Selection and Evaluation of Candidate Reference Genes for Quantitative Real-Time PCR in Aboveground Tissues and Drought Conditions in Rhododendron Delavayi
There has been no systematic identification and screening of candidate reference genes for normalization of quantitative real-time PCR (qRT-PCR) results in Rhododendron delavayi to date. Therefore, the present study used GAPDH, Act, EF1, Tub-, Tub-5, UEC1, TATA, TATA-2, UEP, TIP41, and Ubiquitin to...
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Frontiers Media S.A.
2022-04-01
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| Series: | Frontiers in Genetics |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fgene.2022.876482/full |
| _version_ | 1811309741628981248 |
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| author | Lu Zhang Lu Zhang Yanfei Cai Yanfei Cai Mingchao Zhang Guanghui Du Jihua Wang Jihua Wang |
| author_facet | Lu Zhang Lu Zhang Yanfei Cai Yanfei Cai Mingchao Zhang Guanghui Du Jihua Wang Jihua Wang |
| author_sort | Lu Zhang |
| collection | DOAJ |
| description | There has been no systematic identification and screening of candidate reference genes for normalization of quantitative real-time PCR (qRT-PCR) results in Rhododendron delavayi to date. Therefore, the present study used GAPDH, Act, EF1, Tub-, Tub-5, UEC1, TATA, TATA-2, UEP, TIP41, and Ubiquitin to predict their stabilities on different aboveground tissues (matured leaves (ML), stem tips (STM), and flower buds (FB)) at different developmental stages (young and adult plants) using five statistical algorithms: Delta Ct method, BestKeeper, geNorm, Normfinder, and RefFinder. The findings were confirmed using ML obtained from plants that had been stressed by drought. By using RefFinder with ML samples collected under drought conditions, it was determined that the top five most stable reference genes were GAPDH > UEC1 > Actin > Tubulin- > Tubulin—5, whereas the least stable reference gene was Ubiquitin. In addition, under control conditions, UEC1, UEC2, Actin, and GAPDH were selected as the highest stable potential reference genes at the juvenile stage of R. delavayi with ML and STM. When ML and STM were combined with drought-stressed samples, TIP41, GAPDH, or their combination proved to be the most effective qRT-PCR primers. The findings will aid in the improvement of the precision and reliability of qRT-PCR data and laying the groundwork for future gene functional studies in R. delavayi. |
| first_indexed | 2024-04-13T09:47:33Z |
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| institution | Directory Open Access Journal |
| issn | 1664-8021 |
| language | English |
| last_indexed | 2024-04-13T09:47:33Z |
| publishDate | 2022-04-01 |
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| series | Frontiers in Genetics |
| spelling | doaj.art-6b80ba969d8e45fcb3b2130c9081ca542022-12-22T02:51:42ZengFrontiers Media S.A.Frontiers in Genetics1664-80212022-04-011310.3389/fgene.2022.876482876482Selection and Evaluation of Candidate Reference Genes for Quantitative Real-Time PCR in Aboveground Tissues and Drought Conditions in Rhododendron DelavayiLu Zhang0Lu Zhang1Yanfei Cai2Yanfei Cai3Mingchao Zhang4Guanghui Du5Jihua Wang6Jihua Wang7Flower Research Institute of Yunnan Academy of Agricultural Sciences, Kunming, ChinaNational Engineering Research Center for Ornamental Horticulture, Kunming, ChinaFlower Research Institute of Yunnan Academy of Agricultural Sciences, Kunming, ChinaNational Engineering Research Center for Ornamental Horticulture, Kunming, ChinaSchool of Agriculture, Yunnan University, Kunming, ChinaSchool of Agriculture, Yunnan University, Kunming, ChinaFlower Research Institute of Yunnan Academy of Agricultural Sciences, Kunming, ChinaNational Engineering Research Center for Ornamental Horticulture, Kunming, ChinaThere has been no systematic identification and screening of candidate reference genes for normalization of quantitative real-time PCR (qRT-PCR) results in Rhododendron delavayi to date. Therefore, the present study used GAPDH, Act, EF1, Tub-, Tub-5, UEC1, TATA, TATA-2, UEP, TIP41, and Ubiquitin to predict their stabilities on different aboveground tissues (matured leaves (ML), stem tips (STM), and flower buds (FB)) at different developmental stages (young and adult plants) using five statistical algorithms: Delta Ct method, BestKeeper, geNorm, Normfinder, and RefFinder. The findings were confirmed using ML obtained from plants that had been stressed by drought. By using RefFinder with ML samples collected under drought conditions, it was determined that the top five most stable reference genes were GAPDH > UEC1 > Actin > Tubulin- > Tubulin—5, whereas the least stable reference gene was Ubiquitin. In addition, under control conditions, UEC1, UEC2, Actin, and GAPDH were selected as the highest stable potential reference genes at the juvenile stage of R. delavayi with ML and STM. When ML and STM were combined with drought-stressed samples, TIP41, GAPDH, or their combination proved to be the most effective qRT-PCR primers. The findings will aid in the improvement of the precision and reliability of qRT-PCR data and laying the groundwork for future gene functional studies in R. delavayi.https://www.frontiersin.org/articles/10.3389/fgene.2022.876482/fullgene functiongenomicshousekeeping genemarker-assisted breedingtranscriptomics |
| spellingShingle | Lu Zhang Lu Zhang Yanfei Cai Yanfei Cai Mingchao Zhang Guanghui Du Jihua Wang Jihua Wang Selection and Evaluation of Candidate Reference Genes for Quantitative Real-Time PCR in Aboveground Tissues and Drought Conditions in Rhododendron Delavayi Frontiers in Genetics gene function genomics housekeeping gene marker-assisted breeding transcriptomics |
| title | Selection and Evaluation of Candidate Reference Genes for Quantitative Real-Time PCR in Aboveground Tissues and Drought Conditions in Rhododendron Delavayi |
| title_full | Selection and Evaluation of Candidate Reference Genes for Quantitative Real-Time PCR in Aboveground Tissues and Drought Conditions in Rhododendron Delavayi |
| title_fullStr | Selection and Evaluation of Candidate Reference Genes for Quantitative Real-Time PCR in Aboveground Tissues and Drought Conditions in Rhododendron Delavayi |
| title_full_unstemmed | Selection and Evaluation of Candidate Reference Genes for Quantitative Real-Time PCR in Aboveground Tissues and Drought Conditions in Rhododendron Delavayi |
| title_short | Selection and Evaluation of Candidate Reference Genes for Quantitative Real-Time PCR in Aboveground Tissues and Drought Conditions in Rhododendron Delavayi |
| title_sort | selection and evaluation of candidate reference genes for quantitative real time pcr in aboveground tissues and drought conditions in rhododendron delavayi |
| topic | gene function genomics housekeeping gene marker-assisted breeding transcriptomics |
| url | https://www.frontiersin.org/articles/10.3389/fgene.2022.876482/full |
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