A Y-linked anti-Mullerian hormone type-II receptor is the sex-determining gene in ayu, Plecoglossus altivelis

Whole-genome duplication and genome compaction are thought to have played important roles in teleost fish evolution. Ayu (or sweetfish), Plecoglossus altivelis, belongs to the superorder Stomiati, order Osmeriformes. Stomiati is phylogenetically classified as sister taxa of Neoteleostei. Thus, ayu holds an important position in the fish tree of life. Although ayu is economically important for the food industry and recreational fishing in Japan, few genomic resources are available for this species. To address this problem, we produced a draft genome sequence of ayu by whole-genome shotgun sequencing and constructed linkage maps using a genotyping-by-sequencing approach. Syntenic analyses of ayu and other teleost fish provided information about chromosomal rearrangements during the divergence of Stomiati, Protacanthopterygii and Neoteleostei. The size of the ayu genome indicates that genome compaction occurred after the divergence of the family Osmeridae. Ayu has an XX/XY sex-determination system for which we identified sex-associated loci by a genomewide association study by genotyping-by-sequencing and whole-genome resequencing using wild populations. Genome-wide association mapping using wild ayu populations revealed three sex-linked scaffolds (total, 2.03 Mb). Comparison of whole-genome resequencing mapping coverage between males and females identified male-specific regions in sex-linked scaffolds. A duplicate copy of the anti-Mullerian hormone type-II receptor gene (amhr2bY) was found within these male-specific regions, distinct from the autosomal copy of amhr2. Expression of the Y-linked amhr2 gene was male-specific in sox9b-positive somatic cells surrounding germ cells in undifferentiated gonads, whereas autosomal amhr2 transcripts were detected in somatic cells in sexually undifferentiated gonads of both genetic males and females. Loss-of-function mutation for amhr2bY induced male to female sex reversal. Taken together with the known role of Amh and Amhr2 in sex differentiation, these results indicate that the paralog of amhr2 on the ayu Y chromosome determines genetic sex, and the male-specific amh-amhr2 pathway is critical for testicular differentiation in ayu. Author summary Ayu (or sweetfish), Plecoglossus altivelis, is widely distributed in East Asia. Ayu belongs to the superorder Stomiati and the order Osmeriformes. Stomiati is phylogenetically classified as sister group of Neoteleostei, the largest clade of bony fish including medaka, tuna and cod. The divergence of Protacanthopterygii (salmon and pike) and the common ancestor of Stomiati and Neoteleostei is estimated to have occurred approximately 190 million years ago. Thus, ayu holds an important position in the fish tree of life. We sequenced the ayu genome and constructed linkage maps using a genotyping-by-sequencing approach. Comparative analyses of ayu, medaka and northern pike revealed chromosomal rearrangements in the ayu lineage after the divergence of ayu and northern pike. Association mapping revealed a duplicate copy of the anti-Mullerian hormone type-II receptor gene (amhr2bY) located within a male-specific region. Y-linked amhr2 expression was male-specific in supporting cells in undifferentiated gonads, whereas autosomal amhr2 transcripts were detected in somatic cells of sexually undifferentiated gonads in both. Loss-of-function mutation for amhr2bY induced male-to-female sex reversal. Taken together, these results indicate that the paralog of amhr2 on the Y chromosome determines genetic sex. Our findings support the hypothesis that the male-specific amh-amhr2 pathway is critical for gonadal differentiation in ayu.