Identification and Validation of Reference Genes for Quantitative Gene Expression Analysis in Ophraella communa

Abstract

<i>Ophraella communa</i> is an effective bio-control agent of the invasive common weed. By now, the reference genes in <i>O. communa</i> have not yet been screened and validated. The aim of this study was to screen for the most stable reference genes in different backgrounds, such as different developmental stages, sexes, tissues, and male reproductive system with different body sizes. We selected 12 common housekeeping genes involved in different biological processes, including <i>GAPDH</i>, <i>ACT1</i>, <i>ACT2</i>, <i>ARF1</i>, <i>ARF4</i>, <i>SDH</i>, <i>βTUBC</i>, <i>RPL4</i>, <i>RPL19</i>, <i>RPS18</i>, <i>EF1</i>α, and <i>COX</i> as the candidate reference genes. To analyze the stability of the candidate reference genes, we first used three dedicated algorithms, GeNorm, NormFinder, and BestKeeper, and further comprehensive ranking was provided by ReFinder. The results showed that <i>RPL19</i> and <i>RPL4</i> exhibited the least variation in different developmental stages/sexes and in male reproductive systems with different body sizes. <i>COX</i> proved to be most suitable for normalizing the gene expression levels in different tissues, and coincidentally, <i>RPL19</i> was also found to be second in terms of stability in this study. To the best of our knowledge, this is the first study to identify suitable reference genes for analyzing gene expression in <i>O. communa</i>; thus, this study would lay the foundation for future research on the molecular physiology and biochemistry of <i>O. communa</i> and other insects.