Identifying and validating housekeeping hybrid Prunus spp. genes for root gene-expression studies

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 interspecies (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 PLOS ONE

Relative Telomere Length and Telomerase Reverse Transcriptase (TERT) Expression Are Associated with Age in Almond (Prunus dulcis [Mill.] D.A.Webb)

“…To quantify expression of TERT in age cohort individuals, qRT-PCR was performed in triplicate for each sample. The gene RPII from peach was used as a reference [54,55], and the sequence for the TERT gene was derived from the Texas genome (https://www. rosaceae.org/analysis/295) using the homologous peach gene sequence as a reference [56].…”

Telomere length andTERTexpression are associated with age in almond (Prunus dulcis[Mill.] D.A.Webb)

“…To quantify expression of TERT in age cohort individuals, qRT-PCR was performed in triplicate for each sample. The gene RPII from peach was used as a reference (Bastias et al ., 2020; Tong et al ., 2009), and the sequence for the TERT gene was derived from the ’Texas’ genome (https://www.rosaceae.org/analysis/295) using the homologous peach gene sequence as a reference (Alioto et al ., 2020). Primer sequences are shown in Table 2, and all oligos were synthesized by MilliporeSigma (Burlington, MA) and resuspended to a concentration of 100 µM upon arrival.…”

FEAtl: A Comprehensive web-based Expression Atlas for functional genomics in Tropical and Subtropical Fruit Crops

“…The low tau scores of housekeeping genes indicate that they are expressed at similar levels across different tissues and conditions, which is a key characteristic of these genes (42). Research on housekeeping genes in Prunus rootstocks identi ed several candidates with stable expression levels showed medium expression levels and high stability under different stress conditions (43). Additionally, a study on human housekeeping genes highlighted that a signi cant fraction of genes is broadly expressed in multiple tissues, emphasizing the importance of understanding tissue-speci c gene expression patterns (44).…”