Single‐copy gene‐based chromosome painting in cucumber and its application for chromosome rearrangement analysis in Cucumis

Chromosome painting based on fluorescence in situ hybridization (FISH) has played an important role in chromosome identification and research into chromosome rearrangements, diagnosis of chromosome abnormalities and evolution in human and animal species. However, it has not been applied widely in plants due to the large amounts of dispersed repetitive sequences in chromosomes. In the present work, a chromosome painting method for single‐copy gene pools in Cucumis sativus was successfully developed. Gene probes with sizes above 2 kb were detected consistently. A cucumber karyotype was constructed based on FISH using a cocktail containing chromosome‐specific gene probes. This single‐copy gene‐based chromosome painting (ScgCP) technique was performed by PCR amplification, purification, pooling, labeling and hybridization onto chromosome spreads. Gene pools containing sequential genes with an interval less than 300 kb yielded painting patterns on pachytene chromosomes. Seven gene pools corresponding to individual chromosomes unambiguously painted each chromosome pair of C. sativus. Three mis‐aligned regions on chromosome 4 were identified by the painting patterns. A probe pool comprising 133 genes covering the 8 Mb distal end of chromosome 4 was used to evaluate the potential utility of the ScgCP technique for chromosome rearrangement research through cross‐species FISH in the Cucumis genus. Distinct painting patterns of this region were observed in C. sativus, C. melo and C. metuliferus species. A comparative chromosome map of this region was constructed between cucumber and melon. With increasing sequence resources, this ScgCP technique may be applied on any other sequenced species for chromosome painting research.     

In situ hybridization (FISH) maps chromosomal homologies between Alouatta belzebul (Platyrrhini,Cebidae) and other primates and reveals extensive interchromosomal rearrangements between howler monkey genomes

We hybridized whole human chromosome specific probes to metaphases of the black-and-red howler monkey Alouatta belzebul in order to establish chromosomal homology between humans and black-and-red howlers. The results show that the black-and-red howler monkey has a highly rearranged genome and that the human chromosome homologs are often fragmented and translocated. The number of hybridization signals we obtained per haploid set was 40. Nine human chromosome probes gave multiple signals on different howler chromosomes, showing that their synteny is disturbed in A. Belzebul. Fourteen black-and-red howler autosomes were completely hybridized by one human autosomal paint, six had two signals, three had three signals, and one chromosome had four signals. Howler chromosomes with multiple signals have produced 12 chromosomal syntenies or hybridization associations which differ from those found in humans: 1/2, 2/20, 3/21, 4/15, 4/16, 5/7, 5/11, 8/18, 9/12, 10/16, 14/15, and 15/22. The hybridization pattern was then compared with those found in two red howler taxa and other mammals. The comparison shows that even within the genus Alouatta numerous interchromosomal rearrangements differentiate each taxa: A. belzebul has six unique apomorphic associations, A. seniculus sara and A. seniculus arctoidea share seven derived associations, and additionally A. seniculus sara has four apomorphic associations and A. seniculus arctoideaseven apomorphic associations. A. belzebul appears to have a more conserved karyotype than the red howlers. Both red and black-and-red howlers are characterized by Y-autosome translocations; the peculiar chromosomal sex system found in the red howler taxa could be considered a further transformation of the A. belzebul sex system. The finding that apparently morphologically similar or even identical taxa have such extreme genomic differences has important implications for speciation theory and neotropical primate conservation. Am. J. Primatol. 46:119–133, 1998. © 1998 Wiley-Liss, Inc.     

Chromosome painting in two genera of South American monkeys: species identification, conservation, and management

Cytogenetic studies showed that a number of New World primate taxa, particularly the genera Alouatta, Aotus, and Callicebus, have highly derived karyotypes. Cytogenetics in these primates, at every level of analysis, has contributed to the recognition of species and revealed that their number was certainly underestimated by researchers relying solely on traditional morphological data. Further attention was drawn to Alouatta and Aotus because they are characterized by translocations of the Y chromosome to autosomes, generating multiple sex chromosome systems. Here we present a report on the hybridization of human chromosome-specific paints on metaphases from 4 individuals originally assigned to Alouatta caraya and 1 individual of Aotuslemurinus. This is only the third karyotype studied with chromosome painting out of more than 10 known karyomorphs in Aotus. The banded chromosomes matched those of karyotype II as defined by Ma et al. [1976a], and we were able to more precisely assign the origin of the sample to A. l. griseimembra. Our results on the Argentinean Alouatta caraya samples were generally comparable to the banding and hybridization pattern of previous studies of A. caraya including the presence of an X(1)X(1)X(2)X(2)/X(1)X(2)Y(1)Y(2) sex chromosome system. The karyotype of the Brazilian Alouatta sample labeled as A. caraya differs from the 3 Argentinean samples by at least 10 chromosome rearrangements. The diploid number, G banding, and hybridization pattern of this female cell line was almost identical to previous painting results on Alouatta guariba guariba. Therefore we must conclude that this cell line is actually from an A. guariba guariba individual. The contribution of cytogenetic tools in identifying species or in this case assigning individuals or cell lines to their precise taxonomic allocation is stressed. Gathering further molecular cytogenetic data on New World primates should be conservation and management priorities.     

Heterochromatin and chromosome evolution: a FISH probe of Cebus apella paraguayanus (Primate: Platyrrhini) developed by chromosome microdissection

Neotropical Primate karyotypes are highly variable, particularly in the heterochromatic regions, not only regarding the amount of heterochromatin, but also the composition. G and C banding and FISH techniques provide useful information to characterize interspecific relationships. We used chromosome microdissection to develop a FISH probe of the chromosome 11 heterochromatic block (11qHe+) of Cebus apella paraguayanus (CAPp). Fragments of the 11qHe+ microdissected from fibroblast cell culture were collected in a PCR tube, amplified by degenerate oligonucleotide primer-PCR and subsequently labeled. The specificity of the FISH probe was confirmed in metaphases of some Ceboidea species. Signals were located in the He+ of chromosomes 4, 11, 12, 13, and 19 of CAPp and in the He+ of chromosomes 4, 12 and 13 of C. a. nigritus (CAPn); no signals were observed when other Ceboidea species were analyzed. We propose that the heterochromatin observed in CAPp and CAPn is specific for these species. We consider this C. apella heterochromatin identity as a possible key for the interpretation of chromosomal evolution in these Ceboidea.     

Morphological analysis ofAlouatta seniculus species group (Primates,Cebidae). A comparison with biochemical and karyological data

Fifteen cranial measurements were taken from wild caught specimens ofAlouatta seniculus seniculus, A. s. stramineus andA. s. macconnelli. A morphological analysis showed sex dimorphism in these three groups. A multivariate analysis discriminated among these taxa; males being more clearly discriminated than females. Our data showed that these taxa can be separated on the basis of quantitative cranial traits, biogeographic distribution, karyological differences, and biochemical characters. We therefore propose a new taxonomic arrangement, changing their taxonomic status to the species level (Alouatta seniculus, A. macconnelli, andA. stramineus).     

Cytogenetic mapping of the W chromosome in the genus Leporinus (Teleostei, Anostomidae) using a highly repetitive DNA sequence

The distribution of the Leporinus elongatus LeSpeI repetitive sequence in other Leporinus species was studied in an attempt to elucidate the evolutionary history of sex chromosomes in this genus using chromosome fluorescence in situ hybridization. The presence of fluorescent signals only in species that have differentiated sex chromosomes suggests that this sequence is related to the differentiation of sex chromosomes in this genus. Thus, these data will contribute to a better understanding of chromosome evolution, especially for sex chromosomes, in the Leporinus genus.     

A Novel ZZ/ZW Sex Chromosome System for the Genus Leporinus (Pisces, Anostomidae, Characiformes)

A wide range of sex chromosome mechanisms, including simple and multiple chromosome systems is characteristic of fishes. The Leporinus genus represent a good model to study sex chromosome mechanisms, because an unambiguous ZZ/ZW sex chromosome system was previously described for seven species, while the remaining studied species of the genus do not show differentiated sex chromosomes. The occurrence of sex chromosomes in Leporinus trifasciatus and Leporinus sp2 from the Araguaia river, Amazon basin, Brazil, was here investigated. ZZ/ZW sex chromosomes were detected for both species. The Z and W chromosome morphology of L. trifasciatus is the same as described for other species of the genus Leporinus. However, the Z and W chromosomes of L. sp2 were quite different in their morphology and banding pattern suggesting that the ZW system of this species have originated independently from the ZW system previously described for other Leporinus.     

The master sex-determination locus in threespine sticklebacks is on a nascent Y chromosome

BACKGROUND: Many different environmental and genetic sex-determination mechanisms are found in nature. Closely related species can use different master sex-determination switches, suggesting that these developmental pathways can evolve very rapidly. Previous cytological studies suggest that recently diverged species of stickleback fish have different sex chromosome complements. Here, we investigate the genetic and chromosomal mechanisms that underlie sex determination in the threespine stickleback (Gasterosteus aculeatus). RESULTS: Genome-wide linkage mapping identifies a single chromosome region at the distal end of linkage group (LG) 19, which controls male or female sexual development in threespine sticklebacks. Although sex chromosomes are not cytogenetically visible in this species, several lines of evidence suggest that LG 19 is an evolving sex chromosome system, similar to the XX female/XY male system in many other species: (1) males are consistently heterozygous for unique alleles in this region; (2) recombination between loci linked to the sex-determination region is reduced in male meiosis relative to female meiosis; (3) sequence analysis of X- and Y-specific bacterial artificial chromosome (BAC) clones from the sex-determination region reveals many sequence differences between the X- and Y-specific clones; and (4) the Y chromosome has accumulated transposable elements and local duplications. CONCLUSIONS: Taken together, our data suggest that threespine sticklebacks have a simple chromosomal mechanism for sex determination based on a nascent Y chromosome that is less than 10 million years old. Further analysis of the stickleback system will provide an exciting window into the evolution of sex-determination pathways and sex chromosomes in vertebrates.     

Insights into the meiotic behavior and evolution of multiple sex chromosome systems in spiders

A characteristic feature of spider karyotypes is the predominance of unusual multiple X chromosomes. To elucidate the evolution of spider sex chromosomes, their meiotic behavior was analyzed in 2 major clades of opisthothele spiders, namely, the entelegyne araneomorphs and the mygalomorphs. Our data support the predominance of X(1)X(2)0 systems in entelegynes, while rare X(1)X(2)X(3)X(4)0 systems were revealed in the tuberculote mygalomorphs. The spider species studied exhibited a considerable diversity of achiasmate sex chromosome pairing in male meiosis. The end-to-end pairing of sex chromosomes found in mygalomorphs was gradually replaced by the parallel attachment of sex chromosomes in entelegynes. The observed association of male X univalents with a centrosome at the first meiotic division may ensure the univalents' segregation. Spider meiotic sex chromosomes also showed other unique traits, namely, association with a chromosome pair in males and inactivation in females. Analysis of these traits supports the hypothesis that the multiple X chromosomes of spiders originated by duplications. In contrast to the homogametic sex of other animals, the homologous sex chromosomes of spider females were already paired at premeiotic interphase and were inactivated until prophase I. Furthermore, the sex chromosome pairs exhibited an end-to-end association during these stages. We suggest that the specific behavior of the female sex chromosomes may have evolved to avoid the negative effects of duplicated X chromosomes on female meiosis. The chromosome ends that ensure the association of sex chromosome pairs during meiosis may contain information for discriminating between homologous and homeologous X chromosomes and thus act to promote homologous pairing. The meiotic behavior of 4 X chromosome pairs in mygalomorph females, namely, the formation of 2 associations, each composed of 2 pairs with similar structure, suggests that the mygalomorph X(1)X(2)X(3)X(4)0 system originated by the duplication of the X(1)X(2)0 system via nondisjunctions or polyploidization.     

A phylogeny of howler monkeys (Cebidae: Alouatta) based on mitochondrial, chromosomal and morphological data

The current taxonomic status of the species and subspecies belonging to the genus Alouatta is addressed by combined phylogenetic analysis using morphological, kariotipyc and molecular data (mitochondrial genes cytocrome oxidase II and cytochrome B). Our result demonstrated that Alouatta palliata is the most basal taxon for the genus in concordance with previous studies, as well as showing the validity of the taxon Alouatta sara as a species. Also our analysis shows that the sex chromosome has evolved from a XY/XX system to a X1X2Y1Y2/X1X1X2X2 system within the genus, as well as an increase in the size and complexity of the hioideal bone.     

Flexible chromosome painting based on multiplex PCR of oligonucleotides and its application for comparative chromosome analyses in Cucumis

Chromosome painting is a powerful technique for chromosome and genome studies. We developed a flexible chromosome painting technique based on multiplex PCR of a synthetic oligonucleotide (oligo) library in cucumber (Cucumis sativus L., 2n = 14). Each oligo in the library was associated with a universal as well as nested specific primers for amplification, which allow the generation of different probes from the same oligo library. We were also able to generate double‐stranded labelled oligos, which produced much stronger signals than single‐stranded labelled oligos, by amplification using fluorophore‐conjugated primer pairs. Oligos covering cucumber chromosome 1 (Chr1) and chromosome 4 (Chr4) consisting of eight segments were synthesized in one library. Different oligo probes generated from the library painted the corresponding chromosomes/segments unambiguously, especially on pachytene chromosomes. This technique was then applied to study the homoeologous relationships among cucumber, C. hystrix and C. melo chromosomes based on cross‐species chromosome painting using Chr4 probes. We demonstrated that the probe was feasible to detect interspecies chromosome homoeologous relationships and chromosomal rearrangement events. Based on its advantages and great convenience, we anticipate that this flexible oligo‐painting technique has great potential for the studies of the structure, organization, and evolution of chromosomes in any species with a sequenced genome.     

Independent fusions and recent origins of sex chromosomes in the evolution and diversification of glass knife fishes (Eigenmannia)

The genus Eigenmannia comprises several species groups that display a surprising variety of diploid chromosome numbers and sex-determining systems. In this study, hypotheses regarding phylogenetic relationships and karyotype evolution were investigated using a combination of molecular and cytogenetic methods. Phylogenetic relationships were analyzed for 11 cytotypes based on sequences from five mitochondrial DNA regions. Parsimony-based character mapping of sex chromosomes confirms previous suggestions of multiple origins of sex chromosomes. Molecular cytogenetic analyses involved chromosome painting using probes derived from whole sex chromosomes from two taxa that were hybridized to metaphases of their respective sister cytotypes. These analyses showed that a multiple XY system evolved recently (<7 mya) by fusion. Furthermore, one of the chromosomes that fused to form the neo-Y chromosome is fused independently to another chromosome in the sister cytotype. This may constitute an efficient post-mating barrier and might imply a direct function of sex chromosomes in the speciation processes in Eigenmannia. The other chromosomal sex-determination system investigated is shown to have differentiated by an accumulation of heterochromatin on the X chromosome. This has occurred in the past 0.6 my, and is the most recent chromosomal sex-determining system described to date. These results show that the evolution of sex-determining systems can proceed very rapidly.     

Evolutionary breakpoint regions and chromosomal remodeling in Harttia (Siluriformes: Loricariidae) species diversification

The Neotropical armored catfish genus Harttia presents a wide variation of chromosomal rearrangements among its representatives. Studies indicate that translocation and Robertsonian rearrangements have triggered the karyotype evolution in the genus, including differentiation of sex chromosome systems. However, few studies used powerful tools, such as comparative whole chromosome painting, to clarify this highly diversified scenario. Here, we isolated probes from the X₁ (a 5S rDNA carrier) and the X₂ (a 45S rDNA carrier) chromosomes of Harttia punctata, which displays an X₁X₁X₂X₂/X₁X₂Y multiple sex chromosome system. Those probes were applied in other Harttia species to evidence homeologous chromosome blocks. The resulting data reinforce that translocation events played a role in the origin of the X₁X₂Y sex chromosome system in H. punctata. The repositioning of homologous chromosomal blocks carrying rDNA sites among ten Harttia species has also been demonstrated. Anchored to phylogenetic data it was possible to evidence some events of the karyotype diversification of the studied species and to prove an independent origin for the two types of multiple sex chromosomes, XX/XY₁Y₂ and X₁X₁X₂X₂/X₁X₂Y, that occur in Harttia species. The results point to evolutionary breakpoint regions in the genomes within or adjacent to rDNA sites that were widely reused in Harttia chromosome remodeling.     

Evidence that meiotic sex chromosome inactivation is essential for male fertility

The mammalian X and Y chromosomes share little homology and are largely unsynapsed during normal meiosis. This asynapsis triggers inactivation of X- and Y-linked genes, or meiotic sex chromosome inactivation (MSCI). Whether MSCI is essential for male meiosis is unclear. Pachytene arrest and apoptosis is observed in mouse mutants in which MSCI fails, e.g., Brca1(-/-), H2afx(-/-), Sycp1(-/-), and Msh5(-/-). However, these also harbor defects in synapsis and/or recombination and as such may activate a putative pachytene checkpoint. Here we present evidence that MSCI failure is sufficient to cause pachytene arrest. XYY males exhibit Y-Y synapsis and Y chromosomal escape from MSCI without accompanying synapsis/recombination defects. We find that XYY males, like synapsis/recombination mutants, display pachytene arrest and that this can be circumvented by preventing Y-Y synapsis and associated Y gene expression. Pachytene expression of individual Y genes inserted as transgenes on autosomes shows that expression of the Zfy 1/2 paralogs in XY males is sufficient to phenocopy the pachytene arrest phenotype; insertion of Zfy 1/2 on the X chromosome where they are subject to MSCI prevents this response. Our findings show that MSCI is essential for male meiosis and, as such, provide insight into the differential severity of meiotic mutations' effects on male and female meiosis.     

Fissions,fusions, and translocations shaped the karyotype and multiple sex chromosome constitution of the northeast‐Asian wood white butterfly,Leptidea amurensis

Previous studies have shown a dynamic karyotype evolution and the presence of complex sex chromosome systems in three cryptic Leptidea species from the Western Palearctic. To further explore the chromosomal particularities of Leptidea butterflies, we examined the karyotype of an Eastern Palearctic species, Leptidea amurensis. We found a high number of chromosomes that differed between the sexes and slightly varied in females (i.e. 2n = 118–119 in females and 2n = 122 in males). The analysis of female meiotic chromosomes revealed multiple sex chromosomes with three W and six Z chromosomes. The curious sex chromosome constitution [i.e. W_1–3/Z_1–6 (females) and Z_1–6/Z_1–6 (males)] and the observed heterozygotes for a chromosomal fusion are together responsible for the sex‐specific and intraspecific variability in chromosome numbers. However, in contrast to the Western Palearctic Leptidea species, the single chromosomal fusion and static distribution of cytogenetic markers (18S rDNA and H3 histone genes) suggest that the karyotype of L. amurensis is stable. The data obtained for four Leptidea species suggest that the multiple sex chromosome system, although different among species, is a common feature of the genus Leptidea. Furthermore, inter‐ and intraspecific variations in chromosome numbers and the complex meiotic pairing of these multiple sex chromosomes indicate the role of chromosomal fissions, fusions, and translocations in the karyotype evolution of Leptidea butterflies.     

Genetic variability in four Alouatta species measured by means of nine DNA microsatellite markers: genetic structure and recent bottlenecks

We used microsatellite DNA to study the population genetics of 4 Alouatta species from Central and South America. Our main findings include the following: (1) A. seniculus had the highest level of microsatellite variability while A. caraya and A. palliata had the lowest mean number of alleles per locus and the lowest expected heterozygosity, respectively; (2) the samples of A. seniculus and A. palliata came from different regions and were not in Hardy-Weinberg equilibrium (HWE) which may indicate a Wahlund effect and differentiated gene pools -- in contrast, A. macconnelli and A. caraya were in HWE; (3) the microsatellite genetic heterogeneity of the 4 Alouatta species was similar to the karyotype divergence found among these Alouatta species; the species pair with the lowest level of heterogeneity (genetic differentiation) was A. seniculus/A. caraya, while the Central American species, A. palliata, was highly differentiated from the other 3 South American species; (4) we recommend the establishment of a conservation plan to help protect A. caraya because the Cornuet and Luikart procedure demonstrated a recent bottleneck for this species.     

Chromosomal rearrangements in cattle and pigs revealed by chromosome microdissection and chromosome painting

A pericentric inversion of chromosome 4 in a boar, as well as a case of (2q-;5p+) translocation mosaicism in a bull were analysed by chromosome painting using probes generated by conventional microdissection. For the porcine inversion, probes specific for p arms and q arms were produced and hybridised simultaneously on metaphases of a heterozygote carrier. In the case of the bovine translocation, two whole chromosome probes (chromosome 5, and derived chromosome 5) were elaborated and hybridised independently on chromosomal preparations of the bull who was a carrier of the mosaic translocation. The impossibility of differentiating chromosomes 2 and der(2) from other chromosomes of the metaphases did not allow the production of painting probes for these chromosomes. For all experiments, the quality of painting was comparable to that usually observed with probes obtained from flow-sorted chromosomes. The results obtained allowed confirmation of the interpretations proposed with G-banding karyotype analyses. In the bovine case, however, the reciprocity of the translocation could not be proven. The results presented in this paper show the usefulness of the microdissection technique for characterising chromosomal rearrangements in species for which commercial probes are not available. They also confirmed that the main limiting factor of the technique is the quality of the chromosomal preparations, which does not allow the identification of target chromosomes or chromosome fragments in all cases.     

Karyotype analysis and achiasmatic meiosis in pseudoscorpions of the family Chthoniidae (Arachnida: Pseudoscorpiones)

Karyotypes of pseudoscorpions (Arachnida, Pseudoscorpiones) are largely unknown. Here we describe for the first time karyotypes of the suborder Epiocheirata, represented by 9 European species of two genera of Chthoniidae, Chthonius and Mundochthonius. Diploid chromosome numbers of males range from 21 to 37. Karyotypes of both genera differ substantially. Acrocentric chromosomes predominate in karyotypes of the genus Chthonius, whereas M. styriacus exhibits a predominance of metacentric chromosomes. These differences suggest that the two genera belong probably to distant branches of the family Chthoniidae. It is proposed that karyotype evolution of the genus Chthonius was characterised by a reduction of chromosome numbers by tandem and centric fusions as well as gradual conversion of acrocentric chromosomes to biarmed ones, mostly by pericentric inversions. A tendency towards reduced chromosome numbers is evident in the subgenus Ephippiochthonius. All species display X0 sex chromosome system that is probably ancestral in pseudoscorpions. The X chromosome exhibits conservative morphology. It is metacentric in all species examined, and in the majority of them, a subterminal secondary constriction was found at one of its arms. In contrast to chthoniids, secondary constriction was not reported on sex chromosomes of other pseudoscorpions. Analysis of prophase I chromosomes in males revealed an achiasmatic mode of meiosis. Findings of the achiasmatic meiosis in both genera, Chthonius and Mundochthonius, indicate that this mode of meiosis might be characteristic of the family Chthoniidae. Amongst arachnids, achiasmatic meiosis has only been described in some scorpions, acariform mites, and spiders.