Cytogenetic characterization and description of an X_1X_1X_2X_2/X_1X_2Y sex chromosome system in Collichthys lucidus(Richardson,1844)

The chromosomes of spinyhead croaker Collichthys lucidus（Richardson,1844） were characterized for the first time by fluorescence staining,self genomic in situ hybridization（self-GISH）,and multicolor fluorescence in situ hybridization（FISH） with 18 SrDNA,5 SrDNA and telomeric sequence probes.The female karyotype has exclusively 24 pairs of acrocentric chromosomes（2 n=48 a,NF=48）,while the male one consists of 22 pairs of acrocentric chromosomes,2 monosomic acrocentric chromosomes and a metacentric chromosome（2 n=1 m＋46 a,NF=48）.The difference between female and male karyotypes indicates the presence of a sex chromosome of X1 X1 X2 X2/X1 X2 Y type,where Y is the unique metacentric chromosome in the male karyotype.As revealed by FISH,5 S r DNA and 18 S r DNA sites were mapped at syntenic position of the largest acrocentric chromosome（X_1）,and the short arms of the Y chromosome as well.An X_1-chromosome specific interstitial telomeric signal（ITS） was detected overlapping the 5 S r DNA sites.In addition,self-GISH revealed that the repetitive DNAs accumulated on all the putative sex chromosome.Chromosome fusion accompanied by a partial deletion in the ancestral karyotype（2 n=48 a） is hypothesized for the origin of such multiple sex chromosome system.The present study,as the first description of differentiated sex chromosome in family Sciaenidae,will give clues to the studies on the sex chromosome of other Sciaenids.

Diversity of reptile sex chromosome evolution revealed by cytogenetic and linked-read sequencing

Reptile sex determination is attracting much attention because the great diversity of sex-determination and dosage compensation mechanisms permits us to approach fundamental questions about mechanisms of sex chromosome turnover. Recent studies have made significant progress in better understanding diversity and conservation of reptile sex chromosomes, with however no reptile master sex determination genes identified. Here we describe an integrated genomics and cytogenetics pipeline,combining probes generated from the microdissected sex chromosomes with transcriptome and genome sequencing to explore the sex chromosome diversity in non-model Australian reptiles. We tested our pipeline on a turtle, two species of geckos, and a monitor lizard. Genes identified on sex chromosomes were compared to the chicken genome to identify homologous regions among the four species. We identified candidate sex determining genes within these regions, including conserved vertebrate sex-determining genes pdgfa,pdgfra amh and wt1, and demonstrated their testis or ovary-specific expression. All four species showed gene-by-gene rather than chromosome-wide dosage compensation. Our results imply that reptile sex chromosomes originated by independent acquisition of sex-determining genes on different autosomes,as well as translocations between different ancestral macro-and microchromosomes.We discuss the evolutionary drivers of the slow differentiation and turnover of reptile sex chromosomes.

Detection of Embryo Sex Chromosome by Dual Color Fluorescent In-Situ Hybridization

In order to evaluate the effects of sex chromosomal mosaicism on the accuracy of single-cell gender diagnosis, sex chromosomes of 21 normal fertilized embryos were detected by dual color fluorescent in-situ hybridization (FISH). The results showed that 4 embryos had sex chromosomal mosaicism (19%) and the remaining 17 showed uniformly XX or XY signals in all blastomeres. In conclusion, identification of sex by dual color FISH analysis of a single cell was accurate and efficient , and sex chromosomal mosaicism would not affect preimplantation gender diagnosis.

Knock-in of exogenous sequences based on CRISPR/Cas9 targeting autosomal genes and sex chromosomes in the diamondback moth,Plutella xylostella

Genetic pest control strategies based on precise sex separation and only releasing sterile males can be accomplished by site-specific genome editing.In the current study,we showed that the mutation of single-allele Pxfl(2)d can significantly impair the normal mating behavior and testis development in male adults of the notorious cruciferous insect pest Plutella xylostella,in addition to its known functions in the ovarian development in female adults and egg hatching.Subsequent CRISPR/Cas9-based knock-in experiments revealed that site-specific integration of an exogenous green fluorescent protein(GFP)gene into autosomal Pxfl(2)d for labelling mutants could be achieved.However,this gene is not a suitable target for GFP insertion to establish a genetically stable knock-in strain because of the severe decline in reproductive capacity.We further screened for the W-chromosome-linked and Z-chromosome-linked regions to test the knock-in efficiency mediated by CRISPR/Cas9.The results verified that both types of chromosomes can be targeted for the site-specific insertion of exogenous sequences.We ultimately obtained a homozygous knock-in strain with the integration of both Cas9 and cyan fluorescent protein(CFP)expression cassettes on a Z-linked region in P.xylostella,which can also be used for early sex detection.By injecting the sgRNA targeting Pxfl(2)d alone into the eggs laid by female adults of the Z-Cas9-CFP strain,the gene editing efficiency reached 29.73%,confirming the success of expressing a functional Cas9 gene.Taken together,we demonstrated the feasibility of the knock-in of an exogenous gene to different genomic regions in P.xylostella,while the establishment of a heritable strain required the positioning of appropriate sites.This study provides an important working basis and technical support for further developing genetic strategies for insect pest control.

Pistachio genomes provide insights into nut tree domestication and ZW sex chromosome evolution

Pistachio is a nut crop domesticated in the Fertile Crescent and a dioecious species with ZW sex chromosomes.We sequenced the genomes of Pistacia vera cultivar(cv.)Siirt,the female parent,and P.vera cv.Bagyolu,the male parent.Two chromosome-level reference genomes of pistachio were generated,and Z and W chromosomes were assembled.The ZW chromosomes originated from an autosome following the first inversion,which occurred approximately 8.18 Mya.Three inversion events in the W chromosome led to the formation of a 12.7-Mb(22.8%of the W chromosome)non-recombining region.These W-specific sequences contain several genes of interest that may have played a pivotal role in sex determination and contributed to the initiation and evolution of a ZW sex chromosome system in pistachio.The W-specific genes,including defA,defA-like,DYT1,two PTEN1,and two tandem duplications of six VPS13A paralogs,are strong candidates for sex determination or differentiation.Demographic history analysis of resequenced genomes suggest that cultivated pistachio underwent severe domestication bottlenecks approximately 7640 years ago,dating the domestication event close to the archeological record of pistachio domestication in Iran.We identified 390,211,and 290 potential selective sweeps in 3 cultivar subgroups that underlie agronomic traits such as nut development and quality,grafting success,flowering time shift,and drought tolerance.These findings have improved our understanding of the genomic basis of sex determination/differentiation and horticulturally important traits and will accelerate the improvement of pistachio cultivars and rootstocks.

Maintenance of stem cell self-renewal by sex chromosomal zincfinger transcription factors

In this Editorial review,we would like to focus on a very recent discovery showing the global autosomal gene regulation by Y-and inactivated X-chromosomal transcription factors,zinc finger gene on the Y chromosome(ZFY)and zinc finger protein X-linked(ZFX).ZFX and ZFY are both zinc-finger proteins that encode general transcription factors abundant in hematopoietic and embryonic stem cells.Although both proteins are homologs,interestingly,the regulation of self-renewal by these transcriptional factors is almost exclusive to ZFX.This fact implies that there are some differential roles between ZFX and ZFY in regulating the maintenance of self-renewal activity in stem cells.Besides the maintenance of stemness,ZFX overexpression or mutations may be linked to certain cancers.Although cancers and stem cells are double-edged swords,there is no study showing the link between ZFX activity and the telomere.Thus,stemness or cancers with ZFX may be linked to other molecules,such as Oct4,Sox2,Klf4,and others.Based on very recent studies and a few lines of evidence in the past decade,it appears that the ZFX is linked to the canonical Wnt signaling,which is one possible mechanism to explain the role of ZFX in the self-renewal of stem cells.

The role of sex chromosomes in mammalian germ cell differentiation： can the germ cells carrying X and Y chromosomes differentiate into fertile oocytes？

The sexual differentiation of germ cells into spermatozoa or oocytes is strictly regulated by their gonadal environment, testis or ovary, which is determined by the presence or absence of the Y chromosome, respectively. Hence, in normal mammalian development, male germ cells differentiate in the presence of X and Y chromosomes, and female germ cells do so in the presence of two X chromosomes. However, gonadal sex reversal occurs in humans as well as in other mammalian species, and the resultant XX males and XY females can lead healthy lives, except for a complete or partial loss of fertility. Germ cells carrying an abnormal set of sex chromosomes are efficiently eliminated by multilayered surveillance mechanisms in the testis, and also, though more variably, in the ovary. Studying the molecular basis for sex-specific responses to a set of sex chromosomes during gametogenesis will promote our understanding of meiotic processes contributing to the evolution of sex determining mechanisms. This review discusses the fate of germ cells carrying various sex chromosomal compositions in mouse models, the limitation of which may be overcome by recent successes in the differentiation of functional germ cells from embryonic stem cells under experimental conditions.

The evolution of sex chromosome dosage compensation in animals

The evolution of heteromorphic sex chromosomes shall lead to gene expression dosage problems,as in at least one of the sexes,the sex-linked gene dose has been reduced by half.It has been proposed that the transcriptional output of the whole X or Z chromosome should be doubled for complete dosage compensation in heterogametic sex.However,owing to the variability of the existing methods to determine the transcriptional differences between sex chromosomes and autosomes(S:A ratios)in different studies,we collected more than 500 public RNA-Seq data set from multiple tissues and species in major clades and proposed a unified computational framework for unbiased and comparable measurement of the S:A ratios of multiple species.We also tested the evolution of dosage compensation more directly by assessing changes in the expression levels of the current sex-linked genes relative to those of the ancestral sex-linked genes.We found that in mammals and birds,the S:A ratio is approximately 0.5,whereas in insects,fishes,and flatworms,the S:A ratio is approximately 1.0.Further analysis showed that the fraction of dosage-sensitive housekeeping genes on the X/Z chromosome is significantly correlated with the S:A ratio.In addition,the degree of degeneration of the Y chromosome may be responsible for the change in the S:A ratio in mammals without a dosage compensation mechanism.Our observations offer unequivocal support for the sex chromosome insensitivity hypothesis in animals and suggest that dosage sensitivity states of sex chromosomes are a major factor underlying different evolutionary strategies of dosage compensation.

Molecular analysis of the sex chromosomes of the platyfish Xiphophorus maculatus: Towards the identification of a new type of master sexual regulator in vertebrates

In contrast to mammals and birds,fish display an amazing diversity of genetic sex determination systems,with frequent changes during evolution possibly associated with the emergence of new sex chromosomes and sex-determining genes.To better understand the molecular and evolutionary mechanisms driving this diversity,several fish models are studied in parallel.Besides the medaka(Oryzias latipes Temminck and Schlegel,1846)for which the master sex-determination gene has been identified,one of the most advanced models for studying sex determination is the Southern platyfish(Xiphophorus maculatus,Günther 1966).Xiphophorus maculatus belongs to the Poeciliids,a family of live-bearing freshwater fish,including platyfish,swordtails and guppies that perfectly illustrates the diversity of genetic sex-determination mechanisms observed in teleosts.For X.maculatus,bacterial artificial chro-mosome contigs covering the sex-determination region of the X and Y sex chromosomes have been constructed.Initial molecular analysis demonstrated that the sex-determination region is very unstable and frequently undergoes duplications,deletions,inversions and other rearrangements.Eleven gene candidates linked to the master sex-determining gene have been identified,some of them corresponding to pseudogenes.All putative genes are present on both the X and the Y chromosomes,suggesting a poor degree of differentiation and a young evolutionary age for platyfish sex chromosomes.When compared with other fish and tetrapod genomes,syntenies were detected only with autosomes.This observation supports an independent origin of sex chromosomes,not only in different vertebrate lineages but also between different fish species.

Sex chromosomes behavior and G-banding treatment of male meiosis in nuptial gift-giving spiders of the family Trechaleidae

Preliminary genetic studies in Trechaleidae spider family show high variation in sex chromosomes and high heterocigocity,suggesting high chromatin plasticity.The trechaleids Paratrechalea ornata,Trechalea bucculenta and Trechaleoides biocellata are present in Uruguay.Males offering nuptial gifts during courtship have been reported in P.ornata and T.bucculenta but not in T.biocellata.Nuptial gifts are an inherited trait probably highly affected by environmental factors,which play an important role in gene expression.We hypothesize that this trait could be associated with tissue-specific genes existing in G-bands.We investigate the male meiosis in these 3 species,their sex chromosome system and the effects of G-banding on their chromosomes,and elucidate genetic differences among them.Meiotic stages of the 3 species were submitted to Giemsa-staining and G-banding treatments.We observed a haploid number of n=11 in P.ornata and n=13 in both T.bucculenta and T.biocellata.Males from the 3 species presented an X_(1)X_(2)0 sex chromosome system,which is suggested as ancestral in Araneae.In P.ornata and T.bucculenta,both sex chromosomes were together and aligned in parallel until the segregation during anaphase I.In contrast to these species,sex chromosomes of T.biocellata usually remained distant from each other until diakinesis when they were observed associated in parallel disposition.Interstitial G-bands were similar in P.ornata and T.bucculenta,and they both differed from those in T.biocellata.The special behavior of sex chromosomes in T.biocellata as well as the different G-banding pattern of this species suggests the existence of novel modifications in this species.

Phylogeny and sex chromosome evolution of Palaeognathae

Many paleognaths(ratites and tinamous) have a pair of homomorphic ZW sex chromosomes in contrast to the highly differentiated sex chromosomes of most other birds. To understand the evolutionary causes for the different tempos of sex chromosome evolution, we produced female genomes of 12 paleognathous species and reconstructed the phylogeny and the evolutionary history of paleognathous sex chromosomes.We uncovered that Palaeognathae sex chromosomes had undergone stepwise recombination suppression and formed a pattern of “evolutionary strata”. Nine of the 15 studied species’ sex chromosomes have maintained homologous recombination in their long pseudoautosomal regions extending more than half of the entire chromosome length. We found that in the older strata, the W chromosome suffered more serious functional gene loss. Their homologous Z-linked regions, compared with other genomic regions, have produced an excess of species-specific autosomal duplicated genes that evolved female-specific expression, in contrast to their broadly expressed progenitors. We speculate such “defeminization” of Z chromosome with underrepresentation of female-biased genes and slow divergence of sex chromosomes of paleognaths might be related to their distinctive mode of sexual selection targeting females rather than males, which evolved in their common ancestors.

Offspring Sex Is Not Determined by Gestation Temperature in a Viviparous Lizard(Eremias multiocellata) from the Desert Steppe of Inner Mongolia

Sex-determining systems show a striking diversity not only among species, but also among popula tions. In reptiles, sex-determina tion is a continuum, from tempera ture-dependent sex determination(TSD) to genetic sex determination(GSD).The multi-ocellated racerunner(Eremias multiocellata)is reported to be a cryptic ZZ/ZW chromosomal TSD species, with male-biased sex ratios at high temperatures in two Gansu populations. However, the generality of the sex-determining pattern in different populations of this species remains unclear. To investigate the mode of sex determina tion in a popula tion of E.multiocellata from the desert steppe of Inner Mongolia,we first identified sex chromosomes via comparative genomic hybridization(CGH). We then conducted a thermal manipulation experiment to determine the effect of gestation temperature on offspring sex ratios.From the CGH studies we found that lizards from the Inner Mongolia population possessed ZZ/ZW sex chromosomes. However, our thermal manipulation experiment showed that gestation temperature did not affect the sex ratio of neonates in this population. In combination, these results rule out TSD in the Inner Mongolia population of E. multiocellata, and suggest that there is widespread geographic variation in the sexdetermining system of this species.

SRY-negative 45,X/46,XY adult male with complete masculinization and infertility:A case report and review of literature

BACKGROUND 45,X/46,XY mosaicism is a rare chromosomal abnormality with a wide range ofphenotypes in both males and females, from normal individuals with differentdegrees of genital ambiguity to those who show signs of Turner’s syndrome.More rarely, cases of 45,X/46,XY mosaicism with a normal-appearing malephenotype are not found until a chromosome test is performed to investigate thecause of male infertility.CASE SUMMARY In this study, a 29-year-old male patient with complete azoospermia is reported.Chromosomal analyses of his lymphocytes revealed the karyotype 45,X[93%]/46,X,+mar(Y)[7%]. In addition, Y chromosome-specific markers, such as SRY,ZFY, AZFa, AZFb and AZFc, were not observed in his blood DNA according tomultiplex polymerase chain reaction test. A literature review identified several45,X/46,XY cases with a normal-appearing male phenotype, most of whom werediagnosed during infertility investigation. However, the present case is the firstSRY-negative 45,X/46,XY male case diagnosed during a premarital medicalexamination.CONCLUSION This finding further suggests that sex determination is a complex processregulated by multiple genetic and environmental factors.

Identification of a male-specific region(MSR)in Spinacia oleracea

Dioecy,the presence of male and female individual,has evolved independently from hermaphroditism in multiple flowering plant lineages.Spinach,an important leafy vegetable crop worldwide,is a dioecious species with an XY sex-determination system.Although some markers that are fully linked to male-determination locus were identified,the male-specific region(MSR)remained unclear.In this research,five male individuals and five female lines were resequenced to identify the male-specific region.We identified a region(∼21 kb)on chromosome 4(putative sex chromosome)where the five females had a low reads coverage,while the five males had high coverage.A KASP marker,SponR,developed from a single nucleotide polymorphism(SNP)closely linked to the MSR was co-segregating with the sex determination gene in the population of 958 individuals,suggesting that the MSR might be specifically present in male spinach plants.

Abnormal Karyotype of Pen Shell (Atrina pectinata) During Its Early Embryonic Development in Late Breeding Season

The pen shell,Atrina pectinata,distributes globally.It is one of the most important edible bivalves in east Asian countries.However,there are multiple difficulties in rearing pen shell larvae and juveniles because of their high mortality.To understand the mechanism underlining such high mortality at the early embryonic development stage,we obtained approximately 100 million larvae during later breeding season in June,2017,and checked a large amount of mitotic chromosomal plates of the early embryos and post-spawning gonads tissue slices of their parents.The results showed that most diploid A.pectinata embryos have 17 pairs of chromosomes(2n=34)as their parents do.The first pair of particularly large chromosomes are heterotypic in some diploid embryos while they are homomorphic in others.The primary sex-determination chromosome type is XX/XY.A lot of triploid,pentaploid and aneuploid embryos with different numbers of the largest homomorphic or heteromorphic chromosomes were found due to the degeneration of overmatured parent gonads which hold normal karyotype.These larvae will die even though most of them may develop into the trochophore stage with 34 chromosomes.Genetic deficiency of chromosomes will cause a high rate of mortality in early embryos in late breeding season.These findings should enrich the current knowledge of juvenile pen shell aquaculture.

Application of dual color fluorescence in situ hybridiza tion (D-FISH) to the diagnosis of a 49, XXXXY chromo somal abnormality

To study the technique of D-FISH and its application in the diagnosis of a 49, XXXXY chromosomal abnormality. Methods: Biotin-labeled alpha satellite X chromosome DNA (pBamX7) probe and digoxi-genin-labeled Y chromosome long arm terminal repetitive sequence (pY3.4) probe in situ hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus. After washing, the slides were treated with avidin-FITC, rhodamine-FITC and anti-avidin, amplified with an additional layer and counter-stained with DAPI in an antifade solution. The hy bridization signals and chromosomal or interphase nucleus settings were observed respectively with WIB, WIG and WU filters under fluorescent microscope (Olympus AX-70) and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted. Results: The biotin-labeled pBamX7 probe showed 4 green hybridization signal and the digoxigenin-labeled pY3.4 probe showed 1 red hybridization signal. The chromosome or cytoplasm counter-stained with DAPI showed blue. The positive rate of X chromosome hybridization signal for the 350 metaphase chromosomes and interphase nucleus was 91.43 % and 92. 57 %, respectively, while that of the Y chromosome hybridization signal was 99.5 % and 99.8 %, respectively. Conclusion: D-FISH is a valuable technique in diagnosing 49, XXXXY chromosomal abnormality and other sex chromosomal abnormalities. [Reprod Contracep (in Chinese) 2002; 22: 287]

Sex determination mechanisms and sex control approaches in aquaculture animals

Aquaculture is one of the most efficient modes of animal protein production and plays an important role in global food security.Aquaculture animals exhibit extraordinarily diverse sexual phenotypes and underlying mechanisms,providing an ideal system to perform sex determination research,one of the important areas in life science.Moreover,sex is also one of the most valuable traits because sexual dimorphism in growth,size,and other economic characteristics commonly exist in aquaculture animals.Here,we synthesize current knowledge of sex determination mechanisms,sex chromosome evolution,reproduction strategies,and sexual dimorphism,and also review several approaches for sex control in aquaculture animals,including artificial gynogenesis,application of sex-specific or sex chromosome-linked markers,artificial sex reversal,as well as gene editing.We anticipate that better understanding of sex determination mechanisms and innovation of sex control approaches will facilitate sustainable development of aquaculture.

Regain of sex determination system and sexual reproduction ability in a synthetic octoploid male fish

Polyploids in vertebrates are generally associated with unisexual reproduction,but the direct consequences of polyploidy on sex determination system and reproduction mode remain unknown.Here,we synthesized a group of artificial octoploids between unisexual gynogenetic hexaploid Carassius gibelio and sexual tetraploid Carassius auratus.The synthetic octoploids were revealed to have more than 200 chromosomes,in which 50 chromosomes including the X/Y sex determination system were identified to transfer from sexual tetraploid C.auratus into the unisexual gynogenetic hexaploid C.gibelio.Significantly,a few synthetic octoploid males were found to be fertile,and one octoploid male was confirmed to regain sexual reproduction ability,which exhibits characteristics that are the same to sexual reproduction tetraploid males,such as 1:1 sex ratio occurrence,meiosis completion and euploid sperm formation in spermatogenesis,as well as normal embryo development and gene expression pattern during embryogenesis.Therefore,the current finding provides a unique case to explore the effect of sex determination system incorporation on reproduction mode transition from unisexual gynogenesis to sexual reproduction along with genome synthesis of recurrent polyploidy in vertebrates.

Synteny search identifies carnivore Y chromosome for evolution of male specific genes

The explosive accumulation of mammalian genomes has provided a valuable resource to characterize the evo­lution of the Y chromosome.Unexpectedly,the Y-chromosome sequence has been characterized in only a small handful of species,with the majority being model organisms.Thus,identification of Y-linked scaffolds from un­ordered genome sequences is becoming more important.Here,we used a syntenic-based approach to generate the scaffolds of the male-specific region of the Y chromosome(MSY)from the genome sequence of 6 male car­nivore species.Our results identified 14,15,9,28,14 and 11 Y-linked scaffolds in polar bears,pacific walruses,red pandas,cheetahs,ferrets and tigers,covering 1.55 Mbp,2.62 Mbp,964 Kb,1.75 Mb,2.17 Mbp and 1.84 Mb MSY,respectively.All the candidate Y-linked scaffolds in 3 selected species(red pandas,polar bears and ti­gers)were successfully verified using polymerase chain reaction.We re-annotated 8 carnivore MSYs including these 6 Y-linked scaffolds and domestic dog and cat MSY;a total of 11 orthologous genes conserved in at least 7 of the 8 carnivores were identified.These 11 Y-linked genes have significantly higher evolutionary rates com­pared with their X-linked counterparts,indicating less purifying selection for MSY genes.Taken together,our study shows that the approach of synteny search is a reliable and easily affordable strategy to identify Y-linked scaffolds from unordered carnivore genomes and provides a preliminary evolutionary study for carnivore MSY genes.

Repetitive DNA Sequences from the X Chromosome of the Spiny Eel (Mastacembelus aculeatus)

To investigate the characters of repetitive DNA sequence in the sex chromosomes of the spiny eel （Mastacembelus aculeatus）, the X chromosomal library was screened and a family of repetitive sequence, consisting of Ma 1-Ma 6, was isolated. The fluorescence in situ hybridization （FISH） result confirmed that Ma 1- Ma 5 dispersed over sex chromosomes and all autosomes, whereas, Ma 6 is sex chromosome-specific and distributed only on the C-band positive regions of X chromosome, and Ma 6 maybe the main components of the heterochromatic regions of X chromosome. This study provides additional information about the evolution of sex chromosomes in lower vertebrates such as fish.