The adaptive mechanisms in Agave species enable them to survive and exhibit remarkable tolerance to abiotic stresses. Quantitative real-time PCR is a highly reliable approach for validation of targeted differential gene expression. However, stable housekeeping gene(s) is prerequisite for accurate normalization of expression data by qRT-PCR. Till date, no systematic validation study for candidate housekeeping gene identification or evaluation has been carried-out in Agave species. A total of 17 candidate housekeeping genes were identified from the de novo assembled transcriptomic data of A. sisalana and rigorously analyzed for expression stability assessment under drought, heat, cold and NaCl stress. Different statistical algorithms like geNorm, BestKeeper, NormFinder, and RefFinder on expression data determined the superior housekeeping gene(s) for accurate normalization of the gene of interest (GOI). The comprehensive evaluation revealed the β-Tub 4, WIN-1 and CYC-A as the most stable, while EEF1α, GAPDH, and UBE2 were ranked as the least stable genes in pooled samples. Pairwise combination by geNorm showed that up to two housekeeping genes would be adequate to normalize the GOI expression data precisely. Validation of identified most and least stable housekeeping genes was carried-out by normalizing the expression data of AsHSP20 under abiotic stress conditions. Copy number of AsHSP20 gene supports the reliability of the genes used for normalization. This is the first report on the screening and validation of the housekeeping genes under abiotic stress condition in A. sisalana that would assist to understand the stress tolerance mechanisms by novel gene identification and accurate validation.

中文翻译：

---

鉴定和验证非生物胁迫下龙舌兰（一种CAM植物）中用于qRT-PCR数据归一化的优良管家基因。

龙舌兰物种的适应机制使它们能够生存并表现出对非生物胁迫的显着耐受性。实时定量PCR是验证靶向差异基因表达的高度可靠的方法。但是，稳定的管家基因是通过qRT-PCR准确标准化表达数据的前提。迄今为止，尚未在龙舌兰物种中进行用于候选管家基因鉴定或评估的系统验证研究。从剑麻的从头组装转录组数据中鉴定出总共17个候选管家基因并严格分析干旱，高温，寒冷和NaCl胁迫下的表达稳定性。对表达数据使用不同的统计算法（例如geNorm，BestKeeper，NormFinder和RefFinder）确定了用于优化目的基因（GOI）准确归一化的上等管家基因。综合评价揭示了β -浴池4，WIN - 1和CYC -一个最稳定，而EEF1α，GAPDH，和UBE2在合并的样本中被列为最不稳定的基因。geNorm的成对组合显示，最多两个管家基因足以精确地标准化GOI表达数据。通过在非生物胁迫条件下标准化AsHSP20的表达数据，对鉴定出的最稳定和最不稳定的管家基因进行验证。AsHSP20基因的拷贝数支持用于标准化的基因的可靠性。这是关于在剑麻中非生物胁迫条件下筛选和验证管家基因的第一份报告，该报告将通过新颖的基因鉴定和准确验证来帮助理解胁迫耐受机制。