基于多重PCR靶向测序的烟草1.8K SNP育种液相芯片的开发与应用

Development and Application of a High-Efficiency 1.8K SNP Liquid Chip for Tobacco Breeding Based on Multiplex PCR Sequencing

本研究根据公开发表的烟草K326基因组和烟草430K SNP固相芯片检测数据,以7份种质两两组合之间每条染色体上20个多态标记为目标,基于多重PCR扩增的精准定位测序分型技术(mGPS,Genotyping by Pinpoint Sequencing of multiplex PCR products)开发出烟草1.8K育种液相芯片(YT1.8K.1)。利用该芯片对上述7份种质两两之间杂交的21个杂交组合进行基因分型检测,每个杂交组合之间的平均差异位点数为650个,能同时满足每个组合定向改良筛选高遗传背景回复率单株的需要。利用该芯片对23个烟草品种进行基因型分型检测和聚类分析,聚类分类结果与品种系谱基本吻合;利用该芯片从367个BC2F1群体中筛选出5个背景回复率高于94.96%的单株,高于理论均值87.5%,表明该育种芯片可应用于烟草种质资源聚类分析、定向改良育种的遗传背景筛选。

Based on the publicly available tobacco K326 genome and data from the 430K SNP solid-phase chip for tobacco,this study developed a tobacco 1.8K breeding liquid-phase chip(YT1.8K.1)targeting 20 polymorphic markers on each chromosome between seven pairs of germplasm combinations,utilizing multiplex PCR amplification and pinpoint sequencing-based genotyping technology(mGPS,Genotyping by Pinpoint Sequencing of multiplex PCR products).This chip was used for genotyping in 21 hybrid combinations of the seven germplasms mentioned,with an average of 650 differential loci between each combination,effectively supporting the selective breeding for high genetic background recovery rate in each combination.The chip was further used for genotyping and cluster analysis of 23 tobacco varieties,where the clustering classification results aligned well with the their pedigree information.Additionally,The chip also successfully identified five individual plants with background recovery rates above 94.96%out of 367 BC2F1 populations,surpassing the theoretical mean of 87.5%,indicating that this breeding chip can be applied to cluster analysis of tobacco germplasm resources and genetic background selection for targeted improvement breeding.