Selection of potential reference genes for RT-qPCR in the plant pathogenic fungus Colletotrichum fructicola

Xingzhou Chen; Xinggang Chen; Qian Tan; Yuan He; Zhikai Wang; Guoying Zhou; Junang Liu

doi:10.3389/fmicb.2022.982748

Selection of potential reference genes for RT-qPCR in the plant pathogenic fungus Colletotrichum fructicola

Front Microbiol. 2022 Aug 8:13:982748. doi: 10.3389/fmicb.2022.982748. eCollection 2022.

Authors

Xingzhou Chen^{1

2

3}, Xinggang Chen^{1

2

3}, Qian Tan^{1

2

3}, Yuan He^{1

2

3}, Zhikai Wang^{1

2

3}, Guoying Zhou^{1

2

3}, Junang Liu^{1

2

3}

Affiliations

¹ Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees, Ministry of Education, Central South University of Forestry and Technology, Changsha, China.
² Key Laboratory of National Forestry and Grassland Administration on Control of Artificial Forest Diseases and Pests in South China, Central South University of Forestry and Technology, Changsha, China.
³ Hunan Provincial Key Laboratory for Control of Forest Diseases and Pests, Central South University of Forestry and Technology, Changsha, China.

Abstract

Colletotrichum is widespread, and these pathogenic fungi can cause various plant diseases. Studies have shown that Colletotrichum fructicola cause oil-tea (Camellia oleifera) anthracnose and is widely distributed as a dominant fungus in all Ca. oleifera-producing regions. Real-time quantitative PCR(RT-qPCR) is considered the most reliable technique for simultaneously measuring relative gene expression levels in different tissues. Target genes are typically quantified using RT-qPCR to explore gene function, and reliable RT-qPCR results require data normalization using stable reference genes. No studies have reported a suitable reference gene in C. fructicola. This study has eight candidate reference genes (CfCk, CfRpp, CfUce, CfRrp, CfAdrh, CfDd, CfAct, and CfTub) which were selected from C. fructicola-Ca. oleifera transcriptome data and evaluated and sequenced using geNorm, NormFinder, and BestKeeper algorithms. The results showed that CfRrp had better stability in C. fructicola, both during the growth of pure pathogenic fungi and during the invasion of different oil-tea leaves. After normalization with CfRrp, the differentially expressed target genes were similar to the transcriptome. Our work provides suitable reference genes for future studies to quantify target gene expression levels in C. fructicola.

Keywords: Camellia oleifera; Colletotrichum fructicola; RT-qPCR; reference gene; transcriptome.