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Abstract
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 F2 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 Pl7 resistance gene was developed by analysis of NILs. This CAPS marker should facilitate marker-assisted selection in sunflower.
