苹果小吉丁虫微卫星开发及其种群遗传结构分析

为研究苹果小吉丁虫Agrilus mali Matsumura的种群遗传结构,采用磁珠富集法以生物素标记探针(AC)_(12)和(AG)_(12)构建苹果小吉丁虫微卫星文库,根据阳性克隆测序获得的微卫星侧翼序列设计引物,筛选和开发苹果小吉丁虫多态性微卫星标记,并利用开发的微卫星标记对其不同地理种群的遗传结构进行分析。结果显示,从微卫星文库中的300个阳性克隆中筛选得到248个含有微卫星片段的克隆子,阳性克隆率为82.7%,其中完美型微卫星131个,占52.8%;不完美型90个,占36.3%;混合型27个,占10.9%,表明磁珠富集法开发新微卫星标记的效率较高。从获得的248个苹果小吉丁虫微卫星中筛选获得7个多态性微卫星位点,这7个位点在苹果小吉丁虫8个地理种群样本中的等位基因数为10~23个,有效等位基因数为1.674~12.218个,多态信息含量为0.304~0.796。8个苹果小吉丁虫种群的观测杂合度为0.095~0.676,期望杂合度为0.469~0.755,Shannon指数为0.791~1.621,遗传距离为0.071~0.788。采自新疆维吾尔自治区的7个苹果小吉丁虫种群之间遗传相似度较高,但其与辽宁省种群的遗传相似度较低。

Microsatellite development and genetic diversity analysis of apple buprestid Agrilus mali Matsumura

To research population genetics of apple buprestid Agrilus mali Matsumura, a microsatelliteenriched genomic library of A. mali was constructed by using the magnetic-bead enrichment method. Biotinylated (AC)₁₂ and (AG)₁₂ probes were used to screen the microsatellite sequences of the pest. For screening and developing the polymorphic microsatellite loci, primers were designed based on the conserved flanking sequences of the microsatellites acquired from the sequences of the positive clones. The results showed that 248 of the 300 randomly picked positive clones contained microsatellites, with a screening efficiency of 82.7%. Among the 248 microsatellite locus sequences, 131 loci (accounting for 52.8%) consisted of perfect repeat motifs, 90 loci (36.3%) contained imperfect repeat motifs, and 27 loci (10.9%) had compound repeat motifs, indicating that the magnetic-bead enrichment method was efficient to develop new microsatellites of A. mali. Seven polymorphic microsatellite loci screened from the 248 microsatellites showed 10-23 alleles in A. mali samples from eight geographical regions. The effective number of alleles (Ne), the polymorphic information content (PIC), the observed heterozygosi-ty (Ho), the expected heterozygosity (He), the Shannon index and the genetic distance in those samples were 1.674-12.218, 0.304-0.796, 0.095-0.676, 0.469-0.755, 0.791-1.621 and 0.071-0.788, respectively. The genetic similarity between populations from Xinjiang Uygur Autonomous Region was high and those between populations from Xinjiang Uygur Autonomous Region and Liaoning Province were low.