灌木柳耐盐SNP位点的快速鉴定与标记开发  

Rapid identification and marker development of SNP loci for salt tolerance in shrub willow

作　　者：教忠意[1] 田雪瑶 郑纪伟[1] 王保松[1] 何开跃 何旭东[1] JIAO Zhongyi;TIAN Xueyao;ZHENG Jiwei;WANG Baosong;HE Kaiyue;HE Xudong(Jiangsu Academy of Forestry,Willow Nursery of the Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm,Nanjing 211135,China;College of Biology and the Environment,Nanjing Forestry University,Nanjing 210037,China)

机构地区：[1]江苏省林业科学研究院,江苏省农业种质资源保护与利用平台柳树资源圃,江苏南京211153 [2]南京林业大学生物与环境学院,江苏南京210037

出　　处：《南京林业大学学报（自然科学版）》2023年第5期107-113,共7页Journal of Nanjing Forestry University：Natural Sciences Edition

基　　金：国家自然科学基金项目(31300556)。

摘　　要：【目的】对灌木柳(Salix spp.)耐盐相关位点进行快速鉴定并开发单核苷酸多态性(SNP)标记,为耐盐灌木柳早期鉴定与品种选育提供参考。【方法】以耐盐和敏盐灌木柳无性系为亲本材料建立杂交组合,对子代进行盐胁迫并分别构建耐盐和敏盐混池,利用特定位点参数扩增测定(SLAF-seq)技术对亲本及混池进行简化基因组测序,开发多态性SLAF标签并利用SNP-index法进行关联分析。对候选SLAF标签序列进行功能注释,针对SNP位点设计引物并进行重测序验证。【结果】4个样本(含2个亲本及2个混池)SLAF测序平均Q30值为94.36%,GC含量为39.39%,测序深度为48.57×。测序共获得175468个SLAF标签,其中多态性标签25675个,占比14.63%。关联分析共筛选出与耐盐性状显著关联的SLAF标签18个,包含25个SNP位点。功能注释显示有6、4、3和1个标签序列分别在Nt、NOG、KEGG和Swiss-Prot数据库中有比对结果。重测序结果表明,开发的10个SNP标记在耐盐个体和敏盐个体间发生了碱基位点的替换,与原SLAF测序结果一致。【结论】本研究利用BSA分析方法结合SLAF测序技术可快速、有效地对目标性状进行初步定位与鉴别,可为柳树功能标记的开发及分子标记辅助育种提供理论依据和技术支持。【Objective】Due to the rapid reduction of non-agriculture and non-grain land,the afforestation area in China has been drastically decreased in the past two decades and efforts were extended to the afforestation in saline-alkali wastelands in the coastal region.Thus,cultivation and identification of salt tolerance forest germplasm appear to be of particular the importance.However,the salt tolerance in plants is a complex physiological and biochemical process involving with numerous genes,proteins and other multiple coordinative mechanisms.In order to provide a certain significant reference for an early identification and variety selection of salt tolerance shrub willow,we designed this study to screen the SNP loci related with salt tolerance and to develop corresponding SNP markers.【Method】A hybrid cross was made using salt-tolerance and salt-sensitivity parents identified in the previous study.Cuttings from 1505 selected seedlings were used for the salt tolerance treatment,of which 50 salt-tolerance and 50 salt-sensitivity individuals were screened for the gene mixing-pool construction.Four libraries,including the parents and two gene pools,were established and sequenced using the SLAF strategy on the Illuminna HiSeq 2500 platform.After filtering and mapping,the polymorphic SLAF tags among the two parents were selected and utilized for an association analysis based on the SNP-index method.The sequences of candidate SLAF tags related tightly with salt tolerance were blasted against multiple databases in NCBI for the functional annotation.The primer pairs for the SNP loci in candidate SLAF tags were designed,and the PCR products were re-sequenced for a loci validation.【Result】The mean sequencing depth,Q30 value,and GC content of the four samples including two parents and two gene mixing-pools,were 48.57×,94.36%and 39.39%,respectively.Through the digestion of restriction endonuclease,a total of 175468 SLAF tags were obtained,of which 25675 SLAF tags were polymorphic,accounting for 14.63%of the total SLAF

关 键 词：柳树 耐盐 连锁分析 特定位点参数扩增测定 单核苷酸多态性(SNP) 

分 类 号：S718.46[农业科学—林学]