Genetic diversity of proprietary inbred lines of sunflower, determined by mapped SSR markers and total protein analysis.

Abstract

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.