Exploitation Of EST-SSR Markers And Construction Of Genetic Maps In Flax (Linum Usitatissimum L.)

Flax (Linum usitatissimum L.) as a high-quality fiber crop and oil crop has important economic value in both industry and agriculture. EST-based SSR markers which developed from the coding region not only can be directly used for gene mapping and gene discovery but also have informative, well transferability, easy developed and other advantages. As one of main functional molecular markers, EST -SSR molecular markers have been widely used in many researches of different species, like genetic linkage mapping, comparative mapping, evaluation of genetic diversity, relationship identification and primers transferability research. But the available flax EST-SSR markers too few to meet the currently research needs, so it is an urgent problem to development new flax EST-SSR primer and increase the number of available flax molecular markers. And construct flax genetic map will have great significance for genetic diversity analysis, germplasm identification, gene mapping, marker-assisted breeding and gene cloning of flax, but also provide the theoretical basis for genetic mapping the other hemp crops.In this study, EST sequence downloaded from the NCBI public database was carried out the SSR search and analysis the frequency and distribution of the flax EST-SSR markers. Primers were designed based on conserved sequences at both ends of the EST SSR loci. By polymorphism detected in 20 flax accessions stable available flax EST-SSR markers were developed. And genetic relationship of 20 flax accessions was identified by genetic diversity analysis of EST-SSR markers. The parent accessions CN98816 and Opaline of F2 mapping established populations were also selected from 20 flax accessions. A preliminary flax genetic linkage map has been constructed by EST-SSR makers. A viable development of flax EST-SSR and construction of genetic mapping system has been established. Results as follows:1. Success designed EST-SSR primer and developed EST-SSR markers. In this study, 65 pairs of EST-SSR primers were designed from the 7947 public databases flax EST sequences, of which 32 pairs amplified polymorphic and clear bands and can be used as EST-SSR markers.2. Optimized the flax EST-SSR PCR reaction. Four major components of the system:Mg²⁺, dNTP, primers, Taq polymerase concentration were conducted in single factor experiments to determine the flax EST-SSR PCR reaction optimal system (20μL) as: 1×PCR buffer, 50ng template DNA, Mg²⁺ Concentration1.25mmol/L, dNTP concentration0.125mmol / L, primer concentrations 0.500mmol / L and Taq polymerase 1U.3. The genetic diversity of 20 flax accessions was identified by EST-SSR markers. The 20 flax accessions can be divided into five categories by cluster analysis. Fiber and oil used varieties can be accurately distinguished from the genetic relationship.4. The first domestic genetic map of flax has been built. The map includes three linkage groups, formed by 13 EST-SSR markers, covering 43.4cM length of the genome, with the average distance of 3.3cM. Verify the feasibility of constructing genetic map EST-SSR markers.5. The ESTs sequences corresponding to molecular markers of flax genetic map were analyzed by the programes BLAST of NCBI.After sequence similarity assignment, 9 ESTs have homologous to sequences or proteins with biological functions.