Selection of Reference Genes for Real-Time Quantitative PCR in Pinus massoniana Post Nematode Inoculation

PLoS One. 2016 Jan 22;11(1):e0147224. doi: 10.1371/journal.pone.0147224. eCollection 2016.


Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / biosynthesis
  • ATP-Binding Cassette Transporters / genetics
  • Animals
  • China
  • Disease Resistance / genetics*
  • Gene Expression Profiling
  • Genes, Essential / genetics
  • Genes, Plant / genetics
  • Nuclear Proteins / biosynthesis
  • Nuclear Proteins / genetics
  • Pinus / genetics*
  • Pinus / immunology*
  • Pinus / parasitology
  • Plant Diseases / immunology*
  • Plant Diseases / parasitology
  • Real-Time Polymerase Chain Reaction
  • Reference Standards
  • Ribonucleoproteins / biosynthesis
  • Ribonucleoproteins / genetics
  • Splicing Factor U2AF
  • Tubulin / biosynthesis
  • Tubulin / genetics
  • Tylenchida / immunology*
  • Tylenchida / pathogenicity


  • ATP-Binding Cassette Transporters
  • Nuclear Proteins
  • Ribonucleoproteins
  • Splicing Factor U2AF
  • Tubulin

Grant support

The work was supported by the following: 1. National Science & Technology Pillar Program of China (No. 2012BAD01B02,, Principal Investigator: Zhichun Zhou; 2. National Natural Science Foundation of China (No. 31470670), Principal Investigator: Qinghua Liu; and 3. National High-tech R&D Program of China (863 Program) (No. 2011AA10020302), Principal Investigator: Yi Zhang.