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作物学报 ›› 2008, Vol. 34 ›› Issue (12): 2099-2105.doi: 10.3724/SP.J.1006.2008.02099

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

用磁珠富集法分离亚麻基因组微卫星分子标记

邓欣1; 陈信波1,2,*;龙松华1;王孝纯3;高原1,2;何东锋1,2;王进1;王玉富1   

  1. 1 中国农业科学院麻类研究所，农业部麻类遗传改良与工程微生物重点实验室，湖南长沙410205；2 湖南农业大学作物基因工程湖南省重点实验室，湖南长沙410128；3 黑龙江大学资源与环境研究室，黑龙江哈尔滨150080
  • 收稿日期:2008-05-04 修回日期:2008-07-15 出版日期:2008-12-12 网络出版日期:2008-10-10
  • 通讯作者: 陈信波
  • 基金资助:

    中国农业科学院杰出人才科研启动经费资助

Microsatellite Molecular Marker Enrichment by Magnetic Beads in Flax

DENG Xin1,CHEN Xin-Bo12*,LONG Song-Hua1,WANG Xiao-Chun3,GAO Yuan12,HE Dong-Feng12,WANG Jin1,WANG Yu-Fu1   

  1. 1 Key Laboratory of Genetic Improvement & Engineering Microbiology for Bast Fiber Crops, Ministry of Agriculture / Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410128, Hunan; 2 Crop Gene Engineering Key Laboratory of Hunan Province, Hunan Agricultural University, Changsha 410128, Hunan; 3 College of Agricultural Resources and Environmental Sciences, Heilongjiang University, Harbin 150080, Heilongjiang, China
  • Received:2008-05-04 Revised:2008-07-15 Published:2008-12-12 Published online:2008-10-10
  • Contact: CHEN Xin-Bo

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2157

被引次数

35

摘要：

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300~1 500 bp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。

关键词: 亚麻, 微卫星, 磁珠富集

Abstract:

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 Top 10 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.

Key words: Flax, Microsatellite, Enrichment by magnetic beads

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