The sex-determining region of theMegaselia scalaris (Diptera) Y chromosome

InMegaselia scalaris (Loew) the presence or absence of a male-determining factor, M, is responsible for sex determination. In two wild-type strains, M is located on the homomorphic chromosome pair 2. In the laboratory line Except42 a new Y chromosome was created by recombination between the original Y and the original X chromosome. The Except42 Y chromosome has conserved the sex-determining function and four molecular markers of the original Y chromosome, while 13 original Y markers have been lost. The new Y chromosome, therefore, consists of roughly one-quarter of the original Y chromosome and three-quarters of the original X chromosome. To define the sex-determining region, cosmid clones, one from the original X and one from the original Y chromosome region of the Except42 Y chromosome, were isolated and used as probes for chromosomalin situ suppression (CISS) hybridization. The CISS hybridization signals map the conserved Y segment, including the male-determining factor, to the distal segment of the short arm of the Y chromosome.     

Cytogenetic studies of Hynobiidae (Urodela) XIX. Morphological variation of sex chromosomes pairing behavior of sex lampbrush chromosomes in <Emphasis Type="Italic">Hynobius quelpaertensis</Emphasis> (Mori) from Cheju Island,South Korea

Using Giemsa staining, C-banding and Ag-NOR staining techniques, we analyzed chromosomes in adult male and female Hynobius quelpaertensis and in embryos of this species in egg sacs collected from eight localities of Cheju Island, South Korea. Chromosome pair 21 was consistently homomorphic in male specimens, while it was heteromorphic in female specimens, suggesting the occurrence of ZZ/ZW sex chromosome constitution in this species. The W chromosome, being much larger than the Z chromosome, was of three morphologically distinct types: W^A, W^B and W^C. Lampbrush chromosomes examined in the oocytes of one female specimen having the W^A chromosome showed that the short arm of the W^A chromosome and the long arm of the Z chromosome paired closely and hence are genetically homologous. We also tried to analyze the structural relationship among the three types of W chromosomes based on their C-banding and Ag-NOR patterns.     

An XX/XY sex microchromosome system in a freshwater turtle, Chelodina longicollis (Testudines: Chelidae) with genetic sex determination

Heteromorphic sex chromosomes are rare in turtles, having been described in only four species. Like many turtle species, the Australian freshwater turtle Chelodina longicollis has genetic sex determination, but no distinguishable (heteromorphic) sex chromosomes were identified in a previous karyotyping study. We used comparative genomic hybridization (CGH) to show that C. longicollis has an XX/XY system of chromosomal sex determination, involving a pair of microchromosomes. C-banding and reverse fluorescent staining also distinguished microchromosomes with different banding patterns in males and females in ∼70% cells examined. GTG-banding did not reveal any heteromorphic chromosomes, and no replication asynchrony on the X or Y microchromosomes was observed using replication banding. We conclude that there is a very small sequence difference between X and Y chromosomes in this species, a difference that is consistently detectable only by high-resolution molecular cytogenetic techniques, such as CGH. This is the first time a pair of microchromosomes has been identified as the sex chromosomes in a turtle species.     

Evolution of multiple sex chromosomes in the spider genus <Emphasis Type="Italic">Malthonica</Emphasis> (Araneae: Agelenidae) indicates unique structure of the spider sex chromosome systems

Most spiders exhibit a multiple sex chromosome system, X₁X₂0, whose origin has not been satisfactorily explained. Examination of the sex chromosome systems in the spider genus Malthonica (Agelenidae) revealed considerable diversity in sex chromosome constitution within this group. Besides modes X₁X₂0 (M. silvestris) and X₁X₂X₃0 (M. campestris), a neo-X₁X₂X₃X₄X₅Y system in M. ferruginea was found. Ultrastructural analysis of spread pachytene spermatocytes revealed that the X₁X₂0 and X₁X₂X₃0 systems include a pair of homomorphic sex chromosomes. Multiple X chromosomes and the pair exhibit an end-to-end pairing, being connected by attachment plaques. The X₁X₂X₃X₄X₅Y system of M. ferruginea arose by rearrangement between the homomorphic sex chromosome pair and an autosome. Multiple X chromosomes and the sex chromosome pair do not differ from autosomes in a pattern of constitutive heterochromatin. Ultrastructural data on sex chromosome pairing in other spiders indicate that the homomorphic sex chromosome pair forms an integral part of the spider sex chromosome systems. It is suggested that this pair represents ancestral sex chromosomes of spiders, which generated multiple X chromosomes by non-disjunctions. Structural differentiation of newly formed X chromosomes has been facilitated by heterochromatinization of sex chromosome bivalents observed in prophase I of spider females.     

Additional locus of rDNA sequence specific to the X chromosome of the liverwort, Marchantia polymorpha

The molecular cytogenetic organization of 17S ribosomal RNA genes (17S rDNA), a part of the 45S rDNA repeat, was investigated on the chromosomes of the liverwort Marchantia polymorpha using fluorescence in-situ hybridization (FISH). The numbers of 17S rDNA loci visualized in female and male chromosomes were ten and nine, respectively. This heterogeneous localization was due to the presence of an additional 17S rDNA locus on the X chromosome and its absence on the Y chromosome. The signal on the X chromosome covered almost the entire region of its long arm. The other nine signals were observed on the same loci of respective autosomes in both sexes. Southern hybridization analysis revealed an additional band including 17S rDNA exclusively on EcoRI digested female genomic DNA supporting the existence of an additional 17S rDNA locus on the X chromosome.     

Microdissection of the Y chromosome and fluorescencein situ hybridization analysis of the sex chromosomes of lake trout,Salvelinus namaycush

Lake trout,Salvelinus namaycush, is one of the few salmonids with morphologically differentiated sex chromosomes. Genetic analysis suggested that the sex-determining region of this species lies on the short arm of the Y chromosome. The differential arm of the Y chromosome was microdissected and the resulting DNA amplified in a sequence-independent manner. Amplified DNA was biotin labeled as a probe for fluorescencein situ hybridization (FISH). Strong hybridization signals were seen covering defined regions of both the Y and X chromosomes. Homeologous chromosomes of the ancestrally tetraploid genome were not identified by FISH with the Y probe, indicating diploidization of this region of the genome.     

An XX/XY heteromorphic sex chromosome system in the Australian chelid turtle <Emphasis Type="Italic">Emydura macquarii</Emphasis>: A new piece in the puzzle of sex chromosome evolution in turtles

Chromosomal sex determination is the prevalent system found in animals but is rare among turtles. In fact, heteromorphic sex chromosomes are known in only seven of the turtles possessing genotypic sex determination (GSD), two of which correspond to cryptic sex microchromosomes detectable only with high-resolution cytogenetic techniques. Sex chromosomes were undetected in previous studies of Emydura macquarii, a GSD side-necked turtle. Using comparative genomic hybridization (CGH) and GTG-banding, a heteromorphic XX/XY sex chromosome system was detected in E. macquarii. The Y chromosome appears submetacentric and somewhat larger than the metacentric X, the first such report for turtles. CGH revealed a male-specific chromosomal region, which appeared heteromorphic using GTG-banding, and was restricted to the telomeric region of the p arm. Based on our observations and the current phylogeny of chelid turtles, we hypothesize that the sex chromosomes of E. macquarii might be the result of a translocation of an ancestral Y microchromosome as found in a turtle belonging to a sister clade, Chelodina longicollis, onto the tip of an autosome. However, in the absence of data from an outgroup, the opposite (fission of a large XY into an autosome and a micro-XY) is theoretically equally likely. Alternatively, the sex chromosome systems of E. macquarii and C. longicollis may have evolved independently. We discuss the potential causes and consequences of such putative chromosome rearrangements in the evolution of sex chromosomes and sex-determining systems of turtles in general.     

Molecular characterization and cytological mapping of a non-repetitive DNA sequence region from the W chromosome of chicken and its use as a universal probe for sexing Carinatae birds

A non-repetitive genomic DNA region of about 25 kb was cloned from the W chromosome of chicken using a genomic library prepared from a single W chromosome of the chicken. This region was mapped by fluorescence in situ hybridization (FISH) with mitotic and lampbrush chromosomes to a position between the major EcoRI family and the pericentromeric XhoI family on the W chromosome. A 0.6-kb EcoRI fragment(EE0.6) subcloned from this region consists of a sequence that can be obtained by the exon-trapping procedure and flanking sequences. Sequences, which are closely similar to that of EE0.6, are widely conserved on the W chromosomes of Carinatae birds, as revealed by Southern blot hybridization to HindIII-digested female and male genomic DNAs from 18 species of birds belonging to eight different taxonomic orders. The female sex of those birds can be determined by the presence of an unambiguous female-specific band. For many species of birds, the female sex can also be determined by polymerase chain reaction (PCR) using a set of primers from the flanking sequences in the chicken EE0.6.This revised version was published online in November 2005 with corrections to the Cover Date.     

Heterochromatin Discrimination in Aegilops Speltoides by Simultaneous Genomic In Situ Hybridization

Simultaneous genomic in situ hybridization with probe preannealing (SP-GISH) was used for discriminating Aegilops speltoides chromosome regions by their relatedness to DNA of other species. We used a hybridization mixture of two differently labelled DNAs, one from the species used for chromosome spread preparations and a second from species of different and varying affinity, thus creating a two-colour system showing chromosome regions where alien DNA hybridized. Genomic DNA from A. speltoides was labelled with biotin and preannealed with digoxigenin-labelled total genomic DNA from different accessions of Ae. speltoides, Ae. bicornis, Ae. tauschii and Hordeum spontaneum. The probe mixture was hybridized to mitotic chromosmes of Ae. speltoides. Chromosome regions of preferential hybridization of self-DNA were visualized as green, whereas regions of combined hybridization showed orange–yellow fluorescence. We observed GISH banding patterns with a different degree of green fluorescence along Ae. speltoides chromosomes that directly correlated with evolutionary distance. Small green bands were observed in subtelomeric and telomeric heterochromatic regions using DNA of a different accession of Ae. speltoides, whereas when using DNA of H. spontaneum most regions of the chromosomes, except pericentromeric regions, showed mainly green fluorescence. The resolution and application of the approach to the study of heterochromatin differentiation are discussed.     

A ZZ/ZW microchromosome system in the spiny softshell turtle, Apalone spinifera, reveals an intriguing sex chromosome conservation in Trionychidae

Reptiles display a wide diversity of sex-determining mechanisms ranging from temperature-dependent sex determination (TSD) to genotypic sex determination (GSD) with either male (XY) or female (ZW) heterogamety. Despite this astounding variability, the origin, structure, and evolution of sex chromosomes remain poorly understood. In turtles, TSD is purportedly ancestral while GSD arose multiple times independently. Here we test whether independent (XY or ZW) or morphologically divergent heterogametic sex chromosome systems evolved in tryonichids (Cryptodira) using the GSD spiny softshell turtle, Apalone spinifera, a species with previously unidentified sex chromosomes. A female-specific signal from comparative genomic hybridization (CGH) was detected in a Giemsa/4′,6-diamidino-2-phenylindole faint portion of a microchromosome, indicating the presence of a ZZ/ZW system in A. spinifera. In situ hybridization of a fluorescently labeled 18S rRNA probe identified a large nucleolar organizer region block in the female-specific region of the W (co-localizing with the female-specific CGH signal) and a smaller block on the Z. The heteromorphic ZZ/ZW micro-sex chromosome system detected here is identical to that found in another tryonichid, the Chinese softshell turtle Pelodiscus sinensis, from which A. spinifera diverged ～95 million years ago. These results reveal a striking sex chromosome conservation in tryonichids, compared to the divergent sex chromosome morphology observed among younger XX/XY systems in pleurodiran turtles. Our findings highlight the need to understand the drivers behind sex chromosome lability and conservation in different lineages and contribute to our knowledge of sex chromosome evolution in reptiles and vertebrates.     

Male-biased distribution of the human Y chromosomal genes SRY and ZFY in the lizard Calotes versicolor, which lacks sex chromosomes and temperature-dependent sex determination

In the present investigation on the lizard Calotes versicolor, which lacks temperature-dependent sex determination, all the conventional cytological techniques used failed to resolve a distinguishable pair of sex chromosomes. However, probing of the genome with the human Y-linked genes SRY and ZFY showed sex-specific bias in their distribution. While the SRY probe hybridized to all the males, more than half of the females examined did not show any hybridization. ZFY hybridized to both the sexes, giving two bands; one was common to all the individuals of both sexes, but the other, of the lower molecular length, occurred in all the males but in less than 50% of females. This predominantly male-specific band is named AMF. The SRY-positive females were also positive for the AMF of ZFY. As positive as well as negative females were fertile and none of the males lacked SRY, it appears that SRY is essential for males only and that both the genes are syntenic in this species. This report raises interesting possibilities on the differentiation of the sex chromosomes in C. versicolor and evolution of SRY/ZFY on the Y chromosome of eutherian mammals through the ancestral group(s) that harbour sex-independent SRY- and ZFY-related genes.This revised version was published online in November 2005 with corrections to the Cover Date.     

Molecular cytogenetics of introgressive hybridization in plants

Introgressive hybridization (introgression) is genetic modification of one species by another through hybridization and repeated backcrossing. Introgression is important in the evolution of flowering plants. It is also important in plant breeding where a desirable trait can be transferred from wild to crop species. One of the most recent advances in molecular techniques for studying hybridization and introgression is in situ hybridization of genomic probes to cytological preparations (GISH, genomic in situ hybridization). The present paper describes a successful GISH protocol for detection of intergenomic introgression in breeding materials and in allopolyploid species. In addition, the paper introduces a new possibility of using dispersed repeats to detect introgression and to gain insights into its molecular basis. The approach is referred to as dFISH for dispersed fluorescence in situ hybridization, and the best candidate for this type of probes is probably a retroelement. Southern hybridization data are also presented to support the effectiveness of GISH and dFISH for introgression mapping.     

Very low rate Y-chromosome mosaicism (1∶5,400) detectable by a novel probe enzyme combination

DYZ1 is a repetitive DNA family located on the long arm of the Y chromosome and is the major component of the Q-positive region. DYZ1 consists of about 3,000 copies of a 3.4 kb repeat unit which mainly consists of a tandem array of pentanucleotides, TTCCA. Because of this large number of repeats, DYZ1 has been used as a probe in Southern hybridization for sensitive and rapid detection of the Y chromosome. In cases of XX/XY mosaicism, however, autosomal sequences having homology to DYZ1 hinder the detection of the Y chromosome, especially when the ratio of the Y-bearing cells is low. To solve this problem and improve the detection limit, we have sought the optimum hybridization condition by changing several variables. These variables include the length of probes, the methods of probe labeling, the endonucleases used to digest the genomic DNA and the hybridization buffer. Here we show that theStuI digestion of genomic DNA in combination with the nick translated DYZ1 probe significantly improves the detection limit of the Y-chromosome bearing cells. The presence of Y-chromosome bearing cells was detectable against a background of 5,400-fold female DNA.     

Meiotic chromosomes and stages of sex chromosome evolution in fish: zebrafish, platyfish and guppy

We describe SC complements and results from comparative genomic hybridization (CGH) on mitotic and meiotic chromosomes of the zebrafish Danio rerio, the platyfish Xiphophorus maculatus and the guppy Poecilia reticulata. The three fish species represent basic steps of sex chromosome differentiation: (1) the zebrafish with an all-autosome karyotype; (2) the platyfish with genetically defined sex chromosomes but no differentiation between X and Y visible in the SC or with CGH in meiotic and mitotic chromosomes; (3) the guppy with genetically and cytogenetically differentiated sex chromosomes. The acrocentric Y chromosomes of the guppy consists of a proximal homologous and a distal differential segment. The proximal segment pairs in early pachytene with the respective X chromosome segment. The differential segment is unpaired in early pachytene but synapses later in an ‘adjustment’ or ‘equalization’ process. The segment includes a postulated sex determining region and a conspicuous variable heterochromatic region whose structure depends on the particular Y chromosome line. CGH differentiates a large block of predominantly male-specific repetitive DNA and a block of common repetitive DNA in that region. This revised version was published online in July 2006 with corrections to the Cover Date.     

An XX/XY Sex Chromosome System in a Fish Species, Hoplias malabaricus, with a Polymorphic NOR-Bearing X Chromosome

Cytogenetic studies were carried out in the fish, Hoplias malabaricus, from the Parque Florestal do Rio Doce (Brazil). This population is characterized by 2n = 42 chromosomes for both males and females and an XX/XY sex chromosome system, confirmed through several banding methods. Females show 24 metacentric, 16 submetacentric and 2 subtelocentric chromosomes. Males show 24 metacentric, 17 submetacentric and 1 subtelocentric chromosomes. While the X chromosome is easily recognized (the only subtelocentric element), the Y chromosome is somewhat difficult to identify but appears to correspond to the smallest submetacentric in the male karyotype. In-situ hybridization with an 18S rDNA probe showed 10 well-labeled chromosomes, including the X chromosome. The 5S rDNA is interstitially located in a single metacentric pair independent of the 18S rDNA sites. The NOR on the X chromosome is always active and occurs adjacent to a heterochromatic distal segment on the long arm. Variations in size of the NORs and/or heterochromatic segment correspond to a polymorphic size condition observed in the X chromosome. The present results confirm the XX/XY sex chromosome system in the population analyzed as well as a new cytotype in the Hoplias malabaricus group.     

Localization and Characterization of Nucleotide Sequences From the Canine Y Chromosome

We previously reported the identification of a male-specific 658-bp DNA sequence in dogs. We used a specific primer pair designed for PCR amplification of this fragment with DNA samples from 238 dogs, 6 dingoes and 12 wolves. All 133 male samples amplified the 658-bp sequence, whereas all female samples did not. The sequence was not amplified from male DNA samples representing other wild canids (jackals, coyotes, foxes). A lambda phage was isolated from a canine male genomic library that contained an insert of approximately 15 kb of canine genomic DNA, including the male- specific 658-bp sequence. This lambda phage was used in fluorescence in-situ hybridization experiments. It hybridized to the canine Y chromosome together with a lambda clone containing a segment of the SRY gene and a cosmid clone containing a portion of the pseudoautosomal region. The male-specific 658-bp sequence was located at the end opposite to the pseudoautosomal region while the SRY gene sequence hybridized near the centromere. Additionally, two (CA)-repeat sequences were identified in the lambda clone that contained the 658-bp sequence. Specific primer pairs were designed to amplify each of the repeats. Primer pair MS34 amplified three different alleles from 13 unrelated canine male DNA samples with a PIC value of 0.40. Primer pair MS41 amplified five alleles with a PIC value of 0.71. These microsatellites are the first reported polymorphic sequences in the dog located in the non-recombining portion of the Y chromosome. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparative mapping ofSRY in the great apes

Cytogenetic studies of the primate Y chromosomes have suggested that extensive rearrangements have occurred during evolution of the great apes. We have usedin situ hybridization to define these rearrangements at the molecular level.pHU-14, a probe including sequences from the sex determining geneSRY, hybridizes close to the early replicating pseudoautosomal segment in a telomeric or subtelomeric position of the Y chromosomes of all great apes. The low copy repeat detected by the probeFr35-II is obviously included in Y chromosomal rearrangements during hominid evolution. These results, combined with previous studies, suggest that the Y chromosome in great apes has a conserved region including the pseudoautosomal region and the testis-determining region. The rest of the Y chromosome has undergone several rearrangements in the different great apes.     

Cytogenetic analyses of chromosomal rearrangements in Mus minutoides/musculoides from North-West Zambia through mapping of the telomeric sequence (TTAGGG)n and banding techniques

Three specimens of M. minutoides/musculoides from Zambia were cytogenetically studied through G- and C-banding, DAPI staining and fluorescence in-situ hybridization (FISH) with a (TTAGGG)_n telomeric sequence. Biarmed chromosomes were identified according to the current nomenclature as follows: Rb(2.7), Rb(3.12), Rb(4.5), Rb(6.8), Rb(9.16), and the sex chromosomes Rb(1.X), Rb(1.Y) and Rb(1.X_d), originated from the deleted X chromosome. One female showed the diploid number 2n=24; in the two other individuals, the Rb(9.16) occurred in a heteromorphic condition, and, accordingly, the diploid number was 2n=25. FISH showed the sites of telomeric sequences at telomeres of all the chromosomes, and in an interstitial position at the centromeres of all Robertsonian metacentrics, except one – the Rb(6.8), though the patterns of hybridization varied between chromosomes. Sex chromosome pairs, in the male and females, showed a similar C-banding pattern, but revealed clear differences after FISH. Traces of telomeric sequences were found dispersed in the whole-heterochromatic arm of the Rb(1.X_d). No visible bond between C-positive heterochromatin and telomeric sequences were detected in the other either bi- or uniarmed chromosomes, indicating that they may actually represent retained telomeres in the Robertsonian metacentrics. This revised version was published online in July 2006 with corrections to the Cover Date.     

Meiosis in chromosomally heteromorphic goitered gazelle,Gazella subgutturosa (Artiodactyla,Bovidae)

Chromosomal-pairing behaviour was studied in the spermatocytes of individual goitered gazelles which were heteromorphic for a 14/15 Robertsonian translocation and which possessed an autosome-to-X translocation. Both translocations exhibited trivalent pairing configurations in pachytene and diakinesis/metaphase I nuclei. Synapsis of the sex chromosomes during pachynema was followed by end-to-end association of the X and Y during diakinesis/metaphase I. The only univalents identified were of the Y chromosome; Y univalency ranged from 15.9% at pachynema to 5.7% at diakinesis/metaphase I. Robertsonian trivalents exhited evidence of synaptic adjustment in the paracentromeric region. Chiasmata were formed in most bivalents and trivalents; chiasmata were restricted to the autosomal portion of the autosometo-XY trivalent. Analysis of metaphse II configurations (secondary spermatocytes) revealed no nondisjunction in individuals homozygous or heterozygous for the Robertsonian translocation. These data are consistent with the hypothesis that neither the autosomal nor the gonosomal heteromorphism reduces the meiotic fitness of male goitered gazelles.     

8例性发育异常患者SRY基因分析

目的对8例性发育异常患者进行细胞遗传学及分子遗传学检查以探讨性别发育异常与SRY基因关系.方法用PY3.4,X着丝粒,SRY特异探针进行荧光原位杂交,用于分析性发育异常病人Y染色体及SRY基因异位情况.聚合酶链反应(PCR)扩增SRY基因,直接测序检测SRY基因突变.结果 2例46,XX男性,1例46,XY女性,1例45,X/46,XY嵌合体及1例46,X,t(Y;Y)(p11;q11)男性患者SRY基因均为阳性,直接测序未发现SRY基因阳性患者该基因突变.剩余1例46,XX男性,1例46,XY男性及1例46,XY女性患者SRY基因为阴性.FISH技术证实2例46,XX且SRY基因阳性的男性患者SRY基因易位至X染色体短臂末端.结论 SRY基因是人类性别决定的主导基因,但尚有其他基因参与性别分化.