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采用磁珠富集法及利用生物素标记的(CA)15寡核苷酸探针从波纹唇鱼(Cheilinus undulatus)基因组DNA Bsp143Ⅰ酶切位点的400~1000 bp片段中筛选CA/GT微卫星位点。洗脱的杂交片段克隆到PMD18-T载体上构建富集微卫星基因组文库后,通过PCR筛选出阳性克隆进行测序。在120条序列中有88条序列包含有重复次数不少于5次的微卫星位点,阳性序列比例达到73.33%。其中完美型(perfect)类型微卫星最多的重复次数为26次。在88条非冗长序列中,共有28条(31.82%)微卫星重复序列两端有侧翼序列能够进行引物设计。用波纹唇鱼3个个体的混合基因进行引物筛选,其中的24对具有清晰的扩增条带。将筛选的24对引物对波纹唇鱼1个群体的39个个体进行遗传多样性分析,其中4对引物扩增产物为单态,20对扩增产物呈多态性;20对扩增多态的引物在39个个体中扩增出等位基因数为2~12,平均有效等位基因数为3.5。该波纹唇鱼群体的PIC、Ho、He的平均值分别为0.0782、0.8513、0.5667。这20个微卫星位点适于波纹唇鱼群体遗传结构的研究分析。
The CA / GT microsatellite was screened from the 400-1000 bp fragment of the Bsp143 Ⅰ restriction site of the genomic DNA of Cheilinus undulatus by magnetic bead enrichment and biotinylated (CA) 15 oligonucleotide probes Site. Eluted hybridization fragments were cloned into PMD18-T vector to construct microsatellite genomic enrichment library, and the positive clones were screened by PCR for sequencing. Among the 120 sequences, 88 sequences contained microsatellite loci with repetitions of not less than 5, and the percentage of positive sequences reached 73.33%. Among them, the perfect type of microsatellite has a maximum of 26 repetitions. Of the 88 non-redundant sequences, a total of 28 (31.82%) microsatellite repeats flanked by flanking sequences allowed for primer design. Primers were screened for the mixed genes of 3 individuals of Cormorantidae, of which 24 pairs had a clear amplification band. Twenty-four pairs of primers were selected to analyze the genetic diversity of 39 individuals from a population of Polypodium mollis, of which 4 pairs of primers were single-stranded and 20 pairs of amplified products were polymorphic. Twenty pairs of amplified polymorphisms The number of alleles amplified in 39 individuals was 2 ~ 12, and the average effective number of alleles was 3.5. The average values of PIC, Ho and He in this group of rippled lip fish were 0.0782, 0.8513 and 0.5667, respectively. These 20 microsatellite loci are suitable for the research and analysis of the genetic structure of Populus mollis.