Cargando…
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...
Autores principales: | , , , , |
---|---|
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 |
_version_ | 1784658001264640000 |
---|---|
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. |
format | Online Article Text |
id | pubmed-8875723 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT jiangyongqi applicationofallelespecificpcrinidentifyingoffspringgenotypesofbiallelicsbeiibmutantlinesinrice AT renyinhui applicationofallelespecificpcrinidentifyingoffspringgenotypesofbiallelicsbeiibmutantlinesinrice AT xuxin applicationofallelespecificpcrinidentifyingoffspringgenotypesofbiallelicsbeiibmutantlinesinrice AT wanghao applicationofallelespecificpcrinidentifyingoffspringgenotypesofbiallelicsbeiibmutantlinesinrice AT weicunxu applicationofallelespecificpcrinidentifyingoffspringgenotypesofbiallelicsbeiibmutantlinesinrice |