Generation of a chromosome-level genome assembly for Pacific halibut (Hippoglossus stenolepis) and characterization of its sex-determining genomic region

Abstract

The Pacific halibut (Hippoglossus stenolepis) is a key species in the North Pacific Ocean and Bering Sea ecosystems, where it also supports important fisheries. However, the lack of genomic resources limits our understanding of evolutionary, environmental and anthropogenic forces affecting key life history characteristics of Pacific halibut and prevents the application of genomic tools in fisheries management and conservation efforts. In the present study, we report on the first generation of a high-quality chromosome-level assembly of the Pacific halibut genome, with an estimated size of 602 Mb, 24 chromosome-length scaffolds that contain 99.8% of the assembly and a N₅₀ scaffold length of 27.3 Mb. In the first application of this important resource, we conducted genome-wide analyses of sex-specific genetic variation by pool sequencing and characterized a potential sex-determining region in chromosome 9 with a high density of female-specific SNPs. Within this region, we identified the bmpr1ba gene as a potential candidate for master sex-determining (MSD) gene. bmpr1ba is a member of the TGF-β family that in teleosts has provided the largest number of MSD genes, including a paralogue of this gene in Atlantic herring. The genome assembly constitutes an essential resource for future studies on Pacific halibut population structure and dynamics, evolutionary history and responses to environmental and anthropogenic influences. Furthermore, the genomic location of the sex-determining region in Pacific halibut has been identified and a putative candidate MSD gene has been proposed, providing further support for the rapid evolution of sex-determining mechanisms in teleost fish.

Keywords: Pacific halibut; candidate master sex-determining gene; genome assembly; pool-sequencing; sex-associated genomic region; whole genome sequencing.

FIGURE 1

(a) Photograph of a Pacific halibut (H. stenolepis). Photo credit: Guy Becken. (b) Heatmap of chromosome interaction intensity in the Pacific halibut Hi‐C assembly. The x‐ and y‐axis represent the length of the chromosomes

FIGURE 2

Genome‐wide Manhattan plot visualization of male‐specific (in blue, top) and female‐specific (in red, bottom) single‐nucleotide polymorphisms (SNPs) along the 24 chromosomes of the Pacific halibut genome assembly. Sex‐specific SNPs are represented as dots (total per 50 kb window size) of alternating colours to distinguish their location on adjacent chromosomes. U indicates unplaced chromosomes

FIGURE 3

Distribution of female‐specific SNPs on chromosome 9 (Chr9). F _ST values are shown in (a). SNPs were counted using 50 kb sliding window with an output point every 1000 bp and male (M)‐ and female (F)‐specific SNPs were indicated by blue and red colours, respectively (b, c). A large sex‐determination region was identified on Chr9. Read depth ratio between males and females is shown in (d). The position of the bmpr1ba gene is indicated in (a) by a vertical dashed line. The location of RAD‐tags associated with sex identified by Drinan et al. (2018) are indicated by inverted green triangles in (a). The location of the small 18 kb region within the sex‐determination region that contains an excess of female over male reads is indicated in (d) by an arrow

FIGURE 4

Linkage disequilibrium (r ² ) for RAD‐seq derived SNPs on chromosome 9. Information for females and males is shown in the left and right panels, respectively. Colour represents the median r ² for all comparisons in nonoverlapping 100 kb bins

FIGURE 5

Whole‐genome macrosyntenic relationships between Pacific halibut (H. stenolepis) and Atlantic halibut (H. hippoglossus) (a), Greenland halibut (R. hippoglossoides) (b), turbot (S. maximus) (c), Senegalese sole (S. senegalensis) (d) and half‐smooth tongue sole (C. semilaevis) (e). Orthologous relationships to chromosome 9 of Pacific halibut are highlighted in yellow and orthologous relationship to the sex determining region are highlighted in red. Red asterisks indicate the chromosomes linked to sex in the other flatfish species

FIGURE 6

Syntenic relationship of Pacific halibut (H. stenolepis) chromosome 9 (Chr9) and orthologues in Atlantic halibut (H. hippoglossus) (a) and Greenland halibut (R. hippoglossoides) (b). The sex associated region of Chr9 in Pacific halibut is shaded in light blue, the genomic location of the bmpr1ba gene is indicated in red

FIGURE 7

Syntenic comparison of the genes located around the bmpr1ba gene in Pacific halibut (H. stenolepis), Atlantic halibut (H. hippoglossus), Japanese flounder (P. olivaceus), turbot (S. maximus), Senegalese sole (S. senegalensis) and half‐smooth tongue sole (C. semilaevis). Orthologous genes are identified by colour, a blue line between genes represents a gap in the order of genes (i.e., nonorthologous genes are present in this region of the genome)

FIGURE 8

Maximum likelihood phylogenetic reconstruction of flatfish Bmpr1ba protein sequences, including Pacific halibut (H. stenolepis), Atlantic halibut (H. hippoglossus), Japanese flounder (P. olivaceus), turbot (S. maximus), Senegalese sole (S. senegalensis) and half‐smooth tongue sole (C. semilaevis), compared to zebrafish (D. rerio) and medaka (O. latipes). The values indicate branch support (estimated by aLRT)