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当前已开发的石斛(Dendrobium)SSR标记仅100余对,难以满足研究的需要。为开发更多石斛分子标记,本研究通过生物信息学方法从公共核酸数据库搜索石斛SSR。结果表明,GenBank收录的3599条石斛DNA序列经拼接获得1343条Uni-DNA序列。经搜索,共检测出283个SSR,分布于205条Uni-DNA序列,平均每2815bp含一个SSR。通过序列比对,剔除86条已开发引物的SSR-DNA序列,从剩余的119条序列设计76对引物,并在石斛属32个种间进行可转移性分析,结果显示47对引物得到有效扩增,种间可转移率为51.1%~95.7%,平均为75.9%。有效扩增引物中有46对在石斛种间具多态性,检测出等位基因数2~8个,平均4.0个。选取10对多态性引物扩增60份铁皮石斛资源,每对引物检测出等位基因数2~5个,平均3.4个。根据SSR扩增带型将60份铁皮石斛资源聚为5大类,同一类型内的表型较类群间接近。对DM121扩增产物测序表明铁皮石斛种内的SSR等位变异由SSR重复次数差异造成,而石斛种间的SSR等位变异还包含SSR位点侧翼序列的插入缺失以及替换。
At present, only about 100 SSR markers of Dendrobium have been developed, which is difficult to meet the needs of the research. In order to develop more Dendrobium molecular markers, this study searches for Dendrobium SSR from the public nucleic acid database by bioinformatics methods. The results showed that GenBank contained 3599 pieces of Dendrobium DNA sequences were stitched to obtain 1343 Uni-DNA sequences. After searching, a total of 283 SSRs were detected and distributed in 205 Uni-DNA sequences, with an average SSR of 2815 bp. By sequence alignment, SSR-DNA sequences of 86 developed primers were removed, 76 pairs of primers were designed from the remaining 119 sequences, and the transferability analysis was carried out among 32 species of Dendrobium. The results showed that 47 pairs of primers were effectively expanded Increased, inter-species transfer rate was 51.1% ~ 95.7%, with an average of 75.9%. 46 pairs of effective amplification primers were polymorphic in Dendrobium species, and 2 to 8 alleles were detected, with an average of 4.0. Ten pairs of polymorphic primers were used to amplify 60 Dendrobium candidum resources. The number of alleles detected per pair of primers ranged from 2 to 5 with an average of 3.4. Sixty Dendrobium candidum resources were grouped into five major categories according to the SSR amplification band pattern, and the phenotypes within the same type were closer than those among the groups. Sequencing of the DM121 amplification product showed that the SSR allelic variation in D. officinale species was caused by the difference in the number of SSR repeats, while the SSR allelic variation among D. dendrobium species also included the insertion and deletion of the flanking sequence of the SSR locus.