Comprehensive Identification of Reliable Reference Genes for qRT-PCR Normalization of Fusarium oxysporum-Resistant Genes’ Expressions in Lilium sargentiae Wilson
Source abstract
Abstract
Fusarium wilt (caused by Fusarium oxysporum f. sp. Lilii) is one of the most damaging diseases in lily (Lilium sargentiae Wilson). Although some F. oxysporum-resistant lily varieties have been identified and are being utilized in resistant breeding, the regulation network of the resistance-associated mechanisms is yet to be studied due to the lack of reliable reference genes for qRT-PCR (quantitative reverse transcription PCR) normalization. The reliability of results by qRT-PCR relies mainly on the stability of the reference genes. This study investigated the reliability of nine candidate reference genes (CYP, EF1-α, GAPDH, TUB, UBQ, AQP, HIS, PGK, and RPL13) for qRT-PCR analysis of F. oxysporum-resistant genes. Expression stability analysis via common programs GeNorm, BestKeeper, and NormFinder, at different time points post-inoculation of F. oxysporum, revealed that all nine genes met the basic requirements of reference genes. Amongst them, HIS and GAPDH displayed the highest and the lowest expression stability, respectively. The reliability of HIS was further validated by analyzing the expression levels of four resistance-related candidate genes. The expression patterns of the four target genes were consistent with their responses to pathogenetic fungi in other plants. Our results show that HIS is the most suitable reference gene for accurately normalizing F. oxysporum-resistant genes’ expressions in L. sargentiae.
Source: crossref · Quality: baseline
Research context
OpenAlex topics: Molecular Biology Techniques and Applications · Biological Stains and Phytochemicals · Genomics and Phylogenetic Studies
SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.
Affiliations
Flower Research Institute of Yunnan Academy of Agricultural Sciences, Key Lab of Yunnan Flower Breeding, National Engineering Research Center for Ornamental Horticulture, Kunming 650205, China
Funding
National Key Research and Development Program of China · the Green Food Brand-Build a Special Project (Floriculture) supported by Yunnan Provincial Finance Department · the Joint Lab of Yunnan Seed Industry · Open Fund of National Engineering Research Center for Ornamental Horticulture and Yunnan Key Laboratory of Flower Breeding
References (50)
Show source-provided references
1. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes10.1186/gb-2002-3-7-research0034 ↗
2. Validation of Reference Genes for Quantitative Real-Time PCR during Bicolor Tepal Development in Asiatic Hybrid Lilies (Lilium spp.)10.3389/fpls.2017.00669 ↗
3. Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya10.1186/s13007-019-0455-3 ↗
4. De novo transcriptome sequencing of Momordica cochinchinensis to identify genes involved in the carotenoid biosynthesis10.1007/s11103-012-9919-9 ↗
5. De novo transcriptome analysis of Osmanthus serrulatus Rehd. flowers and leaves by Illumina sequencing10.1016/j.bse.2015.07.027 ↗
6. Cao, A., Shao, D., Cui, B., Tong, X., Zheng, Y., Sun, J., and Li, H. (2019). Screening the Reference Genes for Quantitative Gene Expression by RT-qPCR During SE Initial Dedifferentiation in Four Gossypium hirsutum Cultivars that Have Different SE Capability. Genes, 10.10.3390/genes10070497 ↗
7. Yan, F., Zhu, Y., Zhao, Y., Wang, Y., Li, J., Wang, Q., and Liu, Y. (2020). De novo transcriptome sequencing and analysis of salt-, alkali-, and drought-responsive genes in Sophora alopecuroides. BMC Genom., 21.10.1186/s12864-020-06823-4 ↗
8. Wu, Y., Zhou, J., Liu, Y., Gu, Y., Zhang, H., Ahmad, F., Wang, G., and Ren, L. (2022). Selection and Validation of Reliable Reference Genes for qRT-PCR Normalization of Bursaphelenchus xylophilus from Different Temperature Conditions and Developmental Stages. Appl. Sci., 12.10.3390/app12062880 ↗
9. Real-time RT-PCR normalisation; strategies and considerations10.1038/sj.gene.6364190 ↗
10. Liu, Q., Qi, X., Yan, H., Huang, L., Nie, G., and Zhang, X. (2018). Reference Gene Selection for Quantitative Real-Time Reverse-Transcriptase PCR in Annual Ryegrass (Lolium multiflorum) Subjected to Various Abiotic Stresses. Molecules, 23.10.3390/molecules23010172 ↗
11. Selection and validation of reference genes for quantitative RT-PCR analysis in peach fruit under different experimental conditions10.1016/j.scienta.2017.07.004 ↗
12. Quantification of mRNA using real-time RT-PCR10.1038/nprot.2006.236 ↗
13. Microarray data normalization and transformation10.1038/ng1032 ↗
14. Validation of suitable reference genes for quantitative gene expression analysis in Tripterygium wilfordii10.1007/s11033-019-04867-8 ↗
15. Selection and Validation of Reference Genes for Gene Expression Studies in Codonopsis pilosula Based on Transcriptome Sequence Data10.1038/s41598-020-58328-5 ↗
16. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber10.1016/j.ab.2009.12.008 ↗
17. Heat-stress-dependency and developmental modulation of gene expression: The potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR10.1093/jxb/erg244 ↗
18. Hong, S.-Y., Seo, P.J., Yang, M.-S., Xiang, F., and Park, C.-M. (2008). Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol., 8.10.1186/1471-2229-8-112 ↗
19. Selection of reliable reference genes for gene expression studies in Rhododendron micranthum Turcz10.1016/j.scienta.2012.02.013 ↗
20. Selection of reference genes for real-time RT-PCR normalization in brown alga Undaria pinnatifida10.1007/s10811-018-1576-1 ↗
21. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization10.1016/j.ab.2005.05.022 ↗
22. Selection of reference genes for quantitative real-time RT-PCR analysis in citrus10.1007/s11033-011-0925-9 ↗
23. Identification of valid reference genes for quantitative RT-PCR in Caragana microphylla under salt and drought stresses10.1007/s12298-020-00874-3 ↗
24. Resistance to Fusarium oxysporum f. sp. lilii in Lilium10.17660/ActaHortic.2003.620.38 ↗
25. Identification of Genes Differentially Expressed in a Resistant Reaction to Fusarium Oxysporum in Lilium Regale by SSH10.1016/j.ieri.2013.11.076 ↗
26. In Vitro Selection for Fusarium Resistant Oriental Lily Mutants Using Culture Filtrate of the Fungal Agent10.17660/ActaHortic.2014.1027.22 ↗
27. Resistance Identification and Genetic Diversity Among Lily Varieties Based on Resistance Gene Analogue
28. The PR10 gene family is highly expressed in Lilium regale Wilson during Fusarium oxysporum f. sp. lilii infection10.1007/s13258-014-0185-x ↗
29. Lilium regale Wilson WRKY2 Regulates Chitinase Gene Expression During the Response to the Root Rot Pathogen Fusarium oxysporum10.3389/fpls.2021.741463 ↗
30. Transcriptome Sequencing Analysis Provides Insights Into the Response to Fusarium oxysporum in Lilium pumilum10.1177/1176934319838818 ↗
31. Pollination in Lilium sargentiae (Liliaceae) and the first confirmation of long-tongued hawkmoths as a pollinator niche in Asia10.1111/jse.12419 ↗
32. Van Huylenbroeck, J. (2018). Ornamental Crops, Springer International Publishing.10.1007/978-3-319-90698-0 ↗
33. Genetic variation in resistance to Fusarium oxysporum f.sp. lilii in the genus Lilium10.1111/j.1744-7348.1994.tb04947.x ↗
34. Identification and ITS Sequence Analysis of Pathogen of Lily Wilt
35. Imai, T., Ubi, B.E., Saito, T., and Moriguchi, T. (2014). Evaluation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in Pyrus pyrifolia using different tissue samples and seasonal conditions. PLoS ONE, 9.10.1371/journal.pone.0086492 ↗
36. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations10.1023/B:BILE.0000019559.84305.47 ↗
37. Identification of reference genes provides functional insights into meiotic recombination suppressors in Gerbera hybrida10.1016/j.hpj.2020.09.008 ↗
38. Data Processing System (DPS) software with experimental design, statistical analysis and data mining developed for use in entomological research10.1111/j.1744-7917.2012.01519.x ↗
39. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method10.1006/meth.2001.1262 ↗
40. Application of antagonistic rhizobacteria for control of Fusarium seedling blight and basal rot of lily10.1007/s13313-011-0040-3 ↗
41. A bZIP transcription factor, LrbZIP1, is involved in Lilium regale Wilson defense responses against Fusarium oxysporum f. sp. lilii10.1007/s13258-014-0214-9 ↗
42. Isolation and characterization of a Fusarium oxysporum-resistant gene LrGLP1 from Lilium regale Wilson10.1007/s11627-017-9829-2 ↗
43. Differential gene expression in incompatible interaction between Lilium regale Wilson and Fusarium oxysporum f. sp. lilii revealed by combined SSH and microarray analysis10.1134/S0026893314060144 ↗
44. Evaluation of candidate reference genes for RT-qPCR in lily (Lilium brownii)10.1080/14620316.2014.11513089 ↗
45. Li, X., Cheng, J., Zhang, J., Da Teixeira Silva, J.A., Wang, C., and Sun, H. (2015). Validation of Reference Genes for Accurate Normalization of Gene Expression in Lilium davidii var. unicolor for Real Time Quantitative PCR. PLoS ONE, 10.10.1371/journal.pone.0141323 ↗
46. Resistance Identification of lily Germplasms to Fusarium oxysporum and Screening of the Resistance Identification of lily Germplasms to Fusarium oxysporum and Screening of the Resistance Related Genes
47. Non-host disease resistance response in pea (Pisum sativum) pods: Biochemical function of DRR206 and phytoalexin pathway localization10.1016/j.phytochem.2014.10.013 ↗
48. Transcriptomic changes following the compatible interaction Vitis vinifera-Erysiphe necator. Paving the way towards an enantioselective role in plant defence modulation10.1016/j.plaphy.2013.03.024 ↗
49. Physcomitrella patens Activates Defense Responses against the Pathogen Colletotrichum gloeosporioides10.3390/ijms160922280 ↗
50. Li, L., Sun, W., Zhou, P., Wei, H., Wang, P., Li, H., Rehman, S., Li, D., and Zhuge, Q. (2021). Genome-Wide Characterization of Dirigent Proteins in Populus: Gene Expression Variation and Expression Pattern in Response to Marssonina brunnea and Phytohormones. Forests, 12.10.3390/f12040507 ↗