A Hidden Y Chromosome Emerges: New Genetic Markers Crack the Sex Code of a Fast-Growing Chinese Fish

In the fast-moving world of aquaculture genetics, a modest freshwater fish from southwestern China has just delivered a finding that could reshape how one of its most prized food species is farmed. Acrossocheilus longipinnis, a cyprinid celebrated for its firm flesh, high nutritional value, and rich content of polyunsaturated fatty acids, has long been noted for a striking biological quirk: females grow substantially faster than males. For farmers, that difference represents lost productivity in every mixed-sex pond, and for scientists it has long hinted at an underlying genetic sex-determination system waiting to be decoded. A new study published in BMC Genomics has now done exactly that, identifying male-specific DNA markers and revealing that the species uses an XX/XY sex determination system, the same chromosomal architecture familiar from humans and many other mammals.

The research, led by Zheng-Hang Liu and Fang-Yuan Xiong, with Zhong-Wei Wang as corresponding author, brought together teams from the Scientific Institute of Pearl River Water Resources Protection in Guangzhou and the State Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture at the Institute of Hydrobiology, Chinese Academy of Sciences in Wuhan, along with conservation centers in Guigang dedicated to protecting rare Hongshui River fish. Their motivation was both scientific and practical. Without knowing how sex is determined in A. longipinnis, breeders cannot develop monosex production strategies, the gold standard for exploiting the growth advantage of females. And without a mapped sex chromosome system, the search for the master switch that sets off male or female development remains a needle-in-haystack problem.

The team’s method of choice was 2b-RAD sequencing, a streamlined genotyping approach that has become a favorite tool for fish geneticists working on species with little or no reference genomic information. The technique exploits a specific type of restriction enzyme to slice the genome at thousands of precisely defined positions, capturing short, uniform tags of DNA adjacent to each cut site. Sequencing these tags in a large panel of individuals generates a dense but manageable snapshot of genetic variation across the genome. Because the fragments are so consistent in length and composition, they can be compared across dozens or hundreds of animals to find sequences that appear in one sex and are completely absent in the other, which is precisely the signature expected of DNA residing on a Y chromosome.

Before any sex-linked screening could begin, however, the researchers needed a genomic scaffold on which to place their findings. They assembled a reference genome from a female individual, using k-mer depth distribution analysis to estimate genome size and assess heterozygosity, smudgeplot analysis to confirm ploidy, and BUSCO assessments to verify that the assembly captured a complete set of conserved single-copy genes. Quality and continuity metrics were visualized for two haplotypes, designated Hap-A and Hap-B, and predicted genes were functionally annotated against multiple databases, with circos plots providing global views of each haplotype’s landscape. The assembled female genome contained 50 chromosomes, consistent with the karyology of many cyprinids, and provided the coordinate system into which any male-specific sequence could ultimately be anchored.

The screen itself proved remarkably productive. Out of the many thousands of 2b-RAD tags compared between XY males and XX females, exactly three male-specific sequences emerged, designated ref65115, ref94872, and ref134458. In genomics, three hits might sound like a meager harvest, but in sex chromosome research it is precisely the kind of sparse, consistent signal that indicates a young or recently differentiated sex chromosome rather than a sprawling region of male-limited DNA. Each of the three sequences was detected only in males, never in females, and their joint presence pointed to a single shared origin on a male-specific segment of the genome.

The decisive step came when the researchers mapped these three sequences against the female reference genome using synteny and coverage depth analyses. All three aligned to a single contiguous window between positions 5,724,913 and 5,869,801 base pairs, a stretch of roughly 145 kilobases. Crucially, the alignment pattern indicated that the sequences did not correspond to an allele present in both sexes but instead represented a Y-specific insertion: a block of DNA that has been added to, or retained exclusively on, the Y chromosome and is entirely missing from the X. In other words, the male sex chromosome of A. longipinnis carries a discrete inserted segment at this genomic address, and that insertion is what separates males from females at the molecular level.

Confirmation followed through rigorous population-level validation. Using PCR primers designed from the flanking regions of the three markers, the team generated four diagnostic amplification assays, named ref65115-IN, ref94872-3, ref134458-1, and ref94872-5. On agarose gels, ref65115-IN produced a male-specific band of 217 base pairs, ref94872-3 a male-specific band of 335 base pairs, ref134458-1 a male-specific band of 265 base pairs, and ref94872-5 a shared band of 336 base pairs in both sexes together with a male-specific band of 211 base pairs. Each assay was then tested across 72 cultured individuals drawn from two geographically distinct populations, 18 males and 18 females each from Guangxi and Guizhou Provinces. The male-specific bands appeared in every male and in no female, providing strong evidence that the Y-specific insertion is shared across the species’ range rather than being a local variant confined to one river system.

Those results settle a question that has quietly constrained both the biology and the farming of this species. An XX/XY system means that sex is determined by which chromosome a sperm carries: females inherit two X chromosomes, males one X and one Y. In evolutionary terms, the finding is also intriguing. Many fish lineages show fluid, rapidly evolving sex determination, with different species even within the same genus employing XY, ZW, or environmental systems. Identifying a Y-specific insertion in A. longipinnis adds another data point to this comparative landscape, suggesting that a discrete, recent insertion event on the Y chromosome may have been the founding mutation of male determination in this lineage. That kind of simple origin story is increasingly common in fish genomics, where sex chromosomes are often young enough that the male-specific region is small and tractable.

For the aquaculture industry, the practical implications are immediate and considerable. Because females of A. longipinnis grow significantly faster than males, a monosex, all-female production system would raise yields, shorten production cycles, and reduce feed costs per kilogram of fish, much as all-female Nile tilapia and all-female grass carp culture have done in other systems. The newly validated PCR markers now give breeders a direct molecular test for genotypic sex: rather than waiting months for fish to reach a size at which gonads can be inspected, hatcheries can sex larvae or juveniles from a tiny tissue sample and identify XX females, XY males, and, in future work, potentially phenotypic sex-reversed individuals. Such marker-assisted selection is the foundation on which sex-control breeding programs are built, allowing the production of neomales, or XX individuals carrying testes, whose sperm carry only X chromosomes and who therefore sire all-female offspring when crossed with ordinary XX females.

The study’s authors are careful to frame their work as a beginning rather than an end. The three male-specific markers pin down the sex-determining region to a Y-specific insertion of about 145 kilobases on the female reference assembly, but the region still requires fine mapping against the male genome, and the identity of the master sex-determining gene within or near that insertion remains unknown. Future work will aim at chromosome-scale assignment of the sex chromosomes, high-resolution mapping of the sex-determining region, and ultimately the cloning of the master sex-determining gene, the single locus whose presence or absence tips embryonic development toward testis or ovary. Because the Y-specific region appears compact, that cloning effort is unusually feasible for a non-model fish, and the newly assembled, well-annotated female genome with its 50 chromosomal scaffolds provides exactly the platform such work requires. All experimental procedures were approved by the Animal Ethics Committee of the Institute of Hydrobiology, Chinese Academy of Sciences, and fish were anesthetized with MS-222 before tissue collection, reflecting the ethical standards applied throughout the study. Funded by the Scientific Institute of Pearl River Water Resources Protection, the research is open access under a Creative Commons license, ensuring that breeders, conservationists, and comparative genomics researchers across the field can build on these markers without restriction. For a species prized on dinner tables across southwestern China, and for the farmers who raise it, a 145-kilobase stretch of Y-chromosomal DNA has just become one of the most valuable sequences in the entire genome.

Subject of Research: Identification of male-specific DNA markers and an XX/XY sex determination system in Acrossocheilus longipinnis

Article Title: Identification of male-specific markers via 2b-RAD sequencing reveals an XX/XY sex determination system in Acrossocheilus longipinnis

Article References: Liu, Z.-H., Xiong, F.-Y., Zhou, Y.-L., E, Z.-C., Li, M., Dong, S.-H., Tian, J., Zhen, P., Gui, J.-F., & Wang, Z.-W. (2026). Identification of male-specific markers via 2b-RAD sequencing reveals an XX/XY sex determination system in Acrossocheilus longipinnis. BMC Genomics. https://doi.org/10.1186/s12864-026-13372-9

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13372-9

Keywords: Acrossocheilus longipinnis, sex determination, XX/XY system, 2b-RAD sequencing, male-specific markers, genome assembly, Y-specific insertion, sex-determining region, monosex breeding, aquaculture, marker-assisted selection, cyprinidae