Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/2/2013
Publication Date: 10/1/2013
Citation: Saha, G.C., Vandemark, G.J. 2013. Stability of expression of reference genes among different lentil (Lens culinaris) genotypes subjected to cold stress, white mold disease, and Aphanomyces root rot. Plant Molecular Biology Reporter. 31:1109-1115. Interpretive Summary: Lentils (Lens culinaris) are one of the most ancient and historically important plant sources of protein to human diets, having been domesticated over 8000 years ago by riparian societies along the Euphrates and Tigris rivers. Several factors including cold and diseases cause chronic and occasionally catastrophic losses to lentil production, which requires continual breeding efforts to develop improved varieties. Breeders must be able to identify plants that have beneficial genetic factors (genes) for important traits. RNAs are molecules in plant cells that are used to translate instructions from DNA that result in cellular processes. When different amounts of a specific RNA are detected in plants that are different for a trait, this suggests that the gene may be involved in trait expression. Scientists must use ‘reference’ genes to confirm that other RNAs are present in different amounts. Reference genes should code for RNAs that are present in the same amount under different stages or conditions of plant growth. We tested several possible reference genes in lentils exposed to cold temperatures or white mold disease. We identified two genes; translation initiation factor (TIF), involved in making proteins, and actin, which is part of both muscles and the basic structure of plant cells, that worked very well as reference genes for several different lentil varieties. Five different reference genes were required to examine data obtained from plants infected with white mold but only two reference genes were needed to examine plants subjected to cold stress, which indicates that white mold was a more severe stress. This work will allow us and other scientists to identify genes that are involved in a wide range of important traits in lentil.
Technical Abstract: Lentils have served as an important plant source of dietary protein for over 8000 years. The development of improved lentil varieties is accelerated by a better understanding of the genetic basis of desirable traits, which can be gained by examining patterns of gene expression among phenotypically distinct plants. Precisely quantifying differences in gene expression between plants requires the use of “reference” genes, which should be stably expressed across different lines and treatments, and serve to normalize data for other genes. The objective of this study were to determine the expression stability of several candidate reference genes across five different lentil varieties subjected to cold stress and inoculation with Sclerotinia sclerotiorum, the causal agent of white mold disease. Real-time PCR assays (Sybr Green) were designed for six different genes: TIF, 18S rRNA, actin, ß-tubulin-2, ß-tubulin-3, and GAPDH. Consistent rankings of candidate reference genes were observed for both treatments, with the order from most stable to least stable being TIF, actin, 18S rRNA, ß-tubulin 2, ß-tubulin 3, and GAPDH. Only two reference genes, TIF and actin, were required to normalize data obtained after cold stress but five genes (TIF, actin, 18S rRNA, ß-tubulin 2, and ß-tubulin 3) were required to normalize data obtained from lentils inoculated with S.sclerotiorum. The reference genes reported in this study should have broad applications for examining differential gene expression in lentil in response to a range of abiotic and biotic factors.[National Sclerotinia Initiative]