Evaluation of stability and validation of reference genes for real time PCR expression studies in leaves and roots of Prunus spp. rootstocks under flooding

Abstract Molecular oxygen (O2) is involved in several biochemical reactions of plants. The low availability of oxygen to cells can lead to an energy crisis, since O2 is the main substrate of respiratory energy metabolism in aerobic organisms, acting as the final acceptor of electrons in the mitochondrial transport chain. When oxygen becomes insufficient for respiration, the plants begin a hypoxia condition. This situation can occur naturally due to excessive rainfall and the existence of poorly drained soils, causing soil flooding. This condition occurs in the South region of Rio Grande do Sul State, in Brazil, which is the largest Brazilian producer of stone fruits, mainly peaches, therefore, its production is adversely affected by flooding. Since the production of stone fruits plants (Prunus spp.) in the region occurs using rootstocks, the aim of this study was to identify potential reference genes for normalization of RT-qPCR data in leaves and roots of four rootstock of Prunus spp. (Capdeboscq, NR0170401, Marianna 2624 and Myrabolan 29-C) in the soil flooding condition. Eight candidate genes were evaluated: PLA2 (phospholipase A2), CYP2 (cyclophilin 2), Ef1α (elongantion factor 1-alpha), RPL13 (ribosomal protein L13), TUA (tubulin alpha), TUB (tubulin beta), RPII (RNA polymerase II), and 18S rRNA (18S ribosomal RNA). Some algorithms for the stability analysis were used, such as the ΔCt comparative method, BestKeeper, NormFinder, geNorm, and the RefFinder tool. As result, the CYP2 and RPII genes presented greater stability in the peach roostocks, whereas RPII and RPL13 were more stable in the plum rootstocks. The Ef1α gene showed no expression stability in the rootstocks ‘NR0170401’ (both tissues) and ‘Myrabolan 29-C’ (roots). The 18S rRNA gene was shown to be unstable for ‘Capdeboscq’ and ‘Marianna 2624’ leaves, while the TUB and PLA2 genes were not stable for roots of these cultivars, respectively. Analysis of the expression of LDH1 (Lactate Dehydrogenase) and ADH1 (alcohol Dehydrogenase) proved the importance of validation of the reference genes to obtain accurate results in RT-qPCR. Taken together, this study selected the reference genes suitable for the reliable normalization of gene expression data in Prunus spp. under flooding.