根据生物素与链霉亲和素的亲和原理, 利用磁珠富集法筛选东方田鼠(Microtus fortis)微卫星分子标记。链霉亲和素磁珠捕获生物素标记的微卫星探针, 然后与连有接头的单链限制性酶切片段复性结合, 获得含有微卫星的单链片段, PCR扩增形成双链, 连接T载体并转化感受态细胞, 得到东方田鼠微卫星富集文库。随机挑选70个阳性克隆, 经测序分析, 获得微卫星序列92个。设计合成27对微卫星引物并成功筛选出21对可用引物, 取其中10对引物, 荧光标记后对3个人工驯养及野生东方田鼠种群进行遗传多样性分析。结果显示, 文章所构建的东方田鼠微卫星文库的阳性克隆率较高, 初步筛选的10个微卫星标记均为具有高度多态性的微卫星标记。在3个东方田鼠种群中, 野生湖南种群的观测等位基因数(Na)、有效等位基因数(Ne)、观测杂合度(Ho)、期望杂合度(He)和多态信息含量(PIC)均最高, 人工驯养的湖南种群次之, 人工驯养的宁夏种群最低。
This study was to isolate microsatellite markers from Microtus fortis genome by magnetic beads enrichments. Through hybridization of biotin-labeled microsatellite oligonucleotide probes, which were captured by streptavidin-coated magnetic with the adaptor–ligated enzyme-digested genome fragments, single-stranded DNA fragments containing mi-crosatellites were obtained. After PCR amplification, these fragments were then cloned into T vectors and were transformed into competent cells subsequently. Ninety-two microsatellite sequences were randomly isolated from 70 positive clones. Twenty-one out of 27 pairs of designed microsatellite primers were screened out from the microsatellite sequences, and 10 out of the 21 microsatellite loci were used to investigate the genetic diversity of three populations of M. fortis, Hunan (wild), Hunan (domesticated), and Ningxia (domesticated). All the 10 microsatellite loci used to analyze the genetic diversity exhibited a good level of polymorphism. The values of observed number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphic information content (PIC) were all the highest in the Hunan (wild) population, lower in the Hunan (domesticated) population, and the lowest in the Ningxia (domesticated) population.
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