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通过磁珠富集法筛选东北雅罗鱼的微卫星分子标记。采用限制性内切酶Sau 3AI对东北雅罗鱼基因组DNA进行酶切,选取400～900 bp的片段进行PCR全基因组扩增,并利用生物素标记的(CAG)8、(TGA)8、(AGAT)6、(GATT)6 4个探针进行微卫星片段的富集。将得到的片段与T载体连接后转入DH5α大肠杆菌中,然后以SaulA为引物对长出的菌落进行PCR扩增,挑取扩增后片段大小符合条件的菌落送生物公司测序。结果表明:1 416个阳性克隆中有1 047个克隆含有重复次数大于5次的微卫星序列,完美型占67.99%,非完美型为15.31%;混合性标记占16.70%。从中选出160个微卫星序列设计105对引物并合成,经过筛选,56对引物可扩增出清晰条带,其中23对具有多态性。选取18对多态性引物对达里湖东北雅罗鱼群体进行扩增,结果显示等位基因数为3～11,多态信息含量(PIC)为0.207 7～0.882 0,67%(12对/18对)的位点处于高度多态水平(PIC>0.5)。
Microsatellite Marker Screening of Arroyo in Northeast by Magnetic Enrichment. Genomic DNA was digested with restriction endonuclease Sau 3AI from 400 to 900 bp in the genome of GenBank to amplify the genomic DNA of the genus Arroyo. The genomic DNA was digested with biotinylated (CGA) 8, (TGA) 8, AGAT) 6, (GATT) 64 probes for enrichment of microsatellite fragments. The resulting fragment was ligated with the T vector and transferred into DH5α E. coli. The colonies that grew were then amplified by PCR using SaulA as a primer, and the colonies of the amplified fragments were picked and sequenced. The results showed that among the 1 416 positive clones, 1 047 clones contained microsatellite sequences with repeats of more than 5 times, accounting for 67.99% of the perfect ones and 15.31% of the non-perfect ones. The mixed markers accounted for 16.70%. A total of 105 pairs of primers were designed and synthesized from 160 microsatellite sequences. After screening, 56 pairs of primers amplified clear bands, of which 23 pairs were polymorphic. A total of 18 pairs of polymorphic primers were used to amplify the populations of A. dorsalis from Dali Lake. The results showed that the number of alleles was 3-11 and the polymorphic information content (PIC) was 0.207 7-0.888 0,67% (12 pairs / 18 pairs) were highly polymorphic (PIC> 0.5).