Selection and Validation of Reliable Reference Genes for qRT-PCR Normalization of Bursaphelenchus xylophilus from Different Temperature Conditions and Developmental Stages

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a powerful technique for studying gene expression. The key to quantitative accuracy depends on the stability of the reference genes used for data normalization under different experimental conditions. Pine wood nematode (PWN, Bursaphelenchus xylophilus) is the causal agent of the devastating pine wilt disease (PWD). Extensive and prompt research is needed to understand the molecular mechanism of PWD, but identification of the reference PWN genes for standardized qRT-PCR has not been reported yet. We have analyzed eight candidate reference genes of PWN across different temperature conditions and developmental stages. Delta Ct method, GeNorm, NormFinder, BestKeeper, and RefFinder algorithms were used to evaluate the stability of expression of these genes. Finally, we use heat shock protein 90 (HSP90) in different temperatures and arginine kinase gene (AK) in different developmental stages to confirm the stability of these genes. UBCE and EF1 gamma were most stable across different temperature treatments, whereas EF1 gamma and Actin were most stable across different developmental stages. In general, these results indicate that EF1 gamma is the most stable gene for qRT-PCR under different conditions. The systematic analysis of qRT-PCR reference gene selection will be helpful for future functional analysis and exploration of B. xylophilus genetic resources.