基于RAD-seq的菜心InDel标记开发及应用

Development and Application of InDel Markers in Flowering Chinese Cabbage Based on RAD-seq

【目的】开发多态性丰富的的InDel分子标记,为菜心育种提供研究基础。【方法】以Chiifu-401-42的基因组序列为模板,利用4份菜心材料的RAD-seq重测序数据,在全基因组范围内鉴定InDel位点。利用生物信息学方法筛选4份菜心材料间具有潜在多态性的InDel位点,挑选分布于10条染色体的80个InDel位点设计引物,对55份菜心种质材料进行遗传多样评价。【结果】通过与参考基因组序列比对,在4份菜心材料的重测序数据中共鉴定出84510个InDel位点,其中插入/缺失长度大于5 bp的3609个InDel位点在4份菜心材料间具有潜在多态性。挑选80个InDel位点进行PCR扩增验证,58个(72.5%)在4份测序样品中具有多态性,在55份菜心种质材料中检测到133个等位位点,多态性信息含量(PIC)为0.263~0.618,平均值为0.443。基于InDel标记的检测结果可知,55份菜心种质的遗传相似系数在0.366~0.756,平均值为0.570;在遗传相似系数为0.564时55份菜心种质被分为4个类群,但各类群间的耐热性没有明显差异。【结论】本研究开发的InDel标记具有良好的多态性,能有效地揭示不同菜心种质材料间的遗传距离,可为菜心遗传种质分析、新品种选育等方面提供可利用的标记信息。

【Objective】The study aimed to develop highly polymorphic InDel markers for molecular marker assisted breeding of flowering Chinese cabbage.【Method】The InDel loci were identified by comparing RAD-seq data from four flowering Chinese cabbage accessions with the genome sequence of Chiifu-401-42 as a reference.InDel loci with potential polymorphism were screened out among four accessions by using bioinformatics methods.From these,80 InDel loci evenly distributed across 10 chromosomes were selected for primer design and genetic diversity of 55 flowering Chinese cabbage accessions were evaluated.【Result】By alignment with reference genome sequence,a total of 84 510 InDel loci were identified in RAD-seq data of four accessions.Among them,3 609 InDel loci( > 5 bp) were possibly polymorphic among four flowering Chinese cabbage accessions.Of 80 InDel loci selected for PCR amplification validation,58(72.5%) InDel markers showed polymorphism in the four sequenced accessions,and 133 alleles were detected in 55 accessions.The polymorphic information content(PIC) of these 58 markers ranged from 0.263 to 0.618,with an average of 0.443.Based on the detection results of InDel markers,the genetic similarity coefficient of 55 accessions ranged from 0.366 to 0.756,with an average of 0.570.The 55accessions were classified into four groups with a genetic similarity coefficient of 0.564,and no significant difference was found in heat resistance among different groups.【Conclusion】The nDel markers with good polymorphism developed in this study can effectively reveal the genetic distance among different flowering Chinese cabbage germplasm materials,which could provide useful marker information for genetic germplasm analysis and new variety breeding of flowering Chinese cabbage.