Molecular and Cytogenetic Analysis of rDNA Evolution in Crepis Sensu Lato.

Although Crepis was the first model plant group in which chromosomal changes were considered to play an important role in speciation, their chromosome structure and evolution have been barely investigated using molecular cytogenetic methods. The aim of the study was to provide a better understanding of the patterns and directions of Crepis chromosome evolution, using comparative analyses of rDNA loci number and localisation. The chromosome base number and chromosomal organisation of 5S and 35S rDNA loci were analysed in the phylogenetic background for 39 species of Crepis, which represent the evolutionary lineages of Crepis sensu stricto and Lagoseris, including Lapsana communis. The phylogenetic relationships among all the species were inferred from nrITS and newly obtained 5S rDNA NTS sequences. Despite high variations in rDNA loci chromosomal organisation, most species had a chromosome with both rDNA loci within the same (usually short) chromosomal arm. The comparative analyses revealed several independent rDNA loci number gains and loci repositioning that accompanied diversification and speciation in Crepis. Some of the changes in rDNA loci patterns were reconstructed for the same evolutionary lineages as descending dysploidy.

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.

Organization and evolution of two repetitive sequences, 18-24J and 12-13P, in the genome of Chenopodium (Amaranthaceae).

The abundance and chromosomal organization of two repetitive sequences named 12-13P and 18-24J were analyzed in 24 diploid and nine polyploid species of Chenopodium s.l., with special attention to Chenopodium s.s. Both sequences were predominantly present in species of Chenopodium s.s.; however, differences in the amplification levels were observed among the species. The 12-13P repeat was highly amplified in all of the analyzed Eurasian species, whereas the American diploids showed a marked variation in the amplification levels. The 12-13P repeat contains a tandemly arranged 40 bp minisatellite element forming a large proportion of the genome of Chenopodium (up to 3.5%). FISH revealed its localization to the pericentromeric regions of the chromosomes. The chromosomal distribution of 12-13P delivered additional chromosomal marker for B-genome diploids. The 18-24J repeat showed a dispersed organization in all of the chromosomes of the analyzed diploid species and the Eurasian tetraploids. In the American allotetraploids (C. quinoa, C. berlandieri) and Eurasian allohexaploids (e.g., C. album) very intense hybridization signals of 18-24J were observed only on 18 chromosomes that belong to the B subgenome of these polyploids. Combined cytogenetic and molecular analyses suggests that reorganization of these two repeats accompanied the diversification and speciation of diploid (especially A genome) and polyploid species of Chenopodium s.s.

Molecular and cytogenetic evidence for an allotetraploid origin of Chenopodium quinoa and C. berlandieri (Amaranthaceae).

Most of the cultivated chenopods are polyploids, but their origin and evolutionary history are still poorly understood. Phylogenetic analyses of DNA sequences of four plastid regions, nrITS and nuclear 5S rDNA spacer region (NTS) of two tetraploid chenopods (2n=4x=36), Andean C. quinoa and North American C. berlandieri, and their diploid relatives allowed inferences of their origin. The phylogenetic analyses confirmed allotetraploid origin of both tetraploids involving diploids of two different genomic groups (genomes A and B) and suggested that these two might share very similar parentage. The hypotheses on the origin of the two allopolyploid species were further tested using genomic in situ hybridization (GISH). Several diploid Chenopodium species belonging to the two lineages, genome A and B, suggested by phylogenetic analyses, were tested as putative parental taxa. GISH differentiated two sets of parental chromosomes in both tetraploids and further corroborated their allotetraploid origin. Putative diploid parental taxa have been suggested by GISH for C. quinoa and C. berlandieri. Genome sizes of the analyzed allotetraploids fit nearly perfectly the expected additive values of the putative parental taxa. Directional and uniparental loss of rDNA loci of the maternal A-subgenome was revealed for both C. berlandieri and C. quinoa.

Floral traits and pollination ecology of European Arum hybrids.

Hybridisation is common in plants and can affect the genetic diversity and ecology of sympatric parental populations. Hybrids may resemble the parental species in their ecology, leading to competition and/or gene introgression; alternatively, they may diverge from the parental phenotypes, possibly leading to the colonisation of new ecological niches and to speciation. Here, we describe inflorescence morphology, ploidy levels, pollinator attractive scents, and pollinator guilds of natural hybrids of Arum italicum and A. maculatum (Araceae) from a site with sympatric parental populations in southern France to determine how these traits affect the hybrid pollination ecology. Hybrids were characterised by inflorescences with a size and a number of flowers more similar to A. italicum than to A. maculatum. In most cases, hybrid stamens were purple, as in A. maculatum, and spadix appendices yellow, as in A. italicum. Hybrid floral scent was closer to that of A. italicum, but shared some compounds with A. maculatum and comprised unique compounds. Also, the pollinator guild of the hybrids was similar to that of A. italicum. Nevertheless, the hybrids attracted a high proportion of individuals of the main pollinator of A. maculatum. We discuss the effects of hybridisation in sympatric parental zones in which hybrids exhibit low levels of reproductive success, the establishment of reproductive barriers between parental species, the role of the composition of floral attractive scents in the differential attraction of pollinators and in the competition between hybrids and their parental species, and the potential of hybridisation to give rise to new independent lineages.

High ploidy diversity and distinct patterns of cytotype distribution in a widespread species of Oxalis in the Greater Cape Floristic Region.

BACKGROUND AND AIMS: Genome duplication is widely acknowledged as a major force in the evolution of angiosperms, although the incidence of polyploidy in different floras may differ dramatically. The Greater Cape Floristic Region of southern Africa is one of the world's biodiversity hotspots and is considered depauperate in polyploids. To test this assumption, ploidy variation was assessed in a widespread member of the largest geophytic genus in the Cape flora: Oxalis obtusa. METHODS: DNA flow cytometry complemented by confirmatory chromosome counts was used to determine ploidy levels in 355 populations of O. obtusa (1014 individuals) across its entire distribution range. Ecological differentiation among cytotypes was tested by comparing sets of vegetation and climatic variables extracted for each locality. KEY RESULTS: Three majority (2x, 4x, 6x) and three minority (3x, 5x, 8x) cytotypes were detected in situ, in addition to a heptaploid individual originating from a botanical garden. While single-cytotype populations predominate, 12 mixed-ploidy populations were also found. The overall pattern of ploidy level distribution is quite complex, but some ecological segregation was observed. Hexaploids are the most common cytotype and prevail in the Fynbos biome. In contrast, tetraploids dominate in the Succulent Karoo biome. Precipitation parameters were identified as the most important climatic variables associated with cytotype distribution. CONCLUSIONS: Although it would be premature to make generalizations regarding the role of genome duplication in the genesis of hyperdiversity of the Cape flora, the substantial and unexpected ploidy diversity in Oxalis obtusa is unparalleled in comparison with any other cytologically known native Cape plant species. The results suggest that ploidy variation in the Greater Cape Floristic Region may be much greater than currently assumed, which, given the documented role of polyploidy in speciation, has direct implications for radiation hypotheses in this biodiversity hotspot.

Isolation and characterization of reverse transcriptase fragments of LTR retrotransposons from the genome of Chenopodium quinoa (Amaranthaceae).

KEY MESSAGE: High heterogeneity was observed among conserved domains of reverse transcriptase ( rt ) isolated from quinoa. Only one Ty1- copia rt was highly amplified. Reverse transcriptase sequences were located predominantly in pericentromeric region of quinoa chromosomes. The heterogeneity, genomic abundance, and chromosomal distribution of reverse transcriptase (rt)-coding fragments of Ty1-copia and Ty3-gypsy long terminal repeat retrotransposons were analyzed in the Chenopodium quinoa genome. Conserved domains of the rt gene were amplified and characterized using degenerate oligonucleotide primer pairs. Sequence analyses indicated that half of Ty1-copia rt (51 %) and 39 % of Ty3-gypsy rt fragments contained intact reading frames. High heterogeneity among rt sequences was observed for both Ty1-copia and Ty3-gypsy rt amplicons, with Ty1-copia more heterogeneous than Ty3-gypsy. Most of the isolated rt fragments were present in quinoa genome in low copy numbers, with only one highly amplified Ty1-copia rt sequence family. The gypsy-like RNase H fragments co-amplified with Ty1-copia-degenerate primers were shown to be highly amplified in the quinoa genome indicating either higher abundance of some gypsy families of which rt domains could not be amplified, or independent evolution of this gypsy-region in quinoa. Both Ty1-copia and Ty3-gypsy retrotransposons were preferentially located in pericentromeric heterochromatin of quinoa chromosomes. Phylogenetic analyses of newly amplified rt fragments together with well-characterized retrotransposon families from other organisms allowed identification of major lineages of retroelements in the genome of quinoa and provided preliminary insight into their evolutionary dynamics.

The Use of Ribosomal DNA for Comparative Cytogenetics.

Fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA) sequences provides excellent chromosome markers for comparative cytogenetic analyses, especially in non-model plant species. The tandem repeat nature of a sequence and the presence of a highly conserved genic region make rDNA sequences relatively easy to isolate and clone. In this chapter, we describe the use of rDNA as markers for comparative cytogenetics studies. Traditionally, cloned probes labeled with Nick-translation have been used to detect rDNA loci. Recently, pre-labeled oligonucleotides are also employed quite frequently to detect both 35S and 5S rDNA loci. Ribosomal DNA sequences, together with other DNA probes in FISH/GISH or with fluorochromes such as CMA3 banding or silver staining, are very useful tools in comparative analyses of plant karyotypes.

Tracing the Evolution of the Angiosperm Genome from the Cytogenetic Point of View.

Cytogenetics constitutes a branch of genetics that is focused on the cellular components, especially chromosomes, in relation to heredity and genome structure, function and evolution. The use of modern cytogenetic approaches and the latest microscopes with image acquisition and processing systems enables the simultaneous two- or three-dimensional, multicolour visualisation of both single-copy and highly-repetitive sequences in the plant genome. The data that is gathered using the cytogenetic methods in the phylogenetic background enable tracing the evolution of the plant genome that involve changes in: (i) genome sizes; (ii) chromosome numbers and morphology; (iii) the content of repetitive sequences and (iv) ploidy level. Modern cytogenetic approaches such as FISH using chromosome- and genome-specific probes have been widely used in studies of the evolution of diploids and the consequences of polyploidy. Nowadays, modern cytogenetics complements analyses in other fields of cell biology and constitutes the linkage between genetics, molecular biology and genomics.

Descending Dysploidy and Bidirectional Changes in Genome Size Accompanied Crepis (Asteraceae) Evolution.

The evolution of the karyotype and genome size was examined in species of Crepis sensu lato. The phylogenetic relationships, inferred from the plastid and nrITS DNA sequences, were used as a framework to infer the patterns of karyotype evolution. Five different base chromosome numbers (x = 3, 4, 5, 6, and 11) were observed. A phylogenetic analysis of the evolution of the chromosome numbers allowed the inference of x = 6 as the ancestral state and the descending dysploidy as the major direction of the chromosome base number evolution. The derived base chromosome numbers (x = 5, 4, and 3) were found to have originated independently and recurrently in the different lineages of the genus. A few independent events of increases in karyotype asymmetry were inferred to have accompanied the karyotype evolution in Crepis. The genome sizes of 33 Crepis species differed seven-fold and the ancestral genome size was reconstructed to be 1C = 3.44 pg. Both decreases and increases in the genome size were inferred to have occurred within and between the lineages. The data suggest that, in addition to dysploidy, the amplification/elimination of various repetitive DNAs was likely involved in the genome and taxa differentiation in the genus.

A Chromosome-Scale Assembly of the Garden Orach (Atriplex hortensis L.) Genome Using Oxford Nanopore Sequencing.

Atriplex hortensis (2n = 2x = 18, 1C genome size ∼1.1 gigabases), also known as garden orach and mountain-spinach, is a highly nutritious, broadleaf annual of the Amaranthaceae-Chenopodiaceae alliance (Chenopodiaceae sensu stricto, subfam. Chenopodioideae) that has spread in cultivation from its native primary domestication area in Eurasia to other temperate and subtropical regions worldwide. Atriplex L. is a highly complex but, as understood now, a monophyletic group of mainly halophytic and/or xerophytic plants, of which A. hortensis has been a vegetable of minor importance in some areas of Eurasia (from Central Asia to the Mediterranean) at least since antiquity. Nonetheless, it is a crop with tremendous nutritional potential due primarily to its exceptional leaf and seed protein quantities (approaching 30%) and quality (high levels of lysine). Although there is some literature describing the taxonomy and production of A. hortensis, there is a general lack of genetic and genomic data that would otherwise help elucidate the genetic variation, phylogenetic positioning, and future potential of the species. Here, we report the assembly of the first high-quality, chromosome-scale reference genome for A. hortensis cv. "Golden." Long-read data from Oxford Nanopore's MinION DNA sequencer was assembled with the program Canu and polished with Illumina short reads. Contigs were scaffolded to chromosome scale using chromatin-proximity maps (Hi-C) yielding a final assembly containing 1,325 scaffolds with a N50 of 98.9 Mb - with 94.7% of the assembly represented in the nine largest, chromosome-scale scaffolds. Sixty-six percent of the genome was classified as highly repetitive DNA, with the most common repetitive elements being Gypsy-(32%) and Copia-like (11%) long-terminal repeats. The annotation was completed using MAKER which identified 37,083 gene models and 2,555 tRNA genes. Completeness of the genome, assessed using the Benchmarking Universal Single Copy Orthologs (BUSCO) metric, identified 97.5% of the conserved orthologs as complete, with only 2.2% being duplicated, reflecting the diploid nature of A. hortensis. A resequencing panel of 21 wild, unimproved and cultivated A. hortensis accessions revealed three distinct populations with little variation within subpopulations. These resources provide vital information to better understand A. hortensis and facilitate future study.

Histone modifications associated with both A and B chromosomes of maize.

We report the distribution of several histone modifications along the arms and in centromeric regions of somatic chromosomes of maize, including the supernumerary B chromosome. Acetylated H3 and H4 as well as H3K4me2, modifications associated with euchromatin, were enriched in the distal parts of the A chromosomes, but were progressively depleted toward the centromeres of the A chromosomes and were depleted in the heterochromatic portions of the B chromosome. Classical histone modifications associated with heterochromatin, including H3K9me2, H3K27me1 and H3K27me2, were distributed throughout both A and B chromosomes. However, H3K27me2 showed a reduced level on the B chromosome compared with the A chromosomes and was not associated with some classes of constitutive heterochromatin. We monitored the presence of each histone modification in the centromeric regions using a YFP-tagged centromere-specific histone, CENH3. We observed the presence of H3K9me2 and absence of H3K4me2 in the centromeric regions of both A and B chromosomes of maize, which is in contrast to the presence of H3K4me2 and absence of H3K9me2 in animal centromeres. These results show a diversity of epigenetic modifications associated with centromeric chromatin in different eukaryotes.

Cytogenetic studies of three European species of Centaurea L. (Asteraceae).

Cytogenetic analysis of several populations of Centaurea jacea (2n = 4x = 44), C. oxylepis (2n = 4x = 44) and C. phrygia (2n = 2x = 22) was performed using flow cytometry, differential chromosome staining and FISH. In all species Arabidopsis-type telomeric repeats hybridized only to the terminal part of chromosomes. In C. phrygia three pairs and in C. oxylepis six pairs of chromosomes revealed the hybridization signals of 45S rDNA. Centaurea jacea showed polymorphism in the 45S rDNA loci number, five or six pairs of sites were observed. 5S rDNA loci were located in two pairs of chromosomes in C. phrygia. In C. jacea and C. oxylepis the number and position of 5S rDNA loci were the same: three pairs located interstitially and one terminally. The genome size of the diploid C. phrygia was established as 2.14 pg/2C. The genomes of tetraploid species were nearly two times larger and genome size polymorphism was observed among C. jacea populations.

Karyotype analysis of eight cultivated Allium species.

The karyotypes of Allium, a genus that comprises many crops and ornamental plants, are relatively poorly studied. To extend our knowledge on karyotype structure of the genus, the chromosomal organization of rRNA genes and CMA/DAPI bands was studied. Fluorescence in situ hybridization using 5S and 35S rDNA probes and banding methods (silver staining and CMA3/DAPI staining) were used to analyze the karyotypes of eight cultivated Allium L. species. Analyzed Allium taxa revealed three different basic chromosome numbers (x = 7, 8, 9) and three different ploidy levels (diploid, triploid, and tetraploid). The rDNA sites chromosomal organization is reported the first time for the six species (A. moly, A. oreophilum, A. karataviense, A. nigrum, A. sphaerocephalon, A. porrum). The Allium species that were analyzed showed a high level of interspecies polymorphism in the number and localization of the rDNA sites. The fluorescence in situ hybridization patterns of 35S rDNA sites were more polymorphic than those of the 5S rDNA in the diploid species. Several groups of similar chromosomes could be distinguished among the chromosomes that had rDNA sites in the polyploid species. Each of the groups had three chromosomes (triploid A. sphaerocephalon L.) or four chromosomes (tetraploid A. porrum L.) suggesting their autopolyploid origin. In the genomes of four of the analyzed species, only some of the 35S rDNA sites were transcriptionally active. Fluorochrome banding revealed that the CMA3+ bands were associated with the 35S rDNA sites in all of the species that were analyzed, except A. fistulosum L. in which positive CMA3+ bands were detected in the terminal position of all of the chromosome arms. The rDNA sequences, nucleolar organizer regions (NORs), and CMA/DAPI bands are very good chromosome markers that allowed to distinguished from two to five pairs of homologous chromosomes in analyzed Allium species. The karyotypes of the studied species could be clearly distinguished by the number and position of the rDNA sites, NORs, and CMA/DAPI bands, which revealed high interspecific differentiation among the taxa.

Field cress genome mapping: Integrating linkage and comparative maps with cytogenetic analysis for rDNA carrying chromosomes.

Field cress (Lepidium campestre L.), despite its potential as a sustainable alternative oilseed plant, has been underutilized, and no prior attempts to characterize the genome at the genetic or molecular cytogenetic level have been conducted. Genetic maps are the foundation for anchoring and orienting annotated genome assemblies and positional cloning of candidate genes. Our principal goal was to construct a genetic map using integrated approaches of genetic, comparative and cytogenetic map analyses. In total, 503 F2 interspecific hybrid individuals were genotyped using 7,624 single nucleotide polymorphism markers. Comparative analysis demonstrated that ~57% of the sequenced loci in L. campestre were congruent with Arabidopsis thaliana (L.) genome and suggested a novel karyotype, which predates the ancestral crucifer karyotype. Aceto-orcein chromosome staining and fluorescence in situ hybridization (FISH) analyses confirmed that L. campestre, L. heterophyllum Benth. and their hybrids had a chromosome number of 2n = 2x = 16. Flow cytometric analysis revealed that both species possess 2C roughly 0.4 picogram DNA. Integrating linkage and comparative maps with cytogenetic map analyses assigned two linkage groups to their particular chromosomes. Future work could incorporate FISH utilizing A. thaliana mapped BAC clones to allow the chromosomes of field cress to be identified reliably.

Chromosomal localization of a novel repetitive sequence in the Chenopodium quinoa genome.

In this study, a novel repetitive sequence pTaq10 was isolated from the Taq I digest of the genomic DNA of the pseudocereal Chenopodium quinoa. Sequence analysis indicated that this 286-bp monomer is not homologous to any known retroelement sequence. FISH and Southern blot analysis showed that this sequence is characterized by an interspersed genomic organization. After FISH, hybridization signals were observed as small dots spread throughout all of the chromosomes. pTaq hybridization signals were excluded from 45S rRNA gene loci, but they partly overlapped with 5S rDNA loci. pTaq10 is not a species-specific sequence, as it was also detected in C. berlandieri.

Interspecific somatic hybrids Solanum villosum (+) S. tuberosum, resistant to Phytophthora infestans.

The interspecific somatic hybrids 4x S. villosum (+) 2x S. tuberosum clone DG 81-68 (VT hybrids) were obtained and characterized molecularly and cytogenetically. The morphology of fusion-derived plants was intermediate in relation to the parental species. The expected ploidy level of the regenerants was 6x for the VT hybrids, but the real ploidy of the hybrids varied, with some of them being euploids, and others - aneuploids. The hybridity of the regenerants was verified by random amplified polymorphic DNA (RAPD) analysis. Despite the variation in ploidy, the RAPD patterns of the hybrids were mostly uniform, suggesting similarity of the genotypes of the VT clones. Genomic in situ hybridization (GISH) analysis discriminated between the chromosomes of both parental genomes in VT somatic hybrids and also confirmed their hybridity. The resistance of VT somatic hybrids to Phytophthora infestans was evaluated and all of the hybrids proved to be highly resistant. In search of the mechanisms involved in resistance of the Solanum species to P. infestans, the biochemical reactions occurring early after elicitor treatment were studied. The production of reactive oxygen species (ROS), as one of the earliest reactions induced by pathogens or their elicitors, was examined in the resistant wild species S. villosum, susceptible S. tuberosum clone DG 81-68 and in the VT hybrid, resistant to P. infestans. After treatment of the leaves with elicitor, the relative increase in ROS production was higher in leaves of the susceptible potato clone than in the resistant plants of S. villosum and the somatic hybrid.

Comet-FISH with rDNA probes for the analysis of mutagen-induced DNA damage in plant cells.

We used comet-fluorescence in situ hybridization (FISH) in the model plant species Crepis capillaris following exposure of seedlings to maleic hydrazide (MH). FISH with 5S and 25S rDNA probes was applied to comets obtained under alkaline conditions to establish whether these DNA regions were preferentially involved in comet tail formation. MH treatment induced significant fragmentation of nuclear DNA and of rDNA loci. A 24-h post-treatment recovery period allowed a partial reversibility of MH-induced damage on nuclear and rDNA regions. Analyses of FISH signals demonstrated that rDNA sequences were always involved in tail formation and that 5S rDNA was more frequently present in the tail than 25S rDNA, regardless of treatment. The involvement of 25S rDNA in nucleolus formation and differences in chromatin structure between the two loci may explain the different susceptibility of the 25S and 5S rDNA regions to migrate into the tail. This work is the first report on the application of FISH to comet preparations from plants to analyze the distribution and repair of DNA damage within specific genomic regions after mutagenic treatment. Moreover, our work suggests that comet-FISH in plants may be a useful tool for environmental monitoring assessment.