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

Yajing Wu,Faheem Ahmad,Lili Ren,Jiao Zhou,Guochang Wang,Hongxia Zhang,Yaning Liu,Yutong Gu

doi:10.3390/app12062880

Abstract

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 EF1γ were most stable across different temperature treatments, whereas EF1γ and Actin were most stable across different developmental stages. In general, these results indicate that EF1γ 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.

Highlights

Introduction iationsPine wilt disease (PWD) is a globally quarantined conifer disease and poses a serious threat to forest safety and ecosystem stability in parts of Asia and Europe [1,2]
These comprehensive results showed that the designed primers could accurately amplify the candidate reference genes
In order to obtain an overall representation of primer variability, we examined the expression profiles of the candidate reference genes under different experimental conditions (Figure 2)

Summary

Introduction

Introduction iationsPine wilt disease (PWD) is a globally quarantined conifer disease and poses a serious threat to forest safety and ecosystem stability in parts of Asia and Europe [1,2]. In China, the disease is caused by an invasive species, the pine wood nematode (PWN), Bursaphelenchus xylophilus. The destructiveness of PWN is usually closely related to its native vector beetles and ophiostomatoid fungal species [3]. PWN feeds on the epithelial tissue of the host tree and on ophiostomatoid (blue stain) fungi. Under adverse conditions, such as low food availability and extreme temperatures, PWN will enter a dispersal stage and molt from L2 into third stage dispersal juveniles (LIII ) [7]. These LIII are attracted by cues from their insect vector, Monochamus alternatus, and gather around the beetles’ pupal chambers [2,5].

Methods

Results

Discussion

Conclusion