Genetic linkage map of the Australian barramundi (Lates calcarifer) reveals potential to leverage extreme sex-specific recombination and sequential hermaphrodism for ultimate breeding program control

Genetic linkage map of the Australian barramundi (Lates calcarifer) reveals potential to leverage extreme sex-specific recombination and sequential hermaphrodism for ultimate breeding program control | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genetic linkage map of the Australian barramundi (Lates calcarifer) reveals potential to leverage extreme sex-specific recombination and sequential hermaphrodism for ultimate breeding program control Jessica Hintzsche, Lorenzo Vincenzo Bertola, David B. Jones, Christie Warburton, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8400904/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Lates calcarifer (barramundi or Asian seabass) is a key aquaculture species with a protandrous life cycle, maturing first as male and later as female. As global breeding programs advance to improve traits such as growth and disease resistance, the absence of a high-density genetic linkage map limits progress and constrains understanding of genome architecture and recombination. Such maps are essential for marker-assisted selection, genomic breeding strategies, and comparative genomics, providing a foundation for accelerating genetic improvement. Results Our analysis reveals striking sex-specific recombination differences within the Australian lineage: females recombine only in centromeric regions, while males recombine exclusively in distal regions, with no overlap. This pronounced heterochiasmy has not been observed in Southeast Asian populations. Additionally, we detected 10 chromosomal inversions, expanding known structural variation across the species’ range. Conclusions Given their sequential hermaphroditism, this recombination landscape could allow breeders to strategically utilise sex-specific recombination through the creation of optimal allele combinations, increasing genetic gain and enhancing genetic diversity in L. calcarifer aquaculture populations. Beyond its practical applications, our findings establish L. calcarifer as a model for studying heterochiasmy, as well as providing insights into how this can be leveraged for advanced genomic breeding programs. Heterochiasmy linkage maps hermaphroditism aquaculture breeding recombination landscapes targeted breeding chromosomal inversions evolution Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Lates calcarifer is a rapidly emerging aquaculture species across tropical and subtropical Indo-Pacific regions. Native to India, Sri Lanka, Southeast Asia, northern Australia, and Papua New Guinea, it is commonly called “Asian seabass” around the world but is called “barramundi” in Australia. The species has a complex life history as a protandrous hermaphrodite, maturing first as male before transitioning to female later in life. It is also euryhaline and catadromous, spawning in seawater while juveniles migrate to brackish and freshwater habitats to grow [1]. It is L. calcarifer’s ability to be farmed in diverse culture systems and salinities, along with its favourable production traits, that has resulted to its emergence as a species of growing global aquaculture significance [2] and as a result there are a number of breeding programs underway to improve the species productivity [3-8]. Genomic studies reveal that L. calcarifer exhibits clear genetic structuring across its natural range, with distinct lineages suggesting the presence of incipient or even separate species [2, 9-11]. This divergence reflects an evolutionary history shaped by historical sea-level fluctuations, which created barriers to gene flow between Asian and Australian–Papua New Guinean populations. As a euryhaline teleost, L. calcarifer occupies freshwater systems, estuaries, and nearshore marine environments, making it sensitive to topographical changes. During parts of the Pleistocene, lowered sea levels eliminated the present-day Torres Strait, Arafura Sea, and Java Sea [12], enabling migration from Asia to Australia. Subsequent sea-level rise severed these pathways, isolating populations on the two continents for at least one million years [13, 14]. Evidence of this isolation is seen in Australian populations on either side of the Torres Strait, which differ by approximately 4% in sequence, a divergence attributed to these historical events [13]. Collectively, these genetic differences underscore the need to create population-specific resources and support treating Asian and Australian populations as distinct genetic lineages [9]. Structural variation in the genome provides further evidence for the separation between Asian and Australian L. calcarifer . Further evidence of divergence between Asian and Australian lineages is provided by karyotypic analyses, which reveal that Asian L. calcarifer possess chromosomes with more centrally located centromeres (metacentric and submetacentric), while Australian specimens exhibit chromosomes with more distally positioned centromeres (acrocentric and telocentric) [11, 15]. Given the evolutionary stability of centromeric regions, these structural shifts likely reflect long-term genetic isolation among populations, with centromere repositioning serving as a marker of divergent chromosomal evolution [16]. Therefore, there is a case that development of genomic resources should be conducted independently for Asian and Australian lineages to account for their distinct evolutionary history. A genetic linkage map is an essential genomic resource for any genetically divergent population. These maps are utilised across a range of biological disciplines, including evolutionary biology, conservation, and advanced animal breeding programs due to their ability to determine the relative positions of genetic markers within chromosomes (linkage groups, LG). This information assists with scaffold orientation in genome sequence assembly, quantitative trait locus (QTL) identification, and comparative genomics [17, 18]. Genetic linkage maps also significantly improve the accuracy of selection processes in breeding initiatives. A particularly important application of genetic linkage maps lies in its characterisation of recombination events. Mapping the precise locations and frequencies of chromosomal crossovers gives insight into how alleles are transmitted from parents to offspring, shaping the genetic population structure of future generations. This is especially helpful in aquaculture species, which display remarkable diversity and encompass a wide range of reproductive strategies [19, 20]. The ability to pinpoint crossover events can assist breeders in increasing favourable alleles in the population and ensuring that desired traits are reliably passed on. Genetic linkage maps can be further refined by constructing separate maps for males and females, enabling the characterisation of sex‑specific differences in recombination. These maps are generated by tracking the frequency and chromosomal positions of crossover events (chiasmata) during gametogenesis, using informative meiosis events transmitted from sires, to produce the male map, and from dams, to produce the female map. This information is useful to breeders as this helps breeding programs understand the genetic structure of progeny populations and resolve problems such as linkage drag. Additionally, linkage maps can reveal recombination differences of evolutionary significance, including patterns relevant to studies of heterochiasmy evolution. Over the past two decades, four successive genetic linkage maps have been developed for L. calcarifer , each reflecting incremental gains in marker density and genome coverage, culminating in 8,274 SNPs anchored to the ~700 Mb reference genome [21-25]. These maps have provided important genomic insights, including initial evidence of sex differences in centimorgan-based map lengths [21]. Yet a high-resolution, genome‑wide assessment of sex‑specific recombination patterns remains unexplored. This gap is particularly important given the evolutionary interest in heterochiasmy, especially with a sequentially hermaphroditic species that lacks differentiated sex chromosomes, as well as the practical importance of recombination knowledge for selective breeding. The Australian genetic lineage, despite the species’ prominence to Australian aquaculture and commencement of breeding programs [3, 5, 8, 26], is yet to have a genome-wide genetic linkage map constructed. In this study, we address that gap by generating the first high‑resolution genetic linkage map for Australian L. calcarifer using a custom 70K SNP genotyping array, enabling fine‑scale characterisation of sex‑specific recombination landscapes. This resource offers new opportunities to explore the evolution of heterochiasmy and to design genomic breeding programs optimised for both genetic gain and long‑term diversity. Results Genetic Linkage Map Construction To construct the L. calcarifer genetic linkage map, we analysed 49,885 informative SNP genotypes from 1,952 progeny derived from 7 male and 3 female parents following extensive quality control [8]. Additional filtering was performed using ParentCall2 to identify Mendelian inconsistencies. Although minor errors were detected at a few loci within certain families, sufficient accurate genotypes were present across the full dataset. After silencing these errors within affected families, no markers were removed, ensuring comprehensive marker coverage for map construction. Lep-MAP3 was used for genetic linkage map construction [27]. Varying the marker pair-wise logarithm of odds (LOD) threshold between 10 and 80 in the SeparteChromomses2 module did not affect the number of linkage groups; however, marker retention declined, and the proportion of unplaced markers increased sharply beyond a LOD score of 29 (Supplementary Figure 1). A LOD score of 28 was selected for all subsequent analyses. The resulting 24 linkage groups (LG) (Figure 1) align with the known karyotype of L. calcarifer , which comprises 24 chromosomes [11, 15, 28]. The linkage groups were labelled such that the numbers match the chromosome number from the genome assembly [25]. Sex-specific maps Separate male- and female-only genetic linkage maps were then created to identify any linkage group regions that exhibited differences in recombination rates between the sexes (Supplementary Figure 2). These maps retain the high density of the consensus linkage map, with an average distance between marker loci ranging from 0.13 and 0.25 cM in the males and 0.9 and 0.18 cM in the females (Supplementary Table 1). Both maps had 24 linkage groups. However, there were differences in total marker counts and chromosome lengths (Supplementary Table ). For instance, males exhibited a slightly higher number of mapped markers across most chromosomes, such as chromosome 1, which included 1,957 markers in males compared to 1,551 in females. Similarly, cumulative linkage group lengths tended to be longer in the male map. This reflects the higher number of contributing sires compared to dams to the progeny population, leading to an increase in male-informative markers compared to the female-informative markers (49,885 and 39,398 total markers, respectively). To compare sex-specific recombination patterns, we plotted the genetic positions (in centimorgans) of shared markers in the female linkage map against their positions in the male linkage map for each chromosome. This revealed striking heterochiasmy between the sexes (Figure 2). For every chromosome, male- and female-only regions of recombination were observed, with limited overlap. If recombination were uniform and random across the chromosome, the expectation would be a 1:1 linear relationship between male and female positions. Instead, our Australian L. calcarifer linkage maps showed that female recombination occurs primarily near the centromere, whereas male recombination is concentrated near the distal ends of the acrocentric/telocentric chromosomes (1–11 and 13–72) (Figure 2). This pattern also holds for the single submetacentric (centrally placed centromere) chromosome (12), where female recombination occurs near the centromere and male recombination near the chromosome ends. While heterochiasmy is common in eukaryotes [29-31], complete non-overlap of recombination regions is unusual, with the general exception of sex chromosomes [32-34]. To account for the pronounced heterochiasmy observed in Australian L. calcarifer , we generated two types of consensus maps. The first, an average distance map, which follows the conventional sex-averaged approach by averaging male and female marker positions. However, for this specific circumstance, this method compresses chromosome lengths because intervals in the non-recombining sex are collapsed. To overcome this limitation, we also developed an “additive” map, which sums male and female recombination distances, preserving the contribution of both sexes. For example, chromosome 1 spans 105.87 cM in the additive map compared to 52.93 cM in the mean map (Supplementary Table 2). This additive approach more closely resembles previous consensus map lengths for the species, where there is no sex-specific recombination regions [21], and therefore provides a more biologically meaningful representation of the total recombination potential across sexes in barramundi. The average inter-marker distances for each chromosome for the additive consensus map ranged from 0.09 to 0.18 cM. To our knowledge, this is the first application of an additive consensus map in a species with extreme heterochiasmy, offering a valuable alternative for studies where sex-specific recombination plays a critical role. Comparisons to the Asian Lates calcarifer genome assembly The recombination map of each chromosome was compared against the GCF_001640805.2 version of the physical genome of L. calcarifer from Southeast Asia [25], using a Marey map methodology [35], which visually compares the genetic and physical maps of a chromosome. When examining the recombination patterns of the sexes separately in the Marey maps, some discrepancies between the genetic linkage map and the GCF_001640805.2 assembly in the form of inversions were discovered [25]. In total, 10 inversions were found across the 24 chromosomes. Six inversions were shared between sexes (chromosomes 1, 3, 5, 14, 15, and 23), primarily occurring near crossover points between male- and female-specific recombination regions, suggesting these structural differences may coincide with transitions in recombination landscapes. However, two inversions could only be detected in the females (chromosomes 10 & 16) and two inversions were only detected in males (chromosome 9 & 11) due to the inverted regions falling inside the sex-specific recombination zones. Once the genetic linkage maps were finalised, several regions were observed to contain gaps in centimorgan (cM) positioning. These breaks in the linkage map relative to the physical map were examined in detail. Such gaps can sometimes result from a run of non-informative markers (markers homozygous across all parental samples), but no correlation was observed between gapped regions and marker homozygosity (Supplementary Figure 6). The gap detected on chromosome 6 is also unlikely to be caused by genotyping error. This is supported by the Marey map, which shows the gap in the genetic map (y-axis) but not in the physical map (x-axis) (Figure 3). Furthermore, removing a 1 cM window of markers on either side of the gap and rerunning OrderMarkers2 only increased the gap by 2 cM. These results suggest that the breaks are not due to erroneous or runs of uninformative markers but may instead represent regions of elevated recombination activity, potentially recombination hotspots. Australian versus Asian L. calcarifer chromosomal rearrangements and verifying marker order with LD analyses We used linkage disequilibrium (LD) analysis to validate marker order and confirm potential chromosomal rearrangements relative to the GCF_001640805.2 L. calcarifer reference genome [25]. A block of 36 consecutive SNPs originally assigned to chromosome 9 in the reference genome showed strong LD with markers on chromosome 12 in our linkage map. By calculating linkage disequilibrium (r 2 ) values between the putatively misplaced markers and the markers on chromosome 9 and 12, we were able to assign these markers to chromosome 12 in this population with high confidence (Supplementary Table ) indicating a chromosomal rearrangement between the Australian and Asian lineages, or alternatively a mis-assembly in the L. calcarifer genome. Furthermore, there was one more small potential rearrangement, where 5 markers that were previously assigned to chromosome 2 in the Asian L. calcarifer assembly (GCF_001640805.2) mapped to chromosome 9 in the linkage maps. Discussion This study presents the highest-density genetic linkage map for L. calcarifer to date, as well as the only available map resource for the Australian genetic lineage. Strikingly, our sex specific maps for the species reveal extreme differences in the male and female recombination landscapes, which is intriguing for a protandrous species as this means the locations of meiotic recombination change completely during the individual’s lifetime, something that is not shared by the Asian populations of the species. A key motivation for developing this resource was to create an Australia-specific reference, given the substantial genetic differences observed among L. calcarifer populations from India, Southeast Asia, and Australia/Papua New Guinea, despite their current classification as a single species [2, 9, 11, 13, 15, 36]. Our findings do reveal structural variations between the genomes of fish from these two major regions. When comparing our linkage map to the GCF_001640805.2 L. calcarifer genome assembly, derived from a Southeast Asian individual, we identified several large-scale inversions (Figure 3) [25] . These inversions were not reported in the recent Asian L. calcarifer linkage map, which also used the same reference genome [21]. However, two of these inversions (on chromosomes 3 and 5) were previously noted by Campbell and Hale (2024) [10]. Their study also identified an inversion on chromosome 20 that we did not detect, likely due to their inclusion of Indian-origin fish, which also exhibit distinct genetic profiles compared to Southeast Asian and Australian populations [10]. Campbell and Hale further noted that Indian populations were homozygous for one karyotype, Australian populations were homozygous for the inverted karyotype, and Southeast Asian fish displayed heterozygosity for both arrangements [10]. Notably, our study uncovered additional inversions on chromosomes 1, 9, 10, 11, 14, 15, 16, and 23, which were not reported by Campbell and Hale, 2024 [10]. Beyond these inversions, we observed potential chromosomal rearrangements, or assembly errors, when aligning our linkage map with the reference genome. Previous studies have also reported assembly inconsistencies in the L. calcarifer genome [37]. Using our genetic linkage map, we confirmed the errors identified by Shen et al. (2023) [37] and detected two additional discrepancies. These rearrangements were also present in the last published linkage map for Asian L. calcarifer , suggesting they are more likely due to assembly errors rather than population-specific chromosomal differences [21]. There are likely multiple factors driving the observed genomic inversions. For example, chromosomal inversions can suppress recombination across large genomic regions, preserving advantageous allele combinations that contribute to local adaptation [38]. It is plausible that region-specific selective pressures required such adaptive mechanisms, leading to divergence in genome structure over time. Further research would need to be conducted to verify this hypothesis. It is worth noting that a new iteration of the L calcarifer genome was published during this study (GCA_051027255.1) [39]. When compared to the previous assembly using the Comparative Genome Viewer in NCBI, this new assembly shows inversions on the same chromosomes detected in our analysis [40]. Given that Asian and Australian populations of L. calcarifer have been separated for approximately one million years and have remained reproductively isolated, it is still critical to develop and use genomic resources specific to the population under investigation, in this case, Australian stocks. Importantly, earlier linkage maps for Asian lineages of L. calcarifer and the GCF_001640805.2 genome are concordant and show no evidence of inversions, illustrating the extent of structural diversity across the species’ distribution. These findings highlight that creating genomic resources from randomly sampled individuals across the range could introduce interpretative errors, reinforcing the need for population-specific resources to ensure accuracy in genomic analyses. Chromosomal recombination and heterochiasmy Our high‑resolution genetic linkage map revealed distinct male‑ and female‑specific regions of meiotic recombination, with minimal overlap between the sexes (Figure 2). This spatial segregation of recombination is broadly consistent with patterns reported in a meta‑analysis of 61 teleost fish species [30], which found that, in most cases, male recombination rates are suppressed near the centromere and concentrated toward the telomeres, whereas females tend to exhibit peak recombination near the centromeres, with additional events distributed along the chromosome arms. In our study, this sex‑specific recombination landscape revealed was not apparent in earlier linkage maps for the species, which were created from individuals from the southeast Asian lineage, and found that males and females had similar recombination patterns [21]. Among published examples, the recombination profile most similar to that of the Australian population of L. calcarifer is found in Atlantic halibut ( Hippoglossus hippoglossus ), which also exhibits male‑ and female‑exclusive recombination regions on telocentric chromosomes: female recombination occurs predominantly near centromeres, while male recombination is restricted to distal chromosomal ends [41]. Notably, Atlantic halibut is gonochoristic, with an X/Y sex determination system, in contrast to the sequential hermaphroditism of L. calcarifer . This contrast suggests that the emergence of male‑ and female‑exclusive recombination regions cannot be explained solely by the need to limit recombination around sex‑determining loci. Instead, the drivers of these patterns remain elusive, and the molecular machinery that positions recombination hotspots in L. calcarifer warrants closer investigation. A key gene associated with recombination hotspot specification in vertebrates is PRDM9 , which has numerous orthologs and is present in at least 225 species, including humans, mice, apes, and salmon [42-45]. PRDM9 is among the fastest-evolving genes in vertebrates, and even closely related species differ in the recombination hotspots it determines [43]. Although widespread, many species carry truncated or incomplete PRDM9 variants lacking the KRAB and SSXRD domains, rendering them non-functional [45, 46]. Notably, no PRDM9 ortholog has been identified in the current L. calcarifer genome assembly. A BLAST search using the Atlantic salmon ( Salmo salar ) PRDM9 sequence (NC_059458.1) failed to detect a homolog in barramundi [25], suggesting that this species may lack a functional PRDM9 gene. In species without functional PRDM9 , recombination events are typically conserved and preferentially occur in regions with elevated guanine and cytosine (GC) content and increased chromatin accessibility, such as promoters and CpG islands [21, 47-49]. This pattern indicates that epigenetic factors may play a major role in directing recombination. In Australian L. calcarifer , differential DNA methylation between males and females has been observed, with differentially methylated regions (DMRs) associated with sexual phenotype despite the absence of sex chromosomes [50, 51]. However, current studies have focused primarily on gene-level DMRs, limiting insight into genome-wide patterns. To determine whether epigenetic factors contribute to sex-specific recombination landscapes, chromosome-level DNA methylation profiles should be generated for barramundi. Additionally, structural components of meiosis, such as the synaptonemal complex (SC), may also influence recombination localisation. The SC mediates homologous chromosome pairing and synapsis, and its length and organisation vary among species. In humans, sex-specific differences have been seen in the length of the SC [52]. The difference in SC length, coupled with chromatin packaging during meiotic prophase, allow the SC to preferentially target centromeric or telomeric regions [53]. Differences in SC length have also been detected in teleost fish [54, 55]. These observations suggest that epigenetic differences, combined with mechanical aspects of gametogenesis, such as chromatin and SC binding location, could enable specific recombination regions within the chromosome. The differential methylation of the barramundi genome between the sexes might lead to the expression of different SC homologs, explaining the extreme heterochiasmy of this sequential hermaphroditic species. Lates calcarifer may serve as an excellent model for investigating the evolutionary drivers of heterochiasmy, the phenomenon where recombination rates differ between sexes. A comprehensive review by Sardell and Kirkpatrick (2020) highlights several hypotheses for why heterochiasmy evolves, ranging from mechanistic constraints to adaptive explanations [56]. While some theories suggest that heterochiasmy could arise by chance through processes such as genetic drift acting on sex-averaged recombination rates, this is generally considered unlikely to fully explain the pattern. Mechanistic differences in gametogenesis, such as the distinct processes of sperm and egg formation, may also contribute to sex-specific recombination landscapes. Adaptive explanations, however, provide compelling scenarios. These include roles in female meiotic drive, a process where certain alleles, haplotypes, or even entire chromosomes are preferentially transmitted during meiosis, the selection against aneuploidy and sexually antagonistic selection, where alleles beneficial to one sex are detrimental to the other. Haploid selection, where selection acts on gametes, has also been proposed as a driver of heterochiasmy, although it is rarely observed in female animals [38]. In the context of L. calcarifer , which undergoes sequential hermaphroditism, these adaptive hypotheses take on additional complexity. The transition from male to female during the life cycle could impose unique selective pressures on recombination landscapes, potentially favouring mechanisms that reduce aneuploidy risk or optimise allele transmission across both sexual phases. This makes L. calcarifer a particularly valuable system for testing whether heterochiasmy is shaped by adaptive forces linked to sex-specific roles and life-history strategies. Significance for breeding programs Meiotic recombination is incredibly important to breeding programs as it allows for the creation of new combinations of alleles in the population, and assists in purging deleterious mutations from the population [57]. Additionally, it is important to know where regions of low recombination exist in the genome as these regions will naturally accrue deleterious mutations [58]. Intriguingly in Australian L. calcarifer, a protandrous species, regions of recombination will switch across the individual’s lifetime when the individual switches sex from male to female, conceivably allowing for the chance of recombination across the whole genome during an individual’s lifetime. This heterochiasmy pattern discovered in this genetic linkage map could be strategically applied to breeding programs in two ways. The first possible use of this map in Australian L. calcarifer breeding programs is using it to precisely map QTL. For instance, if we know that there are favourable QTL available in the population that are known to be located in the region near the centromere of the chromosome, an all-female population could be generated using hormonal implants [1] to maximise the recombination occurring in that region of the chromosome. From that, the precision of QTL mapping could be increased. The second use of the genetic linkage map information produced in this study would be to enable efficient stacking of desirable haplotype blocks to create superior progeny [59]. As an example, if multiple desirable haplotype blocks - segments of the chromosome that have high “local” genomic estimated breeding values - exist in the population near the distal end of the chromosome (away from the centromere), we can use these individuals as males so that their recombination occurs in our desired chromosomal region (Figure 4). Additionally, we can use this striking heterochiasmy to introgress traits from wild populations into our farmed fish stocks while minimising linkage drag of unfavourable traits into our elite lines to bolster genetic diversity, disease resistance, and maintain our breeding targets. Conclusion The high-density genetic linkage map developed in this study represents the most comprehensive genetic map for L. calcarifer to date, with 49,885 SNPs mapped to 24 linkage groups. This map provides critical insights into the genetic architecture and meiotic recombination patterns of Australian L. calcarifer . The deviation from the recombination landscape of the Asian L. calcarifer population highlight the genetic divergence of these populations. Furthermore, the observed sex-specific and population-level differences in recombination, alongside the absence of heteromorphic sex chromosomes and the species’ protandrous nature, highlight L. calcarifer ’s potential as a model for investigating the evolution of heterochiasmy. Understanding the spatial distribution of recombination events enables more precise QTL mapping by informing the strategic selection of individuals based on sex and recombination activity. Moreover, the sex-specific recombination architecture presents novel opportunities for targeted breeding in this sequential hermaphrodite. By leveraging the ability to direct reproductive sex, it becomes possible to accelerate the assembly of favourable haplotypes, stacking chromosome blocks associated with traits such as growth, disease resistance, and environmental resilience. This map not only enhances our understanding of L. calcarifer genetics but also unlocks a new frontier in precision breeding, where genomic insights can be directly translated into optimised, sex-informed selection strategies for sustainable aquaculture. Collectively, these findings advance our understanding of L. calcarifer genetics and provide a robust foundation for future research in evolutionary biology, genomics, and aquaculture breeding strategies. METHODS Genotyping Genotyping for this study was sourced from a previous dataset generated by a commercial barramundi aquaculture company (Mainstream Aquaculture Group, Australia), where broodstock in a selective breeding program were fewer than four generations removed from the wild [8]. Briefly, SNP array genotyping was performed using a custom 70K Axiom™ myDesign™ SNP array from ThermoFisher Scientific™, and a subset of the total dataset was used so that the dataset for this study comprised of progeny sampled across nine families derived from three dams and seven sires, resulting in 1,952 F1 offspring [8]. Fin clips from these fish provided DNA for genotyping, which was conducted at the Ramaciotti Centre for Genomics using a GeneTitan™ platform following a stringent quality control pipeline. DNA extraction was carried out with the Chemagic™ DNA Extraction Kit on a Zephyr™ automated workstation. Raw SNP data underwent rigorous filtering to ensure accuracy: a minimum call rate of 0.98, cluster separation score (FLD) of at least 4.0, heterozygous signal strength offset (HetSO) no lower than −0.04, and a minor allele frequency threshold of 0.01. Additionally, sample call rate (≥95%) and Dish QC (≥0.80) were applied as further criteria. After these steps, the dataset retained 52,245 high-quality polymorphic SNPs for linkage mapping. For a full description of these methods, see Jerry et al. (2022) [8]. Genetic Linkage Map Construction The dataset in PLINK bfile format was first converted to the Lep-MAP3 format using custom bash and R scripts in R 4.3.0 [60, 61]. Parental sex was defined as either 1 (male) or 2 (female), while offspring sex was left blank. We used Lep-MAP3 (version 0.5) to construct the genetic linkage maps [27]. First, we filtered the data using the ParentCall2 module to ensure accurate parental genotypes based on Mendelian inheritance patterns. Subsequently, the Filtering2 module removed uninformative markers (removeNonInformative = 1), and markers with high segregation distortion (dataTolerance = 0.00001). The data were then further filtered to remove markers with minor allele frequencies (MAF) (MAFLimit = 0.01) or missing (missingLimit = 0.2) [62]. Markers were first assigned to linkage groups (LGs) using the SeparateChromosomes2 module, testing a range of logarithm of odds (LOD) scores (10–80) with the lodLimit function and setting a minimum of 50 markers per LG. Marker ordering within each LG was performed using OrderMarkers2 with Haldane’s mapping function, running five independent iterations per LG and selecting the run with the highest likelihood. In cases where convergence was not achieved, the hyperPhaser parameter was enabled, and the number of merge and polish iterations was increased to 20 and 5, respectively (from defaults of 6 and 2). With these adjustments, all five replicates were visualised, and the best replicate was chosen based on consistent structure, highest likelihood score, and absence of parallel lines (Supplementary Figure 3). Sex-specific maps were generated using male-informative (informativeMask = 13) or female-informative (informativeMask = 23) markers, including double-heterozygous markers. Male and female maps were then merged in R, ensuring consistent chromosome orientation across sexes and families. Two sex-averaged maps were produced: one by calculating the mean marker position between the sex-specific maps (mean map) and another by summing the two positions (additive map), as recombination occurs in only one sex at a given position. During map construction, some regions could not be resolved by OrderMarkers2, resulting in parallel lines in the sex that was not recombining (Supplementary Figure 4B). These issues were absent in single-family maps (Supplementary Figure 4A) but became apparent as more families were added, with no single family responsible for the discrepancies. We hypothesise that these parallel lines arise when families have differentially informative markers, creating uncertainty in regions of no recombination. Enabling the hyperPhaser parameter resolved these issues (Supplementary Figure 5). Polishing Gaps in some of the linkage groups in the map were interrogated for informativeness (not homozygous) or for misplaced markers. Heterozygosity for the male and female founders was calculated separately using PLINK 1.9 [61]. For the female founders, the flag --freqx generated frequency data, which was then processed using gawk scripts. The gawk scripts were used to calculate the total number of animals by summing the counts of homozygous alleles (A1 and A2), heterozygous alleles, and missing data. Next, gawk was employed to compute heterozygosity by dividing the heterozygosity count by the total count of animals. This process was similarly applied to the male founders. The resulting files for both male and female founders were imported into R for further analysis. In R, the chromosomes of interest—those with gaps and parallel structures—were subsetted from the dataset. Homozygosity was inferred for SNPs where heterozygosity values were either 0 or 1. Finally, these homozygous SNPs were overlaid on heterochiasmy plots to visualize the data effectively. Gaps in the maps were further interrogated for misplaced markers that could be artificially inflating the genetic distance. To do this, a 1 cM window of markers from either side of the gap were removed and OrderMarkers2 was rerun with the new subset. Finally, any markers that could not be placed were removed from the map. Visualisation and Genome Comparison Genetic linkage maps were visualised using ggplot2 v3.4.2 [63], which provided an overview for chromosome-level data. To assess sex-specific recombination differences, marker positions along male and female linkage maps were compared at the chromosome scale, and these comparisons were also implemented and visualised within the ggplot2 framework to ensure consistency and clarity in data representation. To assess synteny between the Australian L. calcarifer linkage maps and the L. calcarifer reference genome assembly (GCF_001640805.2), Marey maps [35, 64] were generated using custom R scripts following the approach described by Akopyan et al. [65]. Centromere positions were inferred by comparing the genome assembly with the published karyotype for the Australian population [11]. The largest chromosome (likely chromosome 12 in GCF_001640805.2) is the only one exhibiting a metacentric/submetacentric centromere, while the remaining 23 chromosomes display acrocentric or telocentric configurations. Recombination patterns on this metacentric chromosome revealed female recombination concentrated in the central region and male recombination restricted to the ends. These observations, combined with previous studies in teleosts [30, 41], allowed us to extrapolate centromere locations and infer recombination landscapes across the genome, under the assumption that sex-specific recombination patterns are consistent across all chromosomes. Linkage Disequilibrium Linkage disequilibrium was calculated by estimating R 2 between all pairs of markers, regardless of the putative chromosome, using PLINK v 1.9 [61]. During the scaffolding process for the genome assembly, chromosome 7 was divided into two separate segments due to minimal supporting evidence for their connection [25]. These segments are now recognised as distinct chromosomes, designated as 7.1 and 7.2 (71 and 72) as well as a chromosome 100, where unplaced markers were collected (--r2 --out ldfile --autosome-num 71 --allow-extra-chr --inter-chr). Discrepancies in the assembly that were detected in the genetic linkage map were then confirmed by observing if the R 2 values were higher in the assembly chromosome or the mapped chromosome. Debugging The use of Large Language Models (LLMs) was used to assist in the debugging of R code when needed. The models used were Copilot, ChatGPT, and Claude. Models were also used to refine the language of written text. Models were not used to generate new text, ideas or in research. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The initial datasets used during the current study are available from the corresponding author on reasonable request. All genetic linkage maps generated during this study are included in this published article [and its supplementary information files]. The code used for all analyses in this manuscript and for the production of figures is available online at the GitHub page website of https://github.com/DigitalFins/Lates_calcarifer_Linkage_map_2025. Competing interests The authors declare that they have no competing interests Funding This project is funded through the Australian Research Council Industrial Transformation Research Program (IH210100014) Supercharging Tropical Aquaculture through Genetic Solutions Authors' contributions JH conceptualised the study, performed data analysis, curated the data, validated results, visualised results, project administration, and drafted the manuscript LVB contributed to methodology, Lep-MAP3 code, data curation, and review and editing of this manuscript DBJ conceptualised the study, assisted with methodology, data curation, provided data resources, and review and editing of this manuscript CW assisted with data curation, review of this manuscript, and supervision OP assisted with data curation, review and editing of this manuscript, and supervision EMR contributed to supervision and review and editing of this manuscript PH contributed the genomic resources for this study HC contributed the genomic resources for this study DJ contributed resources, supervision, review and editing of this manuscript, and acquired funding to support this project BH contributed to the conceptualisation, methodology, validation, supervision, project administration, and review and editing of this manuscript KRZ contributed to the conceptualisation, methodology, validation, supervision, project administration, and review and editing of this manuscript Acknowledgements We gratefully acknowledge Dr Cecile Massault for her invaluable support in transferring data and for generously sharing her expertise in quantitative genetics. Additionally, we would like to thank Hayden B. Ahwan for providing insight into the population structures of Lates calcarifer . Authors' information (optional) References Guppy JL, Marc AF, Jerry DR: Maturation and spawning performance of hormonally-induced precocious female barramundi (Lates calcarifer) and implications of their use in selective breeding. Aquaculture 2022, 552: 737991. Jerry DR: Biology and Culture of Asian Seabass. CRC Press Taylor & Francis Group; 2014. Yue GH, Wang L, Sun F, Yang ZT, Wong J, Wen YF, Pang HY, Lee M, Yeo ST, Liang B, et al: Improving growth, omega-3 contents, and disease resistance of Asian seabass: status of a 20-year family-based breeding program. Reviews in Fish Biology and Fisheries 2023. Yue GH, Wang L, Yang Z, Sun F, Tay YX, Wong J, Yeo S: Genomic resources and their applications in aquaculture of Asian seabass (Lates calcarifer). Reviews in Aquaculture 2023, 15: 853-871. 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Banh QQT, Guppy JL, Domingos JA, Budd AM, Pinto RCC, Marc AF, Jerry DR: Induction of precocious females in the protandrous barramundi (Lates calcarifer) through implants containing 17β-estradiol - effects on gonadal morphology, gene expression and DNA methylation of key sex genes. Aquaculture 2021, 539 . Tease C, Hultén MA: Inter-sex variation in synaptonemal complex lengths largely determine the different recombination rates in male and female germ cells. Cytogenetic and genome research 2004, 107: 208-215. Gruhn Jennifer R, Al-Asmar N, Fasnacht R, Maylor-Hagen H, Peinado V, Rubio C, Broman Karl W, Hunt Patricia A, Hassold T: Correlations between Synaptic Initiation and Meiotic Recombination: A Study of Humans and Mice. The American Journal of Human Genetics 2016, 98: 102-115. Campos-Ramos R, Harvey SC, Penman DJ: Sex-specific differences in the synaptonemal complex in the genus Oreochromis (Cichlidae). Genetica 2009, 135: 325-332. Madison BS, Nath S, Flanagan MK, Dorsey BA, White MA: Sexual dimorphism of synaptonemal complex length among populations of threespine stickleback fish. bioRxiv 2021 : 2020.2007.2030.228825. Sardell JM, Kirkpatrick M: Sex differences in the recombination landscape. The American Naturalist 2020, 195: 361-379. Muller HJ: The relation of recombination to mutational advance. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 1964, 1: 2-9. Yang J, Mezmouk S, Baumgarten A, Buckler ES, Guill KE, McMullen MD, Mumm RH, Ross-Ibarra J: Incomplete dominance of deleterious alleles contributes substantially to trait variation and heterosis in maize. PLOS Genetics 2017, 13: e1007019. Villiers K, Voss-Fels KP, Dinglasan E, Jacobs B, Hickey L, Hayes BJ: Evolutionary computing to assemble standing genetic diversity and achieve long-term genetic gain. bioRxiv 2023 : 2023.2005.2005.539510. R: A Language and Environment for Statistical Computing [https://www.R-project.org/] Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, Sham PC: PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 2007, 81: 559-575. Bertola LV, Hoskin CJ, Jones DB, Zenger KR, McKnight DT, Higgie M: The first linkage map for Australo-Papuan Treefrogs (family: Pelodryadidae) reveals the sex-determination system of the Green-eyed Treefrog (Litoria serrata). Heredity 2023, 131: 263-272. Wickham H: ggplot2. Wiley interdisciplinary reviews: computational statistics 2011, 3: 180-185. Rezvoy C, Charif D, Guéguen L, Marais GA: MareyMap: an R-based tool with graphical interface for estimating recombination rates. Bioinformatics 2007, 23: 2188-2189. Akopyan M, Tigano A, Jacobs A, Wilder AP, Baumann H, Therkildsen NO: Comparative linkage mapping uncovers recombination suppression across massive chromosomal inversions associated with local adaptation in Atlantic silversides. Molecular Ecology 2022, 31: 3323-3341. Additional Declarations No competing interests reported. Supplementary Files LcalcariferLinkagePhysicalsexavgmap.txt SupplementaryInformation.dotx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8400904","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":567914116,"identity":"4be974a9-a305-48c6-b522-74848c202ded","order_by":0,"name":"Jessica Hintzsche","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBACxgYehgNglgQD4wMw4wBRWhLAWpgNiNLCwMADxBAtbBJEaWFu4D14uPBHnT3/7OZj1TxlDHJ8NxIYP/PgdRhfwuEZCWyJM+4cS7vNc47BWPJGArM0fi08Bod5EngSDCRyzG7ztjEkbriRwECMFgl7A4n8b8VALfVALcy/idBiwLhBIoeNGaglweBGAht+W5pBWtISEmfcSDOWnHNOwnDmmYdtlnPwaDFs7zH+zGMDDLEZyQ8/vCmzkec7nnz4xht8WppRuOCoYWzAo4GBQR6Vy4ZX8SgYBaNgFIxQAADy70aW4xgP2gAAAABJRU5ErkJggg==","orcid":"","institution":"The University of Queensland","correspondingAuthor":true,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Hintzsche","suffix":""},{"id":567914117,"identity":"28732684-052e-4f04-badc-d1ea9fdc91ec","order_by":1,"name":"Lorenzo Vincenzo Bertola","email":"","orcid":"","institution":"Australian Institute of Marine Science","correspondingAuthor":false,"prefix":"","firstName":"Lorenzo","middleName":"Vincenzo","lastName":"Bertola","suffix":""},{"id":567914118,"identity":"7cbcbad0-1084-4449-9981-a393befc5803","order_by":2,"name":"David B. 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Zenger","email":"","orcid":"","institution":"James Cook University, ARC Industrial Transformation Research Hub for Supercharging Tropical Aquaculture through Genetic Solutions, James Cook University","correspondingAuthor":false,"prefix":"","firstName":"Kyall","middleName":"R.","lastName":"Zenger","suffix":""}],"badges":[],"createdAt":"2025-12-19 05:38:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8400904/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8400904/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99794704,"identity":"4335e599-d586-438b-8ff0-911484b7f8bb","added_by":"auto","created_at":"2026-01-08 13:36:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":552787,"visible":true,"origin":"","legend":"\u003cp\u003eThe consensus additive (male cM position added to the female cM position) genetic linkage map of Australian barramundi (Lates calcarifer) from a combined family dataset. Each point represents a marker locus. Chromosome numbering follows the current genome assembly, where chromosome 7 was split into two linkage groups (7.1 and 7.2, now 71 and 72, respectively), and chromosomes 16 and 22 were merged into a single linkage group (ASB_LG16_LG22, now just 22). As a result, the map contains 24 linkage groups. Average inter-marker distance ranged from 0.09 to 0.18 cM.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8400904/v1/e0e089dac76753705d7e5e6f.png"},{"id":99795908,"identity":"b690351a-9680-4d73-a643-28da975bcd61","added_by":"auto","created_at":"2026-01-08 13:40:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":258166,"visible":true,"origin":"","legend":"\u003cp\u003eHeterochiasmy between male and female linkage maps\u003cstrong\u003e.\u003c/strong\u003e Each point represents a shared marker, with its position in centimorgans (cM) in the female map on the x-axis and in the male map on the y-axis across all linkage groups. Chromosome numbering reflects the current genome assembly, where chromosome 7 was split into two linkage groups (7.1 and 7.2, shown as 71 and 72) and chromosomes 16 and 22 were merged (ASB_LG16_LG22). As a result, this figure includes 71 and 72 but no chromosome 22, while still representing 24 linkage groups in total.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8400904/v1/7de92778c698d75a94746f55.png"},{"id":99795739,"identity":"92adad52-8d73-4f0d-b172-5d5220d06475","added_by":"auto","created_at":"2026-01-08 13:39:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1660659,"visible":true,"origin":"","legend":"\u003cp\u003eMarey maps comparing the sex-specific genetic linkage maps of Australian L. calcarifer fish (y-axis) with the L. calcarifer genome assembly (GCF_001640805.2) (x-axis) [22]. Each plot shows the relationship between genetic and physical positions of each SNP for a chromosome. Chromosome numbering follows the current assembly, where chromosome 7 was split into two (7.1 and 7.2, shown as 71 and 72) and chromosomes 16 and 22 were merged (ASB_LG16_LG22, shown as 16). All previously reported assembly errors have been corrected, and chromosomes containing markers that were reassigned during this process are indicated with an asterisk (*) to the right of the chromosome number [26]. Chromosomes exhibiting inversions are marked with coloured dots to the left of the chromosome number (green = male inversion; blue = female inversion).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8400904/v1/fd170831d36a3f5082842f66.png"},{"id":99672746,"identity":"bf3b87a7-92b0-4bc8-8ca7-426485a78d57","added_by":"auto","created_at":"2026-01-07 07:26:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":555270,"visible":true,"origin":"","legend":"\u003cp\u003eThe Australian population of Lates calcarifer (barramundi) exhibits distinct regions of meiotic recombination for males and females, allowing breeding programs to exploit the protandrous nature of the species by choosing whether to utilise an individual as a female or male, based on the desired chromosomal reshuffling. This figure illustrates pairs of homologous chromosomes with highlighted chromatids. There are two scenarios in this figure, each representing an individual. The Homologous Chromosomes panel demonstrates two hypothetical individuals (top and bottom) involving favourable (green) and unfavourable (red) haplotypes. The Sex Decision/ Genetic exchange panel indicates the sex the breeding program might elect to use the individual as to control the region of recombination. The Gametes panel presents examples of recombined chromosomes that may be inherited, with desirable gametes marked with a green check. (Figure by Maggie Meiring)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8400904/v1/1f4d8872b43055d3fdba6f98.png"},{"id":99805019,"identity":"a8f21453-3a0b-4185-a509-282674196656","added_by":"auto","created_at":"2026-01-08 14:15:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7239359,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8400904/v1/f962fb00-8c70-4efb-8cae-5c316e91d395.pdf"},{"id":99672747,"identity":"0f40fd27-8277-45e2-95b0-72e9e07a9fe2","added_by":"auto","created_at":"2026-01-07 07:26:28","extension":"txt","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3546399,"visible":true,"origin":"","legend":"","description":"","filename":"LcalcariferLinkagePhysicalsexavgmap.txt","url":"https://assets-eu.researchsquare.com/files/rs-8400904/v1/445a7cc77b6cc289acb22d8b.txt"},{"id":99672742,"identity":"6af8f88f-2a02-43d6-beda-29cdc2cec8e9","added_by":"auto","created_at":"2026-01-07 07:26:26","extension":"dotx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1723290,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.dotx","url":"https://assets-eu.researchsquare.com/files/rs-8400904/v1/7ea1a2bb53cba04d9bebc288.dotx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic linkage map of the Australian barramundi (Lates calcarifer) reveals potential to leverage extreme sex-specific recombination and sequential hermaphrodism for ultimate breeding program control","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cem\u003eLates calcarifer\u003c/em\u003e is a rapidly emerging aquaculture species across tropical and subtropical Indo-Pacific regions. Native to India, Sri Lanka, Southeast Asia, northern Australia, and Papua New Guinea, it is commonly called “Asian seabass” around the world but is called “barramundi” in Australia. The species has a complex life history as a protandrous hermaphrodite, maturing first as male before transitioning to female later in life. It is also euryhaline and catadromous, spawning in seawater while juveniles migrate to brackish and freshwater habitats to grow [1]. It is \u003cem\u003eL. calcarifer’s\u003c/em\u003e ability to be farmed in diverse culture systems and salinities, along with its favourable production traits, that has resulted to its emergence as a species of growing global aquaculture significance\u003cem\u003e \u003c/em\u003e[2] and as a result there are a number of breeding programs underway to improve the species productivity [3-8]. \u003c/p\u003e\n\u003cp\u003eGenomic studies reveal that \u003cem\u003eL. calcarifer\u003c/em\u003e exhibits clear genetic structuring across its natural range, with distinct lineages suggesting the presence of incipient or even separate species [2, 9-11]. This divergence reflects an evolutionary history shaped by historical sea-level fluctuations, which created barriers to gene flow between Asian and Australian–Papua New Guinean populations. As a euryhaline teleost, \u003cem\u003eL. calcarifer\u003c/em\u003e occupies freshwater systems, estuaries, and nearshore marine environments, making it sensitive to topographical changes. During parts of the Pleistocene, lowered sea levels eliminated the present-day Torres Strait, Arafura Sea, and Java Sea [12], enabling migration from Asia to Australia. Subsequent sea-level rise severed these pathways, isolating populations on the two continents for at least one million years [13, 14]. Evidence of this isolation is seen in Australian populations on either side of the Torres Strait, which differ by approximately 4% in sequence, a divergence attributed to these historical events [13]. Collectively, these genetic differences underscore the need to create population-specific resources and support treating Asian and Australian populations as distinct genetic lineages [9].\u003c/p\u003e\n\u003cp\u003eStructural variation in the genome provides further evidence for the separation between Asian and Australian \u003cem\u003eL. calcarifer\u003c/em\u003e. Further evidence of divergence between Asian and Australian lineages is provided by karyotypic analyses, which reveal that Asian \u003cem\u003eL. calcarifer \u003c/em\u003epossess chromosomes with more centrally located centromeres (metacentric and submetacentric), while Australian specimens exhibit chromosomes with more distally positioned centromeres (acrocentric and telocentric) [11, 15]. Given the evolutionary stability of centromeric regions, these structural shifts likely reflect long-term genetic isolation among populations, with centromere repositioning serving as a marker of divergent chromosomal evolution [16]. Therefore, there is a case that development of genomic resources should be conducted independently for Asian and Australian lineages to account for their distinct evolutionary history.\u003c/p\u003e\n\u003cp\u003eA genetic linkage map is an essential genomic resource for any genetically divergent population. These maps are utilised across a range of biological disciplines, including evolutionary biology, conservation, and advanced animal breeding programs due to their ability to determine the relative positions of genetic markers within chromosomes (linkage groups, LG). This information assists with scaffold orientation in genome sequence assembly, quantitative trait locus (QTL) identification, and comparative genomics [17, 18]. Genetic linkage maps also significantly improve the accuracy of selection processes in breeding initiatives.\u003c/p\u003e\n\u003cp\u003eA particularly important application of genetic linkage maps lies in its characterisation of recombination events. Mapping the precise locations and frequencies of chromosomal crossovers gives insight into how alleles are transmitted from parents to offspring, shaping the genetic population structure of future generations. This is especially helpful in aquaculture species, which display remarkable diversity and encompass a wide range of reproductive strategies [19, 20]. The ability to pinpoint crossover events can assist breeders in increasing favourable alleles in the population and ensuring that desired traits are reliably passed on.\u003c/p\u003e\n\u003cp\u003eGenetic linkage maps can be further refined by constructing separate maps for males and females, enabling the characterisation of sex‑specific differences in recombination. These maps are generated by tracking the frequency and chromosomal positions of crossover events (chiasmata) during gametogenesis, using informative meiosis events transmitted from sires, to produce the male map, and from dams, to produce the female map. This information is useful to breeders as this helps breeding programs understand the genetic structure of progeny populations and resolve problems such as linkage drag. Additionally, linkage maps can reveal recombination differences of evolutionary significance, including patterns relevant to studies of heterochiasmy evolution. \u003c/p\u003e\n\u003cp\u003eOver the past two decades, four successive genetic linkage maps have been developed for \u003cem\u003eL. calcarifer\u003c/em\u003e, each reflecting incremental gains in marker density and genome coverage, culminating in 8,274 SNPs anchored to the ~700 Mb reference genome [21-25]. These maps have provided important genomic insights, including initial evidence of sex differences in centimorgan-based map lengths [21]. Yet a high-resolution, genome‑wide assessment of sex‑specific recombination patterns remains unexplored. This gap is particularly important given the evolutionary interest in heterochiasmy, especially with a sequentially hermaphroditic species that lacks differentiated sex chromosomes, as well as the practical importance of recombination knowledge for selective breeding. The Australian genetic lineage, despite the species’ prominence to Australian aquaculture and commencement of breeding programs [3, 5, 8, 26], is yet to have a genome-wide genetic linkage map constructed. In this study, we address that gap by generating the first high‑resolution genetic linkage map for Australian \u003cem\u003eL. calcarifer\u003c/em\u003e using a custom 70K SNP genotyping array, enabling fine‑scale characterisation of sex‑specific recombination landscapes. This resource offers new opportunities to explore the evolution of heterochiasmy and to design genomic breeding programs optimised for both genetic gain and long‑term diversity.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGenetic Linkage Map Construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo construct the \u003cem\u003eL. calcarifer\u003c/em\u003e genetic linkage map, we analysed 49,885 informative SNP genotypes from 1,952 progeny derived from 7 male and 3 female parents following extensive quality control [8]. Additional filtering was performed using ParentCall2 to identify Mendelian inconsistencies. Although minor errors were detected at a few loci within certain families, sufficient accurate genotypes were present across the full dataset. After silencing these errors within affected families, no markers were removed, ensuring comprehensive marker coverage for map construction.\u003c/p\u003e\n\u003cp\u003eLep-MAP3 was used for genetic linkage map construction [27]. Varying the marker pair-wise logarithm of odds (LOD) threshold between 10 and 80 in the SeparteChromomses2 module did not affect the number of linkage groups; however, marker retention declined, and the proportion of unplaced markers increased sharply beyond a LOD score of 29 (Supplementary Figure 1). A LOD score of 28 was selected for all subsequent analyses. The resulting 24 linkage groups (LG) (Figure 1) align with the known karyotype of \u003cem\u003eL. calcarifer\u003c/em\u003e, which comprises 24 chromosomes [11, 15, 28]. The linkage groups were labelled such that the numbers match the chromosome number from the genome assembly [25]. \u003c/p\u003e\n\u003ch3\u003eSex-specific maps\u003c/h3\u003e\n\u003cp\u003eSeparate male- and female-only genetic linkage maps were then created to identify any linkage group regions that exhibited differences in recombination rates between the sexes (Supplementary Figure 2). These maps retain the high density of the consensus linkage map, with an average distance between marker loci ranging from 0.13 and 0.25 cM in the males and 0.9 and 0.18 cM in the females (Supplementary Table 1). Both maps had 24 linkage groups. However, there were differences in total marker counts and chromosome lengths (Supplementary Table ). For instance, males exhibited a slightly higher number of mapped markers across most chromosomes, such as chromosome 1, which included 1,957 markers in males compared to 1,551 in females. Similarly, cumulative linkage group lengths tended to be longer in the male map. This reflects the higher number of contributing sires compared to dams to the progeny population, leading to an increase in male-informative markers compared to the female-informative markers (49,885 and 39,398 total markers, respectively). \u003c/p\u003e\n\u003cp\u003eTo compare sex-specific recombination patterns, we plotted the genetic positions (in centimorgans) of shared markers in the female linkage map against their positions in the male linkage map for each chromosome. This revealed striking heterochiasmy between the sexes (Figure 2). For every chromosome, male- and female-only regions of recombination were observed, with limited overlap. If recombination were uniform and random across the chromosome, the expectation would be a 1:1 linear relationship between male and female positions. Instead, our Australian \u003cem\u003eL. calcarifer\u003c/em\u003e linkage maps showed that female recombination occurs primarily near the centromere, whereas male recombination is concentrated near the distal ends of the acrocentric/telocentric chromosomes (1–11 and 13–72) (Figure 2). This pattern also holds for the single submetacentric (centrally placed centromere) chromosome (12), where female recombination occurs near the centromere and male recombination near the chromosome ends. While heterochiasmy is common in eukaryotes [29-31], complete non-overlap of recombination regions is unusual, with the general exception of sex chromosomes [32-34]. \u003c/p\u003e\n\u003cp\u003eTo account for the pronounced heterochiasmy observed in Australian \u003cem\u003eL. calcarifer\u003c/em\u003e, we generated two types of consensus maps. The first, an average distance map, which follows the conventional sex-averaged approach by averaging male and female marker positions. However, for this specific circumstance, this method compresses chromosome lengths because intervals in the non-recombining sex are collapsed. To overcome this limitation, we also developed an “additive” map, which sums male and female recombination distances, preserving the contribution of both sexes. For example, chromosome 1 spans 105.87 cM in the additive map compared to 52.93 cM in the mean map (Supplementary Table 2). This additive approach more closely resembles previous consensus map lengths for the species, where there is no sex-specific recombination regions [21], and therefore provides a more biologically meaningful representation of the total recombination potential across sexes in barramundi. The average inter-marker distances for each chromosome for the additive consensus map ranged from 0.09 to 0.18 cM. To our knowledge, this is the first application of an additive consensus map in a species with extreme heterochiasmy, offering a valuable alternative for studies where sex-specific recombination plays a critical role.\u003c/p\u003e\n\u003ch3\u003eComparisons to the Asian \u003cem\u003eLates calcarifer\u003c/em\u003e genome assembly\u003c/h3\u003e\n\u003cp\u003eThe recombination map of each chromosome was compared against the GCF_001640805.2 version of the physical genome of \u003cem\u003eL. \u003c/em\u003e\u003cem\u003ecalcarifer\u003c/em\u003e from Southeast Asia [25], using a Marey map methodology [35], which visually compares the genetic and physical maps of a chromosome. When examining the recombination patterns of the sexes separately in the Marey maps, some discrepancies between the genetic linkage map and the GCF_001640805.2 assembly in the form of inversions were discovered [25]. In total, 10 inversions were found across the 24 chromosomes. Six inversions were shared between sexes (chromosomes 1, 3, 5, 14, 15, and 23), primarily occurring near crossover points between male- and female-specific recombination regions, suggesting these structural differences may coincide with transitions in recombination landscapes. However, two inversions could only be detected in the females (chromosomes 10 \u0026amp; 16) and two inversions were only detected in males (chromosome 9 \u0026amp; 11) due to the inverted regions falling inside the sex-specific recombination zones. \u003c/p\u003e\n\u003cp\u003eOnce the genetic linkage maps were finalised, several regions were observed to contain gaps in centimorgan (cM) positioning. These breaks in the linkage map relative to the physical map were examined in detail. Such gaps can sometimes result from a run of non-informative markers (markers homozygous across all parental samples), but no correlation was observed between gapped regions and marker homozygosity (Supplementary Figure 6). The gap detected on chromosome 6 is also unlikely to be caused by genotyping error. This is supported by the Marey map, which shows the gap in the genetic map (y-axis) but not in the physical map (x-axis) (Figure 3). Furthermore, removing a 1 cM window of markers on either side of the gap and rerunning OrderMarkers2 only increased the gap by 2 cM. These results suggest that the breaks are not due to erroneous or runs of uninformative markers but may instead represent regions of elevated recombination activity, potentially recombination hotspots.\u003c/p\u003e\n\u003ch3\u003eAustralian versus Asian \u003cem\u003eL. calcarifer\u003c/em\u003e chromosomal rearrangements and verifying marker order with LD analyses\u003c/h3\u003e\n\u003cp\u003eWe used linkage disequilibrium (LD) analysis to validate marker order and confirm potential chromosomal rearrangements relative to the GCF_001640805.2 \u003cem\u003eL. calcarifer\u003c/em\u003e reference genome [25]. A block of 36 consecutive SNPs originally assigned to chromosome 9 in the reference genome showed strong LD with markers on chromosome 12 in our linkage map. By calculating linkage disequilibrium (r\u003csup\u003e2\u003c/sup\u003e) values between the putatively misplaced markers and the markers on chromosome 9 and 12, we were able to assign these markers to chromosome 12 in this population with high confidence (Supplementary Table ) indicating a chromosomal rearrangement between the Australian and Asian lineages, or alternatively a mis-assembly in the \u003cem\u003eL. calcarifer\u003c/em\u003e genome. Furthermore, there was one more small potential rearrangement, where 5 markers that were previously assigned to chromosome 2 in the Asian \u003cem\u003eL. calcarifer\u003c/em\u003e assembly (GCF_001640805.2) mapped to chromosome 9 in the linkage maps.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study presents the highest-density genetic linkage map for \u003cem\u003eL. calcarifer\u003c/em\u003e to date, as well as the only available map resource for the Australian genetic lineage. Strikingly, our sex specific maps for the species reveal extreme differences in the male and female recombination landscapes, which is intriguing for a protandrous species as this means the locations of meiotic recombination change completely during the individual’s lifetime, something that is not shared by the Asian populations of the species. \u003c/p\u003e\n\u003cp\u003eA key motivation for developing this resource was to create an Australia-specific reference, given the substantial genetic differences observed among \u003cem\u003eL. calcarifer\u003c/em\u003e populations from India, Southeast Asia, and Australia/Papua New Guinea, despite their current classification as a single species [2, 9, 11, 13, 15, 36]. Our findings do reveal structural variations between the genomes of fish from these two major regions. When comparing our linkage map to the GCF_001640805.2 \u003cem\u003eL. calcarifer\u003c/em\u003e genome assembly, derived from a Southeast Asian individual, we identified several large-scale inversions (Figure 3) [25] . These inversions were not reported in the recent Asian \u003cem\u003eL. calcarifer \u003c/em\u003elinkage map, which also used the same reference genome [21]. However, two of these inversions (on chromosomes 3 and 5) were previously noted by Campbell and Hale (2024) [10]. Their study also identified an inversion on chromosome 20 that we did not detect, likely due to their inclusion of Indian-origin fish, which also exhibit distinct genetic profiles compared to Southeast Asian and Australian populations [10]. Campbell and Hale further noted that Indian populations were homozygous for one karyotype, Australian populations were homozygous for the inverted karyotype, and Southeast Asian fish displayed heterozygosity for both arrangements [10].\u003c/p\u003e\n\u003cp\u003eNotably, our study uncovered additional inversions on chromosomes 1, 9, 10, 11, 14, 15, 16, and 23, which were not reported by Campbell and Hale, 2024 [10]. Beyond these inversions, we observed potential chromosomal rearrangements, or assembly errors, when aligning our linkage map with the reference genome. Previous studies have also reported assembly inconsistencies in the \u003cem\u003eL. calcarifer\u003c/em\u003e genome [37]. Using our genetic linkage map, we confirmed the errors identified by Shen et al. (2023) [37] and detected two additional discrepancies. These rearrangements were also present in the last published linkage map for Asian \u003cem\u003eL. calcarifer\u003c/em\u003e, suggesting they are more likely due to assembly errors rather than population-specific chromosomal differences [21].\u003c/p\u003e\n\u003cp\u003eThere are likely multiple factors driving the observed genomic inversions. For example, chromosomal inversions can suppress recombination across large genomic regions, preserving advantageous allele combinations that contribute to local adaptation [38]. It is plausible that region-specific selective pressures required such adaptive mechanisms, leading to divergence in genome structure over time. Further research would need to be conducted to verify this hypothesis.\u003c/p\u003e\n\u003cp\u003eIt is worth noting that a new iteration of the \u003cem\u003eL calcarifer\u003c/em\u003e genome was published during this study (GCA_051027255.1) [39]. When compared to the previous assembly using the Comparative Genome Viewer in NCBI, this new assembly shows inversions on the same chromosomes detected in our analysis [40]. Given that Asian and Australian populations of \u003cem\u003eL. calcarifer\u003c/em\u003e have been separated for approximately one million years and have remained reproductively isolated, it is still critical to develop and use genomic resources specific to the population under investigation, in this case, Australian stocks. Importantly, earlier linkage maps for Asian lineages of \u003cem\u003eL. calcarifer\u003c/em\u003e and the GCF_001640805.2 genome are concordant and show no evidence of inversions, illustrating the extent of structural diversity across the species’ distribution. These findings highlight that creating genomic resources from randomly sampled individuals across the range could introduce interpretative errors, reinforcing the need for population-specific resources to ensure accuracy in genomic analyses.\u003c/p\u003e\n\u003ch3\u003eChromosomal recombination and heterochiasmy\u003c/h3\u003e\n\u003cp\u003eOur high‑resolution genetic linkage map revealed distinct male‑ and female‑specific regions of meiotic recombination, with minimal overlap between the sexes (Figure 2). This spatial segregation of recombination is broadly consistent with patterns reported in a meta‑analysis of 61 teleost fish species [30], which found that, in most cases, male recombination rates are suppressed near the centromere and concentrated toward the telomeres, whereas females tend to exhibit peak recombination near the centromeres, with additional events distributed along the chromosome arms. In our study, this sex‑specific recombination landscape revealed was not apparent in earlier linkage maps for the species, which were created from individuals from the southeast Asian lineage, and found that males and females had similar recombination patterns [21]. Among published examples, the recombination profile most similar to that of the Australian population of \u003cem\u003eL. calcarifer\u003c/em\u003e is found in Atlantic halibut (\u003cem\u003eHippoglossus hippoglossus\u003c/em\u003e), which also exhibits male‑ and female‑exclusive recombination regions on telocentric chromosomes: female recombination occurs predominantly near centromeres, while male recombination is restricted to distal chromosomal ends [41]. Notably, Atlantic halibut is gonochoristic, with an X/Y sex determination system, in contrast to the sequential hermaphroditism of \u003cem\u003eL. calcarifer\u003c/em\u003e. This contrast suggests that the emergence of male‑ and female‑exclusive recombination regions cannot be explained solely by the need to limit recombination around sex‑determining loci. Instead, the drivers of these patterns remain elusive, and the molecular machinery that positions recombination hotspots in \u003cem\u003eL. calcarifer\u003c/em\u003e warrants closer investigation.\u003c/p\u003e\n\u003cp\u003eA key gene associated with recombination hotspot specification in vertebrates is \u003cem\u003ePRDM9\u003c/em\u003e, which has numerous orthologs and is present in at least 225 species, including humans, mice, apes, and salmon [42-45]. \u003cem\u003ePRDM9\u003c/em\u003e is among the fastest-evolving genes in vertebrates, and even closely related species differ in the recombination hotspots it determines [43]. Although widespread, many species carry truncated or incomplete \u003cem\u003ePRDM9\u003c/em\u003e variants lacking the KRAB and SSXRD domains, rendering them non-functional [45, 46]. Notably, no \u003cem\u003ePRDM9\u003c/em\u003e ortholog has been identified in the current \u003cem\u003eL. calcarifer\u003c/em\u003e genome assembly. A BLAST search using the Atlantic salmon (\u003cem\u003eSalmo salar\u003c/em\u003e) \u003cem\u003ePRDM9\u003c/em\u003e sequence (NC_059458.1) failed to detect a homolog in barramundi [25], suggesting that this species may lack a functional \u003cem\u003ePRDM9\u003c/em\u003e gene.\u003c/p\u003e\n\u003cp\u003eIn species without functional \u003cem\u003ePRDM9\u003c/em\u003e, recombination events are typically conserved and preferentially occur in regions with elevated guanine and cytosine (GC) content and increased chromatin accessibility, such as promoters and CpG islands [21, 47-49]. This pattern indicates that epigenetic factors may play a major role in directing recombination. In Australian \u003cem\u003eL. calcarifer\u003c/em\u003e, differential DNA methylation between males and females has been observed, with differentially methylated regions (DMRs) associated with sexual phenotype despite the absence of sex chromosomes [50, 51]. However, current studies have focused primarily on gene-level DMRs, limiting insight into genome-wide patterns. To determine whether epigenetic factors contribute to sex-specific recombination landscapes, chromosome-level DNA methylation profiles should be generated for barramundi. \u003c/p\u003e\n\u003cp\u003eAdditionally, structural components of meiosis, such as the synaptonemal complex (SC), may also influence recombination localisation. The SC mediates homologous chromosome pairing and synapsis, and its length and organisation vary among species. In humans, sex-specific differences have been seen in the length of the SC [52]. The difference in SC length, coupled with chromatin packaging during meiotic prophase, allow the SC to preferentially target centromeric or telomeric regions [53]. Differences in SC length have also been detected in teleost fish [54, 55]. These observations suggest that epigenetic differences, combined with mechanical aspects of gametogenesis, such as chromatin and SC binding location, could enable specific recombination regions within the chromosome. The differential methylation of the barramundi genome between the sexes might lead to the expression of different SC homologs, explaining the extreme heterochiasmy of this sequential hermaphroditic species.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLates calcarifer\u003c/em\u003e may serve as an excellent model for investigating the evolutionary drivers of heterochiasmy, the phenomenon where recombination rates differ between sexes. A comprehensive review by Sardell and Kirkpatrick (2020) highlights several hypotheses for why heterochiasmy evolves, ranging from mechanistic constraints to adaptive explanations [56]. While some theories suggest that heterochiasmy could arise by chance through processes such as genetic drift acting on sex-averaged recombination rates, this is generally considered unlikely to fully explain the pattern. Mechanistic differences in gametogenesis, such as the distinct processes of sperm and egg formation, may also contribute to sex-specific recombination landscapes.\u003c/p\u003e\n\u003cp\u003eAdaptive explanations, however, provide compelling scenarios. These include roles in female meiotic drive, a process where certain alleles, haplotypes, or even entire chromosomes are preferentially transmitted during meiosis, the selection against aneuploidy and sexually antagonistic selection, where alleles beneficial to one sex are detrimental to the other. Haploid selection, where selection acts on gametes, has also been proposed as a driver of heterochiasmy, although it is rarely observed in female animals [38]. In the context of \u003cem\u003eL. calcarifer\u003c/em\u003e, which undergoes sequential hermaphroditism, these adaptive hypotheses take on additional complexity. The transition from male to female during the life cycle could impose unique selective pressures on recombination landscapes, potentially favouring mechanisms that reduce aneuploidy risk or optimise allele transmission across both sexual phases. This makes \u003cem\u003eL. calcarifer\u003c/em\u003e a particularly valuable system for testing whether heterochiasmy is shaped by adaptive forces linked to sex-specific roles and life-history strategies.\u003c/p\u003e\n\u003ch3\u003eSignificance for breeding programs\u003c/h3\u003e\n\u003cp\u003eMeiotic recombination is incredibly important to breeding programs as it allows for the creation of new combinations of alleles in the population, and assists in purging deleterious mutations from the population [57]. Additionally, it is important to know where regions of low recombination exist in the genome as these regions will naturally accrue deleterious mutations [58]. Intriguingly in Australian \u003cem\u003eL. calcarifer,\u003c/em\u003e a protandrous species, regions of recombination will switch across the individual’s lifetime when the individual switches sex from male to female, conceivably allowing for the chance of recombination across the whole genome during an individual’s lifetime. This heterochiasmy pattern discovered in this genetic linkage map could be strategically applied to breeding programs in two ways.\u003c/p\u003e\n\u003cp\u003eThe first possible use of this map in Australian \u003cem\u003eL. calcarifer\u003c/em\u003e breeding programs is using it to precisely map QTL. For instance, if we know that there are favourable QTL available in the population that are known to be located in the region near the centromere of the chromosome, an all-female population could be generated using hormonal implants [1] to maximise the recombination occurring in that region of the chromosome. From that, the precision of QTL mapping could be increased. \u003c/p\u003e\n\u003cp\u003eThe second use of the genetic linkage map information produced in this study would be to enable efficient stacking of desirable haplotype blocks to create superior progeny [59]. As an example, if multiple desirable haplotype blocks - segments of the chromosome that have high “local” genomic estimated breeding values - exist in the population near the distal end of the chromosome (away from the centromere), we can use these individuals as males so that their recombination occurs in our desired chromosomal region (Figure 4). Additionally, we can use this striking heterochiasmy to introgress traits from wild populations into our farmed fish stocks while minimising linkage drag of unfavourable traits into our elite lines to bolster genetic diversity, disease resistance, and maintain our breeding targets.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe high-density genetic linkage map developed in this study represents the most comprehensive genetic map for \u003cem\u003eL. calcarifer\u003c/em\u003e to date, with 49,885 SNPs mapped to 24 linkage groups. This map provides critical insights into the genetic architecture and meiotic recombination patterns of Australian \u003cem\u003eL. calcarifer\u003c/em\u003e. The deviation from the recombination landscape of the Asian \u003cem\u003eL. calcarifer\u003c/em\u003e population highlight the genetic divergence of these populations. Furthermore, the observed sex-specific and population-level differences in recombination, alongside the absence of heteromorphic sex chromosomes and the species’ protandrous nature, highlight \u003cem\u003eL. calcarifer\u003c/em\u003e’s potential as a model for investigating the evolution of heterochiasmy.\u003c/p\u003e\n\u003cp\u003eUnderstanding the spatial distribution of recombination events enables more precise QTL mapping by informing the strategic selection of individuals based on sex and recombination activity. Moreover, the sex-specific recombination architecture presents novel opportunities for targeted breeding in this sequential hermaphrodite. By leveraging the ability to direct reproductive sex, it becomes possible to accelerate the assembly of favourable haplotypes, stacking chromosome blocks associated with traits such as growth, disease resistance, and environmental resilience. This map not only enhances our understanding of \u003cem\u003eL. calcarifer\u003c/em\u003e genetics but also unlocks a new frontier in precision breeding, where genomic insights can be directly translated into optimised, sex-informed selection strategies for sustainable aquaculture. \u003c/p\u003e\n\u003cp\u003eCollectively, these findings advance our understanding of \u003cem\u003eL. calcarifer\u003c/em\u003e genetics and provide a robust foundation for future research in evolutionary biology, genomics, and aquaculture breeding strategies.\u003c/p\u003e"},{"header":"METHODS","content":"\u003ch3\u003eGenotyping\u003c/h3\u003e\n\u003cp\u003eGenotyping for this study was sourced from a previous dataset generated by a commercial barramundi aquaculture company (Mainstream Aquaculture Group, Australia), where broodstock in a selective breeding program were fewer than four generations removed from the wild [8]. Briefly, SNP array genotyping was performed using a custom 70K Axiom™ myDesign™ SNP array from ThermoFisher Scientific™, and a subset of the total dataset was used so that the dataset for this study comprised of progeny sampled across nine families derived from three dams and seven sires, resulting in 1,952 F1 offspring [8]. Fin clips from these fish provided DNA for genotyping, which was conducted at the Ramaciotti Centre for Genomics using a GeneTitan™ platform following a stringent quality control pipeline. DNA extraction was carried out with the Chemagic™ DNA Extraction Kit on a Zephyr™ automated workstation.\u003c/p\u003e\n\u003cp\u003eRaw SNP data underwent rigorous filtering to ensure accuracy: a minimum call rate of 0.98, cluster separation score (FLD) of at least 4.0, heterozygous signal strength offset (HetSO) no lower than −0.04, and a minor allele frequency threshold of 0.01. Additionally, sample call rate (≥95%) and Dish QC (≥0.80) were applied as further criteria. After these steps, the dataset retained 52,245 high-quality polymorphic SNPs for linkage mapping. For a full description of these methods, see Jerry et al. (2022) [8].\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003ch3\u003eGenetic Linkage Map Construction\u003c/h3\u003e\n\u003cp\u003eThe dataset in PLINK bfile format was first converted to the Lep-MAP3 format using custom bash and R scripts in R 4.3.0 [60, 61]. Parental sex was defined as either 1 (male) or 2 (female), while offspring sex was left blank. We used Lep-MAP3 (version 0.5) to construct the genetic linkage maps [27]. First, we filtered the data using the ParentCall2 module to ensure accurate parental genotypes based on Mendelian inheritance patterns. Subsequently, the Filtering2 module removed uninformative markers (removeNonInformative = 1), and markers with high segregation distortion (dataTolerance = 0.00001). The data were then further filtered to remove markers with minor allele frequencies (MAF) (MAFLimit = 0.01) or missing (missingLimit = 0.2) [62].\u003c/p\u003e\n\u003cp\u003eMarkers were first assigned to linkage groups (LGs) using the SeparateChromosomes2 module, testing a range of logarithm of odds (LOD) scores (10–80) with the lodLimit function and setting a minimum of 50 markers per LG. Marker ordering within each LG was performed using OrderMarkers2 with Haldane’s mapping function, running five independent iterations per LG and selecting the run with the highest likelihood. In cases where convergence was not achieved, the hyperPhaser parameter was enabled, and the number of merge and polish iterations was increased to 20 and 5, respectively (from defaults of 6 and 2). With these adjustments, all five replicates were visualised, and the best replicate was chosen based on consistent structure, highest likelihood score, and absence of parallel lines (Supplementary Figure 3). Sex-specific maps were generated using male-informative (informativeMask = 13) or female-informative (informativeMask = 23) markers, including double-heterozygous markers. Male and female maps were then merged in R, ensuring consistent chromosome orientation across sexes and families. Two sex-averaged maps were produced: one by calculating the mean marker position between the sex-specific maps (mean map) and another by summing the two positions (additive map), as recombination occurs in only one sex at a given position.\u003c/p\u003e\n\u003cp\u003eDuring map construction, some regions could not be resolved by OrderMarkers2, resulting in parallel lines in the sex that was not recombining (Supplementary Figure 4B). These issues were absent in single-family maps (Supplementary Figure 4A) but became apparent as more families were added, with no single family responsible for the discrepancies. We hypothesise that these parallel lines arise when families have differentially informative markers, creating uncertainty in regions of no recombination. Enabling the hyperPhaser parameter resolved these issues (Supplementary Figure 5).\u003c/p\u003e\n\u003ch3\u003ePolishing\u003c/h3\u003e\n\u003cp\u003eGaps in some of the linkage groups in the map were interrogated for informativeness (not homozygous) or for misplaced markers. Heterozygosity for the male and female founders was calculated separately using PLINK 1.9 [61]. For the female founders, the flag --freqx generated frequency data, which was then processed using gawk scripts. The gawk scripts were used to calculate the total number of animals by summing the counts of homozygous alleles (A1 and A2), heterozygous alleles, and missing data. Next, gawk was employed to compute heterozygosity by dividing the heterozygosity count by the total count of animals. This process was similarly applied to the male founders. The resulting files for both male and female founders were imported into R for further analysis. In R, the chromosomes of interest—those with gaps and parallel structures—were subsetted from the dataset. Homozygosity was inferred for SNPs where heterozygosity values were either 0 or 1. Finally, these homozygous SNPs were overlaid on heterochiasmy plots to visualize the data effectively.\u003c/p\u003e\n\u003cp\u003eGaps in the maps were further interrogated for misplaced markers that could be artificially inflating the genetic distance. To do this, a 1 cM window of markers from either side of the gap were removed and OrderMarkers2 was rerun with the new subset. Finally, any markers that could not be placed were removed from the map.\u003c/p\u003e\n\u003ch3\u003eVisualisation and Genome Comparison\u003c/h3\u003e\n\u003cp\u003eGenetic linkage maps were visualised using ggplot2 v3.4.2 [63], which provided an overview for chromosome-level data. To assess sex-specific recombination differences, marker positions along male and female linkage maps were compared at the chromosome scale, and these comparisons were also implemented and visualised within the ggplot2 framework to ensure consistency and clarity in data representation.\u003c/p\u003e\n\u003cp\u003eTo assess synteny between the Australian \u003cem\u003eL. calcarifer\u003c/em\u003e linkage maps and the \u003cem\u003eL. calcarifer\u003c/em\u003e reference genome assembly (GCF_001640805.2), Marey maps [35, 64] were generated using custom R scripts following the approach described by Akopyan et al. [65]. Centromere positions were inferred by comparing the genome assembly with the published karyotype for the Australian population [11]. The largest chromosome (likely chromosome 12 in GCF_001640805.2) is the only one exhibiting a metacentric/submetacentric centromere, while the remaining 23 chromosomes display acrocentric or telocentric configurations. Recombination patterns on this metacentric chromosome revealed female recombination concentrated in the central region and male recombination restricted to the ends. These observations, combined with previous studies in teleosts [30, 41], allowed us to extrapolate centromere locations and infer recombination landscapes across the genome, under the assumption that sex-specific recombination patterns are consistent across all chromosomes.\u003c/p\u003e\n\u003ch3\u003eLinkage Disequilibrium\u003c/h3\u003e\n\u003cp\u003eLinkage disequilibrium was calculated by estimating R\u003csup\u003e2\u0026nbsp;\u003c/sup\u003ebetween all pairs of markers, regardless of the putative chromosome, using PLINK v 1.9 [61]. During the scaffolding process for the genome assembly, chromosome 7 was divided into two separate segments due to minimal supporting evidence for their connection [25]. These segments are now recognised as distinct chromosomes, designated as 7.1 and 7.2 (71 and 72) as well as a chromosome 100, where unplaced markers were collected (--r2 --out ldfile --autosome-num 71 --allow-extra-chr --inter-chr). Discrepancies in the assembly that were detected in the genetic linkage map were then confirmed by observing if the R\u003csup\u003e2\u0026nbsp;\u003c/sup\u003evalues were higher in the assembly chromosome or the mapped chromosome.\u003c/p\u003e\n\u003ch3\u003eDebugging\u003c/h3\u003e\n\u003cp\u003eThe use of Large Language Models (LLMs) was used to assist in the debugging of R code when needed. The models used were Copilot, ChatGPT, and Claude. Models were also used to refine the language of written text. Models were not used to generate new text, ideas or in research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003eEthics approval and consent to participate\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eConsent for publication\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eAvailability of data and materials\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe initial datasets used during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eAll genetic linkage maps generated during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003eThe code used for all analyses in this manuscript and for the production of figures is available online at the GitHub page website of https://github.com/DigitalFins/Lates_calcarifer_Linkage_map_2025.\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eCompeting interests\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eFunding\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThis project is funded through the Australian Research Council Industrial Transformation Research Program (IH210100014) Supercharging Tropical Aquaculture through Genetic Solutions\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eAuthors' contributions\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eJH conceptualised the study, performed data analysis, curated the data, validated results, visualised results, project administration, and drafted the manuscript\u003c/p\u003e\n\u003cp\u003eLVB contributed to methodology, Lep-MAP3 code, data curation, and review and editing of this manuscript\u003c/p\u003e\n\u003cp\u003eDBJ conceptualised the study, assisted with methodology, data curation, provided data resources, and review and editing of this manuscript\u003c/p\u003e\n\u003cp\u003eCW assisted with data curation, review of this manuscript, and supervision\u003c/p\u003e\n\u003cp\u003eOP assisted with data curation, review and editing of this manuscript, and supervision\u003c/p\u003e\n\u003cp\u003eEMR contributed to supervision and review and editing of this manuscript\u003c/p\u003e\n\u003cp\u003ePH contributed the genomic resources for this study\u003c/p\u003e\n\u003cp\u003eHC contributed the genomic resources for this study\u003c/p\u003e\n\u003cp\u003eDJ contributed resources, supervision, review and editing of this manuscript, and acquired funding to support this project\u003c/p\u003e\n\u003cp\u003eBH contributed to the conceptualisation, methodology, validation, supervision, project administration, and review and editing of this manuscript\u003c/p\u003e\n\u003cp\u003eKRZ contributed to the conceptualisation, methodology, validation, supervision, project administration, and review and editing of this manuscript\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eAcknowledgements\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eWe gratefully acknowledge Dr Cecile Massault for her invaluable support in transferring data and for generously sharing her expertise in quantitative genetics. Additionally, we would like to thank Hayden B. Ahwan for providing insight into the population structures of \u003cem\u003eLates calcarifer\u003c/em\u003e.\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eAuthors' information (optional)\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGuppy JL, Marc AF, Jerry DR: \u003cstrong\u003eMaturation and spawning performance of hormonally-induced precocious female barramundi (Lates calcarifer) and implications of their use in selective breeding.\u003c/strong\u003e \u003cem\u003eAquaculture \u003c/em\u003e2022, \u003cstrong\u003e552:\u003c/strong\u003e737991.\u003c/li\u003e\n\u003cli\u003eJerry DR: \u003cem\u003eBiology and Culture of Asian Seabass.\u003c/em\u003e CRC Press Taylor \u0026amp; Francis Group; 2014.\u003c/li\u003e\n\u003cli\u003eYue GH, Wang L, Sun F, Yang ZT, Wong J, Wen YF, Pang HY, Lee M, Yeo ST, Liang B, et al: \u003cstrong\u003eImproving growth, omega-3 contents, and disease resistance of Asian seabass: status of a 20-year family-based breeding program.\u003c/strong\u003e \u003cem\u003eReviews in Fish Biology and Fisheries \u003c/em\u003e2023.\u003c/li\u003e\n\u003cli\u003eYue GH, Wang L, Yang Z, Sun F, Tay YX, Wong J, Yeo S: \u003cstrong\u003eGenomic resources and their applications in aquaculture of Asian seabass (Lates calcarifer).\u003c/strong\u003e \u003cem\u003eReviews in Aquaculture \u003c/em\u003e2023, \u003cstrong\u003e15:\u003c/strong\u003e853-871.\u003c/li\u003e\n\u003cli\u003eYe B, Wan Z, Wang L, Pang H, Wen Y, Liu H, Liang B, Lim HS, Jiang J, Yue G: \u003cstrong\u003eHeritability of growth traits in the Asian seabass (Lates calcarifer).\u003c/strong\u003e \u003cem\u003eAquaculture and Fisheries \u003c/em\u003e2017, \u003cstrong\u003e2:\u003c/strong\u003e112-118.\u003c/li\u003e\n\u003cli\u003eYue G, Orban L, Lim H: \u003cstrong\u003eCurrent status of the Asian seabass breeding program. \u003c/strong\u003eIn \u003cem\u003eAquaculture\u003c/em\u003e. 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\u003cstrong\u003e31:\u003c/strong\u003e3323-3341.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Heterochiasmy, linkage maps, hermaphroditism, aquaculture breeding, recombination landscapes, targeted breeding, chromosomal inversions, evolution ","lastPublishedDoi":"10.21203/rs.3.rs-8400904/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8400904/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eBackground\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLates calcarifer \u003c/em\u003e(barramundi or Asian seabass) is a key aquaculture species with a protandrous life cycle, maturing first as male and later as female. As global breeding programs advance to improve traits such as growth and disease resistance, the absence of a high-density genetic linkage map limits progress and constrains understanding of genome architecture and recombination. Such maps are essential for marker-assisted selection, genomic breeding strategies, and comparative genomics, providing a foundation for accelerating genetic improvement.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResults\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOur analysis reveals striking sex-specific recombination differences within the Australian lineage: females recombine only in centromeric regions, while males recombine exclusively in distal regions, with no overlap. This pronounced heterochiasmy has not been observed in Southeast Asian populations. Additionally, we detected 10 chromosomal inversions, expanding known structural variation across the species’ range.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConclusions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGiven their sequential hermaphroditism, this recombination landscape could allow breeders to strategically utilise sex-specific recombination through the creation of optimal allele combinations, increasing genetic gain and enhancing genetic diversity in \u003cem\u003eL. calcarifer\u003c/em\u003e aquaculture populations. Beyond its practical applications, our findings establish \u003cem\u003eL. calcarifer\u003c/em\u003e as a model for studying heterochiasmy, as well as providing insights into how this can be leveraged for advanced genomic breeding programs.\u003c/p\u003e","manuscriptTitle":"Genetic linkage map of the Australian barramundi (Lates calcarifer) reveals potential to leverage extreme sex-specific recombination and sequential hermaphrodism for ultimate breeding program control","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-07 07:26:21","doi":"10.21203/rs.3.rs-8400904/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7c37786b-22ec-4102-a366-7633dc52b43b","owner":[],"postedDate":"January 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-07T07:26:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-07 07:26:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8400904","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8400904","identity":"rs-8400904","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}