Abstract
Prunus rootstock belonging to subgenera Amygdalus (peach), Prunus (plum) and Cerasus (cherry) are either from the same species as the scion or another one. The number of inter-species (including inter-subgenera) hybrids has increased as a result of broadening the genetic basis for stress (biotic and abiotic) resistance/tolerance. Identifying genes associated with important traits and responses requires expression analysis. Relative quantification is the simplest and most popular alternative, which requires reference genes (housekeeping) to normalize RT-qPCR data. However, there is a scarcity of validated housekeeping genes for hybrid Prunus rootstock species. This research aims to increase the number of housekeeping genes suitable for Prunus rootstock expression analysis.Twenty-one candidate housekeeping genes were pre-selected from previous RNAseq data that compared the response of root transcriptomes of two rootstocks subgenera to hypoxia treatment, ‘Mariana 2624’ (P. cerasifera Ehrh.× P. munsoniana W. Wight & Hedrick), and ‘Mazzard F12/1’ (P. avium L.). Representing groups of low, intermediate or high levels of expression, the genes were assayed by RT-qPCR at 72 hours of hypoxia treatment and analyzed with NormFinder software. A sub-set of seven housekeeping genes that presented the highest level of stability were selected, two with low levels of expression (Unknown 3, Unknown 7) and five with medium levels (GTB 1, TUA 3, ATPase P, PRT 6, RP II). The stability of these genes was evaluated under different stress conditions, cold and heat with the hybrid ‘Mariana 2624’ and N nutrition with the hybrids ‘Colt’ (P. avium × P. pseudocerasus Lindl.) and ‘Garnem’ [P. dulcis Mill.× (P. persica L.× P. davidiana Carr.)]. The algorithms of geNorm and BestKeeper software also were used to analyze the performance of these genes as housekeepers.Stability rankings varied according to treatments, genotypes and the software for evaluation, but the gene GBT 1 often had the highest ranking. However, most of the genes are suitable depending on the stressor and/or genotype to be evaluated. No optimal number of reference genes could be determined with geNorm software when all conditions and genotypes were considered. These results strongly suggest that relative RT-qPCR should be analyzed separately with their respective best housekeeper according to the treatment and/or genotypes in Prunus spp. rootstocks.
Highlights
Stone fruit trees (Prunus spp.) are economically important because they produce edible fruits such as peaches, cherries, and apricots
A total of 21 genes were selected for this study to identify housekeeping genes with different levels of expression for RT-qPCR studies of Prunus spp. (Table 1)
This range of gene expression was chosen to identify suitable reference genes for better relative quantification of genes of interest with low, intermediate and high levels of expression. They comprise both widely-used classical reference genes like glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TUB and ACT [19], and ones less commonly used like a cyclophilin-like peptidyl-prolyl cis-trans isomerase
Summary
Stone fruit trees (Prunus spp.) are economically important because they produce edible fruits such as peaches, cherries, and apricots. Most stone fruit trees are grafted on rootstocks (seedlings or clonally propagated) that belong to either the same or other Prunus species [1]. These fruit trees are composed of two parts, the rootstock and scion. Deepening our understanding of the molecular basis of the traits/responses of Prunus rootstocks and identifying the genes involved will be critical steps toward improving rootstock plants by marker-assisted selection (MAS) methods [7]. Other approaches are possible, based on direct modification of key genes associated with traits of interest by genetically engineering Prunus species [8]. The molecular basis of Prunus rootstock traits and responses remain largely unknown. Gene expression studies using NGS ( Generation Sequencing) and/or candidate genes from other model plants represent valuable approaches to identifying key candidate genes underlying traits of interest [9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
