基于SNP标记桃抗蚜性状的基因定位  

Gene Mapping of Aphid-Resistant for Peach Using SNP Markers

作　　者：张南南[1] 鲁振华[1] 崔国朝[1] 潘磊[1] 曾文芳[1] 牛良[1] 王志强[1] 

机构地区：[1]中国农业科学院郑州果树研究所/国家桃葡萄改良中心/农业部果树育种技术重点实验室,郑州450009

出　　处：《中国农业科学》2017年第23期4613-4621,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31470679);中国农业科学院科技创新工程(CAAS-ASTIP-2016-ZFRI);河南省科技计划项目(152102110110)

摘　　要：【目的】挖掘与桃抗蚜性状紧密连锁的SNP位点及候选基因,为桃抗性分子标记辅助选择育种奠定基础,并为进一步揭示桃抗蚜性状的遗传基础和分子机制提供依据。【方法】以来源于‘粉寿星’的抗蚜桃‘01-77-3’为母本,栽培品种‘中油桃13号’为父本,杂交获得F1代分离群体进行基因定位。以‘96-5-1’(抗蚜)为母本,‘10-7’(感蚜)为父本杂交获得F1分离群体作为验证定位位点准确性的材料。参考桃基因组序列(Prunus persicaGenome v2.0.a1)开发基于Sanger测序的SNP标记,在亲本(‘01-77-3’‘中油桃13号’)及各4个子代中PCR扩增后进行Sanger测序,获得候选SNP后,扩大群体验证,实现对抗性基因的初步定位。对亲本(‘01-77-3’‘中油桃13号’‘96-5-1’‘10-7’)进行覆盖度约为70×的全基因组深度测序,并基于重测序数据,在初定位区间内开发基于Sanger测序与HRM分析的SNP标记,筛选多态性标记,对‘01-77-3’ב中油桃13号’杂交后代141株实生苗进行基因分型,以获得与桃抗蚜型基因紧密连锁的分子标记。通过双亲(‘96-5-1’‘10-7’)表型与基因型一致,在精细定位区间内开发与桃抗蚜性状紧密连锁的In Del位点,以验证In Del标记与抗蚜表型是否连锁。最后,参考桃基因组分析定位区间的候选基因。【结果】通过人工接种观察蚜虫对桃F1后代单株新梢危害表明,抗蚜与感蚜比例接近1﹕1(P值为0.556;χ~2为0.348),符合孟德尔遗传规律。基于Sanger测序结果,初步将抗蚜基因定位在桃基因组Pp01_38011783与Pp01_47231340之间,物理距离约为9.22 Mb。亲本全基因组深度测序分别产生Clean data 17.109 Gb,平均覆盖度为75.19×,在Pp01初定位区间内开发基于HRM与Sanger测序的SNP标记共29对,筛选后得到11对紧密连锁的标记。基因分型结果表明,与桃抗蚜基因紧密连锁的标记位于桃基因组Pp01的45.66 Mb和46.12 Mb处,Pp01_45.66处等位基因【Objective】 The objective of this study is to identify the SNP loci tightly linked to peach（ Prunus persica（L.） Batsch） aphid（Myzus persicae（Sulzer）） resistance traits, revealing its genetic basis and laying a foundation for the marker assistant selection in resistance breeding of peach.【Method】In this study, the population used for the mapping study consisted of 141 individuals which were obtained from a cross between female parent（‘01-77-3＇） and male parent（‘CN13＇）. Referencing the peach genome and using Sanger sequencing, single nucleotide polymorphism（SNP） markers were developed in female and male parents and 8 progenies to obtain markers linked to the target loci which were tested on the whole population. Subsequently, using whole genome re-sequencing data of two parents, SNPs for fine mapping were selected based on the genotype of two parents, and employed to conduct genotyping to obtain the SNP marker linked to resistance traits. Ultimately, the fine mapping region was validated by using an In Del marker to verify the genotype of F1 population generated from ‘96-5-1＇ × ‘10-7＇. 【Result】 As a result of phenotype identification of 141 progenies, the segregation ratio of resistance to aphid to susceptible ones showed 1﹕1（P： 0.556; χ~2： 0.348）. Using Sanger sequencing we mapped the resistant gene to an approximate 9.92 Mb physical distance between two SNP markers, Pp01_38011783 and Pp01_47231340 on Pp01. For fine mapping, a total of 17.109 Gb clean data was generated from genome re-sequencing and the average coverage depth is 75.19×. 11 of 29 pairs of primers which were designed based on genome re-sequencing data were effective and linked to target trait. As a result of genotyping, we obtained two SNP makers tightly linked to desired trait, SNP_Pp01_45665389 and SNP_Pp01_46120950, with genetic distance of 1.4 c M and 2.1 c M, respectively. The target locus was between these two markers, an approximate physical distance of 460 Kb, and the gene

关 键 词：桃 SNP标记 抗蚜 基因定位 

分 类 号：S436.621.21[农业科学—农业昆虫与害虫防治]