Onion yellow dwarf virus ∆∆Ct-based relative quantification obtained by using real-time polymerase chain reaction in ‘Rossa di Tropea’ onion

As part of a plant-pathogen interaction study between Onion yellow dwarf virus (OYDV) and onion cultivar Rossa di Tropea, a ΔΔCt-based relative quantification of OYDV was investigated to relate OYDV titer to accumulation of secondary metabolites in onion bulbs. An appropriate reference gene (RG) was required to achieve data normalization. Since no single internal control gene is universally used as an RG, multiple stably expressed reference genes were investigated. In particular, elongation factor (Elf), protein phosphatase 2A (PP2A), helicase (Hel-1), 5.8S rRNA, ubiquitin (UBQ) and s-Actin (s-Act) were compared one to another in both leaf and bulb tissues, at different growth and development stages, and with different infection status (healthy/OYDV-infected). Preliminary gene screening was carried out using an RT-qPCR assay (SYBR chemical), assessing both Ct values and melting curves. Expression stability of the reference genes in the sample sets was independently determined by three different software packages: geNorm, NormFinder and Bestkeeper. In contrast to Elf, PP2A, Hel-1 and s-Act, 5.8S rRNA and UBQ proved to be the most stable RGs. An OYDV specific RT-qPCR TaqMan® assay was also developed and validated for relative quantification of OYDV titer. The assay was shown to be specific and sensitive, able to identify virus presence up to 10−6 dilution, representing a rapid and sensitive diagnostic tool for OYDV detection for application in field surveys. Finally, a ∆∆Ct method was developed, to be applied in future studies describing the molecular interaction between OYDV and onion cv. ‘Rossa di Tropea’. This approach was used to provide relative quantification of OYDV titer in samples obtained from different experimental trials.