Selection and Validation of Reference Genes for qRT-PCR Gene Expression Analysis in Kengyilia melanthera

Junming Zhao; Jian Yang; Xiaoyun Wang; Yanli Xiong; Yi Xiong; Zhixiao Dong; Xiong Lei; Lijun Yan; Xiao Ma

doi:10.3390/genes13081445

Selection and Validation of Reference Genes for qRT-PCR Gene Expression Analysis in Kengyilia melanthera

Genes (Basel). 2022 Aug 14;13(8):1445. doi: 10.3390/genes13081445.

Authors

Junming Zhao¹, Jian Yang¹, Xiaoyun Wang¹, Yanli Xiong¹, Yi Xiong¹, Zhixiao Dong¹, Xiong Lei², Lijun Yan², Xiao Ma¹

Affiliations

¹ College of Grassland Science and Technology, Sichuan Agricultural University, Chengdu 611130, China.
² Sichuan Academy of Grassland Science, Chengdu 611731, China.

Abstract

Kengyilia is a newly established genus. Most species in this genus survive in hash environment, which might be an indicator of an acquirement of stress resistance genes and the potential for molecular breeding in Triticeae species. Quantitative real-time PCR (qRT-PCR) is a widely used technique with varied sensitivity heavily dependent on the optimal level of the reference genes. K. melanthera is a typical psammophyte species which has high drought resistance. The reference genes of K. melanthera are not yet reported. This study aims to evaluate the expression stability of 14 candidate reference genes (EF1A, GAPDH, ACT1, UBI, TUBB3, TIPRL, CACS, PPP2R1B, TUBA1A, EIF4A1, CYPA3, TCTP, ABCG11L, and FBXO6L) under five treatments (drought, heat, cold, salt, and ABA) and find the most stable and suitable one even upon stressed conditions. The software NormFinder, GeNorm, BestKeeper, and RefFinder were used for data analysis. In general, the genes CACS and PPP2R1B are concluded to have the best overall performance under the various treatments. With the ABA treatment, TCTP and TIPRL show the best stability. CACS and TCTP, as well as TIPRL and CYPA3, were most stable under the treatments of cold and salt, respectively. CACS and FBXO6L were ranked the highest with the heat treatment and drought treatment, respectively. Finally, the Catalase-1 (CAT1) gene was used to verify the reliability of the above reference genes. Accordingly, CAT1's expression pattern remained unchanged after normalization with stable reference genes. This study provides beneficial information about the stability and reliability of potential reference genes for qRT-PCR in K. melanthera.

Keywords: Kengyilia melanthera; gene expression; real-time quantitative PCR; reference genes.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Gene Expression
Gene Expression Regulation, Plant*
Genes, Plant*
Poaceae / genetics
Real-Time Polymerase Chain Reaction / methods
Reproducibility of Results
Sodium Chloride
Stress, Physiological / genetics

Substances

Sodium Chloride

Grants and funding

This research was funded by Regional Innovation Cooperation Project of Science and Technology Program of Sichuan Province (grant number 2022YFQ0076), Key R & D Project of Science and Technology Program of Sichuan Province (grant number 2019YFN0170) and Achievements Transformation Project of Science and Technology Program of Sichuan Province (grant number 2022JDZH0002).