云南松基因组微卫星富集文库的序列特征分析

Characteristics of Microsatellite Enrichment Pool from Pinus yunnanensis Genomic DNA

目的：分析基因组微卫星富集文库的序列特征，探索云南松基因组微卫星的分布特征或变异规律。方法：采用磁珠富集法，从云南松基因组DNA的RsaI酶切产物中富集微卫星片段，富集片段经洗脱分离、载体连接及转化等环节，对富集文库鉴定和测序，分析序列特征。结果：在测序获得的159条序列中，有143条序列含有重复次数不少于3次的微卫星序列，序列长度为135～1138bp，平均520bp，克隆比例为89．94％。在这些序列中，以二核苷酸重复类型最多（56．64％），其次是三核苷酸，有51条（35．66％），此外，出现少量（7．70％）重复类型为4—6bp的微卫星序列；各重复单元的碱基组成以探针及其互补序列为主，并出现少量的非探针类型；重复次数介于3—15，随着重复单元长度的增加而出现的频率降低。结论：分析了云南松基因组微卫星富集文库微卫星重复类型、重复单元碱基组成及其序列长度变异，为进一步分析云南松基因组微卫星的分布特征或变异规律奠定基础，进而为开发SSR引物提供基础信息。

Objective： The characteristics of microsatellite enrichment pool from Pinus yunnanensis genomic DNA were analyzed in order to explore the distribution and variation of SSR. Method： For constructing microsatellite repeat sequence of Pinus yunnanensis, genomic DNA was extracted and digested with restriction enzyme Rsa I , then the fragments were hybridized with biotin - labeled simple sequence repeat probes by using magnetic - bead enriched method. The biotinylated hybrids were isolated and cloned. Recombinant clones were screened and tested by PCR. The positive clones were selected for sequencing. The characteristics of fragments were analyzed. Result： Of the 159 sequenced clones, 143 clones （89. 94% ） contained simple sequence repeats with at least 3 times repeat numbers. The sequence size ranged from 135 to 1 138 bp with an average of 520 bp. The repeat units of dinucleotide were the most （56. 64% ）, mainly composed of the biotin - labelled probes base with 3 - 15 repeats. The microsatellite frequency decreased with the increasing of the microsatellite re- peat unit length. Conclusion： The repeat types, base composition and microsatellite repeat unit length were analyzed. This study would be useful for screening of microsatellite characteristics and variation of P. yunnanensis genomic DNA, and the SSR isolation.