Construction of Microsatellite-enriched Library of Pinus yunnanensis

Abstract

Abstract: Microsatellite-enriched libraries in Pinus yunnanensis were constructed using an enrichment method with biotin-labeled probes and streptavidin-coated magnetic beads.Genomic DNAs of P. yunnanensis were extracted and digested with restriction enzyme Rsa 1. Adapters were ligated to the end of restriction fragments. Ligated products were amplified by PCR with primers complementary to adapters. The PCR products were hybridized with biotin-labeled simple sequence repeat probes(AG) (AT) (CG) (GT)12(ACG) , (ACT )12 and(CCA) 12, 12, 12, 12,8. The hybridized complex was caught by magnetic beads coated with streptavidin. The biotinylated hybrids were isolated on the magnetic particles according to the strong affinity between biotin and streptavidin. The selected DNAs were amplified by PCR with primers complementary to adapters and cloned into pMD-19T plasmid vector, and then transformed into E. coli JM109 hosts. SSR enriched genomic DNA library of P. yunnanensis was constructed. A total of 257 positive clones were selected by PCR from 383 transformants in the microsatellite-enriched library,and 143 clones were found to contain simple sequence repeats(SSR)among 159 sequences. Among them, 94(65.73%)were perfect repeat,34 imperfect(23.78%)and 15(10.49%)compound repeat. The results indicated that the method was efficient to construct the microsatelliteenriched library of P. yunnanensis. The library will be useful for further research on the isolation of microsatellite markers and the analysis of genetic diversity..

Xu Yulan, Zhang Ruili, Tian Bin, Cai Nianhui, Kang Xiangyang, Duan Anan. Construction of Microsatellite-enriched Library of Pinus yunnanensis[J]. Biotechnology Bulletin, 2014, 0(1): 93-99.

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