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作物学报 ›› 2012, Vol. 38 ›› Issue (08): 1443-1451.doi: 10.3724/SP.J.1006.2012.01443

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

棉花非冗余性EST-SSR新标记的开发及其评价

王为1,2**，王长彪3,** ，刘方1，陈浩东1,4，王林1，王春英1，张香娣1，王玉红1，王坤波1*   

  1. 1中国农业科学院棉花研究所 / 棉花生物学国家重点实验室，河南安阳455000；2江苏沿海地区农业科学研究所 / 农业部沿海盐碱地科学观测实验站，江苏盐城224002；3 山西省农业科学院棉花研究所，山西运城，044000；4 湖南省棉花科学研究所, 国家杂交棉研究推广中心，湖南常德 415101
  • 收稿日期:2011-12-30 修回日期:2012-04-20 出版日期:2012-08-12 网络出版日期:2012-06-04
  • 通讯作者: 王坤波, E-mail: wkbcri@163.com, wkbcri@cricaas.com.cn
  • 基金资助:

    本研究由国家重点基础研究发展计划项目(973计划) (2010CB126000), 中央级公益性科研院所基本科研业务专项(SJB1105), 国家自然科学基金项目(30900911, 31000729), 江苏省科技支撑计划项目(BE2011304), 江苏沿海所所长基金项目(YHS201103), 山西省基础研究计划项目(青年，2009021032-1)。

Development and Evaluation of New Non-Redundant EST-SSR Markers from Gossypium

WANG Wei1,2,**,WANG Chang-Biao3,**,LIU Fang1,CHEN Hao-Dong1,4,WANG Lin1,WANG Chun-Ying1,ZHANG Xiang-Di1,WANG Yu-Hong1,WANG Kun-Bo1,*   

  1. 1 Cotton Research Institute, Chinese Academy of Agricultural Sciences, State Key Laboratoy of Cotton Biology, Anyang 455000, China; 2 Agricultural Sciences Institute of Coastal Area of Jiangsu, Observation and Experimental Station of Saline Land of Coastal Area, Ministry of Agriculture, Yancheng 224002, China;3 Cotton Research Institute, Shanxi Academy of Agricultural Sciences, Yuncheng 044000; 4 Hunan Cotton Research Institute, NationaL Hybrid Cotton Research Promotion Center, Changde 415101 China
  • Received:2011-12-30 Revised:2012-04-20 Published:2012-08-12 Published online:2012-06-04
  • Contact: 王坤波, E-mail: wkbcri@163.com, wkbcri@cricaas.com.cn

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摘要： 利用Clustal X等软件对公共数据库现有的393 753条棉花EST序列分析，得到349 815条非冗余EST序列，借助自主开发的SSRmine软件共发掘SSR位点11 372个，分布于10 507条EST中，EST-SSR的频率是3%，平均相隔21 kb出现一个SSR。在2~6 bp的重复基元中，三核苷酸和六核苷酸分别占34.1%、40.6%，二、三、四、五和六核苷酸基序分别以AG/CT、AAG/CTT、AAAT/ATTT、AAAAG/CTTTT和AAAAAG/CTTTTT的类型最多。利用去冗余的且在亚洲棉、陆地棉、海岛棉中没有被开发过的410条EST序列设计开发了200对非冗余性SSR引物，利用自主开发的SSRD软件通过SSR引物序列下载、预处理、Blastn、提取相似性分值≥81%的引物编号、提取引物冗余对、冗余引物写成一行等6个步骤去除来源于自身部分同源序列以及与CMD释放的不同棉种相似性SSR引物，得到了非相似性引物，定名为CRIXXX (CRI即Cotton Research Institute)。并分别选用棉花12个种的代表性材料对其中100对进行引物功效评价，包括多态信息含量(polymorphism information content, PIC)及引物通用性研究。结果显示，从自主开发的100对SSR引物筛选出56对均能在12份材料间扩增出稳定明显的条带, 其中多态性引物35对，多态率占35%。引物的PIC变幅为0.097~0.888，平均为0.482；1对海岛棉EST-SSR引物在12份材料间的通用性为100%，25对亚洲棉引物通用性为81%，74对陆地棉引物通用性为80.1%。

关键词: 棉花, EST-SSR标记, 冗余性, 多态性, 通用性

Abstract: A software Clustal X was used to analyse the redundancy of 393 753 ESTs of Gossypium available in public database. By mining 349 815 non-redundant ESTs, a total of 11 372 SSR loci derived from 10 507 ESTs using a software SSRmine developed by ourselves were observed. The frequency of ESTs containing SSRs was 3%, with an average of one SSR in every 21 kb of EST sequence. Besides, trinucleotide and hexanucleotide repeats were found to be the most abundant among 2–6-nucleotide repeat types, accounting for 34.1% and 40.6% respectively. In dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, pentanucleotide repeats and hexanucleotide repeats, AG/CT, AAG/CTT, AAAT/ATTT, AAAAG/CTTTT, AAAAAG/CTTTTT motifs accounted for the highest proportion respectively. Two hundred pairs of new non-redundant EST-SSR primers were developed based on 410 EST sequences removed the redundancy which have not been developed so far in Gossypium arboreum L.,G. hirsutum,and G. barbadense. Andwe used a software SSRmine developed by ourselvesto obtain non-similarity primers, designated CRI (Cotton Research Institute) XXX through six steps, including SSR primer sequences download, pretreatment, Blastn, extraction of primer numbers of similarity score more than 81%, extraction of redundant primers pairs and making redundant primers in a line, to remove homologous sequences from themselves and similar primers released in CMD from different cotton species. Among them, 100 primers were evaluated in polymorphism information content (PIC) and transferability using twelve cotton species including seven representative diploids species and five tetraploid species. The results showed that a total of 56 from the 100 pairs of SSR primers could be amplified the stable and clear polymorphic bands in the 12 accessions mentioned above, moreover, 35 out of 56 pairs of primers were polymorphic, with the primer polymorphism ratio of 35%. PIC of these primers ranged from 0.097 to 0.888, with the average of 0.482. Totally, the transferability among twelve cotton species was 100% for a pair of EST-SSR primers from Gossypium barbadense L., 81% for 25 primers from G. arboreum and 80.1% for 74 primers from G. hirsutum, respectively.

Key words: Gossypium, EST-SSR marker, Redundancy, Polymorphism, Transferability

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