种间转移扩增筛选仙湖苏铁微卫星位点及其遗传多样性研究

Screening of microsatellite loci bycross-species amplification and their use in Cycas fairylakea (Cycadaceae)

运用刺叶苏铁、葫芦苏铁、海南苏铁的SSR引物,在仙湖苏铁中进行种间转移扩增,筛选得到7对引物能扩增出清晰的特异带,其中3对引物的扩增产物具有多态性。为验证微卫星的真实性,扩增产物切胶回收后克隆测序。结果表明：重复单元的数目变化是扩增片段长度多态性的主要来源。运用筛选出的3对SSR标记对4个仙湖苏铁野生种群进行遗传结构研究,等位基因数从2～5,杂合度从0.000～0.667,期望杂合度为0.000～0.610。种群两两遗传分化系数从0～0.382。总体上仙湖苏铁遗传多样性水平低,而种群间遗传分化显著。STRUCTURE分析结果表明,4个野生种群可被分配到3个假想的遗传簇。BOTTLENECK分析结果表明种群近期没有遭遇瓶颈效应。

Simple sequence repeat（SSR）markers developed from Cycas rumphii,C.changjiangensis and C.hain-anensis were cross-species amplified in genomic leaf DNA of C.fairylakea.The positive PCR productions were cloned and sequenced.Seven loci consistently yielded the predicted size ranges.Of these loci,three were polymorphic and confirmed as simple sequence repeat（SSR）loci in C.fairylakea.They were utilized to investigate the genetic di-versity among four wild populations of C.fairylakea.The number of alleles per locus ranged from 2 to 5 ,and the observed and expected heterozygosities varied from 0.000 to 0.667 and from 0.000 to 0.610,respectively.Pairwise Fst varied from 0 to 0.382.In the whole,C.fairylakea has low levels of genetic variation within populations and high variation across populations.The STRUCTURE analysis indicated that the four C.fairylakea populations could be subdivided into three different hypothetical genetic clusters（K）.The BOTTLENECK results suggested that no popu-lations had faced recent significant bottleneck.