Molecular DNA markers have become widely used in all areas of genetic research. The objectives of this thesis were to develop polymorphic markers in sunflower and utilize the markers for genetic and candidate gene analyses. Amplified fragment length polymorphism (AFLP) markers were used to estimate genetic similarities and assess the genetic diversity among 24 public oilseed inbred lines of sunflower (Helianthus annuus L.). A total of 359 AFLP markers were scored by using six AFLP primer combinations. Genetic similarities ranged from 0.70 to 0.91, polymorphism rate ranged from 7 to 24%, and polymorphic information contents (PICs) ranged from 0.0 to 0.5. Principal coordinate and cluster analysis separated the lines into two groups, B-lines and R-lines, illustrating breeding history, basic heterotic pattern and the widespread practice of using each group to develop new lines. ��9 stearoyl-ACP desaturase (SAD) and ��l2 oleate desaturase (OLD) cDNAs were cloned and sequenced. DNA fragment length polymorphism (DFLP), single strand conformational polymorphism (SSCP), and simple sequence repeat (SSR) markers were developed for the SAD6 and SAD17 genes among eight elite inbred lines. PICs for DFLP, SSCP, and SSR markers were 0.18, 0.37, and 0.30, respectively. Length variants were due to long monomeric repeats, insertions, and deletions in intron sequences, thereby producing polymorphic markers. OLD desaturates 18:1-PC (oleoyl phosphatidylcholine) to 18:2-PC, thereby converting oleic to linoleic acid. It is a likely candidate gene to be causing the high oleic phenotype in mutant sunflower. The expression of OLD7 in developing seeds was greatly reduced in mutant as opposed to wildtype backcross-derived lines. The restriction fragment length polymorphism (RFLP) patterns suggest that OLD7 is duplicated and rearranged in mutant lines. Utilizing sunflower SAD gene sequences and 27 inbred lines, intron fragment length polymorphism (IFLP) markers were developed for automated genotyping. These IFLP markers with ~470 to ~850 bp in length had a mean PIC score of 0.414, versus 0.336 for DFLP markers, and 0.582 for SSCP markers. One and two nucleotide length polymorphisms were reliably detected in PCR fragments up to ~150 and ~680 bp, respectively. / Graduation date: 1998

The level of polymorphisms of many biochemical and DNA markers are low in cultivated sunflower (Helianthus annuus L.). The number of mapped public DNA markers is limited. Molecular markers have not been developed for the most important diseases of sunflower, such as downy mildew. The objectives of this study were (i) to help alleviate the problem of low DNA marker polymorphisms by developing simple sequence repeat (SSR) markers, (ii) to build an integrated AFLP-RFLP linkage map by using previously described probes and newly developed AFLPs, and (iii) to clone and characterize candidate disease resistance genes. Forty-four polymorphic SSR markers were developed from a genomic DNA library. Diversity analysis of these SSRs for variability among 10 public inbred lines produced an average of 1.86 alleles per locus and mean heterozygosity of 0.21. The number of alleles ranged from 1 to 5. Trinucleotide SSRs were less polymorphic than dinucleotide and mononucleotide SSRs. Cluster analysis and multidimensional scaling separated elite inbred lines from wild species. There was more polymorphism in wild species than in elite lines. Three hundred and six AFLP markers were developed using 18 primer combinations. Two sets of previously mapped RFLP markers were tested for segregation in an F��� mapping population. A total of 401 markers were assigned to 17 linkage groups covering 1326 cM with a mean spacing of 3.3 cM between adjacent markers. The RFLP markers were well spaced and well distributed throughout the genome. Some linkage groups are sparsely populated with common markers. There were two gaps of 30 or more cM in two linkage groups. We cloned candidate disease resistance genes for downy mildew resistance based on sequence homology among resistance genes in other species. Eleven unique nucleotide binding sequence (NBS) containing clones were isolated and showed similarity to the corresponding domains of cloned disease resistance genes in other plant species. Seven clones mapped to four linkage groups and identified nine loci. A cleaved amplified polymorphic sequence (CAPS) marker that was 3.7 cM from the Pl1 resistance gene was developed by analysis of NILs. This CAPS marker should facilitate marker-assisted selection in sunflower. / Graduation date: 1999

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.