Development Of SSR Primers By Magnetic-Bead Enrichment In Phaseolus Vulgaris

Common bean (Phaseolus vulgaris) is an important annual herb and diploid plant (2n=2X=:22), and can be propagated with seeds. Its nutritional content is plentiful and huge market prospect. Common bean was cultivated mainly in North China for thousands of years and a lot of germplasm was accumulated to set up an enrich gene pool for breeding. However there is variety degradation, mixed bean cultivars and so on. So it is important to analyze common bean germplasm to make full use of the gene pool for breeding.DNA marker is one of the most effective ways for genetic analysis, in which SSR (Simple Sequence Repeat) possessing many advantages such as high polymorphism information content, codominance, good repeatability and stablity, is widely used in a variety of genetic research. The point of this technology under lies the flanking sequence of SSR loci. The objective of this research is to develop common bean SSR primers, which provide molecular markers tool for common bean genetic diversity analysis, and contribute to genetic data collection in conservation and use of germplasm, common bean breeding and protection of ecological environment.In this study, we firstly digested genome DNA, ligated adaptors and preamplified the ligation products. Secondly, the PCR products were hybridized with5’-biotinylated (AG)13probe and captured by streptavidin-coated magnetic beads. The enriched fragments were purified and directly ligated into PMD-T vector and transformed into Escherichia coli cells. Thirdly, we screened the positive clones with PCR using M13universal primers and sequenced the insertion fragments. After analysis of the insertion sequence, the sequences characterized by SSR were selected and primers were designed for microsatellite sequenees. Lastly, the SSR fragments amplified with the putative SSR primers were confirmed in denaturing polyaerylamidegel (8%). As a result, we developed14pairs of SSR primers with high efficient amplification of SSR fragments in common bean.The14pairs of primers were used to test the polymorphisms in the population composed of42common bean varieties.57alleles were amplified in total and each locus contained4.07alleles on average. Of the14locus, the alleles varied from2to6. The minimum one displayed at DH13locus and the maximum at DH12and DH9locus. The observed heterozygosity (Ho) was from0.0270to1.0000and expected heterozygosity (He) from0.2669to0.7821. The polymorphism information content ranged from0.2460to0.7363and the mean content showed0.4730. In the population,13loci deviated from Hardy-Weinberg equilibrium (HWE) except DH13.The results indicates that the14pairs of SSR primers developed with magnetic beads enrichment are available to efficiently amplify SSR fragments in common bean genome DNA, and can be further used for common bean genetic analysis. In addition, this is the first report to develop SSR primers by magnetic beads enrichment in common bean, get the polymorphism SSR primers. It was proved to be a reliable method for isolating abundant microsatellites for common bean.