基于HRM获得与桃Tssd紧密连锁的SNP标记

doi:10.3864/j.issn.0578-1752.2017.08.014

摘要/Abstract

摘要： 【目的】植物中，SNP标记具有分布广泛、分辨率高、共显性和多态性高等特点，是遗传研究的常用分子标记。桃全基因组测序完成，获得了大量SNP位点。利用现有的SNP数据进行简单、快速的SNP基因分型是基因定位、品种鉴定和图谱构建等后续研究的基础。文章拟建立采用高分辨率熔解曲线进行不同类型SNP的基因分型方法，以获得与桃温度敏感半矮生型基因紧密连锁的分子标记。【方法】以普通生长型（ST）单株97-32-46为母本，温度敏感半矮生型单株03-94-2（Tssd）为父本进行人工杂交，利用其分离群体96个后代单株为研究材料。在定位目标基因的区间内开发连锁和不同类型的SNP标记的基础上，采用高分辨率熔解曲线进行SNP的基因分型并获得与目标性状紧密连锁的SNP标记。【结果】明确了DNA模板和Mg²⁺是影响基因分型的关键因子，并确立了反应体系最佳浓度区间。在15 µL反应体系中模板DNA的量低于5.0 ng时或Mg²⁺浓度低于1.6 µmol·L^-1时则不能完成PCR扩增和基因分型；根据亲本基因型和表型一致的SNP位点设计引物，扩增片段长度在140 bp左右。高分辨率熔解曲线分析可对由单个核苷酸变异引起的4种不同类型的SNP（A/T、A/G、A/C和C/G）进行基因分型，并正确区分了温度敏感半矮生型和普通生长型，与进行Sanger测序鉴定的结果一致。采用96孔板对温度敏感半矮生型和普通生长型各48个分离后代单株进行了PCR扩增和基因分型，确定了遗传距离。分型结果表明高分辨率熔解曲线分析技术可以将96个样杂交后代单株分为温度敏感半矮生型和普通生长型2种，正确地区分了A/A基因型和A/T基因型。在96个样品中仅1个没有成功扩增，在温度敏感半矮生型和普通生长型中各存在1个重组单株。获得与温度敏感半矮生型基因紧密连锁的SNP标记，遗传距离为2.11 cM。【结论】建立了基于高分辨率熔解曲线分析的SNP基因分型。尽管高分辨率熔解曲线分析技术无法区分两种不同纯合类型的SNP变异，但仍不失为区分已知变异SNP的有效方法。在已经获得桃大量SNP的基础上，该体系可用于桃的基因定位、遗传多样性和品种鉴定等研究。

关键词: 桃, 高分辨率熔解曲线, SNP基因分型, 温度敏感半矮生型

Abstract: 【Objective】 SNP marker is characterized as highly distribution, high resolution and co-domination in plant organism, which is considered as a promising molecular marker. The achievements of peach whole genome sequencing generated massive amounts of SNPs. How to establish a sensitive and effective method for identifying genotyping of different SNP genotypes is a base of further research on gene mapping, cultivar identification and linkage map construction. 【Method】 The aim of this study was to determine if HRM can detect the genotyping of different SNP genotypes and identify a SNP marker tightly linked to PpTssd gene. The segregation population of semi-dwarf progeny was selected with parents 97-32-46 (ST) × 03-94-2 (Tssd). According to gene mapping, different types of SNP markers within mapping region were developed, and the HRM analysis was employed to conduct SNP genotyping and the SNP marker linked to desired traits were generated.【Result】As the key factors for genotyping, the proper concentrations of temperate DNA and Mg²⁺ were established. In the 15 µL PCR reaction system, genotyping could not be complete when template DNA was less than 5 ng and Mg²⁺ was less than 1.6 µmol·L^-1. The primers were designed based on the phenotype and genotype, spanning each desired SNP to amplify DNA fragments shorter than 150 bp. HRM analysis could discriminate four types of SNPs (A/T, A/G, A/C, and C/G) occurred via single nucleotide mutation and the result was validated by Sanger sequence. HRM analysis divided temperature-sensitive semi-dwarf and standard type individuals into two groups. A SNP tightly linked to Tssd gene was identified in 96 individuals consisting of 48 Tssd and 48 ST, respectively. The HRM technique distinguished the Tssd and ST into two groups except for one individual with null amplification. Ultimately, the homozygous A/A and heterozygous A/T were identified, and generated a SNP tightly linked to Tssd gene with 2.11cM with two recombinants.【Conclusion】SNP genotyping of different SNPs were established based on HRM analysis. Although HRM could not distinguish two types of homozygote, HRM analysis still can be a effective method for SNP genotyping and can be used for gene mapping, genetic diversity and cultivar identification in peach based on this study.

Key words: peach, high resolution melting (HRM), SNP genotyping, temperature-sensitive semi-dwarf type

鲁振华，牛良，张南南，崔国朝，潘磊，曾文芳，王志强. 基于HRM获得与桃Tssd紧密连锁的SNP标记[J]. 中国农业科学, 2017, 50(8): 1505-1513.

LU ZhenHua, NIU Liang, ZHANG NanNan, CUI GuoChao, PAN Lei, ZENG WenFang, WANG ZhiQiang. SNP Marker Tightly Linked to Tssd for Peach Using High Resolution Melting Analysis[J]. Scientia Agricultura Sinica, 2017, 50(8): 1505-1513.

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