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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 total of 349815 non-redundant ESTs were obtained from 393753 ESTs of common database using ClustalX software. A total of 11372 SSR loci were found in 10,507 ESTs by using self-developed SSRmine software. EST-SSR The frequency is 3%, an average of 21kb separated by an SSR. In the 2 ~ 6bp repeat motifs, trinucleotide and hexanucleotide accounted for 34.1% and 40.6%, respectively. The two, three, four, five and six nucleotide motifs were AG / CT, AAG / CTT , AAAT / ATTT, AAAAG / CTTTT and AAAAAG / CTTTTT type the most. 200 pairs of non-redundant SSR primers were designed and developed using 410 EST sequences that were de-redundant and were not developed in Asian cotton, upland cotton and sea island cotton. The SSR primers were downloaded by SSRD software developed by ourselves. Blastn was used to extract the primer number with the similarity score≥81%. The redundant pairs of primers were extracted and the redundant primers were written in a single line. Six steps were used to remove SSRs from different homologous sequences and different cotton varieties released from CMD Primers were obtained with non-similar primers designated CRIXXX (CRI Cotton Research Institute). 100 pairs of representative materials were selected from 12 representative materials of cotton to evaluate the efficacy of primers, including polymorphism information content (PIC) and primer versatility. The results showed that 56 pairs of 100 pairs of SSR primers independently developed could stably amplify bands between 12 materials, of which 35 pairs were polymorphic and the polymorphism rate was 35%. The PIC amplitudes of the primers ranged from 0.097 to 0.888 with an average of 0.482. The commonality of EST-SSR primer pair was 12% for 12 pairs of materials, 81% for 25 pairs of cotton primers, 74 pairs of upland cotton primers Universal is 80.1%.