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目的:利用磁珠富集法构建霍山石斛的微卫星文库,用筛选出的微卫星引物对安徽霍山居群的24株野生霍山石斛进行遗传多样性分析。方法:根据链亲和素磁珠和生物素特异结合的特性,用3’端生物素标记的探针与两端连接已知序列人工接头的霍山石斛基因组DNA酶切片段杂交,杂交复合物结合到包被有链亲和素的磁珠上,洗脱并收集吸附在磁珠上的含有微卫星的片段。经过克隆和测序,再根据微卫星两侧的保守序列设计引物,最后利用从中筛选出的微卫星引物对霍山石斛居群进行遗传学分析。结果:本实验共筛选得到12对多态性位点丰富、带型清晰、重复性好的引物,并检测到65个等位基因,每个位点的等位基因数从2到8个不等,平均预期杂合度(HE)和平均观察杂合度(HO)分别为0.638,0.500。12个多态性位点的哈温平衡检测结果显示有2个位点显著偏离哈温平衡(P<0.05),可能是由于存在无效等位基因。连锁不平衡检测结果表明有3对位点显著连锁不平衡(P<0.05);结论:这些微卫星标记可以用于霍山石斛的居群遗传学分析。
OBJECTIVE: To construct microsatellite DNA library of Dendrobium huoshanense by enrichment with magnetic beads and analyze the genetic diversity of 24 wild Dendrobium huoshanense populations in Anhui Huoshan population using the selected microsatellite primers. Methods: According to the characteristic of the specific binding between streptavidin beads and biotin, the biotin-labeled probe was used to hybridize with the DNA fragment of Dendrobium huoshanense, whose both ends were connected to the artificial linker of known sequence. The hybridization complex was bound To the magnetic beads coated with streptavidin, eluting and collecting the microsatellite-containing fragments adsorbed on the magnetic beads. After cloning and sequencing, the primers were designed according to the conserved sequences on both sides of microsatellite. Finally, the microsatellite DNA primers were used to carry out the genetic analysis of D. huoshanensis population. Results: A total of 12 pairs of polymorphic loci were screened in this experiment, with clear bands and good repeatability. 65 alleles were detected, and the number of alleles per locus ranged from 2 to 8 The results of the Havan equilibrium test showed that there were two sites significantly deviated from the Havan equilibrium (P <0.01), and the average expected heterozygosity (HE) and average observed heterozygosity (HO) were 0.638 and 0.500 respectively. 0.05), probably due to the presence of null alleles. The results of linkage disequilibrium test showed that there are three pairs of loci with significant linkage disequilibrium (P <0.05). Conclusion: These microsatellite markers can be used for population genetic analysis of Dendrobium huoshanense.