Contribution of the satellitome to the exceptionally large genome of dinoflagellates: The case of the harmful alga Alexandrium minutum.

Dinoflagellates are known to possess an exceptionally large genome organized in permanently condensed chromosomes. Focusing on the contribution of satellite DNA (satDNA) to the whole DNA content of genomes and its potential role in the architecture of the chromosomes, we present the characterization of the satellitome of Alexandriun minutum strain VGO577. To achieve this, we analyzed Illumina reads using graph-based clustering and performed complementary bioinformatic analyses. In this way, we discovered 180 satDNAs occupying 17.38 % of the genome. The 12 most abundant satDNAs represent the half of the satellitome but no satDNA is overrepresented, with the most abundant contributing ∼1.56 % of the genome. The largest repeat unit is 517 bp long but more than the half of the satDNAs (101) have repeat units shorter than 20 bp. We used FISH to map a selected set of 26 satDNAs. Although some satDNAs generate discrete hybridization signals at specific chromosomal locations (hybridization sites, HS), our cytological analysis showed that most satDNAs are dispersed throughout the genome, probably forming short arrays. Two satDNAs co-localize with the 45S rDNA. With the exception of telomeric DNA, no other satDNA yields HS on all chromosomes. In addition, we analyzed nine satDNAs yielding HS in VGO577 in four other A. minutum strains. Polymorphism at the intraspecific level was found for the presence/absence and/or abundance of some satDNAs, suggesting the amplification/deletion of these satDNAs following geographic separation or during culture maintenance of the strains. We also discuss how these results contribute to the understanding of chromosome architecture and evolution of dinoflagellate genomes.

Differential Spreading of Microsatellites in Holocentric Chromosomes of Chagas Disease Vectors: Genomic and Evolutionary Implications.

This study focused on analyzing the distribution of microsatellites in holocentric chromosomes of the Triatominae subfamily, insect vectors of Chagas disease. We employed a non-denaturing FISH technique to determine the chromosomal distribution of sixteen microsatellites across twenty-five triatomine species, involving five genera from the two principal tribes: Triatomini and Rhodniini. Three main hybridization patterns were identified: strong signals in specific chromosomal regions, dispersed signals dependent on microsatellite abundance and the absence of signals in certain chromosomal regions or entire chromosomes. Significant variations in hybridization patterns were observed between Rhodniini and Triatomini species. Rhodniini species displayed weak and scattered hybridization signals, indicating a low abundance of microsatellites in their genomes. In contrast, Triatomini species exhibited diverse and abundant hybridization patterns, suggesting that microsatellites are a significant repetitive component in their genomes. One particularly interesting finding was the high abundance of GATA repeats, and to a lesser extent AG repeats, in the Y chromosome of all analyzed Triatomini species. In contrast, the Y chromosome of Rhodniini species did not show enrichment in GATA and AG repeats. This suggests that the richness of GATA repeats on the Y chromosome likely represents an ancestral trait specific to the Triatomini tribe. Furthermore, this information can be used to elucidate the evolutionary relationships between Triatomini and other groups of reduviids, contributing to the understanding of the subfamily's origin. Overall, this study provides a comprehensive understanding of the composition and distribution of microsatellites within Triatominae genomes, shedding light on their significance in the evolutionary processes of these species.

Migration of repetitive DNAs during evolution of the permanent translocation heterozygosity in the oyster plant (Tradescantia section Rhoeo).

Due to translocation heterozygosity for all chromosomes in the cell complement, the oyster plant (Tradescantia spathacea) forms a complete meiotic ring. It also shows Rabl-arrangement at interphase, featured by polar centromere clustering. We demonstrate that the pericentromeric regions of the oyster plant are homogenized in concert by three subtelomeric sequences: 45S rDNA, (TTTAGGG)n motif, and TSrepI repeat. The Rabl-based clustering of pericentromeric regions may have been an excellent device to combine the subtelomere-pericentromere sequence migration (via inversions) with the pericentromere-pericentromere DNA movement (via whole arm translocations) that altogether led to the concerted homogenization of all the pericentromeric domains by the subtelomeric sequences. We also show that the repetitive sequence landscape of interstitial chromosome regions contains many loci consisting of Arabidopsis-type telomeric sequence or of TSrepI repeat, and it is extensively heterozygous. However, the sequence arrangement on some chromosomal arms suggest segmental inversions that are fully or partially homozygous, a fact that could be explained if the inversions started to create linkages already in a bivalent-forming ancestor. Remarkably, the subterminal TSrepI loci reside exclusively on the longer arms that could be due to sharing sequences between similarly-sized chromosomal arms in the interphase nucleus. Altogether, our study spotlights the supergene system of the oyster plant as an excellent model to link complex chromosome rearrangements, evolution of repetitive sequences, and nuclear architecture.

First record of the spatial organization of the nucleosome-less chromatin of dinoflagellates: The nonrandom distribution of microsatellites and bipolar arrangement of telomeres in the nucleus of Gambierdiscus australes (Dinophyceae).

Dinoflagellates are a group of protists whose exceptionally large genome is organized in permanently condensed nucleosome-less chromosomes. In this study, we examined the potential role of repetitive DNAs in both the structure of dinoflagellate chromosomes and the architecture of the dinoflagellate nucleus. Non-denaturing fluorescent in situ hybridization (ND-FSH) was used to determine the abundance and physical distribution of telomeric DNA and 16 microsatellites (1- to 4-bp repeats) in the nucleus of Gambierdiscus australes. The results showed an increased relative abundance of the different microsatellite motifs with increasing GC content. Two ND-FISH probes, (A)20 and (AAT)5 , did not yield signals whereas the remainder revealed a dispersed but nonrandom distribution of the microsatellites, mostly in clusters. The bean-shaped interphase nucleus of G. australes contained a region with a high density of trinucleotides. This nuclear compartment was located between the nucleolar organizer region (NOR), located on the concave side of the nucleus, and the convex side. Telomeric DNA was grouped in multiple foci and distributed in two polarized compartments: one associated with the NOR and the other peripherally located along the convex side of the nucleus. Changes in the position of the telomeres during cell division evidenced their dynamic distribution and thus that of the chromosomes during dinomitosis. These insights into the spatial organization of microsatellites and telomeres and thus into the nuclear architecture of G. australes will open up new lines of research into the structure and function of the nucleosome-less chromatin of dinoflagellates.

Temperature-dependent growth and sexuality of the ciguatoxin producer dinoflagellate Gambierdiscus spp. in cultures established from the Canary Islands.

Benthic dinoflagellates of the genus Gambierdiscus produce ciguatoxins, compounds that when metabolized in fish and consumed by humans cause ciguatera poisoning (CP). This syndrome, which is widespread in tropical and subtropical regions, has recently been reported also in subtropical-temperate latitudes such as the Canary Islands where CP events have been regularly detected since 2004. This study examined the effect of temperature on the growth of Gambierdiscus isolated from Canary waters: G. australes, G. caribaeus, G. carolinianus, G. excentricus, and G. silvae. From the temperature vs. growth curves, the maximum growth (µm), optimum temperature range for growth (Topt), and the temperature yielding maximum growth (Tm) were estimated for each species. The results revealed temperature-dependent differences in the growth parameters. G. caribaeus had the highest Tm and Topt, followed by G. australes, G. carolinianus, G. silvae, and G excentricus. G. australes tolerated the widest range of temperatures (from 15 °C to 29 °C), which may explain its broader geographic distribution, both worldwide and across the Canary archipelago. Neither G. excentricus nor G. silvae survived at 29 °C whereas G. caribaeus reached mean growth rates (± standard deviation) up to 0.19 ± 0.01 div.day-1 at that temperature, followed by G. australes (0.16 ± 0.01 div.day-1) and G. carolinianus (0.14 ± 0.04 div.day-1). G. caribaeus showed no measurable growth at 19°C, whereas G. excentricus and G. silvae along with G. australes appeared as the species better adapted to lower temperatures. In an intraspecific variability study of 12 strains of G. australes, the mean (± standard deviation) of µm and Tm were 0.17 ± 0.01 div.day-1 and 27.7 ± 0.5 °C, respectively. An analysis of the shapes and position of the cell nuclei at the different temperatures showed that nuclei characteristic of vegetative cells appeared mainly at 26 °C but extreme temperatures resulted in nuclei with a more variable morphology. The presence of putative zygotes at extreme temperatures (17 °C, 19 °C and 29 °C) suggests that sexual reproduction is promoted as an adaptive strategy which could play an important role in the expansion of geographic distribution of Gambierdiscus species.

The 5S rRNA genes in Alexandrium: their use as a FISH chromosomal marker in studies of the diversity, cell cycle and sexuality of dinoflagellates.

Chromosomal markers of the diversity and evolution of dinoflagellates are scarce because the genomes of these organisms are unique among eukaryotes in terms of their base composition and chromosomal structure. Similarly, a lack of appropriate tools has hindered studies of the chromosomal localization of 5S ribosomal DNA (rDNA) in the nucleosome-less chromosomes of dinoflagellates. In this study, we isolated and cloned 5S rDNA sequences from various toxin-producing species of the genus Alexandrium and developed a fluorescence in situ hybridization (FISH) probe that allows their chromosomal localization. Our results can be summarized as follows: 1) The 5S rDNA unit is composed of a highly conserved 122-bp coding region and an intergenic spacer (IGS), the length and sequence of which are variable even within strains. 2) Three different IGS types, one containing the U6 small nuclear RNA (snRNA) gene, were found among four of the studied species (A. minutum, A. tamarense, A. catenella and A. pacificum). 3) In all strains investigated by FISH (A. minutum, A. tamarense, A. pacificum, A. catenella, A. andersonii and A. ostenfeldii), 5S rDNA gene arrays were separate from the nucleolar organizer region, which contains the genes for the large 45S pre-ribosomal RNA. 4) One to three 5S rDNA sites per haploid genome were detected, depending on the strains/species. Intraspecific variability in the number of 5S rDNA sites was determined among strains of A. minutum and A. pacificum. 5) 5S rDNA is a useful chromosomal marker of mitosis progression and can be employed to differentiate vegetative (haploid) vs. planozygotes (diploid) cells. Thus, the FISH probe (oligo-Dino5Smix5) developed in this study facilitates analyses of the diversity, cell cycle and life stages of the genus Alexandrium.

Integrative genetic map of repetitive DNA in the sole Solea senegalensis genome shows a Rex transposon located in a proto-sex chromosome.

Repetitive sequences play an essential role in the structural and functional evolution of the genome, particularly in the sexual chromosomes. The Senegalese sole (Solea senegalensis) is a valuable flatfish in aquaculture albeit few studies have addressed the mapping and characterization of repetitive DNA families. Here we analyzed the Simple Sequence Repeats (SSRs) and Transposable elements (TEs) content from fifty-seven BAC clones (spanning 7.9 Mb) of this species, located in chromosomes by multiple fluorescence in situ hybridization (m-BAC-FISH) technique. The SSR analysis revealed an average density of 675.1 loci per Mb and a high abundance (59.69%) of dinucleotide coverage was observed, being 'AC' the most abundant. An SSR-FISH analysis using eleven probes was also carried out and seven of the 11 probes yielded positive signals. 'AC' probes were present as large clusters in almost all chromosomes, supporting the bioinformatic analysis. Regarding TEs, DNA transposons (Class II) were the most abundant. In Class I, LINE elements were the most abundant and the hAT family was the most represented in Class II. Rex/Babar subfamily, observed in two BAC clones mapping to chromosome pair 1, showed the longest match. This chromosome pair has been recently reported as a putative sexual proto-chromosome in this species, highlighting the possible role of the Rex element in the evolution of this chromosome. In the Rex1 phylogenetic tree, the Senegalese sole Rex1 retrotransposon could be associated with one of the four major ancient lineages in fish genomes, in which it is included O. latipes.

Chromosomal markers in the genus Karenia: Towards an understanding of the evolution of the chromosomes, life cycle patterns and phylogenetic relationships in dinoflagellates.

Dinoflagellates are a group of protists whose genome is unique among eukaryotes in terms of base composition, chromosomal structure and gene expression. Even after decades of research, the structure and behavior of their amazing chromosomes-which without nucleosomes exist in a liquid crystalline state-are still poorly understood. We used flow cytometry and fluorescence in situ hybridization (FISH) to analyze the genome size of three species of the toxic dinoflagellate genus Karenia as well the organization and behavior of the chromosomes in different cell-cycle stages. FISH was also used to study the distribution patterns of ribosomal DNA (45S rDNA), telomeric and microsatellites repeats in order to develop chromosomal markers. The results revealed several novel and important features regarding dinoflagellate chromosomes during mitosis, including their telocentric behavior and radial arrangement along the nuclear envelope. Additionally, using the (AG)10 probe we identified an unusual chromosome in K. selliformis and especially in K. mikimotoi that is characterized by AG repeats along its entire length. This feature was employed to easily differentiate morphologically indistinguishable life-cycle stages. The evolutionary relationship between Karenia species is discussed with respect to differences in both DNA content and the chromosomal distribution patterns of the DNA sequences analyzed.

A novel FISH technique for labeling the chromosomes of dinoflagellates in suspension.

Dinoflagellates possess some of the largest known genomes. However, the study of their chromosomes is complicated by their similar size and their inability to be distinguished by traditional banding techniques. Dinoflagellate chromosomes lack nucleosomes and are present in a liquid crystalline state. In addition, approaches such as fluorescent in situ hybridization (FISH) are problematic because chromosomes are difficult to isolate from the nuclear membrane, which in dinoflagellates remains intact, also during mitosis. Here we describe a novel, reliable and effective technique to study dinoflagellate chromosomes by physical mapping of repetitive DNA sequences in chromosomes in suspension (FISH-IS), rather than on a microscope slide. A suspension of non-fixed chromosomes was achieved by lysing the cells and destabilizing the nuclear envelope. This treatment resulted in the release of the permanently condensed chromosomes in a high-quality chromosomal suspension. Nevertheless, slide preparations of the chromosomes were not suitable for conventional FISH because the nuclear integrity and chromosomal morphology was destroyed. Our newly developed, simple and efficient FISH-IS technique employs fluorescently labeled, synthetic short sequence repeats that are hybridized with suspended, acetic-acid-pretreated chromosomes for 1 h at room temperature. The method can be successfully used to discriminate single chromosomes or specific chromosomal regions, depending on the specificity of the repeat sequences used as probes. The combination of FISH-IS and flow sorting will improve genomic studies of dinoflagellates, overcoming the difficulties posed by their huge genomes, including long stretches of non-coding sequences in multiple copies and the presence of high-copy-number tandem gene arrays.

Comparative repeatome analysis on Triatoma infestans Andean and Non-Andean lineages, main vector of Chagas disease.

Triatoma infestans is the most important Chagas disease vector in South America. Two main evolutionary lineages, named Andean and non-Andean, have been recognized by geographical distribution, phenetic and genetic characteristics. One of the main differences is the genomic size, varying over 30% in their haploid DNA content. Here we realize a genome wide analysis to compare the repetitive genome fraction (repeatome) between both lineages in order to identify the main repetitive DNA changes occurred during T. infestans differentiation process. RepeatExplorer analysis using Illumina reads showed that both lineages exhibit the same amount of non-repeat sequences, and that satellite DNA is by far the major component of repetitive DNA and the main responsible for the genome size differentiation between both lineages. We characterize 42 satellite DNA families, which are virtually all present in both lineages but with different amount in each lineage. Furthermore, chromosomal location of satellite DNA by fluorescence in situ hybridization showed that genomic variations in T. infestans are mainly due to satellite DNA families located on the heterochromatic regions. The results also show that many satDNA families are located on the euchromatic regions of the chromosomes.

On the allopolyploid origin and genome structure of the closely related species Hordeum secalinum and Hordeum capense inferred by molecular karyotyping.

Background and Aims: To provide additional information to the many phylogenetic analyses conducted within Hordeum , here the origin and interspecific affinities of the allotetraploids Hordeum secalinum and Hordeum capense were analysed by molecular karyotyping. Methods: Karyotypes were determined using genomic in situ hybridization (GISH) to distinguish the sub-genomes and , plus fluorescence in situ hybridization (FISH)/non-denaturing (ND)-FISH to determine the distribution of ten tandem repetitive DNA sequences and thus provide chromosome markers. Key Results: Each chromosome pair in the six accessions analysed was identified, allowing the establishment of homologous and putative homeologous relationships. The low-level polymorphism observed among the H. secalinum accessions contrasted with the divergence recorded for the sub-genome of the H. capense accessions. Although accession H335 carries an intergenomic translocation, its chromosome structure was indistinguishable from that of H. secalinum . Conclusion: Hordeum secalinum and H. capense accession H335 share a hybrid origin involving Hordeum marinum subsp. gussoneanum as the genome donor and an unidentified genome progenitor. Hordeum capense accession BCC2062 either diverged, with remodelling of the sub-genome, or its genome was donated by a now extinct ancestor. A scheme of probable evolution shows the intricate pattern of relationships among the Hordeum species carrying the genome (including all H. marinum taxa and the hexaploid Hordeum brachyantherum ).

Sequencing of long stretches of repetitive DNA.

Repetitive DNA is widespread in eukaryotic genomes, in some cases making up more than 80% of the total. SSRs are a type of repetitive DNA formed by short motifs repeated in tandem arrays. In some species, SSRs may be organized into long stretches, usually associated with the constitutive heterochromatin. Variation in repeats can alter the expression of genes, and changes in the number of repeats have been linked to certain human diseases. Unfortunately, the molecular characterization of these repeats has been hampered by technical limitations related to cloning and sequencing. Indeed, most sequenced genomes contain gaps owing to repetitive DNA-related assembly difficulties. This paper reports an alternative method for sequencing of long stretches of repetitive DNA based on the combined use of 1) a linear vector to stabilize the cloning process, and 2) the use of exonuclease III for obtaining progressive deletions of SSR-rich fragments. This strategy allowed the sequencing of a fragment containing a stretch of 6.2 kb of continuous SSRs. To demonstrate that this procedure can sequence other kinds of repetitive DNA, it was used to examine a 4.5 kb fragment containing a cluster of 15 repeats of the 5S rRNA gene of barley.

Allopolyploidy and the complex phylogenetic relationships within the Hordeum brachyantherum taxon.

Hordeum brachyantherum Nevski includes two subspecies: the diploid (2×) subsp. californicum, and subsp. brachyantherum, which itself includes a tetraploid (4×) and a hexaploid (6×) cytotype. The phylogenetic relationships between these taxa and the origin of the polyploids remain controversial. To provide additional information to the many molecular phylogenetic analyses conducted within Hordeum, FISH-based karyotypes were produced for all subspecies/cytotypes within H. brachyantherum. Chromosomes of H. roshevitzii and H. marinum subsp. gussoneanum were also analysed since these species are potentially involved in the origin of the polyploids. For karyotyping, ten repetitive DNA sequences were screened to indentify repeats showing sufficient diversity in terms of copy number and localisation that they might serve as physical markers for distinguishing between each mitotic chromosome pair in all accessions. Genomic in situ hybridisation (GISH) was used to distinguish between subgenomes in polyploids. The karyotype maps allowed the assessment of the chromosomal diversity within species/cytotypes and the identification of possibly homoeologous chromosomes. The results show a wide divergence between the chromosomes of subsp. californicum and H. roshevitzii, and with their supposed derivatives in subsp. brachyantherum 4×. One of the three subgenomes of subsp. brachyantherum 6× is derived from subsp. gussoneanum with no genomic reorganisation (i.e., neither amplification nor loss of the repetitive DNA sequences analysed). It is generally accepted that subsp. brachyantherum 4× is the other progenitor of subsp. brachyantherum 6×, but the present results suggest this to be unlikely. The present findings thus show the cytogenetic diversity and genomic structure of H. brachyantherum, and reveal its complex evolutionary history, in which chromosomal diversification and allopolyploidy have played important roles.

The Hidden Sexuality of Alexandrium Minutum: An Example of Overlooked Sex in Dinoflagellates.

Dinoflagellates are haploid eukaryotic microalgae in which rapid proliferation causes dense blooms, with harmful health and economic effects to humans. The proliferation mode is mainly asexual, as the sexual cycle is believed to be rare and restricted to stressful environmental conditions. However, sexuality is key to explaining the recurrence of many dinoflagellate blooms because in many species the fate of the planktonic zygotes (planozygotes) is the formation of resistant cysts in the seabed (encystment). Nevertheless, recent research has shown that individually isolated planozygotes in the lab can enter other routes besides encystment, a behavior of which the relevance has not been explored at the population level. In this study, using imaging flow cytometry, cell sorting, and Fluorescence In Situ Hybridization (FISH), we followed DNA content and nuclear changes in a population of the toxic dinoflagellate Alexandrium minutum that was induced to encystment. Our results first show that planozygotes behave like a population with an "encystment-independent" division cycle, which is light-controlled and follows the same Light:Dark (L:D) pattern as the cycle governing the haploid mitosis. Resting cyst formation was the fate of just a small fraction of the planozygotes formed and was restricted to a period of strongly limited nutrient conditions. The diploid-haploid turnover between L:D cycles was consistent with two-step meiosis. However, the diel and morphological division pattern of the planozygote division also suggests mitosis, which would imply that this species is not haplontic, as previously considered, but biphasic, because individuals could undergo mitotic divisions in both the sexual (diploid) and the asexual (haploid) phases. We also report incomplete genome duplication processes. Our work calls for a reconsideration of the dogma of rare sex in dinoflagellates.

Nuclear and cell morphological changes during the cell cycle and growth of the toxic dinoflagellate Alexandrium minutum.

Elucidation of the cell cycle of dinoflagellates is essential to understand the processes leading to their massive proliferations, known as harmful algal blooms. In this study, we used imaging flow cytometry (IFC) to monitor the changes in DNA content and nuclear and cell morphology that occur during clonal growth of the toxic species Alexandrium minutum Halim. Our results indicate that the population was in S phase (C→2C DNA content) during the light period, whereas haploid cells with a C DNA content peaked only during a short interval of the dark period. The timing of the phases, identified based on the nuclear morphology and cytoplasmic-to-nuclear (CNR) ratio of the cells, suggests that the length of the G2/M phase is regulated by nutrient levels whereas the beginning of S phase is clock controlled. In addition we found that up to 7% of individual cells achieved a DNA content higher than 2C, indicative of either zygote formation and replication (homothallism), or of double-haploid cells able to divide (polyploid forms). Cells belonging to different cell cycle phases (G1-S-G2/M) could be readily discriminated based on nuclear size. Our study provides evidence of cell-cycle plasticity during clonal growth and unambiguously characterizes the cell-cycle phases of this dinoflagellate species.

Next generation sequencing and FISH reveal uneven and nonrandom microsatellite distribution in two grasshopper genomes.

Simple sequence repeats (SSRs), also known as microsatellites, are one of the prominent DNA sequences shaping the repeated fraction of eukaryotic genomes. In spite of their profuse use as molecular markers for a variety of genetic and evolutionary studies, their genomic location, distribution, and function are not yet well understood. Here we report the first thorough joint analysis of microsatellite motifs at both genomic and chromosomal levels in animal species, by a combination of 454 sequencing and fluorescent in situ hybridization (FISH) techniques performed on two grasshopper species. The in silico analysis of the 454 reads suggested that microsatellite expansion is not driving size increase of these genomes, as SSR abundance was higher in the species showing the smallest genome. However, the two species showed the same uneven and nonrandom location of SSRs, with clear predominance of dinucleotide motifs and association with several types of repetitive elements, mostly histone gene spacers, ribosomal DNA intergenic spacers (IGS), and transposable elements (TEs). The FISH analysis showed a dispersed chromosome distribution of microsatellite motifs in euchromatic regions, in coincidence with chromosome location patterns previously observed for many mobile elements in these species. However, some SSR motifs were clustered, especially those located in the histone gene cluster.

Ribosomal DNA organization patterns within the dinoflagellate genus Alexandrium as revealed by FISH: life cycle and evolutionary implications.

Dinoflagellates are a group of protists whose genome differs from that of other eukaryotes in terms of size (contains up to 250pg per haploid cell), base composition, chromosomal organization, and gene expression. But rDNA gene mapping of the active nucleolus in this unusual eukaryotic genome has not been carried out thus far. Here we used FISH in dinoflagellate species belonging to the genus Alexandrium (genome sizes ranging from 21 to 170 pg of DNA per haploid genome) to localize the sequences encoding the 18S, 5.8S, and 28S rRNA genes. The results can be summarized as follows: 1) Each dinoflagellate cell contains only one active nucleolus, with no hybridization signals outside it. However, the rDNA organization varies among species, from repetitive clusters forming discrete nuclear organizer regions (NORs) in some to specialized "ribosomal chromosomes" in other species. The latter chromosomes, never reported before in other eukaryotes, are mainly formed by rDNA genes and appeared in the species with the highest DNA content. 2) Dinoflagellate chromosomes are first characterized by several eukaryotic features, such as structural differentiation (centromere-like constrictions), size differences (dot chromosomes), and SAT (satellite) chromosomes. 3) NOR patterns prove to be useful in discriminating between cryptic species and life cycle stages in protists.

Chromosomal characterization of the three subgenomes in the polyploids of Hordeum murinum L.: new insight into the evolution of this complex.

Hordeum murinum L. is a species complex composed of related taxa, including the subspecies glaucum, murinum and leporinum. However, the phylogenetic relationships between the different taxa and their cytotypes, and the origin of the polyploid forms, remain points of controversy. The present work reports a comparative karyotype analysis of seven accessions of the H. murinum complex representing all subspecies and cytotypes. The karyotypes were determined by examining the distribution of the repetitive Triticeae DNA sequences pTa71, pTa794, pSc119.2, pAs1 and pHch950, the simple sequence repeats (SSRs) (AG)10, (AAC)5, (AAG)5, (ACT)5, (ATC)5, and (CCCTAAA)3 via in situ hybridization. The chromosomes of the three subgenomes involved in the polyploids were identified. All tetraploids of all subspecies shared the same two subgenomes (thus suggesting them to in fact belong to the same taxon), the result of hybridization between two diploid ancestors. One of the subgenomes present in all tetraploids of all subspecies was found to be very similar (though not identical) to the chromosome complement of the diploid glaucum. The hexaploid form of leporinum came about through a cross between a tetraploid and a third diploid form. Exclusively bivalent associations among homologous chromosomes were observed when analyzing pollen mother cells of tetraploid taxa. In conclusion, the present results identify all the individual chromosomes within the H. murinum complex, reveal its genome structure and phylogeny, and explain the appearance of the different cytotypes. Three cryptic species are proposed according to ploidy level that may deserve full taxonomic recognition.

The evolutionary history of sea barley (Hordeum marinum) revealed by comparative physical mapping of repetitive DNA.

BACKGROUND AND AIMS: Hordeum marinum is a species complex that includes the diploid subspecies marinum and both diploid and tetraploid forms of gussoneanum. Their relationships, the rank of the taxa and the origin of the polyploid forms remain points of debate. The present work reports a comparative karyotype analysis of six H. marinum accessions representing all taxa and cytotypes. METHODS: Karyotypes were determined by analysing the chromosomal distribution of several tandemly repeated sequences, including the Triticeae cloned probes pTa71, pTa794, pAs1 and pSc119·2 and the simple sequence repeats (SSRs) (AG)10, (AAC)5, (AAG)5, (ACT)5 and (ATC)5. KEY RESULTS: The identification of each chromosome pair in all subspecies and cytotypes is reported for the first time. Homologous relationships are also established. Wide karyotypic differences were detected within marinum accessions. Specific chromosomal markers characterized and differentiated the genomes of marinum and diploid gussoneanum. Two subgenomes were detected in the tetraploids. One of these had the same chromosome complement as diploid gussoneanum; the second subgenome, although similar to the chromosome complement of diploid H. marinum sensu lato, appeared to have no counterpart in the marinum accessions analysed here. CONCLUSIONS: The tetraploid forms of gussoneanum appear to have come about through a cross between a diploid gussoneanum progenitor and a second, related-but unidentified-diploid ancestor. The results reveal the genome structure of the different H. marinum taxa and demonstrate the allopolyploid origin of the tetraploid forms of gussoneanum.

Cytogenetic diversity of SSR motifs within and between Hordeum species carrying the H genome: H. vulgare L. and H. bulbosum L.

Non-denaturing FISH (ND-FISH) was used to compare the distribution of four simple sequence repeats (SSRs)-(AG) n , (AAG) n , (ACT) n and (ATC) n -in somatic root tip metaphase spreads of 12 barley (H. vulgare ssp. vulgare) cultivars, seven lines of their wild progenitor H. vulgare ssp. spontaneum, and four lines of their close relative H. bulbosum, to determine whether the range of molecular diversity shown by these highly polymorphic sequences is reflected at the chromosome level. In both, the cultivated and wild barleys, clusters of AG and ATC repeats were invariant. In contrast, clusters of AAG and ACT showed polymorphism. Karyotypes were prepared after the identification of their seven pairs of homologous chromosomes. Variation between these homologues was only observed in one wild accession that showed the segregation of a reciprocal translocation involving chromosomes 5H and 7H. The two subspecies of H. vulgare analysed were no different in terms of their SSRs. Only AAG repeats were found clustered strongly on the chromosomes of all lines of H. bulbosum examined. Wide variation was seen between homologous chromosomes within and across these lines. These results are the first to provide insight into the cytogenetic diversity of SSRs in barley and its closest relatives. Differences in the abundance and distribution of each SSR analysed, between H. vulgare and H. bulbosum, suggest that these species do not share the same H genome, and support the idea that these species are not very closely related. Southern blotting experiments revealed the complex organization of these SSRs, supporting the findings made with ND-FISH.