Identification of candidate reference genes in tropical bamboos stable across species, tissues, and developmental stages

Abstract

Bamboo possesses many unique physiological characteristics, but the molecular understanding of many of these processes remains poorly understood till to date. One major reason is unavailability of sufficient sequence and expression data. Selection of suitable reference genes is pivotal to initiate any gene expression analyses. Although, suitable reference genes have been identified in the temperate bamboo Phyllostachys edulis, it has not been done for tropical bamboo. In this study, expression stability of 10 candidate reference genes were investigated in 4 widely grown tropical bamboo species (Bambusa tulda, B. balcooa, B. bambos, and B. vulgaris), different organs (young leaves from flowering and non flowering culms, flag leaf (leaf just below the mature inflorescence), possible flag leaf (leaf covering the immature inflorescence), culm sheath, internode, root, rhizome, and inflorescence bud), different parts (basal, middle, and tip regions of leaf; internodes located in the basal, middle, and tip region of the branch, and developmental stages early, middle, and late inflorescence buds) by using 3 reliable computational tools (geNorm, NormFinder, and RefFinder). A universal single reference gene for normalization of gene expression data was not identified. However, the eukaryotic initiation factor 4α (eIF4α), clathirin adaptor complexes medium subunit (CAC), and nucleotide tract-binding protein (NTB) were found stable in the selected organs across different bamboo species. On the other hand, eIF4α ranked top when different organs and peptidyl prolyl cis-trans isomerase/cyclophilin (CYP), eukaryotic elongation factor 1α (eEF1α) and ubiquitin 5 (UBQ5) ranked top when different developmental stages of B. tulda were analyzed. Taken together, this study not only identifies reference gene/s that are stable across species, organs, and developmental stages of bamboo, but it also assesses the impacts of major contributing factors regulating expression stability of the reference genes.