Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic <i>SbeIIb</i> Mutant Lines in Rice
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 populat...
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MDPI AG
2022-02-01
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| Series: | Plants |
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| Online Access: | https://www.mdpi.com/2223-7747/11/4/524 |
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| author | Yongqi Jiang Yinhui Ren Xin Xu Hao Wang Cunxu Wei |
| author_facet | Yongqi Jiang Yinhui Ren Xin Xu Hao Wang Cunxu Wei |
| author_sort | Yongqi Jiang |
| collection | DOAJ |
| description | 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 <i>starch branching enzyme IIb</i> 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. |
| first_indexed | 2024-03-09T21:12:18Z |
| format | Article |
| id | doaj.art-a0b23a1120df47afa482f6fddd611162 |
| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-03-09T21:12:18Z |
| publishDate | 2022-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Plants |
| spelling | doaj.art-a0b23a1120df47afa482f6fddd6111622023-11-23T21:42:46ZengMDPI AGPlants2223-77472022-02-0111452410.3390/plants11040524Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic <i>SbeIIb</i> Mutant Lines in RiceYongqi Jiang0Yinhui Ren1Xin Xu2Hao Wang3Cunxu Wei4Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Yangzhou University, Yangzhou 225009, ChinaKey Laboratory of Crop Genetics and Physiology of Jiangsu Province, Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Yangzhou University, Yangzhou 225009, ChinaKey Laboratory of Crop Genetics and Physiology of Jiangsu Province, Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Yangzhou University, Yangzhou 225009, ChinaKey Laboratory of Crop Genetics and Physiology of Jiangsu Province, Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Yangzhou University, Yangzhou 225009, ChinaKey Laboratory of Crop Genetics and Physiology of Jiangsu Province, Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Yangzhou University, Yangzhou 225009, ChinaBi-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 <i>starch branching enzyme IIb</i> 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.https://www.mdpi.com/2223-7747/11/4/524clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systembi-allelic mutation lineallele specific PCRgenotype identification |
| spellingShingle | Yongqi Jiang Yinhui Ren Xin Xu Hao Wang Cunxu Wei Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic <i>SbeIIb</i> Mutant Lines in Rice Plants clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system bi-allelic mutation line allele specific PCR genotype identification |
| title | Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic <i>SbeIIb</i> Mutant Lines in Rice |
| title_full | Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic <i>SbeIIb</i> Mutant Lines in Rice |
| title_fullStr | Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic <i>SbeIIb</i> Mutant Lines in Rice |
| title_full_unstemmed | Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic <i>SbeIIb</i> Mutant Lines in Rice |
| title_short | Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic <i>SbeIIb</i> Mutant Lines in Rice |
| title_sort | application of allele specific pcr in identifying offspring genotypes of bi allelic i sbeiib i mutant lines in rice |
| topic | clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system bi-allelic mutation line allele specific PCR genotype identification |
| url | https://www.mdpi.com/2223-7747/11/4/524 |
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