A de novo transcriptome and valid reference genes for quantitative real-time PCR in Colaphellus bowringi

PLoS One. 2015 Feb 18;10(2):e0118693. doi: 10.1371/journal.pone.0118693. eCollection 2015.

Abstract

Background: The cabbage beetle Colaphellus bowringi Baly is a serious insect pest of crucifers and undergoes reproductive diapause in soil. An understanding of the molecular mechanisms of diapause regulation, insecticide resistance, and other physiological processes is helpful for developing new management strategies for this beetle. However, the lack of genomic information and valid reference genes limits knowledge on the molecular bases of these physiological processes in this species.

Results: Using Illumina sequencing, we obtained more than 57 million sequence reads derived from C. bowringi, which were assembled into 39,390 unique sequences. A Clusters of Orthologous Groups classification was obtained for 9,048 of these sequences, covering 25 categories, and 16,951 were assigned to 255 Kyoto Encyclopedia of Genes and Genomes pathways. Eleven candidate reference gene sequences from the transcriptome were then identified through reverse transcriptase polymerase chain reaction. Among these candidate genes, EF1α, ACT1, and RPL19 proved to be the most stable reference genes for different reverse transcriptase quantitative polymerase chain reaction experiments in C. bowringi. Conversely, aTUB and GAPDH were the least stable reference genes.

Conclusion: The abundant putative C. bowringi transcript sequences reported enrich the genomic resources of this beetle. Importantly, the larger number of gene sequences and valid reference genes provide a valuable platform for future gene expression studies, especially with regard to exploring the molecular mechanisms of different physiological processes in this species.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Coleoptera / embryology
  • Coleoptera / genetics*
  • Female
  • Gene Expression Profiling / methods*
  • Genome, Insect
  • Insect Proteins / genetics
  • Insect Proteins / standards*
  • Male
  • Real-Time Polymerase Chain Reaction / methods*
  • Reference Standards
  • Sequence Analysis, DNA

Substances

  • Insect Proteins

Grants and funding

The research was supported by grants of the Natural Science Foundation of China (31272045), the Program for New Century Excellent Talents in University (NCET-12-0864) and the Fundamental Research Funds for the Central Universities (2012ZYTS044, 2013PY028). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.