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.

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.

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.

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.

Bos taurus-indicus hybridization correlates with intralocus sexual-conflict effects of PRDM9 on male and female fertility in Holstein cattle.

BACKGROUND: Crossover localization during meiotic recombination is mediated by the fast-evolving zinc-finger (ZnF) domain of gene PRDM9. To study its impact on dairy cattle performance, we compared its genetic variation between the relatively small Israeli (IL) Holsteins and the North American (US) Holsteins that count millions. RESULTS: Initially, we analyzed the major BTA1 haplotypes present in IL Holsteins based on the 10 most telomeric SNPs of the BovineSNP50 BeadChip. Sequencing of representative haplotype carriers indicated that for all frequent haplotypes (> 6%), the variable PRDM9 ZnF array consisted of seven tandem ZnF repeats. Two rare haplotypes (frequency < 4%) carried an indicine PRDM9, whereas all others were variants of the taurine type. These two haplotypes included the minor SNP allele, which was perfectly linked with a previously described PRDM9 allele known to induce unique localization of recombination hotspots. One of them had a significant (p = 0.03) negative effect on IL sire fertility. This haplotype combined the rare minor alleles of the only SNPs with significant (p < 0.05) negative substitution effects on US sire fertility (SCR). Analysis of telomeric SNPs indicated general agreement of allele frequencies (R = 0.95) and of the substitution effects on sire fertility (SCR, R = 0.6) between the US and IL samples. Surprisingly, the alleles that had a negative impact on male fertility had the most positive substitution effects on female fertility traits (DPR, CCR and HCR). CONCLUSIONS: A negative genetic correlation between male and female fertility is encoded within the BTA1 telomere. Cloning the taurine PRDM9 gene, which is the common form carried by Holsteins, we encountered the infiltration of an indicine PRDM9 variant into this population. During meiosis, in heterozygous males, the indicine PRDM9 variant may induce incompatibility of recombination hotspots and male infertility. However, this variant is associated with favorable female fertility, which would explain its survival and the general negative correlation (R = - 0.3) observed between male and female fertility in US Holsteins. Further research is needed to explain the mechanism underlying this positive effect and to devise a methodology to unlink it from the negative effect on male fertility during breeding.

Quantitative trait loci on LGs 9 and 14 affect the reproductive interaction between two Oreochromis species, O. niloticus and O. aureus.

Effective farming of tilapia requires all-male culture, characterized by uniformity and high growth rate. Males of O. aureus (Oa) and females of O. niloticus (On) produce all-male offspring, but there is a behavioral reproductive barrier between the two species that prevents mass production. In crosses between Oa and On broodstocks, few hybrid females are attracted to the Oa male nests (denoted responders), and if they harbor the On alleles for the sex determination (SD) sites on linkage groups (LGs) 1, 3, and 23, all-male progeny are produced. Yet, without controlling for the alleles underlying SD, the parental stocks gradually lose their capability for all-male production. Hypothesizing that marker-assisted selection for female responders would allow production of sustainable broodstocks, we applied genotyping-by-sequencing to generate 4983 informative SNPs from 13 responding and 28 non-responding females from two full-sib families. Accounting for multiple comparisons in a genome-wide association study, seven SNPs met a false discovery rate of 0.061. Lowest nominal probabilities were on LGs 9 and 14, for which microsatellite DNA markers were designed within the candidate genes PTGDSL and CASRL, respectively. By increasing the sample size to 22 responders and 47 non-responders and by genotyping additional established microsatellites, we confirmed the association of these LGs with female responsiveness. The combined effects of microsatellites GM171 and CARSL-LOC100690618 on LGs 9 and 14 explained 37% of the phenotypic variance of reproductive interaction (p < 0.0001). Based on these findings, we propose a strategy for mass production of all-male tilapia hybrids through selection for genomic loci affecting SD and female responsiveness.

Unveiling genomic regions that underlie differences between Afec-Assaf sheep and its parental Awassi breed.

BACKGROUND: Sheep production in Israel has improved by crossing the fat-tailed local Awassi breed with the East Friesian and later, with the Booroola Merino breed, which led to the formation of the highly prolific Afec-Assaf strain. This strain differs from its parental Awassi breed in morphological traits such as tail and horn size, coat pigmentation and wool characteristics, as well as in production, reproductive and health traits. To identify major genes associated with the formation of the Afec-Assaf strain, we genotyped 41 Awassi and 141 Afec-Assaf sheep using the Illumina Ovine SNP50 BeadChip array, and analyzed the results with PLINK and EMMAX software. The detected variable genomic regions that differed between Awassi and Afec-Assaf sheep (variable genomic regions; VGR) were compared to selection signatures that were reported in 48 published genome-wide association studies in sheep. Because the Afec-Assaf strain, but not the Awassi breed, carries the Booroola mutation, association analysis of BMPR1B used as the test gene was performed to evaluate the ability of this study to identify a VGR that includes such a major gene. RESULTS: Of the 20 detected VGR, 12 were novel to this study. A ~7-Mb VGR was identified on Ovies aries chromosome OAR6 where the Booroola mutation is located. Similar to other studies, the most significant VGR was detected on OAR10, in a region that contains candidate genes affecting horn type (RXFP2), climate adaptation (ALOX5AP), fiber diameter (KATNAl1), coat pigmentation (FRY) and genes associated with fat distribution. The VGR on OAR2 included BNC2, which is also involved in controlling coat pigmentation in sheep. Six other VGR contained genes that were shown to be involved in coat pigmentation by analyzing their mammalian orthologues. Genes associated with fat distribution in humans, including GRB14 and COBLL1, were located in additional VGR. Sequencing DNA from Awassi and Afec-Assaf individuals revealed non-synonymous mutations in some of these candidate genes. CONCLUSIONS: Our results highlight VGR that differentiate the Awassi breed from the Afec-Assaf strain, some of which may include genes that confer an advantage to Afec-Assaf and Assaf over Awassi sheep with respect to intensive sheep production under Mediterranean conditions.

Identification of male-specific amh duplication, sexually differentially expressed genes and microRNAs at early embryonic development of Nile tilapia (Oreochromis niloticus).

BACKGROUND: The probable influence of genes and the environment on sex determination in Nile tilapia suggests that it should be regarded as a complex trait. Detection of sex determination genes in tilapia has both scientific and commercial importance. The main objective was to detect genes and microRNAs that were differentially expressed by gender in early embryonic development. RESULTS: Artificial fertilization of Oreochromis niloticus XX females with either sex-reversed ΔXX males or genetically-modified YY 'supermales' resulted in all-female and all-male embryos, respectively. RNA of pools of all-female and all-male embryos at 2, 5 and 9 dpf were used as template for a custom Agilent eArray hybridization and next generation sequencing. Fifty-nine genes differentially expressed between genders were identified by a false discovery rate of p < 0.05. The most overexpressed genes were amh and tspan8 in males, and cr/20ß-hsd, gpa33, rtn4ipl and zp3 in females (p < 1 × 10-9). Validation of gene expression using qPCR in embryos and gonads indicated copy number variation in tspan8, gpa33, cr/20ß-hsd and amh. Sequencing of amh identified a male-specific duplication of this gene, denoted amhy, differing from the sequence of amh by a 233 bp deletion on exonVII, hence lacking the capability to encode the protein motif that binds to the transforming growth factor beta receptor (TGF-ß domain). amh and amhy segregated in the mapping family in full concordance with SD-linked marker on LG23 signifying the QTL for SD. We discovered 831 microRNAs in tilapia embryos of which nine had sexually dimorphic expression patterns by a false discovery rate of p < 0.05. An up-regulated microRNA in males, pma-mir-4585, was characterized with all six predicted target genes including cr/20ß-hsd, down-regulated in males. CONCLUSIONS: This study reports the first discovery of sexually differentially expressed genes and microRNAs at a very early stage of tilapia embryonic development, i.e. from 2 dpf. Genes with sexually differential expression patterns are enriched for copy number variation. A novel male-specific duplication of amh, denoted amhy, lacking the TGF-ß domain was identified and mapped to the QTL region on LG23 for SD, thus indicating its potential role in SD.

Re-Evaluation of Genotyping Methodologies in Cattle: The Proficiency of Imputation.

In dairy cattle, identifying polymorphisms that contribute to complex economical traits such as residual feed intake (RFI) is challenging and demands accurate genotyping. In this study, we compared imputed genotypes (n = 192 cows) to those obtained using the TaqMan and high-resolution melting (HRM) methods (n = 114 cows), for mutations in the FABP4 gene that had been suggested to have a large effect on RFI. Combining the whole genome sequence (n = 19 bulls) and the cows' BovineHD BeadChip allowed imputing genotypes for these mutations that were verified by Sanger sequencing, whereas, an error rate of 11.6% and 10.7% were encountered for HRM and TaqMan, respectively. We show that this error rate seriously affected the linkage-disequilibrium analysis that supported this gene candidacy over other BTA14 gene candidates. Thus, imputation produced superior genotypes and should also be regarded as a method of choice to validate the reliability of the genotypes obtained by other methodologies that are prone to genotyping errors due to technical conditions. These results support the view that RFI is a complex trait and that searching for the causative sequence variation underlying cattle RFI should await the development of statistical methods suitable to handle additive and epistatic interactions.

Comparing BeadChip and WGS Genotyping: Non-Technical Failed Calling Is Attributable to Additional Variation within the Probe Target Sequence.

Microarray-based genomic selection is a central tool to increase the genetic gain of economically significant traits in dairy cattle. Yet, the effectivity of this tool is slightly limited, as estimates based on genotype data only partially explain the observed heritability. In the analysis of the genomes of 17 Israeli Holstein bulls, we compared genotyping accuracy between whole-genome sequencing (WGS) and microarray-based techniques. Using the standard GATK pipeline, the short-variant discovery within sequence reads mapped to the reference genome (ARS-UCD1.2) was compared to the genotypes from Illumina BovineSNP50 BeadChip and to an alternative method, which computationally mimics the hybridization procedure by mapping reads to 50 bp spanning the BeadChip source sequences. The number of mismatches between the BeadChip and WGS genotypes was low (0.2%). However, 17,197 (40% of the informative SNPs) had extra variation within 50 bp of the targeted SNP site, which might interfere with hybridization-based genotyping. Consequently, with respect to genotyping errors, BeadChip varied significantly and systematically from WGS genotyping, introducing null allele-like effects and Mendelian errors (<0.5%), whereas the GATK algorithm of local de novo assembly of haplotypes successfully resolved the genotypes in the extra-variable regions. These findings suggest that the microarray design should avoid polymorphic genomic regions that are prone to extra variation and that WGS data may be used to resolve erroneous genotyping, which may partially explain missing heritability.

Characterisation of the chromosome fusions in Oreochromis karongae.

Oreochromis karongae, one of the "chambo" tilapia species from Lake Malawi, has a karyotype of 2n = 38, making it one of the few species investigated to differ from the typical tilapia karyotype (2n = 44). The O. karongae karyotype consists of one large subtelocentric pair of chromosomes, four medium-sized pairs (three subtelocentric and one submetacentric) and 14 small pairs. The five largest pairs could be distinguished from each other on the basis of size, morphology and a series of fluorescence in situ hybridisation (FISH) probes. The largest pair is easily distinguished on the basis of size and a chromosome 1 (linkage group 3) bacterial artificial chromosome (BAC) FISH probe from Oreochromis niloticus. BAC clones from O. niloticus chromosome 2 (linkage group 7) hybridised to one of the medium-sized subtelocentric chromosome pairs (no. 5) of O. karongae, distinguishing the ancestral medium-sized pair from the three other medium-sized chromosome pairs (nos. 2, 3 and 4) that appear to have resulted from fusions. SATA repetitive DNA hybridised to the centromeres of all 19 chromosome pairs and also revealed the locations of the relic centromeres in the three fused pairs. Telomeric (TTAGGG)(n) repeats were identified in the telomeres of all chromosomes, and an interstitial telomeric site (ITS) was identified in three chromosomal pairs (no. 2, 3 and 4). Additionally, two ITS sites were identified in the largest chromosome pair (pair 1), confirming the origin of this chromosome from three ancestral chromosomes. SATA and ITS sites allowed the orientation of the fusions in pairs 2, 3 and 4, which all appear to have been in different orientations (q-q, p-q and p-p, respectively). One of these fusions (O. karongae chromosome pair no. 2) involves a small chromosome (equivalent to linkage group 1), which in O. niloticus carries the main sex-determining gene. 4',6-Diamidino-2-phenyloindole staining of the synaptonemal complex in male O. karongae revealed the presumptive positions of the kinetochores, which correspond well to the centromeric positions observed in the mitotic karyotype.

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.

Analysis of copy loss and gain variations in Holstein cattle autosomes using BeadChip SNPs.

BACKGROUND: Copy number variation (CNV) has been recently identified in human and other mammalian genomes, and there is a growing awareness of CNV's potential as a major source for heritable variation in complex traits. Genomic selection is a newly developed tool based on the estimation of breeding values for quantitative traits through the use of genome-wide genotyping of SNPs. Over 30,000 Holstein bulls have been genotyped with the Illumina BovineSNP50 BeadChip, which includes 54,001 SNPs (~SNP/50,000 bp), some of which fall within CNV regions. RESULTS: We used the BeadChip data obtained for 912 Israeli bulls to investigate the effects of CNV on SNP calls. For each of the SNPs, we estimated the frequencies of occurrence of loss of heterozygosity (LOH) and of gain, based either on deviation from the expected Hardy-Weinberg equilibrium (HWE) or on signal intensity (SI) using the PennCNV "detect" option. Correlations between LOH/CNV frequencies predicted by the two methods were low (up to r = 0.08). Nevertheless, 418 locations displayed significantly high frequencies by both methods. Efficiency of designating large genomic clusters of olfactory receptors as CNVs was 29%. Frequency values for copy loss were distinguishable in non-autosomal regions, indicating misplacement of a region in the current BTA7 map. Analysis of BTA18 placed major quantitative trait loci affecting net merit in the US Holstein population in regions rich in segmental duplications and CNVs. Enrichment of transporters in CNV loci suggested their potential effect on milk-production traits. CONCLUSIONS: Expansion of HWE and PennCNV analyses allowed estimating LOH/CNV frequencies, and combining the two methods yielded more sensitive detection of inherited CNVs and better estimation of their possible effects on cattle genetics. Although this approach was more effective than methodologies previously applied in cattle, it has severe limitations. Thus the number of CNVs reported here for the Holstein breed may represent as little as one-tenth of inherited common structural variation.

Basal Levels of CD18 Antigen Presenting Cells in Cow Milk Associate with Copy Number Variation of Fc Gamma Receptors.

Differentiation of cells by flow cytometry provides informative somatic cell counts (SCCs) that allow analyzing leukocyte population patterns in udder infections of different etiologies. Postulating that this approach also enhances the statistical power to detect genetic variants linked to cell levels in milk of healthy mammary glands, we used monoclonal antibodies anti-CD18, anti-CD4, anti--CD14, and anti-PMN to count cells presenting these surface antigens, and performed a genome-wide association study of these counts in 125 Israeli Holsteins genotyped using SNP BeadChips. We identified an informative haplotype of 15 SNPs in the centromeric end of BTA3 that was strongly associated with CD18 cells (p < 2.3 × 10-9). Within this region, examination of the network of genes interacting with ITGB2 (CD18) indicated an Fc-γ-receptor gene cluster, including FCGR2A (CD32). Sanger-sequence analysis of FCGR2s-linked exon 3 variation to CD18 counts. Meta-analysis of RNA-Seq data revealed a significant negative correlation (R = -0.51) between expression of CD32 and CD18 in milk. Assembly of DNA-Seq reads uncovered FCGR copy-number variation and a variant, designated V7, was abundant in dairy cattle, probably reflecting adaptation to selection pressure for low SCC in Holstein milk.

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.

Mapping of the Sex Determining Region on Linkage Group 12 of Guppy (Poecilia reticulata).

Poecilia reticulata is one of the most popular ornamental fish species with a higher demand for males due to their large colorful fins. The objectives of this study were mapping of the sex-determining (SD) region on linkage group 12 of guppy, and identification of a sex specific marker. We generated eight polymorphic microsatellite markers distributed along the distal 5.4 Mbp sequence of the previously identified SD region on linkage group (LG) 12. The markers were tested for association with sex in 156 individuals of the Red Blonde and Flame strains, and 126 progeny of four full-sibs Red Blonde families. A male-specific allele was found for microsatellite gu1066 at position of 25.3 Mbp on LG12 for both strains, and gu832 at position 24.4 Mbp for the Flame strain. Thus, a region of 0.9 Mbp between these markers, harboring 27 annotated genes, was selected for analysis. Based on association of copy number variation and sex determination we mapped a duplicated region between LGs 9 and 12, of 1.26 Mbp, containing 59 genes on LG12. The common interval between the segment bounded by gu1066 and gu832, and the duplicated region of 0.43 Mbp on LG12 harbors 11 genes of which 6 have multiple copies (54%). Growth arrest and DNA damage inducible gamma-like (GADD45G-like) is a plausible positional and functional candidate gene for its role in male fertility. We characterized the genomic structure of the gene in guppy, and found two isoforms; but no sex-biased differences were evident in genomic sequence and gene expression.

Male-specific protein (MSP): a new gene linked to sexual behavior and aggressiveness of tilapia males.

MSP is a male-specific protein initially identified in the serum of sexually active Sarotherodon galilaeus males, and is shown herein to be present in the serum of sexually mature males, but not females, of three other tilapia species. Cloning of the MSP cDNA and analysis of its predicted amino-acid sequence revealed that it is an outlier lipocalin that contains a signal peptide in its N-terminal region. The abundance of highly homologous sequences found in fish and the monophyletic relationship to tetrapod Alpha-1-acid glycoprotein (AGP) places it as a clade XII lipocalin. MSP was shown to undergo major N-glycosylation, characteristic of many lipocalins. The expression pattern of MSP, as determined at both the RNA and protein levels, points to the liver, head kidney and testis as production tissues, and resembles a pattern typical of some hormones. We found that MSP is secreted in urine and seminal fluids, and is present in the skin mucus of socially dominant males. Moreover, we discovered a positive correlation between MSP levels in the serum and the dominance and aggressive behavior displayed by socially dominant males. Based on these data, we suggest that MSP is a novel male-specific lipocalin that may function in intra and inter-sex communication.

Amh and Dmrta2 genes map to tilapia (Oreochromis spp.) linkage group 23 within quantitative trait locus regions for sex determination.

Recent studies have revealed that the major genes of the mammalian sex determination pathway are also involved in sex determination of fish. Several studies have reported QTL in various species and strains of tilapia, regions contributing to sex determination have been identified on linkage groups 1, 3, and 23. Genes contributing to sex-specific mortality have been detected on linkage groups 2, 6, and 23. To test whether the same genes might control sex determination in mammals and fishes, we mapped 11 genes that are considered putative master key regulators of sex determination: Amh, Cyp19, Dax1, Dmrt2, Dmrta2, Fhl3l, Foxl2, Ixl, Lhx9, Sf1, and Sox8. We identified polymorphisms in noncoding regions of these genes and genotyped these sites for 90 individuals of an F2 mapping family. Mapping of Dax1 joined LG16 and LG21 into a single linkage group. The Amh and Dmrta2 genes were mapped to two distinct regions of LG23. The Amh gene was mapped 5 cM from UNH879 within a QTL region for sex determination and 2 cM from UNH216 within a QTL region for sex-specific mortality. Dmrta2 was mapped 4 cM from UNH848 within another QTL region for sex determination. Cyp19 was mapped to LG1 far from a previously reported QTL region for sex determination on this chromosome. Seven other candidate genes mapped to LG4, -11, -12, -14, and -17.

Sequence motifs capable of forming DNA stem-loop structures act as a replication diode.

Calculating peak-height ratios between single-nucleotide polymorphisms (SNP) alleles in sequencing chromatograms is a practical method for estimating their copy number proportions (CNPs). However, it is surprising that sequencing DNA from different directions might yield different results. We analyzed three adjacent SNPs within the ovine period circadian-clock 2 (PER2) gene that displayed such behavior. We compared Sanger and DNA-seq sequencing for this locus and applied high-resolution melt and MFOLD analyses to point to the DNA secondary structure that underlined this phenomenon. A synthetic system of oligonucleotides cloned into plasmids was used to further test the effect of such structures on sequencing. Our analyses indicated that a stem-loop structure capable of G-T pairing mediated the orientation bias by stabilizing this structure for specific alleles in heterozygous situations. We propose that this wobble-like pairing hinders DNA polymerase passage on one strand while, on the complementary strand, the nonpaired A-C nucleotide counterparts allow unobstructed replication. Experimentation with synthetic amplicons that form similar stem-loop structures supported our hypothesis. We coined the term 'replication diode' for this effect and demonstrated that we can minimize it by lowering DNA and salt concentration. We also demonstrated that common genomic palindromes might induce the replication diode effect by applying bidirectional sequencing to an amplicon containing the palindrome within the human miRNA 1-1 gene. Hence, to obtain reliable peak-height ratios, bidirectional sequencing should be practiced at the lowest possible ionic strength whenever estimating CNPs. Further research is needed to determine whether the observed variable stem-loop structures affect PER2 regulation in vivo.

Gene Augmentation Therapy for a Missense Substitution in the cGMP-Binding Domain of Ovine CNGA3 Gene Restores Vision in Day-Blind Sheep.

Purpose: Applying CNGA3 gene augmentation therapy to cure a novel causative mutation underlying achromatopsia (ACHM) in sheep. Methods: Impaired vision that spontaneously appeared in newborn lambs was characterized by behavioral, electroretinographic (ERG), and histologic techniques. Deep-sequencing reads of an affected lamb and an unaffected lamb were compared within conserved genomic regions orthologous to human genes involved in similar visual impairment. Observed nonsynonymous amino acid substitutions were classified by their deleteriousness score. The putative causative mutation was assessed by producing compound CNGA3 heterozygotes and applying gene augmentation therapy using the orthologous human cDNA. Results: Behavioral assessment revealed day blindness, and subsequent ERG examination showed attenuated photopic responses. Histologic and immunohistochemical examination of affected sheep eyes did not reveal degeneration, and cone photoreceptors expressing CNGA3 were present. Bioinformatics and sequencing analyses suggested a c.1618G>A, p.Gly540Ser substitution in the GMP-binding domain of CNGA3 as the causative mutation. This was confirmed by genetic concordance test and by genetic complementation experiment: All five compound CNGA3 heterozygotes, carrying both p.Arg236* and p.Gly540Ser mutations in CNGA3, were day-blind. Furthermore, subretinal delivery of the intact human CNGA3 gene using an adeno-associated viral vector (AAV) restored photopic vision in two affected p.Gly540Ser homozygous rams. Conclusions: The c.1618G>A, p.Gly540Ser substitution in CNGA3 was identified as the causative mutation for a novel form of ACHM in Awassi sheep. Gene augmentation therapy restored vision in the affected sheep. This novel mutation provides a large-animal model that is valid for most human CNGA3 ACHM patients; the majority of them carry missense rather than premature-termination mutations.