Identification of the master sex determining gene in Northern pike (Esox lucius) reveals restricted sex chromosome differentiation

Abstract

Teleost fishes, thanks to their rapid evolution of sex determination mechanisms, provide remarkable opportunities to study the formation of sex chromosomes and the mechanisms driving the birth of new master sex determining (MSD) genes. However, the evolutionary interplay between the sex chromosomes and the MSD genes they harbor is rather unexplored. We characterized a male-specific duplicate of the anti-Müllerian hormone (amh) as the MSD gene in Northern Pike (Esox lucius), using genomic and expression evidences as well as by loss-of-function and gain-of-function experiments. Using RAD-Sequencing from a family panel, we identified Linkage Group (LG) 24 as the sex chromosome and positioned the sex locus in its sub-telomeric region. Furthermore, we demonstrated that this MSD originated from an ancient duplication of the autosomal amh gene, which was subsequently translocated to LG24. Using sex-specific pooled genome sequencing and a new male genome sequence assembled using Nanopore long reads, we also characterized the differentiation of the X and Y chromosomes, revealing a small male-specific insertion containing the MSD gene and a limited region with reduced recombination. Our study depicts an unexpected level of limited differentiation within a pair of sex chromosomes harboring an old MSD gene in a wild population of teleost fish, highlights the pivotal role of genes from the amh pathway in sex determination, as well as the importance of gene duplication as a mechanism driving the turnover of sex chromosomes in this clade.Author SummaryIn stark contrast to mammals and birds, teleosts have predominantly homomorphic sex chromosomes and display a high diversity of sex determining genes. Yet, population level knowledge of both the sex chromosome and the master sex determining gene is only available for the Japanese medaka, a model species. Here we identified and provided functional proofs of an old duplicate of anti-Müllerian hormone (Amh), a member of the Tgf-β family, as the male master sex determining gene in the Northern pike, Esox lucius. We found that this duplicate, named amhby (Y-chromosome-specific anti-Müllerian hormone paralog b), was translocated to the sub-telomeric region of the new sex chromosome, and now amhby shows strong sequence divergence as well as substantial expression pattern differences from its autosomal paralog, amha. We assembled a male genome sequence using Nanopore long reads and identified a restricted region of differentiation within the sex chromosome pair in a wild population. Our results provide insight on the conserved players in sex determination pathways, the mechanisms of sex chromosome turnover, and the diversity of levels of differentiation between homomorphic sex chromosomes in teleosts.

Multiple evidences suggest sox2 as the main driver of a young and complex sex determining ZW/ZZ system in turbot (Scophthalmus maximus)

“…In this regard, turbot's would be among the youngest sex chromosome pairs found in fish at a stage similar to that of the pufferfish (Fugu rubripes; Kamiya et al, 2012), where the only difference between X and Y chromosomes was a single differential SNP at the promoter of the amh receptor. Very recently, a similar picture has been reported in E. lucius, where a small male-specific insertion containing the SD gene, has been documented (Pan et al, 2019). Although examples of mature SD systems such as those reported in the genus Characidium and…”

“…It is worth a reflection on why SD of fish is so unstable in evolutionary terms, where the classical model of two well differentiated sex chromosomes is scarce. The high turnover of SD in fish suggests that different genomic regions can be recruited to replace the previous SD system following the so called "hot-potato" model (Gammendinger and Kocher, 2018), although data strongly suggest the suitability of some genes like dmY or amh, recruited independently in several fish species to drive sex differentiation (Matsuda et al, 2002;Hattori et al, 2012;Pan et al, 2019). In this regard, sox2 and the putative involvement of sox2ot lncRNA on its regulation, would add a new master gene and a novel regulatory mechanism confirming the multiple options to drive sex across the fish genomes.…”

“…Regardless of their SD system, chromosome heteromorphisms are rare in this vertebrate group (Cioffi et al, 2017). Underlying this diversity, an important number of different SDg have been reported, both involving classical transcription factors, such as dmy (Y-specific DM-domain) (Matsuda et al, 2002) or sox3 (SRYrelated HMG-box) (Takehana et al, 2014), as well as transforming growth factorrelated genes, such as gsdf (gonadal soma-derived growth factor on the Y chromosome) (Myosho et al, 2012) or amh (anti-Mullerian hormone) (Hattori et al, 2012;Pan et al, 2019) and its receptor amhr2 (Kamiya et al, 2012), but also other unexpected genes, such the interferon-related sdY (Yano et al, 2013), and more recently, bcar1 (Bao et al, 2019) and hsd17b1 (Koyama et al, 2019), related to the steroidogenic pathway.…”

Sex determining gene transposition as an evolutionary platform for chromosome turnover

“…Genetic sex determination can be polymorphic (Bulmer and Bull 1982;Liew et al 2012; Moore and Roberts 2013) but it may also be controlled by a single master sex determining gene (Meise et al 1998;Pan et al 2019). Such genes are likely to function by initiating the developmental cascade which ends in the development of ovaries or testes.…”

Sex chromosome and sex locus characterization in the goldfish, Carassius auratus

“…74 Unlike birds and mammals, sex determination in teleost is highly dynamic, with frequent 75 turnovers of both sex determination (SD) systems [8] and master sex determining genes (MSD) 76 [9,10]. Currently about half a dozen different master sex determining genes have been 77 identified in teleosts, including dmrt1 in the Japanese medaka, Oryzias latipes [11], sdY in 78 rainbow trout [12], amh in Northern pike, Nile tilapia and pejerrey [13][14][15], amhr2 in yellow 79 perch and the Takifugu pufferfish [16,17], gsdf in sablefish and Luzon medaka, O. luzonensis 80 [18,19], gsdf6a in the turquoise killifish [20] and sox3 in the Indian ricefish O. dancena [21]. 81 MSD turnover can be evolutionarily rapid as has been shown for instance in various ricefish 82 species [22].…”