How diverse a monocentric chromosome can be? Repeatome and centromeric organization of Juncus effusus (Juncaceae).

Juncus is the largest genus of Juncaceae and was considered holocentric for a long time. Recent findings, however, indicated that 11 species from different clades of the genus have monocentric chromosomes. Thus, the Juncus centromere organization and evolution need to be reassessed. We aimed to investigate the major repetitive DNA sequences of two accessions of Juncus effusus and its centromeric structure by employing whole-genome analyses, fluorescent in situ hybridization, CENH3 immunodetection, and chromatin immunoprecipitation sequencing. We showed that the repetitive fraction of the small J. effusus genome (~270 Mbp/1C) is mainly composed of Class I and Class II transposable elements (TEs) and satellite DNAs. Three identified satellite DNA families were mainly (peri)centromeric, with two being associated with the centromeric protein CENH3, but not strictly centromeric. Two types of centromere organization were discerned in J. effusus: type 1 was characterized by a single CENH3 domain enriched with JefSAT1-155 or JefSAT2-180, whereas type 2 showed multiple CENH3 domains interrupted by other satellites, TEs or genes. Furthermore, while type 1 centromeres showed a higher degree of satellite identity along the array, type 2 centromeres had less homogenized arrays along the multiple CENH3 domains per chromosome. Although the analyses confirmed the monocentric organization of J. effusus chromosomes, our data indicate a more dynamic arrangement of J. effusus centromeres than observed for other plant species, suggesting it may constitute a transient state between mono- and holocentricity.

Oligo-barcode illuminates holocentric karyotype evolution in Rhynchospora (Cyperaceae).

Holocentric karyotypes are assumed to rapidly evolve through chromosome fusions and fissions due to the diffuse nature of their centromeres. Here, we took advantage of the recent availability of a chromosome-scale reference genome for Rhynchospora breviuscula, a model species of this holocentric genus, and developed the first set of oligo-based barcode probes for a holocentric plant. These probes were applied to 13 additional species of the genus, aiming to investigate the evolutionary dynamics driving the karyotype evolution in Rhynchospora. The two sets of probes were composed of 27,392 (green) and 23,968 (magenta) oligonucleotides (45-nt long), and generated 15 distinct FISH signals as a unique barcode pattern for the identification of all five chromosome pairs of the R. breviuscula karyotype. Oligo-FISH comparative analyzes revealed different types of rearrangements, such as fusions, fissions, putative inversions and translocations, as well as genomic duplications among the analyzed species. Two rounds of whole genome duplication (WGD) were demonstrated in R. pubera, but both analyzed accessions differed in the complex chain of events that gave rise to its large, structurally diploidized karyotypes with 2n = 10 or 12. Considering the phylogenetic relationships and divergence time of the species, the specificity and synteny of the probes were maintained up to species with a divergence time of ~25 My. However, karyotype divergence in more distant species hindered chromosome mapping and the inference of specific events. This barcoding system is a powerful tool to study chromosomal variations and genomic evolution in holocentric chromosomes of Rhynchospora species.

Comparative analysis of repetitive DNA in dysploid and non-dysploid Phaseolus beans.

Structural karyotype changes result from ectopic recombination events frequently associated with repetitive DNA. Although most Phaseolus species present relatively stable karyotypes with 2n = 22 chromosomes, the karyotypes of species of the Leptostachyus group show high rates of structural rearrangements, including a nested chromosome fusion that led to the dysploid chromosome number of the group (2n = 20). We examined the roles of repetitive landscapes in the rearrangements of species of the Leptostachyus group using genome-skimming data to characterize the repeatome in a range of Phaseolus species and compared them to species of that group (P. leptostachyus and P. macvaughii). LTR retrotransposons, especially the Ty3/gypsy lineage Chromovirus, were the most abundant elements in the genomes. Differences in the abundance of Tekay, Retand, and SIRE elements between P. macvaughii and P. leptostachyus were reflected in their total amounts of Ty3/gypsy and Ty1/copia. The satellite DNA fraction was the most divergent among the species, varying both in abundance and distribution, even between P. leptostachyus and P. macvaughii. The rapid turnover of repeats in the Leptostachyus group may be associated with the several rearrangements observed.

High rates of structural rearrangements have shaped the chromosome evolution in dysploid Phaseolus beans.

KEY MESSAGE: Karyotypes evolve through numerical and structural chromosome rearrangements. We show that Phaseolus leptostachyus, a wild bean, underwent a rapid genome reshuffling associated with the reduction from 11 to 10 chromosome pairs, but without whole genome duplication, the highest chromosome evolution rate known for plants. Plant karyotypes evolve through structural rearrangements often associated with polyploidy or dysploidy. The genus Phaseolus comprises ~ 90 species, five of them domesticated due to their nutritional relevance. Most of the species have 2n = 22 karyotypes and are highly syntenic, except for three dysploid karyotypes of species from the Leptostachyus group (2n = 20) that have accumulated several rearrangements. Here, we investigated the degrees of structural rearrangements among Leptostachyus and other Phaseolus groups by estimating their chromosomal evolution rates (CER). For this, we combined our oligo-FISH barcode system for beans and chromosome-specific painting probes for chromosomes 2 and 3, with rDNA and a centromeric probe to establish chromosome orthologies and identify structural rearrangements across nine Phaseolus species. We also integrated the detected rearrangements with a phylogenomic approach to estimate the CERs for each Phaseolus lineage. Our data allowed us to identify translocations, inversions, duplications and deletions, mostly in species belonging to the Leptostachyus group. Phaseolus leptostachyus showed the highest CER (12.31 rearrangements/My), a tenfold increase in contrast to the 2n = 22 species analysed. This is the highest rate known yet for plants, making it a model species for investigating the mechanisms behind rapid genome reshuffling in early species diversification.

Repeat-based phylogenomics shed light on unclear relationships in the monocentric genus Juncus L. (Juncaceae).

The repetitive fraction (repeatome) of eukaryotic genomes is diverse and usually fast evolving, being an important tool for clarify plant systematics. The genus Juncus L. comprises 332 species, karyotypically recognized by having holocentric chromosomes. However, four species were recently described as monocentric, yet our understanding of their genome evolution is largely masked by unclear phylogenetic relationships. Here, we reassess the current Juncus systematics using low-coverage genome skimming data of 33 taxa to construct repeats, nuclear rDNA and plastome-based phylogenetic hypothesis. Furthermore, we characterize the repeatome and chromosomal distribution of Juncus-specific centromeric repeats/CENH3 protein to test the monocentricity reach in the genus. Repeat-base phylogenies revealed topologies congruent with the rDNA tree, but not with the plastome tree. The incongruence between nuclear and plastome chloroplast dataset suggest an ancient hybridization in the divergence of Juncotypus and Tenageia sections 40 Myr ago. The phylogenetic resolution at section level was better fitted with the rDNA/repeat-based approaches, with the recognition of two monophyletic sections (Stygiopsis and Tenageia). We found specific repeatome trends for the main lineages, such as the higher abundances of TEs in the Caespitosi and Iridifolii + Ozophyllum clades. CENH3 immunostaining confirmed the monocentricity of Juncus, which can be a generic synapomorphy for the genus. The heterogeneity of the repeatomes, with high phylogenetic informativeness, identified here may be correlated with their ancient origin (56 Mya) and reveals the potential of comparative genomic analyses for understanding plant systematics and evolution.

Cytogenetic characterization and karyotype evolution in six Macroptilium species (Leguminosae).

Macroptilium (Benth.) Urb. is a neotropical legume genus from the subtribe Phaseolinae. The investigated species present a stable chromosome number (2n = 22), but differ in their karyotype formulae, suggesting the presence of chromosome rearrangements. In this work, we comparatively analysed the karyotypes of six species (Macroptilium atropurpureum, Macroptilium bracteatum, Macroptilium erythroloma, Macroptilium gracile, Macroptilium lathyroides, and Macroptilium martii) from the two main clades that form the genus. Heterochromatin distribution was investigated with chromomycin A3 (CMA)/4',6-diamidino-2-phenylindole (DAPI) staining and fluorescent in situ hybridization was used to localize the 5S and 35S ribosomal DNA (rDNA) sites. Single copy bacterial artificial chromosomes (BACs) previously mapped in the related genera Phaseolus L. and Vigna Savi were used to establish chromosome orthologies and to investigate possible rearrangements among species. CMA+/DAPI- bands were observed, mostly associated with rDNA sites. Additional weak, pericentromeric bands were observed on several chromosomes. Although karyotypes were similar, species could be differentiated mainly by the number and position of the 5S and 35S rDNA sites. BAC markers demonstrated conserved synteny of the main rDNA sites on orthologous chromosomes 6 and 10, as previously observed for Phaseolus and Vigna. The karyotypes of the six species could be differentiated, shedding light on its karyotype evolution.

Differential Repeat Accumulation in the Bimodal Karyotype of Agave L.

The genus Agave presents a bimodal karyotype with x = 30 (5L, large, +25S, small chromosomes). Bimodality within this genus is generally attributed to allopolyploidy in the ancestral form of Agavoideae. However, alternative mechanisms, such as the preferential accumulation of repetitive elements at the macrochromosomes, could also be important. Aiming to understand the role of repetitive DNA within the bimodal karyotype of Agave, genomic DNA from the commercial hybrid 11648 (2n = 2x = 60, 6.31 Gbp) was sequenced at low coverage, and the repetitive fraction was characterized. In silico analysis showed that ~67.6% of the genome is mainly composed of different LTR retrotransposon lineages and one satellite DNA family (AgSAT171). The satellite DNA localized at the centromeric regions of all chromosomes; however, stronger signals were observed for 20 of the macro- and microchromosomes. All transposable elements showed a dispersed distribution, but not uniform across the length of the chromosomes. Different distribution patterns were observed for different TE lineages, with larger accumulation at the macrochromosomes. The data indicate the differential accumulation of LTR retrotransposon lineages at the macrochromosomes, probably contributing to the bimodality. Nevertheless, the differential accumulation of the satDNA in one group of macro- and microchromosomes possibly reflects the hybrid origin of this Agave accession.

Investigating the diversification of holocentromeric satellite DNA Tyba in Rhynchospora (Cyperaceae).

BACKGROUND AND AIMS: Satellite DNAs (satDNAs) are repetitive sequences composed by tandemly arranged, often highly homogenized units called monomers. Although satDNAs are usually fast evolving, some satDNA families can be conserved across species separated by several millions of years, probably because of their functional roles in the genomes. Tyba was the first centromere-specific satDNA described for a holocentric organism, until now being characterized for only eight species of the genus Rhynchospora Vahl. (Cyperaceae). Here, we characterized Tyba across a broad sampling of the genus, analysing and comparing its evolutionary patterns with other satDNAs. METHODS: We characterized the structure and sequence evolution of satDNAs across a robust dadated phylogeny based on Hybrid Target-Capture Sequencing (hyb-seq) of 70 species. We mined the repetitive fraction for Tyba-like satellites to compare its features with other satDNAs and to construct a Tyba-based phylogeny for the genus. KEY RESULTS: Our results show that Tyba is present in the majority of examined species of the genus, spanning four of the five major clades and maintaining intrafamily pairwise identity of 70.9% over 31 Myr. In comparison, other satellite families presented higher intrafamily pairwise identity but are phylogenetically restricted. Furthermore, Tyba sequences could be divided into 12 variants grouped into three different clade-specific subfamilies, showing evidence of traditional models of satDNA evolution, such as the concerted evolution and library models. Besides, a Tyba-based phylogeny showed high congruence with the hyb-seq topology. Our results show structural indications of a possible relationship of Tyba with nucleosomes, given its high curvature peaks over conserved regions and overall high bendability values compared with other non-centromeric satellites. CONCLUSIONS: Overall, Tyba shows a remarkable sequence conservation and phylogenetic significance across the genus Rhynchospora, which suggests that functional roles might lead to long-term stability and conservation for satDNAs in the genome.

Plastome sequencing of South American Podocarpus species reveals low rearrangement rates despite ancient gondwanan disjunctions.

BACKGROUND: Historical reconstructions within Podocarpaceae have provided valuable information to disentangle biogeographic scenarios that begun 65 Mya. However, early molecular phylogenies of Podocarpaceae failed to agree on the intergeneric relationships within the family. The aims of this study were to test whether plastome organization is stable within the genus Podocarpus, to estimate the selective regimes affecting plastome protein-coding genes, and to strengthen our understanding of the phylogenetic relationships and biogeographic history. METHODS AND RESULTS: We sequenced the plastomes of four South American species from Patagonia, southern Yungas, and Brazilian subtropical forests. We compared their plastomes to those published from Brazil, Africa, New Zealand, and Southeast Asia, along with representatives from other genera within Podocarpaceae as outgroups. The four newly sequenced plastomes ranged in size between 133,791 bp and 133,991 bp. Gene content and order among chloroplasts from South American, African and Asian Podocarpus were conserved and different from the plastome of P. totara, from New Zealand. Most genes showed substitution patterns consistent with a conservative selective regime. Phylogenies inferred from either complete sequences or protein coding regions were mostly congruent with previous studies, but showed earlier branching of P. salignus, P. totara and P. sellowii. CONCLUSIONS: Highly similar and conserved plastomes of African, South American and Asian species suggest that P. totara plastome should be revised and compared to other species from Oceanic distribution. Furthermore, given such structural conservation, we suggest plastome sequencing is not useful to test whether genomic order can be climatically or geologically structured.

Repeat-based holocentromeres influence genome architecture and karyotype evolution.

The centromere represents a single region in most eukaryotic chromosomes. However, several plant and animal lineages assemble holocentromeres along the entire chromosome length. Here, we compare genome organization and evolution as a function of centromere type by assembling chromosome-scale holocentric genomes with repeat-based holocentromeres from three beak-sedge (Rhynchospora pubera, R. breviuscula, and R. tenuis) and their closest monocentric relative, Juncus effusus. We demonstrate that transition to holocentricity affected 3D genome architecture by redefining genomic compartments, while distributing centromere function to thousands of repeat-based centromere units genome-wide. We uncover a complex genome organization in R. pubera that hides its unexpected octoploidy and describe a marked reduction in chromosome number for R. tenuis, which has only two chromosomes. We show that chromosome fusions, facilitated by repeat-based holocentromeres, promoted karyotype evolution and diploidization. Our study thus sheds light on several important aspects of genome architecture and evolution influenced by centromere organization.

Comparative cytogenomics reveals genome reshuffling and centromere repositioning in the legume tribe Phaseoleae.

The tribe Phaseoleae includes several legume crops with assembled genomes. Comparative genomic studies have evidenced the preservation of large genomic blocks among legumes, although chromosome dynamics during Phaseoleae evolution has not been investigated. We conducted a comparative genomic analysis to define an informative genomic block (GB) system and to reconstruct the ancestral Phaseoleae karyotype (APK). We identified GBs based on the orthologous genes between Phaseolus vulgaris and Vigna unguiculata and searched for GBs in different genomes of the Phaseolinae (P. lunatus) and Glycininae (Amphicarpaea edgeworthii) subtribes and Spatholobus suberectus (sister to Phaseolinae and Glycininae), using Medicago truncatula as the outgroup. We also used oligo-FISH probes of two P. vulgaris chromosomes to paint the orthologous chromosomes of two non-sequenced Phaseolinae species. We inferred the APK as having n = 11 and 19 GBs (A to S), hypothesizing five chromosome fusions that reduced the ancestral legume karyotype to n = 11. We identified the rearrangements among the APK and the subtribes and species, with extensive centromere repositioning in Phaseolus. We also reconstructed the chromosome number reduction in S. suberectus. The development of the GB system and the proposed APK provide useful approaches for future comparative genomic analyses of legume species.

The Evolution of Cytogenetic Traits in Cuscuta (Convolvulaceae), the Genus With the Most Diverse Chromosomes in Angiosperms.

Karyotypes are characterized by traits such as chromosome number, which can change through whole-genome duplication and dysploidy. In the parasitic plant genus Cuscuta (Convolvulaceae), chromosome numbers vary more than 18-fold. In addition, species of this group show the highest diversity in terms of genome size among angiosperms, as well as a wide variation in the number and distribution of 5S and 35S ribosomal DNA (rDNA) sites. To understand its karyotypic evolution, ancestral character state reconstructions were performed for chromosome number, genome size, and position of 5S and 35S rDNA sites. Previous cytogenetic data were reviewed and complemented with original chromosome counts, genome size estimates, and rDNA distribution assessed via fluorescence in situ hybridization (FISH), for two, seven, and 10 species, respectively. Starting from an ancestral chromosome number of x = 15, duplications were inferred as the prevalent evolutionary process. However, in holocentric clade (subgenus Cuscuta), dysploidy was identified as the main evolutionary mechanism, typical of holocentric karyotypes. The ancestral genome size of Cuscuta was inferred as approximately 1C = 12 Gbp, with an average genome size of 1C = 2.8 Gbp. This indicates an expansion of the genome size relative to other Convolvulaceae, which may be linked to the parasitic lifestyle of Cuscuta. Finally, the position of rDNA sites varied mostly in species with multiple sites in the same karyotype. This feature may be related to the amplification of rDNA sites in association to other repeats present in the heterochromatin. The data suggest that different mechanisms acted in different subgenera, generating the exceptional diversity of karyotypes in Cuscuta.

Karyotype asymmetry in Cuscuta L. subgenus Pachystigma reflects its repeat DNA composition.

Cuscuta is a cytogenetically diverse genus, with karyotypes varying 18-fold in chromosome number and 127-fold in genome size. Each of its four subgenera also presents particular chromosomal features, such as bimodal karyotypes in Pachystigma. We used low coverage sequencing of the Cuscuta nitida genome (subgenus Pachystigma), as well as chromosome banding and molecular cytogenetics of three subgenus representatives, to understand the origin of bimodal karyotypes. All three species, C. nitida, C. africana (2n = 28) and C. angulata (2n = 30), showed heterochromatic bands mainly in the largest chromosome pairs. Eighteen satellite DNAs were identified in C. nitida genome, two showing similarity to mobile elements. The most abundant were present at the largest pairs, as well as the highly abundant ribosomal DNAs. The most abundant Ty1/Copia and Ty3/Gypsy elements were also highly enriched in the largest pairs, except for the Ty3/Gypsy CRM, which also labelled the pericentromeric regions of the smallest chromosomes. This accumulation of repetitive DNA in the larger pairs indicates that these sequences are largely responsible for the formation of bimodal karyotypes in the subgenus Pachystigma. The repetitive DNA fraction is directly linked to karyotype evolution in Cuscuta.

Oligo-FISH barcode in beans: a new chromosome identification system.

KEY MESSAGE: An Oligo-FISH barcode system was developed for two model legumes, allowing the identification of all cowpea and common bean chromosomes in a single FISH experiment, and revealing new chromosome rearrangements. The FISH barcode system emerges as an effective tool to understand the chromosome evolution of economically important legumes and their related species. Current status on plant cytogenetic and cytogenomic research has allowed the selection and design of oligo-specific probes to individually identify each chromosome of the karyotype in a target species. Here, we developed the first chromosome identification system for legumes based on oligo-FISH barcode probes. We selected conserved genomic regions between Vigna unguiculata (Vu, cowpea) and Phaseolus vulgaris (Pv, common bean) (diverged ~ 9.7-15 Mya), using cowpea as a reference, to produce a unique barcode pattern for each species. We combined our oligo-FISH barcode pattern with a set of previously developed FISH probes based on BACs and ribosomal DNA sequences. In addition, we integrated our FISH maps with genome sequence data. Based on this integrated analysis, we confirmed two translocation events (involving chromosomes 1, 5, and 8; and chromosomes 2 and 3) between both species. The application of the oligo-based probes allowed us to demonstrate the participation of chromosome 5 in the translocation complex for the first time. Additionally, we detailed a pericentric inversion on chromosome 4 and identified a new paracentric inversion on chromosome 10. We also detected centromere repositioning associated with chromosomes 2, 3, 5, 7, and 9, confirming previous results for chromosomes 2 and 3. This first barcode system for legumes can be applied for karyotyping other Phaseolinae species, especially non-model, orphan crop species lacking genomic assemblies and cytogenetic maps, expanding our understanding of the chromosome evolution and genome organization of this economically important legume group.

Plastome evolution in the Caesalpinia group (Leguminosae) and its application in phylogenomics and populations genetics.

MAIN CONCLUSION: The chloroplast genomes of Caesalpinia group species are structurally conserved, but sequence level variation is useful for both phylogenomic and population genetic analyses. Variation in chloroplast genomes (plastomes) has been an important source of information in plant biology. The Caesalpinia group has been used as a model in studies correlating ecological and genomic variables, yet its intergeneric and infrageneric relationships are not fully solved, despite densely sampled phylogenies including nuclear and plastid loci by Sanger sequencing. Here, we present the de novo assembly and characterization of plastomes from 13 species from the Caesalpinia group belonging to eight genera. A comparative analysis was carried out with 13 other plastomes previously available, totalizing 26 plastomes and representing 15 of the 26 known Caesalpinia group genera. All plastomes showed a conserved quadripartite structure and gene repertoire, except for the loss of four ndh genes in Erythrostemon gilliesii. Thirty polymorphic regions were identified for inter- or intrageneric analyses. The 26 aligned plastomes were used for phylogenetic reconstruction, revealing a well-resolved topology, and dividing the Caesalpinia group into two fully supported clades. Sixteen microsatellite (cpSSR) loci were selected from Cenostigma microphyllum for primer development and at least two were cross-amplified in different Leguminosae subfamilies by in vitro or in silico approaches. Four loci were used to assess the genetic diversity of C. microphyllum in the Brazilian Caatinga. Our results demonstrate the structural conservation of plastomes in the Caesalpinia group, offering insights into its systematics and evolution, and provides new genomic tools for future phylogenetic, population genetics, and phylogeographic studies.

Aiming off the target: recycling target capture sequencing reads for investigating repetitive DNA.

BACKGROUND AND AIMS: With the advance of high-throughput sequencing, reduced-representation methods such as target capture sequencing (TCS) emerged as cost-efficient ways of gathering genomic information, particularly from coding regions. As the off-target reads from such sequencing are expected to be similar to genome skimming (GS), we assessed the quality of repeat characterization in plant genomes using these data. METHODS: Repeat composition obtained from TCS datasets of five Rhynchospora (Cyperaceae) species were compared with GS data from the same taxa. In addition, a FISH probe was designed based on the most abundant satellite found in the TCS dataset of Rhynchospora cephalotes. Finally, repeat-based phylogenies of the five Rhynchospora species were constructed based on the GS and TCS datasets and the topologies were compared with a gene-alignment-based phylogenetic tree. KEY RESULTS: All the major repetitive DNA families were identified in TCS, including repeats that showed abundances as low as 0.01 % in the GS data. Rank correlations between GS and TCS repeat abundances were moderately high (r = 0.58-0.85), increasing after filtering out the targeted loci from the raw TCS reads (r = 0.66-0.92). Repeat data obtained by TCS were also reliable in developing a cytogenetic probe of a new variant of the holocentromeric satellite Tyba. Repeat-based phylogenies from TCS data were congruent with those obtained from GS data and the gene-alignment tree. CONCLUSIONS: Our results show that off-target TCS reads can be recycled to identify repeats for cyto- and phylogenomic investigations. Given the growing availability of TCS reads, driven by global phylogenomic projects, our strategy represents a way to recycle genomic data and contribute to a better characterization of plant biodiversity.

Large vs small genomes in Passiflora: the influence of the mobilome and the satellitome.

MAIN CONCLUSIONS: While two lineages of retrotransposons were more abundant in larger Passiflora genomes, the satellitome was more diverse and abundant in the smallest genome analysed. Repetitive sequences are ubiquitous and fast-evolving elements responsible for size variation and large-scale organization of plant genomes. Within Passiflora genus, a tenfold variation in genome size, not attributed to polyploidy, is known. Here, we applied a combined in silico and cytological approach to study the organization and diversification of repetitive elements in three species of this genus representing its known range in genome size variation. Sequences were classified in terms of type and repetitiveness and the most abundant were mapped to chromosomes. We identified long terminal repeat (LTR) retrotransposons as the most abundant elements in the three genomes, showing a considerable variation among species. Satellite DNAs (satDNAs) were less representative, but highly diverse between subgenera. Our results clearly confirm that the largest genome species (Passiflora quadrangularis) presents a higher accumulation of repetitive DNA sequences, specially Angela and Tekay elements, making up most of its genome. Passiflora cincinnata, with intermediate genome and from the same subgenus, showed similarity with P. quadrangularis regarding the families of repetitive DNA sequences, but in different proportions. On the other hand, Passiflora organensis, the smallest genome, from a different subgenus, presented greater diversity and the highest proportion of satDNA. Altogether, our data indicates that while large genomes evolved by an accumulation of retrotransposons, the smallest genome known for the genus has evolved by diversification of different repeat types, particularly satDNAs.

BAC- and oligo-FISH mapping reveals chromosome evolution among Vigna angularis, V. unguiculata, and Phaseolus vulgaris.

Cytogenomic resources have accelerated synteny and chromosome evolution studies in plant species, including legumes. Here, we established the first cytogenetic map of V. angularis (Va, subgenus Ceratotropis) and compared this new map with those of V. unguiculata (Vu, subgenus Vigna) and P. vulgaris (Pv) by BAC-FISH and oligopainting approaches. We mapped 19 Vu BACs and 35S rDNA probes to the 11 chromosome pairs of Va, Vu, and Pv. Vigna angularis shared a high degree of macrosynteny with Vu and Pv, with five conserved syntenic chromosomes. Additionally, we developed two oligo probes (Pv2 and Pv3) used to paint Vigna orthologous chromosomes. We confirmed two reciprocal translocations (chromosomes 2 and 3 and 1 and 8) that have occurred after the Vigna and Phaseolus divergence (~9.7 Mya). Besides, two inversions (2 and 4) and one translocation (1 and 5) have occurred after Vigna and Ceratotropis subgenera separation (~3.6 Mya). We also observed distinct oligopainting patterns for chromosomes 2 and 3 of Vigna species. Both Vigna species shared similar major rearrangements compared to Pv: one translocation (2 and 3) and one inversion (chromosome 3). The sequence synteny identified additional inversions and/or intrachromosomal translocations involving pericentromeric regions of both orthologous chromosomes. We propose chromosomes 2 and 3 as hotspots for chromosomal rearrangements and de novo centromere formation within and between Vigna and Phaseolus. Our BAC- and oligo-FISH mapping contributed to physically trace the chromosome evolution of Vigna and Phaseolus and its application in further studies of both genera.

Multiple and independent rearrangements revealed by comparative cytogenetic mapping in the dysploid Leptostachyus group (Phaseolus L., Leguminosae).

Polyploidy and dysploidy have been reported as the main events in karyotype evolution of plants. In the genus Phaseolus L. (2n = 22), a small monophyletic group of three species, the Leptostachyus group, presents a dysploid karyotype with 2n = 20. It was shown in Phaseolus leptostachyus that the dysploidy was caused by a nested chromosome fusion (NCF) accompanied by several translocations, suggesting a high rate of karyotype evolution in the group. To verify if this karyotype restructuring was a single event or occurred progressively during the evolution of this group, we analysed P. macvaughii, sister to Phaseolus micranthus + P. leptostachyus. Twenty-four genomic clones of P. vulgaris previously mapped on P. leptostachyus, in addition to the 5S and 35S rDNA probes, were used for fluorescence in situ hybridization. Only a single rearrangement was common to the two species: the nested chromosome fusion (NCF) involving chromosomes 10 and 11. The translocation of chromosome 2 is not the same found in P. leptostachyus, and pericentric inversions in chromosomed 3 and 4 were exclusive of P. macvaughii. The other rearrangements observed in P. leptostachyus were not shared with this species, suggesting that they occurred after the separation of these lineages. The presence of private rearrangements indicates a progressive accumulation of karyotype changes in the Leptostachyus group instead of an instant genome-wide repatterning.

How diverse is heterochromatin in the Caesalpinia group? Cytogenomic characterization of Erythrostemon hughesii Gagnon & G.P. Lewis (Leguminosae: Caesalpinioideae).

MAIN CONCLUSION: Cytogenomic characterization of Erythrostemon hughesii reveals a heterogeneity of repeats in its subtelomeric heterochromatin. Comparative analyses with other Caesalpinia group species reveal a significant reduction in the abundance of Ty3-gypsy/Chromovirus Tekay retrotransposons during its evolution. In numerically stable karyotypes, repetitive DNA variability is one of the main causes of genome and chromosome variation and evolution. Species from the Caesalpinia group (Leguminosae) are karyotypically characterized by 2n = 24, with small chromosomes and highly variable CMA+ heterochromatin banding patterns that correlate with environmental variables. Erythrostemon hughesii differs from other species of the group examined to date for having subtelomeric CMA+ bands; this contrasts with most species in the group which have proximal bands. Here we analyse the repeatome of E. hughesii using genome skimming and chromosomal mapping approaches to characterize the identity of the most abundant repetitive elements and their physical location. The repetitive fraction of E. hughesii comprises 28.73% of the genome. The most abundant elements were retrotransposons (RT) with long terminal repeats (LTR-RT; 9.76%) and satellite DNAs (7.83%). Within the LTR-RTs, the most abundant lineages were: Ty1/copia-Ale (1%), Ty3/gypsy CRM (0.88%) and Ty3/gypsy Athila (0.75%). Using fluorescent in situ hybridization four satellite DNAs and several LTR-RT elements were shown to be present in most subtelomeric CMA+ bands. These results highlight how the repeatome in E. hughesii, a species from Oaxaca state in Mexico, is clearly distinct from Northeast Brazilian species of the Caesalpinia group, mainly due to its high diversity of repeats in its subtelomeric heterochromatic bands and low amount of LTR-RT Ty3/gypsy-Tekay elements. Comparative sequence analysis of Tekay elements from different species is congruent with a clade-specific origin of this LTR-RT after the divergence of the Caesalpinia group. We hypothesize that repeat-rich heterochromatin may play a role in leading to faster genomic divergence between individuals, increasing speciation and diversification.