Journal
PLANTS-BASEL
Volume 11, Issue 4, Pages -Publisher
MDPI
DOI: 10.3390/plants11040524
Keywords
clustered regularly interspaced short palindromic repeats (CRISPR); CRISPR associated (Cas) system; bi-allelic mutation line; allele specific PCR; genotype identification
Categories
Funding
- National Natural Science Foundation of China [32071927]
- Innovation Program for College Students [202011117054Y, C202011117001Z]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
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This study established a rapid and accurate method using AS-PCR and agarose gel electrophoresis to identify the offspring genotypes of rice bi-allelic mutant lines, providing a reference for the quick screening of homozygous mutant plants.
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.
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