椭圆叶花锚简化基因组的SSR信息分析及SSR引物开发

基于RAD-seq简化基因组测序技术获得椭圆叶花锚（Halenia ellipitica D.Don）简化基因组水平上的序列信息并开发SSR分子标记。利用SR search软件检测而得到双端各有至少100 bp的五种类型的SSR（二、三、四、五、六核苷酸）位点共6 201个，并成功设计其中3 865个SSR引物。在能成功设计引物的SSR位点中，三核苷酸SSR位点最多；重复序列长度包括17种（12~36 bp）；重复序列的基序共达316种，其中五核苷酸基序种类最多（91种）。从中挑选65对可对应五种SSR类型的引物，经梯度PCR检验后，利用椭圆叶花锚的4个居群32个个对可扩增的引物进行PCR和聚丙烯酰氨凝胶电泳检测，其中14对引物能在绝大多数个体中扩增，且13对具多态性。13个多态性位点的等位基因数量均值为5.462，多态性较高且不连锁（P<0.05）；其中10个位点在多数居群中偏离哈迪温伯格平衡（P<0.01）且存在较高的纯合子数量（观测杂合度H_o均值0.226）；近交系数在-0.443~1，均值为0.656；基因流N_m为0.474。

Simplified Genome SSR Information and Development of SSR Primers of Halenia ellipitica(Gentianaceae)

The restriction-site associated DNA sequencing(RAD-seq) technology was used to analyze simple sequence repeats(SSR) information of Halenia ellipitica D. Don; SSR markers for this species were developed based on RAD-seq data. We identified 6 201 SSR loci with at least 100 bp at two ends, including five types(di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide, and hexa-nucleotide). Primers of 3 865 loci were designed successfully. Among which, amount of tri-nucleotide SSR loci is the most. Repeat motif type number is 316, and type number of penta-nucleotide motif is the most(91). Afterward, 65 designed primers were randomly selected to represent the five SSR types, and tested using annealing temperature gradient PCR. Subsequently, 32 individuals from four natural populations of H. elliptica were employed to estimate the SSR primers that can be amplified. One monomorphic and 13 polymorphic SSR loci were developed according to the result of PCR and Polyacrylamide gel electrophoresis. The number of alleles of the 13 polymorphic SSR loci is high(mean 5.462), and no linkage relation was found(P<0.01); ten loci deviate from HWE(P<0.01) and have many homozygotes(observed heterozygosity mean of 0.226); the range of inbreeding coefficient is in -0.443-1(mean of 0.656); the gene flow N_m is 0.474.