5S rDNA Probes Research Articles

Chromosomal mapping of 5S rDNA in two species of the genus Acanthocephalus (Echinorhynchida)

Chromosomal mapping of 5S rDNA in two Acanthocephala species was performed for the first time using fluorescence in situ hybridization (FISH) with a 5S rDNA probe. The 5S rDNA PCR products from the genomes of both species were sequenced and aligned and an identical 141 bp long coding region was determined. The same patterns of 5S rDNA gene cluster distribution were observed, with FISH signal restricted to a single autosomal chromosome pair. A preference for distal positioning on the chromosomes (subtelomeric position) was observed in both species. In addition, two-color FISH was performed to examine the mutual positions of 5S and 18S rDNA on the chromosomes. Our knowledge of the organization of the Acanthocephala genome is extremely limited and its chromosomes are poorly studied. Any new information about the location of chromosomal markers as important features of the respective karyotype may be useful in solving evolutionary questions.

Comparative molecular and conventional cytogenetic analyses of three species of Rhinella (Anura; Bufonidae).

The genus Rhinella corresponds to a group of anurans characterized by numerous taxonomic and systemic challenges, leading to their organization into species complexes. Cytogenetic data for this genus thus far are limited to the diploid number and chromosome morphology, which remain highly conserved among the species. In this study, we analyse the karyotypes of three species of the genus Rhinella (Rhinella granulosa, Rhinella margaritifera, and Rhinella marina) using both classical (conventional staining and C-banding) and molecular (FISH-fluorescence in situ hybridization with 18S rDNA, telomeric sequences, and microsatellite probes) cytogenetic approaches. The aim of this study is to provide data that can reveal variations in the distribution of repetitive sequences that can contribute to understanding karyotypic diversification in these species. The results revealed a conserved karyotype across the species, with 2n = 22 and FN = 44, with metacentric and submetacentric chromosomes. C-banding revealed heterochromatic blocks in the pericentromeric region for all species, with a proximal block on the long arms of pairs 3 and 6 in R. marina and on the short arms of pairs 4 and 6 in R. margaritifera. Additionally, 18S rDNA probes hybridized to pair 5 in R. granulosa, to pair 7 in R. marina, and to pair 10 in R. margaritifera. Telomeric sequence probes displayed signals exclusively in the distal region of the chromosomes, while microsatellite DNA probes showed species-specific patterns. These findings indicate that despite a conserved karyotypical macrostructure, chromosomal differences exist among the species due to the accumulation of repetitive sequences. This variation may be attributed to chromosome rearrangements or differential accumulation of these sequences, highlighting the dynamic role of repetitive sequences in the chromosomal evolution of Rhinella species. Ultimately, this study emphasizes the importance of the role of repetitive DNAs in chromosomal rearrangements to elucidate the evolutionary mechanisms leading to independent diversification in the distinct phylogenetic groups of Rhinella.

Mapping of Repetitive Sequences in Brachyhypopomus brevirostris (Hypopomidae, Gymnotiformes) from the Brazilian Amazon.

Brachyhypopomus (Hypopomidae, Gymnotiformes) is a monophyletic genus consisting of 28 formally described species. Karyotypic data are available for 12 species. The same karyotype is described for two species (B. brevirostris and B. hamiltoni), as well as different karyotypes for the same species from distinct locations (B. brevirostris). In this context, B. brevirostris may constitute a cryptic species complex. Thus, in the present study, we analyzed the karyotype of B. brevirostris, from Santarém, Pará, and Tefé, Amazonas, using classical cytogenetics (conventional staining and C-banding) and molecular techniques (fluorescence in situ hybridization using 18S rDNA, 5S rDNA, U2 snRNA, and telomeric probes). The results show that samples from both locations present 2n = 38, with all chromosomes being acrocentric (FC = 38a). In both populations, 18S rDNA sequences are present on only one pair of homologous chromosomes and telomeric sequences occur only at the ends of the chromosomes. In the Tefé sample, the 5S rDNA occurs in two pairs, and the U2 snRNA in three pairs. These results are the first descriptions of these sequences for B. brevirostris samples from the Tefé locality, as well as the first karyotypic description for the Santarém locality. Future cytotaxonomic studies of this genus can benefit from these results.

Comparative Cytogenetics in Tyrannidae (Aves, Passeriformes): High Genetic Diversity despite Conserved Karyotype Organization

Introduction: Passeriformes has the greatest species diversity among Neoaves, and the Tyrannidae is the richest in this order with about 600 valid species. The diploid number of this family remains constant, ranging from 2n = 76 to 84, but the chromosomal morphology varies, indicating the occurrence of different chromosomal rearrangements. Cytogenetic studies of the Tyrannidae remain limited, with approximately 20 species having been karyotyped thus far. This study aimed to describe the karyotypes of two species from this family, Myiopagis viridicata and Sirystes sibilator. Methods: Skin biopsies were taken from each individual to establish fibroblast cell cultures and to obtain chromosomal preparations using the standard methodology. The chromosomal distribution of constitutive heterochromatin was investigated by C-banding, while the location of simple repetitive sequences (SSRs), 18S rDNA, and telomeric sequences was found through fluorescence in situ hybridization. Results: The karyotypes of both species are composed of 2n = 80. The 18S rDNA probes hybridized into two pairs of microchromosomes in M. viridicata, but only a single pair in S. sibilator. Only the telomeric portions of each chromosome in both species were hybridized by the telomere sequence probes. Most of the SSRs were found accumulated in the centromeric and telomeric regions of several macro- and microchromosomes in both species, which likely correspond to the heterochromatin-rich regions. Conclusion: Although both species analyzed showed a conserved karyotype organization (2n = 80), our study revealed significant differences in their chromosomal architecture, rDNA distribution, and SSR accumulation. These findings were discussed in the context of the evolution of Tyrannidae karyotypes.

Karyological Study of Acanthocephalus lucii (Echinorhynchida): The Occurrence of B Chromosomes in Populations from PCB-Polluted Waters

In this study, we performed a cytogenetic analysis of Acanthocephalus lucii specimens from three sites with different levels of environmental pollution. Standard and fluorochrome staining (CMA3/DAPI), fluorescence in situ hybridization (FISH) with 18S rDNA and histone H3 probes, and silver impregnation were performed. Chromosome complements of 2n = 7/8 (male/female), n = 1m + 2sm + 1a (X), and CMA3-positive bands in all chromosomes were found in all three populations. FISH revealed one 18S rDNA locus on the X chromosome and one locus of H3 histone genes on the first chromosome pair. At the intraspecific level, the populations differed in the presence of supernumerary B chromosomes, which were found in all specimens from Zemplínska Šírava and in 89.4% of specimens from the Laborec River, but not at the reference site. The first two sites are considered to be water bodies with high toxin contamination. Based on this fact, we assume an increased frequency of chromosome breaks leading to the formation of DNA fragments that have the potential to form B chromosomes. The present results add to the very limited data on the organization of multigene families in the genome of Acanthocephala and suggest a possible causal link between water pollution and the occurrence of B chromosomes in fish parasites.

Initial Steps of XY Sex Chromosome Differentiation in the Armored Catfish Hypostomus albopunctatus (Siluriformes: Loricariidae) Revealed by Heterochromatin Accumulation.

In fish species, heterochromatinization is one process that could trigger sex chromosome differentiation. The present article describes a nascent XX/XY sex chromosome system evidenced by heterochromatin accumulation and microsatellite (GATA)8 in Hypostomus albopunctatus from two populations of the Paraná River basin. The specimens of H. albopunctatus from the Campo and Bossi Rivers share the same karyotype. The species exhibits 74 chromosomes (8m+14sm +16st +36a, fundamental number = 112). The C-banding technique suggests male heterogamety in H. albopunctatus, where the Y-chromosome is morphologically like the X-chromosome but differs from it for having long arms that are entirely heterochromatic. Double fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes confirmed the Ag-nucleolus organizer region sites in a single pair for both populations, and minor rDNA clusters showed interpopulational variation. FISH with the microsatellite (GATA)8 probe showed a dispersed pattern in the karyotype, accumulating these sequences of sex chromosomes of both populations. FISH with microsatellite (CGC)10 probe showed interpopulational variation. The absence of differentiated sex chromosomes in H. albopunctatus is described previously, and a new variant is documented herein where XY chromosomes can be seen in an early stage of differentiation.

Comparative karyotypic study of fifteen cyprinids (Cyprinidae, Cyprininae) species. An insight into the chromosomal evolution of the tribe Systomini.

The family Cyprinidae is the largest freshwater fish group with 377 genera and over 3,000 described species. However, this group of fish has very limited cytogenetics and advanced molecular cytogenetics information. Therefore, in this study the karyotypes and other chromosomal characteristics of 15 species in the tribe Systomini (Cyprininae) were examined using Ag-NOR staining along with fluorescence in situ hybridization (5S and 18S rDNA). All species share a similar karyotype (2n = 50; NF = 88-100) in both sexes and no differentiated sex chromosome was observed. Chromosomes bearing NOR sites ranged from one to four pairs among the species, mostly mapped adjacent to telomeres in the short arms of distinct pairs in all analyzed species. This difference indicates an extensive rearrangement of chromosomes including genomic differences. The use of the 5S and 18S rDNA probe confirmed the Ag-NOR sites interstitially located in the telomeric regions of distinct chromosomes, characterizing an interspecies variation of these sites. In most of its analyzed species, the signals of 18S rDNA probe corresponded to the Ag-NOR regions, except in Barbonymus altus, B. gonionotus, B. schwanenfeldii and Puntius brevis having these signals on the same as Ag-NOR regions and other sites.

Epigenetic histone H3 phosphorylation marks discriminate between univalent- and bivalent-forming chromosomes during canina asymmetrical meiosis.

Dogroses (Rosa sect. Caninae) are mostly pentaploid, bearing 2n = 5x = 35 chromosomes in somatic cells. They evolved a unique form of asymmetrical meiosis characterized by two types of chromosomes: (1) chromosomes forming bivalents and distributed in the normal sexual way; and (2) chromosomes occurring as univalents and transferred by a female gamete only. In the mature pollen of pentaploid species, seven bivalent-derived chromosomes are transmitted to offspring, and 21 unpaired univalent chromosomes are eliminated during microsporogenesis. To discriminate between bivalent- and univalent-forming chromosomes, we studied histone H3 phosphorylation patterns regulating meiotic chromosome condensation and segregation. We analysed histone modification patterns during male canina meiosis in two representative dogrose species, 5x Rosa canina and 5x Rosa rubiginosa, by immunohistochemical and molecular cytogenetics approaches. Immunostaining of meiotic cells included α-tubulin, histone H3 phosphorylation (H3S10p, H3S28p and H3T3p) and methylation (H3K4me3 and H3K27me3) marks. In addition, fluorescent in situ hybridization was carried out with an 18S rDNA probe. In the first meiotic division, univalent chromosomes underwent equational division into chromatids, while homologues in bivalents were segregated as regular dyads. In diakinesis, bivalent chromosomes displayed strong H3 phosphorylation signals in proximal regions, spreading to the rest of the chromosome. In contrast, in univalents, the H3 phosphorylation signals were weaker, occurring mostly outside proximal regions largely overlapping with the H3K4me3 signals. Reduced phosphorylation was associated with relative under-condensation of the univalent chromosomes, particularly at early diakinesis. We hypothesize that the absence of pairing and/or recombination in univalent chromosomes negatively affects the histone H3 phosphorylation of their chromatin and perhaps the loading of meiotic-specific cohesins. This apparently destabilizes cohesion of sister chromatids, leading to their premature split in the first meiotic division.

Contributions of species Rineloricaria pentamaculata (Loricariidae:Loricariinae) in a karyoevolutionary context

Species of Rineloricaria demonstrate an interesting evolutionary history from a cytogenetic point of view, due to the occurrence of extensive variation in diploid number (2n=36 -70 chromosomes), with Robertsonian rearrangements mostly responsible for this karyotypic diversity. In this study we present the karyotypic data for a population of Rineloricaria pentamaculata, collected in the Itiz stream, a tributary of the Paraná River Basin (Paraná, Brazil), which exhibited 2n=56 chromosomes distributed in 8m/sm+48st/a (number fundamental equal to 64) and simple NOR system revealed by fluorescent in situ hybridization (FISH) with 18S rDNA probe, silver nitrate and positive C band, located on the first submetacentric chromosome pair (pair 5). In addition, the NOR pair showed a size heteromorphism for this region, rich in GC composition (positive CMA3). Clusters of 5S rDNA were located in 14 chromosomes and the FISH with a telomeric probe was used to map possible evidence of chromosomal fusions, however, it showed only telomeric sites. These results corroborate the data for the species R. pentamaculata and the genus Rineloricaria, showing that they are similar to most of the populations analyzed. About the cytogenetic data of R. pentamaculata, we reaffirm that most populations were conserved, but in those with derived characteristics, Robertsonian chromosomal rearrangements probably contributed to the karyotypic evolution of the group.

Chromosome Mapping of Repetitive DNAs in the Picasso Triggerfish (Rhinecanthus aculeatus (Linnaeus, 1758)) in Family Balistidae by Classical and Molecular Cytogenetic Techniques

This work presents the cytogenetic analysis conducted on the Picasso triggerfish (Rhinecanthus aculeatus (Linnaeus, 1758)) from Thailand. Mitotic chromosomes were prepared from the anterior kidney. The cell suspensions were harvested by in vivo colchicine treatment. The present study includes the chromosomal investigation on R. aculeatus, using conventional (Giemsa staining, Ag-NOR and C-banding) and molecular approaches (in situ mapping of five different repetitive DNA classes including 18S rDNA, 5S rDNA, (CA)15, (GA)15 and (CAA)10 as markers.) The results showed that R. aculeatus has karyotypes formed exclusively by telocentric chromosomes (44t; NF=44). The C-positive heterochromatic blocks are preferentially located in the centromeric and telomeric regions of some chromosomal pairs. The Ag-NOR sites occupy the interstitial position of the long arms of the largest telocentric pair (pair 1). The exclusive location of the major ribosomal sites in these pairs was confirmed by hybridization with 18S rDNA probes. However, the 5S rDNA genes are not located on 18S rDNA-bearing chromosomes, but instead located exclusively in the subcentromeric region of pair 4. The mapping of (CA)15, (GA)15 and (CAA)10 microsatellites are sparsely dispersed along all the chromosomes. The karyotype formula of R. aculeatus is 2n (44) = 44t.

Cytotaxonomy and karyotype evolution in Neotropical Meliponini (Hymenoptera: Apidae) inferred by chromosomal mapping of 18S rDNA and five microsatellites

The Neotropical Meliponini bees, commonly known as stingless bees, are phylogenetically subdivided into three clades in which the chromosome numbers vary from n = 8 to n = 17. The goal of this study was to identify the major chromosomal rearrangements that occurred during the Neotropical Meliponini (Apidae) karyotypic evolution. In this way, we mapped 18S rDNA and five microsatellites in 33 stingless bee species collected from different Brazilian regions. The species belonged to 15 genera and showed six different chromosome numbers: n = 8, n = 9, n = 11, n = 14, n = 15, and n = 17. The 18S rDNA probe showed a variation from 2 to 12 marked chromosomes in different positions (terminal, subterminal, or centromeric), including 2 B chromosomes out of the 7 B found in Tetragonisca fiebrigi. The microsatellite (GA)15, (GAG)10, (CAA)10, and (TCAGG)6 probes formed clusters on the euchromatic regions of the chromosomes and were useful in the identification of putative Robertsonian fusion events that led to the decrease in the chromosome number during the evolution of the Neotropical Meliponini clade. (TTAGG)6 constituted the telomeric sequence of the analyzed species. The ancestral state of the three Neotropical Meliponini clades is difficult to infer, although, the putative ancestral karyotype probably had a single pair of 18S rDNA cistrons, and the decrease in chromosome number and increase in the 18S rDNA sites occurred independently between genera.

Exploring Fish Miscellany from Biodiversity Hotspot Region of Northeast India Through Chromosome Profiling

Abstract North Eastern states of India are blessed with extensive ecoclimatic conditions and much of India’s endemic flora and fauna. This region is rich in fish diversity with plenty of species of ornamental importance, including model species zebrafish. Among the potential ornamental fishes, Ctenops nobilis is designated as Near Threatened and Danio dangila is included in the Least Concern list by IUCN. Cytogenetic profiling was undertaken here to provide complementary data for precise species identification and to study genetic inheritance, genome organization and evolution of the species. The cytogenetic analyses revealed diploid chromosome numbers 44 and 50 with karyotype formula 8m+20sm+10st+6t (FN=72) and 20m+24sm+6st (FN=94), respectively, in C. nobilis and D. dangila. The staining of nucleolar organizer regions revealed the presence of Ag-NORs, CMA3 sites and 18S rDNA probe signals on one pair sub-telocentric chromosome and one pair sub-metacentric chromosome in C. nobilis and D. dangila, respectively. These biomarkers are an important resource for molecular taxonomy, evolutionary/phylogenetic studies and conservation genetics of C. nobilis and D. dangila.

Comparative molecular cytogenetics in five species of stingless bees (Hymenoptera, Apidae)

Repetitive DNA sequences constitute a large portion of the eukaryotic genome. Despite their importance, only a few Meliponini species, especially those from the Melipona Illiger, 1806 genus, had some microsatellite sequences and the rDNA genes physically mapped. Therefore, in order to expand our knowledge on this genomic component of neotropical bees, in the present study we investigated the distribution of microsatellite sequences, including the telomeric repeat (TTAGG)6 and rDNA clusters, in the chromosomes of five stingless species, Cephalotrigona capitata (Smith, 1854), Partamona auripennis Pedro & Camargo, 2003, P. cupira (Smith, 1863), P. helleri (Friese 1900), and Tetragona elongata (Lepeletier & Serville, 1828). The microsatellite probes (GA)15, (GAG)10, and (CAA)10, revealed hybridization signals in the euchromatic regions of most chromosome pairs. The (GAG)10 probe, however, also yielded a positive signal in the heterochromatic arm of one chromosome pair of T. elongata. The telomeric (TTAGG)6 sequence hybridized to the ends of chromosome pairs, while signals for the 18S rDNA probe varied among the species. Here was demonstrated the karyotypic diversity that exists among five species of stingless bees in terms of repetitive sequences. These cytogenetic marker provides new information about the chromatin composition and rDNA cluster locations for five Meliponini species (three genera) and highlights the contribution of repetitive sequences to the karyotypic evolution of this tribe.

Chromosomal Diversification in Pseudacanthicus Species (Loricariidae, Hypostominae) Revealed by Comparative Mapping of Repetitive Sequences.

Simple SummaryThe fishes of the Loricariidae family have a huge genetic diversity, mainly involving variations in the number and shape of chromosomes. The recognition of the species genus Pseudacanthicus is complex due to the large diversity of forms and limited knowledge of their genetic diversity. In this study, the karyotypes of three Pseudacanthicus species were comparatively analyzed using classical and molecular methods. They presented the same diploid number, but with different compositions of repetitive DNA sequences. Such information can be useful for the recognition of distinct species, in addition to providing important insights into the real biodiversity of this important group of Neotropical fish.Pseudacanthicus is a genus of Neotropical fish with eight valid species, in addition to numerous lineages not formally identified. It occurs along the Amazon and Tocantins River basins, in Suriname and in the Guiana shield. There are no karyotypic data in the literature for species of this genus. Here, the karyotypes of three Pseudacanthicus species (P. spinosus, P. leopardus and Pseudacanthicus sp.) were comparatively analyzed by classical cytogenetics and fluorescence in situ hybridization using 18S and 5S rDNA probes, U2 snDNA and telomeric sequences. The analyzed species presented 52 chromosomes and KF = 18 m + 34 sm. Constitutive heterochromatin occurred in blocks on a few chromosomes. The 18S rDNA occurred in a single pair; interestingly, P. leopardus presented only one locus of this sequence in its diploid genome. The 5S rDNA sequence occurred in only one pair in P. leopardus, and in multiple sites in Pseudacanthicus sp. and P. spinosus. The snDNA U2 occurred in only one pair in all analyzed species. Telomeric sequences did not show interstitial sites. Although Pseudacanthicus species share the same 2n and KF, repetitive sequence analysis revealed karyotypic diversity among these species. The occurrence of DNA double-strand breaks related to fragile sites, unequal crossing over and transpositions is proposed as the mechanism of karyotypic diversification, suggesting that the conservation of the karyotypic macrostructure is only apparent in this group of fish.

The Chromosome Number and rDNA Loci Evolution in Onobrychis (Fabaceae).

The evolution of chromosome number and ribosomal DNA (rDNA) loci number and localisation were studied in Onobrychis Mill. Diploid and tetraploid species, as well as two basic chromosome numbers, x = 7 and x = 8, were observed among analysed taxa. The chromosomal distribution of rDNA loci was presented here for the first time using fluorescence in situ hybridisation (FISH) with 5S and 35S rDNA probes. Onobrychis species showed a high polymorphism in the number and localisation of rDNA loci among diploids, whereas the rDNA loci pattern was very similar in polyploids. Phylogenetic relationships among the species, inferred from nrITS sequences, were used as a framework to reconstruct the patterns of basic chromosome number and rDNA loci evolution. Analysis of the evolution of the basic chromosome numbers allowed the inference of x = 8 as the ancestral number and the descending dysploidy and polyploidisation as the major mechanisms of the chromosome number evolution. Analyses of chromosomal patterns of rRNA gene loci in a phylogenetic context resulted in the reconstruction of one locus of 5S rDNA and one locus of 35S rDNA in the interstitial chromosomal position as the ancestral state in this genus.

Chromosome characterization and physical mapping of 18S rDNA in Lilium longiflorum originated interspecific hybrids using combined genomic and fluorescent in situ hybridization

This study was aimed to differentiate parental genomes, examining intergenomic composition, and mapping mitotic metaphase chromosomes by localizing parental and 18S rDNA probes in seven interspecific hybrid progenies that originated from Lilium longiflorum. Since single method of in situ hybridization has been widely used in interspecific lily breeding, while in this study, flow cytometry was used in conjunction with genomic and fluorescent in situ hybridization to determine the genomic contribution of each parent to the interspecific progenies. A significant variation was observed in the DNA content, chromosome length, and 18S loci in F1 as compared to the female and male parents. L. longiflorum showed nearly two times higher DNA content than the male parents and L. longiflorum × Asiatic progenies, but eight times higher than L. longiflorum × L. hansonii. Genomic in situ hybridization results revealed that both female and male parents contributed an equal number of chromosomes to their interspecific F1 offspring. Fluorescent in situ hybridization mapping revealed that 18S rDNA had 8, 6 and 7 loci in L. longiflorum parents, i.e., White heaven, Bright tower, and White tower, respectively, whereas each Asiatic cultivar and L. hansonii used as male showed 8 and 12 loci respectively. Interspecific progenies showed 8 and 7 loci in LA, and 10–11 in LM hybrids. These cytogenetic results implied equal genetic and chromosomal contribution from both parents to their intergenomic progenies. Therefore, this combined cytogenetic method has the potential to be genome specific and time-saving approach in lily breeding that could determine the status of hybrids and their genomic origin while achieving physical mapping and detecting genes in different genomes.

The Divergence of Chromosome Structures and 45S Ribosomal DNA Organization in Cucumis debilis Inferred by Comparative Molecular Cytogenetic Mapping.

Cucumis debilis W.J.de Wilde & Duyfjes is an annual and monoecious plant. This species is endemic to Southeast Asia, particularly Vietnam. However, C. debilis is rarely studied, and no detailed information is available regarding its basic chromosome number, 45S ribosomal DNA (rDNA) status, and divergence among other Cucumis species. In this study, we characterized the morphological characters and determined and investigated the basic chromosome number and chromosomal distribution of 45S rDNA of C. debilis using the fluorescent in situ hybridization (FISH) technique. A maximum likelihood tree was constructed by combining the chloroplast and internal transcribed spacer of 45S rDNAs to infer its relationship within Cucumis. C. debilis had an oval fruit shape, green fruit peel, and protrusion-like white spots during the immature fruit stage. FISH analysis using 45S rDNA probe showed three pairs of 45S rDNA loci located at the terminal region in C. debilis, similar to C. hystrix. Meanwhile, two, two, and five pairs of 45S rDNA loci were observed for C. melo, C. metuliferus, and C. sativus, respectively. One melon (P90) and cucumber accessions exhibited different chromosomal localizations compared with other members of Cucumis. The majority of Cucumis species showed the terminal location of 45S rDNA, but melon P90 and cucumber exhibited terminal–interstitial and all interstitial orientations of 45S rDNA loci. Based on molecular cytogenetics and phylogenetic evidence, C. debilis is more closely related to cucumber than melon. Therefore, C. debilis may serve as a potential parental accession for genetic improvement of cucumber through interspecific hybridization.

ESTUDIO CITOGENÉTICO EN ARAÑAS DE ARENA (SICARIIDAE) DE LA CAATINGA BRASILEÑA: DIVERSIDAD DEL SISTEMA DE CROMOSOMAS SEXUALES EN ESPECIES ESTRECHAMENTE RELACIONADAS

In this study, we investigated the chromosomes of three species of Sicarius spiders from the Brazilian Caatinga, using classical and molecular cytogenetic techniques. Based on the phylogenetic approach, we also discussed about the variation of diploid number, types of sex chromosome system and changes in the localization of ribosomal genes of Scytodoidea. Sicarius are Synspermiata spiders that together with the genera Loxosceles and Hexophthalma constitute the family Sicariidae. In this group, the available cytogenetic data showed a low diploid number range (2n♂=18 to 2n♂=23) and the presence of only multiple sex chromosome systems (X1X2Y and X1X20). Mitotic metaphase cells exhibited 2n♂=16+X1X2Y for Sicarius cariri and S. ornatus, and 2n♂=18+XY for S. tropicus. In these species, silver impregnation revealed nucleolar organizer region (Ag-NOR) on the terminal region of pair 1. In S. ornatus and S. tropicus, the results obtained with fluorescent in situ hybridization (FISH) using 18S rDNA probe were similar to Ag-NOR, however in S. cariri, the ribosomal sites were localized in the terminal region of the X1 sex chromosome. In this work, we presented the first description of a simple sex chromosome system for Sicariidae, helping to understand how the XY sex chromosome system evolved from the X1X2Y system. Additionally, FISH data incongruous with Ag-NOR indicate that the cytogenetic studies in Sicariidae allow investigating the relation between the karyotype evolution and the distribution and the activity of rDNA genes. Key words: karyotype, mitosis, nucleolar organizer region, rDNA, Sicarius

Unraveling the DNA Methylation in the rDNA Foci in Mutagen-Induced Brachypodium distachyon Micronuclei.

Many years have passed since micronuclei were first observed then accepted as an indicator of the effect of mutagens. However, the possible mechanisms of their formation and elimination from the cell are still not fully understood. Various stresses, including mutagens, can alter gene expression through changes in DNA methylation in plants. In this study we demonstrate for the first time DNA methylation in the foci of 5S and 35S rDNA sequences in individual Brachypodium distachyon micronuclei that are induced by mutagenic treatment with maleic acid hydrazide (MH). The impact of MH on global epigenetic modifications in nuclei and micronuclei has been studied in plants before; however, no in situ analyses of DNA methylation in specific DNA sequence sites are known. To address this problem, we used sequential immunodetection of 5-methylcytosine and fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes on the non-dividing cells of B. distachyon. Such investigations into the presence or absence of DNA methylation within specific DNA sequences are extremely important in plant mutagenesis in the light of altering gene expression.

Study and Physical Mapping of the Species-Specific Tandem Repeat CS-237 Linked with 45S Ribosomal DNA Intergenic Spacer in Cannabis sativa L.

Hemp (Cannabis sativa L.) is a valuable crop and model plant for studying sex chromosomes. The scientific interest in the plant has led to its whole genome sequencing and the determination of its cytogenetic characteristics. A range of cytogenetic markers (subtelomeric repeat CS-1, 5S rDNA, and 45S rDNA) has been mapped onto hemp’s chromosomes by fluorescent in situ hybridization (FISH). In this study, another cytogenetic marker (the tandem repeat CS-237, with a 237 bp monomer) was found, studied, and localized on chromosomes by FISH. The signal distribution and karyotyping revealed that the CS-237 probe was localized in chromosome 6 with one hybridization site and in chromosome 8 with two hybridization sites, one of which colocalizes with the 45S rDNA probe (with which a nucleolus organizer region, NOR, was detected). A BLAST analysis of the genomic data and PCR experiments showed that the modified CS-237 monomers (delCS-237, 208 bp in size) were present in the intergenic spacers (IGSs) of hemp 45S rDNA monomers. Such a feature was firstly observed in Cannabaceae species. However, IGS-linked DNA repeats were found in several plant species of other families (Fabaceae, Solanaceae, and Asteraceae). This phenomenon is discussed in this article. The example of CS-237 may be useful for further studying the phenomenon as well as for the physical mapping of hemp chromosomes.