The cultivated sunflower (Helianthus annuus L., x=17) is one of the most important annual oilseed crops in the world. There are very few publicly shared sequence-based DNA markers and genetic maps in sunflower, even though molecular DNA markers and genetic maps have become widely used in all areas of genetic research and breeding in plant species. The objectives of this study were to develop sequence-based molecular markers and utilize the markers for genetic analyses and constructing maps in the cultivated sunflower. A total of 131 functional simple sequence repeat (SSR) markers were developed for 16 elite inbred lines using a small insert genomic library enriched for short simple sequence repeats. The polymorphism information content (PIC) estimated from 74 polymorphic SSR markers ranged from 0.0 to 0.93 with mean value of 0.55. Tetranucleotide repeats were significantly more polymorphic than dinucleotide and trinucleotide repeats, and no obvious correlation was found between repeat numbers and PIC scores. Genetic distance among 16 inbred lines, estimated from 74 polymorphic SSR markers ranged 0.175 to 0.543. Principal coordinate and cluster analyses of the genetic distance matrix well explained the difference between oilseed lines and confectionery lines, and sterility maintainer lines and fertility restorer lines. A total of 1,090 SSR markers were screened for polymorphism between the parents of two mapping populations. The two genetic maps were constructed by genotyping 94 recombinant inbred lines from a cross between PHA and PHB (276 SSR loci covering 1377.4 cM with mean distance of 4.99 cM), and 94 F��� progeny from a cross between HA370 and HA372 (122 SSR loci integrated into the existing RFLP framework map covering 1348.0 cM with mean distance of 6.77 cM). Ninety-three percent of the SSR markers were mapped to single loci and 56.5% of the loci were co-dominant. Clustering of SSR loci was observed near centromeric regions and most of the distorted loci were mapped to centromeric or distal regions. A concerted effort to develop SSR markers and generate highresolution SSR maps will enhance future fingerprinting analyses, fine-scale genome analyses and molecular breeding in the cultivated sunflower. / Graduation date: 2002

Graduation date: 2005

Single nucleotide polymorphisms (SNPs) are the most common DNA polymorphisms in plant and animal genomes. SNPs were identified in the allele sequences of up to 12 sunflower (Helianthus annuus L.) genotypes for a genome-wide sample of 81 loci originally mapped using restriction fragment length polymorphism (RFLP) markers. The RFLP loci anchor a dense RFLP linkage map and second-generation linkage maps constructed using simple sequence repeats and other high-throughput DNA markers. The goal of this study was to develop high-throughput SNP markers for the cDNA-RFLP loci for linkage mapping, diversity analysis, and molecular breeding and genomics research. The specific objectives were to: (i) develop FP-TDI or fluorescent primer-extension SNP assays for the cDNA-RFLP loci; (ii) validate the SNP markers by screening 12 sequenced (reference) genotypes; and (iii) assess the utility and polymorphism rate of the SNP markers across a genetically diverse panel of unsequenced wild and domesticated sunflower genotypes. SNP markers were successfully developed for 44 of the targeted 49 cDNA-RFLP loci. Thirty-one of the SNP markers were further selected for a diversity analysis across a genetically diverse panel of wild and domesticated sunflower genotypes. The mean heterozygosity (H) score for the loci was 0.41, which nears the maximum H score of 0.5 for biallelic markers. SNPs in this study exhibited polymorphism rates close to those of RFLP and SSR markers in inbred sunflower lines. / Graduation date: 2004

Genotype by environment interaction (GxE) is the tendency of the phenotypic performance of two or more plant genotypes in one environment to not be predictive of their relative performance in another environment. To discover the importance of GxE in this region, a large set of USDA and commercial hybrids were tested in the regions of practical significance to sunflower production in order to produce recommendations regarding mega-environments for yield and oil. Rank changes for oil content occurred among hybrids and two common factors accounted for 68.6% of the total GxE variation. Breeding programs testing pre-commercial hybrids in multiple environments for oil content could be beneficial. Yield covariates for lodging, bird damage, and disease were significant but occurred in different locations with variable severity each year making it difficult to divide the growing region into mega-environments for yield.

Sunflower (Helianthus annuus) consists of high levels of polyunsaturated fatty acids, making its oil susceptible to oxidation. Tocopherols can retard or prevented oxidation. The aim of this study is to determine the phenotypic tocopherol (vitamin E) composition and genetic diversity of the biosynthetic pathway genes. Seeds were characterized for fatty acid and tocopherol content. A positive correlation was found between oleic acid, γ (r =0.17) and δ (r =0.23) tocopherol but none between linoleic acid and all four tocopherol derivatives. Vitamin E gene homologues were identified and a concomitant pathway constructed, with genes of interest sequenced to determine their genetic variation. A sunflower gene database was developed for these genes and used to obtain 489 SNPs and 145 indels from the accessions evaluated. Only 139 of these SNPs were located in the exon regions of the gene candidates. These exon-based SNPs may influence tocopherol flow through possible enzyme structural modifications / School of Agriculture and Life Sciences / M. Sc. (Life Sciences)

This study compared DNA based SSR markers with total seed protein markers, used to evaluate genetic diversity of sunflower. The multiplex-ability, cost effectiveness and applicability of microsatellites as molecular markers for a genetic diversity study were investigated and evaluated based on pedigree data of the sunflower germplasm. A solution for oil and fat interference in ultrathin iso-electric focusing gels was investigated, in order to make imaging and interpretation easier and clearer. Total protein analysis was utilized for the determination of genetic diversity on the same inbred material used for the DNA analysis. Finally a correlation is made between the data obtained on DNA vs Protein compared with phenotype and expected pedigree data. A set of 73 SSR markers with known mapped positions were utilized to determine genetic similarity in a group of sunflower inbred lines. Cluster analysis of genetic similarity revealed an excellent correlation with the breeding background and source information obtained from breeders on all inbred lines used in this study. Cluster analysis gave a clear differentiation between B and R-lines, showing clearly defined heterotic groups of the proprietary set of inbred lines. The most outstanding single-locus SSR markers in the set used for this study were identified and used as a core set. Multiplex assays were designed and optimized for the most cost and time effective method for rapid variety identification. The selected markers produced robust PCR products, amplified a single locus each, were polymorphic among the elite inbred lines and supplied a good, genome-wide framework of completely co-dominant, single-locus DNA markers for molecular breeding. The use of a fluorescent-tailed primer technique resulted in a considerable cost saving. Furthermore, the SSR markers can be multiplexed through optimization, in order to avoid undesirable primer-primer interactions and non-specific amplification. First stage iso-electric focusing of total protein extracts were used to analyze sunflower looking at genetic purity and genetic variety verification on diverse sunflower germplasm. Severe visual interference was visible on most seed storage protein extracts of sunflower. This interference was visible as a distortion in the gel matrix on the anodal end of the gel, and caused important proteins to denature in the presence of heightened field strength and the absence of a uniform matrix. Adjustment of the extraction solutions removed this interference. Total protein profiles were generated with the use ultrathin layer iso-electric focusing (UTLIEF) to assess the level of genetic diversity on the same set of sunflower lines used for the SSR analysis. Finally, the genetic diversity of the sunflower germplasm was analysed by comparing proteomic, genomic and pedigree data from the same germplasm. A total of 295 alleles were amplified with a set of 73 SSR markers with known mapped positions. These were utilized to determine the genetic relatedness of a group of B-lines and R-lines of sunflower. In parallel, a total of 68 protein bands were visualized using protein samples of two types of seed storage proteins derived from exactly the same sunflower lines. Cluster analysis clearly differentiated between the B-lines and R-lines, identifying defined heterotic groups of this proprietary set of lines. The comparison of DNA and protein data for the application of genetic diversity studies is analysed, as well as the general comparison on the use of the two different molecules as markers. / Thesis (Ph.D)-University of KwaZulu-Natal, Pietermaritzburg, 2008.