Screening and validation of reference genes for qPCR in different tissues of blackspot tuskfish (Choerodon schoenleinii)

Blackspot tuskfish (Choerodon schoenleinii) is an economically important coral reef fish with high aquaculture value and a unique protogynous hermaphroditic reproductive pattern. Quantitative real-time PCR (qPCR) is widely used for gene expression analysis, but its accuracy depends on the use of stable reference genes. However, suitable reference genes for C. schoenleinii have not yet been systematically evaluated. To screen for stably expressed reference genes in different tissues of C. schoenleinii, eighteen candidate reference genes (eif2s2, ube2a, actc1, rpl7l1, eif1, eif3a, ppia, rps18, rps27a, snrpd2, taf13, tor1a, tubb4b, rps27l, dera, gapdh, actg1, and hsp90ab1) were selected. Their expression levels in gonad, liver, spleen, kidney, intestine, heart, brain, muscle, skin, and gill tissues were analyzed using qPCR, and expression stability was evaluated using BestKeeper, NormFinder, geNorm, and RefFinder. Among the 18 candidate genes, all except hsp90ab1 exhibited acceptable amplification efficiencies (90% - 103%) and high coefficients of determination (R² > 0.98), indicating good amplification specificity. C_T value analysis revealed variation in expression across tissues, with eif2s2 showing the least variation and rps27a the greatest. Comprehensive analysis using the four algorithms showed generally consistent results, identifying eif3a, eif2s2, tubb4b, and rps27l as the most stable genes, while actc1 and gapdh were the least stable. geNorm analysis indicated that V4/5 = 0.143, suggesting that four reference genes are sufficient for normalization across multiple tissues, whereas two reference genes are adequate for single-tissue analyses. Overall, tubb4b, eif2s2, eif3a, and rps27l were stably expressed across the ten tissues and are recommended as a reference gene combination for qPCR studies in different tissues of C. schoenleinii. For studies focusing on a single tissue, a dual-reference gene strategy is recommended. This study provides reliable tools for gene expression analysis and functional research in C. schoenleinii.