Selection and validation of reference genes for target gene analysis with quantitative RT-PCR in leaves and roots of bermudagrass under four different abiotic stresses.

Abstract

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is an effective method for quantifying expression levels of target genes. The accuracy of qRT-PCR results is largely dependent on the selection of stable reference genes. The stability of reference gene expression may vary with plant species and environmental conditions. The objective of this study was to select stable reference genes for qRT-PCR analysis of target genes in different organs under different abiotic stresses for a perennial grass species, bermudagrass (Cynodon dactylon). The stability of eight potential reference genes (TUB, ACT, GAPDH, EF1α, TIP41, PP2A, CACS and UPL7) was evaluated under four different abiotic stresses (salt, drought, cold and heat) and in leaves and roots of bermudagrass. Four programs (geNorm, NormFinder, BestKeeper and RefFinder) were employed to evaluate the stability of reference gene expression and to identify the most stable reference genes for bermudagrass. Eight potential reference genes exhibited differential expression stability in leaves and roots under salt, drought, cold and heat stress. The expression levels of PP2A and CACS were stable in roots and leaves under salt stress, in leaves under drought stress and in roots exposed to cold and heat stress. EF1α and TIP41 expression was stable in roots of drought-stressed plants. UPL7, TUB and GAPDH were stably expressed in leaves under cold stress. Expression levels of PP2A and TIP41 were stable in leaves under heat stress. The use of the reference genes identified as internal controls for examination of gene expression patterns and quantification of expression levels of target genes will enable accurate qRT-PCR analysis in bermudagrass.