Microsatellite Loci Isolation Using Microsatellite-enriched Libraries From Tibetan Antelope

To isolate a set of microsatellite primer-pairs, the microsatellite-enrichedlibraries of Tibetan antelope were constructed in this study. The results suggest theseisolated microsatellites will be useful for studies involving gene flow, geneticdiversity analysis and genetic structure within Tibetan antelope populations on theQinghai-Tibetan Plateau (QTP).Enriched microsatellite libraries for Tibetan Antelope were constructed usingFast isolation by AFLP of sequences containing repeats (FIASCO). Four DNAsamples (No.118and No.132in Qinghai, No.14in Tibet, No.62in Xinjiang)collected from three geographical units were mixed with equal quality. Then mixedDNA was digested overnight at37℃with MseⅠ, and the fragment of300-1000bpwere separated by agrose gel electrophoresis. After purifing these fragments anddenaturating them, single-strand DNA fragments were enriched for microsatellites byhybridization with5’-biotinylated (AG)12、(AC)12、(AT)12、(GC)12、(AAC)8、(TGC)8and (AGT)8probe. Each set of fragments captured by the probes wereligated into a pCR@2.1vector and transformed into DH5α compent cells. By PCRdetection, positive clone which presented2bands were selected and sequenced. Themain results were as follows:1. Tibetan antelope microsatellite enrichment library was established by theligation into a pCR@2.1and cloning transformation with biotin-labeledoligo-nucleotide probes (AG)12,(AC)12,(AAC)8,(TGC)8,(AGT)8. By PCRdetection, the positive cloning rate is respectively50.7%,40.7%,31.1%,50%,41.6%.The Tibetan antelope genome microsatellite enrichment library with the relativelyhigh positive rates indicated a good quality library.2. Using the method of magnetic beads enrichment screened successfully Tibetanantelope microsatellite locis, received144microsatellite sequences and repeat timesranged from5to39.The Tibetan antelope microsatellite enrichment library wasn’tenhanced the microsatellite types of (GC)n and (AT)n within7kinds of biotinylatedprobes.3. Primers were designed with Primer5.0version software and compounded107pairs of primers. The24Tibetan antelope genomic DNA samples of the threegeographical populations were amplifed and22polymorphic primers were obtained with the method of magnetic beads enrichment.4. Tibetan antelope microsatellite enhanced library was constructed for the firsttime and some sequences encompassed SSRs were submitted to GenBank (GenBankAccession Number: KC422272-KC422315).