U2 snDNA Research Articles

Conserved number of U2 snDNA sites in Piabina argentea, Piabarchus stramineus and two Bryconamericus species (Characidae, Stevardiinae)

ABSTRACT The chromosomal location of 5S rRNA and U2 snRNA genes of Piabina argentea, Piabarchus stramineus and two Bryconamericus species from two different Brazilian river basins were investigated, in order to contribute to the understanding of evolutionary characteristics of these repetitive DNAs in the subfamily Stevardiinae. The diploid chromosome number was 2n = 52 for Bryconamericus cf. iheringii, Bryconamericus turiuba, Piabarchus stramineus and Piabina argentea. The 5S rDNA clusters were located on one chromosome pair in P. stramineus and B. cf. iheringii, and on two pairs in B. turiuba and P. argentea. The U2 snDNA clusters were located on the one pair in all species. Two-color FISH experiments showed that the co-localization between 5S rDNA and U2 snDNA in P. stramineus can represent a marker for this species. Thus, the present study demonstrated that the number of U2 snDNA clusters observed for the four species was conserved, but particular characteristics can be found in the genome of each species.

Chromosomal locations of U2 snDNA clusters in Megaleporinus, Leporinus and Schizodon (Characiformes: Anostomidae)

The U small nuclear RNA (U snRNA) genes comprise a multigene family and are required for splicing of pre-mRNA. In this paper, we aimed to study the chromosomal location of the U2 snRNA gene in Megaleporinus, Leporinus and Schizodon species, which constitute interesting models for the study of repetitive DNA and genomic evolution in fish once the group comprises species with and without heteromorphic sex chromosomes. The all six species showed 2n = 54 chromosomes: Megaleporinus elongatus, Megaleporinus macrocephalus, Leporinus striatus, Leporinus friderici, Schizodon borelli and Schizodon isognathus. The U2 snDNA clusters were evident in only one medium-sized submetracentric pair in all analyzed species and this may represent a condition shared by Anostomidae family.

Chromosome caracterization of Melipona scutellaris (Hymenoptera, Apidae, Meliponini) based on repetitive DNAs

Repetitive DNAs constitute a significant part of the genomes and are used as cytogenetic markers for the understanding of chromosome evolution. Bees of the genus Melipona presents conserved chromosome number but high variability concerning heterochromatin distribution, dividing the genus into two groups: (i) species with low quantity of heterochromatin and (ii) species with high amount of heterochromatin. Here aiming the understanding of repetitive DNAs chromosomal organization in Melipona we mapped through fluorescence in situ hybridization (FISH) some repetitive DNAs in M. scutellaris. For this, FISH were performed with the amplification products of the 18S rDNA, U2 snDNA and DOP-PCR, as well as the product of the C0t fraction and different microsatellites. The mitotic chromosomes used in FISH were obtained by squashing the brain ganglia, and in this material, being performed also the C-band and CMA/DAPI. The distribution of the C0t fraction in other species of Melipona was verified with membrane hybridization. Chromosome number of n = 9 and 2n = 18 (male and female, respectively) was confirmed and a large amount of heterochromatin was observed along the chromosomes, except at the terminals that were characterized as euchromatic and only rich in different microsatellites. The heterochromatic regions were observed with richness in A + T and C + G. The FISH experiments showed signals from the 18S rDNA and U2 snDNA probes on distinct chromosomes. This demonstrates that these multigenic families followed different evolutionary paths in the chromosome evolution of the species. The DOP-PCR and C0t fraction showed signs of hybridization in the heterochromatic regions, and the C0t fraction of M. scutellaris was also observed in the genomes of M. rufiventris, M. rufiventris rufiventris and M. seminigra, evidencing the dispersion of similar repetitive elements in genomes of these three species. Thus, it was possible to characterize the karyotype of M. scutellaris with different cytogenetic markers. In addition, the results with membrane hybridization reinforce the hypothesis of the repetitive DNA sharing among nearby species, suggesting that there are different repetitive elements present in the genome of the genus Melipona.

Population study of the B chromosome frequency in the grasshopper Abracris flavolineata

Additional parasitic elements known as B chromosomes have been described in approximatey 15% of eukaryotic species. They have primary characteristics that define their origin, composition, ways of accumulation and evolution, such as irregular modes of inheritance, pairing incapacity with standard A chromosomes during meiosis and accumulation of distinct repetitive DNAs. The grasshopper Abracris flavolineata presents diploid number 2n = 23, X0 (male) and 2n = 24, XX (female) and presence of one or two B submetacentric chromosomes were reported exclusively in Rio Claro/SP population, varying in frequency temporally from 13.5% to 31.5% in males. In this work aiming to understand the spatial variation of B chromosome we investigated presence and frequency of B chromosomes in males belonging to three populations, Santa Bárbara do Pará/PA, Cabo/PE and Posadas/Misiones/Argentina using 61, 19 and 14 individuals, respectively. For Argentina population the frequency estimated was only 7,14% (one individual), while for Pernambuco it was higher with about 21,05% (four individuals), and for Pará none of the individuals presented B chromosomes. None of the populations presented individuals with 2B chromosome as reported in Rio Claro/SP, suggesting differential drive and accumulation. The fluctuation of B chromosome frequency and accumulation in these natural populations could be explained by several factors, like differences in parasitism rate, genetic drift or else by negative effects for host development. Moreover local environment characteristics, associated to possible reproductive isolation, caused by vicariance processes, could also influence B chromosome frequency variation. Here we expanded the knowledge of B chromosome geographic distribution in A. flavolineata and the next step is to check if the B chromosome is the same variant in distinct populations, using for example the U2 snDNA as probe, wich is present in the B chromosome from Rio Claro/SP population.

Genomic organization of 5S rDNAs in Triportheus (Triportheidae, Characiformes): U1 snRNAs linkage and evolutionary divergence among species

The 5S rDNA array is composed of multiple copies of a conserved transcriptional unit and a variable non-transcribed spacer (NTS). The NTS may include other multigene families, such as small nuclear RNAs (snRNAs) and transposable elements (TEs). Here, we integrated genetic and cytogenetic analyses to better understand the evolutionary patterns of 5S rDNAs in Triportheus. A total of 66 clones containing 5S rDNAs of eight Triportheus species were sequenced. The evolutionary divergence between species and the phylogenetic analyzes of 5S rDNAs were achieved by DnaSP and SplitsTree, respectively. We performed the fluorescence in situ hybridization (FISH) method using 5S rRNA and U1 snRNA genes as probes. The 5S rRNA gene in Triportheus has a coding region of 120 pb and a NTS of ~1500 pb. Inside the NTS, a high homology with the U1 snRNAs was revealed by BLASTn, evidencing that 5S rRNA and U1 snRNA sequences are linked in the same array. Different types of retrotransposons and DNA transposon fragments were also identified in the NTS. The coding region presented a low evolutionary divergence among species, thus contrasting with the much higher one of the NTS, especially for Triportheus auritus in which high values were found. In addition, the neighbor-network showed a clear clustering for most species. The 5S rDNA signals were localized in one or two chromosome pairs in all species, again with the exception of T. auritus which carried ten sites. Besides, the U1 snDNA was mapped in co-localization with one 5S rDNA site, confirming the sequence analyzes. Finally, the genetic and cytogenetic data evidenced the linkage between U1 and 5S rDNA, as well as the differential evolutionary trends of T. auritus in relation to other Triportheus species. In fact, according to previous phylogenetic studies, T. auritus is representative of the first lineage that differentiated in the genus, corresponding to the sister group of all Triportheus species. The high NTS evolutionary divergence among species highlights its variability, probably due to TEs insertion in this genomic region. A mix of the concerted and birth-and-death models is probably related to the 5S rDNA evolutionary process in Triportheus.

Next-generation sequencing analysis reveals novel insights of B chromosome evolution of the grasshopper Abracris flavolineata

B chromosomes are additional elements in a normal karyotype that has been considered frequently as parasitic and dispensable. These elements do not recombine with A chromosomes, could be accumulated and present non-mendelian patterns of inheritance. In most of the cases they comprise an enrichment of heterochromatic repetitive DNA, such as Satellite DNAs (satDNA). To contribute to understanding about origin, composition and evolutionary patterns of B chromosome in the grasshopper Abracris flavolineata, in this work we characterized through Illumina sequencing and graph-based clustering the most abundant tandem repeats of A. flavolineata, karyotype 2n = 22,X0+B?.The analyses revealed the occurrence of ten families of satDNAs comprising about 6.279% of the male genome harboring one B chromosome. For the X chromosome and autosomes, distinct patterns of hybridization was observed between the satDNAs showing variability among them, like scattered blocks on euchromatic areas or specific ones on heterochromatic regions of the chromosomes. Five of the satDNAs recovered were distributed on centromeric and both terminal regions of the B chromosome, reinforcing the hypothesis of B chromosome origin through isochromosome formation. Two satDNA families were located only in centromere, while three families were not present in B chromosome. CL220 satDNA was the unique family shared between the B chromosome and only one A element, the chromosome 1, supporting strongly the hypothesis of its origin from this autosomal pair, also noticed previously by U2 snDNA mapping. In addition, this analysis provides new information regarding the composition and evolution of B chromosome of A. flavolineata. Furthermore, the non-spreading and specific location of satDNAs and other repetitive sequences restrict to telomeres and centromere reinforces the possible idea that this B chromosome could harbor unknown euchromatic sequences along chromosomal arms and do not present intense amplification of heterochromatic ones, as have been commonly described for other species.

Identification of a New Mullet Species Complex Based on an Integrative Molecular and Cytogenetic Investigation of Mugil hospes (Mugilidae: Mugiliformes).

Mullets are very common fishes included in the family Mugilidae, (Mugiliformes), which are characterized by both a remarkably uniform external morphology and internal anatomy. Recently, within this family, different species complexes were molecularly identified within Mugil, a genus which is characterized by lineages that sometimes show very different karyotypes. Here we report the results of cytogenetic and molecular analyses conducted on Mugil hospes, commonly known as the hospe mullet, from Ecuador. The study aims to verify whether the original described species from the Pacific Ocean corresponds to that identified in the Atlantic Ocean, and to identify species-specific chromosome markers that can add new comparative data about Mugilidae karyotype evolution. The karyotype of M. hospes from Ecuador is composed of 48 acrocentric chromosomes and shows two active nucleolar organizer regions (NORs). In situ hybridization, using different types of repetitive sequences (rDNAs, U1 snDNA, telomeric repeats) as probes, identified species-specific chromosome markers that have been compared with those of other species of the genus Mugil. Cytochrome c oxidase subunit I (COI) sequence analysis shows only 92–93% similarity with sequences previously deposited under this species name in GenBank, all of which were from the Atlantic Ocean. Phylogenetic reconstructions indicate the presence of three well-supported hospe mullet lineages whose molecular divergence is compatible with the presence of distinct species. Indeed, the first lineage includes samples from Ecuador, whereas the other two lineages include the Atlantic samples and correspond to M. brevirostris from Brazil and Mugil sp. R from Belize/Venezuela. Results here provided reiterate the pivotal importance of an integrative molecular and cytogenetic approach in the reconstruction of the relationships within Mugilidae.

First Chromosomal Analysis in Hepsetidae (Actinopterygii, Characiformes): Insights into Relationship between African and Neotropical Fish Groups.

Hepsetidae is a small fish family with only the genus Hepsetus, with six described species distributed throughout the South, Central and Western regions of Africa, showing a close relationship with the Alestidae and some Neotropical fish families. However, no cytogenetic information is available for both Hepsetidae and Alestidae species, thus preventing any evolutionary comparative studies at the chromosomal level. In the present study, we are providing new cytogenetic data for Hepsetus odoe, including the standard karyotype, C-banding, repetitive DNAs mapping, comparative genomic hybridization (CGH) and whole chromosome painting (WCP), providing chromosomal patterns and subsidies for comparative cytogenetics with other characiform families. Both males and females H. odoe have 2n = 58 chromosomes (10m + 28sm + 20st/a), with most of the C-band positive heterochromatin localized in the centromeric and subtelomeric regions. Only one pair of chromosomes bears proximal 5S rDNA sites in the short arms, contrasting with the 18S rDNA sequences which are located in the terminal regions of four chromosome pairs. Clear interstitial hybridization signals are evidenced for the U1 and U2 snDNA probes, but in only one and two chromosome pairs, respectively. Microsatellite motifs are widely distributed in the karyotype, with exception for the (CGG)10, (GAA)10 and (GAG)10 probes, which highlight conspicuous interstitial signals on an unique pair of chromosomes. Comparative data from conventional and molecular cytogenetics, including CGH and WCP experiments, indicate that H. odoe and some Erythrinidae species, particularly Erythrinus erythrinus, share similar chromosomal sequences suggesting some relatedness among them, although bearing genomic specificities in view of their divergent evolutionary histories.

U1 snDNA chromosomal mapping in ten spittlebug species (Cercopidade, Auchenorrhyncha, Hemiptera).

Spittlebugs, which belong to the family Cercopidae (Auchenorrhyncha, Hemiptera), form a large group of xylem-feeding insects that are best known for causing damage to plantations and pasture grasses. The holocentric chromosomes of these insects remain poorly studied in regards to the organization of different classes of repetitive DNA. To improve chromosomal maps based on repetitive DNAs and to better understand the chromosomal organization and evolutionary dynamics of multigene families in spittlebugs, we physically mapped the U1 snRNA gene with fluorescence in situ hybridization (FISH) in 10 species of Cercopidae belonging to three different genera. All the U1 snDNA clusters were autosomal and located in interstitial position. In seven species, they were restricted to one autosome per haploid genome, while three species of the genus Mahanarva showed two clusters in two different autosomes. Although it was not possible to precisely define the ancestral location of this gene, it was possible to observe the presence of at least one cluster located in a small bivalent in all karyotypes. The karyotype stability observed in Cercopidae is also observed in respect to the distribution of U1 snDNA. Our data are discussed in light of possible mechanisms for U1 snDNA conservation and compared with the available data from other species.

Contrasting Evolutionary Paths Among Indo-Pacific Pomacentrus Species Promoted by Extensive Pericentric Inversions and Genome Organization of Repetitive Sequences.

Pomacentrus (damselfishes) is one of the most characteristic groups of fishes in the Indo-Pacific coral reef. Its 77 described species exhibit a complex taxonomy with cryptic lineages across their extensive distribution. Periods of evolutionary divergences between them are very variable, and the cytogenetic events that followed their evolutionary diversification are largely unknown. In this respect, analyses of chromosomal divergence, within a phylogenetic perspective, are particularly informative regarding karyoevolutionary trends. As such, we conducted conventional cytogenetic and cytogenomic analyses in four Pomacentrus species (Pomacentrus similis, Pomacentrus auriventris, Pomacentrus moluccensis, and Pomacentrus cuneatus), through the mapping of repetitive DNA classes and transposable elements, including 18S rDNA, 5S rDNA, (CA)15, (GA)15, (CAA)10, Rex6, and U2 snDNA as markers. P. auriventris and P. similis, belonging to the Pomacentrus coelestis complex, have indistinguishable karyotypes (2n = 48; NF = 48), with a peculiar syntenic organization of ribosomal genes. On the other hand, P. moluccensis and P. cuneatus, belonging to another clade, exhibit very different karyotypes (2n = 48, NF = 86 and 92, respectively), with a large number of bi-armed chromosomes, where multiple pericentric inversions played a significant role in their karyotype organization. In this sense, different chromosomal pathways followed the phyletic diversification in the Pomacentrus genus, making possible the characterization of two well-contrasting species groups regarding their karyotype features. Despite this, pericentric inversions act as an effective postzygotic barrier in many organisms, which appear to be also the case for P. moluccensis and P. cuneatus; the extensive chromosomal similarities in the two species of P. coelestis complex suggest minor participation of chromosomal postzygotic barriers in the phyletic diversification of these species.

Chromosomal organization of four classes of repetitive DNA sequences in killifish Orestias ascotanensis Parenti, 1984 (Cyprinodontiformes, Cyprinodontidae).

Orestias Valenciennes, 1839 is a genus of freshwater fish endemic to the South American Altiplano. Cytogenetic studies of these species have focused on conventional karyotyping. The aim of this study was to use classical and molecular cytogenetic methods to identify the constitutive heterochromatin distribution and chromosome organization of four classes of repetitive DNA sequences (histone H3 DNA, U2 snRNA, 18S rDNA and 5S rDNA) in the chromosomes of O. ascotanensis Parenti, 1984, an endemic species restricted to the Salar de Ascotán in the Chilean Altiplano. All individuals analyzed had a diploid number of 48 chromosomes. C-banding identified constitutive heterochromatin mainly in the pericentromeric region of most chromosomes, especially a GC-rich heterochromatic block of the short arm of pair 3. FISH assay with an 18S probe confirmed the location of the NOR in pair 3 and revealed that the minor rDNA cluster occurs interstitially on the long arm of pair 2. Dual FISH identified a single block of U2 snDNA sequences in the pericentromeric regions of a subtelocentric chromosome pair, while histone H3 sites were observed as small signals scattered in throughout the all chromosomes. This work represents the first effort to document the physical organization of the repetitive fraction of the Orestias genome. These data will improve our understanding of the chromosomal evolution of a genus facing serious conservation problems.

The U2 snDNA Is a Useful Marker for B Chromosome Detection and Frequency Estimation in the Grasshopper Abracris flavolineata

In this study, we describe a strategy to determine the presence of B chromosomes in the living grasshopper Abracris flavolineata by FISH using U2 snDNA as a probe in interphase hemolymph nuclei. In individuals without B chromosomes, (0B) 2 dot signals were noticed, corresponding to A complement U2 snDNA clusters. In +1B and +2B individuals, 4 or 8 additional signals were noticed, respectively. In all cases, the absence or presence of 1 or 2 B chromosomes correlated in hemolymph and in somatic or germline tissues, validating the efficiency of the marker. Our data suggest that the B chromosome of A. flavolineata is present in all somatic tissues. B-carrying individuals showed the same number of B chromosomes in germ and somatic cells, suggesting that the B is mitotically stable. The marker was used to compare B chromosome frequency in the analyzed population with a sample collected previously, in order to test for B frequency changes and differences of B chromosome prevalence among sexes, but no statistically significant differences were noticed. The identification of living animals harboring B chromosomes will be very useful in future studies of B chromosome transmission, as well as in functional studies involving RNA analysis, thus contributing to the understanding of evolutionary history and the possible role of the B chromosome in A. flavolineata.

Chromosomal evolutionary dynamics of four multigene families in Coreidae and Pentatomidae (Heteroptera) true bugs

Previous chromosome mapping of multigene families in Pentatomomorpha (Heteroptera) insects, which was restricted to the major rDNA, revealed remarkable conservation of number of clusters and chromosomal positions. Aiming to understand the chromosomal organization and evolutionary patterns of multigene families in karyotypes of Heteroptera, we performed a chromosomal mapping using four distinct multigene families in representatives of Coreidae (ten species) and Pentatomidae (five species). A single pair of the major rDNA cluster (18S rDNA probe) and a single pair of the minor rDNA cluster (5S rDNA probe), both terminally located were primarily observed, being, in most species, located in distinct chromosomes. However, some alternative patterns were also observed. In species in which the U2 snDNA and H4 gene clusters were mapped, they were mainly located in one autosomal pair each, wherein the H4 gene cluster was located in different positions. Our data suggest that the karyotype diversity reported in Coreidae is not reflected in the distribution diversity of multigene families. This contrasts with the data for Pentatomidae, with a conserved gross karyotype but a discrete diversity in the location of the clusters of multigene families, indicating genome dynamics for these markers. The findings are discussed to shed light on the possible causes for the conservation or variation observed and to assist in understanding the chromosomal evolutionary trends in the group.

Highly Similar Morphologies Between Chromosomes Bearing U2 snRNA Gene Clusters in the Group Astyanax Baird and Girard, 1854 (Characiformes, Characidae): An Evolutionary Approach in Species with 2n = 36, 46, 48, and 50.

Repetitive sequences and their chromosomal locations have been widely studied in species of the Astyanax genus. However, the chromosomal organization of U2 snDNA remains largely unknown. The aims of this study were to examine the chromosomal contexts of U2 snRNA and 5S rRNA genes in Astyanax species and determine the degree of chromosome morphological similarity between species with different diploid numbers. Clusters of U2 snDNA and 5S rDNA were determined in nine species of Astyanax, including two karyomorphs of Astyanax fasciatus Cuvier, 1819. All species exhibited U2 snDNA clusters on two chromosome pairs, except Astyanax mexicanus De Filippi, 1853 (one pair). The 5S rDNA clusters were located on one chromosome pair in Astyanax altiparanae Garutti and Britski, 2000, and Astyanax marionae Eigenmann, 1911, two pairs in Astyanax abramis Jenyns, 1842, Astyanax asuncionensis Géry, 1972, Astyanax bockmanni Vari and Castro, 2007, Astyanax eigenmanniorum Cope, 1894, A. fasciatus (karyomorphs I and II), and Astyanax schubarti Britski, 1964, and four pairs in A. mexicanus. The relationships between the repetitive sequences in different species suggest that A. schubarti and A. mexicanus exhibit an unusual U2 snDNA chromosomal format as a result of events occurring in the evolutionary history of the Astyanax group.

Organization of some repetitive DNAs and B chromosomes in the grasshopper Eumastusia koebelei koebelei (Rehn, 1909) (Orthoptera, Acrididae, Leptysminae).

B chromosomes occur in approximately 15% of eukaryotes and are usually heterochromatic and rich in repetitive DNAs. Here we describe characteristics of a B chromosome in the grasshopper Eumastusia koebelei koebelei (Rehn, 1909) through classical cytogenetic methods and mapping of some repetitive DNAs, including multigene families, telomeric repeats and a DNA fraction enriched with repetitive DNAs obtained from DOP-PCR. Eumastusia koebelei koebelei presented 2n=23, X0 and, in one individual, two copies of the same variant of a B chromosome were noticed, which are associated during meiosis. The C-positive blocks were located in the pericentromeric regions of the standard complement and along the entire length of the B chromosomes. Some G+C-rich heterochromatic blocks were noticed, including conspicuous blocks in the B chromosomes. The mapping of 18S rDNA and U2 snDNA revealed only autosomal clusters, and the telomeric probe hybridized in terminal regions. Finally, the DOP-PCR probe obtained from an individual without a B chromosome revealed signals in the heterochromatic regions, including the entire length of the B chromosome. The possible intraspecific origin of the B chromosomes, due to the shared pool of repetitive DNAs between the A and B chromosomes and the possible consequences of their association are discussed.

Repetitive DNA Sequences and Evolution of ZZ/ZW Sex Chromosomes in Characidium (Teleostei: Characiformes).

Characidium constitutes an interesting model for cytogenetic studies, since a large degree of karyotype variation has been detected in this group, like the presence/absence of sex and supernumerary chromosomes and variable distribution of repetitive sequences in different species/populations. In this study, we performed a comparative cytogenetic analysis in 13 Characidium species collected at different South American river basins in order to investigate the karyotype diversification in this group. Chromosome analyses involved the karyotype characterization, cytogenetic mapping of repetitive DNA sequences and cross-species chromosome painting using a W-specific probe obtained in a previous study from Characidium gomesi. Our results evidenced a conserved diploid chromosome number of 2n = 50, and almost all the species exhibited homeologous ZZ/ZW sex chromosomes in different stages of differentiation, except C. cf. zebra, C. tenue, C. xavante and C. stigmosum. Notably, some ZZ/ZW sex chromosomes showed 5S and/or 18S rDNA clusters, while no U2 snDNA sites could be detected in the sex chromosomes, being restricted to a single chromosome pair in almost all the analyzed species. In addition, the species Characidium sp. aff. C. vidali showed B chromosomes with an inter-individual variation of 1 to 4 supernumerary chromosomes per cell. Notably, these B chromosomes share sequences with the W-specific probe, providing insights about their origin. Results presented here further confirm the extensive karyotype diversity within Characidium in contrast with a conserved diploid chromosome number. Such chromosome differences seem to constitute a significant reproductive barrier, since several sympatric Characidium species had been described during the last few years and no interespecific hybrids were found.

High similarity of U2 snDNA sequence between A and B chromosomes in the grasshopper Abracris flavolineata.

B chromosomes are frequently enriched for a wide variety of repetitive DNAs. Among grasshoppers in the species Abracris flavolineata (Ommatolampidinae) the B chromosomes are submetacentric, C-negative and harbor repetitive DNAs such as, U2 snDNA, C 0 t-1 DNA, two Mariner-like elements and some microsatellites. Here, we provide evidence showing the intragenome similarity between the B chromosome and the A complement in A. flavolineata, combining analysis of microdissection and chromosome painting and B chromosome-specific amplification through polymerase chain reaction (PCR) of U2 snDNA. Chromosome painting revealed signals spread through the C-negative regions, including the A and B chromosomes. Moreover, significant clustered signals forming bands were observed in some A chromosomes, and for the B chromosome, significant signals were located on both arms, which could be caused by accumulation of repetitive DNA sequences. The C-positive regions did not reveal any signals. Sequence comparison of U2 snDNA between that obtained from a genome without the B chromosome and that from µB-DNA revealed high similarity with the occurrence of four shared haplotypes, one of them (i.e., Hap1) being highly prevalent and putatively ancestral. The highest divergence from Hap1 was observed for Hap3, which was caused by only six mutational steps. These data support an intraspecific origin of the B chromosome in A. flavolineata that is highly similar with the A complement, and the low U2 snDNA sequence diversity observed in the B chromosome could be related to its recent origin, besides intrachromosomal concerted evolution for U2 snDNA repeats in the B chromosome.

Repetitive DNA chromosomal organization in the cricket Cycloptiloides americanus: a case of the unusual X1X 20 sex chromosome system in Orthoptera.

A common placement for most sex chromosomes that is involved in their evolutionary histories is the accumulation of distinct classes of repetitive DNAs. Here, with the aim of understanding the poorly studied repetitive DNA organization in crickets and its possible role in sex chromosome differentiation, we characterized the chromosomes of the cricket species Cycloptiloides americanus, a species with the remarkable presence of the unusual sex chromosome system X1X20♂/X1X1X2X2♀. For these proposes, we used C-banding and mapping through the fluorescence in situ hybridization of some repetitive DNAs. The C-banding and distribution of highly and moderately repetitive DNAs (C 0t-1 DNA) varied depending of the chromosome. The greater accumulation of repetitive DNAs in the X2 chromosome was evidenced. The microsatellites were spread along entire chromosomes, but (AG)10 and (TAA)10 were less enriched, mainly in the centromeric areas. Among the multigene families, the 18S rDNA was spread throughout almost all of the chromosomes, except for pair 5 and X2, while the U2 snDNA was placed exclusively in the largest chromosome. Finally, the 5S rDNA was exclusively located in the short arms of the sex chromosomes. The obtained data reinforce the importance of chromosomal dissociation and inversion as a primary evolutionary mechanism to generate neo-sex chromosomes in the species studied, followed by the repetitive DNAs accumulation. Moreover the exclusive placement of 5S rDNA in the sex chromosomes suggests the involvement of this sequence in sex chromosome recognition throughout meiosis and, consequently, their maintenance, in addition to their avoiding degeneration.

U1 snDNA clusters in grasshoppers: chromosomal dynamics and genomic organization.

The spliceosome, constituted by a protein set associated with small nuclear RNA (snRNA), is responsible for mRNA maturation through intron removal. Among snRNA genes, U1 is generally a conserved repetitive sequence. To unveil the chromosomal/genomic dynamics of this multigene family in grasshoppers, we mapped U1 genes by fluorescence in situ hybridization in 70 species belonging to the families Proscopiidae, Pyrgomorphidae, Ommexechidae, Romaleidae and Acrididae. Evident clusters were observed in all species, indicating that, at least, some U1 repeats are tandemly arrayed. High conservation was observed in the first four families, with most species carrying a single U1 cluster, frequently located in the third or fourth longest autosome. By contrast, extensive variation was observed among Acrididae, from a single chromosome pair carrying U1 to all chromosome pairs carrying it, with occasional occurrence of two or more clusters in the same chromosome. DNA sequence analysis in Eyprepocnemis plorans (species carrying U1 clusters on seven different chromosome pairs) and Locusta migratoria (carrying U1 in a single chromosome pair) supported the coexistence of functional and pseudogenic lineages. One of these pseudogenic lineages was truncated in the same nucleotide position in both species, suggesting that it was present in a common ancestor to both species. At least in E. plorans, this U1 snDNA pseudogenic lineage was associated with 5S rDNA and short interspersed elements (SINE)-like mobile elements. Given that we conclude in grasshoppers that the U1 snDNA had evolved under the birth-and-death model and that its intragenomic spread might be related with mobile elements.

Comparative Chromosome Mapping of U2 snRNA and 5S rRNA Genes in Gymnotus Species (Gymnotiformes, Gymnotidae): Evolutionary Dynamics and Sex Chromosome Linkage in G. pantanal

A comparative mapping of U2 small nuclear RNA (snRNA) and 5S ribosomal RNA (rRNA) genes was performed in 6 Gymnotus species. All species analyzed presented the U2 snDNA organized in conspicuous blocks and not co-located with rRNA genes. In addition, 5 species showed the U2 snDNA located in a single pair of chromosomes, which seems to be a conserved trait in this genus. Conversely, G. pantanal was the only species displaying several terminal signals in different chromosome pairs, including the X₁ sex chromosome but not the Y chromosome. This is the first report of U2 snRNA genes in sex chromosomes of fishes. The absence of sites in the Y chromosome of G. pantanal indicates a possible loss of terminal segments of the chromosomes involved in the Y formation.