球茎甘蓝SSR分子标记开发及指纹图谱构建

SSR molecular markers development and fingerprints construc⁃tion in kohlrabi(Brassica oleracea var.gongylodes L.)

【目的】开发球茎甘蓝SSR分子标记,并构建球茎甘蓝的指纹图谱,以期从分子水平了解球茎甘蓝品种间的遗传多样性及亲缘关系,为球茎甘蓝育种材料选择提供参考。【方法】基于转录组数据,利用生物信息学方法开发球茎甘蓝的SSR标记并分析其分布特征,PCR检测SSR引物的有效性和多态性,筛选出多态性引物,用于分析18份球茎甘蓝材料的亲缘关系并构建指纹图谱。【结果】通过球茎甘蓝转录组测序共获得196642条Unigenes,其中85468条含有SSR位点,SSR出现频率43.46%;单一型SSR位点以单核苷酸(44.74%)为主要类型,其次为二核苷酸(26.37%)和三核苷酸(27.35%)类型。SSR位点有112种重复基序,其中单核苷酸重复基序以A/T为主(占44.16%),二核苷酸重复基序以AG/CT为主(占18.11%)。PCR筛选获得29对引物能扩增出86条多态性条带,平均每对引物扩增出3.28条。根据扩增出的多态性条带对18份球茎甘蓝材料进行聚类分析,结果显示在遗传距离为0.61处将其分为五大类,同时还构建了18份球茎甘蓝DNA指纹图谱数据库,建立了相应的识别二维码。【结论】开发获得29个球茎甘蓝SSR分子标记,基于这些分子标记构建的球茎甘蓝指纹图谱可用于种质资源的多样性分析、品种选育及开发利用。

【Objective】To develop SSR molecular markers and construct fingerprints of kohlrabi(Brassica oleracea var.gongylodes L.),in order to understand genetic diversity difference and genetic relationship of kohlrabi varieties at the molecular level and to provide reference for breeding materials selection of kohlrabi.【Method】Based on transcriptomic data,SSR markers were developed and their distribution characteristics were analyzed by bioinformatics methods.PCR method was used to detect validity and polymorphism of SSR primers to screen polymorphic primers that were used to analyze the relationship among 18 kohlrabi materials and construct fingerprints.【Result】A total of 196642 unigenes were obtained by transcriptome sequencing,of which the number of 85468 unigenes contained SSR sites,accounting for 43.46%.The dominant type among the SSR sites was mononucleotides(44.74%),followed by dinucleotides(26.37%)and trinucleotides(27.35%).The SSR site analysis revealed 112 repeat motifs,of which mononucleotide repeat motifs were mainly A/T(44.16%),and dinucleotide repeat motifs were mainly AG/CT(18.11%).Through PCR,29 pairs of primers were screened,which could amplify 86 clear bands of high polymorphism and with an average amplification of 3.28 primers per pair.Clustering analysis of 18 kohlrabi materials was conducted with polymorphic bands and the results showed that the kohlrabi materials were divided into 5 categories at the genetic distance of 0.61.At the same time,DNA fingerprint database of 18 kohlrabi materials were constructed based on polymorphic bands and the corresponding identification two-dimensional code was established.【Conclusion】Twenty-nine SSR molecular marker of kohlrabi are developed in this study,and kohlrabis fingerprint based on these molecular marker can be applied in diversity analysis of germplasm resources,variety breeding,development and utilization of kohlrabis.