Optimal Reference Genes in Different Tissues, Gender, and Gonad of Yellow River Carp (Cyprinus carpio var) at Various Developmental Periods

Yellow River carp is one of the most important breeding fish in China owing to its economic value in aquaculture, and has been extensively studied using quantitative real-time PCR. As many of the reference genes commonly used in the studies on Yellow River carp significantly vary among the species, it is important to determine their stability. To assess an appropriate housekeeping gene that is stably expressed across different tissues, gender, and gonad of Yellow River carp at various developmental stages, six reference genes (beta-2 microglobulin (B2M), beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF-1 alpha), 18S ribosomal RNA (18S rRNA), and 40S ribosomal protein (40S)) were evaluated using descriptive analysis and three software programs (geNorm, BestKeeper, and NormFinder), along with RNA-seq statistics. The results showed that 40S is the best candidate gene for normalization in 2 -year-old adult Yellow River carps; 18S rRNA and ACTB are the best candidate genes for normalization in juvenile carps; and GAPDH and 40S are the best candidate genes for normalization in carp gonad at different developmental stages. These findings were in accordance with the RNA-seq transcripts pertaining to the expression level and stability of these reference genes. Furthermore, EF-l alpha and 40S had the most stable expression levels in all the tissues of the juvenile and adult carps. Thus, the results of the present study could be valuable, for further investigation and better molecular genetic analyses of optimal reference genes in Yellow River carps.