高丹草EST-SSR标记的开发及其遗传多样性

Development of EST-SSR marker and genetic diversity analysis in Sorghum bicolor×Sorghum sudanenes

对NCBI数据库中210 878条高粱EST序列进行处理,得到57 498条无冗余EST序列,经SSR搜索,发现3 338个SSR分布于3 116条EST序列中,分布频率为1/11.28 kb,包括215种基元重复类型。其中三核苷酸重复最高,占68.33%,二核苷酸重复占17.97%。3 338条SSR序列中有1 694条序列能够设计出引物,所占比例为50.75%。选取14对引物进行合成,对50份高丹草、7份高粱和3份苏丹草材料进行了EST-SSR扩增,共检测到72个等位变异,平均每对引物检测出5.14个基因位点。每对引物多态性指数范围为0.54~0.93,遗传距离的变化范围0.1646~0.6398。结果显示：供试材料具有较丰富的遗传多样性,根据EST-SSR数据的聚类分析,将供试材料按亲缘关系远近分为5大类,来源相同的品种大致聚在一类,呈现出一定的地域性分布规律。同时发现4个特异分子标记,其中引物D1763只对314A和白壳苏丹草杂交后代GB-4-2高丹草审定品种产生特异性,此标记已作为该材料的特异性标记用于种质资源的鉴定中,同时表明,EST-SSR标记是高丹草遗传多样性及特异性研究的一种有效方法。

A total of 57 498 non-redundant ESTs were identified from 210 878 ESTs of Sorghum in NCBI by sequence analysis. In all, 3 338 SSRs were distributed in 3 116 ESTs with an average frequency of one SSR per 11.28 kb, which included 215 SSR motifs. Analysis of SSR motifs revealed that the trinucleotides were major motifs, accounting for 68.33%.The dinucleofides motifs accounted for 17.97%. There were 1 694 sequences from 3 338 EST-SSR sequences could be designed into primers and the proportion was 50.75%. Fourteen primers were selected to amplify EST-SSR loci with 50 collections of Sorghum bicolor × S. sudanenes, 7 collections of S. bicolor and 3 collections of S. sudanenes. Seventy-two allele Variations were detected and the frequency was 5.14 gene loci per primer. The polymorphism index of each primer was in the range of 0.54-0.93. The genetic distance ranged from 0.1646 to 0.6398. This showed abundant genetic diversity in the materials. The materials were divided into 5 groups with clustering analysis of EST-SSR data. Each group included the varieties with similar parents or similar regional distribution. Meanwhile, 4 specific molecular markers were found. Primer D1763 was specific in the registered variety GB-4-2 which was the progeny of S. bicolor 314A × S. sudanenes White Skull. The marker was specific in justification of the germ difference. These results showed that the EST-SSR was an effective marker for genetic diversity analysis and specificity studies on S. bicolor ×S. sudanenes.