Monoploid potato genotypes represent plant material that is free from the "genetic load" of lethal and severely deleterious alleles normally present in the highly heterozygous cultivated potato species. Field evaluations enabled the identification of agronomically superior monoploid potato genotypes from a population of more than 100 anther-derived monoploids. Chemical fusion and electrofusion between pairs selected from 31 superior monoploids resulted in the production of three different groups of intermonoploid somatic hybrids. The hybridity of somatic hybrid plants and calluses was confirmed through PCR-based amplification of simple sequence repeat (SSR) sequences in the potato genome. Polymorphic SSR loci between the monoploid parents of a particular group of somatic hybrids were used to separate true somatic hybrids (heterozygous at the loci) from parental somaclones regenerating from unfused protoplasts (homozygous for one parental band at the loci). One group of somatic hybrids (SH1, SH2 and SH2B) was of particular interest because it resulted from the fusion of a S. phureja monoploid to a high acetylleptinidine-producing monoploid derived from an F1 hybrid between S. chacoense and S. phureja. The leptine acetylleptinidine (ALD) is produced only by some accessions of S. chacoense Bitt. and provides resistance to feeding by the Colorado potato beetle (Leptinotarsa decemlineata Say) when present in sufficient concentrations. The somatic hybrids produced moderate levels of ALD in leaves and stems (roughly 60% that of a high ALD-producing S. chacoense clone). Pollinations of SH1, SH2 and SH2B by several diploid and tetraploid potato clones resulted in three fruit on SH2, one fruit on SH2B and no fruit on SH1. Two resulting progeny populations of SH2 [SH2A = SH2 × S. andigena 8-1 (4x); SH2P = SH2 × S. phureja 66AP11-53 (2x)] expressed higher fertility than the original somatic hybrids and were sexually crossed as both male and female parents to S. tuberosum cv. Atlantic. All of the SH2 progeny populations expressed acetylleptinidines, albeit at lower levels than the SH2 somatic hybrid, providing strong evidence that the genes controlling acetylleptinidine production are dominant. Variation for ALD expression in the SH2 progeny indicated one or a few genes with additive effect controlling the ALD trait. In addition, the choice of male parent in sexual crosses to SH2 affected subsequent ALD expression in progeny populations. The SH2 progeny represent an important first step towards transferring acetylleptinidines to cultivated potato. SH1, SH2 and SH2B appeared to be negatively affected by an unusually high ploidy (hexaploid, 6x). Field-grown plants produced many tubers (mean = 35) of low weight (mean = 10.4 g) and were stunted in appearance. Anther culture of SH2 yielded triploid regenerants (3x). These regenerants may be more phenotypically normal than the original somatic hybrids because of lower ploidy. Segregation of SSR alleles in the triploid anther culture regenerants provided evidence that the hexaploid somatic hybrid SH2 genome is comprised of four homologous genomes of CP2-103 (the high leptine-producing monoploid) and two homologous genomes of 13-14 203 (the S. phureja monoploid). / Master of Science

Submitted to the faculty of the School of Informatics in partial fulfillment of the requirements for the degree Master of Science in Bioinformatics in the School of Informatics,Indiana University July, 2004 / DNA markers have revolutionized the field of genetics by increasing the pace of genetic analysis. Simple sequence repeats (SSRs) are repetitions of nucleotide motifs of 1 to 5 bases and are currently the markers of choice in many plant and animal genomes due to their abundant distribution in the genomes, hypervariable nature and suitability for high-throughput analysis. While SSRs, once developed, are extremely valuable, their development is time consuming, laborious and expensive. Sequences from many genomes are continuously made freely available in the public databases and mining of these sources using computational approaches permits rapid and economical marker development. Expressed sequence tags (ESTs) are ideal candidates for mining SSRs not only because of their availability in large numbers but also due to the fact that they represent expressed genes. Large scale SSR mining efforts in plants to date focused on monocotyledonous plants. In this project, an efficient SSR identification tool was developed and used to mine SSRs from more than 53 dicotyledonous species. A total of 92,648 non-redundant ESTs or 6.0% of the 1.54 million dicotyledonous ESTs investigated in this study were found to contain SSRs. The frequency of non-redundant-ESTs containing SSRs among the species investigated ranged from 2.65% to 16.82%. More than 80% of the non-redundant ESTs having SSRs contained a single SSR repeat while others contained 2 or more SSRs. An extensive analysis of the occurrence and frequencies of various SSR types revealed that the A/T mononucleotide, AG/GA/CT/TC dinucleotide, AAG/AGA/GAA/CTT/TTC/TCT trinucleotide and TTTA and TTAA tetranucleotide repeats are the most abundant in dicotyledonous species. In addition, an analysis of the number of repeats across species revealed that majority of the mononucleotide SSRs contained 15-25 repeats while majority of the di- and tri-nucleotide SSRs contained 5-10 repeats. By providing valuable information on the abundance of SSRs in ESTs of a large number of dicotyledonous species, this study demonstrates the potential of computational mining approach for rapid discovery of SSRs towards the development of markers for genetic analysis and related applications.

We investigated the transferability of 20 Fragaria vesca microsatellite primer pairs to 13 Fragaria vesca accessions, six Fragaria species and ten commercially important species in Rosaceae. Genetic diversity studies were carried among 16 diploid Fragaria accessions using these polymorphic microsatellites. The average number of alleles amplified for a polymorphic locus was 4.7 with maximum being 8.0 and minimum being 3.0. Observed heterozygosity ranged from 0.00 to 0.84 with an average of 0.28. Expected heterozygosity ranged from 0.33 to 0.91 with an average of 0.76. Power of discrimination varied from 0.43 to 0.92 with an average of 0.78. Transferability of microsatellites to F. orientalis (4x) and F. Ã ananassa (8x) was high, i.e., 18 (90%) primers produced amplicons. Cross species amplification within Rosaceae using these primers showed limited transference. Four microsatellites showed amplification for different species in Rosaceae. Products generated by UDF-003 and UDF-018 primers were sequenced. Sequencing results for UDF-018 showed that three species, i.e., Pyrus calleryana, Prunus persica and Rubus idaeus contained the expected microsatellite whereas another four, i.e., Cotoneaster salicifolius, Rosa rugosa, Amelanchier arborea and Potentilla fruticosa had conserved regions resulting in generation of amplicons. For UDF 003, Spirea xbumalda and Prunus persica did not contain a microsatellite although there was some sequence similarity with Fragaria. Size homoplasy, i.e., alleles of identical size with different numbers of repeats within the SSR was observed among Fragaria and Rosaceae species for primer UDF-018, suggesting a need for caution when interpreting SSR variation from band migration in the absence of DNA sequences. / Master of Science

Microsatellites are strings of short DNA motifs (≤6 bp) repeated in tandem across genomes of both prokaryotes and eukaryotes. In 20 years, they became popular genetic markers, successfully employed in the field of genetic mapping and gene hunting, as well as to address various biological questions at the individual, family, population and species level. However, evolutionary and demographic inferences from microsatellite polymorphism are hampered by controversy and ambiguity in the mutational processes of microsatellite sequences. Drawing on new data from genome projects, I review in Chapter 1 the concept of a microsatellite life cycle, which hypothesizes that microsatellites follow a life cycle from birth, through expansion, contraction, death and potentially resurrection. To document and understand this integrative concept of evolution, which could help improve current models of microsatellite evolution, there is an implicit need to study the evolution of microsatellites above the species level. A prerequisite of such comparative studies is therefore to find microsatellite loci that are conserved between different species. The near or full completion of many vertebrate genomes and their alignment against one another offer the ultimate approach to find genomic elements conserved over a large evolutionary scale. In Chapter 2, I present a new comprehensive method to find conserved microsatellites in whole genomes. Using the multiple-alignment of the human genome against those of 11 mammalian and five non-mammalian vertebrates, I examine the genomewide conservation of microsatellites, and challenge the general assumption that microsatellites are too labile to be maintained in distant species. In Chapter 3, I present similar results using the alignment of the newly sequenced platypus genome against those of three mammals, the chicken and the lizard, and incorporate these data into the framework created by the 17-genome analysis. This enlarged dataset was ground for attempting to reconstruct a vertebrate phylogeny from the presence/absence of microsatellites in the different genomes. Maximum parsimony analyses resulted in a tree much similar to that of the current view of the vertebrate phylogeny, while Bayesian analyses showed some discrepancies. This work opens a way for novel theoretical developments regarding the inference of ancestral states of microsatellites. In Chapter 4, I show how knowledge on conserved microsatellite sites can help for the development of a set of comparative primers useful across the Mammalia; implementing a similar protocol, nine conserved dinucleotide repeats were genotyped in 20 unrelated individuals of 18 species (nine sister species) encompassing the mammalian phylogeny, including marsupials and monotremes, and four microsatellites were sequenced in 4 individuals per species. My results emphasize conserved microsatellites as a new resource for genetic mapping and population studies. Finally, in Chapter 5, I recount the unexpected extent of structural change among mammalian orthologous microsatellites, including change of complexity, motif replacement and overall length variability. Altogether, these findings provide a comprehensive framework that may help in many areas of research, including molecular ecology, genome mapping, population genetics, and genome and microsatellite evolution.

<em>Cladophora</em> is a widespread freshwater filamentous cholorophyte genus and is frequently observed in eutrophic waters where it can produce large nuisance blooms. These blooms can have direct impacts on water intake for power generation, irrigation canals and can be aesthetically unpleasant. Much of the ecological and physiological studies on <em>Cladophora</em> have assumed that the populations of this genus in North America belong to the species <em>Cladophora glomerata</em>. However, this has never been tested despite that it is well documented that identifying freshwater <em>Cladophora</em> to the species level is difficult due morphological variability under different ecological conditions. In addition, the species epithets for freshwater <em>Cladophora</em> are based on European collections and it is not clear if these should be applied to North America. This study examines approximately 40 collections of <em>Cladophora</em> from the Laurentian Great Lakes and 43 from various locations in North America ranging from the Northwest Territories to Puerto Rico. Initially we determined the nucleotide sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal cistron and observed sequence divergence to be low (0-3%), demonstrating an inability for this marker to resolve species delineation as divergence of this region was low. Amplification of the inter-simple sequence repeat (ISSR) regions were used to analyze microsatellite motif frequency throughout the genome to evaluate the biogeography relationships, including diversity, of freshwater <em>Cladophora</em> sp. five different primers were used on 70 individuals. UPGMA analyses of the presence/absence of bands demonstrate that each of the Great Lake populations separate into groups according to the Lake they were initially sampled from. However, collections from North America are highly variable and do not form well supported biogeographic clades. In addition, these collections appear to be distinct from type cultures of freshwater <em>Cladophora</em> from Europe. Supplementary morphological analysis using suggested taxonomically valid criterion (length and diameter of main axis, ultimate branch, and apical cell) none were able to differentiate Great Lake populations.

<em>Cladophora</em> is a widespread freshwater filamentous cholorophyte genus and is frequently observed in eutrophic waters where it can produce large nuisance blooms. These blooms can have direct impacts on water intake for power generation, irrigation canals and can be aesthetically unpleasant. Much of the ecological and physiological studies on <em>Cladophora</em> have assumed that the populations of this genus in North America belong to the species <em>Cladophora glomerata</em>. However, this has never been tested despite that it is well documented that identifying freshwater <em>Cladophora</em> to the species level is difficult due morphological variability under different ecological conditions. In addition, the species epithets for freshwater <em>Cladophora</em> are based on European collections and it is not clear if these should be applied to North America. This study examines approximately 40 collections of <em>Cladophora</em> from the Laurentian Great Lakes and 43 from various locations in North America ranging from the Northwest Territories to Puerto Rico. Initially we determined the nucleotide sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal cistron and observed sequence divergence to be low (0-3%), demonstrating an inability for this marker to resolve species delineation as divergence of this region was low. Amplification of the inter-simple sequence repeat (ISSR) regions were used to analyze microsatellite motif frequency throughout the genome to evaluate the biogeography relationships, including diversity, of freshwater <em>Cladophora</em> sp. five different primers were used on 70 individuals. UPGMA analyses of the presence/absence of bands demonstrate that each of the Great Lake populations separate into groups according to the Lake they were initially sampled from. However, collections from North America are highly variable and do not form well supported biogeographic clades. In addition, these collections appear to be distinct from type cultures of freshwater <em>Cladophora</em> from Europe. Supplementary morphological analysis using suggested taxonomically valid criterion (length and diameter of main axis, ultimate branch, and apical cell) none were able to differentiate Great Lake populations.

One hundred fifty-five peach (Prunus persica) cultivars, from Asia, Brazil, Europe, and the USA, were examined using eleven Simple Sequence Repeats (SSRs) to study the genetic relationships among low chill as compared to high chill peach germplasm. Data was analyzed by NTSYSpc to form a similarity matrix using Nei and Li’s Dice similarity coefficient. This similarity matrix was then subjected to a cluster analysis and a dendrogram was constructed using the UPGMA (Unweighted Pair-Group Method, Arithmetic Mean) method. A wide range of diversity was detected, from 0.33 coefficient of similarity amongst the Thai peaches to 0.97 between two Brazilian peaches. The most distant clusters were the low chill peaches from Thailand and Taiwan and the local cultivars (both fruit and ornamental types) from China. Among the improved germplasm, there were distinct clusters for the Chinese/Japanese cultivars, three clusters for the Brazilian cultivars and one for the cultivars from the USA and Europe. The Brazilian materials clustered according to breeding programs in São Paulo and Pelotas reflecting the different sets of local cultivars used in the breeding efforts. The largest group investigated was the European/USA peaches. This group subdivided into three distinct clusters, with a general clustering of the low chill germplasm. The low chill accessions from Asia were genetically distant from the improved low chill peaches from the USA or Brazil. The low chill peaches from the Americas were more closely related to the high chill peaches developed in the USA and China/Japan due to the introgression of this germplasm into a low chill background.

Brassica carinata is being actively pursued as a new industrial oil crop platform for the Canadian Prairies. A genetic assessment of B. carinata was performed to elucidate its evolutionary origins and create a genetic map to assist in locating genes and traits of interest that would help in marker-assisted breeding. First, genetic analysis using simple sequence repeat (SSR) markers, previously tested on B. juncea and B. napus, was performed, to examine the genetic diversity of 37 B. carinata lines. SSR analysis revealed world accessions were more diverse than lines conditioned to grow in the prairies. Diversity analysis revealed that the parental lines of a double haploid (DH) population, 179 and 345, obtained from the John Innes Centre (JIC), were among the more genetically diverse lines, supporting the use of this population for linkage mapping. Genetic markers created from 3’ targeted SNP discovery between 179 and 345, were tested on the DH population resulting in the generation of a B. carinata genetic linkage map essentially with no prior sequence data knowledge. This genetic map contained 341 SNP and 86 SSR loci identifying eight linkage groups belonging to the B genome, nine belonging to the C genome and two unidentified groups spanning 2041 cM. Comparative mapping of polymorphic markers identified in the amphidiploid B. carinata indicated the orientation of B and C genomes coincide with that of other Brassica species, and the two genomes have remained essentially unaltered, with no major chromosomal rearrangements since the formation of B. carinata. A lesser number of polymorphic markers were detected in the C genome, which suggested the B genome is more genetically diverse in B. carinata. Limited field trials of the 179 x 345 DH population were performed during the 2011 and 2012 growing seasons. Preliminary quantitative trait loci (QTLs) for agronomic traits including flowering time (FT), plant height (PH), and seed quality were identified.

Microsatellites are strings of short DNA motifs (≤6 bp) repeated in tandem across genomes of both prokaryotes and eukaryotes. In 20 years, they became popular genetic markers, successfully employed in the field of genetic mapping and gene hunting, as well as to address various biological questions at the individual, family, population and species level. However, evolutionary and demographic inferences from microsatellite polymorphism are hampered by controversy and ambiguity in the mutational processes of microsatellite sequences. Drawing on new data from genome projects, I review in Chapter 1 the concept of a microsatellite life cycle, which hypothesizes that microsatellites follow a life cycle from birth, through expansion, contraction, death and potentially resurrection. To document and understand this integrative concept of evolution, which could help improve current models of microsatellite evolution, there is an implicit need to study the evolution of microsatellites above the species level. A prerequisite of such comparative studies is therefore to find microsatellite loci that are conserved between different species. The near or full completion of many vertebrate genomes and their alignment against one another offer the ultimate approach to find genomic elements conserved over a large evolutionary scale. In Chapter 2, I present a new comprehensive method to find conserved microsatellites in whole genomes. Using the multiple-alignment of the human genome against those of 11 mammalian and five non-mammalian vertebrates, I examine the genomewide conservation of microsatellites, and challenge the general assumption that microsatellites are too labile to be maintained in distant species. In Chapter 3, I present similar results using the alignment of the newly sequenced platypus genome against those of three mammals, the chicken and the lizard, and incorporate these data into the framework created by the 17-genome analysis. This enlarged dataset was ground for attempting to reconstruct a vertebrate phylogeny from the presence/absence of microsatellites in the different genomes. Maximum parsimony analyses resulted in a tree much similar to that of the current view of the vertebrate phylogeny, while Bayesian analyses showed some discrepancies. This work opens a way for novel theoretical developments regarding the inference of ancestral states of microsatellites. In Chapter 4, I show how knowledge on conserved microsatellite sites can help for the development of a set of comparative primers useful across the Mammalia; implementing a similar protocol, nine conserved dinucleotide repeats were genotyped in 20 unrelated individuals of 18 species (nine sister species) encompassing the mammalian phylogeny, including marsupials and monotremes, and four microsatellites were sequenced in 4 individuals per species. My results emphasize conserved microsatellites as a new resource for genetic mapping and population studies. Finally, in Chapter 5, I recount the unexpected extent of structural change among mammalian orthologous microsatellites, including change of complexity, motif replacement and overall length variability. Altogether, these findings provide a comprehensive framework that may help in many areas of research, including molecular ecology, genome mapping, population genetics, and genome and microsatellite evolution.

Gopher tortoise (Gopherus polyphemus) population sizes have drastically declined in the past 100 years. Much of this decline has been attributed to past human predation, to habitat loss from human development, and potentially to the recently discovered upper respiratory tract disease. An understanding of the genetic structure among populations is critical for the long-term success of relocation and other management strategies. This research focuses on the development of a suite of genetic markers and the answers they provided to questions concerning present day population genetics and its use in management. In addition, this study provides inference on historical refugia and dispersal patterns of the gopher tortoise through the Pleistocene. Nine microsatellite loci were identified, optimized, and characterized from a G. polyphemus microsatellite-enriched DNA library. These loci are applicable for population level analysis along with parentage analysis in all Gopherus species. In addition, a few of the loci also work in other Testudinies. Application of these markers to eighteen Florida and two Georgia populations of gopher tortoises reveal considerable amount of genetic diversity within the species and substantial genetic subdivision among populations, especially in the northern part of the Florida peninsula and southern Georgia. Admixture and genetic homogenization in central Florida may be attributed to past human mitigation events as much of this area has been substantially developed. These data indicate a more conservative approach to relocation is necessary if the goal is to maintain the genetic distinctiveness of these areas. Lastly, these genetic data, in conjunction with historical geological, climactic, and fossil records, were used to identify gopher tortoise refugia, and dispersal patterns during the Pleistocene. Within Florida, four major genetic assemblages were determined that correspond to four Pleistocene ridges that would have been present at high sea levels: Lake Wales Ridge, Brooksville Ridge, Southern Atlantic Coastal Ridge, and Mt. Dora Ridge. In addition, these data indicate that tortoises that dispersed into southeastern Florida after the fall in sea level were most closely related to tortoises from the Brooksville Ridge. Likewise, tortoises in northwestern Florida and southern Georgia were most closely related to tortoises from the Mt. Dora Ridge.