基于万寿菊转录组测序的SSR标记开发

Development of SSR Molecular Markers Based on Transcriptome Sequencing of Tagetes erecta

万寿菊(Tagetes erecta L.)花蕾转录组测序共获得48 953条Unigene,利用MISA软件检测出20 666个SSR位点,分布于13 849条Unigene中,出现频率为28.29%,平均分布距离为2.51 kb。优势重复基序为三核苷酸、四核苷酸,分别占总SSR位点的50.16%和20.94%。ATG/ATG和AAAC/GTTT分别是三核苷酸、四核苷酸的优势重复基元,占总SSR重复类型的13.82%和3.66%。随机选取不同主导重复基元类型并合成SSR引物36对,以20份万寿菊自交系的基因组DNA为模板,对引物有效性和多态性进行了验证,30对引物可以扩增到清晰稳定的目标条带,有效扩增率为80.56%;其中13对引物具有多态性,平均H_e和PIC分别为0.275和0.608。以上结果表明,万寿菊转录组测序产生的Unigene信息可作为开发SSR标记的有效来源,获得的大批量SSR标记可为万寿菊的遗传多样性分析和遗传图谱构建提供可靠的标记选择。

A total of 48 953 Unigenes were obtained by transcriptome sequencing of flower buds of Tagetes erecata. MISA found 20 666 SSRs located in 13 849 unigenes with the frequency of 28.29% and a mean distance of 2.51 kb per one. Trinucleotide and tetranucleotide were major types with the percentages of 50.16% and 20.94%,respectively. ATG/ATG and AAAC/GTTT were most frequent motifs in trinucleotide and tetranucleotide repeats,accounting for 13.82% and 3.66%,respectively. Based on different dominant motifs types,36 primers were chosen randomly and verified with 20 different inbred lines of Tagetes erecta. The results showed that 30 primers were effective with a valid rate of 80.56%,and 13 primers showed polymorphism with H_e and PIC mean values of 0.275 and 0.608,respectively. These data indicated that the Unigenes generated from transcriptome sequencing can be used as an effective source to develop SSR markers,and the large quantities of SSR markers will provide reliable resources for analysis on genetic polymorphism and constructing genetic map in Tagetes erecta.