Abstract
Abstract in English, Chinese The autotetraploid Carassius auratus (4nRR, 4 n=200, RRRR) is derived from whole-genome duplication of Carassius auratus red var. (RCC, 2 n=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 scarb1, 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 scarb1 homeologs in 4nRR: two original scarb1 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 scarb1 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 scarb1, with all scarb1 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. 同源四倍体鲫（autotetraploid Carassius auratus，4nRR， 4n=200，RRRR）来源于红鲫（ Carassius auratus red var.，RCC， 2n=100, RR）的全基因组复制。该研究表明，皮肤色素细胞和色素的改变是4nRR体色形成和变异的直接原因(RCC为红色，4nRR为棕黄色)。为进一步探索4nRR体色形成和变异的分子调控机制，对RCC和4nRR进行转录组测序分析和分子功能验证。转录组分析结果表明，参与类胡萝卜素代谢相关的 scarb1基因在4nRR中的表达极显著低于RCC（ P<0.01）。此外，有效编辑该候选色素基因表明 scarb1在RCC体色中起重要作用。4nRR存在4个遗传组成不同的 scarb1同源基因：两个来自RCC的原始 scarb1同源基因和两个重复 scarb1同源基因。值得注意的是，其中的三个同源基因均具有两个高度保守的等位基因，且在皮肤中表现出明显的偏倚和等位基因特异性表达。此外，通过对原始和复制 scarb1同源基因及其等位基因进行精确编辑后，结果表明单突变和多突变4nRR个体表型均表现为从棕黄色皮肤到青灰色的转变。同时，青灰色区域的比例区域面积表现出基因剂量效应。该研究结果证实重复 scarb1存在亚功能化，且 scarb1同源基因协同平等地参与了4nRR体色的形成。这是关于同源多倍体鱼类中复制同源基因功能分化的第一篇报道，极大地丰富了对同源多倍体体色形成和变异的了解。.