Molecular Cytogenetic Analysis of the European Hake Merluccius merluccius (Merlucciidae,Gadiformes): U1 and U2 snRNA Gene Clusters Map to the Same Location

The European hake (Merluccius merluccius) is a highly valuable and intensely fished species in which a long-term alive stock has been established in captivity for aquaculture purposes. Due to their huge economic importance, genetic studies on hakes were mostly focused on phylogenetic and phylogeographic aspects; however chromosome numbers are still not described for any of the fifteen species in the genus Merluccius. In this work we report a chromosome number of 2n = 42 and a karyotype composed of three meta/submetacentric and 18 subtelo/telocentric chromosome pairs. Telomeric sequences appear exclusively at both ends of every single chromosome. Concerning rRNA genes, this species show a single 45S rDNA cluster at an intercalary location on the long arm of subtelocentric chromosome pair 12; the single 5S rDNA cluster is also intercalary to the long arm of chromosome pair 4. While U2 snRNA gene clusters map to a single subcentromeric position on chromosome pair 13, U1 snRNA gene clusters seem to appear on almost all chromosome pairs, but showing bigger clusters on pairs 5, 13, 16, 17 and 19. The brightest signals on pair 13 are coincident with the single U2 snRNA gene cluster signals. Therefore, the use of these probes allows the unequivocal identification of at least 7 of the chromosome pairs that compose the karyotype of Merluccius merluccius thus opening the way to integrate molecular genetics and cytological data on the study of the genome of this important species.     

Evolutionary dynamics of 5S rDNA location in acridid grasshoppers and its relationship with H3 histone gene and 45S rDNA location

We analyze the chromosomal location of 5S rDNA clusters in 29 species of grasshoppers belonging to the family Acrididae. There was extensive variation among species for the number and location of 5S rDNA sites. Out of 148 sites detected, 75% were proximally located, 21.6% were interstitial, and only 3.4% were distal. The number of 5S rDNA sites per species varied from a single chromosome pair (in six species) to all chromosome pairs (in five species), with a range of intermediate situations. Thirteen chromosomes from eight species carried two 5S rDNA clusters. At intraspecific level, differences among populations were detected in Eyprepocnemis plorans, and some heteromorphisms have also been observed in some species. Double FISH for 5S rDNA and H3 histone gene DNA, performed on 17 of these 29 species, revealed that both markers are sometimes placed in a same chromosome but at different location, whereas they appeared to co-localize in five species (Calliptamus barbarus, Heteracris adpersa, Aiolopus strepens, Oedipoda charpentieri and O. coerulescens). Double fiber-FISH in A. strepens and O. coerulescens showed that the two DNAs are closely interspersed with variable relative amounts of both classes of DNA. Finally, no correlation was observed between the number of 5S and 45S rDNA clusters in 23 species where this information was available. These results are discussed in the light of possible mechanisms of spread that led to the extensive variation in the number of clusters observed for both rDNA types in acridid grasshoppers.     

Chromosomal locations of U2 snDNA clusters in <Emphasis Type="Italic">Megaleporinus</Emphasis>, <Emphasis Type="Italic">Leporinus</Emphasis> and <Emphasis Type="Italic">Schizodon</Emphasis> (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.     

Molecular cytogenetic analysis and the establishment of a cell culture in the fish species Hollandichthys multifasciatus (Eigenmann & Norris, 1900) (Characiformes,Characidae)

Hollandichthys is a fish genus of the family Characidae that was until recently considered to be monotypic, with cytogenetic, morphological, and molecular data being restricted to a few local populations. In the present study, the karyotype of a population of Hollandichthys multifasciatus was analyzed using classical and molecular cytogenetic approaches for the investigation of potential markers that could provide new perspectives on the cytotaxonomy. H. multifasciatus presented a diploid number of 2n=50 chromosomes and a karyotype formula of 8m+10sm+32st. A single pair of chromosomes presented Ag-NORs signals, which coincided with the 18S rDNA sites visualized by FISH, whilst the 5S rDNA sequences were mapped in two chromosome pairs. The distribution of the U snRNA genes was mapped on the Hollandichthys chromosomes for the first time, with the probes revealing the presence of the U1 snDNA on the chromosomes of pair 20, U2 on pairs 6 and 19, U4 on pair 16, and U6 on the chromosomes of pair 11. The results of the present study indicated karyotypic differences in comparison with the other populations of H. multifasciatus studied previously, reinforcing the need for further research to identify isolated populations or the potential existence of cryptic Hollandichthys species.     

Archolaemus janeae (Gymnotiformes,Teleostei): First insights into karyotype and repetitive DNA distribution in two populations of the Amazon

Archolaemus, one of the five genera of Neotropical freshwater fish of the family Sternopygidae (Gymnotiformes), was long considered a monotypic genus represented by Archolaemus blax. Currently, it consists of six species, most of them occurring in the Amazon region. There are no cytogenetic data for species of this genus. In the present study, we used classical cytogenetics (conventional staining and C‐banding) and molecular cytogenetics (probes of telomeric sequences and multigenic families 18S rDNA, 5S rDNA, and U2 snDNA) to study the karyotype of Archolaemus janeae from Xingu and Tapajós rivers in the state of Pará (Brazil). The results showed that the two populations have identical karyotypes with 46 chromosomes: four submetacentric and 42 acrocentric (2n = 46; 4m/sm + 42a). Constitutive heterochromatin occurs in the centromeric region of all chromosomes, in addition to small bands in the interstitial and distal regions of some pairs. The 18S rDNA occurs in the distal region of the short arm of pair 2; the 5S rDNA occurs in five chromosome pairs; and the U2 snDNA sequence occurs in chromosome pairs 3, 6, and 13. No interstitial telomeric sequence was observed. These results show karyotypic similarity between the studied populations suggesting the existence of a single species and are of great importance as a reference for future cytotaxonomic studies of the genus.     

Chromosomal Mapping of Repetitive DNAs in the Grasshopper Abracris flavolineata Reveal Possible Ancestry of the B Chromosome and H3 Histone Spreading

Supernumerary chromosomes (B chromosomes) occur in approximately 15% of eukaryote species. Although these chromosomes have been extensively studied, knowledge concerning their specific molecular composition is lacking in most cases. The accumulation of repetitive DNAs is one remarkable characteristic of B chromosomes, and the occurrence of distinct types of multigene families, satellite DNAs and some transposable elements have been reported. Here, we describe the organization of repetitive DNAs in the A complement and B chromosome system in the grasshopper species Abracris flavolineata using classical cytogenetic techniques and FISH analysis using probes for five multigene families, telomeric repeats and repetitive C₀t-1 DNA fractions. The 18S rRNA and H3 histone multigene families are highly variable and well distributed in A. flavolineata chromosomes, which contrasts with the conservation of U snRNA genes and less variable distribution of 5S rDNA sequences. The H3 histone gene was an extensively distributed with clusters occurring in all chromosomes. Repetitive DNAs were concentrated in C-positive regions, including the pericentromeric region and small chromosomal arms, with some occurrence in C-negative regions, but abundance was low in the B chromosome. Finally, the first demonstration of the U2 snRNA gene in B chromosomes in A. flavolineata may shed light on its possible origin. These results provide new information regarding chromosomal variability for repetitive DNAs in grasshoppers and the specific molecular composition of B chromosomes.     

Pairing Competition between Identical and Homologous Chromosomes in Rye and Grasshoppers

Meiotic pairing preferences between identical and homologous but not identical chromosomes were analyzed in spontaneous tetraploid/diploid chimeras of three male grasshoppers (Eyprepocnemis plorans) whose chromosome pair 11 were heterozygous for C-banding pattern and in four induced tetraploid/diploid chimaeral rye plants (Secale cereale) heterozygous for telomeric heterochromatin C-bands in chromosomes 1R and 2R. In the grasshoppers, a preference for identical over homologous pairing was observed, whereas in rye both a preference for homologous rather than identical pairing and random pairing between the four chromosomes of the set was found. From the results in rye, it can be deduced that pairing preferences do not depend exclusively on the similarities between chromosomes involved. It is suggested that genotypic or cryptic structural differences between the homologous chromosomes of each pair analyzed might be responsible for the pairing preferences found. This hypothesis can also explain the results obtained in grasshoppers, although the possibility of premeiotic association cannot be excluded in this material.     

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.     

Chromosomal mapping of the major and minor ribosomal genes, (GATA)<Subscript>n</Subscript> and U2 snRNA gene by double-colour FISH in species of the Batrachoididae family

In the present study dual-colour fluorescence in situ hybridization (FISH) was performed to study the chromosomal distribution of 18S and 5S rDNAs, (GATA)_n and 5S rDNA, and U2 snRNA and 18S rDNA in four species of Batrachoididae family: Amphichthys cryptocentrus, Batrachoides manglae, Porichthys plectrodon and Thalassophryne maculosa. The 18S rDNA signals were present in only one pair of chromosomes in all the four Batrachoididae species. The 5S rDNA was mapped on one pair of chromosomes, except in B. manglae, which showed a hybridization signal in two pairs. The two ribosomal genes are located on different chromosome pairs, except in A. cryptocentrus, in which they appear co-located. In all the cases, the (GATA)_n probe produced disperse hybridization signals in all four species. The U2 snRNA signals appear very widely scattered in A. cryptocentrus, P. plectrodon, but show a degree of clustering in a specific chromosome pair in B. manglae. In T. maculosa, they are thinly dispersed and strong hybridization signals are observed co-located to the 18S rDNA-bearing chromosomes. Finally, a double-colour FISH with U2 snRNA and 5S rDNA probes was performed in B. manglae, and this showed that these genes were not co-located. These results have been compared with those from another Batrachoididae species, and evolutive processes of these species are discussed.     

Complete mitochondrial genomes of three Oxya grasshoppers (Orthoptera) and their implications for phylogenetic reconstruction

Oxya is a genus of grasshoppers (Orthoptera: Acridoidea) attacking rice and other gramineous plants in Africa and Asia. In the present study, we characterized complete mitochondrial genomes (mitogenomes) of three species, Oxya japonica japonica (15,427 bp), Oxya hainanensis (15,443 bp) and Oxya agavisa robusta (15,552 bp) collected from China. The three mitogenomes contained a typical gene set of metazoan mitogenomes and shared the same gene order with other Acridid grasshoppers, including the rearrangement of tRNA^Asp and tRNA^Lys. Analyses of pairwise genetic distances showed that ATP8 was the least conserved gene, while COI the most conserved. To determine the position of Oxya grasshoppers in the phylogeny of Acrididae, we reconstructed phylogenetic trees among 64 species from across 11 subfamilies using nucleotide sequences of mitogenomes. While the tree confirms traditional classifications of Acrididae at major higher-levels, it suggests a few modifications for classifications at lower-levels.     

Post-meiotic B chromosome expulsion,during spermiogenesis,in two grasshopper species

Most supernumerary (B) chromosomes are parasitic elements carrying out an evolutionary arms race with the standard (A) chromosomes. A variety of weapons for attack and defense have evolved in both contending elements, the most conspicuous being B chromosome drive and A chromosome drive suppression. Here, we show for the first time that most microspermatids formed during spermiogenesis in two grasshopper species contain expulsed B chromosomes. By using DNA probes for B-specific satellite DNAs in Eumigus monticola and Eyprepocnemis plorans, and also 18S rDNA in the latter species, we were able to count the number of B chromosomes in standard spermatids submitted to fluorescence in situ hybridization, as well as visualizing B chromosomes inside most microspermatids. In E. plorans, the presence of B-carrying microspermatids in 1B males was associated with a significant decrease in the proportion of B-carrying standard spermatids. The fact that this decrease was apparent in elongating spermatids but not in round ones demonstrates that meiosis yields 1:1 proportions of 0B and 1B spermatids and hence that B elimination takes place post-meiotically, i.e., during spermiogenesis, implying a 5–25% decrease in B transmission rate. In E. monticola, the B chromosome is mitotically unstable and B number varies between cells within a same individual. A comparison of B frequency between round and elongating spermatids of a same individual revealed a significant 12.3% decrease. We conclude that B chromosome elimination during spermiogenesis is a defense weapon of the host genome to get rid of parasitic chromosomes.     

Comparative mitogenome analysis of three species and monophyletic inference of Catantopinae (Orthoptera: Acridoidea)

To enrich the genomic database of Catantopinae (Orthoptera: Acrididae), mitogenomes of three species from different genera, Traulia nigritibialis (15,701 bp), Choroedocus capensis (16,293 bp) and Stenocatantops splendens (15,574 bp), were characterized and compared with those of other grasshoppers in the subfamily. All 13 protein-coding genes (PCGs) were initiated by ATN codons except COI with ACC (C. capensis and S.splendens) and ND6 with TTG (S. splendens). All transfer RNA (tRNA) genes had a typical clover-leaf structure, except tRNA^Ser(AGN) in which the base pairs of the dihydrouridine (DHU) arm were reduced. The phylogenetic relationships were constructed among 22 species from four subfamiles of Acrididae by classical classifications based on two datasets of their mitogenomes using both Bayesian Inference (BI) and Maximum Likelihood (ML). The phylogenetic analysis confirmed the monophyly of the three other subfamilies, but did not provided support of the monophyly of Catantopinae.     

Analysis of three multigene families as useful tools in species characterization of two closely-related species, Dicentrarchus labrax, Dicentrarchus punctatus and their hybrids

By analyzing three multigene families, two closely related and commercially important species, Dicentrarchus labrax and Dicentrarchus punctatus, were characterized by cytogenetic and molecular methods. The interspecies hybrid Dicentrarchus labrax (♀) × Dicentrarchus punctatus (♂) was also analyzed. The multigene families studied were the 5S rDNA, 45S rDNA and the U2 snRNA. A microsatellite GTT motif was found within the non transcribed spacers (NTS) of the 5S rDNA from the two species. However, hexanucleotide duplication next to this microsatellite was observed in the D. labrax and hybrid clones, but not in D. punctatus. The U2 snRNA appeared to be linked to the U5 gene and showed two variant sequences, in both D. labrax and D. punctatus. They differed in one insertion/deletion of 7 nucleotides. The first internal transcribed spacer (ITS-1) region showed higher nucleotide variability in D. punctatus than in D. labrax. Nucleotide polymorphism within species and also nucleotide divergence between species were determined in the different gene regions. In a FISH analysis we obtained three chromosomal markers, because the 5S rDNA, 18S rDNA and U2 snRNA probes hybridized each in three different chromosome pairs. Hence none of them was co-localized. The 5S rDNA cluster and U2 snRNA were localized in acrocentric chromosome pairs, while the 18S rRNA gene probe hybridized in a subtelocentric pair. Finally, the usefulness of the results in developing tools for phylogenetic analysis and species identification are discussed in relation to other fish species.     

Microsatellite Organization in the Grasshopper Abracris flavolineata (Orthoptera: Acrididae) Revealed by FISH Mapping: Remarkable Spreading in the A and B Chromosomes

With the aim of acquiring deeper knowledge about repetitive DNAs chromosomal organization in grasshoppers, we used fluorescent in situ hybridization (FISH) to map the distribution of 16 microsatellite repeats, including mono-, di-, tri- and tetra-nucleotides, in the chromosomes of the species Abracris flavolineata (Acrididae), which harbors B chromosome. FISH revealed two main patterns: (i) exclusively scattered signals, and (ii) scattered and specific signals, forming evident blocks. The enrichment was observed in both euchromatic and heterochromatic areas and only the motif (C)₃₀ was absent in heterochromatin. The A and B chromosomes were enriched with all the elements that were mapped, being observed in the B chromosome more distinctive blocks for (GA)₁₅ and (GAG)₁₀. For A complement distinctive blocks were noticed for (A)₃₀, (CA)₁₅, (CG)₁₅, (GA)₁₅, (CAC)₁₀, (CAA)₁₀, (CGG)₁₀, (GAA)₁₀, (GAC)₁₀ and (GATA)₈. These results revealed an intense spreading of microsatellites in the A. flavolineata genome that was independent of the A+T or G+C enrichment in the repeats. The data indicate that the microsatellites compose the B chromosome and could be involved in the evolution of this element in this species, although no specific relationship with any A chromosome was observed to discuss about its origin. The systematic analysis presented here contributes to the knowledge of repetitive DNA chromosomal organization among grasshoppers including the B chromosomes.     

Chromosome evolution in ground beetles: localization of the rDNA loci in the tribe Harpalini (Coleoptera,Carabidae)

The pattern of localization of the ribosomal genes was studied by means of fluorescence in situ hybridization in 39 species of the tribe Harpalini. Most of them show one pair of autosomes carrying the ribosomal genes in a distal position of a single chromosome arm. This pattern is hypothesized to be ancestral for the whole tribe. Both, chromosome number and the number and localization of rDNA loci, show little variation and are therefore of little phylogenetic value. Only in the subtribe Ditomina is there enough variation to characterize phyletic relationships. The stability of rDNA loci is even higher than the constancy of chromosome number, as most species of Ditomina (genera Dixus, Eocarterus, Carterus, Odontocarus and Ditomus) have the usual pair of autosomes with rDNA loci, in spite of remarkable differences in the diploid number. Only Dixus sphaerocephalus and Dixus clypeatus have two autosomal pairs with a fluorescent signal. These results do not support the hypothesis that the high chromosome numbers found within Ditomina are the result of polyploid change from the ancestral 2n = 37 karyotype of the tribe Harpalini. Chromosomal translocations or the presence of mobile genetic elements are plausible sources of the few cases of intraspecific polymorphism in the rDNA loci found in species of Harpalus.     

Molecular cytogenetic analysis reveals the existence of two independent neo-XY sex chromosome systems in Anatolian Pamphagidae grasshoppers

Background

Neo-XY sex chromosome determination is a rare event in short horned grasshoppers, but it appears with unusual frequency in the Pamphagidae family. The neo-Y chromosomes found in several species appear to have undergone heterochromatinization and degradation, but this subject needs to be analyzed in other Pamphagidae species. We perform here karyotyping and molecular cytogenetic analyses in 12 Pamphagidae species from the center of biodiversity of this group in the previously-unstudied Anatolian plateau.

Results

The basal karyotype for the Pamphagidae family, consisting of 18 acrocentric autosomes and an acrocentric X chromosome (2n♂?=?19, X0; 2n♀?=?20, XX), was found only in G. adaliae. The karyotype of all other studied species consisted of 16 acrocentric autosomes and a neo-XY sex chromosome system (2n♂♀?=?18, neo-XX♀/neo-XY♂). Two different types of neo-Y chromosomes were found. One of them was typical for three species of the Glyphotmethis genus, and showed a neo-Y chromosome being similar in size to the XR arm of the neo-X, with the addition of two small subproximal interstitial C-blocks. The second type of the neo-Y chromosome was smaller and more heterochromatinized than the XR arm, and was typical for all Nocarodeini species studied. The chromosome distribution of C-positive regions and clusters of ribosomal DNA (rDNA) and telomeric repeats yielded additional information on evolution of these neo-XY systems.

Conclusion

Most Pamphagidae species in the Anatolian region were found to have neo-XY sex chromosome systems, belonging to two different evolutionary lineages, marked by independent X-autosome fusion events occurred within the Trinchinae and Pamphaginae subfamilies. The high density of species carrying neo-XY systems in the Anatolian region, and the different evolutionary stage for the two lineages found, one being older than the other, indicates that this region has a long history of neo-XY sex chromosome formation.

     

A cytogenetic analysis in Psophus stridulus (L.) (Orthoptera: Acrididae): B-chromosomes and abnormal spermatid nuclei

The male chromosome complement of Psophus stridulus (L.) (Orthoptera: Acrididae) has been analyzed by using orcein staining, C-banding and silver impregnation. During spermatogenesis only one pair of autosomes (M₉) shows an active nucleolar organizer region located in a C-banded constriction. There are other chromosome pairs with constrictions but these do not show nucleolar activity. The relationship between these constrictions and the C-banding pattern exhibited by this species is analyzed.In a sample of 83 males from five populations, two different supernumerary chromosomes were observed. Four males had a metacentric B-chromosome (B^m) similar in size to the sex chromosome and mitotically stable. Its meiotic behaviour indicates that it is an isochromosome. An additional small B-chromosome (B⁸) was also found in a single follicle of one individual carrying the B^m.A high rate of abnormal spermatids (macrospermatids) was scored in the individuals carrying B's. This proportion is notably higher in the follicle containing both the B^m and the B⁸.     

Polymorphisms in bovine immune genes and their associations with somatic cell count and milk production in dairy cattle

Background

Chromosome rearrangements are an important part of the speciation process in many taxa. The study of chromosome evolution in bivalves is hampered by the absence of clear chromosomal banding patterns and the similarity in both chromosome size and morphology. For this reason, obtaining good chromosome markers is essential for reliable karyotypic comparisons. To begin this task, the chromosomes of the mussels Brachidontes puniceus and B. rodriguezi were studied by means of fluorochrome staining and fluorescent in situ hybridization (FISH).

Results

Brachidontes puniceus and B. rodriguezi both have 2n = 32 chromosomes but differing karyotype composition. Vertebrate-type telomeric sequences appear at both ends of every single chromosome. B. puniceus presents a single terminal major rRNA gene cluster on a chromosome pair while B. rodriguezi shows two. Both mussels present two 5S rDNA and two core histone gene clusters intercalary located on the long arms of two chromosome pairs. Double and triple-FISH experiments demonstrated that one of the 5S rDNA and one of the major rDNA clusters appear on the same chromosome pair in B. rodriguezi but not in B. puniceus. On the other hand, the second 5S rDNA cluster is located in one of the chromosome pairs also bearing one of the core histone gene clusters in the two mussel species.

Conclusion

Knowledge of the chromosomal distribution of these sequences in the two species of Brachidontes is a first step in the understanding of the role of chromosome changes on bivalve evolution.