基于磁珠富集法的绣线菊SSR分子标记分离与筛选

Isolation and screening of molecular genetic marker SSR in Spiraea based on magnetic beads enriched

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。

Simple sequence repeat（ SSR） markers are high polymorphic and codominant,which have become one of the most popular molecular markers used with applications in many different fields. In this study,SSR markers from Spiraea alpina based on magnetic beads enriched were isolated and screened. The total DNA of S. alpina was extracted and digested with restriction enzyme Mse I. Then the digested fragments were ligated with adaptors and hybridized with biotinylated（ AC）15and（ AG）15probes. The tentative SSR DNA was isolated by streptavidin-coated magnetic beads from the hybridized mixture. After elution,amplification and transformation,the SSR-enriched library was constructed. The second PCR screening was performed to screen positive clones,with the primers of probes and vector＇s primers. Sixty sequences were randomly choosed to design the primers from 112 sequences,obtained from positive clones.Finally,16 pairs of polymorphic primers were used to amplify 92 individual from 4 different populations of S. alpina and Spiraea mongolica. Results showed that all populations have a high level of number of alleles per locus,expected and observed heterozygosities.Twenty six of total 64 loci（ 4 population × 16 locus） showed significant deviation from Hardy-Weinberg equilibrium. Results suggested that these SSR makers could be used for genetic diversity,evolution and genetic relationship of S. alpina and S. mongolica,and enrich molecular markers for genetic diversity of Spiraea.