Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya
Abstract Background A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in quantitative real-time PCR (qRT-PCR) analyses. However, there is no absolute universality in reference genes among different species. It’s hard to find an ideal reference gene...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2019-07-01
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| Series: | Plant Methods |
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| Online Access: | http://link.springer.com/article/10.1186/s13007-019-0455-3 |
| _version_ | 1819095801082675200 |
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| author | Canbin Chen Jingyu Wu Qingzhu Hua Noemi Tel-Zur Fangfang Xie Zhike Zhang Jianye Chen Rong Zhang Guibing Hu Jietang Zhao Yonghua Qin |
| author_facet | Canbin Chen Jingyu Wu Qingzhu Hua Noemi Tel-Zur Fangfang Xie Zhike Zhang Jianye Chen Rong Zhang Guibing Hu Jietang Zhao Yonghua Qin |
| author_sort | Canbin Chen |
| collection | DOAJ |
| description | Abstract Background A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in quantitative real-time PCR (qRT-PCR) analyses. However, there is no absolute universality in reference genes among different species. It’s hard to find an ideal reference gene to fit for different tissues and growth periods. Pitaya (Hylocereus) is commercially produced as a new fruit crop at a large scale in tropical and subtropical regions. To date, there is no report on the identification of the most reliable reference genes for qRT-PCR normalization in pitaya. Results In this study, six candidate reference genes i.e. Actin(1), GAPDH, UBC(1), UBC(2) EF1-α(1) and histone(1) were selected from thirty-nine typical candidate reference genes to determine the most stable reference genes for qRT-PCR normalization in different tissues, temperature stresses and fruit developmental stages of pitaya. Among the six candidate reference genes, Actin(1) and EF1-α(1) were the most stable gene according to calculations of three statistical methods (GeNorm, NormFinder and BestKeeper) while UBC(1) and UBC(2) showed the lowest expression stability. The six candidate reference genes were further validated by comparing expression profiles of key genes related to betalain biosynthesis at flesh coloration stages of Guanhuahong (Hylocereus monacanthus) and Guanhuabai (H. undatus) pitayas. Actin(1) was recommended the best reference gene for accurate normalization of qRT-PCR data. Conclusions In this study, the stability of the selected reference genes for normalizing the qRT-PCR data were identified from pitaya. Actin(1) was the most stably expressed genes in different tissues and fruit developmental stages in pitaya. The present work provides the first data of reference gene identification for pitaya and will facilitate further studies in molecular biology and gene function on Hylocereus and other closely related species. |
| first_indexed | 2024-12-21T23:49:04Z |
| format | Article |
| id | doaj.art-a08de996932d46bf96b71c956e38b991 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-21T23:49:04Z |
| publishDate | 2019-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-a08de996932d46bf96b71c956e38b9912022-12-21T18:46:00ZengBMCPlant Methods1746-48112019-07-0115111210.1186/s13007-019-0455-3Identification of reliable reference genes for quantitative real-time PCR normalization in pitayaCanbin Chen0Jingyu Wu1Qingzhu Hua2Noemi Tel-Zur3Fangfang Xie4Zhike Zhang5Jianye Chen6Rong Zhang7Guibing Hu8Jietang Zhao9Yonghua Qin10State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Key Laboratory of South China Horticultural Crop Biology and Germplasm Enhancement, Ministry of Agriculture, College of Horticulture, South China Agricultural UniversityState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Key Laboratory of South China Horticultural Crop Biology and Germplasm Enhancement, Ministry of Agriculture, College of Horticulture, South China Agricultural UniversityState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Key Laboratory of South China Horticultural Crop Biology and Germplasm Enhancement, Ministry of Agriculture, College of Horticulture, South China Agricultural UniversityFrench Associates Institute for Agriculture and Biotechnology of Drylands, The J. Blaustein Institutes for Desert Research, Ben-Gurion University of the NegevState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Key Laboratory of South China Horticultural Crop Biology and Germplasm Enhancement, Ministry of Agriculture, College of Horticulture, South China Agricultural UniversityState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Key Laboratory of South China Horticultural Crop Biology and Germplasm Enhancement, Ministry of Agriculture, College of Horticulture, South China Agricultural UniversityState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Key Laboratory of South China Horticultural Crop Biology and Germplasm Enhancement, Ministry of Agriculture, College of Horticulture, South China Agricultural UniversityState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Key Laboratory of South China Horticultural Crop Biology and Germplasm Enhancement, Ministry of Agriculture, College of Horticulture, South China Agricultural UniversityState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Key Laboratory of South China Horticultural Crop Biology and Germplasm Enhancement, Ministry of Agriculture, College of Horticulture, South China Agricultural UniversityState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Key Laboratory of South China Horticultural Crop Biology and Germplasm Enhancement, Ministry of Agriculture, College of Horticulture, South China Agricultural UniversityState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Key Laboratory of South China Horticultural Crop Biology and Germplasm Enhancement, Ministry of Agriculture, College of Horticulture, South China Agricultural UniversityAbstract Background A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in quantitative real-time PCR (qRT-PCR) analyses. However, there is no absolute universality in reference genes among different species. It’s hard to find an ideal reference gene to fit for different tissues and growth periods. Pitaya (Hylocereus) is commercially produced as a new fruit crop at a large scale in tropical and subtropical regions. To date, there is no report on the identification of the most reliable reference genes for qRT-PCR normalization in pitaya. Results In this study, six candidate reference genes i.e. Actin(1), GAPDH, UBC(1), UBC(2) EF1-α(1) and histone(1) were selected from thirty-nine typical candidate reference genes to determine the most stable reference genes for qRT-PCR normalization in different tissues, temperature stresses and fruit developmental stages of pitaya. Among the six candidate reference genes, Actin(1) and EF1-α(1) were the most stable gene according to calculations of three statistical methods (GeNorm, NormFinder and BestKeeper) while UBC(1) and UBC(2) showed the lowest expression stability. The six candidate reference genes were further validated by comparing expression profiles of key genes related to betalain biosynthesis at flesh coloration stages of Guanhuahong (Hylocereus monacanthus) and Guanhuabai (H. undatus) pitayas. Actin(1) was recommended the best reference gene for accurate normalization of qRT-PCR data. Conclusions In this study, the stability of the selected reference genes for normalizing the qRT-PCR data were identified from pitaya. Actin(1) was the most stably expressed genes in different tissues and fruit developmental stages in pitaya. The present work provides the first data of reference gene identification for pitaya and will facilitate further studies in molecular biology and gene function on Hylocereus and other closely related species.http://link.springer.com/article/10.1186/s13007-019-0455-3HylocereusReference genesIdentificationqRT-PCRNormalizationCytochrome P450 gene |
| spellingShingle | Canbin Chen Jingyu Wu Qingzhu Hua Noemi Tel-Zur Fangfang Xie Zhike Zhang Jianye Chen Rong Zhang Guibing Hu Jietang Zhao Yonghua Qin Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya Plant Methods Hylocereus Reference genes Identification qRT-PCR Normalization Cytochrome P450 gene |
| title | Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya |
| title_full | Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya |
| title_fullStr | Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya |
| title_full_unstemmed | Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya |
| title_short | Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya |
| title_sort | identification of reliable reference genes for quantitative real time pcr normalization in pitaya |
| topic | Hylocereus Reference genes Identification qRT-PCR Normalization Cytochrome P450 gene |
| url | http://link.springer.com/article/10.1186/s13007-019-0455-3 |
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