Evidence for multiple sex-determining loci in Tasmanian Atlantic salmon (Salmo salar)

Abstract

Phenotypic sex in salmonids is determined primarily by a genetic male heterogametic system; yet, sex reversal can be accomplished via hormonal treatment. In Tasmanian Atlantic salmon aquaculture, to overcome problems associated with early sexual maturation in males, sex-reversed females are crossed with normal females to produce all female stock. However, phenotypic distinction of sex-reversed females (neo-males) from true males is problematic. We set out to identify genetic markers that could make this distinction. Microsatellite markers from chromosome 2 (Ssa02), to which the sex-determining locus (SEX) has been mapped in two Scottish Atlantic salmon families, did not predict sex in a pilot study of seven families. A TaqMan 64 SNP genome-wide scan suggested SEX was on Ssa06 in these families, and this was confirmed by microsatellite markers. A survey of 58 families in total representing 38 male lineages in the SALTAS breeding program found that 34 of the families had SEX on Ssa02, in 22 of the families SEX was on Ssa06, and two of the families had a third SEX locus, on Ssa03. A PCR test using primers designed from the recently published sdY gene is consistent with Tasmanian Atlantic salmon having a single sex-determining gene that may be located on at least three linkage groups.

Figure 1

Lineage containing the four sires of the seven families in the initial pilot study. All of these families were identified to have SEX located on Ssa06. Individuals (males) are shown in the format, Year class (YC), _, individual ID, (for example, 2003_0032; YC 2003, ID 0032). Families are classified in the same way (for example, 2003_13; YC 2003, family ID 13). Pedigrees of all 40 lineages can be found in Supplementary Figure S1. The four sires are shown in bold.

Figure 2

An abstract of the second largest family (2009_32), with the sex locus mapped Ssa03, to illustrate the comparisons of phenotype to sdY phenotype predictions and haplotype predictions of phenotype. AS-1 (ancestral sire-1) represents the paternal or Y-linked allele housed by the grandsire (2002_0013), while AS-2 represent the maternal or X-linked allele housed by the grandsire. Likewise S-1 (sire-1), inherited from the AS, and S-2, inherited from the ancestral dam (AD) represent the Y-linked and X-linked alleles housed by the sire (2007_0001), respectively. Offspring ID's run in the same row where Individual ID's are shown in the format year class_IDXXXX (for example, 2009_0598 is the 2009 Year class ID 0598). Haplotype offspring sex predictions that do not match the sex call from phenotype are highlighted in green. The sdY predictions are based on the presence of exon 2 and exon 4 (predicting male) indicated by a 1 or absence indicated by a 0. Fabp6b represents a quality control of DNA, where a 1 indicates presence and 0 indicates absence of an amplified product. Full details of this family can be found in Supplementary Table S3.

Figure 3

sdY triplex PCR test with primers (see Materials and Methods) from exon 2 (∼110 bp), exon 4 (∼250 bp) and fabp6b (∼450 bp) (Y. Lai personal communication) comparing sires with dams from families with SEX on Ssa02/Linkage group 1, Ssa06/ Linkage group 4 and Ssa03/Linkage group 11. The 100-bp ladder is shown in the first and last lanes.

Figure 4

Mechanisms of sdY movement during meiosis of a sire with SEX located on Ssa02. Each major pathway is designated a different number: (1) homologous recombination including sdY between X and Y; (2) transposition or (3) translocation of sdY to Ssa03 or Ssa06; or (4) translocation or transposition of sdY to another chromosome within the genome (labeled N). Upon transposition to a new chromosome for example, Ssa06 or Ssa03 the homologous pair becomes a new X and Y (designated neo X and neo Y) after which point segregation can produce different phenotype/sdY/genotype predictions. Note that case 4 could not be observed because individuals in which sex likely jumped were harvested, and thus had no offspring that could be analyzed. Following any of these three major events chromosomal segregation further alters the haplotype/sdY/phenotype sex predictions (shown at bottom). Homeologous recombination could also occur between the paralogous chromosomes Ssa06 and Ssa03 (outcomes the same as in 2.) This figure also does not include sex reversal due to temperature (see Table 2).