大豆长片段插入/缺失标记的开发与应用

Development and Application of Large-size Insertion/Deletion Markers in Soybean

为了开发稳定可靠、操作简便的大豆分子标记,以12份地理来源不同的大豆种质资源为材料,通过基因组10倍深度重测序开发长片段插入/缺失(InDel)标记,并应用2018年黄淮海大豆多点鉴定96份参试品系DNA指纹图谱构建。结果表明,在参试材料中,共检测到插入/缺失片段长度大于20 bp的InDel标记66561个,平均每条染色体InDel标记的数量为3262个;位于内含子的InDel占比12.35%,位于基因上游序列和下游序列的InDel占比分别为25.83%,20.44%,位于基因间隔区的InDel占比34.39%,位于外显子的InDel占比0.19%,位于5′-UTR和3′-UTR的InDel占比分别为0.93%和1.51%;片段长度介于20~40 bp的InDel数量为42453个,41~60 bp为13044个,61~80 bp为5034个,81~100 bp为2285个,大于100 bp为2413个;从检测到的66561个InDel位点中,根据插入/缺失片段长度,在基因组中随机选区160个InDel位点,利用引物设计、PCR和琼脂糖凝胶电泳等技术,在55℃的退火温度条件下,开发出32个有且仅有2个等位变异、基于1%琼脂糖凝胶电泳技术易于分辨的长片段插入/缺失标记。应用新开发的32个标记,构建了2018年黄淮海大豆多点鉴定96个参试品系DNA指纹图谱,参试的大豆材料纯度为96.84%,没有同物异名现象发生。研究开发的InDel标记稳定可靠、操作简便,可应用于大豆DNA指纹图谱构建、种子纯度检测等工作。

The objective of this work was to develop novel,convenient,and reliable molecular marker in soybean.Twelve soybean germplasm accessions from different geography region were used to develop large-size InDel markers based on 10 depth genomic re-sequencing.Developed InDel markers were used to construct DNA fingerprint of 96 varieties in Huanghuaihai Uniform Test in 2018.The results showed that a total of 66561 InDel markers,with insertion/deletion fragment size larger than 20 bp,were discovered among the 12 accessions.The distribution pattern among the genome of these InDel markers were also illustrated.The ratio of the InDel in intron,upstream of the gene,downstream of the gene,intergenic region,intragenic region,5′-UTR and 3′-UTR were 12.35%,25.83%,20.44%,34.39%,0.19%,0.93%,1.51%,respectively.There were 42453 InDels yield insertion/deletion fragment size between 20 to 40 bp,13044 InDels between 41-60 bp,5034 InDels between 61-80 bp,2285 InDels between 81-100 bp,and 2413 InDels larger than 100 bp.Then,160 InDel markers were randomly selected according to the insertion/deletion fragment size throughout the whole genome.As results,thirty-two InDel markers,that only yield 2 alleles in for each locus and could be distinguished easily on agarose gel,were developed and validated through primer design,PCR and agarose gel electrophoresis.Additionally,DNA fingerprint of 96 varieties in Huanghuaihai Uniform Test in 2018,were constructed using the 32 developed InDel markers.As results,the average purity of the tested soybean varieties was 96.84%,and no varieties shared the same name.The InDel markers developed in this study were stable,reliable and user friendly.They could be used to construct DNA fingerprint and test seed purity of soybean.