Studies On Est-ssr And Srap Markers In Flax

EST-SSR is a new type of molecular marker developed from the databank of sequence of expression sequence tags (EST) and/or cDNAs.As a new kind of molecular marker,EST-SSR markers have more advantages than the traditional genomic-derived SSRs because they are part of expressed genes.Therefore,EST-SSRs might be involved in gene functioning directly orindirectly.The ability of SSR markers will be greatly enhanced.In this study,we design primer pairs based on the searching the EST database of NCBI. Then using selected primers for SSR Markers of fertility gene for thermo-sensitivity male-sterile line of flax. The Studying exploited flax SRAP markers, and establish an optimum SRAP system. The main results are as follows:1 In this research,222 SSR from 7941 EST in NCBI are obtained, representing 2.73% of total ESTs. Trinucleotide repeats, accounting for 72.1% of EST-SSRs, are dominant. Di- and Tri-nucleotide repeats are similar with frequency, accounting for 14.4% and 13.5% of SSRs, respectively. AGAA is the most frequent motif, about 67.67% in tetranucleotide repeats.221 pairs of EST-SSR primers, using the 222 available sequences, were exploited according to primer designed principle.3 Optimization of SSR PCR reaction system in Flax. The results showed that the best concentration was 25mmol Mg2+1.9mmol/L, 10mmol dNTPs 0.55mmol/L, 1.5UTaq enzyme,50ng/μL template DNA 100ng,25ng/μL primer 75ng in 20μL reaction system.418 pairs from 21 designed primer pairs of EST-SSRs show the amplification under a suitable PCR system in 10 flax germplasm,14 primers amplify clear bands with high SSR polymorphism.Based on EST-SSR markers,dendrogram analysis could divide 10 flax germplasm into 3 groups.5 With bulked segregant analysis(BSA),bulked male-fertile DNA pool and bulkedmale-sterile DNA pool were used for EST-SSR analysis which were respectively consistedof 10 fertile plant individuals and 10 sterile plants from F2 generations of 1S×81A350.PCR amplification was carried out with 14 primers.After many experiments,The primer SSR757 was founded that produced polymorphism DNA fragment between two bulked DNA pools.The polymorphism amplification fragment appeared in bulked male-fertile DNA pool and possibly linked to male-fertilitygene for the thermo-sensitivity male-sterile line of Flax.6 The Studying exploited flax SRAP markers, and establish an optimum SRAP system. In 20μL reaction system, the results showed that the best concentration was 1.5mmol Mg2+,0.3mmoldNTP,1.5UTaq enzyme,30ng/μL template DNA90ng, 25ng/L primer 100ng. Using ten flax varieties to test this optimized SRAP system, tested by 10% polyacrylamide gel,the result showed that the SRAP reaction system was steady sand reproducible.