Identification and effective regulation of gene involved in pigmentation change in autotetraploid .

The autotetraploid (4nRR, 4 =200, RRRR) is derived from whole-genome duplication of red var. (RCC, 2 =100, RR). In the current study, we demonstrated that chromatophores and pigment changes directly caused the coloration and variation of 4nRR skin (red in RCC, brownish-yellow in 4nRR). To further explore the molecular mechanisms underlying coloration formation and variation in 4nRR, we performed transcriptome profiling and molecular functional verification in RCC and 4nRR. Results revealed that , associated with carotenoid metabolism, underwent significant down-regulation in 4nRR. Efficient editing of this candidate pigment gene provided clear evidence of its significant role in RCC coloration. Subsequently, we identified four divergent homeologs in 4nRR: two original homeologs from RCC and two duplicated ones. Notably, three of these homeologs possessed two highly conserved alleles, exhibiting biased and allele-specific expression in the skin. Remarkably, after precise editing of both the original and duplicated homeologs and/or alleles, 4nRR individuals, whether singly or multiply mutated, displayed a transition from brownish-yellow skin to a cyan-gray phenotype. Concurrently, the proportional areas of the cyan-gray regions displayed a gene-dose correlation. These findings illustrate the subfunctionalization of duplicated , with all genes synergistically and equally contributing to the pigmentation of 4nRR. This is the first report concerning the functional differentiation of duplicated homeologs in an autopolyploid fish, substantially enriching our understanding of coloration formation and change within this group of organisms.