用磁珠富集法分离亚麻基因组微卫星分子标记

Microsatellite Molecular Marker Enrichment by Magnetic Beads in Flax

应用Dynal磁珠-生物素标记的微卫星探针(CT)15与亚麻基因组DNA酶切片段杂交,捕获300～1500bp含有微卫星序列的DNA片段,连接到pMD18-T载体中,构建富集微卫星序列的小片段插入文库。利用接头引物和根据微卫星核心序列设计的引物VRV(CT)15使用PCR方法直接对文库筛选,从422个转化子中获得了104个阳性克隆,对其进行测序分析,获得了97个微卫星序列,微卫星序列的富集效率达到22.99%,PCR扩增筛选效率93.27%。对97个微卫星序列进行比对分析,其中51个重复序列的两端序列高度相似,据其设计的特异引物对阳性克隆进行2次筛选,能淘汰相似度高的同类序列,提高筛选亚麻微卫星标记的效率。

Flax （Linum usitatissimum L.） is one of the important oil and fiber crops in the world and can be used as a model plant for bast fiber genomics. The objectives of this study were to set up an efficient protocol to develop microsatellite markers for flax genetic linkage map construction, gene mapping, and marker-assisted selection （MAS）. The 300-1 500 bp flax DNA fractions containing microsatellite sequences were captured by hybridizating the digested genomic DNA fragments with the oligonucleotide probes （CT）15 attached to streptavadin coated magnetic beads （Dynal）. The enriched DNA fragments were ligated into pMD18-T vector and then transformed into E. coli Top10 competent cells to form an enriched microsatellite sequence library. PCR screening using adaptor primer and VRV （CT）15 as primers identified 104 microsatellite clones from 422 transformants in the libraries. Sequence analysis of these positive clones confirmed 97 microsatellite sequences, with a high enrichment efficiency of 22.99% and PCR screening efficiency of 93.27%. Comparative analysis of the 97 microsatellite sequences showed that 51 among them were of high similarity for microsatellite sequences. PCR amplification using a pair of primers designed from these 51 sequences could successfully identify clones with these high similar microsatellite sequences before sequencing. This method can be used as an efficient tool to eliminate high copy microsatellite clones in screening microsatellite library.