Using RAD‐seq to recognize sex‐specific markers and sex chromosome systems

Next‐generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. 2010 ). Among the most impressive of these sequencing innovations is restriction site‐associated DNA sequencing or RAD‐seq (Baird et al. 2008 ; Andrews et al. 2016 ). RAD‐seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD‐seq data has been to identify sex‐specific genetic markers, markers found in one sex but not the other (Baxter et al. 2011 ; Gamble & Zarkower 2014 ). Sex‐specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon 1998 ; Mossman & Waser 1999 ), the management and breeding of endangered species (Taberlet et al. 1993 ; Griffiths & Tiwari 1995 ; Robertson et al. 2006 ) and sexing embryonic material (Hacker et al. 1995 ; Smith et al. 1999 ). Furthermore, sex‐specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank 2010 ; Gamble & Zarkower 2014 ). Thus, species with male‐specific markers have male heterogamety (XY) while species with female‐specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi ( 2016 ) illustrate the ease by which RAD‐seq data can generate sex‐specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD‐seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig.  1 ), Fowler & Buonaccorsi ( 2016 ) uncover shared sex‐specific markers and a conserved sex chromosome system.     

Determination of the optimal tissue source and number of microsatellites for detection of zygotic origin of cattle twins

Abstract

Twenty pairs of cattle twins were genotyped for 3 to 12 microsatellites each using semen, blood, milk and hair roots. Chimaerism was recognized in 19 pairs by discrepancies in microsatellite analysis from milk and blood as opposed to semen and hair, or by detection of more than 2 alleles per genotype from milk and blood. Chimaerism of 2, 3 or 4 alleles was demonstrated in genotypes of twins from blood as compared to 1 or 2 alleles only from hair. The appearance of predominant bands in genotypes from blood or milk representing alleles of only one of the co‐twins was not consistent among the different microsatellites for the same twins. No evidence for germ cell chimaerism was found in semen of dizygotic male twins although our PCR system could detect cell mixes as small as a 1:100 ratio. Genotyping from either hair or semen for 4 microsatellites are sufficient to confirm zygotic origin of twins at .98 accuracy. Researchers should be aware of the possibility of erroneous genotyping when analyzing DNA from twins derived from blood or milk and the potential of chimaerism as an experimental model to study different immunological characteristics of cattle co‐twins.     

Within‐population Y‐linked genetic variation for lifespan in Drosophila melanogaster

The view that the Y chromosome is of little importance for phenotypic evolution stems from early studies of Drosophila melanogaster. This species’ Y chromosome contains only 13 protein‐coding genes, is almost entirely heterochromatic and is not necessary for male viability. Population genetic theory further suggests that non‐neutral variation can only be maintained at the Y chromosome under special circumstances. Yet, recent studies suggest that the D. melanogaster Y chromosome trans‐regulates hundreds to thousands of X and autosomal genes. This finding suggests that the Y chromosome may play a far more active role in adaptive evolution than has previously been assumed. To evaluate the potential for the Y chromosome to contribute to phenotypic evolution from standing genetic variation, we test for Y‐linked variation in lifespan within a population of D. melanogaster. Assessing variation for lifespan provides a powerful test because lifespan (i) shows sexual dimorphism, which the Y is primarily predicted to contribute to, (ii) is influenced by many genes, which provides the Y with many potential regulatory targets and (iii) is sensitive to heterochromatin remodelling, a mechanism through which the Y chromosome is believed to regulate gene expression. Our results show a small but significant effect of the Y chromosome and thus suggest that the Y chromosome has the potential to respond to selection from standing genetic variation. Despite its small effect size, Y‐linked variation may still be important, in particular when evolution of sexual dimorphism is genetically constrained elsewhere in the genome.     

Female‐only sex‐linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex‐reversed females (neomales). To provide molecular evidence for the proposed system, novel sex‐linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male‐specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female‐specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female‐specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female‐linked SCAR markers can be applied for rapid detection of prawn gender. These sex‐specific SCAR markers and sex‐associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).     

Genomic characterization of sex‐identification markers in Sebastes carnatus and Sebastes chrysomelas rockfishes

Fish have evolved a variety of sex‐determining (SD) systems including male heterogamy (XY), female heterogamy (ZW) and environmental SD. Little is known about SD mechanisms of Sebastes rockfishes, a highly speciose genus of importance to evolutionary and conservation biology. Here, we characterize the sex determination system in the sympatrically distributed sister species Sebastes chrysomelas and Sebastes carnatus. To identify sex‐specific genotypic markers, double digest restriction site – associated DNA sequencing (ddRAD‐seq) of genomic DNA from 40 sexed individuals of both species was performed. Loci were filtered for presence in all of the individuals of one sex, absence in the other sex and no heterozygosity. Of the 74 965 loci present in all males, 33 male‐specific loci met the criteria in at least one species and 17 in both. Conversely, no female‐specific loci were detected, together providing evidence of an XY sex determination system in both species. When aligned to a draft reference genome from Sebastes aleutianus, 26 sex‐specific loci were interspersed among 1168 loci that were identical between sexes. The nascent Y chromosome averaged 5% divergence from the X chromosome and mapped to reference Sebastes genome scaffolds totalling 6.9Mbp in length. These scaffolds aligned to a single chromosome in three model fish genomes. Read coverage differences were also detected between sex‐specific and autosomal loci. A PCR‐RFLP assay validated the bioinformatic results and correctly identified sex of five additional individuals of known sex. A sex‐determining gene in other teleosts gonadal soma‐derived factor (gsdf) was present in the model fish chromosomes that spanned our sex‐specific markers.     

New‐age ideas about age‐old sex: separating meiosis from mating could solve a century‐old conundrum

Ever since Darwin first addressed it, sexual reproduction reigns as the ‘queen’ of evolutionary questions. Multiple theories tried to explain how this apparently costly and cumbersome method has become the universal mode of eukaryote reproduction. Most theories stress the adaptive advantages of sex by generating variation, they fail however to explain the ubiquitous persistence of sexual reproduction also where adaptation is not an issue. I argue that the obstacle for comprehending the role of sex stems from the conceptual entanglement of two distinct processes – gamete production by meiosis and gamete fusion by mating (mixis). Meiosis is an ancient, highly rigid and evolutionary conserved process identical and ubiquitous in all eukaryotes. Mating, by contrast, shows tremendous evolutionary variability even in closely related clades and exhibits wonderful ecological adaptability. To appreciate the respective roles of these two processes, which are normally linked and alternating, we require cases where one takes place without the other. Such cases are rather common. The heteromorphic sex chromosomes Y and W, that do not undergo meiotic recombination are an evolutionary test case for demonstrating the role of meiosis. Substantial recent genomic evidence highlights the accelerated rates of change and attrition these chromosomes undergo in comparison to those of recombining autosomes. I thus propose that the most basic role of meiosis is conserving integrity of the genome. A reciprocal case of meiosis without bi‐parental mating, is presented by self‐fertilization, which is fairly common in flowering plants, as well as most types of apomixis. I argue that deconstructing sex into these two distinct processes – meiosis and mating – will greatly facilitate their analysis and promote our understanding of sexual reproduction.     

Conflict over condition‐dependent sex allocation can lead to mixed sex‐determination systems

Theory suggests that genetic conflicts drive turnovers between sex‐determining mechanisms, yet these studies only apply to cases where sex allocation is independent of environment or condition. Here, we model parent–offspring conflict in the presence of condition‐dependent sex allocation, where the environment has sex‐specific fitness consequences. Additionally, one sex is assumed to be more costly to produce than the other, which leads offspring to favor a sex ratio less biased toward the cheaper sex in comparison to the sex ratio favored by mothers. The scope for parent–offspring conflict depends on the relative frequency of both environments: when one environment is less common than the other, parent–offspring conflict can be reduced or even entirely absent, despite a biased population sex ratio. The model shows that conflict‐driven invasions of condition‐independent sex factors (e.g., sex chromosomes) result either in the loss of condition‐dependent sex allocation, or, interestingly, lead to stable mixtures of condition‐dependent and condition‐independent sex factors. The latter outcome corresponds to empirical observations in which sex chromosomes are present in organisms with environment‐dependent sex determination. Finally, conflict can also favor errors in environmental perception, potentially resulting in the loss of condition‐dependent sex allocation without genetic changes to sex‐determining loci.     

Sex-linked markers and microsatellite locus duplication in the cichlid species Oreochromis tanganicae

Cichlid species of the genus Oreochromis vary in their genetic sex-determination systems. In this study, we used microsatellite DNA markers to characterize the sex-determination system in Oreochromis tanganicae. Markers on linkage group 3 were associated with phenotypic sex, with an inheritance pattern typical of a female heterogametic species (WZ-ZZ). Further, locus duplication was observed for two separate microsatellite markers on the sex chromosome. These results further advance our understanding of the rapidly evolving sex-determination systems among these closely related tilapia species.     

The origin of the non‐recombining region of sex chromosomes in Carica and Vasconcellea

Carica and Vasconcellea are two closely related sister genera in the family Caricaceae, and were once classified as two sections under Carica. Sex chromosomes have been found in papaya and originated approximately 2–3 million years ago. The objectives of this study were to determine whether sex chromosomes have evolved in Vasconcellea. Six X/Y gene pairs were cloned, sequenced and analyzed from three dioecious, one trioecious and one monoecious species of Vasconcellea. The isolation of distinctive X and Y alleles in dioecious and trioecious species of Vasconcellea demonstrated that sex chromosomes have evolved in this genus. Phylogenetic analyses indicated a monophyletic relationship between the X/Y alleles of Carica and those of Vasconcellea. Distinctive clusters of X/Y alleles were documented in V. parviflora and V. pulchra for all available gene sequences, and in V. goudatinana and V. cardinamarcensis for some X/Y alleles. The X and Y alleles within each species shared most single nucleotide polymorphism haplotypes that differed from other species. Limited evidence of gene conversion was documented among the X/Y alleles of some species, but was not sufficient to cause the evolutionary patterns reported herein. The Carica and Vasconcellea sex chromosomes may have originated from the same autosomes bearing the X allelic form that still exist in the monoecious species V. monoica, and have evolved independently after the speciation event that separated Carica from Vasconcellea. Within Vasconcellea, sex chromosomes have evolved at the species level, at least for some species.     

The relationship between gene dosage,gene expression,and sex in Drosophila

In Drosophila, the ratio of the number of X chromosomes to sets of other chromosomes initiates a series of events which result in sexual differentiation. In addition, this ratio establishes dosage compensation, a mechanism which equalizes the products of X-linked genes in males and females. The present review discusses possible genetic entities responsible for the interpretation of chromosomal sex and subsequent sex-mediated regulation during development.     

Co-occurrence of multiple, supposedly incompatible modes of sex determination in a lizard population

Sex is determined genetically in some species (genotypic sex determination, or GSD) and by the environment (environmental sex determination, or ESD) in others. The two systems are generally viewed as incompatible alternatives, but we have found that sex determination in a species of montane lizard ( Bassiana duperreyi , Scincidae) in south-eastern Australia is simultaneously affected by sex chromosomes and incubation temperatures, as well as being related to egg size. This species has strongly heteromorphic sex chromosomes, and yet incubation at thermal regimes characteristic of cool natural nests generates primarily male offspring. We infer that incubation temperatures can over-ride genetically determined sex in this species, providing a unique opportunity to explore these alternative sex-determining systems within a single population.     

Characterization of sex chromosomes in rainbow trout and coho salmon using fluorescence in situ hybridization (FISH)

With the aim of characterizing the sex chromosomes of rainbow trout (Oncorhynchus mykiss) and to identify the sex chromosomes of coho salmon (O. kisutch), we used molecular markers OmyP9, 5S rDNA, and a growth hormone gene fragment (GH2), as FISH probes. Metaphase chromosomes were obtained from lymphocyte cultures from farm specimens of rainbow trout and coho salmon. Rainbow trout sex marker OmyP9 hybridizes on the sex chromosomes of rainbow trout, while in coho salmon, fluorescent signals were localized in the medial region of the long arm of one subtelocentric chromosome pair. This hybridization pattern together with the hybridization of a GH2 intron probe on a chromosome pair having the same morphology, suggests that a subtelocentric pair could be the sex chromosomes in this species. We confirm that in rainbow trout, one of the two loci for 5S rDNA genes is on the X chromosome. In males of this species that lack a heteromorphic sex pair (XX males), the 5S rDNA probe hybridized to both subtelocentrics This finding is discussed in relation to the hypothesis of intraspecific polymorphism of sex chromosomes in rainbow trout.     

A supernumerary “B-sex” chromosome drives male sex determination in the Pachón cavefish,Astyanax mexicanus

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High‐density linkage maps fail to detect any genetic component to sex determination in a Rana temporaria family

Sex chromosome differentiation in Rana temporaria varies strikingly among populations or families: whereas some males display well‐differentiated Y haplotypes at microsatellite markers on linkage group 2 (LG₂), others are genetically undistinguishable from females. We analysed with RADseq markers one family from a Swiss lowland population with no differentiated sex chromosomes, and where sibship analyses had failed to detect any association between the phenotypic sex of progeny and parental haplotypes. Offspring were reared in a common tank in outdoor conditions and sexed at the froglet stage. We could map a total of 2177 SNPs (1123 in the mother, 1054 in the father), recovering in both adults 13 linkage groups (= chromosome pairs) that were strongly syntenic to Xenopus tropicalis despite > 200 My divergence. Sexes differed strikingly in the localization of crossovers, which were uniformly distributed in the female but limited to chromosome ends in the male. None of the 2177 markers showed significant association with offspring sex. Considering the very high power of our analysis, we conclude that sex determination was not genetic in this family; which factors determined sex remain to be investigated.     

A new marker based on the avian spindlin gene that is able to sex most birds,including species problematic to sex with CHD markers

We have developed a new marker (Z43B) that can be successfully used to identify the sex of most birds (69%), including species difficult or impossible to sex with other markers. We utilized the zebra finch Taeniopygia guttata EST microsatellite sequence (CK309496) which displays sequence homology to the 5′ untranslated region (UTR) of the avian spindlin gene. This gene is known to be present on the Z and W chromosomes. To maximize cross‐species utility, the primer set was designed from a consensus sequence created from homologs of CK309496 that were isolated from multiple distantly related species. Both the forward and reverse primer sequences were 100% identical to 14 avian species, including the Z chromosome of eight species and the chicken Gallus gallus W chromosome, as well as the saltwater crocodile Crocodylus porosus. The Z43B primer set was assessed by genotyping individuals of known sex belonging to 61 non‐ratite species and a single ratite. The Z and W amplicons differed in size making it possible to distinguish between males (ZZ) and females (ZW) for the majority (69%) of non‐ratite species tested, comprising 10 orders of birds. We predict that this marker will be useful for obtaining sex‐typing data for ca 6,869 species of birds (69% of non‐ratites but not galliforms). A wide range of species could be sex‐typed including passerines, shorebirds, eagles, falcons, bee‐eaters, cranes, shags, parrots, penguins, ducks, and a ratite species, the brown kiwi, Apteryx australis. Those species sexed include species impossible or problematic to sex‐type with other markers (magpie, albatross, petrel, eagle, falcon, crane, and penguin species). Zoo Biol. 35:533–545, 2016. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc.     

Occurrence of ZZ/ZW sex chromosomes in <Emphasis Type="Italic">Thoracocharax stellatus</Emphasis> fish (Characiformes,Gasteropelecidae) from the Araguaia River,South America

The karyotypic and chromosomal characteristics of the hatchetfish Thoracocharax stellatus from the Araguaia River, Brazil (Araguaia-Tocantins basin) were analyzed using Giemsa, AgNO(3), and CMA(3) fluorescent staining, and C-banding. The diploid chromosome number was 54 and the karyotypes of females and males were composed of six metacentrics, six submetacentrics, six subtelocentrics and 36 acrocentrics. Two unpaired acrocentric chromosomes were detected in the female karyotype. C-banding showed heterochromatic blocks at several chromosomes and an entirely heterochromatic acrocentric chromosome in females that was lacking in the male karyotype. This discovery indicated a heteromorphic sex chromosome system of the ZZ/ZW type. Ag-staining and CMA(3) fluorescence revealed one major chromosome pair bearing the NORs with the presence of additional signals in some metaphases. Both heterochromatic segments associated with Ag-NORs and the W chromosome were positively stained by CMA(3). Considering the present data and previous findings it is hypothesized that the occurrence of ZW sex chromosome system is widespread in the genus Thoracocharax.