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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

PDF

2480

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

[1]Akagi H, Yokozeki Y, Inagali A, Fujimura T. Microsatellite DNA markers for rice chromosomes. Theor Appl Genet, 1996, 93: 1071-1077
[2]Tautz D. Hyper variability of simple sequence as a general source for polymorphic DNA markers. Nucl Acids Res, 1989, 17: 6463-6471
[3]Liu F(刘峰), Dong-Fang Y(东方阳), Zou J-J(邹继军), Chen S-Y(陈受宜), Zhuang B-C(庄炳昌). Soybean germplasm di-versity and genetic variance detected by microsatellite mark-ers. Acta Genet Sin (遗传学报), 2000, 27(7): 628-633(in Chinese with English abstract)
[4]Chu M-X(储明星), Wang J-Z(王吉振), Wang A-G(王爱国), Li L(李宁), Fu J-L(傅金恋). Cloning and sequencing of four microsatellite loci in small tail han sheep. Acta Genet Sin (遗传学报), 2002, 29(5): 402-405(in Chinese with English ab-stract)
[5]Xue H-B(薛华柏), Qiao Y-S(乔玉山), Xu K-Y(许宽勇), Zhang Z(章镇). DNA fragment isolation of genomic mi-crosatellite in Prunus salicina by magnesphere. Acta Bot Bo-reali-Occident Sin (西北植物学报), 2006, 26(7): 1320-1325 (in Chinese with English abstract)
[6]Chai S-C(柴守诚), Yuan H-Y(员海燕). Characteristics of various repeated DNA sequences in higherplants. Acta Bot Boreali-Occident Sin (西北植物学报), 1999, 19(3): 555-563 (in Chinese with English abstract)
[7]Xu L(徐莉), Zhao G-F(赵桂仿). Microsatellite DNA marker and its application in genetic diversity research. Acta Bot Bo-reali-Occident Sin (西北植物学报), 2002, 22(3): 714-722(in Chinese with English abstract)
[8]Gao G-Q(高国庆), He Guohao, Li Y-R(李杨瑞). Microsatel-lite enrichment from AFLP fragments by magnetic beads. J Peanut Sci (花生学报), 2003, 32 (suppl): 272-276(in Chinese with English abstract)
[9]Roose-Amsaleg C, Cariou-Pham E, Vautrin D, Tavernier R, Solignac M. Polymorphic microsatellite loci in Linum usita-tissimnm. Mol Ecol Notes, 2006, 6: 796-799
[10]Brown J, Hardwick L J, Wright A F. A simple method for rapid isolation of microsatellites from yeast artificial chro-mosomes. Mol Cell Probes, 1995, 9: 53-58
[11]Carol A R, Arcangeli L, Block A B. Development of 11 mir-crosatellite loci for population studies in the swordfish Xiphias gladius (Teleostei: Scombridae). Mol Ecol Notes, 2003, 3: 147-149
[12]Zane L, Bargelloni L, Patarnello T. Strategies for microsatel-lite isolation: A review. Mol Ecol, 2002, 11: 1-16
[13]Xue H(薛辉), Wu X-B(吴孝兵), Yan P(晏鹏). Application of microsatellite DNA in molecular ecology and strategies for loci isolation. Chin J Appl Ecol (应用生态学报), 2005, 16(2): 385-389 (in Chinese with English abstract)
[14]Lu C-Y(鲁翠云), Sun X-W(孙效文), Cao J(曹洁), Liang L-Q(梁利群). Microsatellite enrichment by magnetic beads in silver carp (Hypophthalmichtys molitrix). J Agric Biotechnol (农业生物技术学报), 2005, 13(6): 772-776(in Chinese with English abstract)
[15]Shen F-J(沈富军), Phill W, Zhang Z-H(张志和), Stephanie S, Kemp S J, Yue B-S(岳碧松). Enrichment of giant panda mi-crosatellite markers using dynal magnet beads. Acta Genet Sin (遗传学报), 2005, 32(5): 457-462(in Chinese with English abstract)
[16]Sun X-W(孙效文), Lu C-Y(鲁翠云), Liang L-Q(梁利群). Isolation of microsatellite with enrichment by magnetic beads in grass carp, Ctenopharyngodon idella. J Fish China (水产学报), 2005, 29(4): 482-486(in Chinese with English ab-stract)
[17]Deng X(邓欣), Chen X-B(陈信波), Long S-H(龙松华), Wang Y-F(王玉富). Genetic diversity analysis of ten flax cultivars with RAPD markers. Plant Fiber Sci China (中国麻业科学), 2007, 29(4): 184-188(in Chinese with English abstract)
[18]Lunt D H, Hutchinson W F, Carvalho G R. An efficient method for PCR-based isolation of microsatellite arrays (PIMA). Mol Ecol, 1999, 8: 891-894
[19]Quan Y-C(全迎春), Sun X-W(孙效文), Liu W(刘伟), Liang L-Q(梁利群), Lu C-Y(鲁翠云). Microsatellite enrichment by magnetic beads in Silurus meriaionalis. J Fish China (水产学报), 2006, 30(2): 185-191(in Chinese with English abstract)
[20]Zane L, Bargelloni L, Patarnello T. Strategies for microsatel-lite isolation:a review. Mol Ecol, 2002, 11: 1-16
[21]Weber J L. Informativeness of human (dC-dA)n·(dG-dT)n polymorphisms. Genomics, 1990, 7: 524-530
[22]Orit G, Pearse D E, Avise J. Phylogenetic assessment of length variation at a microsatellite locus. Proc Natl Acad Sci USA, 1997, 94: 745-749
[23]Smulders M J M, Bredemeijer G, Rus-Kortekaas W, Arens P, Vosman B. Use of short microsatellites from database se-quences to generate polymorphisms among Lycopersicon es-culentum cultivars and accessions of other Lycopersicon spe-cies. Theor Appl Genet, 1997, 97: 264-272
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