基于SNP标记桃抗蚜性状的基因定位
发布时间：2018-04-22 05:18
本文选题：桃 + SNP标记 ； 参考：《中国农业科学》2017年23期
【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㑳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处等位基因为T和C,Pp01_46.12处等位基因为G和C,遗传距离分别为1.4 c M、2.1 c M;与抗蚜基因完全连锁的标记SNP_Pp01_45712702,位于桃基因组Pp01_45.71处,等位基因为G和T。基于亲本(‘96-5-1’‘10-7’)重测序数据,在精细定位区间内开发紧密连锁的In Del标记KYYZ_Pp01_45799758,位于Pp01_45.79处。对验证群体92个单株进行PCR扩增,经聚丙烯酰胺凝胶电泳检测表明,仅1个单株基因型与表型不符,分子鉴定准确率为98.91%。【结论】利用亲本深度测序与SNP标记技术相结合的方法,精细定位了控制桃抗蚜性状的基因,将抗蚜基因缩小到物理区间460 Kb内,共包含56个转录本,包括52个候选基因。
[Abstract]:[objective] to explore the SNP loci and candidate genes closely linked to aphid resistance traits in peach, so as to lay a foundation for the breeding of peach resistance molecular marker assisted selection. In order to further reveal the genetic basis and molecular mechanism of aphid resistance in peach, [methods] the aphid resistant peach was used as the female parent, and the cultivar 'Zhong Nectarine 13' was used as the male parent. F _ 1 segregation population was obtained by hybridization for gene mapping. F _ 1 segregated population was used as the material to verify the accuracy of locus loci by crossing 10-7 (sensitive aphids) with 96-5-1 (aphids resistant) as the female parent. Referring to the Prunus persicaGenome v2.0.a1), the SNP marker based on Sanger sequencing was developed, and the Sanger sequence was amplified by PCR amplification in the parent Prunus 01-77-3 'Nectarine 13' and four progenies of Prunus persicaGenome v2.0.a1). After obtaining candidate SNP, the population validation was expanded. To achieve the preliminary location of the resistance gene. The complete genome depth sequencing was carried out with coverage of about 70 脳, and SNP markers based on Sanger sequencing and HRM analysis were developed in the initial location interval. In order to obtain the molecular markers closely linked to the aphid-resistant gene, 141 seedlings were genotyped by screening the polymorphic markers. The in Del loci closely linked to aphid resistance traits were developed within the fine mapping interval to verify whether in Del markers were linked to aphids resistance phenotypes. Finally, the candidate genes were analyzed with reference to the loci of peach genome. [results] the harm of aphids to the single shoot of F1 progenies was observed by artificial inoculation. The results showed that the ratio of aphids resistance to aphids to aphid resistance to aphids was close to 1 ~ (-1) P = 0.556, 蠂 ~ (2) = 0.348, which was in accordance with Mendelian inheritance rule. Based on the results of Sanger sequencing, the aphid resistance gene was preliminarily located between the genomic Pp01_38011783 and Pp01_47231340 of peach, and the physical distance was about 9.22 Mb. Clean data 17.109 GB was generated by deep sequencing of parent genome, with an average coverage of 75.19 脳. A total of 29 pairs of SNP markers based on HRM and Sanger sequencing were developed in the initial Pp01 mapping region. Eleven pairs of closely linked markers were obtained after screening. The genotyping results showed that, The marker closely linked to aphid resistance gene is located at 45.66Mb and 46.12Mb in the peach genomic Pp01. The alleles T and C Pp011are G and C respectively, and the genetic distance is 1.4c MN 2.1c; the marker completely linked to aphid resistance gene is Pp01Mb. The SNPs Pp01s, located at the peach genome Pp01_45.71, The alleles were G and T. Based on the resequenced data of the parent, the closely linked in Del marker KYYZPp0145799758 was developed in the fine mapping region, which is located at Pp01_45.79. PCR amplification was carried out on 92 individual plants in a validated population. The results of polyacrylamide gel electrophoresis showed that only one individual genotype did not match the phenotype. The accuracy of molecular identification was 98.91. [conclusion] by combining parental deep sequencing with SNP marker technique, the gene controlling aphid resistance in peach was mapped carefully, and the gene was reduced to a physical interval of 460Kb, containing 56 transcripts. There are 52 candidate genes.
【作者单位】： 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;
【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)
【分类号】：S436.621.21
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本文编号：1785771
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