Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon

PLoS One. 2014 Jan 27;9(1):e87197. doi: 10.1371/journal.pone.0087197. eCollection 2014.

Abstract

Melon (Cucumis melo. L) is not only an economically important cucurbitaceous crop but also an attractive model for studying many biological characteristics. Screening appropriate reference genes is essential to reverse transcription quantitative real-time PCR (RT-qPCR), which is key to many studies involving gene expression analysis. In this study, 14 candidate reference genes were selected, and the variations in their expression in roots and leaves of plants subjected to biotic stress, abiotic stress, and plant growth regulator treatment were assessed by RT-qPCR. The stability of the expression of the selected genes was determined and ranked using geNorm and NormFinder. geNorm identified the two most stable genes for each set of conditions: CmADP and CmUBIep across all samples, CmUBIep and CmRPL in roots, CmRAN and CmACT in leaves, CmADP and CmRPL under abiotic stress conditions, CmTUA and CmACT under biotic stress conditions, and CmRAN and CmACT under plant growth regulator treatments. NormFinder determined CmRPL to be the best reference gene in roots and under biotic stress conditions and CmADP under the other experimental conditions. CmUBC2 and CmPP2A were not found to be suitable under many experimental conditions. The catalase family genes CmCAT1, CmCAT2, and CmCAT3 were identified in melon genome and used as target genes to validate the reliability of identified reference genes. The catalase family genes showed the most upregulation 3 days after inoculation with Fusarium wilt in roots, after which they were downregulated. Their levels of expression were significantly overestimated when the unsuitable reference gene was used for normalization. These results not only provide guidelines for the selection of reference genes for gene expression analyses in melons but may also provide valuable information for studying the functions of catalase family genes in stress responses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agriculture / methods*
  • Catalase / genetics
  • Catalase / metabolism
  • Cucumis melo / genetics*
  • DNA Primers / genetics
  • Fusarium
  • Gene Expression Profiling / methods*
  • Gene Expression Regulation, Plant / genetics*
  • Genes, Plant / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*

Substances

  • DNA Primers
  • Catalase

Grant support

This work was founded by the earmarked fund for Modern Agro-industry Technology Research System (CARS-26-16), National Science & Technology Pillar Program during the 12th Five-year Plan Period (2012BAD02B03-16) and Special Fund for Agro-scientific Research in the Public Interest (201003066-6).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.