Identification and validation of qRT-PCR reference genes for analyzing grape infection with gray mold.
Grape gray mold
QRT-PCR
Reference genes
Journal
BMC genomics
ISSN: 1471-2164
Titre abrégé: BMC Genomics
Pays: England
ID NLM: 100965258
Informations de publication
Date de publication:
24 Oct 2024
24 Oct 2024
Historique:
received:
02
06
2024
accepted:
09
10
2024
medline:
25
10
2024
pubmed:
25
10
2024
entrez:
25
10
2024
Statut:
epublish
Résumé
Grapes are highly valued for their nutritional and economic benefits, and have been widely studied for their biological attributes such as fruit development, quality formation, and stress resistance. One significant threat to grape quality is gray mold, caused by Botrytis cinerea, which can infect the flowers, fruits, leaves, and stems. The quantitative real-time PCR (qRT-PCR), known for its high sensitivity and quantitative accuracy, is an essential tool for analyzing gene expression related to the pathogenesis of gray mold, thereby providing deeper insights into the disease. In this study, we aim to identify stable internal reference genes crucial for accurate gene expression analysis via qRT-PCR. Utilizing transcriptome data from grapes under various disease stresses, we identified twelve candidate reference genes with consistently high expression levels. The stability of these genes was assessed through delta-CT, geNorm, NormFinder, BestKeeper, and RefFinder analyses after establishing the cycling thresholds (Ct) in different grape varieties treated with Botrytis cinerea. Our findings reveal that VIT-17s0000g02750 and VIT-06s0004g04280 exhibit stable expression and are suitable as new reference genes. This foundational work supports further research into the molecular mechanisms of grape biological processes.
Sections du résumé
BACKGROUND
BACKGROUND
Grapes are highly valued for their nutritional and economic benefits, and have been widely studied for their biological attributes such as fruit development, quality formation, and stress resistance. One significant threat to grape quality is gray mold, caused by Botrytis cinerea, which can infect the flowers, fruits, leaves, and stems. The quantitative real-time PCR (qRT-PCR), known for its high sensitivity and quantitative accuracy, is an essential tool for analyzing gene expression related to the pathogenesis of gray mold, thereby providing deeper insights into the disease.
RESULT
RESULTS
In this study, we aim to identify stable internal reference genes crucial for accurate gene expression analysis via qRT-PCR. Utilizing transcriptome data from grapes under various disease stresses, we identified twelve candidate reference genes with consistently high expression levels. The stability of these genes was assessed through delta-CT, geNorm, NormFinder, BestKeeper, and RefFinder analyses after establishing the cycling thresholds (Ct) in different grape varieties treated with Botrytis cinerea.
CONCLUSIONS
CONCLUSIONS
Our findings reveal that VIT-17s0000g02750 and VIT-06s0004g04280 exhibit stable expression and are suitable as new reference genes. This foundational work supports further research into the molecular mechanisms of grape biological processes.
Identifiants
pubmed: 39448910
doi: 10.1186/s12864-024-10889-9
pii: 10.1186/s12864-024-10889-9
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
997Subventions
Organisme : The Science and Technology Project of Anhui Province
ID : 2022j11020010
Organisme : The Science and Technology Project of Anhui Province
ID : 2022j11020010
Organisme : The Science and Technology Project of Anhui Province
ID : 2022j11020010
Organisme : The Science and Technology Project of Anhui Province
ID : 2022j11020010
Organisme : The Science and Technology Project of Anhui Province
ID : 2022j11020010
Organisme : The Science and Technology Project of Anhui Province
ID : 2022j11020010
Organisme : The Science and Technology Project of Anhui Province
ID : 2022j11020010
Informations de copyright
© 2024. The Author(s).
Références
Choi NR, Kim JN, Kwon MJ, Lee JR, Kim SC, Lee MJ, Choi WG, Kim BJ. Grape seed powder increases gastrointestinal motility. Int J Med Sci. 2022;19(5):941–51.
doi: 10.7150/ijms.72529
pubmed: 35693751
pmcid: 9149643
Li Z, Jiang E, Liu M, Sun Q, Gao Z, Du Y. Effects of coverlys TF150
doi: 10.3390/ijms242316659
pubmed: 38068982
pmcid: 10706710
Shen F, Wu W, Han X, Wang J, Li Y, Liu D. Study on the occurrence law and green control of grape gray mold from the perspective of ecological balance. Bioengineered. 2021;12(1):779–90.
doi: 10.1080/21655979.2021.1888578
pubmed: 33645432
pmcid: 8806280
Zhu L, Yang C, You Y, Liang W, Wang N, Ma F, Li C. Validation of reference genes for qRT-PCR analysis in peel and flesh of six apple cultivars (Malus domestica) at diverse stages of fruit development. Sci Hort. 2019;244:165–71.
doi: 10.1016/j.scienta.2018.09.033
Demidenko NV, Penin AA. Comparative analysis of Gene expression level by quantitative real-time PCR has limited application in objects with different morphology. PLoS One. 2012;7(5):e38161.
doi: 10.1371/journal.pone.0038161
pubmed: 22666472
pmcid: 3364230
Huggett J, Dheda K, Bustin S, Zumla A. Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 2005;6(4):279–84.
doi: 10.1038/sj.gene.6364190
pubmed: 15815687
Wan H, Zhao Z, Qian C, Sui Y, Malik AA, Chen J. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem. 2010;399(2):257–61.
doi: 10.1016/j.ab.2009.12.008
pubmed: 20005862
Zhang W, Xu J, Wang Q, Li J, Li Y, Dong M, Sun H. Transcriptome-based identification of the optimal reference genes for quantitative real-time polymerase chain reaction analyses of Lingonberry fruits throughout the growth cycle. Plants-Basel. 2023;12(24):4180.
doi: 10.3390/plants12244180
pubmed: 38140507
pmcid: 10748091
Zhang K, Fan W, Chen D, Jiang L, Li Y, Yao Z, Yang Y, Qiu D. Selection and validation of reference genes for quantitative gene expression normalization in Taxus spp. Sci Rep. 2020;10(1):22205.
doi: 10.1038/s41598-020-79213-1
pubmed: 33335184
pmcid: 7747704
Zhao J, Zhou M, Meng Y. Identification and validation of reference genes for RT-qPCR analysis in Switchgrass under heavy metal stresses. Genes. 2020;11(5):502.
doi: 10.3390/genes11050502
pubmed: 32375288
pmcid: 7291066
Wang L, Qiao F, Geng G, Lu Y. Evaluation of candidate reference genes for gene expression analysis in Wild Lamiophlomis rotata. Genes. 2023;14(3):573.
doi: 10.3390/genes14030573
pubmed: 36980847
pmcid: 10048348
Su X, Lu L, Li Y, Zhen C, Hu G, Jiang K, Yan Y, Xu Y, Wang G, Shi M, et al. Reference gene selection for quantitative real-time PCR (qRT-PCR) expression analysis in Galium aparine L. PLoS One. 2020;15(2):e0226668.
doi: 10.1371/journal.pone.0226668
pubmed: 32017769
pmcid: 6999859
Liu Y, Zhang C, Harijati N, Diao Y, Liu E, Hu Z. Selection and evaluation of reference genes for RT-qPCR analysis in amorphophallus konjac based on transcriptome data. Genes. 2023;14(8):1513.
doi: 10.3390/genes14081513
pubmed: 37628565
pmcid: 10454643
Li C, Xu J, Deng Y, Sun H, Li Y. Selection of reference genes for normalization of cranberry (Vaccinium macrocarpon Ait.) gene expression under different experimental conditions. PLoS One. 2019;14(11):e0224798.
doi: 10.1371/journal.pone.0224798
pubmed: 31715627
pmcid: 6850891
Lian C, Zhang B, Yang J, Lan J, Yang H, Guo K, Li J, Chen S. Validation of suitable reference genes by various algorithms for gene expression analysis in Isodon rubescens under different abiotic stresses. Sci Rep. 2022;12(1):19599.
doi: 10.1038/s41598-022-22397-5
pubmed: 36380055
pmcid: 9666634
Chen C, Wu J, Hua Q, Tel-Zur N, Xie F, Zhang Z, Chen J, Zhang R, Hu G, Zhao J, et al. Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya. Plant Methods. 2019;15:70.
doi: 10.1186/s13007-019-0455-3
pubmed: 31333756
pmcid: 6613322
Wang W, Zhang X, Xu X, Xu X, Fu L, Chen H. Systematic identification of reference genes for qRT-PCR of Ardisia Kteniophylla A. DC under different experimental conditions and for anthocyanin-related genes studies. Front Plant Sci. 2023;14:1284007.
doi: 10.3389/fpls.2023.1284007
pubmed: 38023897
pmcid: 10656778
Xie X, Wang Y. VqDUF642, a gene isolated from the Chinese grape Vitis Quinquangularis, is involved in berry development and pathogen resistance. Planta. 2016;244(5):1075–94.
doi: 10.1007/s00425-016-2569-4
pubmed: 27424038
Kumar V, Sharma R, Trivedi PC, Vyas GK, Khandelwal V. Traditional and novel references towards systematic normalization of qRT-PCR data in plants. Aust J Crop Sci. 2011;5(11):1455–68.
Gou T, Yang L, Hu W, Chen X, Zhu Y, Guo J, Gong H. Silicon improves the growth of cucumber under excess nitrate stress by enhancing nitrogen assimilation and chlorophyll synthesis. Plant Physiol Biochem. 2020;152:53–61.
doi: 10.1016/j.plaphy.2020.04.031
pubmed: 32388420
Zhou Y, Zhang Y, Mu D, Lu Y, Chen W, Zhang Y, Zhang R, Qin Y, Yuan J, Pan L, et al. Selection of Reference Genes in Evodia rutaecarpa var. officinalis and Expression Patterns of Genes Involved in Its Limonin Biosynthesis. Plants-Basel. 2023;12(18):3197.
doi: 10.3390/plants12183197
pubmed: 37765365
pmcid: 10534417
Chen Y, Tan Z, Hu B, Yang Z, Xu B, Zhuang L, Huang B. Selection and validation of reference genes for target gene analysis with quantitative RT-PCR in leaves and roots of bermudagrass under four different abiotic stresses. Physiol Plant. 2015;155(2):138–48.
doi: 10.1111/ppl.12302
pubmed: 25331743
Liu H, Liu L, Han H, Liu K, Wang H. Panel of suitable reference genes and its gender differences of fetal rat liver under physiological conditions and exposure to dexamethasone during pregnancy. Reprod Toxicol. 2021;100:74–82.
doi: 10.1016/j.reprotox.2021.01.005
pubmed: 33453333
Andersen CL, Jensen JL, Orntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004;64(15):5245–50.
doi: 10.1158/0008-5472.CAN-04-0496
pubmed: 15289330
Luo M, Gao Z, Li H, Li Q, Zhang C, Xu W, Song S, Ma C, Wang S. Selection of reference genes for miRNA qRT-PCR under abiotic stress in grapevine. Sci Rep. 2018;8:4444.
doi: 10.1038/s41598-018-22743-6
pubmed: 29535408
pmcid: 5849727
Song Y, Hanner RH, Meng B. Probing into the effects of grapevine leafroll-associated viruses on the physiology, fruit quality and gene expression of grapes. Viruses-Basel. 2021;13(4):593.
doi: 10.3390/v13040593
Kong Q, Deng R, Li X, Zeng Q, Zhang X, Yu X, Ren X. Based on RNA-Seq analysis identification and expression analysis of Trans-Scripusina synthesize-related genes of UV-treatment in postharvest grape fruit. Arch Biochem Biophys. 2020;690:108471.
Chen M, Wang Z, Hao Z, Li H, Feng Q, Yang X, Han X, Zhao X. Screening and validation of appropriate reference genes for real-time quantitative PCR under PEG, NaCl and ZnSO
Zhu X, Wang B, Wang X, Wei X. Screening of stable internal reference gene of Quinoa under hormone treatment and abiotic stress. Physiol Mol Biology Plants. 2021;27(11):2459–70.
doi: 10.1007/s12298-021-01094-z
Bakshi A, Moin M, Gayatri MB, Reddy ABM, Datla R, Madhav MS, Kirti PB. Involvement of target of rapamycin (TOR) signaling in the regulation of crosstalk between ribosomal protein small subunit 6 Kinase-1 (RPS6K-1) and ribosomal proteins. Plants-Basel. 2023;12(1):176.
doi: 10.3390/plants12010176
pubmed: 36616305
pmcid: 9824793
Wang L, Qiu T, Yue J, Guo N, He Y, Han X, Wang Q, Jia P, Wang H, Li M, et al. Arabidopsis ADF1 is regulated by MYB73 and is involved in response to salt stress affecting actin filament organization. Plant Cell Physiol. 2021;62(9):1387–95.
doi: 10.1093/pcp/pcab081
pubmed: 34086948