Linkage Disequilibrium Mapping Of A Verticillium Dahliae Resistance Gene And Construction Of A SSH Library In Cotton

Cotton Verticillium wilt, a disease of cotton caused by Verticillium dahliae, is a kind of worldwide spread disease that is difficult to be controlled in cotton production. Development of cotton resistant cultivars can provide an effective and economical method for controlling Verticillium wilt. Therefore, researches on deeply understanding of expression rules about resistance gene and the molecular mechanism about interactions between Verticillium dahliae and the host would provide theoretical foundation for developing resistant cultivars.The objectives of the research were attempted to screen molecular marker and candidate genes associated with resistance gene. (1) thirty-four cotton cultivars coming from different sources were selected to detect the SSR molecular marker linked with Verticillium wilt resistance gene, including twenty-two resist cultivars and twelve susceptible cultivars. The resistance of the cultivars was determined in the field disease nursery, and the result verifies the original resistance. Five hundred pairs of SSR primers covering the whole cotton genome were selected to genotype all cultivars. Out of these primers, three hundred and eighteen pairs of SSR primers showed polymorphic between the cultivars. By cluster analysis with regional fuzzy clustering algorithm, the tested varieties could be divided into two groups, and the result is related to resistance of every cultivar. Then -linkage disequilibrium mapping method was used to test an association between SSR markers and Verticillium resistance gene. with SAS software, the result showed that fourteen markers are significantly related to resistance gene at P<0.001 level. (2) To obtain resistant-related candidate genes of Verticillium wilt from upland cotton, a high Verticillium wilt resistant cotton cultivar(changkangmian) was used to construct a SSH library. The cDNAs from the inoculated seedlings of 48 hours were used as the tester and those from the control seedlings as the driver. The total RNA and mRNA were successfully isolated using a method of modified CTAB, followed the synthesis of double cDNA. After two rounds of PCR, the up-regulated fragments for induction of Verticillium wilt were accumulated in the library. The purified PCR products were ligated to the vector and transferred into Escherichia coli. There are 434 clones in the library and the length of inserts ranges from 0.45kb to 0.7kb. All the clones have been classified into 20 kinds by fingerprinting and each clone of every kind was selected to be sequenced. Sequence similarity searches were performed with the Blast. Most of them showed high homology to genes or ESTs from the SSH libraries induced by other pathogens. Their amino acid sequences showed homology to some resistant-related genes, such as cytochrome P450 mono-oxygenase, Thaumatin-like Protein, chitinase and PR proteins. The results would be helpful to isolate the Verticillium wilt resistant-related genes and understand the molecular mechanisms of disease response in cotton.