A duplicated copy of id2b is an unusual sex-determining candidate gene on the Y chromosome of arapaima (Arapaima gigas)

Abstract

Arapaima gigas is one of the largest freshwater fish species of high ecological and economic importance. Overfishing and habitat destruction are severe threats to the remaining wild populations. By incorporating a chromosomal Hi-C contact map, we improved the arapaima genome assembly to chromosome-level, revealing an unexpected high degree of chromosome rearrangements during evolution of the bonytongues (Osteoglossiformes). Combining this new assembly with pool-sequencing of male and female genomes, we identified id2bbY, a duplicated copy of the inhibitor of DNA binding 2b (id2b) gene on the Y chromosome as candidate male sex-determining gene. A PCR-test for id2bbY was developed, demonstrating that this gene is a reliable male-specific marker for genotyping. Expression analyses showed that this gene is expressed in juvenile male gonads. Its paralog, id2ba, exhibits a male-biased expression in immature gonads. Transcriptome analyses and protein structure predictions confirm id2bbY as a prime candidate for the master sex-determiner. Acting through the TGFβ signaling pathway, id2bbY from arapaima would provide the first evidence for a link of this family of transcriptional regulators to sex determination. Our study broadens our current understanding about the evolution of sex determination genetic networks and provide a tool for improving arapaima aquaculture for commercial and conservation purposes.

Figure 1

Characterization of the sex chromosome and sex-locus of Arapaima gigas. (A, B) Genome-wide Manhattan plot visualization of male-specific single-nucleotide polymorphisms (SNVs) along the 28 chromosomes (Chr) of the female (A) and male (B) A. gigas genome assemblies. Male-specific SNVs are represented as dots (total per 50 kb window size) of alternating colors to distinguish their location on adjacent chromosomes. The maximum genome-wide values of female-specific SNVs are shown on the plots (red lines). (C) Zoomed view of the sex-specific SNVs (total per 5 kb window size) on the sex-biased region of Chr05 with the location of the inhibitor of DNA binding 2 b gene (id2b) and of the kinase D interacting substrate 220 b gene (kidins220b). (D) Zoomed view of the normalized coverage depth (average per 5 kb window size) of the sex-biased Chr05 homologous region on male Chr26 with location of the duplicated id2bbY copy of the Chr05 id2b gene and of the truncated duplicated kidins220bΔ of the Chr05 kidins220b.E. Multiple alignment plots of the percentage (%) of sequence identity between the id2bbY locus on Chr26, the coding sequences (CDS) of id2b and kidins220b and the corresponding autosomal Chr05 region used as a reference.

Figure 2

Phylogenetic analyses of Id2 (Id2a) and Id2b and Id2bbY amino acid sequences in vertebrates. The topology of the gene tree follows the expected phylogenetic relationships of the organisms. Red arrows show the Id2 sequences in arapaima. Blue arrows indicate the species that lost the id2b gene.

Figure 3

Primary and tertiary structure of Id2 proteins. (A) Amino acid sequence alignment of Id2 in vertebrates and localization of the HLH domain. Red stars display the amino acid changes exclusive for Id2bbY. Structure prediction of Id2a (B, C), Id2ba (D, E) and Id2bbY (F, G) of arapaima.

Figure 4

Transcriptome analyses of sex-related genes in juvenile and adult gonads of male and female. (A) Relative mRNA expression comparison between id2a, id2ba and id2bbY. (B) Heat map of sex-related genes showing higher expression levels in yellow and lower expression in blue. Arrows indicate transcription factors and growth factor genes important for testis development.

Figure 5

Evolutionary origin of the arapaima Y-chromosome. Schematic representation of a local DNA duplication on chromosome (Chr) 5 followed by an insertion of the duplicated fragment into Chr26 (sex chromosomes) of arapaima. The duplication contains the male-specific marker id2bbY and a truncated copy of kidins220b gene, kidins220bΔ. Created in BioRender.com.

Figure 6

Conceptual links between TGF-β signaling, id2 and known sex-determining genes. Ligands belonging to the TGF-β superfamily bind to their respective type 2 receptor, which in turn recruits the type 1 receptor activating the SMAD factors and leading to expression of id2. The Id2 protein interacts directly with E proteins therefore preventing their binding to DNA in the E Box sequence. Different components of the TGF-β signaling pathway recurrently became the sex-determining gene, where Id2b would be in a downstream position in the pathway. Created in BioRender.com.