Jiang Yongqi, Ren Yinhui, Xu Xin, Wang Hao, Wei Cunxu
Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Yangzhou University, Yangzhou 225009, China.
Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu Province, Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou 225009, China.
Plants (Basel). 2022 Feb 15;11(4):524. doi: 10.3390/plants11040524.
Bi-allelic mutant lines induced by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems are important genetic materials. It is very important to establish a rapid and cheap method in identifying homozygous mutant plants from offspring segregation populations of bi-allelic mutant lines. In this study, the offspring genotypes of rice bi-allelic mutant lines were identified using the allele specific PCR (AS-PCR) method. The target sequences of two alleles were aligned from their 5' to 3' ends, and the first different bases were used as the 3' ends of mismatch primers. Another mismatched base was introduced at the third nucleotide from the 3' end of mismatch primer. The PCR reaction mixture and amplification program were optimized according to the differences of mutation target sequence and mismatch primers. The offspring plant genotypes of bi-allelic mutant lines could be accurately identified using the amplified DNA fragments by agarose gel electrophoresis. This study could provide a method reference for the rapid screening of homozygous mutant plants from offspring segregation population of heterozygous and bi-allelic mutant lines.
由成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）系统诱导产生的双等位基因突变系是重要的遗传材料。建立一种快速且廉价的方法，从双等位基因突变系的后代分离群体中鉴定纯合突变体植株非常重要。在本研究中，利用等位基因特异性PCR（AS-PCR）方法鉴定水稻双等位基因突变系的后代基因型。将两个等位基因的目标序列从其5'端到3'端进行比对，并将第一个不同的碱基用作错配引物的3'端。在错配引物3'端的第三个核苷酸处引入另一个错配碱基。根据突变目标序列和错配引物的差异优化PCR反应混合物和扩增程序。通过琼脂糖凝胶电泳利用扩增的DNA片段可准确鉴定双等位基因突变系的后代植株基因型。本研究可为从杂合和双等位基因突变系的后代分离群体中快速筛选纯合突变体植株提供方法参考。
