Facultad de Ciencias de la Salud, Instituto de Ciencias de la Salud, Universidad Autónoma de Chile, Avenida Pedro de Valdivia, Santiago, Chile.
Instituto de Investigaciones Agropecuarias (INIA) CRI Rayentué, Sector Los Choapinos, Rengo, Chile.
PLoS One. 2020 Mar 18;15(3):e0228403. doi: 10.1371/journal.pone.0228403. eCollection 2020.
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.
李属砧木属于李亚属（桃）、李属（李）和樱属（樱），与接穗同属或不同属。由于扩大了对生物和非生物胁迫（biotic and abiotic）抗性/耐受性的遗传基础，种间（包括亚种间）杂种的数量有所增加。鉴定与重要性状和响应相关的基因需要进行表达分析。相对定量是最简单、最受欢迎的方法，需要参考基因（管家）来标准化 RT-qPCR 数据。然而，对于杂种李属砧木物种，缺乏经过验证的管家基因。本研究旨在增加适合李属砧木表达分析的管家基因数量。从比较两种砧木亚种根转录组对缺氧处理反应的 RNAseq 数据中预选了 21 个候选管家基因，这两种砧木亚种分别为'马里亚纳 2624'（P. cerasifera Ehrh.×P. munsoniana W. Wight & Hedrick）和'Mazzard F12/1'（P. avium L.）。这些基因代表低、中或高水平表达的基因，在缺氧处理 72 小时后通过 RT-qPCR 进行检测，并使用 NormFinder 软件进行分析。选择了一组七个表达稳定性最高的管家基因，其中两个基因表达水平较低（未知 3、未知 7），五个基因表达水平中等（GTB 1、TUA 3、ATPase P、PRT 6、RP II）。在不同的胁迫条件下，包括冷和热胁迫下的杂种 '马里亚纳 2624'以及氮营养胁迫下的杂种 'Colt'（P. avium × P. pseudocerasus Lindl.）和 'Garnem' [P. dulcis Mill.×（P. persica L.× P. davidiana Carr.）]评估了这些基因的稳定性。geNorm 和 BestKeeper 软件的算法也用于分析这些基因作为管家的性能。根据处理、基因型和评估软件，稳定性排名有所不同，但基因 GTB 1 通常排名最高。然而，根据要评估的胁迫因子和/或基因型，大多数基因都是适用的。当考虑所有条件和基因型时，geNorm 软件无法确定最佳参考基因数量。这些结果强烈表明，在李属砧木中，相对 RT-qPCR 应根据处理和/或基因型分别与各自的最佳管家基因进行分析。
