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

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.

Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Colletotrichum fructicola from Litchi

“…Previous research has consistently identified RP-encoding genes (like RPL and RPS) as optimal reference genes for studying gene expression. For example, in C. kahawae samples, RPL18 was revealed served as the optimal reference gene [24]. Based on our results, the stability of RPS5 as a reference gene in experiments related to different culture mediums is unparalleled.…”

“…Figueiredo determined that three reference genes were sufficient for the entire dataset, while four reference genes were sufficient for Catimor 88 and two or three reference genes were sufficient for Caturra hypocotyls inoculated with Colletotrichum kahawae [43]. However, utilizing an excessive number of reference genes may create practical challenges during the execution and operation of a study [24]. Therefore, to ensure reliable normalization, it is optimal to choose a minimum of two highly accurate reference genes, and this has been confirmed in previous studies [48,50].…”

“…To identify the most suitable reference genes, it is necessary to systematically design experiments to assess the stability of various candidate reference genes across different species and experimental conditions. There were five reports of suitable reference genes for RT-qPCR analysis in Colletotrichum spp., but only one for those in C. fructicola [23][24][25][26][27], which has restricted further investigations into the functional genes of C. fructicola. Thus, it is necessary to identify suitable reference genes in C. fructicola under different experimental conditions.…”

Selection and Validation of Reference Genes for Reverse-Transcription Quantitative PCR Analysis in Sclerotium rolfsii

“…RT-qPCR is a widely used technique to quantify the gene expression profiles in species [16]. At present, suitable reference genes have been screened and validated in many fungal species [17][18][19][20]. However, there are no available reference genes reported for S. rolfsii.…”