Comparative Analysis of the Shared Sex-Determination Region (SDR) among Salmonid Fishes

Abstract

Salmonids present an excellent model for studying evolution of young sex-chromosomes. Within the genus, Oncorhynchus, at least six independent sex-chromosome pairs have evolved, many unique to individual species. This variation results from the movement of the sex-determining gene, sdY, throughout the salmonid genome. While sdY is known to define sexual differentiation in salmonids, the mechanism of its movement throughout the genome has remained elusive due to high frequencies of repetitive elements, rDNA sequences, and transposons surrounding the sex-determining regions (SDR). Despite these difficulties, bacterial artificial chromosome (BAC) library clones from both rainbow trout and Atlantic salmon containing the sdY region have been reported. Here, we report the sequences for these BACs as well as the extended sequence for the known SDR in Chinook gained through genome walking methods. Comparative analysis allowed us to study the overlapping SDRs from three unique salmonid Y chromosomes to define the specific content, size, and variation present between the species. We found approximately 4.1 kb of orthologous sequence common to all three species, which contains the genetic content necessary for masculinization. The regions contain transposable elements that may be responsible for the translocations of the SDR throughout salmonid genomes and we examine potential mechanistic roles of each one.

Keywords: comparative genomics; retrotransposition; salmonid; sex-chromsome; sex-determination; transposable element.

Fig. 1.—

Sequence alignments of GenBank accessions DQ393568.1 (OtY3), KC756279 (Chinook salmon sdY), and KJ908737 along the predicted Ots_SDR scaffold (16,722 bp). KJ908737 represents novel sequence generated by genome-walking methods in this study.

Fig. 2.—

Pairwise sliding window alignments between each SDR contig. Evolutionary Conserved Regions (ECRs) were confirmed independently through phylogenetic analysis and are reported in blue. Red alignment blocks have greater than 50% sequence identity, however are not related by descent. Omy, O. mykiss, Ots, O. tshawytscha, Ssa, S. salar. The sdY mRNA sequences are almost entirely contained within the orthologous sequence shared by all three species in each comparison.

Fig. 3.—

Major conserved domains of three candidate TEs described in the text. The shared repetitive element containing several unnamed proteins did not retrieve any conserved domains in BLASTx searches. In panels A, B, and C, only scaffolds from species with the highest BLASTx score to the respective gene are represented. The scaffold lengths correspond to the length of alignment to the TE of interest. In panel D, rainbow trout was used as a scaffold to show the difference in alignment length on the 5′-end of the sequence, which may be explained by incomplete reverse transcription. In panels B and C, there are domains found in different ORFs. Light blue regions of scaffolds have been masked by low complexity filter SEG.

Fig. 4.—

Comparative homology among three SDR scaffolds. Important genetic elements within or surrounding the shared SDR are mapped. This includes sex-linked genetic markers, sdY mRNAs from each species, and TEs possibly involved in transposition of the region. Accession numbers for the TEs are found in the text. Accession numbers for other sex-linked elements are as follows: S. salar sdY (GB:JF826020), O. mykiss sdY (GB:NM_001281416), O. tshawytscha sdY (GB:KF006343), OmyY1 (GB:JQ995497), OtY2 (GB:GU181208). OmyY1 and OtY2 are both found in Chinook salmon, however OtY2 is not found in rainbow trout and neither marker is found in Atlantic salmon. This explains the well-documented success of the particular markers to accurately amplify in males in the respective species.

Fig. 5.—

Neighbor-joining tree for TC1-like transposase. Tree was generated from multiple sequence alignments performed by ClustalW. Input sequences include elements within each SDR contig, and the top ten alignments in the rainbow trout and Atlantic salmon genomes to each SDR-related sequence. Colored circles represent the source species for genomic sequences and colored arrows represent the elements found in the three SDRs.

Fig. 6.—

Neighbor-joining tree for pol-like protein. Tree was generated from multiple sequence alignments performed by ClustalW. Input sequences include elements within each SDR contig, and the top ten alignments in the rainbow trout and Atlantic salmon genomes to each SDR-related sequence. Colored circles represent the source species for genomic sequences and colored arrows represent the elements found in the three SDRs.

Fig. 7.—

Neighbor-joining tree for Unnamed proteins. Tree was generated from multiple sequence alignments performed by ClustalW. Input sequences include elements within each SDR contig, and the top ten alignments in the rainbow trout and Atlantic salmon genomes to each SDR-related sequence. Colored circles represent the source species for genomic sequences and colored arrows represent the elements found in the three SDRs.

Fig. 8.—

Neighbor-joining tree for RNA-directed DNA polymerase from mobile element jockey-like. Tree was generated from multiple sequence alignments performed by ClustalW. Input sequences include elements within each SDR contig, and the top ten alignments in the rainbow trout and Atlantic salmon genomes to each SDR-related sequence. Colored circles represent the source species for genomic sequences and colored arrows represent the elements found in the three SDRs.

Fig. 9.—

A simplified hypothetical model of retrotransposition of the SDR facilitated by RNA-directed DNA polymerase from mobile element jockey-like. (A) A promoter upstream of sdY acts as recognition site for an RNA polymerase, and (B) transcription occurs until a termination site in the retrotransposon is recognized. All internal transcripts in the SDR are naturally transcribed in the opposite direction. (C) The retro-TE flanks the SDR transcript and recognizes a target insertion site elsewhere in the genome. (D) The SDR is reverse transcribed, (E) a template jump occurs, and the complementary DNA strand is synthesized. (F) The nonhomologous flaps of DNA are removed, gaps are filled in and ligated by an unknown series of enzymes. The result is duplication of the SDR with the flanking retrotransposon, and a formation of a new Y chromosome. Males with two Y chromosomes may pass one, both, or neither to offspring with a 25% chance of normal XY male offspring with a new sex-chromosome pair.