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Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic SbeIIb 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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Detalles Bibliográficos
Autores principales: Jiang, Yongqi, Ren, Yinhui, Xu, Xin, Wang, Hao, Wei, Cunxu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8875723/
https://www.ncbi.nlm.nih.gov/pubmed/35214855
http://dx.doi.org/10.3390/plants11040524
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author Jiang, Yongqi
Ren, Yinhui
Xu, Xin
Wang, Hao
Wei, Cunxu
author_facet Jiang, Yongqi
Ren, Yinhui
Xu, Xin
Wang, Hao
Wei, Cunxu
author_sort Jiang, Yongqi
collection PubMed
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 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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spelling pubmed-88757232022-02-26 Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic SbeIIb Mutant Lines in Rice Jiang, Yongqi Ren, Yinhui Xu, Xin Wang, Hao Wei, Cunxu Plants (Basel) Article 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. MDPI 2022-02-15 /pmc/articles/PMC8875723/ /pubmed/35214855 http://dx.doi.org/10.3390/plants11040524 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Jiang, Yongqi
Ren, Yinhui
Xu, Xin
Wang, Hao
Wei, Cunxu
Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic SbeIIb Mutant Lines in Rice
title Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic SbeIIb Mutant Lines in Rice
title_full Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic SbeIIb Mutant Lines in Rice
title_fullStr Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic SbeIIb Mutant Lines in Rice
title_full_unstemmed Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic SbeIIb Mutant Lines in Rice
title_short Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic SbeIIb Mutant Lines in Rice
title_sort application of allele specific pcr in identifying offspring genotypes of bi-allelic sbeiib mutant lines in rice
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8875723/
https://www.ncbi.nlm.nih.gov/pubmed/35214855
http://dx.doi.org/10.3390/plants11040524
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