双向等位基因特异性PCR技术在烟草SNP分型中的应用

林世锋; 王仁刚; 潘飞; 王自力; 元野; 李尊强; 任学良; 龙明锦; 张吉顺; 曾吉凡; 史跃伟

doi:10.13496/j.issn.1007-5119.2022.01.002

双向等位基因特异性PCR技术在烟草SNP分型中的应用

1. 贵州省烟草科学研究院, 烟草行业分子遗传重点实验室, 贵阳 550081;
2. 上海农科种子种苗有限公司, 上海 201106;
3. 黑龙江省烟草公司牡丹江烟草科学研究所, 哈尔滨 150076

基金项目:

中国烟草总公司贵州省公司科技项目（201601、201830、201901）；中国烟草总公司黑龙江省公司科技项目（202030000200126）

详细信息

作者简介:
林世锋(1978-),男,副研究员,博士,研究方向为烟草遗传育种与分子生物学。E-mail:linshifeng1978@163.com

通讯作者:
王仁刚,E-mail:rengangwang@126.com

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出版历程
- 收稿日期: 2021-06-21
- 修回日期: 2021-10-24
- 网络出版日期: 2023-11-30
- 刊出日期: 2022-02-27

Application of Bi-directional PCR Amplification of Specific Alleles in Single Nucleotide Polymorphism Genotyping of Tobacco

1. Guizhou Academy of Tobacco Science, Key Laboratory of Molecular Genetics, CNTC, Guiyang 550081, China;
2. ShanghaiAgricultural Scientific Seed & Seedling Co., Ltd, Shanghai 201106, China;
3. Mudanjiang Tobacco Science Research Institute, Heilongjiang Provincial Tobacco Company, Harbin 150076, China

摘要

摘要: 为提高抗PVY烟草品种的分子育种效率,本研究针对抗病种质资源半坤村晒烟中隐性抗病基因eIF4E1的SNP位点G149C建立一种简单快速的双向等位基因特异性PCR (Bi-directional PCR amplification of specific alleles,Bi-PASA)检测方法,并对该检测方法的特异性、准确度及实际应用效果进行验证。结果表明,建立的Bi-PASA检测方法能够在一个PCR反应中有效区分eIF4E1基因G149C位点3种基因型:野生型GG、杂合突变型GC、纯合突变型CC。利用Bi-PASA检测方法可以对K326×半坤村晒烟F₂分离群体的基因型进行有效鉴别,且鉴定结果与普通的等位基因特异性PCR (allele-specific PCR,AS-PCR)以及直接测序法的鉴定结果一致。综上所述,本研究建立的Bi-PASA检测方法特异性强、准确度高、操作简便,可更好地应用于eIF4E1基因的分子标记辅助育种。
- 烟草 /
- eIF4E1基因 /
- 双向等位基因特异性PCR /
- SNP分型
Abstract: In order to improve the efficiency of molecular marker assisted breeding for PVY-resistant tobacco varieties,we dedicated to establish a simple and rapid bi-directional PCR amplification of specific alleles (Bi-PASA) method for detecting the single nucleotide polymorphism (SNP) G149C of the recessive PVY resistance gene eIF4E1 of the resistant germplasm Bankuncunshaiyan,and to verify the specificity,accuracy and practicality of the established detection method.It turned out that the established Bi-PASA detection method can effectively distinguish the three different genotypes for the G149C SNP of the eIF4E1 gene in a single PCR reaction: wildtype (GG),heterozygous mutant (GC) and homozygous mutant (CC).The genotypes of eIF4E1 in the F₂ population derived from K326/Bankuncunshaiyan were successfully identified by the Bi-PASA method.In addition,the genotypes of the F₂ population identified by the established Bi-PASA method were all the same with that identified by the conventional allele-specific PCR (AS-PCR) and direct sequencing,which suggested that the established Bi-PASA method is characterized by strong specificity,high accuracy and simple operation.Therefore,it can be better applied in the eIF4E1 gene marker assisted breeding.
- tobacco /
- eIF4E1 /
- bi-directional PCR amplification of specific alleles /
- SNP genotyping

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参考文献(27)

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