Master-Key Regulators of Sex Determination in Fish and Other Vertebrates-A Review.

In vertebrates, mainly single genes with an allele ratio of 1:1 trigger sex-determination (SD), leading to initial equal sex-ratios. Such genes are designated master-key regulators (MKRs) and are frequently associated with DNA structural variations, such as copy-number variation and null-alleles. Most MKR knowledge comes from fish, especially cichlids, which serve as a genetic model for SD. We list 14 MKRs, of which dmrt1 has been identified in taxonomically distant species such as birds and fish. The identification of MKRs with known involvement in SD, such as amh and fshr, indicates that a common network drives SD. We illustrate a network that affects estrogen/androgen equilibrium, suggesting that structural variation may exert over-expression of the gene and thus form an MKR. However, the reason why certain factors constitute MKRs, whereas others do not is unclear. The limited number of conserved MKRs suggests that their heterologous sequences could be used as targets in future searches for MKRs of additional species. Sex-specific mortality, sex reversal, the role of temperature in SD, and multigenic SD are examined, claiming that these phenomena are often consequences of artificial hybridization. We discuss the essentiality of taxonomic authentication of species to validate purebred origin before MKR searches.

Absence of Figla-like Gene Is Concordant with Femaleness in Cichlids Harboring the LG1 Sex-Determination System.

Oreochromis niloticus has been used as a reference genome for studies of tilapia sex determination (SD) revealing segregating genetic loci on linkage groups (LGs) 1, 3, and 23. The master key regulator genes (MKR) underlying the SD regions on LGs 3 and 23 have been already found. To identify the MKR in fish that segregate for the LG1 XX/XY SD-system, we applied short variant discovery within the sequence reads of the genomic libraries of the Amherst hybrid stock, Coptodon zillii and Sarotherodon galilaeus, which were aligned to a 3-Mbp-region of the O. aureus genome. We obtained 66,372 variants of which six were concordant with the XX/XY model of SD and were conserved across these species, disclosing the male specific figla-like gene. We further validated this observation in O. mossambicus and in the Chitralada hybrid stock. Genome alignment of the 1252-bp transcript showed that the figla-like gene's size was 2664 bp, and that its three exons were capable of encoding 99 amino acids including a 45-amino-acid basic helix-loop-helix domain that is typical of the ovary development regulator-factor-in-the-germline-alpha (FIGLA). In Amherst gonads, the figla-like gene was exclusively expressed in testes. Thus, the figla-like genomic presence determines male fate by interrupting the female developmental program. This indicates that the figla-like gene is the long-sought SD MKR on LG1.

Cross-species conservation of a transposase-linked element enables genetic sexing of commercial populations of Russian sturgeon (Acipenser gueldenstaedtii).

All-female culture of sturgeon is essential for efficient caviar production. However, Russian sturgeon (Acipenser gueldenstaedtii) does not exhibit external sexual dimorphism, and therefore, commercial farms apply gonadal endoscopy or ultrasound at the earliest age of 4-5 years to separate the sexes, with ~90% accuracy. Recently, a dominant genomic marker (AllWSEX2) has been found with association to femaleness in sturgeons. We developed a duplex PCR (dAllWSEX2) with the adjacent bmp7 gene as an internal control, to validate an effective PCR. Robust amplification of control fragments was observed for all samples of our commercial A. gueldenstaedtii stock (n = 337). The dAllWSEX2 assay was significantly associated with sex (n = 43, p < 1.6 × 10-8 ), yet four (18%) of the endoscopy-determined females were genetic males. To examine whether some females display a male genetic profile, we tested 96 egg-producing females, which were all verified as genetic females, indicating that the observed mismatches may be attributed to wrong sexing by endoscopy. Application of dAllWSEX2 on 100 7-month-old fish showed no sex-dependent differences in body weight, indicating that weighing is not an applicable tool for sorting females at a young age. Sanger sequencing of the bmp7 fragment revealed octaploidy and sex-independent variation, suggesting that the critical sex-determining region harboring AllWSEX2 is small. In keeping with a model of a single-ploidy encoding female determination, AllWSEX2 showed no variation despite being a transposase-linked repetitive element. Cross-species conservation of AllWSEX2, and absence of annotated sex-determination genes in this region suggests that, in sturgeons, the sex-determining mechanism is different from mechanisms identified in other fish.

Gene Variant of Barrier to Autointegration Factor 2 (Banf2w) Is Concordant with Female Determination in Cichlids.

Oreochromis fishes exhibit variability of sex-determination (SD) genes whose characterization contributes to understanding of the sex differentiation network, and to effective tilapia farming, which requires all-male culture. However, O. niloticus (On) amh is the only master-key regulator (MKR) of SD that has been mapped (XY/XX SD-system on LG23). In O. aureus (Oa), LG3 controls a WZ/ZZ SD-system that has recently been delimited to 9.2 Mbp, with an embedded interval rich with female-specific variation, harboring two paics genes and banf2. Developing genetic markers within this interval and using a hybrid Oa stock that demonstrates no recombination repression in LG3, we mapped the critical SD region to 235 Kbp on the orthologous On physical map (p < 1.5 × 10-26). DNA-seq assembly and peak-proportion analysis of variation based on Sanger chromatograms allowed the characterization of copy-number variation (CNV) of banf2. Oa males had three exons capable of encoding 90-amino-acid polypeptides, yet in Oa females, we found an extra copy with an 89-amino-acid polypeptide and three non-conservative amino acid substitutions, designated as banf2w. CNV analysis suggested the existence of two to five copies of banf2 in diploidic Cichlidae. Disrupting the Hardy-Weinberg equilibrium (p < 4.2 × 10-3), banf2w was concordant with female determination in Oa and in three cichlids with LG3 WZ/ZZ SD-systems (O. tanganicae, O. hornorum and Pelmatolapia mariae). Furthermore, exclusive RNA-seq expression in Oa females strengthened the candidacy of banf2w as the long-sought LG3 SD MKR. As banf genes mediate nuclear assembly, chromatin organization, gene expression and gonad development, banf2w may play a fundamental role inducing female nucleus formation that is essential for WZ/ZZ SD.

Assessing adaptive requirements and breeding potential of spelt under Mediterranean environment.

The rising demand for spelt wheat (Triticum aestivum ssp. spelta) as a high-value grain crop has raised interest in its introduction into non-traditional spelt growing areas. This study aimed to assess adaptive constrains of spelt under short Mediterranean season. At first screening of a wide spelt collection for phenology and allelic distribution at the photoperiod (PPD) and vernalization (VRN) loci was done. In addition an in-depth phenotypic evaluation of a selected panel (n = 20) was performed, including agronomically important traits and concentration of grain mineral (GMC) and grain protein (GPC) content. Results from both wide screening and in-depth in panel (group of 18 spelt lines and two bread wheat lines) evaluation shows that the major adaptive constraint for spelt under Mediterranean conditions is late heading, caused by day length sensitivity, as evident from phenology and allelic profile (PPD and VRN). All lines carrying the photoperiod-sensitive allele (PPD-D1b) were late flowering (> 120DH). Based on the panel field evaluations those consequently suffer from low grain yield and poor agronomic performances. As for minerals, GMC for all but Zn, significantly correlated with GPC. In general, GMC negatively correlated with yield which complicated the assessment of GMC per-se and challenge the claim for higher mineral content in spelt grains. The exceptions were, Fe and Zn, which did not correlate with yield. Spelt lines showing high Fe and Zn concentration in a high-yield background illustrate their potential for spelt wheat breeding. Improving spelt adaptation to Mediterranean environments could be mediated by introducing the insensitive-PPD-D1a allele to spelt wheat background. Following this breeding path spelt could better compete with bread wheat under short season with limited and fluctuating rain fall.

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

Various master key regulators (MKRs) that control a binary switch of sex determination (SD) have been found in fish; these provide an excellent model for the study of vertebrate genetic SD. The SD region in flathead grey mullet has been previously mapped to a 1 Mbp region harboring 27 genes, of which one is follicle-stimulating hormone receptor (fshr). Although this gene is involved in gonad differentiation and function, it has not been considered as an MKR of SD. We systematically investigated polymorphism in mullet fshr using DNA shotgun sequences, and compared them between males and females. Capable of encoding nonconservative amino acid substitutions, c.1732G>A and c.1759T>G exhibited association with sex on a population level (N = 83; P ≤ 6.7 × 10-19). Hence, 1732 A and 1759 G represent a male-specific haplotype of the gene, designated as "fshry." Additional flanking SNPs showed a weaker degree of association with sex, delimiting the SD critical region to 143 nucleotides on exon 14. Lack of homozygotes for fshry, and the resulting divergence from Hardy-Weinberg equilibrium (N = 170; P ≤ 3.9 × 10-5), were compatible with a male heterogametic model (XY/XX). Capable of replacing a phenylalanine with valine, c.1759T>G alters a conserved position across the sixth transmembrane domain of vertebrate FSHRs. Amino acid substitutions in this position in vertebrates are frequently associated with constant receptor activation and consequently with FSH/FSHR signaling alteration; thus, indicating a potential role of fshr as an MKR of SD.

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

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.

Construction of a microsatellites-based linkage map for the white grouper (Epinephelus aeneus).

The white grouper (Epinephelus aeneus) is a promising candidate for domestication and aquaculture due to its fast growth, excellent taste, and high market price. A linkage map is an essential framework for mapping quantitative trait loci for economic traits and the study of genome evolution. DNA of a single individual was deep-sequenced, and microsatellite markers were identified in 177 of the largest scaffolds of the sequence assembly. The success rate of developing polymorphic homologous markers was 94.9% compared with 63.1% of heterologous markers from other grouper species. Of the 12 adult mature fish present in the broodstock tank, two males and two females were identified as parents of the assigned offspring by parenthood analysis using 34 heterologous markers. A single full-sib family of 48 individuals was established for the construction of first-generation linkage maps based on genotyping data of 222 microsatellites. The markers were assigned to 24 linkage groups in accordance to the 24 chromosomal pairs. The female and male maps consisting of 203 and 202 markers spanned 1053 and 886 cM, with an average intermarker distance of 5.8 and 5.0 cM, respectively. Mapping of markers to linkage groups ends was enriched by using markers originating from scaffolds harboring telomeric repeat-containing RNA. Comparative mapping showed high synteny relationships among the white grouper, kelp grouper (E. bruneus), orange-spotted grouper (E. coioides), and Nile tilapia (Oreochromis niloticus). Thus, it would be useful to integrate the markers that were developed for different groupers, depending on sharing of sequence data, into a comprehensive consensus map.