石斛SSR标记的开发及可转移性分析

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％N95．7％，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DMl21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。

Development and Transfer Analysis of SSR in Dendrobium

To date, only 100 SSR markers in Dendrobium have been developed, which are far from sufficient for research applications. To develop molecular markers, we mined SSR of Dendrobium from public nucleotide data through bioinformation methods. Some 1343 Uni-DNA sequences were assembled from the 3599 DNA sequences of Dendrobium from GenBank. By scanning the Uni-DNA sequences, 283 SSRs were distributed in 205 Uni-DNA sequences, with an average frequency of 1 SSR per 2815 bp. Sequence alignment indicated that 86 of the 205 SSR-DNA sequences had already been used to design primers. In this study, 76 primer pairs were designed from the remaining 119 sequences for transferability analysis among 32 Dendrobium species. Results showed that 47 primer pairs were amplified effectively with transfer rates ranging from 51.1% to 95.7% （average 75.9%）. Of which, 46 primer pairs were able to detect polymorphism among the Dendrobium species with 2-8 alleles （average 4.0 alleles）. Ten pairs of polymorphic primers were selected to detect polymorphism in 60 accessions of D. officinale, and 2-5 alleles （average 3.4 alleles） were found per SSR locus. Based on the SSR amplification pattern, the 60 accessions of D. officinale were clustered into five clusters, and phenotypes were closer within clusters than between clusters. The sequencing of the amplified fragment of DM121 revealed that allele variation within D. officinale was attributed mainly to the variation of SSR repeat numbers, whereas allele variations among Dendrobium species were also caused by a single base indel and substitution in the microsatellite flankinQ reaion.