Stability of Expression of Reference Genes Among Different Lentil (Lens culinaris) Genotypes Subjected to Cold Stress, White Mold Disease, and Aphanomyces Root Rot

Abstract

Precise quantification of differences in gene expression between plants requires the use of “reference” genes, which are stably expressed across different lines and treatments and serve as endogenous controls for normalizing gene expression data. The objectives of this study were to determine the expression stability of several reference genes across five different lentil varieties subjected to either cold stress, inoculation with Sclerotinia sclerotiorum, the causal agent of white mold disease, or inoculation with Aphanomyces euteiches, the causal agent of Aphanomyces root rot. Expression stability was examined in the stems and leaves of plants subjected to cold stress or inoculation with S. sclerotiorum and in the roots of plants inoculated with A. euteiches. Real-time PCR assays (SYBR Green) were designed for six different genes: translation initiation factor (TIF), 18S rRNA, actin, β-tubulin-2, β-tubulin-3, and glyceraldehyde 3-phosphate dehydrogenase. TIF, actin, and 18S rRNA tended to be the most stably expressed genes, with expression stability (M) values less than 0.5 during cold stress and inoculation with A. euteiches. Two reference genes were required to normalize data from plants exposed to cold stress or inoculated with A. euteiches. The reference genes exhibited the lowest expression stability in plants inoculated with S. sclerotiorum, for which five reference genes were required to normalize data. The reference genes reported in this study appear to have a promise for examining gene expression in lentil foliar and root tissues in response to diverse abiotic and biotic factors.