsimony and other methods (software). Sinauer Associates, Sunderland, MA. Taliaferro CM. (1995) Diversity and vulnerability of bermuda turfgrass species. Crop Sci. 35:327-332. Temnykh S, Park WD, Ayres N, Cartinhour S, Hauck N, Lipovich L, Cho YG, Ishii T, McCouch SR. (2000) Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.). Theor Appl Genet. 100:697-712. Thompson, JD, Higgins DG, and Gibson TJ. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.

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98 Trenholm LE Cisar JL, Unruh JB. (2000) Bermudagrass for Florida lawns. University of Florida Fact Sheet ENH19. http://edis.ifas.ufl.edu (Visited Apr .30, 2003) Vermeulen PH, Beard JB, Jussey MA, Green RL. (1991) Starch-gel electrophoresis used for identification of turf type Cynodon genotypes. Crop Sci. 31: 223-227. Welsh J, McClelland M. (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleid Acids Res. 19:303-306. Williams JG, Kubelik AR, Livak KJ, Rafalski JA Tingey SV. (1990)DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531-6535. Xu WW, Sleper DA. (1991) A survey of restriction fragment length polymorphisms in tall fescue and its relatives. Genome vol. 34:686-692. Xu WW, Sleper DA. (1993) Phylogeny of tall fescue and related species using RFLPs. Theor Appl Genet. 88:685-690. Xu WW, Sleper DA, Chao S. (1992) Detection of RFLPs in perennial ryegrass, using heterologous probes from tall fescue. Crop Sci. 32:1366-1370. Xu WW, Sleper DA, Krause GF. (1994) Genetic diversity of tall fescue germplasm based on RFLPs. Crop Sci. 34:246-252. Yemnykh S, Park WD, Ayres N, Cartinhour S, Hauck N, Lipovich L, Cho YG, Ishii T, McCouch SR. (2000) Mapping and genome organization of microsatellite sequences in rice(Oryza sativa L.) Theor Appl Genet. 100:697-712. Zhang LH, Ozias-Akins P, Kochert G, Kresovich S, Dean R, Hanna W. (1999) Differentiation of bermudagrass (Cynodon spp.) genotypes by AFLP analyses. Theor Appl Genet. 98:895-902.

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BIOGRAPHICAL SKETCH Neil Williams was born in Buhl, Idaho and grew up on his familys small farm. Raising a large garden encouraged experimentation with new varieties of vegetables and flowers. During high school, Neil was involved in Future Farmers of America where he participated in speaking and leadership competitions. Upon graduation he enrolled in the local community college for a year before taking a leave from schoolwork to fulfill a 2-year service mission to Moscow, Russia for the Church of Jesus Christ of Latter-day Saints. Picking up at Ricks College in Rexburg, Idaho after his mission Neil earned his Associates degree in general education. From there Neil entered Utah State University in Logan, Utah and spent 4 years working on his Bachelor of Science degree in horticulture (with a minor in Russian language). While in Logan, he married his wife Suzanne Camphuysen and they had two children, Genavieve and Tyler. On the encouragement of Grant Vest, Neil accepted the offer from Dr. Brian Scully and Christine Chase (at the University of Florida) to work on a molecular marker study. Throughout his high-school and college experience Neil was involved with the horticulture industry (in retail, as a manager; and in the seed industry, as a farm helper and crew chief). Sales and speaking are his strengths in these areas. Neil also enjoys running, reading, and playing with his children. Collin Michael Williams, his third child, was born shortly after the completion of his Master of Science degree in horticulture at the University of Florida. 99


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PCR-BASED POLYMORPHISMS IN BERMUDAGRASS (Cynodon spp.)


By

NEIL RAY WILLIAMS


















A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2003

































Copyright 2003

by

Neil Ray Williams




























I dedicate this thesis and the research it represents to Suzanne C. Williams who has
supported my efforts and made possible all that you see here through her sacrifice and
loving support.















ACKNOWLEDGMENTS

I appreciate the contributions of my supervisory committee (Drs. Christine Chase,

Brian Scully, Laurie Trenholm and Terry Kamps). I thank Victor Ortega and the Chase

Lab; and Ginger Clark in the ICBR for their invaluable input and help. I also

acknowledge the financial support of the Horticultural Sciences Department at the

University of Florida; the G. C. Horn Fellowship, awarded by the Florida Turfgrass

Association; the Davidson Travel Grant; and the Dade County Scholarship. I especially

appreciate my family (Suzanne, Genavieve, Tyler Ian, and soon Collin Micheal Williams

as well as mothers, fathers, brothers and sisters). I acknowledge the hand of Jesus Christ

in this work.
















TABLE OF CONTENTS
page

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

LIST OF TABLES ............................................... ...................... vii

LIST OF FIGU RE S ........ ........ ........................................ .. ................. viii

A B ST R A C T ......... ..... ............................................................................. ........

CHAPTER

1 REVIEW OF LITERATURE ...................................................... .....................

P problem .................................................. 1
In tro d u ctio n ............................................ ................................ 1
B re e d in g ................................................................................................................ 4
M olecular M arkers ....................................................... 6
Iso zy m e s .................. .. ... ........... .. ......... ............. .... .. 7
Random Fragment Length Polymorphism (RFLP)......................................7
Random Amplified Polymorphic DNA (RAPD) ...........................................9
DNA-Amplification Fingerprinting (DAF) ..................................................10
Amplification Fragment Length Polymorphism (AFLP).............................11
Sim ple Sequence R epeat (SSR ) ........................................ ....................12

2 CROSS-TAXA PRIMERS APPLIED TO BERMUDAGRASS ............................22

In tro d u ctio n ............................................................................................................ 2 2
M materials and M methods ........................................................................ ..................24
P lant M material ...............................................................2 4
D N A P reparation .............................................................. .......... .............. .. 25
C ro ss T ax a P rim ers........... ............................................................. .... ........ .. 2 5
P C R ......................... ....................................................................................... 2 5
PCR Product Sequence A nalysis...................................... ......................... 26
R e su lts ...........................................................................................2 7
D isc u ssio n .............................................................................................................. 2 9

3 SPECIES-SPECIFIC PRIM ER S ............................................ .......................... 49

In tro du ctio n ...................................... ................................................ 4 9
M materials and M methods ........................................................................ ..................5 1


v









P lan t M material ...............................................................5 1
DN A Preparation .............................................. .... .. ................. 52
Enriched Library Construction........................................... ......... ... ............... 52
Prim er D evelopm ent......... ................. ................... ................... ............... 54
A n aly sis ............. ................. ................................................................ 5 5
Results ............... ...... ............ ............. ...............55
D iscu ssio n ......... .................................... ............................5 8

4 C O N C L U SIO N S ................................................................82

APPENDIX

A INDIVIDUAL GENETIC SIMILARITY MATRICES FOR CROSS-TAXA
PRIMER SETS ZMADH2N AND LPSSRHO1A10 ..................................... 83

B NUCLEOTIDE SEQUENCE FOR FUTURE PRIMER DEVELOPMENT .............85

C INDIVIDUAL GENETIC SIMILARITY MATRICES FOR BER 1, BER9, AND
B E R 11 ................................................................................ 9 1

LIST OF REFEREN CES ................................................................................... 94

B IO G R A PH IC A L SK E T C H ....................................................................................... 99
















LIST OF TABLES


Table p

2.1 Origins and uses of bermudagrass cultivars evaluated for simple sequence
re p e a ts ........................................................................ 3 3

2.2 Origins of cross-taxa primer pairs used in PCR reactions on bermudagrass
te m p late s ...................................................... ................ 3 4

2.3 Basic step-down PCR programs used to test primer amplification........................36

2.4 Sorghum bicolor cross-taxa amplicon sizes estimated in base pairs........................37

2.5 Loliumperenne cross-taxa amplicon sizes estimated in base pairs .........................38

2.6 Zea mays cross-taxa amplicon sizes estimated in base pairs .................................39

2.7 Genetic similarity matrix including combined data from primer sets
LPSSRHO1A10 and ZMADH2N ............................. ... ............... 40

3.1 Origins and uses of bermudagrass cultivars evaluated for simple sequence
re p e a ts ........................................................................ 6 2

3.2 Cynodon spp.-specific primers developed from the sequencing of fragments in
the enriched D N A library ........................................................... ............... 63

3.3 Basic step-down PCR programs used to test primer amplification........................64

3.4 BER1 PCR product length on 23 bermudagrass DNA templates from gel
#20027 (Figure3.4) ........ ............................... ..... ...... .... .. ............ 65

3.5 BER9 PCR product length on 23 bermudagrass DNA templates from gel
# 2 0 7 3 5 (F igu re 3 .5) ............ ....................................................................... .. ...... .. 6 6

3.6 BER11 PCR product length on 23 bermudagrass DNA templates from gel
# 2 0 7 6 3 (F igu re 3 .6)............ ....................................................................... .. ...... .. 6 7

3.7 Genetic similarity matrix including combined data from all BER primer sets........68

A 1 ZM ADH2N genetic similarity m atrix. ........................................... .....................83

A.2 LPSSRHO1A10 genetic similarity matrix.........................................84
















LIST OF FIGURES


Figure pge

1.1 Anatomical features of a grass stem and leaf section used in grass taxonomy and
separation of one species from another. .............. ..................................... ......... 18

1.2 Two closely related turf varieties that appear similar. ............. ..................19

1.3 Diagram of the PCR process by which small amounts of DNA can quickly be
copied by DNA polymerase to produce large numbers of a single fragment ..........20

1.4 Diagram of ideal SSR marker system showing fragments are visualized and how
they can show allele inheritance....................... .... ............................ 21

2.1 SB4-22 primer pair amplification of 23 bermudagrass DNA templates ................41

2.2 SB6-36 primer pair amplification of 23 bermudagrass DNA templates ................42

2.3 LPSSRHO1A10 primer pair amplification of 23 bermudagrass DNA templates....43

2.4 LPSSRHO1AO7 primer pair amplification of 23 bermudagrass DNA templates...44

2.5 CLUSTAL W (1.81) alignment of LPSSRHO1AO7 amplification product
se q u en ce s.............................. ......................................................... ............... 4 5

2.6 MZEPPDKA2 primer pair amplification of 23 bermudagrass DNA templates.......46

2.7 ZMADH2N primer pair amplification of 23 bermudagrass DNA templates...........47

2.8 Sequence of ZMADH2N amplification products from Figure 2.6 aligned by
C L U ST A L W (1.8 1).. .......................................... .. .... .......... ...... ............48

3.1 Dot-blot hybridization of dilution series of enriched libraries to verify
enrichm ent for (CA )n repeats.. ............................ ...............................................69

3.2 Colony transfer and hybridization of initial bacteria transformation and
hybridization to fluorescent probe to show transformation success rate.. ...............70

3.3 Nucleotide sequences of the clones from which species-specific primers were
developed. ...........................................................................7 1









3.4 BER1 amplification products of replicated PCR reactions using 23 bermudagrass
DNA templates, fractionated on agarose gels, showing the reproduction of
fragm ent length patterns ........................................................................... .... ..... 73

3.5 BER9 amplification products of replicated PCR reactions using 23 bermudagrass
DNA templates, fractionated on agarose gels, showing the reproduction of
fragm ent length patterns........................................................................... ..... .... 74

3.6 BER11 amplification products of replicated PCR reactions using 23 bermudagrass
DNA templates, fractionated on agarose gels, showing the reproduction of
fragm ent length patterns ........................................................................... .... ..... 75

3.7 Products of BER9 primer pair amplification on bermudagrass DNA fractionated
on polyacrylamide gels showing the amplicon pattern for the bermudagrass
tem plate show n at the top each lane .............................................. ............... 76

3.8 Gel #16088-2, showing amplification of BER 1 on a different group of DNA
tem plates ............................................................................ ........ ....... 77

3.9 Phylogenetic analysis of BER1 fragment length data from gel #20027
using P A U P ........................................................................... 78

3.10 Phylogenetic analysis of BER9 fragment length data from gel #20735 using
P A U P ............................... ........... ..............................................7 9

3.11 Phylogenetic analysis of BER11 fragment length data from gel #20763 using
P A U P ................................................... ..................... ................ 8 0

3.12 Phylogenetic analysis of the collective fragment length data from Tables 3.5-3.7
using P A U P ........................................................................... 81

B. 1 Nucleotide sequences of the clones from which future species-specific primers
m ay be developed ............... ...................... .................. ........... 85















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

PCR-BASED POLYMORPHISM IN BERMUDAGRASS (Cynodon spp.)

By

Neil Ray Williams

December 2003

Chair: Brian Scully
Cochair: Chris Chase
Major Department: Horticultural Sciences

Hybrid bermudagrass is an important turf-type grass in Florida and other southern

states. Cultivars with similar appearance, but differences in economic value-added traits,

make genetic markers desirable. For this reason, a microsatellite DNA marker system

that will provide immediate cultivar identification was studied. Two methods of primer

development were used to identify microsatellite DNA polymorphisms. In the first,

thirty-five primer pairs developed for other grasses in the Poaceae family resulted in only

two patterns that reproduced reliably. In the second, a genomic library enriched for

(AC)n repeats was developed and sequences were used for primer development. This

method of microsatellite amplification provided three primer pairs that reliably amplified

useful polymorphisms on bermudagrass DNA templates.














CHAPTER 1
REVIEW OF LITERATURE

Problem

Commercial, clonally propagated varieties of improved bermudagrass used for turf

are not easily distinguished by morphological and physiological characteristics. Varieties

exhibit only subtle morphological differences that are subject to environmental

influences. While similar in appearance, varieties may differ significantly in cost because

of reactions to drought, high foot traffic, fertilizers, and herbicides. Thus, distinguishing

among varieties becomes important for enforcement of uniform commercial code (which

states that people will receive what they have paid for). The purpose of this study was to

develop a consistent, easily analyzed microsatellite marker system for identification of

improved and hybrid bermudagrass varieties. This was attempted in two phases 1) the

trial and use of published, cross-taxa microsatellite primers and 2) the development of

new, species-specific primers.

Introduction

The genus Cynodon is a member of the family Poaceae; group Chlorideae.

Cynodon contains nine species, all of which are characterized as sod forming, perennial,

warm-season grasses with a broad economic influence in tropical to transitional regions

(Beard 1973; Taliaferro 1995). The turf-type species of interest to this study are

Cynodon dactylon (L.) Pers (C. dactylon); Cynodon transvaalensis (Burtt-Davy) (C.

transvaalensis); and their hybrids









Cynodon dactylon, known in the U.S. as common bermudagrass, is a tetraploid

with a chromosome number of 2n=4x=36. It is widely distributed between 45N and

45S latitudes with some cold-hardy varieties reaching farther north (Taliaferro 1995). It

is generally characterized by a folded vernation, no auricle, and a fringe of hairs on the

ligule, using the diagram in Figure 1.1 to reference these features. It is rhizomatous,

stoloniferous, and produces seed excessively on 4-5 digitate spikes raised above the leaf

canopy. Specifically, leaf character is highly variable, with color ranging from light to

dark green; and texture ranging from medium to coarse. Leaves turn light brown to white

during dormancy; and the species tolerates neither shade nor low temperatures well. It

does tolerate drought, heat, and high foot traffic; and forms an aggressive sod on a wide

range of soil types. The strong, creeping growth habit makes bermudagrass a weed in

ornamental beds (and with turfgrasses of finer textures). Because of its versatility and

specifically selected enhancements (such as durability, drought tolerance, and uniformity

of leaf texture), C. dactylon is fit for use as a turfgrass; or for utility, soil stabilization, or

low-maintenance playing fields and golf course roughs (Beard 1973).

The African Bermudagrass (C. transvaalensis), is a diploid species with a

chromosome number of 2n=2x=18 (Beard 1973). It is confined to the South African

Transvaal, its center of origin (Taliaferro 1995). General characteristics resemble C.

dactylon, but leaf color is light green to yellow with fine, dense leaf texture. It spreads by

rhizomes and stolons, although less aggressively than C. dactylon; and seed production

(though poor), is also above the canopy. In lower temperatures, the yellow-green leaves

and stolons turn reddish-purple (Beard 1973). Because of uniform morphology

maintained within the varieties and its ability to breed interspecifically with C. dactylon,









it has become important in the U.S. turfgrass industry, where it is maintained specifically

for breeding purposes. In Africa it is used as a turfgrass and for forage (Beard 1973;

Taliaferro 1995).

The hybrid of C. dactylon X C. transvaalensis is a sterile triploid with 2n=3x=27

chromosomes. Hybrids are used in the southern US and in subtropical climates

throughout the world that do not drop below -12o C during dormancy. Cold is the main

limiting factor to bermudagrass success (Beard 1973; Taliaferro 1995). Morphologically

similar to C. dactylon auricless, ligules, etc.), hybrids can be selected with a combination

of desirable turf traits from the two species (Taliaferro 1995). It spreads by rhizomes and

stolons more quickly than C. transvaalensis, but has shorter intemodes and denser growth

than C. dactylon. Mowing heights vary by cultivar and use, ranging from below 1s" to

1". In Florida it is preferred by golfers because it provides a smooth playing surface; and

by golf course superintendents because it keeps a medium to dark green color during

dormancy, eliminating the need to overseed. It is also improved to meet the needs of the

golf course industry's demanding requirements for salt tolerance and lower fertilization

rates (Busey and Dudeck 1999). It is intolerant to shade, and root systems are shortened

by low mowing, making it susceptible to drought stress when not properly maintained.

These triploid hybrid varieties are propagated vegetatively from root and shoot cuttings

(Taliaferro 1995). Hybrid bermudagrass is used extensively on golf courses and athletic

fields, but because of its medium to higher maintenance requirements, bermudagrass is

not recommended for home turf (Beard 1973).

Hybrid and improved bermudagrass is an economically important grass in the state

of Florida. According to the 2000 economic report, 59 million rounds of golf were









played on the 1,334 golf courses statewide. Golf courses cover 207,582 acres of the state

and were responsible for $4.4 billion in revenue (Haydu and Hodges 2002). Forty-one of

these golf courses were new, supporting the $307.2 million sod industry (Haydu et al.

2002) that covers 80,347 acres of state and private land (Haydu et al. 2002).

Bermudagrass accounts for over half of the golf course grasses or 136,000 acres (Hodges

and Haydu 2002) and covers 6% of the sod industry production acreage, or 4,556 acres

total. Turf bermudagrass garnered $16.3 million at $0.114ft-2, which accounted for 19%

of the sod industry revenue that year (Haydu et al. 2002).

Breeding

Breeding and selection of bermudagrass has been almost continuous over the past

century. Because of the genetic variability of C. dactylon (and its wide geographic

adaptability), seeded varieties (both named and unnamed) are common. Cynodon

transvaalensis selection has been centered in South Africa, from which much of the first

germplasm for hybrid bermudagrass was obtained through cooperative breeding efforts

between Africa and the U.S. (Busey and Dudeck 1999; Taliaferro 1995).

Busey and Dudeck (1999 pg. 97) describe both seeded and vegetatively propagated

bermudagrass as "a complex of interbreeding species undergoing rapid evolution through

natural and human intervention." Developments in seeded varieties have yielded grasses

that are more cold tolerant, have lower fertilization requirements, are finer textured, and

provide more uniform plant quality than the wild relatives. Cynodon dactylon is

currently the main source of seeded varieties in the U.S. (Taliaferro 1995). Numex,

Sahara, and Mirage are a few of the limited number of seeded bermudagrass varieties

raised in the Southern U.S. Seeded varieties are more vigorous and more heterogeneous

than hybrids, making encroachment and contamination by unimproved varieties less of a









problem (Busey and Dudeck 1999). Other C. dactylon varieties are released for

vegetative propagation although they do produce some seed. Examples of these are

Floratex (Trenholm et al. 2000), GN-1 (Greg Norman Turf), and Tiflawn (Hein 1953).

Hybrid bermudagrass is the result of the interspecific cross of C. dactylon X C.

transvaalensis, which provides uniformity of leaf texture, color, foot-traffic resistance,

and winter color that are of great value to athletic fields and golf courses. Hybrids used

for golf courses are blocked into two major selection groups 1) greens grasses and 2)

fairway grasses. Much of the original turf-type Cynodon spp. material was imported

from South Africa in the 1940s and 1950s (Taliaferro 1995). The crossing of vigor and

variable green colors with fine texture and uniform performance has produced grasses

that are fine and short enough for closely mown greens. Other varieties grow vigorously

with coarser-textured leaves and are better adapted to well-traveled fairways and football,

soccer, and baseball fields (Beard 1973). Tifgreen (Hein 1961) was the first such hybrid

developed by the USDA-ARS in Tifton, Georgia; followed by Tiffine (Busey and

Dudeck 1999) in 1953; and Tifway 419 (Burton 1966b) in 1960. Interspecific crosses

resulted in Midiron (Anderson et al. 1988) from Kansas State University, and MS Choice

(Krans et al. 1995) from the Mississippi Agriculture and Forestry Experiment Station in

1995. Tifgreen and Tifway are interspecific hybrids that were subjected to induced

mutation by gamma irradiation, and resulted in Tifgreen II (Busey and Dudeck 1999) and

Tifway II (Burton 1985) respectively. Tifway II was then irradiated to produce TifEagle

(Zhang et al. 1999). Midiron was also irradiated and Tifsport (Haring 1998) was

selected. Natural mutation, contamination, or somatic variations have given rise to

Tifdwarf (Burton 1966a) and MS Supreme (Krans et al. 1995) from Tifgreen. Each of









these mutations have been compared to existing varieties and selected based on foot

traffic resistance; shade tolerance; fertilizer, water, and mowing requirements; and winter

hardiness and leaf color and texture.

Taliaferro (1995) discussed the decreasing diversity of germplasm that may lead to

disease problems if not addressed. A lack of readily visible morphological variability

presents another problem within categories of grasses (Figure 1.2). While the varieties

appear similar, their reaction to drought, wear, fertilizers, and herbicides can be

sufficiently different (warranting significant differences in cost). As a limited genetic

base of parent material is used to produce new cultivars, homopheneity is unavoidable.

For this reason, technology using a genetic marker system was sought and the various

systems were compared and analyzed for usefulness in this task. This will help enforce

uniform commercial code, which protects the buyer from receiving something other than

what he or she paid for.

Molecular Markers

There are many types of marker systems available to the researcher in the

molecular world, but we will discuss two categories: those not based on the polymerase

chain reaction (PCR); and PCR-based systems. Molecular markers not based on PCR

include isozymes (which show protein polymorphisms) and restriction fragment length

polymorphisms (RFLPs), which are DNA based. PCR-dependent markers include

random amplified polymorphic DNA (RAPD), DNA amplification fingerprinting (DAF),

amplified fragment length polymorphism (AFLP), and simple sequence repeats (SSR)

also known as microsatellites. The purpose of these systems is to detect polymorphism

(mutation) within the genome of an organism. All rely on electrophoresis for separation

of polymorphic proteins or DNA fragments.









Isozymes

Isozyme markers use the positive and negative charges in the amino acids as the

driving force for separation. Because protein molecules of the same size differ in charge

because of one or more amino acid differences, plants with differing polymorphisms may

be distinguished (Hartl 2000). Sample preparation is as simple as lysing cells to release

the proteins from the cytoplasm. These cellular contents are then fractionated by a gel

matrix in the presence of an electrical current and stained for enzyme activity.

Vermeulen et al. (1991) ran starch gel electrophoresis on crude protein of 21

bermudagrass cultivars that were not closely related and successfully distinguished all of

them. Other studies were able to distinguish 7 forage type bermudagrass varieties (Dabo

et al. 1990), indicating that isoenzymes could be used to identify different cultivars.

Concern was raised because of the plant growth/environment influence associated

with this technique. There is no apparent connection between morphological

characteristics or botanic species and isozyme banding patterns in bermudagrass

(Vermeulan et al. 1991). There are fewer protein polymorphisms than DNA

polymorphisms because replacement of an encoded amino acid is required for

polymorphisms to be seen. Furthermore, isozyme analysis is restricted to proteins that

can be identified by an activity stain. Protein polymorphisms are also more difficult to

interpret because a change in amino acid sequence may reflect a number of DNA

mutation events (Hartl 2000).

Random Fragment Length Polymorphism (RFLP)

RFLPs are DNA markers based on the premise that some DNA molecules in a

population contain a particular restriction site, whereas others lack it because of mutation

(Hartl 2000). This polymorphism is shown by placing highly purified nuclear DNA in









contact with an enzyme recognizing a sequence of 4-8 nucleotides. Fractionating the

fragments on a gel and doing a southern blot (Hartl 2000) allows hybridization of a radio

labeled or fluorescent probe. The probe will reveal a pattern of DNA fragments based on

the number of restriction sites present or absent along its sequence or flanking sequence.

Xu and Sleper (1991) applied RFLP analysis on Festuca spp., using 174 cDNA,

species-specific probes to differentiate between cultivars. With this information, a

linkage map was developed that provides an indirect selection tool for agronomic traits.

From the development of these probes, a cross-taxa experiment involving Lolium perenne

(L.) (L. perenne) was conducted with 2/3 ofFestuca spp. (L.) probes cross-hybridizing to

L. perenne DNA. Of the probes that successfully hybridized, 69% were polymorphic on

5 genotypes (Xu et al. 1992). This study shows the successful application of cross-taxa

RFLP work in gene tracking through interspecific and intergenic hybridization and

breeder selection (Xu and Sleper 1993).

RFLPs require sufficient highly purified genomic DNA from each of a large

number of individuals. They also require a library of probes, and radio-labeling for the

most sensitive detection. For these reasons many researchers have turned to PCR based

methods (Reiter 1994).

PCR utilizes oligonucleotide primers as priming sites for polymerase from Thermus

aquaticus to begin the in vitro amplification of DNA (Figure 1.3). Using nucleotides,

magnesium, and repeated heating and cooling cycles to copy a small segment of the

genomic DNA, one molecule of template DNA can be amplified into microgram

quantities within hours. PCR itself is technically simple, because reagents are easily

mixed and heating is done by a programmed machine. Primer information can be









transferred from one lab to another without the need to transfer live cultures or libraries.

The transfer of sequence information and the ease of oligonucleotide construction

increase the speed of information transfer (Reiter 1994).

Random Amplified Polymorphic DNA (RAPD)

RAPD is a sequence-arbitrary PCR method used in genetic analysis, which was

developed by two groups simultaneously (Welsh and McClelland; 1990Williams et al.

1990). RAPDs use short (10 mer) oligonucleotide primers in the PCR reaction allowing

multiple amplicons when a primer pair binds in opposite orientation in close proximity.

When analyzed by gel electrophoresis, a fairly simple fragment pattern is detectable after

ethidium bromide staining. The fragment length patterns of individuals are compared

and differences or similarities noted (Reiter 1994).

RAPD markers have been used for a variety of studies in turf and other grasses.

Huff et al. (1993) showed RAPD variation within and among natural populations of

Buchloe dactyloides (Nutt.) Engelm, an out crossing species, by means of statistical

evaluation to eliminate within population polymorphism. Research has been done

following migration in Triticum dicoccoides (L.) (Fahima et al. 1999) and also in Poa

annua (L.) (Sweeney and Dannegerger 1995) using RAPDs to disprove isolation theories

and to follow gene flow. Golembiewski et al. (1997) used RAPDs to test Agrostis spp.

(L.) bulk seed samples and were able to identify most with assurance. This information

is useful for researchers, breeders, and seed producers who are interested in more precise

monitoring and control of germplasm sources in open-pollinated varieties. Sweeney and

Dannegerger (2000) tried to modernize RFLP by combining it with RAPD on L. perenne,

first amplifying RAPD products and then performing RFLP analysis on these products.









While theoretically it was possible to obtain more information, only one reliable probe

was discovered.

Of particular interest to the present study, Busey et al. (2000) attempted the use of

RAPDs to distinguish off-types within the variety Tifdwarf. Analysis was sensitive

enough to distinguish between Tifdwarf and a contaminant of different morphology,

proving that it was contaminated and not merely mutated. Tifdwarf and Tifgreen proved

to be too closely related for separation using RAPD analysis.

As with other arbitrarily primed PCR systems, RAPD markers have low

reproducibility because multiple amplicons are competing for available enzyme and

substrate, and because low stringency thermal cycling allows mismatch annealing

between primer and substrate. RAPD polymorphism is seen when priming sites are

present or absent and products are scored by a plus/minus system revealing mainly

dominant markers (Reiter 1994).

DNA-Amplification Fingerprinting (DAF)

Caetano-Anolles et al. (1997) developed a second arbitrarily primed, PCR-based

marker method termed DNA amplification fingerprinting (DAF). DAF uses a single,

short oligonucleotide primer of 5-8 nucleotides long, at high concentration in

combination with either low (40-50) or high stringency annealing temperature, high

resolution fragment separation on polyacrylamide gels, and sensitive fragment detection

with silver staining.

DAF was used in a genetic stability evaluation (Caetano-Annoles et al. 1995) to

show that Tifway 419 off-types are planting contaminations, and not mutations, based on

the genetic stability among repetitions of Tifway 419 plantings. Tifway and Tifway II,

however, were indistinguishable using this technique. In a later study (Caetano-Annoles









1998) Tifdwarf and Tifgreen off-types were shown to be somatic mutations, generally

having similar, but distinct genotypes compared to the true types. Anderson et al. (2001)

also used DAF to determine homogeneity of a named grass and proved that U-3

bermudagrass populations have undergone genetic drift while Tifway 419 populations

remain more genetically similar.

DAF produces a banding pattern that is highly complex, containing many more

fragment lengths than RAPD. This allows an increased chance of observing

polymorphism, but is still unable to show heterozygosis. Because it is arbitrarily primed,

it is also susceptible to low reproducibility.

Amplification Fragment Length Polymorphism (AFLP)

Amplified fragment length polymorphism (AFLP) is a PCR-based, sequence

independent method wherein genomic DNA is first digested with one or more restriction

enzymes, and an adapter of known sequence is ligated to the fragments. PCR primers

have sequence specificity for the adaptor with one or more arbitrary, selective bases at

their 3' ends. The arbitrary base limits the number of amplification products, and

polymorphisms are detected as differences in the restriction sites and in the sequences

flanking those sites. Adapter specific primers allow for increased stringency making

AFLP more robust than the arbitrarily primed methods (Reiter 1994).

Bert et al. (1999) developed a high density linkage map of L. perenne using AFLP.

This map can be used in genetic studies and marker-assisted breeding, two important uses

of molecular markers. The cultivars were grouped as previously expected from RFLP

and RAPD work, but showed a wider distribution of markers for linkage to quantitative

and qualitative traits. Manifesto et al. (2001) did a similar study in Triticum aestivum

(L.), studying the private and public breeders stocks in Argentina. The genetic diversity









was lower than expected, indicating great similarities in selection of germplasm and the

use of released lines in breeding programs.

Zhang et al. (1999) at Tifton, GA studied 27 cultivars of Cynodon spp. using AFLP

markers. Testing 18 primer sets with three replications of successful markers, they were

able to separate all cultivars with a high level of confidence. They showed a small

dissimilarity coefficient of 0.08 between Tifgreen and its mutant Tifdwarf, but were able

to successfully distinguish them. AFLP produced some unexpected results, separating

Tifway, its mutant Tifway II, and a subsequent mutant, TifEagle, into different groups on

the dendrogram. In contrast, DAF was unsuccessful in separating them (Caetano-

Annoles 1998). Also of interest, two seeded varieties had dissimilarity coefficients of

0.05, which was the lowest in the subset.

The use of radio-labeling was originally required to visualize AFLP bands, but has

now been replaced by fluorescent markers and capillary electrophoresis. AFLPs reveal

primarily dominant markers because of moderately complex banding patterns.

Fluorescent labels, computer analysis, and capillary electrophoresis units present a

considerable expense and require a high level of technical expertise (Reiter 1994).

Simple Sequence Repeat (SSR)

SSR markers belong to a class of molecular markers whose primers are sequence

dependent. Known also as microsatellites, SSRs are ubiquitous DNA motifs repeated

tandemly (Figure 1.4). Factors influencing the success of a particular motif include:

frequency throughout genome, structural constraints of the DNA helix, and the size of the

fragment to be amplified. Di-, tri-, or tetra nucleotide motifs are repeated perfectly at

least three times, with the shorter motifs being more likely to mutate in most organisms

(Cho et al. 2000). These additions or deletions cause the number of repeats to vary









greatly between alleles of a given locus. The variability in the number of repeat units is

the basis of observed polymorphism (Reiter 1994). SSRs have become popular because

they show easily distinguished co-dominance, are more easily reproduced than sequence

independent markers, assay robustness, high rate of polymorphism, and distribution

throughout eukaryotic genomes. Primer sequence is easily transferred between labs and

primer synthesis is readily available. Currently, they are used for marker-assisted

breeding, genetic analysis, and to obtain relationship, evolution, and migration

information for an organism (Gethi et al. 2002). Primers are designed using the sequence

of the more highly conserved region flanking a region of the repeat motif. Mutation in

the primer site and insertion and deletion events of the repeat motif, are the sources of

polymorphism, thus the choice for repeat motif composition is important. Variables for

motif success consist of mutation rate, overcrowding, structural functions, and size. The

ideal size for an amplification product is between 100-300 bp in length, allowing for the

most rapid fractionation (Reiter 1994).

A partial list of molecular marker studies in monocots using SSRs include: L.

perenne (Kubik et al. 1999; Jones et al. 2001), Oryza sativa (L.) (0. sativa) (Chen et al.

1997; Temnykh et al. 2000; Cho et al. 2000), Zea mays (L.) (Z. mays) (Chin et al. 1996;

Gethi et al. 2002; Matsuoke et al. 2002; Senior et al. 1998), Paspalum vaginatum

(Swartz) (P. vaginatum) (Liu et al. 1995), Saccharum officinarum (L.) (Cordiero et al.

2000), Hordeum vulgare (L.) (Sanchez et al. 1996), and Secale cereale (L.) (Saal and

Wricke 1999). Comparisons between SSR and RAPD markers (Sanchez et al. 1996),

SSRs and RFLPs, (Chen et al. 1997), and SSRs and isozymes (Senior et al. 1998) found

SSRs to be either comparable or superior in their results for cultivar differentiation. The









advantage of simple co-dominance makes this genetic tool ideal for following parentage

or trait inheritance.

The drawback inherent with sequence-dependent primer development is that

genetic material must be sequenced. Sequence information of 0. sativa and Z. mays is

readily available in large databases. From sequences of both microsatellites and the

regions flanking them, studies have been developed to identify microsatellite type,

abundance, and length. There is also sufficient length of sequence information on either

side of the repeat motif to provide ample choice for primer selection. Database mining is

the least expensive method of primer design but requires large quantities of genome

information to be available. Cho et al. (2000) indicated that sequence information

available on GenBank produces fewer polymorphisms than sequence information

obtained from a genomic library.

For organisms that are not highly studied, it is theoretically feasible to use primer

sets developed in the study of closely related species. First used in the study of animals

(Moore et al. 1991), and later in plants (Kubik et al. 1999), cross-taxa SSR technology

has been applied successfully between closely related species, generally where one

species is better characterized genetically, or is more economically valuable. Such is the

case with Festuca spp. and L. perenne, wherein RFLP probes (Xu et al. 1992) and SSR

primer pairs have been successfully applied to follow the inheritance of traits through

inter-genic crosses. In the SSR study of Kubik et al. (1999), thirteen primers developed

from L. perenne genomic DNA were used to amplify DNA templates from species of

varying genetic relatedness. Primer sets developed for L. perenne successfully amplified

products from genomes of Festuca spp. and Lolium multiflorum (L.), closely related









grasses, and produced polymorphic information. This primer development technique

relies on evolutionary conservation of the primer region, and is more expensive than

database searching because of the uncertainty of amplification and the possibility of

lower rates of polymorphism with increasing genetic distance from the source species

(Kubik et al. 1999). Kubik et al. (1999) tested the use of three primers sets developed for

SSR amplification of P. vaginatum on L. perenne template DNA and was unsuccessful.

In a study using primer pairs characterized on L. perenne, SSR primers (Jones et al.

2001) were applied to Phalaris spp. (L.) and Avena sativa (L.), members of the tribe

Aveneae, with 38% and 12% efficiency of cross-taxa amplification, respectively. Similar

results were obtained when primers developed for Z mays were used on Sorghum bicolor

(L.) (Moench) (S. bicolor). One hundred polymorphic primer sets were developed from

Z mays information on GenBank. Of these 12 primer sets amplified products on S.

bicolor DNA templates, but only 2-3% of the 100 primer pairs were reliable and

polymorphic (Brown et al. 1996).

Kamps and Okagaki (Maximizing Taxonomical Transfer in Molecular Marker

Development and Cross-taxa SSRs for Tripsacum. Used by permission.) developed a

universally applied stringency modification to reveal cross-taxa SSR markers using a set

of four touchdown PCR programs. Starting at 65-550 C with progressively lower

stringency, 59 cross-taxa primers from Z mays and 32 from S. bicolor were applied to

Tripsacum dactyloides (L.) (T. dactyloides). A five degree drop in stringency for PCR

protocols revealed 14% more polymorphism from Z mays primers and 19% more from S.

bicolor primers. Zea mays and T. dactyloides are both in tribe Maydeae, while S. bicolor









is in tribe Andropogoneae. All three are in super tribe Andropogonodae (Devos and Gale

1997).

When attempting cross-taxa amplification, PCR products must conform to the

following requirements: only amplification products found within a specified length of

the parental polymorphic or monomorphic fragment sizes and containing a region of

repeat motif should be considered. This is because of the possibility of mispriming under

the reduced stringency programs (Jones et al. 2001).

Development, sequencing, and primer selection from genomic libraries is the most

expensive and time consuming method of SSR primer development, but has been shown

to yield more informative markers than GenBank and is more reliable than the use of

cross-taxa primer sets in showing polymorphism. The high cost of primer development

arises from the low number of primer sets that can be developed from the sequence

available and the low percentage of those that show polymorphism. This is true even in

highly enriched libraries. Jones et al. (2001), in their study of L. perenne, sequenced

1,853 clones using multiplex enrichment and identified 859 that contained viable SSRs.

Only 366 of these had flanking sequence long enough to develop 16-24 bp primers.

From these 100 primer pairs were found to be polymorphic. Thus, only 5% of sequenced

clones produce polymorphic information.

While set-up is costly and time consuming, once primers have been identified, the

actual running of the SSR reactions is relatively easy. PCR products can be fractionated

on an inexpensive, high-concentration agarose gel and analyzed with as little equipment

as an ultraviolet box, ethidium bromide staining and a camera. Comparison of PCR









fragments to a marker ladder of known molecular lengths is done so that accurate sizing

of the products may be determined by fractionation on a crude (agarose) gel system.

SSR markers were developed for this study ofbermudagrass cultivar identification.

Two sources for primer sets were used. First, SSR primer pairs developed in the study of

other members in the Poaceae family will be applied to bermudagrass DNA templates to

determine if this less expensive method may be used. Second, an enriched genomic

library will be developed and sequencing of that library done to develop bermudagrass-

specific SSR primer sets. In this initial study it is expected that the grasses will be

distinguished as groups, but further research will be needed to individually identify each

cultivar. Statistical and phylogenetic analysis of the PCR product length will be done and

the gel fractionation pictures compared to determine the success of these initial primer

sets. The ultimate goal is to uniquely identify each named cultivar and the new breeding

lines that are to be released so that a genetic method of cultivar identification may affirm

phenotypic assessment. Future applications of this research may include genetic analysis,

marker-assisted breeding, relationship, evolution, and migration information for Cynodon

spp.































Figure 1.1 Anatomical features of a grass stem and leaf section used in grass taxonomy
and separation of one species from another (from http://www.weeds.montana.
edu/crop/jgg.htm; Visited April 30, 2003).
























Figure 1.2 Two closely related turf varieties that appear similar. Despite their
appearance, they have slightly different growth habits, which may only be
seen after two or more years of growth and maintenance. Left: Champion
bermudagrass. Right: Floradwarf.






20


S95 deg


--Primers


DNA Synthesis


95 deg


Primers

nt h
*Synthe


o e

sisj Jr


Figure 1.3 Diagram of the PCR process by which small amounts of DNA can quickly be
copied by DNA polymerase to produce large numbers of a single fragment
(from http://members.aol.com/BearFlag45/BiologylA/LectureNotes/LNPics
/Recomb/pcr.gif; Visited April 30, 2003).


4ro

C


SIDNA
Jl r
i Jl0"'


r


I


r










PCR amplfkltkn of an S kw na:
(CA),
hi three diplold plnnt genotypa:
P1 P2, and the F O P1 x P2


Cmruarvd PCR pdmer sdtes



P (CA)n-2

F (CA)n


(CA)n_2 .


Polycrylamidse I el tophrwlls
and viuallzation of PCR produel
via autoradogrphy or fluorecence


P1 F1 P2



112


Figure 1.4 Diagram of ideal SSR marker system showing fragments are visualized and
how they can show allele inheritance. Notice that there is one solid band
surrounded by lighter extraneous bands (from http://www.nal.usda.gov/
pgdic/Probe/v2nl/chart.gif; Visited April 30, 2003).














CHAPTER 2
CROSS-TAXA PRIMERS APPLIED TO BERMUDAGRASS

Hybrid bermudagrass is an important turf-type grass in Florida and other southern

states. Cultivars with similar appearance, but differences in economic value-added traits,

make genetic markers desirable. A microsatellite DNA marker system to provide

immediate cultivar identification was studied using thirty-five cross-taxa primer pairs

developed for SSR study on other grasses in the Poaceae family. Cross-taxa primer

application resulted in only two patterns that reproduced reliably applied to all 23

bermudagrass DNA templates. This method of PCR amplification was successfully able

to group grasses into major grass types, but did not show microsatellites in the

amplification product.

Introduction

Commercial, clonally propagated varieties of improved bermudagrass used for turf

are not easily distinguished by morphological and physiological characteristics because

their subtle differences are subject to environmental influences. Because of the limited

genetic base of parent material used to produce new cultivars, physical similarity is

unavoidable. While the varieties appear similar, their reaction to drought, foot traffic,

fertilizers, and herbicides can be significantly different. In order to distinguish between

commercially important varieties, technology using a genetic marker system was sought.

Simple sequence repeat (SSR) markers, known also as microsatellites, are one to

eight nucleotide DNA motifs repeated three times or more. Polymorphisms in SSRs are

because of the difference in the number of times the motif occurs at a given locus (Reiter









1994). Once characterized and tested SSR primer pair sequences are easily transferable

between labs, are easily synthesized, and allow higher stringency than random amplified

DNA (RAPD) or DNA amplification fingerprinting (DAF) during PCR. Furthermore,

the analysis is technically simpler than amplified fragment length polymorphism (AFLP)

and SSRs set up for PCR with minimal DNA manipulation. AFLPs require the additional

steps of restriction digestion and adapter ligation and must be fractionated on more labor-

intensive DNA sequencing gels.

Cross-taxa molecular markers have been successfully used among closely related

plant species but tend to break down as the distance from the species of origin increases.

One previous study in cross-taxa application used restriction fragment length

polymorphism (RFLP) molecular markers developed for Festuca spp. (L.) (Xu and Sleper

1992). In this study probes from Festuca spp. were applied to Loliumperenne (L.) (L.

perenne), a closely related monocot, and interspecific crosses were tracked for

inheritance of important traits. SSRs have also been applied in cross-taxa studies of

closely related species including the use of 16 L. perenne primer sets on Festuca spp.

(Kubik et al. 1999). Another study applied 59 cross-taxa primer sets from Zea mays (L.)

(Z. mays) and 32 from Sorghum bicolor (L.) (Moench) (S. bicolor), to Tripsacum

dactyloides (L.) (T. dactyloides) using lower stringency of annealing temperature to

increase PCR amplifications (Kamps and Okagaki, in prep).

In a previous AFLP study researchers were successful in distinguishing 27 named

bermudagrass cultivars and breeding lines (Zhang et al. 1999). In this study we

attempted to apply SSRs to named bermudagrass cultivars to confirm the unusual

placement of Tifeagle, which is an irradiated mutant of Tifway II (Zhang et al. 1999)









with the greens grasses. Primer development being the largest cost in SSR primer

research (Kubik et al. 1999), cross-taxa primers published for other grasses were used as

an inexpensive source of primer sets.

The hypotheses of this study are that when cross-taxa primers from other members

of the Poaceae family are applied to bermudagrass DNA templates amplification products

will occur, homologous regions will be amplified, SSRs will occur within these products

and the use of step-down PCR will increase the events of cross-taxa polymorphism. In

the event that polymorphism is observed amplification products must be within 100 base

pairs (bp) of the size of the positive control, contain a confirmed SSR, and be clearly and

reliably reproduced. To test these hypotheses, we used 36 cross-taxa primers developed

for Saccharum officinarum (L.) (S. officinarum), S. bicolor, Z mays, L. perenne, and

Oryza sativa (L.) (0. sativa) all of which are in different subfamilies of Poaceae (Devos

and Gale 1997). The genus Cynodon is the only member of the subfamily Chloridoideae

represented in this study.

Materials and Methods

Plant Material

The names or identification numbers of the 23 genotypes tested in this study are

listed in Table 2.1. Named cultivars were obtained as breeders or foundation seed from

the same sources used by the AFLP study ofZhang et al. (1999). Grass samples were

maintained in black, one and two gallon plastic containers, using a soil less mixture of

three parts sand to one part peat and one part perlite, in a greenhouse on the University of

Florida campus.









DNA Preparation

Using the method of Dellaporta et al. (1983) DNA was extracted from two grams

of frozen leaf tissue. DNA concentrations were read by a SmartSpec 3000 (BIO-RAD,

Hercules, CA). Genomic DNA samples were diluted to lug/ul and were maintained at -

200 C. The following positive control DNAs were graciously provided for this research:

L. perenne by Dr. C.M. Styles, 0. sativa, by Dr. W.Y. Song, S. bicolor by Dr. D. Pring,

and S. officinarum, and Z mays by Dr. C.D. Chase.

Cross Taxa Primers

Thirty six primer pairs were taken from published studies within the family

Poaceae (Table 2.2). These cross-taxa primers were first tested on six plant introductions

(PI) (Table 2.1) of Cynodon spp. with four step-down PCR programs (Table 2.3) of

varied stringency starting as high as 65C and going as low as 45C. The annealing

temperature in the first ten cycles was lowered by one degree per cycle, followed by

thirty cycles at the lowest annealing temperature in order to increase cross-taxa

amplification. Primers amplifying DNA fragments of 60-700 bp were subsequently used

to test 23 different bermudagrass templates, including utility, greens, fairway, and ball

field types as well as breeding accessions (BA) and PIs (Table 2.1). Sizes of PCR

products were estimated based on 100 bp and 25 bp marker ladders (Invitrogen, Carlsbad,

CA) run on each gel. PCR amplification of the entire set of genomic samples was done at

least twice and reproducibility was determined based on the comparison of the results.

PCR

All PCR amplifications were done in a PTC-100 Peltier-effect thermal cycler (MJ

Research, Inc.; Waltham, MA). PCR was performed using 1.25 units Hot Start

Polymerase (Promega), 1 X supplied buffer (50 mM KC1, 10 mM Tris HC1 (pH 8.0),









1.63 mM (volume/volume) Triton X 100), 0.2 mM of each dNTP, 1.5 mM MgCl2,

1.0 uM of each primer (forward and reverse), and 1.0 .g of template DNA, in a total

volume of

50 IL. The PCR products were fractionated on 3% Agarose 1000 (Invitrogen; Carlsbad,

CA) gels in IX TBE buffer (1.2% Trizma base, 0.6% Boric acid, 0.07% EDTA) at 110 V

for four hours and stained with a 0.1 [g/ mL ethidium bromide solution for 30 min. Gels

were visualized over a UV light box and images captured on the Alphalmager 2200

system (Alpha Innotech Corp. San Leandro, CA).

PCR Product Sequence Analysis

PCR products for cross-taxa alignment were either sequenced directly, or cloned

and reamplified prior to sequencing. In the latter case, PCR products were ligated into

P-Gem T Easy Vector (Promega) and transformed into JM-109 (Promega) competent

cells according to manufacturer's recommendations. Colonies were grown on LB media

with 100 [g/ mL ampicillin, 80 [g/ mL x-gal, and 0.5 mM IPTG then selected based on

blue-white color according to instructions for JM-109 competent cells. Cloned inserts

were amplified by PCR using M13 forward (5'-GTTTTCCCAGTCACGAC) and reverse

(5'-CAGGAAACAGCTATGAC) primers. Inserts were then sequenced by the ICBR

DNA Sequencing Core Laboratory at the University of Florida, through use of ABI 373a

Stretch/377 DNA sequencers (http://www.biotech.ufl.edu/DNASequencing/

services.html). DNA sequences were aligned with CLUSTAL W (1.81) Multiple

Sequence Alignments (Thompson et al. 1994) on the San Diego Workbench

(http://workbench.sdsc.edu/ Visited April 30, 2003). Statistical analysis of fragment

patterns was done using the genetic similarity formula GS=m/(m+n) where GS=genetic









similarity between individuals, m=matches, and n=fragments not matching (Senior et al.

1998).

Results

After successful amplification of product on their species of origin, thirty six

primer pairs (Table 2.2) were used to screen six bermudagrass PIs (Table 2.1). Eighteen

primer pairs (50%) showed no cross-taxa amplification, twelve (33%) amplified

fragments without polymorphism or with multiple amplification products that were too

numerous to discern easily. Both of these observations are designated as "not

informative". Six (17%) primer sets initially produced informative polymorphic

amplification products (Table 2.2) and two of these showed reproducible fragment length

patterns. All primer sets were screened using four touchdown PCR programs (Table 2.3),

KAM4NW most often producing amplification products.

From the five species that contributed primers the S. officinarum (Cordiero et al.

2000) and 0. sativa (Chen et al. 1997; Temnykh et al. 2000) primer sets either did not

amplify from bermudagrass templates at all, or amplified products that were judged not

informative on either the subset of six bermudagrass PIs or the full set of 23 genotypes

(Table 2.2).

Five of the seven S. bicolor primer pairs amplified products on bermudagrass DNA

templates (Table 2.2). One amplified only monomorphic products, two amplified

products that were difficult to discern, and two primer sets were initially informative

(Table 2.4). Primer pair SB4-22 (Figure 2.1) failed to show amplification product after

the initial trial even though it occasionally revealed polymorphisms and amplification

products of a size similar to the positive control. The amplification products of SB6-36

on bermudagrass templates were also occasionally polymorphic, but more often appeared









monomorphic (Figure 2.2) with lengths of 500-600 bp, three fold larger than the size of

the positive control amplicon.

Three of the seven L. perenne primer pairs amplified PCR products on

bermudagrass DNA templates (Table 2.2). Only two primer sets revealed polymorphism

when tested on all templates. Of these two, LPSSRHO1A10 amplified multiple

polymorphic products as well as some faint, inconsistent amplicons (Figure 2.3). The

fragments that were common between gels (Table 2.5) placed grasses into the groups

indicated by Zhang et al. (1999) and were used to calculate genetic similarity. Primer

pair LPSSRHO1A07 did not predictably amplify products in the named cultivars, but the

amplification products from the PIs, while faint, were usually distinguishable (Figure

2.4). Sequencing showed that the LPSSRHO1AO7 positive control product was 251 bp

and contained a (GA)io SSR. Fragments of 108 bp and 452 bp in length from BA 475

(Figure 2.4) were also sequenced and contained no SSR (Figure 2.5). When aligned,

sequences showed a 21 bp region of conserved sequence in the three amplicons near the

3' end. The LPSSRHO1A07 control template sequence was submitted to BLASTN

(Altschul et al. 1990) and subjected to a redundancy search against all plant sequences in

GenBank using default settings, and the closest match was on chromosome 12 in O.

sativa.

A total of eight Z. mays primer pairs were tested on the bermudagrass templates.

Three pairs did not yield detectable amplification products and another three sets

amplified only monomorphic products on the 23 bermudagrass templates (Table 2.2).

MZEPPDKA2 (Figure 2.6) amplified inconsistently, but amplified products of the same

approximate size for a specific cultivar (Table 2.6). Primer pair ZMADH2N amplified









consistently, showing two or more polymorphic fragments, and placed the grasses into

groups predicted by Zhang et al. (1999). Fragment length patterns showed that the

breeding lines were individually distinguishable one from another. Greens grasses and

fairway grasses formed separate groups, but grasses within each group were similar

(Figure 2.7, Table 2.6).

Sequencing of the ZMADH2N primer pair (Figure 2.8) positive control product

revealed a 128 bp fragment containing an SSR of (AG)s. Fragments of 174 bp and 339

bp from PI 289-916 DNA template (Figure 2.7) were also sequenced, but contained no

SSR. Alignment showed that Random base pairs matched between fragments, but no

extended sequence was conserved among all three. The sequence of the positive control

for Z. mays was submitted to BLAST (Altschul et al. 1990) against all plant sequences in

GenBank and, as expected, showed homology to the gene for alcohol dehydrogenase II.

Genetic similarity analysis of LPSSRHO1A10 (Table 2.5) and ZMADH2N (Table

2.7) fragment length patterns showed high similarities within the greens grasses and three

of the fairway grasses (Table 2.7). By this statistical analysis Midiron and MS Choice

were considered dissimilar from each other and from the other fairway grasses. The

greens grasses were distinct from the other groups and individuals when matrices were

combined. Similarity matrices for individual primer sets are in Appendix A.

Discussion

We assessed cross-taxa primers developed for Poaceae species that are not closely

related to bermudagrass, but were hoped to provide an inexpensive method of primer

development. For this study, we designate successful cross-taxa amplicons as being

within 100 bp of the length of the positive control, containing SSRs, and repeatable

through PCR amplification. In the study ofKamps and Okagaki (In prep), primer pairs









from Z mays and S. bicolor successfully amplified polymorphic regions from T

dactyloides DNA. The occurrence of amplification events was increased by using

annealing temperatures below that of the species-specific primers. All three of these

plants are in super tribe Andropogonodae, with Z mays and T. dactyloides in the same

tribe of Maydeae, while S. bicolor is from tribe Andropogoneae (Devos and Gale 1997).

In correlating work done by Jones et al. (2001) using primer pairs derived from L.

perenne, polymorphic amplifications were found between L. perenne, Lolium multiflorum

(L.), and the Festuca spp. (Schreb), the same genus and same tribe (Poeae) respectively.

When L. perenne primers were applied to Poa spp. (L.), Phalaris spp. (L.), and Avena

sativa (L.), the last being a member of a different tribe, lower levels of cross-

amplification were detected.

In general, most of the primer pairs used in this study amplified products that were

300 bp larger than the positive control and failed to reproduce successfully through

repeated trials. Many of the primer sets designated as "informative" separated the grasses

into expected groups of phenotypic similarity such as fairways and greens grasses when

they successfully amplified. Amplification products that were many times larger than the

positive control amplicon may be PCR artifacts where only one primer sat down, but

even in some of those cases the breeding lines were distinguishable one from another. In

the case of MZEPPDKA2, amplification products were the same size when trials were

repeated, but no sequencing was done to confirm presence or absence of an SSR or the

cause of erratic amplification patterns. It seems possible that further manipulation of

PCR conditions may uncover the means of reliably amplifying polymorphic product.









It is encouraging that the amplification pattern of the two primer pairs that

amplified similar products twice on all DNA templates, ZMADH2N and

LPSSRHO1A10, grouped the grasses as expected from the AFLP study done by Zhang et

al. (1999). Knowing that the Z. mays primer pair ZMADH2N, which successfully

amplified products from S. bicolor (Brown et al. 1996), also amplifies a product from

bermudagrass templates encourages the possibility of success with certain primers from

organisms that are distantly related. The lack of SSR is disappointing, but

understandable given the genetic distance. Primer set LPSSRHO1A10 (Figure 2.3) also

produced a repeatable pattern through subsequent amplifications on bermudagrass DNA,

but fragments of multiple lengths suggested that mispriming is a concern with this primer

pair. It was interesting to note that bermudagrass amplicons from the ZMADH2N and

LPSSRHO1AO7 primer pairs showed no SSRs and poor homology with the SSR

containing positive controls when aligned by CLUSTALW (Thompson et al. 1994)

(Figures 2.5, 2.8). Lack of SSR in cross-taxa amplification indicates that the lower

stringencies in the PCR reactions allow cross-taxa SSR primers to function as little more

than long RAPD primers.

The genetic similarity analysis shows that while the grasses are grouped together,

there is still similarity between the fairway grasses and the breeding lines. This system is

a beginning, but eventually we would like to be able to find much lower similarities and

much larger differences between cultivars and groups.

Based on the findings of this and other studies (Jones et al. 2001; Brown et al.

1996), the use of cross-taxa primers from distantly related species for inexpensive, simple

detection of polymorphic SSRs is not supported. Lower stringency PCR programs









increased the number of cross-taxa primer pair amplification events from bermudagrass

DNA, but the number and quality of polymorphic products were unacceptably low to

encourage further investigation for quick, confident identification of cultivars. Perhaps,

as more closely related species are studied, and their genomic sequences become

available, cross-taxa amplification success will increase. Primer pairs developed for

Cynodon spp. will also be useful to other members of the Chlorideae tribe. These

indications suggest that the development of a species-specific set of primers used for the

detection of SSR polymorphism would be advantageous for hybrid bermudagrass and its

parents for marker assisted breeding, cultivar identification, and genetic relatedness

studies.














Table 2.1 Origins and uses of bermudagrass cultivars evaluated for simple sequence repeats


Type Identification
Fairway MS Choice
Midiron
Tifsport
Tifway II
Tifway 419
Greens Tifgreen
Tifdwarf
Tifeagle
MS Supreme
Champion
Floradwarf
Turf/ GN-1
Ball field Floratex
Tiflawn
Tifton 10
Breeding BA 481
Accessions BA 157
BA 475
Plant *PI 290-868
Introductions *PI 290-895
*PI 289-916
*PI 290-900
*PI 290-894


Ploidy
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
ND
36 (4n)
36 (4n)
54 (6n)
ND
ND
ND
36 (4n)
36 (4n)
27 (3n)
27 (3n)
18 (2n)


Taxon
txd
txd
txd
txd
txd
txd
txd
txd
txd
txd
txd
ND
d
d
d
ND
ND
ND
d
d
txd
txd
t


Source
J. Kranz & W. Philley, MSU
J. Fry, Kansas State University
W. Hanna, USDA-ARS. Tifton, GA
W. Hanna, USDA-ARS. Tifton, GA
W. Hanna, USDA-ARS. Tifton, GA
W. Hanna, USDA-ARS. Tifton, GA
W. Hanna, USDA-ARS. Tifton, GA
W. Hanna, USDA-ARS. Tifton, GA
J. Kranz & W. Philley, MSU, MI
Mike Brown, Coastal Turf, TX
Al Dudeck, Dept. Env. Hort. UF, FL
Jimmy Doublle, GNT, Florida
Al Dudeck, Dept. Env. Hort. UF, FL
W. Hanna, USDA-ARS. Tifton, GA
W. Hanna, USDA-ARS. Tifton, GA
B. Scully, EREC-IFAS-UF, FL
B. Scully, EREC-IFAS-UF, FL
B. Scully, EREC-IFAS-UF, FL
PI collection, Athens, GA
PI collection, Athens, GA
PI collection, Athens, GA
PI collection, Athens, GA
PI collection, Athens, GA


Reference
Krans et al. 1995
Anderson et al. 1988
Zhang et al. 1999
Burton 1985
Burton 1966b
Hein 1961,
Burton 1966a
Zhang et al. 1999
Krans et al. 1999
www.championdwarf.com
Dudeck 1991
www.shark.com/gnturf/introduction
Dudeck 1991
Hein 1953
Hanna et al. 1990
EREC, Breeding line
EREC, possible 'Wintergreen'
EREC, Breeding line
'Royal Cape' South Africa
'Reitz' South Africa
'Magennis' South Africa
'Damascus' South Africa
'Sekapplos Fine' South Africa


C. dactylon is designated d, C. transvaalensis is designated t, and their hybrid is designated txd. MSU: Mississippi State University,
UF: University of Florida, GNT: Gregg Norman Turf, MS: Mississippi, EREC: Everglades Research and Education Center, ND: not
determined. Five templates used as subset plus PI 290-905 (2n), which was later dropped, for a total of six designates by (*).









Table 2.2 Origins of cross-taxa primer pairs used in PCR reactions on bermudagrass
templates


Primers from L. perenne
LPSSRHO1A02
LPSSRHO1A07
LPSSRHO1A10
LPSSRHO1E10
LPSSRH01H06
LPSSRH02C11
LPSSRKO1A11
Primers from S. bicolor
Sb4-32
Sb5-236
Sb5-85
Sb6-36
Sb6-84
Sb4-22


Sb4-15
Primers from S.
SMC222CG
SMC226CG
SMC248CG
SMC319CG
SMC477CG
SMC863CG
SMC1039CG


Amplification


Results
not informative
informative
informative


not informative
not informative
not informative
informative

informative


officinarum


not informative



not informative


not informative
not informative
not informative


Primers from 0. sativa
RM110
RM280
RM349
RM23
RM44
RM227
RM256


Program
(Table 2.3)

KAM4NW
KAM3NW


KAM3NW

KAM4NW









Table 2.2. Continued
Primers from Z mays
MZE-PPDKA2
ZMADH2N
CMBMC1893
ZMBNLG1633
p-umc2042
p-bnlgl252
p-dupssrl
p-umcl308


Amplification Results
+ informative
+ informative


Program
KAM4NW
KAM4NW


not informative
not informative


not informative


Cross-taxa primers applied to bermudagrass DNA. Informative fragment length patterns
were bright, polymorphic in nature, and clear. Not informative patterns were
monomorphic under more stringent conditions, but under less stringent conditions the
fragments amplified are too numerous to discern easily. Primer sources are L. perenne
(Jones et al. 2001); S. bicolor (Brown et al. 1996); S. officinarum (Cordiero et al. 2000);
0. sativa (Temnykh et al. 2000; Chen et al. 1997); Z. mays (Brown et al. 1996); and
Maize Genome Database mapping to chromosome 2 (http://www.agron.missouri.edu/).









Table 2.3 Basic step-down PCR programs used to test primer amplification
Step Minutes Temp (degrees C)
1 5 95
2 1 95
3 1 annealing temp
4 1.5 72
5 Repeat steps 2-4 ten times,


6


lowering annealing temp 1 degree
in each cycle.
1 95


7 1 lowest annealing temp
8 1.5 72
9 Repeat steps 6-8 thirty times
using lowest annealing temp.
10 5 72
11 cool down 10


The four step-down annealing temperatures (65-55, 60-50, 55-45, and 50-40) used for
this project were originally developed by Kamps and Okagaki (In prep). These programs
were identified in this project as KAM1NW: 65-55, KAM2NW: 60-50, KAM3NW 55-
45, and KAM4NW: 50-40 respectively.










Table 2.4 Sorghum bicolor cross-taxa amplicon sizes estimated in base pairs
Accession DNA Amplification Products

SB4-22 primers SB6-36 primers

Gel#19570-2 Gel#19872 Gel#19621
MS Choice 550 650 600
Midiron 300 650
Tifsport 300 550 650 600
Tifway II 550
Tifway 419 550 650
Tifgreen 300 550 650+ 600
Tifdwarf 300 550 650+ 600
Tifeagle 550 600
MS Supreme 300 550 600
Champion 550
Floradwarf 300 550 600
GN-1 300 600 550 650 600
Floratex 300 550 650 600
Tiflawn 550 650 600
Tifton 10 300 550 650 600
BA 481 300 550 650 600
BA 157 300 550 650
BA475 300 550 650 600
PI290-905 550 600
PI289-916 550 650 600
PI290-900 300 500
PI290-868 300 400 550 650 600
PI 290-895 300 400 550 650+ 600
S. bicolor 300 200

This table describes the major products amplified from the entire set of genotypes to
determine reproducibility. Estimated length of PCR products is given in nucleotide base
pairs based on 100 bp and 25 bp marker ladders (Invitrogen) run on each gel. Based on
sequencing lengths vs. visual determination, an error of -50 bp was determined for
fragments because of resolution of agarose gels and human error. Positive control PCR
product was the only clear amplification in gel # 19924 therefore no results are reported.










Table 2.5 Lolium perenne cross-taxa amplicon sizes estimated in base pairs
Accession DNA Amplification Products

LPSSRHO1A10 primers LPSSRHO 1A7 primers

Gel#19570-1 Gel#19950 Gel#19621 Gel#19898-1
MS Choice 250 300 250 300
Midiron 300 600 300 600
Tifsport 150 300 150 300 600
TifwayII 150 300 600
Tifway 419 150 300 600
Tifgreen 225 600 600 600
Tifdwarf 225 600
Tifeagle 225 600
MS Supreme 225 600 600 600 600
Champion 600 600 600
Floradwarf 225 600 600 600
GN-1 225 300 700
Floratex 300 300
Tiflawn 250 300 250 110 500 500
Tifton10 200 250 300 200 250 300 110
BA481 200 300 200 250 300 110 500 500
BA157 200 300 200 250 300 110 500 500
BA475 300 250 300 110 500 500
PI 290-905 150 300 150 200 300 600 600
PI 289-916 150 300 150 300 600 600
PI 290-900 125 300 300 410
PI 290-868 200 300 200 400 600 400 600
PI 290-895 250 300 250 600 410 600
L. perenne 150 200 150 200 200 200

Length of PCR products is given in nucleotide base pairs, and was scored by estimation
based on 100 bp and 25 bp marker ladders (Invitrogen) run on each gel. Based on
sequencing lengths vs. visual determination, an error of -50 bp was determined for
fragments because of resolution of agarose gels and human error. This table describes
the major products amplified on the entire set of genotypes to determine reproducibility.










Table 2.6 Zea mays cross-taxa amplicon sizes estimated in base pairs
Accession DNA Amplification Products

MZEPP-DKA2 primers ZMADH2N primers
Gel
#19364-2 Gel #20001 Gel #19975 Gel #19364
MS Choice 500 650 275 400 275 400
Midiron 175 175 650 275 400 600 275 400
Tifsport 650 650 275 600 275 600
Tifway II 650 275 600
Tifway 419 650 275 600
Tifgreen 550 650 200 400 200 400
Tifdwarf 550 650 200 400 200 400
Tifeagle 550 650 200 400 400
MS
Supreme 550 650 200 400 200 400
Champion 550 650 200 400 400
Floradwarf 550 650 200 400 200 400
GN-1 400 400
Floratex 650 275 400 275 400
Tiflawn 650 275 275
Tifton 10 275 275 275 400 275 400
BA 481 650 275 400 275 400
BA 157 650 400 400
BA 475 650 275 400 275 400
PI 290-905 650 600 600
PI289-916 650 200 400 200 400
PI290-900 650
PI 290-868 650 275 400 275 400
PI 290-895 650 275 600 275 600
Z. mays 125 125 150 150

Length of PCR products is given in nucleotide base pairs, and was scored by estimation
based on 100 bp and 25 bp marker ladders (Invitrogen) run on each gel. Based on
sequencing lengths vs. visual determination, an error of -50 bp was determined for
fragments because of resolution of agarose gels and human error. This table describes
the major products amplified on the entire set of genotypes to determine reproducibility.














Table 2.7 Genetic similarity matrix including combined data from primer sets LPSSRHO1A10 and ZMADH2N
0 0 -n 0 -1 a 33 3 P
C-) -H 0 "- 0 0

( (D (D (D do d o
3 (D i< C --- 0 ) g (0
s C( W -


0.33 1
0.11 0.11 1
0.11 0.11 1 1
0.11 0.11 1 1 1
0 0.11 0 0 0 1
0 0.11 0 0 0 1 1
0 0.17 0 0 0 1 1 1
0 0.11 0 0 0 1 1 1 1
0 0.25 0 0 0 0.5 0.5 0.5 0.5 1
0 0.17 0 0 0 1 1 1 1 0.5 1
0.13 0.13 0 0 0 0.13 0.13 0.13 0.13 0 0.13 1
0.5 0.5 0.17 0.17 0.17 0 0 0 0 0 0 0.17 1
0.5 0.17 0.17 0.17 0.17 0 0 0 0 0 0 0 0.17 1
0.66 0.25 0.08 0.08 0.08 0 0 0 0 0 0 0.1 0.25 0.33 1
0.33 0.33 0.11 0.11 0.11 0 0 0 0 0 0 0 0.33 0.17 0.66 1
0.17 0.17 0 0 0 0 0 0 0 0 0 0.25 0.17 0 0.33 0.5 1
0.5 0.5 0.11 0.11 0.11 0 0 0 0 0 0 0.17 0.5 0.25 0.33 0.5 0.25 1
0 0 0.33 0.33 0.33 0 0 0 0 0 0 0 0 0 0 0 0 0 1
0.17 0.17 0 0 0 0 0 0 0 0 0 0.13 0.17 0 0.13 0.17 0.17 0.25 0 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
0.33 0.33 0.11 0.11 0.11 0 0 0 0 0 0 0.13 0.33 0.17 0.66 1 0.5 0.5 0 0.11 0 1
0.33 0.11 0.33 0.33 0.33 0 0 0 0 0 0 0 0.33 0.5 0.22 0.11 0 0.17 0.17 0 0 0.11 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1


Genetic similarity was determined by the formula GS=m/ (m+n) where GS=genetic similarity, m=matches, and n=fragments not
matching.


MS Choice
Midiron
Tifsport
Tifway II
Tifway419
Tifgreen
Tifdwarf
Tifeagle
MS Supreme
Champion
Floradwarf
GN-1
Floratex
Tiflawn
Tifton 10
BA 481
BA 157
BA 475
PI 290-905
PI 289-916
PI 290-900
PI 290-868
PI 290-895
POSITIVE













Greens

Et

(D O
S,, EO "
7- F- j= 2 o 2 T L


Lawn


-o -
a)

C-4 7-7


Breeding Lines

0-0


<<<--


co ln a
CO CO )O

CM CM CI) 0o
- 0 a) C
0- a: 0- z


Figure 2.1 SB4-22 primer pair amplification of 23 bermudagrass DNA templates. The
positive control PCR reaction was performed on S. bicolor DNA template.
The negative control consists of the entire PCR reaction mix, including
primers, but without template DNA.


Fairway



o o c
ci~ ci


~
~




~", _I


8




~













Fairway Greens

0aE Ct
ao

i.i. i .I i1 i.


Lawn

C)
cX0
L)
COO

C ro I-
inoE
n~oj -I-


Breeding Lines

0-0


SN C CM C


w
CO Mn a

M5 M5 CtO -0-
- 0 a)> o:
0- 0- 0- z -^


rp I TW


Figure 2.2 SB6-36 primer pair amplification of 23 bermudagrass DNA templates. The
positive control PCR reaction was performed on S. bicolor DNA template.
The negative control consists of the entire PCR reaction mix, including
primers, but without template DNA.


C I2

Oj


O










Fairway Greens
01E ,- t

a i. 1 ti i rC
S^^^^E^Ma)
0 0 >' >' iCOu-C


Lawn
C)
cX0
L)
COO
C ro I-
inoE
n~oj -I-


Breeding Lines

0-0

<<<--


w
CO M a)
CO CO a)
M ML CO -0

cool a
0- 0- 0 0


Figure 2.3 LPSSRHO1A10 primer pair amplification of 23 bermudagrass DNA
templates. The positive control PCR reaction was performed on L. perenne
DNA template. The negative control consists of the entire PCR reaction mix,
including primers, but without template DNA.


Co
O)
0






I












Fairway Greens
0L E t


o ",Io"
4M- WW =M 2M ii
7-7 7 - 0 L


Lawn
CC
cX 0
a)~
L CO 0
LO q
Cni4*Ir*Ir


=


Breeding Lines

0-0
0) 0) 0)
co C in )0
<<<--


6 6 E C,
Of
CO0n )

CM M CC 0)
- a) 0:)
0- : 0- z -z
sows


,1


19898e1


Ti7


Figure 2.4 LPSSRHO1AO7 primer pair amplification of 23 bermudagrass DNA
templates. The positive control PCR reaction was performed on L. perenne
DNA template. The negative control consists of the entire PCR reaction mix,
including primers, but without template DNA.


U t


-.

0-





0




.0
g .__













LPSSRHO1AO7 F
UPPER
LOWER
CONTROL


UPPER
LOWER
CONTROL


UPPER
LOWER
CONTROL


UPPER
LOWER
CONTROL


UPPER
LOWER
CONTROL


UPPER
LOWER
CONTROL


UPPER
LOWER
CONTROL


UPPER
LOWER
CONTROL
LPSSRHO1AO7 R

UPPER
LOWER
CONTROL
LPSSRHO1AO7 R


-TGGAGGGCTCGTGGAGAAGT--------------------------------------
-TGGAGGGCTCGTGGAGAAGTCCAGTAAAGAAACTCACCATCAAAAATGGGGTGGGCTC
-TGGAGGGCTCGTGGAGAAGT--------------------------------------
TTGGAGGGCTCGTGGAGAAGT--------------------------------------


CCTCAAGGGGCGCCGCCAGTCACAATGAGTTGCCTAAGCTGGAACTATCATGGACTTGGC




AATGCCGCGACAGTTAAAGAACTTCGCGATCTTGTGAAGCGTTTTGCCCCATCTGTGTTG




TGCGTTCAAGAAACTTAGATTCGTAACTCGCGGGTGGAAAGTTTGAAAAATATACTTGGC


ACATCGACC


TTTGACC-AAGCGTTTGTTGTTAGTAGTCAACGCCGTAGTGGAGGCTTAGGAATCTTCTA


CGCAGCCGAAGACTTCGGTGTT


TCGGGAGGCGAGAGAGAGAGAGAGAGAGATGCA


GAATAATAATATAAGAATGACTATTTTACCGTACTCGCAATATCATATTGACATGATTTT
----------------------------------------------AAAGACTGGGTG--
GGTGGTGAATGCGACGCATGCGCTTTGGACGTGGCACCGGAACGACGACGACGAGCCT--


CAACGAGGGGCAAGCTGATCCGTGGAGGTTGACATGCGTGTACGGGGAGGCGTAGACAAG
-GATCAAGGTAAATGTTG-ATGGCGCATTTGTGGCACAAACAGATCAAGCAGGT-GCAAG
--GTGGTGGCCGATCAAGTGTGGATCACCAGCCTAGCATCCAAGCCGGCTTGTA-ACAAG


TGAGTGGCTCGTCCTAATCACTAGTGCGGCCGCCTGCAGGTCGACCATATGGGAGCGGTT
TGAGTGGCTCGTCCTACTTCTC---------------------------------CGGTT
TGAGTGGCTCGTCCTAATCACTAGTGCGGCCGCCTGCAGGTCGACCATATGGGAGCGGTT
-------------------------------------------------------CGGTT

CCCACGCCTTGC
CCCACGCCTTGC
CCCACGCCTTGC
CCCACGCCTTGC
AAAAAAAAAAAA


Figure 2.5 CLUSTAL W (1.81) alignment of LPSSRHO1AO7 amplification product

sequences. The forward primer is not seen in the product designated 'Lower'

because this product was sequenced directly with the LPSSRHO1AO7

forward primer. The other products were first cloned into the pGemT plasmid

vector (Promega) and sequenced using the M13 forward primer. Sequence

was then trimmed to include only those sequences within the LPSSRHO1AO7

forward and reverse primers. Bases that are conserved between fragments are

marked by (*). The positive control SSR is underlined and the primer

sequences are designated by (A).













Fairway Greens

2E ,- t2

I= .=,o Ia1 i
S i-i i-i- -i-s U o


Lawn

C)
cX 0
L)
COO
CO


LO q-I- =-
C-4 7_7


Breeding Lines

0-0


<<<--


w
CO M a)
CO CO a)
M ML CO -0
c ol -
(N4 (N CM) cD
- 0 a) 0:
0 00

A


, -'V--W --- 4 -W. 4r.


20001 Ii

I::AILi k IA.jI:


Figure 2.6 MZEPPDKA2 primer pair amplification of 23 bermudagrass DNA templates.
The positive control PCR reaction was performed on Z. mays DNA template.
The negative control consists of the entire PCR reaction mix, including
primers, but without template DNA.


L
-
o -
U
I


M! _


0-.~F~~"i1









Fairway Greens
EWt
=E =a) a.CO

|.i. a
0 0 >' >' CO
CL CO CO 0)
Q) Iro ~
4M-WW M 2 -


Lawn
CD
w
coX
in c
C- I- C
(NuC FO
'0~~~


Breeding Lines I
LnOO CD o LO
M )M )MT)COCO a)
(N CN CN CN CN C) CO
-- 0 0Co
CO0COQ-0-0-0-a- z -


tHU


r


~I


Figure 2.7 ZMADH2N primer pair amplification of 23 bermudagrass DNA templates.
The positive control PCR reaction was performed on Z. mays DNA template.
The negative control consists of the entire PCR reaction mix, including
primers, but without template DNA.































Figure 2.8 Sequence of ZMADH2N amplification products from Figure 2.6 aligned by
CLUSTAL W (1.81). Amplification products on bermudagrass and the
product amplified on the positive control template are shown, as well as the
alignment of the reverse primer. These fragments were directly sequenced
using the forward primer in a single pass from PCR product. The positive
control SSR is underlined. Bases that are conserved between fragments are
marked by (*). Reverse primer is designated by (A) and forward primer
sequence is (5' TGCGAAG
AAAGCAGTAGCAAA).














CHAPTER 3
SPECIES-SPECIFIC PRIMERS

Hybrid bermudagrass is an important turf-type grass in Florida and other southern

states. Cultivars with similar appearance, but differences in economic value-added traits,

make genetic markers desirable. For this reason, a microsatellite DNA marker system

that will provide immediate cultivar identification was studied. A genomic library was

enriched for (AC)n repeats for primer development. This method of microsatellite primer

development provided three primer pairs that reliably amplified polymorphisms on

bermudagrass DNA templates. Unique genetic fingerprinting in many cultivars was also

shown using a simple genetic similarity matrix and phylogenetic analysis.

Introduction

Cynodon dactylon (L.) Pers. (C. dactylon), known in the U.S. as common

bermudagrass, is a tetraploid with chromosome number of 2n=4x=36 (Taliaferro 1995).

Generally C. dactylon is described as having a folded vernation, no auricle, and a fringe

of hairs on the ligule. It is rhizomatous, stoloniferous, and produces seed excessively on

4-5 digitate spikes raised above the leaf canopy. Cynodon transvaalensis (Burtt-Davy)

(C. transvaalensis), known as African bermudagrass, is a diploid with a chromosome

number of 2n=2x=18 (Beard 1973). Cynodon transvaalensis is used in the U.S. turfgrass

industry because of its uniform morphology maintained within varieties and its ability to

cross interspecifically with C. dactylon (Taliaferro 1995; Beard 1973).

The hybrid of C. dactylon X C. transvaalensis, is a sterile triploid with 2n=3x=27

chromosomes (Taliaferro 1995; Beard 1973). Morphologically similar to C. dactylon









auricless, ligules, etc.), the hybrid possesses a desirable combination of turf traits from

the two species (Taliaferro 1995). In Florida, bermudagrass accounts for over half of the

golf course grasses or 136,000 acres (Haydu et al. 2002a) and 6% of the sod industry

production acreage, or 4,556 acres. Turf bermudagrass garnered $16.3 million at $0.114

ft-2, which accounted for 19% of the sod industry revenue in 2000 (Haydu et al. 2002b).

Commercial, clonally propagated varieties of improved bermudagrass used for turf

are not easily distinguished by morphological and physiological characteristics because

of subtle differences subject to environmental influences. Although the varieties appear

similar, their reaction to drought, foot traffic, fertilizer application rates, and herbicides

can be significantly different. A genetic marker system was sought in order to more

quickly distinguish between economically valuable cultivars.

Simple sequence repeat (SSR) markers, also known as microsatellites, are one, two

or three nucleotide DNA motifs repeated three times or more (Edwards et al. 1996). The

variation in the number of times the motif occurs between alleles of a given locus is the

basis of observed polymorphism. SSRs have become popular because they show easily

distinguished co-dominance, are more easily reproduced than sequence independent

markers, assay robustness, high rate of polymorphism, and distribution throughout

eukaryotic genomes. Primer sequence is easily transferred between labs and primer

synthesis is readily available. Currently, they are used for marker-assisted breeding,

genetic analysis, and to obtain relationship, evolution, and migration information for an

organism (Gethi et al. 2002). One major drawback to SSR markers is that large amounts

of genomic sequence is needed to produce primers that flank a high copy number of the

repeat (Reiter 1994). According to previous studies, the rate of mutation observed at a









given locus is dependant on the type of motif, thus di-nucleotide repeats mutate more

frequently than tri- or tetra-nucleotide repeats (Cho et al. 2000). SSR primers are 16-20

base pair (bp) long, which allows higher stringency during the polymerase chain reaction

(PCR) than the 10 bp random amplified polymorphic DNA (RAPD) primers or the 5-8 bp

DNA amplification fingerprinting (DAF) primers. Furthermore, SSR analysis is

technically simpler than amplified fragment length polymorphism (AFLP) analysis.

SSRs require minimal DNA manipulation while AFLPs require restriction digestion and

adapter ligation. Size fractionation of SSRs is done on inexpensive agarose gels, which

are easy to set up and use (Reiter 1994).

AFLP markers were successful in distinguishing 27 named bermudagrass cultivars

and breeding lines (Zhang et al. 1999). Studies in Zea mays (Gethi et al. 2002) have

shown that 44 primer pairs detect small genetic variations within inbred lines, which are

presumably genetically identical. In this study we attempted to apply species-specific

SSRs to many of the same cultivars to confirm the unusual placement of Tifeagle, which

is an irradiated mutant of Tifway II (Zhang et al. 1999). We will seek to determine if it is

possible to differentiate between closely related species of bermudagrass using primers

developed from bermudagrass genomic sequence enriched for (CA/GT)n repeats, the

third most common SSR in plant genomes (Morgante and Olivieri 1993).

Materials and Methods

Plant Material

The 23 genotypes tested in this study are listed in Table 3.1 along with their origin,

ploidy, and use. Named cultivars were obtained as breeders' or foundation seed from the

same sources used by the AFLP study of Zhang et al. (1999). GN-1, which is generally

classified as a fairway grass, is more cytogenetically similar to the lawn grasses and was









therefore grouped with them. Grass samples were maintained in black, one and two

gallon size plastic containers, using a soil-less mixture of three parts sand to one part peat

and one part perlite, in a greenhouse on the University of Florida campus. Dr. B. Scully

graciously provided additional stocks of Tifsport, Tifway 419, Tifway II, Tifdwarf,

Tifeagle, Champion, MS Supreme, and Floradwarf, from his breeding program at the

Everglades Research and Education Center, University of Florida.

DNA Preparation

DNA was extracted from two grams of frozen leaf tissue by the method of

Dellaporta et al. (1983). Nucleotide concentration was read on a SmartSpec 3000 (BIO-

RAD. Hercules, CA). DNAs were diluted to 1.0 pg/ul and stocks maintained at -200C.

Enriched Library Construction

An enriched microsatellite library (Kandpal et al. 1994) was developed for Tifway

419 and plant introduction (PI) 290-984 DNA for (CA)n repeats. The Tifway 419 library

was used for all primer development in this project. Library enrichment was

accomplished by digesting 5.0 tg of genomic DNA with Sau 3A I enzyme overnight and

fragments between 0.4-1.5 kilobase pairs in length were isolated by passing restricted

DNA through Chroma Spin columns (Clontech Laboratories, Palo Alto, CA). Sau 3A I

(Forward Sau-L-A: 5' GCGGTACCCGGGAAGCTTGG; Reverse Sau-L-A: 5'

GATCCCAAGCTTCCCGGGTACCGC) linkers were ligated to the fragments and the

ligation mixture was washed through Chroma Spin columns (Clontech Laboratories) to

remove excess linker. Fragments were amplified by PCR using primers annealing to the

Sau 3A-I linkers with random base pair overhangs into the genomic sequence and

15.0 iL of the product was denatured and hybridized to a biotinylated probe [5'-(CA)15

TATAAGATA-Biotin]. This hybridization solution was washed using the VECTREX









Avidin D matrix (Vector Laboratories, Burlingame, CA), which binds to the biotinylated

probe. In the wash solution (0.5 M phosphate), only DNA fragments hybridized to the

probe remain attached to the matrix, but when eluted with distilled water, the DNA

fragments were released from the probe and removed. Both the impoverished fraction

and the enriched fraction were saved and tested for (CA)n enrichment by dot blotting a

10% dilution series (from 100% down to 0.1%) of both fractions on 3MM paper and

hybridizing with a fluorescent probe. Film was exposed to the paper for 2 hrs (Figure

3.1). Fragments of enriched library were again amplified by PCR using the

Sau 3A I primers, ligated into a vector and transformed into Escherichia coli (One

Shot, Invitrogen; Carlsbad, CA) using the TOPO TA Cloning vector (Invitrogen).

Transformed bacterial colonies were raised on blue/white inducing media (LB media

with 80 [g/ mL x-gal, and 0.5 mM IPTG) and were also tested for repeat presence by

colony lift on 3MM paper and hybridization of a fluorescent probe (Figure 3.2). Those

colonies fluorescing and showing white color were chosen.

After the initial test transformation, subsequent ligations used P-Gem T Easy

Vector (Promega Corp.; Madison, WI) transformed into JM-109 (Promega) competent

cells. Transformed colonies were grown on LB media with 100 [ig/ mL ampicillin, 80

[g/ mL x-gal, and 0.5 mM IPTG and selection was based on blue/white color according

to manufacturer's instructions. Cloned inserts were amplified by PCR using M13

forward (5' GTTTTCCCAGTCACGAC) and reverse (5' CAGGAAACAGCTATGAC)

primers. Inserts were then sequenced by the University of Florida ICBR DNA

Sequencing Core using ABI 373a Stretch/377 DNA sequencers

(http://www.biotech.ufl.edu/DNASequencing/services.html).









Primer Development

The genomic insert sequences that contained a microsatellite of at least five repeats,

and sufficient flanking sequence to develop primer pairs (Figure 3.3), were chosen and

primer pairs were developed using Primer 3 (Rozen and Skaletsky 2000) on the San

Diego Workbench (http://workbench.sdsc.edu/). The criteria used for primer selection

was a 58 C annealing temperature and a primer length of 16-20 bp (Table 3.2).

Species-specific primers were first tested for amplification on plant introduction

(PI) 290-905, PI 290-900, PI 290-895, and Tifway 419 (Table 3.1). Primer amplification

was tested using four step-down PCR programs (Table 3.3) of varied stringencies, which

consisted often cycles with one degree lower annealing temperature each cycle. These

were followed by 30 cycles at the lowest annealing temperature. The primer sets that

amplified DNA fragments were then used to test 23 different bermudagrass genotypes,

including fairway, greens, turf/ ball field types, and breeding material (Table 3.1). All

PCR was amplified in a PTC-100 Peltier-effect thermal cycler (MJ Research, Inc.;

Waltham, MA). TaqBead Hot Start Polymerase (Promega) was used in all reactions with

final concentrations of reactants being: 1.0 X Promega reaction buffer (50 mM KC1,

10 mM Tris HC1, 1.63 mM (volume/volume) Triton X 100), 0.2 mM of each dNTP,

1.5 mM MgC12, 1.0 uM of each primer (forward and reverse), 1.0 pg of template DNA,

and 1.25 units of Taq polymerase in a total volume of 50 IL. The PCR products were

fractionated on 3% Agarose 1000 (Invitrogen) gels in IX TBE buffer (1.2% Trizma base,

0.6% Boric acid, 0.07% EDTA) at 110 V for four hours and stained with a 0.1 [g/ mL

ethidium bromide solution for 30 min. To obtain a more precise estimate of fragment

size, polymorphic PCR products were fractionated on 6% polyacrylamide gels and then

stained with a 0.1 [g/ mL ethidium bromide solution for 10 min. Gels were visualized









over a UV light box and the images were captured on the Alphalmager 2200 system

(Alpha Innotech Corp. San Leandro, CA). PCR product length was determined by

estimation based on a 100 bp and 25 bp marker ladder (Invitrogen) run on each gel. The

amplification products of the entire set of template DNAs from at least two separate PCR

reactions were fractionated on separate gels to confirm reproducibility of the results.

Analysis

Fragment lengths for phylogenetic tree construction and genetic similarity were

determined by computer (Tables 3.4-3.6), using Kodak Digital Science ID version 2.0.2

(Eastman Kodak Co. New Haven, CO) and Excel was used to present the results as

0 (absence) or 1 (presence) of a fragment within a range of 10 bp. The phylogenetic trees

were constructed using PAUP Version: 4.0 beta 10 (Swofford 2000), run on Power

Macintosh (PPC). Genetic similarities were calculated using the formula GS=m/(m+n)

where GS=genetic similarity between individuals, m=matches, and n=fragments not

matching (Senior et al. 1998).

Results

An SSR enriched library was developed using Tifway 419 as the source of genomic

DNA. DNA fragments were enriched for (CA)n repeats and a dilution series hybridized

to a fluorescent probe (Figure 3.1) to attempt quantification. Following amplification and

cloning of the library, bacterial colonies were screened for the presence of the repeat

motif (Figure 3.2). Out of 250 colonies on the test plate, 178 contained the (CA)n repeat.

After ligation and transformation were optimized, 50% of the colonies chosen by

blue/white color selection contained genomic inserts of> 600 bp, based on PCR

amplification of the insert.









From 96 inserts of> 600 bp, 56 yielded sequences longer than 100 bp because of

problems with sequencing. Of these, 21 genomic fragments carried SSRs with (CA)n and

n > 5. Eleven fragments contained sufficient flanking sequence for primer development

(Figure 3.3; Table 3.2). There were other imperfect SSR motifs represented within

several sequences. Predicted amplicon sizes ranged from 97 to 612 bp. For future study,

106 additional clones were sequenced yielding 74 that contained inserts, and 27 that

contained confirmed SSRs and enough flanking sequence for primer development. These

sequences are reported in Appendix B and are presently being exploited.

All 11 primer pairs developed from cloned sequences (Table 3.2) were tested for

amplifications on a subset of DNA templates (Table 3.1) using PCR programs

KAM1NW, KAM2NW, and KAM3NW (Table 3.3). PCR products were scored for

clarity and brightness of fragments on the agarose gels. Only three of the 11 primer pairs

amplified a product over the three PCR protocols. The program producing the clearest

amplicons was then used to amplify from the entire 23 templates (Figures 3.4-3.7).

Replicated trials of each primer set amplification were fractionated on separate agarose

gels and higher resolution polyacrylamide gels. BER1 and BER11 amplified products

that were smaller than expected, but products amplified using BER9 were within 75 bp of

the expected size (Table 3.2).

Tables 3.4-3.6 list estimated amplicon sizes using primers BER1 (Figure 3.4),

BER9 (Figure 3.5), and BER11 (Figure 3.6) to amplify from the 23 bermudagrass

templates. The five fairway grasses (Table 3.1) were separated into smaller groups

consisting of Midiron and MS Choice, which were unique from each other and from the

other fairway grasses. Tifway 419, Tifway II, Tifsport consistently showed similar









amplicons. Amplicons from greens grasses (Table 3.1) showed little variation. Tifeagle

may have a longer BER9 amplification product visible on the polyacrylamide gel (Figure

3.7), but the pattern of fragment length remained undistinguishable on the agarose gels

(Figure 3.5). The four lawn grasses were differentiated into three groups by

amplification of product using the BER1 primer pair (Figures 3.4). BER9 (Figures 3.5)

showed no product for Tiflawn or Tifton 10, but GN-1 and Floratex appeared similar.

BER11 appeared able to distinguish all four lawn grasses by agarose fractionation (Figure

3.6). The breeding lines, which consist of the breeding accessions (BAs) and PIs, were

separated into a group containing BA 481 and BA 157, while the PIs were individually

distinguished by unique PCR products. To verify the grouping of Tifeagle with the

greens grasses and Tifsport with Tifway 419 and Tifway II, DNA was isolated from

another sample of these grasses, and BER1 was used to amplify products from all named

varieties (Figure 3.10). The same results were obtained.

BER1 amplicons fractionated on gel #20027 (Figure 3.4) were scored (Table 3.4)

and used to produce a phylogenetic tree (Figure 3.9). Grasses from different categories

were grouped together by this method. PI 290-900, Floratex, Tifway 419, Tifway II

Tifsport, and MS Choice were all placed in one group. Greens grasses were grouped

together, as were BA 475, BA 157, BA 481, Tifton 10, and Tiflawn. Two groups of

interest were formed, GN-1 with PI 290-895 and Midiron with PI 289-916.

BER9 amplicons fractionated on gel #20735 (Figure 3.5) were scored (Table 3.5),

and showed two groups of six (Figure 3.10). The first group consisted of Tifton 10,

PI 290-868, BA 475, BA 157, BA 481, and Tiflawn while the greens grasses formed the









second. MS Choice and Midiron were widely separated, with Midiron being closer to the

three other fairway grasses. PI 289-916 and PI 290-895 were grouped together.

BER 11 amplicons from gel #20763 (Figure 3.6) were scored (Table 3.6) and

grouped the greens grasses together, with Midiron closer to them than to a group of the

three fairway grasses. Both groups were distinguished from MS Choice. BA 475 was

separated from BA 481 and BA 157 by these amplicons.

A combined phylogenetic analysis (Figure 3.13) showed that the six greens grasses

were indistinguishable, as were the two groups of the fairway grasses and the BAs. Each

of the other grasses had unique amplicons from one or another primer pair.

A genetic similarity matrix (Table 3.7) was produced from the results shown in

Tables 3.4 to 3.6. By this means also greens grasses were clearly similar, as were the

three fairway grasses and BA 481 and BA 157. The largest similarity outside of these

groups was 33% between MS choice and PI 290-868. Individual matrices for each

primer set are in Appendix C.

Discussion

Enrichment for (CA)n repeats in the bermudagrass genome was successful.

Although problems occurred with sequencing we obtained three polymorphic primers

from 96 sequences, similar to numbers from other enriched libraries (Kubik et al. 1999;

Cordiero et al. 2000). PCR amplification using primers developed from the enriched

library usually showed single or double amplification products with no apparent

correspondence to the ploidy level. From the amplicon patterns produced by each primer

pair, phylogenetic and statistical analyses showed distinct groups, and when combined,

showed great dissimilarity and uniquely identified many of the lawn grasses, breeding

lines, and fairway grasses.









Nine out of the eleven primer pairs developed failed to produce amplification

products. It is possible that some of the regions were inaccessible to normal PCR

reactions and would be available if the DNA were first restriction digested as it was in the

building of the enriched library. It is also possible that there were problems in the editing

of the sequence so the primers themselves did not function. Perhaps variations in the

PCR program and PCR reaction mixture would allow amplification to occur. PCR

products produced by BER1 and BER11 from Tifway 419 bermudagrass DNA were

smaller than expected (Table 3.3). In this case it is possible that stem-loop formations

occurred or primers amplified different alleles of the site that was sequenced.

Sequencing of products may explain these size discrepancies.

The generally low level of microsatellite polymorphism within the groups of

fairway grasses and greens grasses is in agreement with previous studies (Busey et al.

2000; Taliaferro 1995; Caetano-Annoles 1998). Taliaferro (1995) proposed that the

restricted background in turfgrass breeding programs may become a serious problem in

agreement with our results revealing identical grouping of BA 157 and BA 481. Midiron,

MS Choice, the lawn grasses, and the PIs are hybrids from different breeding programs

and were different for the SSR markers.

The relationships between mutants and irradiated parents came into question by the

use of SSR and AFLP markers. The documented histories state that the greens grasses

are mutants of Tifgreen, and that Tifeagle is a mutant of Tifway II (Taliaferro 1995;

Zhang et al. 1999). This explains the difficulty in distinguishing the greens grasses by

molecular markers. However there are questions about the origin of Tifeagle. Both in

this work and in the work of Zhang et al. (1999) Tifeagle is more similar to the greens









grasses than it is to its irradiated parent. In a similar case, Tifway 419 and Tifway II are

related (Burton 1985) but have shown similarity to Tifsport, which is a mutant of

irradiated Midiron (Zhang et al. 1999). Both genetic similarity and phylogenetic trees

produced from the gel pictures fail to group these grasses with their irradiated parents.

When DNA samples from other individuals of these cultivars were tested our

observations were sustained (Figure 3.10).

Because Tifeagle came from the irradiation of a fairway grass it would be expected

to appear similar to Tifway II. The same is true of Tifsport. We observed that Tifeagle

was not similar to Tifway II, but was similar to the greens grasses, which would require

at least five independent changes. Tifsport also showed no similarity to Midiron by

genetic similarity (Table 3.7). More probably, mislabeling or contamination of seed

material occurred at some point in the irradiation and selection process.

Although closely related cultivars were not distinguished, this study represents the

first attempt to use microsatellites for the identification ofbermudagrass cultivars. The

(CA)n repeat is the third most common repeat in plant genomes (Morgante and Olivieri

1993), and was chosen to avoid overcrowding during the library enrichment steps.

Sequencing and primer development of fragments from the Tifway 419 library

(Appendix B) are currently being exploited to continue this work. When this library is

exhausted the PI 290-984 library could be exploited or new libraries could attempt

enrichment for (AG)n, which is more common in plants. Sequencing of fragments

enriched for (CA)n revealed that microsatellites contain numerous motifs (Figure 3.3).

As more primer pairs are characterized, and more than one individual from a cultivar is

used, unweighted pair group method with arithmetic mean (UPGMA), Nei's genetic






61


distance, and other statistical analyses may be conducted. A larger number of SSR loci

would separate groups that are unresolved in this study.














Table 3.1 Origins and uses of bermudagrass cultivars evaluated for simple sequence repeats


Type Identification
Fairway MS Choice
Midiron
Tifsport
Tifway II
*Tifway 419
Greens Tifgreen
Tifdwarf
Tifeagle
MS Supreme
Champion
Floradwarf


Ploidy
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)
27 (3n)


Taxon
txd
txd
txd
txd
txd
txd
txd
txd
txd
txd
txd


Source
J. Kranz & W. Philley, MSU
J. Fry, Kansas State University
W. Hanna, USDA-ARS. Tifton, GA
W. Hanna, USDA-ARS. Tifton, GA
W. Hanna, USDA-ARS. Tifton, GA
W. Hanna, USDA-ARS. Tifton, GA
W. Hanna, USDA-ARS. Tifton, GA
W. Hanna, USDA-ARS. Tifton, GA
J. Kranz & W. Philley, MSU, MI
Mike Brown, Coastal Turf, TX
Al Dudeck, Dept. Env. Hort. UF, FL


Reference
Krans et al. 1995
Anderson et al. 1988
Zhang et al. 1999
Burton 1985
Burton 1966b
Hein 1961,
Burton 1966a
Zhang et al. 1999
Krans et al. 1999
www.championdwarf.com
Dudeck,1991


Turf/ GN-1 ND ND Jimmy Doublle, GNI, Flonda www.shark.com/gnturf/introduction
Ball field Floratex 36 (4n) d Al Dudeck, Dept. Env. Hort. UF, FL Dudeck 1991
Tiflawn 36 (4n) d W. Hanna, USDA-ARS. Tifton, GA Hein 1953
Tifton 10 54 (6n) d W. Hanna, USDA-ARS. Tifton, GA Hanna et al. 1990
Breeding BA 481 ND ND B. Scully, EREC-IFAS-UF, FL EREC, Breeding line
Accessions BA 157 ND ND B. Scully, EREC-IFAS-UF, FL EREC, possible 'Wintergreen'
BA 475 ND ND B. Scully, EREC-IFAS-UF, FL EREC, Breeding line
Plant *PI 290-868 36 (4n) d PI collection, Athens, GA 'Royal Cape' South Africa
Introductions PI 290-895 36 (4n) d PI collection, Athens, GA 'Reitz' South Africa
*PI 289-916 27 (3n) txd PI collection, Athens, GA 'Magennis' South Africa
PI 290-900 27 (3n) txd PI collection, Athens, GA 'Damascus' South Africa
*PI 290-894 18 (2n) t PI collection, Athens, GA 'Sekapplos Fine' South Africa
Cynodon dactylon is designated d, C. transvaalensis is designated t, and their hybrid is designated txd. MSU: Mississippi State
University, UF: University of Florida, GNT: Gregg Norman Turf, MS: Mississippi, EREC: Everglades Research and Education
Center, ND: not determined. The four templates used as a subset are designated by (*).










Table 3.2 Cynodon spp.-specific primers developed from the sequencing of fragments in
the enriched DNA library
Repeat
Product and
Primer Sequence Results Program Size Length
BER1 F 5'-GCTATACACAATGTGCAGCGT Polymorphic Kam2nw 256 bp (CA)19
R 5'-GATGATTTCGAGCACAGCAG
BER2 F 5'-ACCAATAGAGGGGGTGTAGC No amplification 171 bp (GT)16
R 5'-CACCCATATTTGTATTGGGATT


BER3 F 5'-AAAGCGAGCCCCCACTA
R 5'-CGAAGAGTTGACCTCCGAA
BER4 F 5'-CCCCATGTGGTTTTCTGTATC
R 5'-TACGGACCCCGACTCAT
BER5 F 5'-TCTCGTGTCAATAAGGCACA
R 5'-ATCATGTAGCAGCTCCCAAC
BER6 F 5'-GAGCCTTCACAAGAAGTGGA
R 5'-ATGACACATGGAGAGGGAAG
BER7 F 5'-CGACCTTTGGCCACTATAAA
R 5'-ATGACACATGGAGAGGGAAG
BER8 F 5'-CTAAAGGCGCCATCTCGTAG
R 5'-GTTCAAACCCAGCTCTCTGC
BER9 F 5'-TCTTAATCCATGATGCCGAT
R 5'-GGAACCTAACACCGTGAGTG
BER10F 5'-CATCAGACAACAGGGTGACA
R 5'-ACATTCATTTGCATTGCCTT
BER11F 5'-TTCCTGAAGTCGATGGGTAA
R 5'-TGACTTGGAGACATGAGCAA


No amplification


No amplification


No amplification


No amplification


No amplification


No amplification


Polymorphic


No amplification


Polymorphic


97 bp (GT)12


279 bp (GT)6


612bp (CA)lo


231 bp (GT)30


246 bp (CA)19
(GT)31
127 bp (GT)11


Kam3nw 293 bp


(CA)22


270 bp (GT)24


Kam3nw 456 bp


(CA)10


Sequence of the eleven primer pairs developed from Tifway 419 genomic DNA (Figure
3.3) for use in this project. Primers that successfully amplified are underlined. Also
indicated are the results of amplification on a subset of 4 genotypes (Table 3.1), the PCR
programs (Table 3.3) that were used to successfully amplify products on bermudagrass
templates, the expected product length, and the length of the largest (CA/GT)n repeat.
Product size indicates the length predicted by Primer 3 (Rozen and Skaletsky 2000).









Table 3.3 Basic step-down PCR programs used to test primer amplification
Step Minutes Temp (degrees C)
1 5 95
2 1 95
3 1 annealing temp
4 1.5 72
5 Repeat steps 2-4 ten times,
lowering annealing temp 1 degree
in each cycle.
6 1 95
7 1 lowest annealing temp
8 1.5 72
9 Repeat steps 6-8 thirty times
using lowest annealing temp.
10 5 72
11 cool down 10

The four step-down annealing temperatures (65-55 o, 60-50 55-45 and 50-40 o) used
for this project were originally developed by Kamps and Okagaki (In prep). They were
identified in this project as KAM1NW: 65-55 o, KAM2NW: 60-50 KAM3NW 55-45 ,
and KAM4NW: 50-40 o respectively.









Table 3.4 BER1 PCR product length on 23
#20027 (Figure3.4)
Grass ID
MS Choice
Midiron
Tifsport
Tifway II
Tifway 419
Tifgreen
Tifdwarf
Tifeagle
MS Supreme
Champion
Floradwarf
GN-1
Floratex
Tiflawn
Tifton 10
BA 481
BA 157
BA 475
PI 290-905
PI 289-916
PI 290-900
PI 290-868
PI 290-895


bermudagrass DNA templates from gel


Product size
105
130


108






105 141
130
103
100


PCR product length (in bp) was determined by the computer program Kodak Digital
Science ID version 2.0.2 (Eastman Kodak Co.) based on 100 bp and 25 bp marker
ladders (Invitrogen) run on each gel. This information was used to produce phylogenetic
tree information (Figure 3.9) and compute genetic similarity (Table 3.7).









Table 3.5 BER9 PCR product length on 23
#20735 (Figure 3.5)
Grass ID
MS Choice
Midiron
Tifsport
Tifway II
Tifway 419
Tifgreen
Tifdwarf
Tifeagle
MS Supreme
Champion
Floradwarf
GN-1
Floratex
Tiflawn
Tifton 10
BA 481
BA 157
BA 475
PI 290-905
PI 289-916
PI 290-900
PI 290-868
PI 290-895


bermudagrass DNA templates from gel

Product size
330
345
298 345
298 345
298 345
307
307
307
307
307
307
316 350
303 330


286 321
316
290


316


PCR product length (in bp) was determined by the computer program Kodak Digital
Science ID version 2.0.2 (Eastman Kodak Co.) based on 100 bp and 25 bp marker
ladders (Invitrogen) run on each gel. This information was used to produce phylogenetic
tree information (Figure 3.10) and compute genetic similarity (Table 3.7).









Table 3.6 BER11 PCR product length on 23 bermudagrass DNA templates from gel
#20763 (Figure 3.6)


Grass ID
MS Choice
Midiron
Tifsport
Tifway II
Tifway 419
Tifgreen
Tifdwarf
Tifeagle
MS Supreme
Champion
Floradwarf
GN-1
Floratex
Tiflawn
Tifton 10
BA 481
BA 157
BA 475
PI 290-905
PI 289-916
PI 290-900
PI 290-868
PI 290-895


Product size
209 236
224
248
251
251
209 254
209 251
209 251
209 254
206 251
209 251
230 251
206 260


222 248
236
236
214


214
230


260
245
257


PCR product length (in bp) was determined by the computer program Kodak Digital
Science ID version 2.0.2 (Eastman Kodak Co.) based on 100 bp and 25 bp marker
ladders (Invitrogen) run on each gel. This information was used to produce phylogenetic
tree information (Figure 3.11) and compute genetic similarity (Table 3.7).










Table 3.7 Genetic similarity matrix including combined data from all BER primer sets


0 a
8


j 0j, ^ o n _
UI 9 1 s -0 T -P -P 9i SP

CDO ri a oi M ao c


1
0 1


0 0 1
0 0 1
0 0 1
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0.25 0.25 0
0.25 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0.33 0 0
0 0 0


MS Choice
Midiron
Tifsport
Tifway II
Tifway419
Tifgreen
Tifdwarf
Tifeagle
MS Supreme
Champion
Floradwarf
GN-1
Floratex
Tiflawn
Tifton 10
BA 481
BA157
BA475
PI 290-905
PI 289-916
PI 290-900
PI 290-868
PI 290-895


0 0 0 0 1
0 0 0 0 0 1
0 0.25 0 0 0 0 1
0 0 0 0 0.25 0 0
0 0 0.25 0.25 0 0 0
0 0 0 0 0 0 0


1 1
0 0 1
0 0 1 1
0 0 1 1 1
0 0 1 1 1 1
0 0 1 1 1 1 1
0 0 1 1 1 1 1 1
0 0 0 0 0 0 0 0 1
0 0 0 0 0 0 0 0 0 1
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0.17 0
0 0 0 0 0 0 0 0 0.17 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0.11 0
0 0 0 0 0 0 0 0 0 0


Genetic similarity was determined by the formula GS=m/(m+n) where GS=genetic similarity, m=matches, and n=fragments not
matching.


1
0 1
0 0












Impoverished
genomic fraction


Enriched
genomic fraction


100% 10% 1% 0.1%



Figure 3.1 Dot-blot hybridization of dilution series of enriched libraries to verify
enrichment for (CA)n repeats. Two rows of dark spots in the enriched
genomic fraction indicate genomic libraries from Tifway 419 and PI 290-894.
The dark spots show that the fluorescent probe has hybridized to the
microsatellite in the sample. Two spots corresponding to the two
impoverished fractions were placed directly above the enriched fraction.
Going from left to right a 10% dilution rate of enriched DNA was tested to
show that sufficient concentration of DNA library had occurred. Inaccuracies
in pipetting account for the darker spots in lower concentrations.




























Figure 3.2 Colony transfer and hybridization of initial bacteria transformation and
hybridization to fluorescent probe to show transformation success rate.
Bacterial colonies were checked for the presence of repeat of interest in
correspondence with blue/white color selection. Most of the colonies that
fluoresced after hybridization were also white, indicating the presence of
insert. Only those colonies that were both white and had fluoresced were
chosen for amplification and sequencing.









71



14441 C1 BER1
4GCCTGGGAAGCTTGTGGATCGCACGAAACGCTATACACAATGTGCAGCGT
ACACACACACACACACACACACCACACACACACACACACAC GCACACACAA
3TGAAATGCAAATATATACTTTCATTAAGAATACACACACAAATTAGCTTA


FTCTCTCTCT


CTCTCTCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT


AGAGGGTGTGTGTGTGTGTGGTGTGTGTGCAGGAAATTATATTCGGAGGTCAACTCTTCGGT

Plate 14441 F7 BER4
AAATGATGCCCCATGTGGTTTTCTGTATCTTATGCTGGTTTGTGTATACTCCTACACATGAATCAGGAGT
ACTTACAAGTTTGCTTGGATAAGTTNTGTTACCAAGAAAGAACCGGACGACGTTTCTACAGTATCACAAA
GAAAAAACAAAGAGGAATGCCTGTCCGTGTGTGTGTGTTGAAGAACCACGGACGTACTCCTGCTCGTA
GCAGAGGCAGAGCACGCAGCAGCAGGCGTTCGCAGCGGAACAGCAGCGACGTGATGAGTCTACTCAGCCC
CAGGCATGGGGTCCGTACCCTCCGACGCCG

Plate 14441 F10 BER5
AGCTATTATCAAATGTCATTTCTCTTATTACGATAAATTATCCATTTCAGCTCCAGCTCTCGTGTCAATA
AGGCACACACCCACTCCCATTCACTGCCCCTCTCTCTCTCTCTCTCTCTCTCTTTCTCTCTCTCTCTCTC
TCTCTCTCTCTCTCTACACACACACACACACACACTCAAAAAGAAGCAATCTTCCCTCTTTCACATAGT


'AATATAT


FTATATATAA


TTCACAAGTTCAAAGAAAGTGCTTGAGCCTTCACAAGAAGTGGATAACAGCCCACGTCCCTCTCTCTCT
CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCATCACTCTCTGTGTGTGTGTGTGTGTGTGTGTGTGTG
TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGAGAGAGAGAGAGAGAGAGAGGGGGGAGCATCCGGGC


ACACACACACACACACACP


GGGTCACAT TCTTCACTCCAACACACACACACACACACACAC
CATATATAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG


TGTGTGTGTGTGTGTGTGTGTGTGTGAGAGAGAGAGAGAGAGAGA


Figure 3.3 Nucleotide sequences of the clones from which species-specific primers were

developed. Underlined sequences show the location of SSR primers, and

bolded sequences indicate all of the potential microsatellites.



















TGTGTGTC


TGTGTGTGTGTGTGTGP
GAGAGAGAGAGAGAGAG


CAGCTCCCAACCACTACGGCGTCCATGTCGTTTTTGTGTGTGTGTGTGTG
GGAGGCTCGATTTGGAAGCAGAGAGCTGGGTTTGAACTGTGGAGTATTGC
TCGGTTTGAAATTCGAATGAAACGCATGGTTTCGTCGCTGCAAAAGGCAA
ACAGTCCACAAGCAAGCAAGCAAAAGAGGGAAGTGCTATATCGGGCACATGCTA
AGCATTTACTTTTTAAATACGCAGGAAAAATTACTTTATTTGGTTCTTCC
ATAAAAAAAAGGTCTAGTATAGTCTTTTCAATATCTTTCTTTTAGCACCA
GATTTTTACCTCTTGTTAATTGACGTGTTGATAATTAGATGGGTGTTGAT
AACCATAGTACAAACTAAATACATAACACTATGAGTACATACAAGCACAA
AATTTACAAACTTATCACATATGCCTCGTTATTGATGGAGCCATCACTAA
TGAATTTAGGGCTCCAAAATAGGATCCCAAGCTTCCCGGGTACCGCAAGG
GGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGGCGT



GGATCTCCTTCTTAATCCATGATGCCGATATGACTCAATAAATAATTC
TCTTGGTCTATCACTAGTTGGTGCAATATTCTTGCACTTACAGTTCAAAC
ACTATATCTAGTAGTTTGGATACTTGGTAAGACAAGAAATATATTTTTTG
CACACACAGCACACACACACACACACACACACACACACACACACAATACA
ATCACTCACGGTGTTAGGTTCCGTGATATTATGCAGTGCGCGAGAAAATC
.CCACACGTGACTGATTGATTCTCACCTCAATATGACAAACTTTACCAAGG
TTATCTTTAAGGTGCTGTTTGGCAATGTGTCAATCCAAAAACTCAGGAGG
GAAAATAGTGAGGT zCACAAATGCAAATAAAGAAACAAAGTGCAGCAAAT
TTATTTACGAGGGTCAACAATGGTTGCATGTGGTGGTTTCTACTAAATAA
ATTAGCTTTCCTGCTAGGGAGTTGGTGTGCAGACTGGAAAGTCTCCACAT
CGGGTACCGCAAGGGCGAATTCCAGCACACTGGCGGCCGTTACTAGTGGA




GTGCGGTTGCGTGGCTATATCTAGCATTCCTTTTGCAACACTTCAGTTTT
CTATTGCGGCAATCGATACTAACTATAACAACATTTTGTATCAATTGCGA
ATCAGACAACAGGGTGACAATAACATCGCCCTAAGTACAACGCACATATC
TTATTGCAATTAAGTGTGTGTTTGTGTGTGTGTGTGTGTGTGTGTGTGTG
CGCTCTCGCGTGTGCTCCAGGAGAGAGAGAGAGAGAGAGAGAGAGA


TCTCTCTCTCTCTCTCACACACACACACACACACAC
TAATTTCCAATTCCTAGTGGAATTTAGTCTAGATTGk
GGTATGGCACTTCTATTCCCTTTATGTATGACTTTG(
TGCTTATGATATATGTTCCTTGTTTGGTTATATATA;


'CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC
TCAAAGAGAAGCAAACATTCTCTCTCTCACATAG
AGCCTTGCTCATGTCTCCAAGTCATAGGAGTATT
CTCTATTTCAATAATATATTTCTTTCATATAGTG


Figure 3.3 Continued












Fairway Greens Lawn Breeding Lines

Eat ELO COO LO
co -r woc o-ooo -
CD~t 0D( M a
.0 a)2 0 CO OD M M o MO COO a)
0 0>'>'Q)CO- ) I- -06 66
oD *U)~~ C(U OC C OCN (N (N (N CN V )C
TT -FFF~hHHH H~O~LLmmma....CCLCCa
C0 '2U 0 O0C- .00C
.0 .............. ................... ... ....*




oTO-


Figure 3.4 BER1 amplification products of replicated PCR reactions using 23
bermudagrass DNA templates, fractionated on agarose gels, showing the
reproduction of fragment length patterns. Names of genotypes are at the top
of each lane, 100 bp and 25 bp marker ladder (Invitrogen) was run on each gel
with bands of significant length indicated. The middle marker lane is 25 bp in
the top gel and 100 bp in the lower gel. The positive control PCR reaction in
gel #20027 was run on Tifway 419 DNA template. Tifway 419 DNA was
used as the positive control in the top gel, but was dropped because it is one of
the 23 DNA templates. The negative controls consist of the entire PCR
reaction mix, including primers, but without template DNA.


WAN


"o


nn,
8












Fairway Greens


L N O E t0


I7 i I i 1 i i i l 2 E


Lawn I Breeding Lines


0


In
0


(Q
mo


w
co on S
0) L) O aO-
CM CM a) ao

M M CO CO
- a) a) ZD
0- E- Z Z -
0.0.ZZ-i~i


Figure 3.5 BER9 amplification products of replicated PCR reactions using 23
bermudagrass DNA templates, fractionated on agarose gels, showing the
reproduction of fragment length patterns. Names of genotypes are at the top
of each lane as well as 100 bp and 25 bp marker ladder (Invitrogen), which
was run on each gel with bands of significant length indicated. The middle
marker lane is 100 bp in the top gel and 25 bp in the lower gel. The negative
controls consist of the entire PCR reaction mix, including primers, but without
template DNA.


Oo
to



Oo
B r
a C













Greens

a)
(D -

3: m E CO

i- 7 2 E:-
"3oz
HF.mcD,


Lawn I Breeding Lines


0


In

rn-

mo


CO Ln a
CO CO a)

M M CO -0
c ol -
CN CN ) C o
- 0 W o
00.0.Q-Z -


Figure 3.6 BER11 amplification products of replicated PCR reactions using 23
bermudagrass DNA templates, fractionated on agarose gels, showing the
reproduction of fragment length patterns. Names of genotypes are at the top
of each lane, 100 bp and 25 bp marker ladder (Invitrogen) was run on each gel
with a band of significant length indicated. The middle marker lane is 100 bp
in the top gel and 25 bp in the lower gel. The negative controls consist of the
entire PCR reaction mix, including primers, but without template DNA.


Fairway

0

o o
C) C O
U) 2 '2
2 2 7


0


0
0D




Qn-

co
S _
o











Fairway I I Greens
a) 0 CO-a
= l-lo
.^o ll -l^-
S~ciE^ ^,0
r i-i i-i F-i-ou-oj


Lawn I Breeding Lines
LOO CD LOL
x CD- C
a3 1 I 6

u- 1- M) 0. O. 0. 0.
O C0 (N C(N(N (N
7-mm ooo


21049 21050


Figure 3.7 Products of BER9 primer pair amplification on bermudagrass DNA
fractionated on polyacrylamide gels showing the amplicon pattern for the
bermudagrass template shown at the top each lane. A 25 bp marker ladder
(Invitrogen) marks the approximate length of DNA fragments. This confirms
reproduction of DNA fragments of a predictable length for this primer set.


-~0

LO U
C- 2T













2n


5 o o), o?,
0S) CO 0T) CT)
Q-0 0) CO) 0

--- L E. .


oo aaa B


3n or
An 4n


a)
E
E t
,o CO
() CO CO *
^ ri i- TT i= O


CO M
CO CO CO CO -
0 ID
EL -L E- z z


CL)
L 7- L ( r in
i z<<<


Figure 3.8 Gel #16088-2, showing amplification ofBER 1 on a different group of DNA
templates. Tifsport is grouped with the fairway grasses and Tifeagle is
grouped with the greens grasses as in the amplification of the other samples of
these grasses. Tifgreen is grouped with the fairway grasses, but this proved to
be an error in the sample of that cultivar. Breeder's seed was obtained to
insure correct identification and grouped Tifgreen appropriately with the
greens grasses.


I I I'l













Tifgreen

Tifdwarf

Tifeagle

MS Supreme

Champion

Floradwarf


Midiron

PI 289-916

GN-1

P 1290-895

PI 290-868


* BA 475

* BA 157

* BA 481

* Tifton 10

STiflawn


PI 290-905

PI 290-900

Floratex

Tifway 419

Tifway II

Tifsport

MS Choice





Figure 3.9 Phylogenetic analysis of BER1 fragment length data (Table 3.5) from gel
#20027 (Figure 3.4) using PAUP (Swofford 2000).


~I












Tifsport

Tifway II

Tifway 419

Midiron

GN-1

PI 289-916

PI 290-895

Tifton 10

PI 290-868

BA 475

BA 157

BA 481

Tiflawn

Tifgreen

Tifdwarf

Tifeagle

MS Supreme

Champion

Floradwarf

PI 290-905

SPI 290-900

Floratex

MS Choice




Figure 3.10 Phylogenetic analysis of BER9 fragment length data (Table 3.6) from gel
#20735 (Figure 3.5) using PAUP (Swofford 2000).












Champion

Floradwarf

MS Supreme

Tifeagle

Tifdwarf

Tifgreen


Tifsport

Tifway II

Tifway 419


GN-1

BA 475


Tifton 10

SPI 289-916

PI 290-868

PI 290-895

Midiron

Tiflawn

PI 290-905

BA 481

BA 157

MS Choice




Figure 3.11 Phylogenetic analysis of BER11 fragment length data (Table 3.7) from gel
#20763 (Figure 3.6) using PAUP (Swofford 2000).








81


Tifgreen

Tifdwarf

Tifeagle

MS Supreme

Champion

Floradwarf

Midiron

Tifton 10

l ---- PI 290-868

PI 289-916

GN-1

PI 290-895

BA 481

BA 157

Tiflawn

BA 475

Tifsport

Tifway II

Tifway 419

PI 290-900

PI 290-905

Floratex

MS Choice




Figure 3.12 Phylogenetic analysis of the collective fragment length data from Tables 3.5-
3.7 (Figures 3.3-3.5) using PAUP (Swofford 2000).














CHAPTER 4
CONCLUSIONS

In summary, primers amplifying three SSR containing products with repeats of >5

were isolated from the genomic library of C. dactylon X C.transvaalensis, var. Tifway

419, one of the 23 varieties tested in this study. Species-specific primers yielded

reliability of reproduction from one PCR reaction to another and consistently amplified

products of an acceptable length. On the whole, cross-taxa primer pairs would require

more optimization in order to exhibit reliable amplification. As other markers are found

it is likely that even tightly linked groups may be distinguishable.

Two varieties, Tifeagle and Tifsport, were not similar to their irradiated parents.

This brings into question the source of their origin and should be the topic of further

research into the relationship of commercial varieties of bermudagrass.

















APPENDIX A
INDIVIDUAL GENETIC SIMILARITY MATRICES FOR CROSS-TAXA PRIMER
SETS ZMADH2N AND LPSSRHO1A10

Table A. 1 ZMADH2N genetic similarity matrix.



Midiron ) 1n 1 f f f -1
--- a o ) --- 0 C-O O-S -O C-> C- C
o. < .^ u a B e a z .- .. Co 'o co o o _,

Tifgreen 0.33 0.33 0 0 0 1,
C' (0 3 = <- ,o5 o 0: < m

MS Choice 1
Midiron 1 1
Tifsport 0.5 0.5 1
Tifwayll 0 0 0 1
Tifway419 0 0 0 0 1
Tifgreen 0.33 0.33 0 0 0 1
Tifdwarf 0.33 0.33 0 0 0 1 1
Tifeagle 0.5 0.5 0 0 0 0.5 0.5 1
MSSupreme 0.33 0.33 0 0 0 1 1 0.5 1

Champion 0.5 0.5 0 0 0 0.5 0.5 1 0.5 1 0.5 1
Floradwarf 0.331 10.33 0 0 0 0.5 03 0.5 1
Tiflawn-1. 5 0 0.5 1 0 0 0 0 0 0 0 0 0 0.5 1
Tifton 10ratex 1 1 0.5 0 0 0.33 0.33 0.5 0.33 0.5 0.33 0.5 1 0.5 1
Tiflawn 0.5 0.5 0 0 0 0 0.5 1 0.5 1 0.5 0 0.5 1
Tift 157 1 1 0.5 0 0 0.33 0.33 0.5 0.33 0.5 0.33 0.5 1 0.5 1 0.5 1
BA475 1 1 0.5 0.5 0 0 0 0.33 0.5 0. 33 0.5 0.33 0.5 1 0.5 0 0.5 1 1
BA 157 1 1 0.5 0 0 0.33 0.33 0.5 0.33 0.5 0.33 0.5 1 0.5 1 0.5 1
BA 475 1 1 0.5 0 0 0.33 0.33 0.5 0.33 0.5 0.33 0.5 1 0.5 1 0.5 1 1
PI290-905 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
PI289-916 0.33 0.33 0 0 0 1 1 0.5 1 0.5 1 0.5 0.33 0 0.33 0.5 0.33 0.33 0 1
PI290-900 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
PI290-868 1 1 0.5 0 0 0.33 0.33 0.5 0.33 0.5 0.33 0.5 1 0.5 1 0.5 1 1 0 0.33 0 1
PI290-895 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
POSITIVE 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1







84


Table A.2 LPSSRHO1A10 genetic similarity matrix.




D -i -0 -n -0 -0 -0 -0

o 0 = ) C C ) x CDo C B -,o 0 -C Co Co
(D C


MS Choice 1
Midiron 0.33 1
Tifsport 0.33 0.33 1
Tifwayll 0 0 0 1
Tifway419 0 0 0 0 1
Tifgreen 0 0.5 0 0 0 1
Tifdwarf 0 0 0 0 0 0 1
Tifeagle 0 0 0 0 0 0 0 1
MS Supreme 0 0.5 0 0 0 1 0 0 1
Champion 0 0.5 0 0 0 1 0 0 1 1
Floradwarf 0 0 0 0 0 0 0 0 0 0 1
GN-1 0 0 0 0 0 0 0 0 0 0 0 1
Floratex 0.5 0.5 0.5 0 0 0 0 0 0 0 0 0 1
Tiflawn 0.5 0 0.33 0 0 0 0 0 0 0 0 0 0 1
Tifton 10 0.25 0.25 0.25 0 0 0 0 0 0 0 0 0 0.33 0.33 1
BA481 0.33 0.33 0.33 0 0 0 0 0 0 0 0 0 0.5 0 0.66 1
BA157 0.33 0.33 0.33 0 0 0 0 0 0 0 0 0 0.5 0 0.66 1 1
BA475 0.5 0.5 0.5 0 0 0 0 0 0 0 0 0 1 00.33 0.5 0.5 1
PI290-905 0.33 0.33 1 0 0 0 0 0 0 0 0 0 0.5 0 0.25 0.33 0.33 0.5 1
PI289-916 0.33 0.33 1 0 0 0 0 0 0 0 0 0 0.5 0 0.25 0.33 0.33 0.5 1 1
PI290-900 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
PI290-868 0 0 0 0 0 0 0 0 0 0 0 0 0 00.33 0.5 0.5 0 0 0 0 1
PI290-895 0.5 0 0 0 0 0 0 0 0 0 0 0 0 1 0.33 0 0 0 0 0 0 0 1
POSITIVE 0 0 0.33 0 0 0 0 0 0 0 0 0 0 0 0.25 0.33 0.33 0 0.33 0.33 0 0 0 1















APPENDIX B
NUCLEOTIDE SEQUENCE FOR FUTURE PRIMER DEVELOPMENT


Plate 20972 A2
Gatctagagttctagacaaagatagtgctactagaacacacacacacacacacacacacacacacacacacacacacacaca
cacacacacagagcggaccaccctagccagggacccaggagggaaatcaaatctttaaccttttgtccgaagcaagctaaggg
aaccacatgaacagtgacacagtgtcatgcatgcatgcctgcgttgcggcctcgacaaagggaagggcagctataataatggc
gccaaaggcacgcagcaatgcagctcaatgcatcgtattatgcgttgcgtgcgcgcctggaaagagagcagagatgcttgggt
cgccacggtacggcacggcaatggctgctgcatgtagacggagacctggcccggccgggcggccggcagggggaggccc
agcctgggccctgaagcgtaggccgtgccaggcc

Plate 20972 A4
Ccgggaagcttgggatctgctttgcgtttttggttcacaagacatcacaaagtaaaaaatcatgcatccaaagtctcaacatcgtgt
tacattctccgccccagcctcatcaaagtaagaggagattgaatttaactaacattaggcagcaacaatgcagacatttgtttttttttt
ttgtgtgtgtgtgtgtgtgtgtgtgtgtcaatttggtgtagaaaacgtgaaagacgcgatgctgctagggattcttgggactgctgtg
atgttttaattttgacataccacaaatacatggacctgccagtagtttgtcagcagtgttgtattaaagatacaagcggttttagccttg
ttaagagttacttcattcgtaagtatagtttaagttgttaaatctcttgtacaaactcttttaataacatttttctcttggtcattcatgtcaac
tttctttctcttgctcattaat

Plate 20972 A5
Gatccgttgtttaccccaagagtatcttgtcccatccgagaaatcatgatagagttagtagtattcactggaacattaaccccccttt
aattggcaaaaaaaggtgtattctttcttagtgcttaaggagtagctaggcatgcaggtgatagaggtgatatgcagcaacacatct
tgtctgctctgtctcttccctctatagaagcttttgttgacttgtgtggaatatggtatgcctgagattcaagctcactatgatacctgat
gagtcaggctcctcaagttgcttgtgcctgcttctttacttgtcatttgctctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtggtgtgt
gtgtgacataatctttatgtaactgcaacactacagaacccaccaattattggaacatgcatgaaagatttcgtgtaattattggcaa
gagatcccaagcttcccgggtgcaatcactagtgcggccgcctgcaggtcgaccatatgggagagctcccaacgcgttggatg
catagcttgagtattctatagtgtcacctaaatagcttggcgtaatcatggtcatagctgtttcctg

Plate 20972 A12
Gatctttcgggtcgaaactactgtgaactagagtgttatagtcaacccccttaagggatgatgccctcagtagcgcaccacacac
acacacacacacacacacacacacacacacacacacagagagagagagagagagagagagagagagagggagagagag
agagcaagctgatacccagaatcttctcagtacaagaccgtgttgcgggtctatccacacgcatacgtagacatgcaatgaaa
gttaatgctctgaaaccactgtaacacctaccatccaaactacgtttttcttctagccataccatatatgggagggtatgcctattcttc
catagcacatcttttatattttcgttctcgttttatgtaaggggttaattcgaaggtgcaatgattggaacaacgatgattttatgcttacc
cttacccactaaacttctgttatgggattaatcacatctctacttcactttattggccttctgagccaaaactactactactacgggttttc
aatgggttgggatgttacaccttattagtgctcaagtattgtgagtatgtccaatatggtgtaaaacatatgaaaacacactgaaaat
atacgcaagtagaatccagta

Figure B.1 Nucleotide sequences of the clones from which future species-specific primers
may be developed. Motif greater than five repeats underlined.











Plate 20972 B2
Acggccagtgaattgtaatacgactcactatagggcgaattgggcccgacgtcgcatgctcccggccgccatggccgcggga
ttgcggtgcccgggaagcttggctctcaagattgtaaaagactgtcccaggcaaagaacctaacagggttgatgaccgcgagg
gagtgactctcacggtctcatcgcatctctataatgatatatcccctccaccccatgcacgcgcgcgcacacacacacacacaca
cacacacacacctgctggccataagacccaacaaaattcacactatcacatataatcattaattatgttaaattccctgcaagtcggt
ataattttaatctacaaagcagagttcaaagttcaaacacaaacaaaaagagagaatgttcaaaatggaaaaactattcatttaggg
acaaaaggaatgcatagatttctacacgtggtattattttccctttgccatatggcaaatgggaatgaaaagcgaataactaagtga
agcaactaatttttttgctattttgcagactgaattctctccccatcccatccatttcccggattcctaaagagagtaagaacaaaaatt
agcacagggaagt

Plate 20972 B4
ggatcaataggcagaaaagaaagagcctagaacttgtgcattattgtactagcacaagataaaagtaacacacacacacacaca
taaaagtcagttctcttatcatcgagagaacaatagaagtcagaaggaagagaggagagaaatacttagttcgtcatgatgcaag
gagctgcatctcaggcccaaggtatatttattggtataatacatgagagaatgtcgcacatattctatccggaaaatatacgacaag
ctctatatcacagagtgtaatatattgttatataaggttaaatcagtcttatgctaacat

Plate 20972 B5
gatcgtctagggatgcttaaacagttagatgttcatgtcgttatgtctcaagacacatatctgaaaaacaaagcgacgagcccctac
ttttgatgcaaatgctgcatatcgctcgagcgagatgaacacacacacacacacacacaccctcgagcaagtgcattacatcacg
gtgatgttcctttgtctccgtccccggccgaccgtgcatgcctctgcatgccaccaaagcctttctacctttaatcagtgcactacga
aaaggagaggccggcattaccgaaaatttgtccaaggattttcgaccgatggtgatgacagaggagagcaaaggctggcttttg
cttccacagggaaatgtttcagagcaggcagccaggctctctcaacttttgcatggtaatgccaacggttagacaaggtgttggcc
tgttggcactacatgactagctatttggttaagtaccacaactgagattgatgagtacggtttattgttggtaatgagctgaaacaata
actgtagcgctagcacgctcagaagaatatatatactaaagaaatatgtgcatggtgtcagtagatgtcacgaaagttcgcagcg
gaacatatttgtagaaa

Plate 20972 B6
ccgggaagcttggatctaagaaaacaaataaatgagctagaaacagttaatttttttattacatcaacagtgttcaattgactacgtttt
tttcattgaagaacaaagatggcaccttttcaacatacgagtgcaagagaactactcctttattggaggtgagtttttgtgtgtgtgtg
tgtgtgtgtgtgtgtgtactgcaaaaagtaggcttatttgttaccttatttgctacgttattgttgcattggtgaatatgcccctaactagg
attttttttgttcctttttct

Plate 20972 B7
gatcccctgtcgactgccgggcgcgcccgggtggtagagcacccggggcccgggtcatgtaaccccctcctccttggtctata
aaaggaggaggcgagcaccgtgtaaaaggacagagaaagaaatatactcagacaagcaggacgtagggtgttacgcatcaa
gcgacccgaacctgggtaaaacacgcgtgttcctacatccgttcgcctgaccacccgagaccgggatccgagctccccataac
ctttcccaagggcggccttgcagacccccatttggaggtgctcacacacacacacacaccgacgacaacactcaaagaaaacg
tttcatgagaagcatgtctgacaaatgtcacgggcccaacggcagaaggcaaaactgctgcatcaccgctgacacatcagggct
gtgacgagggctaaccctgcacttaacgcagtgcatctccgtgtgtctgccagagcgcccaggagcctagttaatactactgtatt
atgaagtgtactagtaatagtaatagcatggcgatactacatatgagctttgaaattcctcactgccaaggtttttttttttgaggcgctt
ctcactgccagtgggtcatcagcgtccgactttgagaggaggaggcttgaagcggcggaagggcctaagagctgtcggtccg


Figure B.1 Continued









Plate 20972 B12
atacgactcactatagggcgaattgggcccgacgtcgcatgctcccggccgccatggccgcgggattattaataatattggtgca
aatggaaagcctgattcaaaaacatgtttagaagttttaagaaattcttcaagaaacggtataagtatttgagtggtgaccaatatgt
acactaacattatacaataacttaacaatgaaaacttaaagtgttaatcctgtagttatttctcttctctctagtgtgaacagatatgcaa
ccctgtttcaaaaacacgtttagaagtttccaatttattgtgaaaaattggcataagtatttgagtatactaacttaatagaatgactag
ctactaatgacaatttaaagtgttcatctttggggtggaaaccaatactataacaagggcacagaaccaatagtgttgtgcgtgtgt
gtgtgtgtgtctggggggctcaagcccccgctccattagtgcatctgctgctcccttcacttgaacactatttacgccatatgcaaa
cttgtcaatgtttaatctgctctgattcaaatacctcaaaggtatacgtattaaatgatgctacctcaaatatcactttttgtttctgccac
cccaagaatcatatagtccttttttctctatcattttgtatattttatttacttgcgttggctaccttataaataataagatatatggatgtggt
catgtaatagtttatcttaccctagtttaagatatataatcgattcctttaccttggtt

Plate 20972 C2
gatccagcgtcacccgaagctgcgacgaccagatctggctaccccctctctggcggcgcggcggcacatcccctcctctcagg
cggtgcagcatgttcaacggtgtggcaggacccatagtggacccaaacacatttttttttgtttattaatcagactagtagcagcgg
gtgatattaccacctgtcgctacaaacatctttaaagcggcgggtaaccgctactgctgctacaactcgcgatttgtagtttcacaa
gcatagcggcggtgtattgctgtagtgtgcaatacaatattttttttctacttctaggagttgttactttcacatatatgtatctcacaaag
tagagtgtatctcttcatacttgaggtttctaaaaaaacatctacatatatttgacttttaaaagtagtatatgaaaatacttttataagaa
atgtgatatatcacatgatgcaaaaatatatgagttttgctcaaagtttttcgttgtttatcactttatatatataaatgttgctatcttattttt
aaatcactatacacgcacacacacacacacacacacacacacacacacacacactcacctttctataagtgtggcgtgagagatt
tattagctcaaactatagaacataggagtatatagtatatacctacatagatgtttatgccaacaggttaaataatt

Plate 20972 C4
gatcagaattattcgcaaattccagcgccatgggttgtaaagtgcttgcttcttctcacccgggcctccgctatcggatattgcgac
atcggcagcgcatggcgaggactccccctacttcaccggttggaaagcctacgacgagaacccctacgactccgtcaccaacc
ccgctggtgtcatccagatgggccttgcagagaaccaggtttgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtt
gtgtgtgtgtgtgtgtgtttgaccttgcacagcactgcaaaatgcacatcatcgtcatgacgaagctgattgattagcgatggccgt
gcaggtgtcctttgatcccaagcttc

Plate 20972 C6
aatacgactcactatagggcgaattgggcccgacgtcgcatgctcccggccgccatggccgcgggattttatctcaatgtttgttt
ctgtgtgtgtgtggtgtgtgtgtgtgtgtgtgtgtgtgtgtggtgtgtgtgtgtgtgtgtaagatgttagacataacccctttgtctgc
agcttatttctcctttattgttggcgcacttggagggtacattttcctattattaagtcgataaagaagaatgtagaggatgccgcttgtt
tctcacttgagattaagacaaaggggataaaaagggttggccgtatgatggccttcgtgcatggtatctttccatatttttggtatcca
ccatccatgtcacgaagaatgatatgaggtcagtctttgctgtcctgtctatgtccttatagcctgcaggttatgtgaagtttgaattat
gttagtgcagtatgtatgctagtaactcgtaagtcatgacagttgtgttaactggtctgcatttatatttatcccgaccaattctctg
attgaccgattctcctgaccatttgtattccacttttatttaaagaatattgtttgctctctaatgctatgtattccacttttattctcccgatt
ctcttgcattaattaccaggctctaatttgtggtctttgtgtcagggtatggttg

Plate 20972 C9
gatcgcccccaaacttgacgcagactagtttcgtgcaatccctagcggcaagaccaagctcgatggtggggcaagacagttttc
gtcctatctcgttggagtccatgacaatgccgaccgccttgtaaaaaggaactcccatgttttagaaaaacaaatggtgtgtgtgtg
tgtgtgtgtgtgtgtgttgtgtgtgtgtgtgtgtgtgtttgtgtgtgtgtgtgtgtgtgtgtgttgtgtgtgtgtgtgcgtgtgtgtgt
gtgtgtgtgtgtgatgattntctactttggtttagtgcgaagtaccatcgaagccgctttcttggaatgaacatgctcatattccttcaa


Figure B.1 Continued









Plate 20972 C9
ataacttcagaaccacataatattgttattatatctatataagttgaataatgttatcttctatatttgcacttccctcacagggattatagg
catacaatccgtacaaaaccagaagtttttttctttgtgaatcatcccaaacctgaagttgtgcacttgagtcatgccaacctactgcg

Plate 20972 C10
gatccttcaccactgcacaatagagaaaggaaaaagggtagaggaaatgtgaagttcacatgctattacccctgggtgcactcct
atcatccgtgttagccagatattttccatatgtcgtaatagtgaaatgtatggacatataactatcaaatgaagacatacatagtaaaa
gagtatagctggttaggttttttgtgatgtgacctacccacttcagtttttattattctacagctctgaatgagtgctagcatttacaagtg
attattttttcattggcaggggaatatattacccattaaatctcctttaggtgtttgtgtgtgtgtgtgtgtgtgtttgtggtggtggtg
gtggtacttgtgcgtatgaccatgtctatactcttcttctgaatgaaatatcaagggatgcggaaaagaactatacaacatgcgttta
ctttaagacatgaccaaatatttcctacgacattcctttgtggtggctaccgagagatgtagatatagagttagcttc

Plate 20972 C12
ggatcaataggcagaaaagaaagagcctagaacttgtgcattattgtactagcacaagataaaagtaacacacacacacacaca
taaaagtcagttctcttatcatcgagagaacaatagaagtcagaaggaagagaggagagaaatacttagttcgtcatgatgcaag
gagctgcatctcaggcccaaggtatatttattggtataatacatgagagaatgtcgcacatattctatccggaaaatatacgacaag
ctctattcacagagtgtaatatattgttatataaggttaaatcagtcttatgctaacat

Plate 20972 Dl
gatctggtatttgcaggcttgcaactatggtggagataaagcaagcagaaatgaagttagaagggtgttaccagtcaacatagca
actgtaattagctttagtttctttacggtcttgtagcgattatgtaagagcagccacactctttcggtcgtttgaacatcaaatcagattt
atagaagcaccgttgcctcgattttatgcttgcccacagtcataacaccgaaacttgaacaaaaaaactgaccccacaaagaata
aaacgagacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacagagagaga
gagttattttcaagcatcgggtgaatgttgagcaccgatgctctgtttttgagttgaagcacctgtggtttcaacggaaagcatcgat
gcttgaacaaaaaagtagagaaaaagttggttaagacacggatgactgtgggtgctctaaaatgttgccgaccgtatagtggaaa
gcatgtttttgcatatctgatatcccttctagcgtgcatattaatgttgcatccatgatttctgatttttttttaaatgatttctaaagttggtta
agtcaatggaatgatgcaaactgactgtgattgatgccatattcatgtagta

Plate 20972 D2
gatcctgtgggaattatcttaattgccttgacgagcaagcagcccaaaggttacttccccaacaaagccttcatccgaaacacaga
aaatcacagttcagattactgtgttggctagcaaagacaggtgcctaaagatgatgagaaaagtaaacacagaattaaacacaca
cacacacacacacacacacacaca cacacacacacacacacacacacaaaacagaataagatgtcttataaagggtacaa
aagaacttaccagccagtcttttgttgaaaatgcaccgaatcagccacatatgagatatgactggcaaacaatcactctgctcaatc
ggcctcctctgtcagtcatcaagatgttctcaaagcctggccatagagttttccgcctttcttttggcatattcatgaactatgctagtt
gtgctctccctgtggcgcaagtagaactcagcaagcaatattctattttcttcaggtaaga

Plate 20972 D5
atacgactcactatagggcgaattgggcccgacgtcgcatgctcccggccgccatggccgcgggattcagcagtaacgtggct
actagagacgattaaatgggacatgatgactgttggcatccgggtatgccatacctttttttttaaaaaaaataatgggtaggagtat
gccatacgctgaccgcattgaaccgaatttgcgtggtcaagcaaaaaataatgggtagtagtagcaggttggtggtggttacttttt
ttttctcgaaaacgcgggacagttaacaggacaagaacccgcaacggcaccaacaatacaaaaaacaccacgacacaacac
acacagcacaaacacatacgaacaacgatgaaacgcacgcacaaatacacgacacacacacaggacaggacaccgtgaggt
taagggttgcttcaagatggcgccttcaagaaggtcatgacacttagacgccaccgccatccgatccggcgagccggatccca

Nucleotide sequences of the clones from which future species-specific primers may be
developed.









Plate 20972 D9
gatcaattgatacccgcctgactcccacctgactctagctgctcaagcaggaacgtacatcacatgcccctcaagaagcttttgttt
tgataatcaaatggttcattcaaattataagacaatgagatttcatattaccgtcagctggaaaggtggtgggtacaaaataattttaa
aagaaaatcatctattacaattcgggtgtactcttatttgcaatggtagggcgaaaacaaaatcctcatatgggtgcatgatacaatt
aattgggtaatttgaaagaaacaaatgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgagagagagagagagagagagagagaga
gagagagagagatgcttctgcctcctgtcgggactgcatgtgagtccagcttcatagtctgtcggctgcatatatataattctctcg
gtttccaccggcagaaaacacgacaggacgcttgaagttgctacaattctaacgacgcctacgtgaagttaattccctaattattgg
tagaagcatactatattttagaatttaaattagggtagaattgttttaaatatttttataatgacagaggtggtta

Plate 20972 D10
ggatcaaacgttgtcactgaactttaaacacgagtgagggggaaattaagcactcaggagtccttgtgtgacggctcaaatcgaa
accaataaagatgatgacgaattgaggatagaaccaacaactgcaattgaagtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtt
gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgttttgcgccttgtgcttggtgaacatgcaatgattcac
accaactgcctcatttcttctgatactctaggatagtcgattgcgatcccaagcttc

Plate 20972 D12
ccgggaagcttgggatcaagcaaaatagtcccaaaactgtaaaccacactctctgggcttactctacctgcagaaacatatagca
gttgatgaagaacattagtatcgtgtgtgcgcgcgcgcgcgtgcgtgggtgtgtgtgtgtgtgcctgtgtgtgtgtgtgtgtgtgtg
tgtgtgtgtgtgtgtgtgtgtgtgtgtctgtgtgtgtgttacacttgtctgtcttaaggnactctggaggtgtgaatgctaagtttgtact
gtaactctttccgtcacggctgttcttcatcagaccgaaacaagatagtctagggttaccatnctacaattcaccatggaaaaaagg
attagtgattctgtaagtgtatgcaaggtctgagaagaaatcaaggnatacacttgaaggctcaccanatcgaagacgactctata
tgcatgcaggtcatgataaagcgctctctctttg

Plate 20972 Fl
atcgtatcttttctagattgtatctttttatatgttttctgttgtaaactccttaacttggcatgtactccaatccatagtcttggtcttctcgta
cttcaagtcgtgcacgaccatgccgggggctgttcccggtcatataaatgaaatgacatcaggttcattcattttggcccgacaaat
aaaaatcaatctagcacgacatcttgcacgcacaggtccagccgtccaggttgtgtttctagagttcatcagaccctgctgaccca
aaggggtgcaccttaattacatagaggtagcatgggcacgatgacaacacacacacacacacacacacacacacacacacac
acacacacacaaaatgatgattcactttagtagaaacgaactggcggcagcaattactctggcagt

Plate 20972 F 11
Aacgacggccagtgaattgtaatacgactcactatagggcgaattgggcccgacgtcgcatgctcccggccgccatggccgc
gggattagaacccgtaacggccccgacaaaacatacaaacccacgacacacacatggacacaaacaaacaacacacacaca
cgacgcacacacacacacacgaacccacatacacatacaccacgaggacactgcgagattaggggttgctcccagatgacgc
ctttaggaaggacatgacgtttagacgccatcgccatct

Plate 20972 F12
tacgactcactatagggcgaattgggcccgacgtcgcatgctcccggccgccatggccgcgggattattttgttggtgtgtgtg
tgtgtgtgtgtgtgtgtgtgtgtgtgttgtggggtggaaggttacttcttggagctaagaaatacatcgacaaccactttgagccatg
gtcctatctttgcagattcaagttttgttaattgggagatgaggttaagagttcaagcgccagaccgcttcaatcccatcgttgagaa
gaaacacatcaaatttcttatcgagggcatccatgacctatggtgtgccatgataactttatgaagactttgatttgcctctgcaagg
aaataaatcaagagtgaaatgcatggtgaacatgcattcctttgtgaagatttgggttgaaagatactctactctttatgatccccca
aaagagatgtaggaactgtatttatgg


Figure B.1 Continued









Plate 20972 G4
gatcgagagcattagtgaggatggcgaagcaacattactaacgctaatatgtggacgtacaaaaggagctcatgcatgtgtcatc
gtgcgataggcttggccccaggtgaacgatttcaacgttgatgacgacttgggtgatttcacagtgtgtgtgtgtgtg ttttcatcc
ctgacattggggtgacatggactagtaatatcgtacagagaggagcaccatgtgtaaatatacaaaatgtaaaataaggtgcgtg
agtatgaggggtacagactaattgcaattataacctaataatcttttgtattgctgaacttaatttcttcttaaataaaacagcggtgctc
ctgcctatttctcgaaaacaaagagggtgtgtcatgttggatagtcatgtgatggataagtcacaattatacgtataacacatcatca
aaatacatggtaatgaccgtgttgatactaagaagcgagcttcatggccacaatttgcagtttcatgaaattaaggatcccaagctt

Plate 20972 H5
gatccatggacatgcacaaggagcattccgctgtccatggtgtcaagggacggcatgaggtaggagcgctcattgaagccagt
gcatactaactcacctgtaatcattgctacttacctgaagcctggccagcacttgttgagttccacagtgaggacgtcgctggcata
tgtcgcgacacagaagtggcctcgcacggagaggcccccgccctgaatgagtggcaggtcctcgtcctcctcttcatgtacagc
cgcagttcatgaactctagtgggacccaatgatcgcccccaaacttgacgcagactagtttcgtgcaatccctagcggcaagacc
aagctcgatggtggggcaagacagtttcatcctatctcgtccgagcccatgacaatgccgaccgccttgtaaaaaggaactccc
atgtttaggaaaaacaaatggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcgtgtgtgtgtgtgtgtgtgtgtgatgattttctactt
cggtttagtgcgaagtaccatctaagctgaataatgtggaatgaacatgctcatattccttcaaataacttcagaaccacataatattt
ttattttatctttataagttgaataatgttatcttctttatttgcacttccctcaca


Figure B.1 Continued