Use of quantitative real-time PCR to select reference genes in the testis and ovary of Apostichopus japonicus during the breeding period

Abstract

Gonads play a critical role in the reproduction of sea cucumbers, and their development is regulated by a large number of genes. To date, the ideal reference genes (RGs) for studying sea cucumber gonadal development have not been reported. In this study, we selected ten RGs and conducted quantitative real-time PCR analysis to identify the most stable among Apostichopus japonicus gonads at four different stages of gonadal development. We used ∆Cq, GeNorm, NormFinder, BestKeeper, and RefFinder to evaluate the stability of candidate genes. EF1β and Grb2 together were the preferred combination of stable RGs among all of the gonad samples and for the ovaries alone. UBQ combined with RPS4 was the best combination of stable RGs in the testes. This study aimed to provide a basis for analysis of differences in gene expression related to gonadal development in sea cucumbers, and the results provide valuable information for further studies of the molecular mechanisms involved in sea cucumber gonadal development.

Keywords

• Expression stability
• Gonads
• Quantitative real-time PCR
• Reference genes
• Sea cucumber breeding period