Genome-wide mapping of the sex-linked region and development of co-dominant InDel markers for sex identification in mud carp (Cirrhinus molitorella)

Mud carp ( Cirrhinus molitorella ) is an important freshwater aquaculture species in southern China and Southeast Asia, but the absence of reliable external sexual dimorphism before maturity limits early sex identification. We resequenced 89 fish (40 females and 49 males) from seven sampling groups and combined genome-wide association analysis with independent assessments of between-sex F ST , sex-specific heterozygosity, genotype segregation, sequencing depth, and linkage disequilibrium (LD). The principal association cluster was refined to a 2.38-Mb sex-linked region (SLR) on chromosome 6 (Chr06:37,500,000–39,882,941 bp), containing 2129 genome-wide significant SNPs and 645 significant InDels. The lead SNP (Chr06:39,869,941; P = 5.5 × 10 −90 ), elevated male heterozygosity, and a predominantly female-homozygous/male-heterozygous genotype pattern supported an XX/XY system. Two co-dominant markers, CM-SexInDel-1 and CM-SexInDel-2, were concordant with phenotypic sex in 200 independently sampled cultured juveniles from two validation cohorts. RNA-seq of seven stage-IV ovaries and seven stage-IV testes characterized differentiated-gonad expression within the mapped interval, where nine of 20 detectably expressed SLR genes were differentially expressed. RT-qPCR using six female and six male biological replicates supported ovary-biased expression of nr4a2 , acvr1 , and actr5 and testis-biased expression of ccdc148 . These genomic and expression results prioritize four focal genes for early-developmental and functional follow-up. The refined SLR and simple PCR-based markers provide genomic resources for sex identification and future breeding applications in mud carp.