【摘要】 为明确海蓬子EST序列中SSR的总体特征,开发海蓬子EST-SSR引物,从NCBI网站下载海蓬子及其近缘植物EST序列,利用Cross match等软件过滤掉载体序列、polyA/T和过短或过长的低质量序列,经CAP3软件拼接后获得非冗余EST,然后用在线软件SSRIT从这些序列中查找SSR,采用primer5.0软件设计SSR引物,并进行PCR扩增,对引物适用性进行评价。结果表明,截止2013年5月,NCBI数据库共有海蓬子相关EST序列1 439条,经软件处理后获得无冗余EST序列1 139条,总长542 084 bp,从这些序列中搜索到210个SSR,分布于167条EST中,出现频率为14.66%。SSR平均分布距离为2.58 kb,平均长度为14.56 bp;三核苷酸重复是主导类型,占ESTSSRs总数的40.95%,其次为四核苷酸和五核苷酸重复,分别占29.52%和13.33%;二核苷酸占11.90%,六核苷酸最少,仅占4.29%。在所有类型的重复基元中,ACT/AGT类型最多,占10.95%,其次为AAT/ATT和ACG/CGT,分别占8.10%和6.67%。随机挑选了30个SSR位点,根据其两侧保守序列,设计并合成了相应的EST-SSR引物,对海蓬子DNA进行PCR检测分析,结果有14对引物获得了清晰的条带,进一步将其中的5对引物对10个样本小群体进行了验证,结果显示有较好的多态性。该研究结果表明海蓬子EST序列中含有高频率的SSR位点,ESTSSR标记开发效率较高,为进一步开展海蓬子分子标记研究提供了一批适用的EST-SSR引物。更多还原

【Abstract】 To analyze the characteristics of SSRs derived from Salicornia plants ESTs(expressed sequence tag),and to develop EST-SSR markers,all ESTs sequences from Salicornia plants and closely related species were down loaded with from NCBI database.A set of software such as cross match was used for the removal of vector sequence,poly A/T,low-quality sequences,segments shorter than 100 bp or longer than 700 bp in over-long EST sequence.Non-redundant ESTs was obtained by sequence assembly using CAP3 software,and analyzed online by SSRIT,a web tool for SSR hunting.Primer 5.0 software was used to design PCR primers and estimate the usability of EST-SSR primers.The PCR products were subsequently analyzed by running non-denaturing PAGE gel and silver-staining method.The results indicated that 1 139 non-redundant ESTs sequences with a total length of 542 084 bp were identified from 1 439 ESTs up to May 12,2013 from NCBI database.210 SSRs was hunted in 167 non-redundant ESTs,with a frequency of 14.66% of SSR in all ESTs.The average SSR density was one SSR per 2.58 kb of EST sequences,and the average SSR length was 14.56 bp.The tri-nucleotide repeat dominated the repeat type with a percentage up to 40.95% of the total,followed by the tetranucleotide,which accounted for 29.52%.The frequency of the pentanucleotide repeats were close to the dinucleotide,being 13.33% and 11.90,respectively,and the hexanucleotide repeats,accounting for 4.29% in all SSRs,were the least repeat type.Repeat motif ACT/AGT,comprising 10.95% of all,was the most abundant types,and AAT/ATT and ACG/CGT followed,with the percentages of 8.10% and 6.67%.30 SSR loci were chosen randomly and corresponding PCR primers were designed according to the information of flank conserved sequence.14 pairs of primers showed clear band patterns,and 5 of which displayed polymorphism in a mini-population composed of 10 Salicornia plants samples.The ESTSSR primers obtained in this study could be used for further studies of molecular marker of Salicornia plants.更多还原