三色堇转录组SSR分析及分子标记开发

Development of Genic-SSR Markers by Transcriptome Sequencing in Viola × wittrockiana

为解决三色堇(Viola×wittrockiana)缺乏共显性DNA标记的问题,利用MISA软件对其转录组测序获得的167 576条unigene进行筛选,共检测出23 791个SSR位点,出现频率为14.20%,平均分布距离为6.75 kb。SSR位点中主导类型为三核苷酸重复,占总SSR的28.31%;其次是二核苷酸重复,占总SSR的12.86%。AG/CT与GAA/TTC分别是二核苷酸与三核苷酸的优势重复基元,分别占总SSR重复类型的4.01%和1.85%。利用Primer 3共设计出6 863对SSR引物。随机选择30对引物进行PCR扩增,其中18对引物可扩增出清晰、可重复条带,并在42份三色堇资源中表现多态性。基于扩增的多态性SSR信息,42份三色堇种质资源的聚类结果与其遗传背景基本一致。本研究中获得的18对SSR引物可为三色堇遗传图谱构建、功能基因定位等提供有力工具。

To develop co-dominant DNA markers in pansy(Viola×wittrockiana),167 576 unigenes from pansy transcriptomic database were screened by MISA software.A total of 23 791 simple sequence repeat(SSR)loci were detected in the unigenes with the frequency of 14.20%,and the average distribution distance of SSR was 6.75 kb.The predominant type of SSR was trinucleotide repetition amounting for28.31%of total SSRs,followed by dinucleotide repetition accounting for 12.86%.AG/CT and GAA/TTC are the dominant elements in dinucleotide and trinucleotide repetitions,accounting for 4.01%and 1.85%of the total SSRs,respectively.Primer 3 was employed to design SSR primers and a total of 6 863 pairs of SSR primers were obtained.Thirty pairs of primers were randomly selected for PCR in pansies,in which 18 pairs of primers amplified clear and repeatable bands and showed polymorphism in 42 pansy germplasm accessions.Based on the amplified polymorphic SSR data,the dendrogram results were in accordance with the genetic backgrounds of 42 accessions.Therefore,a large amount of SSR markers can be developed from transcriptome sequencing and employed in genetic linkage map construction and functional gene mapping in pansy.