应用T载体连接PCR技术分离黄嘴白鹭微卫星位点

Isolation of Microsatellite Loci in Chinese Egret(Egretta eulophotes) Using T-vector-ligation PCR

传统的微卫星分离策略对于低丰度的物种如鸟类而言,通常是低效的.探讨了一种改良的T载体PCR序列获取法用于黄嘴白鹭(Egretta eulophotes)微卫星DNA的分离,称为T载体连接PCR(T-vector-ligation PCR,TVL-PCR).在获取第一侧微卫星序列的71个阳性克隆中,59个克隆含有重复序列,设计了48对位点特异巢式引物.经过两轮TVL-PCR后,31个位点的扩增产物为预期大小,经测序后获得21个完整的微卫星位点.除去侧翼序列过短和小卫星位点外,设计16个微卫星位点进行多态信息检测,其中7个位点显示出多态性,平均等位基因数为2～20,观察杂合度(HO)和期望杂合度(HE)分别为0.375～0.958和0.477～0.956,所有位点都符合哈迪-温伯格平衡(HWE)和连锁平衡.说明TVL-PCR技术对低丰度微卫星物种的位点分离具有一定的应用价值.

Traditional enriched strategies for microsatellite isolation are low effective when dealing with taxa with a low frequency of microsatellite such as birds.Here,an improved and efficient PCR method for microsatellite isolation in Chinese egret（Egretta eulophotes）,called T-vector-ligation PCR（TVL-PCR）,was presented.Among the 71 positive clones obtained from one flanking sequences of microsatellites,59 contained repeat motif and 48 pairs of locus specific nested primers were designed.After two round TVL-PCR,products of 31 loci were in the expected size and 21 complete microsatellite loci were obtained.After elimination of one minisatellite and four sequences with short flanking regions,16 pairs of primers were designed and tested.Seven loci revealed polymorphism,the number of alleles per locus ranged from 2 to 20,with the observed and expected heterozygosity ranging from 0.375 to 0.958 and 0.477 to 0.956.All loci conform to Hardy-Weinberg equilibrium and no pairs showed evidence of linkage disequilibrium.These results suggested that this method can be a useful alternative of microsatellite isolation for taxa with a low frequency of microsatellite.