EST-SSR Molecular Marker Development And Construction Of Genetic Map In Stylosanthes

Stylosanthes.spp belongs to leguminosae, is annual or perenial herbal plant. Originated in the Americas and the Caribbean.1960s for the first time introduced to our country in Hainan Island. Stylosanthes is an important tropical forage in South China with high economic value which could be used for feeding domestic animals and plays a role in preserving water and soil and regulating ecosystem. In recent years, with the rapid development of molecular marker technology, molecular marker technology has been widely used in crop breeding, biological research. However, the genetic background of the Stylosanthes is less, and the lack of molecular markers has seriously hampered the molecular breeding and genetic diversity of the column. In this study, we developed 2008 pairs of molecular markers based on the EST sequences achieved from transcriptome sequencing technology and established genetic map via F2 population, which is aimed at revealing the genetic background of stylosanthes spp.The main results are as follows:1. For aquiring effective molecular markers and applying them in the genetics study of stylosanthes, in this experiment, we used MISA and PRIMER 3.0 software to exploit EST-SSR marker and analyzed their characters from the 36558 EST sequences obtained by transcriptome sequencing, then randomly synthesized 523 pairs of SSR markers and evaluated their validity and utility. Finally we got 2008 pairs of new EST-SSR markers of stylosanthes, among them dinucleotide and trinucleotide are the main repeat types accounting for 30.50% and 50.33% respectively and AG/CT and AAG/CTT are the most frequent motifs in dinucleotide and trinucleotide repeats.2. In the 523 pairs of primers randomly synthesized,472 pairs of them had amplification products and our amplification rate reached 90.25%, among them 179 pairs of primers were of polymorphism accounting for 37.92% in amplification products, suggesting our primers have relatively high polymorphism. Analysis of new EST-SSR marker demonstrated that 2 F1 single plants are both true hybrid and F2 population single plants could be applied to gene identification via the new EST-SSR marker. Our new EST-SSR marker will lay the foundation for establishing genetic map, gene clone and building fingerprint chromatography.3. Stylosanthes hippocompoides and S. guianensis stylo 1979 were cross-fertilized artifi-cially to achieve F1 single plant and F2 population were built via selfing of F1. The new developed EST-SSR marker was applied in the gene identification of F2 population and the Mapmarker 3.0 software was used to build genetic map of stylosanthes. The map included 13 linkage groups with full-length of 730.1 cM,57markers were labeled in 90 markers, the LOD value was 3.0 and the average map distance among markers was 12.8 cM.