伊犁鲈微卫星位点的筛选及近缘物种通用性

Isolation of Microsatellite Loci from Perca schrenkii and its Universal in Related Species

为开发伊犁鲈(Perca schrenkii)分子标记用于鲈属鱼类种质资源保护,以伊犁鲈为材料,应用磁珠富集法进行了微卫星标记的筛选。从伊犁鲈尾鳍提取总DNA,进行酶切、接头连接、PCR扩增,再采用生物素标记(CA)15探针及生物素标记(TG)15探针对扩增产物进行杂交富集,经再次PCR扩增及T-A克隆,成功构建了伊犁鲈基因组微卫星富集文库。采用重复序列引物筛选获得阳性克隆,随机选取48个阳性克隆进行测序,测得序列46个,其中38个克隆含有微卫星序列,41个位点的微卫星重复数在8次以上。根据测得序列设计17对微卫星引物,均能在伊犁鲈群体中扩增获得目的条带。采用该17对引物对河鲈(P.fluviatilis)及黄金鲈(P.flavescens)群体样本进行扩增,10对引物具有通用性,其中6对在河鲈中具有高度多态性(PIC>0.5),5对在黄金鲈中具有高度多态性。

Microsatellites DNA was isolated and enriched from genome of Perca schrenkii by magnetic beads, and were also applied to the other two species in the same genus, i.e.P, fluviatilis and P. flavescens, which aimed to get effective microsatellite primers in the germplasm resources conservation of the species in Perca. Genomic DNA of tail fin from P. schrenkii was extracted and digested with restriction enzyme. Restriction fragments were ligated with linkers and amplified with primers. PCR products were enriched by hybridization with biotin-labeled （CA）15 and biotin-labeled （TG）15 probes. After the selected DNAs amplified, cloned into T/A vectors and transformed into E. coli DHSα, the microsatellite-enriched library of P. schrenkii was successfully constructed. 48 random clones were selected with repeat-sequence primers from the library and were sequenced. As a result, 38 microsatellite sequences were isolated, and the repeat times of motifs in 41 microsatellite loci were much more than 8. 17 pairs of primers were designed from the microsatellite loci all of which were polymorphism for P. schrenkii. These primers were also amplified in P. fluviatilis and P. flavescens, among which 10 pairs could be used, 6 pairs in P. flavescens and 5 in P. flavescens were high polymorphic （PIC 〉 0.5）.