摘要
利用ClustalX等软件对公共数据库现有的393753条棉花EST序列分析,得到349815条非冗余EST序列,借助自主开发的SSRmine软件共发掘SSR位点11372个,分布于10507条EST中,EST-SSR的频率是3%,平均相隔21kb出现一个SSR。在2~6bp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%。
A software Clustal X was used to analyse the redundancy of 393753 ESTs of Gossypium available in public database.By mining 349815 non-redundant ESTs,a total of 11372 SSR loci derived from 10507 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 proportions,respectively.Two hundred pairs of new non-redundant EST-SSR primers were de-veloped based on 410 EST sequences removed the redundancy which have not been developed so far in Gossypium arboreum,G.hirsutum,and G.barbadense.We used a software SSRmine developed by ourselves to obtain non-similarity primers,designated CRI(Cotton Research Institute)XXX through six steps,including SSR primer sequences download,pretreatment,Blastn,extrac-tion 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 12 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 the 12 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,re-spectively.
作者
王为
王长彪
刘方
陈浩东
王琳
王春英
张香娣
王玉红
王坤波
WANG Wei;WANG Chang-Biao;LIU Fang;CHEN Hao-Dong;WANG Lin;WANG Chun-Ying;ZHANG Xiang-Di;WANG Yu-Hong;WANG Kun-Bo(Cotton Research Institute,Chinese Academy of Agricultural Sciences/State Key Laboratoy of Cotton Biology,Anyang 455000,China;Agricultural Sciences Institute of Coastal Area of Jiangsu/Observation and Experimental Station of Saline Land of Coastal Area,Ministry of Agri-culture,Yancheng 224002,China;Cotton Research Institute,Shanxi Academy of Agricultural Sciences,Yuncheng 044000,China;Hunan Cotton Research Institute/National Hybrid Cotton Research Promotion Center,Changde 415101,China)
出处
《作物学报》
CAS
CSCD
北大核心
2012年第8期1443-1451,共9页
Acta Agronomica Sinica
基金
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)
江苏省农业科技自主创新基金(自由探索类)
江苏沿海所所长基金项目(YHS201103)
山西省基础研究计划项目(2009021032-1)
