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微卫星序列(SSR)具有多态性高、共显性遗传等特点,是一种极具价值的分子遗传标记。采用磁珠富集法从高山绣线菊基因组DNA中分离和筛选SSR标记。高山绣线菊基因组经限制性内切酶Mse I酶切后与接头连接,并与生物素标记SSR探针(AC)15和(AG)15杂交,然后通过链霉亲和素磁珠富集、洗脱、PCR扩增、克隆,完成微卫星文库构建。利用载体通用引物和探针序列引物进行PCR扩增,筛选重组克隆并测序,获得112条序列。随机挑选其中60条序列设计的引物,经初期筛选获得多态性引物16对。用所得16对引物对4个居群92个个体的蒙古绣线菊和高山绣线菊进行PCR扩增。统计分析PCR产物的毛细管电泳结果,发现4个居群的平均等位基因数、平均期望杂合度及平均观测杂合度都比较高。64个数据系列(4个居群×16个位点)中的26个显著偏离HardyWeinberg平衡,推测可能由于无效等位基因的存在所引起。分析显示研究开发的16对多态性SSR引物可以用于后续遗传多样性、物种进化与亲缘关系等方面研究,丰富了绣线菊遗传多样性研究的分子标记。
Microsatellite sequence (SSR) is characterized by high polymorphism and codominant inheritance, which is a valuable molecular genetic marker. SSR markers were isolated and screened from genomic DNA of Tamarix chinensis by magnetic bead enrichment. The Tamarix chinensis genome was digested with restriction endonuclease Mse I and ligated to the linker and hybridized with biotin-labeled SSR probes (AC) 15 and (AG) 15 and then by streptavidin beads enrichment , Elution, PCR amplification, cloning, microsatellite library construction completed. The PCR amplification was carried out by using the universal primers and the probe primer of the vector, and the recombinant clones were screened and sequenced to obtain 112 sequences. Randomly selected 60 of them were designed primers, and 16 pairs of polymorphic primers were obtained through initial screening. Sixteen pairs of primers were used for PCR amplification of four populations of 92 species of Spiraea polymorpha and Takoyaki. The results of capillary electrophoresis analysis of PCR products were statistically analyzed. The average number of alleles, average expected heterozygosity and average observed heterozygosity of the four populations were found to be relatively high. Twenty-six of the 64 data series (4 populations × 16 loci) deviated significantly from the Hardy-Weinberg equilibrium, presumably due to the presence of null alleles. The analysis showed that the 16 pairs of polymorphic SSR primers developed by this study can be used for subsequent genetic diversity, species evolution and phylogenetic relationships, which enriches the molecular markers of the genetic diversity of Spiraea.