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

Authors: Chao, Wun; WANG, HONGXIA - Oak Ridge Institute For Science And Education (ORISE); Horvath, David; Anderson, James

Submitted to: Industrial Crops and Products
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/6/2018
Publication Date: 12/17/2018
Citation: Chao, W.S., Wang, H., Horvath, D.P., Anderson, J.V. 2019. 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. Industrial Crops and Products. 129:495-502. https://doi.org/10.1016/j.indcrop.2018.12.017.
DOI: https://doi.org/10.1016/j.indcrop.2018.12.017

Interpretive Summary: Gene expression can provide insights into the nature and behavior of genetic networks, and quantitative real-time polymerase chain reaction (qRT-PCR) is an important tool for measuring gene expression due to its accuracy, specificity, and sensitivity. The accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. Several strategies have been applied for normalizing real-time PCR data, and a method using stably-expressed endogenous reference gene(s) is the most accepted and frequently used. The aim of this study was to find internal reference genes for qRT-PCR analysis in different organs and vernalized tissues of camelina (Camelina sativa). After evaluated nineteen stably expressed candidate reference genes using a web-based tool, we have identified SEC3A, which encodes a component of the exocyst complex, to be the most appropriate reference gene for accurate normalization of gene expression data in camelina. In this study, we subsequently used SEC3A to normalize the expression of specific alleles of FLOWERING LOCUS C (FLC), which encodes a repressor protein of flowering, using the two newly designed allele-specific markers in a total of 30 summer- and winter-biotypes. We found that these two allele-specific markers can accurately (100%) differentiate summer- and winter-biotypes.

Technical Abstract: Quantitative real-time polymerase chain reaction (qRT-PCR) is an important tool for measuring gene expression due to its accuracy, specificity, and sensitivity. The accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in different organs and vernalized tissues of camelina (Camelina sativa). Nineteen stably expressed candidate reference genes were selected from among 22,157 genes identified by RNAseq analysis of pre- and post-vernalized tissues of a summer- (CO46) and winter- (Joelle) biotype. Two additional candidate reference genes were selected from orthologs of arabidopsis reference gene family members encoding ACT2 and NIS. We also evaluated the transcript levels of three camelina plant genes, SOC1, FLC, and MAF2, which are known to be differentially-regulated in response to cold temperatures. The stability of gene expression was ranked using NormFinder, geNorm, BestKeeper, and Comparative 'CT software. Our analyses revealed SEC3A, UbOxRed, and RUB to be the most appropriate reference genes for accurate normalization of gene expression data 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.