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Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments

Cryptomeria fortunei has become one of the main timber afforestation species in subtropical high-altitude areas of China due to its fast growth, good material quality, and strong adaptability, showing broad application prospects. Quantitative real-time PCR (qRT-PCR) is the most accurate and widely u...

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Autores principales: Zhang, Yingting, Zhu, Lijuan, Xue, Jinyu, Yang, Junjie, Hu, Hailiang, Cui, Jiebing, Xu, Jin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8224294/
https://www.ncbi.nlm.nih.gov/pubmed/34064253
http://dx.doi.org/10.3390/genes12060791
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author Zhang, Yingting
Zhu, Lijuan
Xue, Jinyu
Yang, Junjie
Hu, Hailiang
Cui, Jiebing
Xu, Jin
author_facet Zhang, Yingting
Zhu, Lijuan
Xue, Jinyu
Yang, Junjie
Hu, Hailiang
Cui, Jiebing
Xu, Jin
author_sort Zhang, Yingting
collection PubMed
description Cryptomeria fortunei has become one of the main timber afforestation species in subtropical high-altitude areas of China due to its fast growth, good material quality, and strong adaptability, showing broad application prospects. Quantitative real-time PCR (qRT-PCR) is the most accurate and widely used gene expression evaluation technique, and selecting appropriate reference genes (RGs) is essential for normalizing qRT-PCR results. However, suitable RGs for gene expression normalization in C. fortunei have not been reported. Here, we tested the expression stability for 12 RGs in C. fortunei under various experimental conditions (simulated abiotic stresses (cold, heat, drought, and salinity) and hormone treatments (methyl jasmonate, abscisic acid, salicylic acid, and gibberellin) and in different tissues (stems, tender needles, needles, cones, and seeds) using four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper). Then, geometric mean rankings from these algorithms and the RefFinder program were used to comprehensively evaluate RG stability. The results indicated CYP, actin, UBC, and 18S as good choices for studying C. fortunei gene expression. qRT-PCR analysis of the expression patterns of three target genes (CAT and MAPK1/6) further verified that the selected RGs were suitable for gene expression normalization. This study provides an important basis for C. fortunei gene expression standardization and quantification.
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spelling pubmed-82242942021-06-25 Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments Zhang, Yingting Zhu, Lijuan Xue, Jinyu Yang, Junjie Hu, Hailiang Cui, Jiebing Xu, Jin Genes (Basel) Article Cryptomeria fortunei has become one of the main timber afforestation species in subtropical high-altitude areas of China due to its fast growth, good material quality, and strong adaptability, showing broad application prospects. Quantitative real-time PCR (qRT-PCR) is the most accurate and widely used gene expression evaluation technique, and selecting appropriate reference genes (RGs) is essential for normalizing qRT-PCR results. However, suitable RGs for gene expression normalization in C. fortunei have not been reported. Here, we tested the expression stability for 12 RGs in C. fortunei under various experimental conditions (simulated abiotic stresses (cold, heat, drought, and salinity) and hormone treatments (methyl jasmonate, abscisic acid, salicylic acid, and gibberellin) and in different tissues (stems, tender needles, needles, cones, and seeds) using four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper). Then, geometric mean rankings from these algorithms and the RefFinder program were used to comprehensively evaluate RG stability. The results indicated CYP, actin, UBC, and 18S as good choices for studying C. fortunei gene expression. qRT-PCR analysis of the expression patterns of three target genes (CAT and MAPK1/6) further verified that the selected RGs were suitable for gene expression normalization. This study provides an important basis for C. fortunei gene expression standardization and quantification. MDPI 2021-05-21 /pmc/articles/PMC8224294/ /pubmed/34064253 http://dx.doi.org/10.3390/genes12060791 Text en © 2021 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
Zhang, Yingting
Zhu, Lijuan
Xue, Jinyu
Yang, Junjie
Hu, Hailiang
Cui, Jiebing
Xu, Jin
Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments
title Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments
title_full Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments
title_fullStr Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments
title_full_unstemmed Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments
title_short Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments
title_sort selection and verification of appropriate reference genes for expression normalization in cryptomeria fortunei under abiotic stress and hormone treatments
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8224294/
https://www.ncbi.nlm.nih.gov/pubmed/34064253
http://dx.doi.org/10.3390/genes12060791
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