Massoniana Ssr Primer Development

The objectives of this work were to seek new microsatellite markers for Pinus massoniana, using a construction enriched microsatellites library that was a rapid and efficient method. Microsatellites were enriched by hybridizing biotin-labeled SSR probes with AFPL fragments and captured the hybridized SSR-containing fragments by magnetic beads coated with streptavidin. After washing to remove the non-SSR fragments, the eluted single-stranded DNAs were enriched from preamplified (single elective base primer pairs) AFLP fragments. Those became doule-stranded by PCR amplification, ligated into T vector, then transformed into E.coli competent cells to produce a microsatellite enriched library. After selected, 58 positive clones were sequenced, then submitted 27 sequences to GENEBANK.Thirteen SSR-containing clones were identified in non-homology plaques.Analysising the sequences by SSRHunter, the dimotif(TG/AG)n,(GA/CT)n were the most abundant microsatellites. There existed other motifsincludeing trinucleotide repeats (AAG)n,(CTT)n,(AAG)n.The ESTs database of Pinus taeda was screened by using the home-made software. Ten microsatellite sequences were obtained, about 2.0% in whole database.SSRs were designed as primers by using Primer5.0 and tested for 7 individuals of Pinus massoniana. Seventeen primers revealed polymorphism and then tested for 5 individuals of Pinus thumbergii and 5 individuals of Cedrus deodara: M1 and M15 revealed polymorphism among 5 individuals of Cedrus deodara; M1,M6 and M14 revealed polymorphism among 5 individuals of Pinus thumbergii.Enriched microsatellite librarie to get SSR primer in Pinus massoniana, got SSR from ESTs of Pinus taeda and successed to get 17 primers were studied for the firs time. It was proved to be a reliable method for isolating abundant microsatellites for Pinus massonian.