Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress

Lolium temulentum L. (Darnel ryegrass) is a valuable model grass species for the study of stress in forage and turf grasses. Gene expression analysis by quantitative real-time RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in real-time quantitative RT-PCR for abiotic stress studies in L. temulentum. Partial sequences of nine L. temulentum housekeeping genes were obtained by RT-PCR using degenerate primers designed from the corresponding genes in closely related species. Primers for quantitative RT-PCR were designed based on these partial sequences. The housekeeping genes were evaluated for their expression stability in samples from plants subjected to different forms of abiotic stress. The analysis found that eEF-1α and UBQ5 were the most stable and ACT11 was the least stable of the genes tested. Analysis by geNorm indicated that the two most stably expressed housekeeping genes (eEF-1α and UBQ5) should be utilized and are sufficient for normalization of gene expression during stress-related studies in L. temulentum.