Systematic identification and validation of the reference genes from 447 transcriptome datasets of moso bamboo (Phyllostachys edulis)

Abstract

Bamboo was one of the first plants to be cultivated in China and is widely used in industry and daily life. The study of gene function has become an important part of bamboo breeding, whereas quantitative real-time PCR (qRT-PCR) is a powerful tool for gene expression analysis. The accuracy of qRT-PCR results largely depends on suitable reference genes. In this study, a transcriptome-wide identification of reference genes was conducted based on 447 transcriptome datasets, comprising 200 tissue samples, 107 treated samples, and 140 samples from various moso bamboo (Phyllostachys edulis) forms. A total of 3444, 1013, and 3962 stably expressed genes were identified from these three groups, respectively. Functional enrichment analysis revealed significant enrichment of these genes in pathways, including the spliceosome, proteasome, and oxidative phosphorylation. Eight candidate genes (ADPRE, GAPDH, TRX, TUBA, NRP, MBF, UNK, and CAM1), were selected for qRT-PCR validation using 112 samples. To assess their stability, five statistical methods (geNorm, NormFinder, BestKeeper, Delta-Ct, and RefFinder) were employed. The most suitable reference genes were ADPRE and GAPDH for different tissues, GAPDH and CAM1 for different treatments, and GAPDH and TRX for various moso bamboo forms. Overall, ADPRE and GAPDH were the most stable reference genes across all conditions, while TUBA and TRX were the least stable reference genes. In addition, a significant negative correlation was found between the Ct values of RT-qPCR and the log2TPM values from the transcriptome data (Ct = −1.534x + 37.221), providing a potential method for estimating gene expression levels. The identified reference genes, particularly ADPRE and GAPDH, provide a robust set of references for gene expression studies in moso bamboo.