Selection of endogenous reference genes for qRT-PCR analysis in Camelina sativa and identification of FLOWERING LOCUS C allele-specific markers to differentiate summer- and winter-biotypes

Abstract

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis greatly relies on transcript normalization using stably expressed reference genes. This study identifies reference genes for qRT-PCR analysis in different organs and vernalized tissues of camelina (Camelina sativa). Nineteen stably expressed candidate reference genes from 22,157 genes were identified by RNAseq analysis of pre- and post-vernalized tissues of a summer- (CO46) and winter- (Joelle) biotype. Two additional candidate reference genes were also selected from orthologs of arabidopsis encoding ACTIN2 (ACT2) and NOT INDUCED BY SCLEROTINIA INFECTION (NIS). We also evaluated the transcript levels of three camelina plant genes, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), FLOWERING LOCUS C (FLC), and MADS AFFECTING FLOWERING 2 (MAF2), which are known to be differentially-regulated in response to cold temperatures. The stability of transcript abundance was ranked using NormFinder, geNorm, BestKeeper, and Comparative ΔCT software. EXOCYST COMPLEX COMPONENT SEC3A (SEC3A), UBIQUINONE OXIDOREDUCTASE (UbOxRed), and RING-U-BOX domains-containing protein (RUB) were the most suitable reference genes for normalization of gene expression in camelina. In this study, SEC3A was used to normalize qRT-PCR expression data obtained using two sets of allele-specific FLC markers to differentiate summer- and winter-biotypes across 30 accessions of camelina.