甘蔗EST-SSR标记的开发和多样性分析

Data-mining and Diversity Analysis of EST-SSRs from Sugarcane

运用生物信息学分析方法对甘蔗(Saccharum spp.)的282 809条表达序列标签(ESTs)进行分析,共获得Unigene 138 590条,发掘出分布于10 086条Unigene的10 505个微卫星(EST-SSR)位点,SSR发生频率为7.26%,平均9.22 kb出现1个SSR。在7个杂交甘蔗品种中,三核苷酸基元的SSR数量最多(74.95个/Mb),其次为二核苷酸基元的SSR(22.95个/Mb),二者占总数的90.27%。以CCG/CGG、AG/CT为重复基元的SSR是最常见的EST-SSR类型,富含G/C的EST-SSR占据极明显的优势。与拟南芥和水稻cDNA序列内SSR的比较表明,富含G/C的SSR可能是禾本科植物SSR的一个共同特点。研究发现,SSR在EST与基因组序列之间以及在不同甘蔗品种之间的分布存在差别,具有丰富的多样性。此外,从EST数据分析获得杂交甘蔗品种的16个保守基因,从中发掘了9个SSR位点。最后,利用Primer3和e-PCR软件设计了一批具有物种特异性和通用性的EST-SSR PCR引物,相关数据可以通过http://59.50.66.49/SSR/sugarcane/sugarcane.EST-SSR.tgz链接从因特网下载。该研究结果可为开展甘蔗遗传多样性分析、种质资源鉴定和分子辅助育种提供参考。

282 809 expressed sequence tags （ESTs） from sugarcane （Saccharum ofcinarum L.） were analyzed by bioinformatics methods. ESTs were assembled into 138 590 Unigenes. 10 505 simple sequence repeats （SSRs） were mined from 10 086 Unigenes （7.26% of total）, on average, 1 EST-SSR per 9.22 Kb. Amongst EST-SSRs of 7 Saccharum sp. cultivated varieties, trinucleotide repeats were the most abundant SSRs （74.95/Mb） and dinucleotide SSRs were the second most ones （22.95/Mb）, which accounting for 90.27% of total SSRs. EST-SSRs with CCG/CGG and AG/CT repeat motifs are the most abundant. G/C rich EST-SSRs have a very significant advantage. Results from comparing sugarcane EST-SSRs with SSRs in the eDNA sequences of Arabidopsis thaliana and Oryza sativa showed that G/C rich SSRs may be a common characteristics in plants of the family Gramineae. Results showed that there were obvious differences between SSRs from ESTs and genome sequences, between SSRs from different sugarcane varieties, which suggested that sugarcane SSRs were of rich diversities. Besides, 16 homologues gene clusters from Saccharum sp. cultivated varieties were obtained by the analysis of ESTs, from which 9 SSR loci were derived. Lastly, a set of either species-specific or species-transferable EST-SSR PCR primers were designed by Primer3 and e-PCR softwares. Data could be obtained through the following intemet links： http：//59.50.66.67/SSR/sugarcane/sugarcane.EST-SSR.tgz. Results provided in this study could facilitate the genetic diversity analysis, germplasm identification and molecular assisted selection in sugarcane.