Screening of reference genes in real-time PCR for Radopholus similis
Jun-Yi Li,
Wan-Zhu Chen,
Si-Hua Yang,
Chun-Ling Xu,
Xin Huang,
Chun Chen,
Hui Xie
Affiliations
Jun-Yi Li
Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, Department of Plant Pathology, Guangdong Province Key Laboratory of Microbial Signals and Disease Control, College of Agriculture, South China Agricultural University, Guangzhou, Guangdong Province, People’s Republic of China
Wan-Zhu Chen
Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, Department of Plant Pathology, Guangdong Province Key Laboratory of Microbial Signals and Disease Control, College of Agriculture, South China Agricultural University, Guangzhou, Guangdong Province, People’s Republic of China
Si-Hua Yang
Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, Department of Plant Pathology, Guangdong Province Key Laboratory of Microbial Signals and Disease Control, College of Agriculture, South China Agricultural University, Guangzhou, Guangdong Province, People’s Republic of China
Chun-Ling Xu
Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, Department of Plant Pathology, Guangdong Province Key Laboratory of Microbial Signals and Disease Control, College of Agriculture, South China Agricultural University, Guangzhou, Guangdong Province, People’s Republic of China
Xin Huang
Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, Department of Plant Pathology, Guangdong Province Key Laboratory of Microbial Signals and Disease Control, College of Agriculture, South China Agricultural University, Guangzhou, Guangdong Province, People’s Republic of China
Chun Chen
Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, Department of Plant Pathology, Guangdong Province Key Laboratory of Microbial Signals and Disease Control, College of Agriculture, South China Agricultural University, Guangzhou, Guangdong Province, People’s Republic of China
Hui Xie
Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, Department of Plant Pathology, Guangdong Province Key Laboratory of Microbial Signals and Disease Control, College of Agriculture, South China Agricultural University, Guangzhou, Guangdong Province, People’s Republic of China
Six candidate reference genes were chosen from the transcriptome database of Radopholus similis using the bioinformatics method, including four conventional reference genes (actin, Eukaryotic translation initiation factor 5A (eIF5A), Tubulin alpha (a-tubulin), ubiquitin (UBI)) and two new candidate reference genes (Ribosomal protein S21 (Rps21) and Serine/threonine protein phosphatase PP1-β catalytic subunit (β-PP1)). In addition, a traditional reference gene 18S ribosomal RNA (18S rRNA) obtained from NCBI databases was also added to the analysis. Real-time PCR was used to detect the expression of seven candidate reference genes in six populations of R. similis and four developmental stages (female, male, larva and egg) of a population. The stability of the expression of candidate genes was evaluated by three software programs, BestKeeper, geNorm and NormFinder. The results showed that eIF5A is the most suitable reference gene for gene functional research of different populations, while both Rps21 and eIF5A are the most suitable reference genes for different developmental stages of a population. Therefore, eIF5A is the best reference gene for studying R. similis. However, one defect of this study is that only seven candidate reference genes were analyzed; ideally, more genes should be tested.
