Identification of reference genes via real-time quantitative PCR for investigation of the transcriptomic basis of the squalene biosynthesis in different tissues on olives under drought stress

• UBC1 was the best reference gene across different tissues and cultivars in olives under drought stress. • Under drought stress, the amount of squalene in the leaves increased dramatically, as the genes related to squalene production were also significantly up-regulated. Squalene plays a crucial role in plant growth, development, and stress tolerance. To select the best reference genes (RGs) for expression profile analysis of genes involved in squalene biosynthesis in olives under stress tolerance, the expression stability of 22 candidate RGs across four tissues (root, stem, leaf, and fruit) and five representative cultivars under drought stress was assessed using six methods. Our study showed that, ubiquitin-conjugating enzyme1 ( UBC1 ) and 60S ribosomal protein L18-2-like ( 60S ) were the most stable RGs across five different cultivars and various tissues. Additionally, elongation factor 1-alpha-like ( EF1-α2) and UBC1 were the most stable RG in olives under drought stress. UBC1 was the appropriate RG for further study, qPCR analysis showed that five certain genes involved in the squalene biosynthesis exhibited significant differential expression under varying conditions. Specially, the expression level of squalene synthase (SQS) in the leaves was highest, and HPLC analysis showed that squalene content was the highest in leaves. Likewise, the expression levels of SQS and farnesyl diphosphate synthase ( FPPS ) in the leaves of the cultivar Arbosana and Chelmsford Lal were significantly higher than those of the other cultivars, respectively, and HPLC analysis also showed that squalene content was highest in the two cultivars. Interestingly, the amount of squalene increased dramatically in olive under drought stress, as lupeol synthase ( LUPS ) was significantly up-regulated. To date, this study firstly provided the comprehensive analysis of RGs related to squalene biosynthesis in various cultivars and tissues of olives under drought stress.