Preferential Mapping of Sex-Biased Differentially-Expressed Genes of Larvae to the Sex-Determining Region of Flathead Grey Mullet (Mugil cephalus)

Abstract

Flathead gray mullet ( Mugil cephalus ) is a cosmopolitan mugilid species popular in fishery and aquaculture with an economic preference for all-female population. However, it displays neither sexual dimorphisms nor heteromorphic sex chromosomes. We have previously presented a microsatellite-based linkage map for this species locating a single sex determination region (SDR) on linkage group 9 (LG9) with evidence for XX/XY sex determination (SD) mechanism. In this work, we refine the critical SDR on LG9, and propose positional- and functional- candidate genes for SD. To elucidate the genetic mechanism of SD, we assembled and compared male and female genomic sequences of 19 syntenic genes within the putative SDR on mullet’s LG9, based on orthology to tilapia’s LG8 (tLG8) physical map. A total of 25 sequence-based markers in 12 genes were developed. For all markers, we observed association with sex in at least one of the two analyzed M. cephalus full-sib families, but not in the wild-type population. Recombination events were inferred within families thus setting the SDR boundaries to a region orthologous to ∼0.9 Mbp with 27 genes on tLG8. As the sexual phenotype is evident only in adults, larvae were assigned into two putative sex-groups according to their paternal haplotypes, following a model of XY/XX SD-system. A total of 107 sex-biased differentially expressed genes in larvae were observed, of which 51 were mapped to tLG8 (48% enrichment), as compared to 5% in random control. Furthermore, 23 of the 107 genes displayed sex-specific expression; and 22 of these genes were positioned to tLG8, indicating 96% enrichment. Of the 27 SDR genes, BCCIP , DHX32A , DOCK1 , and FSHR ( GTH-RI ) are suggested as positional and functional gene candidates for SD.

A novel c.1759T>G variant in follicle-stimulating hormone-receptor gene is concordant with male determination in the flathead grey mullet (Mugil cephalus)

“…A caudal fin sample was taken from each of the adult specimens and preserved in ethanol (90–96%). Fin samples were also taken from two groups of 28 dph larvae (Group A, n = 7; Group B, n = 8, Dor et al 2020 ), and from 87 nonsexed fingerlings of a wild population caught in year 2019 in river estuaries along the Israeli Mediterranean coast.…”

“…Mugil cephalus fshr raw reads and previously built SOAPdenovo scaffolds ( Dor et al 2016 , 2020 ) of a male and female were assembled separately. The Gap5 ( Staden et al 2000 ) wrapper and the BWA program were used for alignment of reads and scaffolds against the masked gene sequence of Dicentrarchus labrax Fshr (GenBank accession no.…”

“…Based on synteny with the Nile tilapia ( Oreochromis niloticus ) genome, the SD region of M. cephalus was initially mapped to a 1.8 Mbp region ( Dor et al 2016 ). A follow-up study fine-mapped the SD region down to a 1.0 Mbp region on mullet LG9, harboring 27 genes with an enriched set of genes showing sex-biased gene expression ( Dor et al 2020 ). Although 25 sequence-based markers in 12 of these genes were analyzed, on a population level, none of these polymorphisms exhibited association with sex.…”

“…Although 25 sequence-based markers in 12 of these genes were analyzed, on a population level, none of these polymorphisms exhibited association with sex. One of the genes mapped in the region was Follicle-stimulating hormone receptor ( fshr) , indicating it as a candidate MKR gene for SD, based on its putative position and relevant function in sex differentiation ( Murozumi et al 2014 ; Dor et al 2020 ). Although the use of the candidate gene approach in the analysis of the genetic control of human diseases has not been effective ( Altshuler et al 2008 ), the fact that the majority of discovered SD genes in vertebrates have a known function in reproduction, suggests that this approach may be suitable for the search for MKRs of SD ( Shirak et al 2006 ; Eshel et al 2014 ).…”

“…In this study, we used our established genomic libraries ( Dor et al 2016 , 2020 ) and resequencing data of fshr in M. cephalus to investigate its role in SD. A panel of wild type (WT) fish and full-sib families was used to test the association of fshr polymorphism with sex.…”