基于磁珠富集法开发并筛选西川红景天的微卫星标记

利用FIASCO磁珠富集法,开发和筛选青藏高原特有珍贵植物西川红景天(Rhodiola alsia)多态性微卫星标记。结果表明:用(AG)15和(AC)15两种微卫星标记探针构建富集文库,共获得阳性克隆约2500个;从中随机挑取1200检测,发现具有多态性的阳性克隆达400个;随机挑取200多态性的阳性克隆进行测序,从中获得105个SSR位点,用在线软件primer3-2.3.4成功设计得SSR引物105对;其中45对可以成功扩增,而13对所扩增的片段在相距较远的4个自然居群的24个个体中显示较高的遗传多态性。用4个居群的80个个体检测这13对引物发现,平均等位基因数(A)约为9.192,观测杂合度(Ho)和期望杂合度(He)均值分别约为0.712和0.734,如此高的多态性已经满足后期研究的需要;数个位点在某些居群中显著偏离哈迪温伯格平衡(P0.01),这可能是实际研究的居群并不能达到哈迪—温伯格定律所假设的无限大等理想状态所致。结合此前基于表达序列标签(Expressed Sequence Tag,EST)序列开发的SSR多态性位点,该研究结果为今后利用SSR位点开展西川红景天的居群遗传结构分析和其他研究提供了一组有效工具。

Development and screening of SSR markers in Rhodiola alsia with magnetic beads method

FIASCO magnetic beads method was utilized to develop polymophic SSR markers of Rhodiola alsia, a precious species endemic to the Qinghai-Xizang Plateau. Two SSR marker probes,(AG)15 and(AC)15, were used to construct enriched SSR library and 2 500 positive clones were obtained. Then 1 200 positive clones were randomly selected to check and within which 400 clones were found with polymorphism. Half of the polymorphic clones were randomly chosen for sequencing and 105 SSR loci were achieved. Subsequently, 105 pairs ofprimers were designed by online software, primer 3-2.3.4, for amplifying the SSR sequences. Among which, a total of 45 pairs of primers were positive for amplification and the sequences amplified by thirteen pairs showed high genetic polymorphism when detected in four far apart natural populations with 24 individuals. Afterwards, the whole of 80 individuals in the four populations were employed to check the thirteen SSR loci. The average number of alleles per locus(A)was around 9.192, the mean of observed heterozygosity(Ho)and the expected heterozygosity(He)were around 0.712 and around 0.734, respectively. Such an extraordinary polymorphism was enough to meet the needs of future research. However, several loci were significantly deviated from Weinberg-Hardy equilibrium(P<0.01)in some populations, which may be due to the fact that the population of the actual study could not reach the ideal state of Weinberg-Hardy''s law. Combining SSR polymorphic loci developed previously based on EST(Expressed Sequence Tag)sequences, this study provides a set of serviceable tools for population genetic structure analysis on R. alsia and other researches based on SSR markers.