Abstract
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 starch branching enzyme IIb 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.
Highlights
Accepted: 14 February 2022The studies of gene function and its expression regulation in plants can reveal the molecular mechanism of plant growth and development and the physiological function of resistance to biotic and abiotic stresses through forward and reverse genetics [1,2,3].The plant mutants may serve as the most important genetic materials to carry out the above studies
Twenty-one T1 lines were obtained from rice cultivar Kitaake with SBEIIb mutation through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system
The present study indicated that the Allele specific PCR (AS-PCR) could rapidly and accurately detect the homozygous and heterozygous mutant plants with base substitution, insertion, and detection from a large number of offspring segregation populations of heterozygous and bi-allelic mutant lines induced by CRISPR/Cas9 system
Summary
Accepted: 14 February 2022The studies of gene function and its expression regulation in plants can reveal the molecular mechanism of plant growth and development and the physiological function of resistance to biotic and abiotic stresses through forward and reverse genetics [1,2,3].The plant mutants may serve as the most important genetic materials to carry out the above studies. The studies of gene function and its expression regulation in plants can reveal the molecular mechanism of plant growth and development and the physiological function of resistance to biotic and abiotic stresses through forward and reverse genetics [1,2,3]. The mutations of plants are induced by physical or chemical mutagens, which can create a large number of genetic mutants and new alleles within a short period for forward genetics [4,5]. The reverse genetics can reveal the gene function through the mutation or expression regulation of target gene [1,2]. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system can mutate and edit the target gene, and has been widely used in plant sciences and crop improvement [6,7,8,9,10].
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