Selection of optimal reference genes for quantitative RT-PCR transcript abundance analysis in white clover (Trifolium repens L.)

Abstract

Quantitative reverse transcription PCR (qRT-PCR) is a widely used method for transcript abundance analyses in plants. Relative quantification by qRT-PCR requires the use of a stably expressed reference gene. There are many ‘housekeeping’ genes reported in different plant species that are used as reference genes. However, it is important that the steady-state mRNA levels of these housekeeping genes are confirmed across different conditions and tissues in each species studied. Prior to this study, no comprehensive work had been performed in identifying optimal reference genes in white clover (Trifolium repens L.). To identify suitable reference genes in white clover, we analysed the transcript abundance stability of seven candidate genes in two organs (leaves and stolons) across two treatments (water-limited and well-watered). ΔCt, NormFinder and ANOVA tests were carried out to evaluate the mRNA level stability of candidate reference genes. According to the ΔCt results, the genes with the most stable mRNA levels were EF1α and ACT11. When stability among groups was evaluated by NormFinder, UBQ was the most stable across all organs and treatments. By multiple criteria, EF1α, followed by ACT11 and UBQ, was the most stably-expressed gene across organs and treatments, and each of these are recommended as reference genes for transcript abundance studies in white clover.