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Schematic BAC-FISH comparative analysis, using BACs from P. vulgaris and Vigna unguiculata (*), for six chromosomes of V. aconitifolia (Vac; present work) and V. unguiculata (Vu; Vasconcelos et al. 2015), Phaseolus vulgaris (Pv; Pedrosa-Harand et al. 2009; Fonsêca et al. 2010), P. lunatus (Plu; Bonifácio et al. 2012; Almeida and Pedrosa-Harand 2013), and P. microcarpus (Pm; Fonsêca and Pedrosa-Harand 2013), involved in the major rearrangements in relation to P. vulgaris, considering the proposed phylogeny for the genera Vigna and Phaseolus (adapted from
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Comparative cytogenetic mapping is a powerful approach to gain insights into genome organization of orphan crops, lacking a whole sequenced genome. To investigate the cytogenomic evolution of important Vigna and Phaseolus beans, we built a BAC-FISH (fluorescent in situ hybridization of bacterial artificial chromosome) map of Vigna aconitifolia (Vac...
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... Pedrosa-Harand 2013) and P. lunatus ( Bonifácio et al. 2012; Almeida and PedrosaHarand 2013). The cytogenetic comparison is schematically represented in a simplified phylogenetic tree (adapted of Delgado-Salinas et al. 2011), which helped us to understand the macrosynteny, collinearity, and chromosome alterations among Vigna and Phaseolus species (Fig. 4). Inversions were the only rearrangement type found in Phaseolus genus. One paracentric and one pericentric inversion were identified between clades A and B. Within clade B (P. vulgaris and P. lunatus), three pericentric inversions were previously described. Altogether, the results described above associated with these previous BAC-FISH ...
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... 221J10 (Pv4) and 190C15 (Pv4) in Vac4. h 5S rDNA and BAC M002E09 (Vu5) in Vac5. Bars in a and h represent 5 μm We also expanded our comparison to the literature data for P. lunatus ( Bonifácio et al. 2012;Almeida and Pedrosa-Harand 2013) and P. microcarpus (Fonsêca and Pedrosa-Harand 2013), including the chromosomes involved in rearrangements (Fig. 4). In Phaseolus, P. vulgaris, P. lunatus, and P. microcarpus showed conserved synteny, being identified by BAC-FISH only inversions involving four chromosomes (3, 6, 9, and 10, but only Chr3 is represented in Fig. 4) resulting in breaks of collinearity, in addition to one duplication involving Pm3 and Pm6 (Pv3 marker) ( Fonsêca et al. ...
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... and Pedrosa-Harand 2013) and P. microcarpus (Fonsêca and Pedrosa-Harand 2013), including the chromosomes involved in rearrangements (Fig. 4). In Phaseolus, P. vulgaris, P. lunatus, and P. microcarpus showed conserved synteny, being identified by BAC-FISH only inversions involving four chromosomes (3, 6, 9, and 10, but only Chr3 is represented in Fig. 4) resulting in breaks of collinearity, in addition to one duplication involving Pm3 and Pm6 (Pv3 marker) ( Fonsêca et al. 2010;Bonifácio et al. 2012;Fonsêca and Pedrosa-Harand ...
Citations
... characterization of the heterochromatin and repetitive sequence distribution pattern in Phaseolus and related genera, such as Vigna (Fonsêca and Pedrosa-Harand 2017, Oliveira et al. 2020, Ribeiro et al. 2020). Almeida and Pedrosa-Harand (2013) demonstrated that lima bean and common bean (P. ...
... Vigna), and P. vulgaris. These species are closely related, with an estimated divergence around 9.7 my (Li et al. 2013;Vasconcelos et al. 2015;Oliveira et al. 2020;do Vale Martins et al. 2021;de Oliveira Bustamante et al. 2021). ...
... On the other hand, BACs H080A10 and 169G16 were detected in the opposite arm of these species (Table S2 and Fig. S1d, h, l). Similar findings regarding the location on different arms of chromosome 8 was also reported by Oliveira et al. (2020) for V. aconitifolia and V. unguiculata (BACs 221F15 and 169G16) and by do Vale Martins et al. (2021) for V. angularis and V. unguiculata (BACs M057N05 and H080A10). ...
... Our work, using V. unguiculata as a reference, confirmed karyotype changes previously detected in V. aconitifolia and V. angularis (both from V. subg. Ceratotropis) (Vasconcelos et al. 2015;Oliveira et al. 2020;do Vale Martins et al. 2021). We successfully identified additional chromosome rearrangements in several new Vigna species, especially in V. vexillata, belonging to the African V. subg. ...
Inversions and translocations are the major chromosomal rearrangements involved in Vigna subgenera evolution, being Vigna vexillata the most divergent species. Centromeric repositioning seems to be frequent within the genus.
Oligonucleotide-based fluorescence in situ hybridization (Oligo-FISH) provides a powerful chromosome identification system for inferring plant chromosomal evolution. Aiming to understand macrosynteny, chromosomal diversity, and the evolution of bean species from five Vigna subgenera, we constructed cytogenetic maps for eight taxa using oligo-FISH-based chromosome identification. We used oligopainting probes from chromosomes 2 and 3 of Phaseolus vulgaris L. and two barcode probes designed from V. unguiculata (L.) Walp. genome. Additionally, we analyzed genomic blocks among the Ancestral Phaseoleae Karyotype (APK), two V. unguiculata subspecies (V. subg. Vigna), and V. angularis (Willd.) Ohwi & Ohashi (V. subg. Ceratotropis). We observed macrosynteny for chromosomes 2, 3, 4, 6, 7, 8, 9, and 10 in all investigated taxa except for V. vexillata (L.) A. Rich (V. subg. Plectrotropis), in which only chromosomes 4, 7, and 9 were unambiguously identified. Collinearity breaks involved with chromosomes 2 and 3 were revealed. We identified minor differences in the painting pattern among the subgenera, in addition to multiple intra- and interblock inversions and intrachromosomal translocations. Other rearrangements included a pericentric inversion in chromosome 4 (V. subg. Vigna), a reciprocal translocation between chromosomes 1 and 5 (V. subg. Ceratotropis), a potential deletion in chromosome 11 of V. radiata (L.) Wilczek, as well as multiple intrablock inversions and centromere repositioning via genomic blocks. Our study allowed the visualization of karyotypic patterns in each subgenus, revealing important information for understanding intrageneric karyotypic evolution, and suggesting V. vexillata as the most karyotypically divergent species.
... Somta et al. (2018), suggested that markers CEDG261 and DMB-SSR160 should be useful for marker-assisted selection for C. chinensis resistance in moth bean. A list of genetic linkage maps in Vigna aconitifolia is depicted in Table 7. Oliveira et al. (2020) presented the first physical map of moth bean and compared chromosomes with other Vigna and Phaseolus species. As with Yundaeng et al. (2019), a high magnitude of genome synteny was observed between moth bean and other related pulse crops such as mung bean (Vigna radiata), adzuki bean (V. ...
... umbellata), yard long bean (V. unguiculata) as well as common bean (Phaseolus vulgaris) (Oliveira et al., 2020). As a result, molecular markers and genomic resources developed in these crops will be highly useful in moth bean crop improvement programmes. ...
Moth bean (Vigna aconitifolia) is an orphan legume of Vigna genus, exhibiting wide adaptability and has the potential to grow well in arid and semi-arid areas, predominantly across different eco-geographical regions of Asia, particularly the Indian subcontinent. The inherent adaptive attributes of this crop have made it more tolerant towards a diverse array of abiotic and biotic stresses that commonly restrain yield among other Vigna species. Additionally, the legume is recognized for its superior nutritional quality owing to its high protein content as well as amino acid, mineral and vitamin profile and is utilized as both food and fodder. Moth bean can play a vital role in sustaining food grain production, enhancing nutritional security as well as provide a source of income to resource-poor farmers amid rise in global temperatures and frequent drought occurrences, particularly in rain-fed cropping systems which accounts for about 80% of the world’s cultivated land. However, this minor legume has remained underutilized due to over-exploitation of major staple crops. With the exception of a few studies involving conventional breeding techniques, crop improvement in moth bean for traits such as late maturity, indeterminate growth habit, shattering and anti-nutritional factors has not garnered a lot of attention. Recent advances in sequencing technologies, modern breeding approaches and precision phenotyping tools, in combination with the available crop gene pool diversity in gene banks, can accelerate crop improvement in moth bean and lead to the development of improved cultivars. Considering the recent surge in awareness about the development of climate-smart crops for sustainable agricultural future, collective effort towards effective utilization of this hardy, neglected legume is the need of the hour.
... The three genera share the same chromosome number for most of their species (2n = 22), with small chromosomes and mostly symmetrical karyotypes (Forni-Martins1989;Mercado-Ruaro and Delgado-Salinas 1998). Nevertheless, detailed comparative genomics and chromosome maps using bacterial artificial chromosome (BAC) and oligo-fluorescent in situ hybridization (FISH) probes revealed inversions and translocations in Phaseolus and Vigna (Fonsêca and Pedrosa-Harand 2013;Vasconcelos et al. 2015;Fonsêca et al. 2016;Lonardi et al. 2019;Oliveira et al. 2020; de Oliveira Bustamante et al. 2021;Montenegro et al. 2022). In the reconstruction of the ancestral Phaseoleae karyotype (APK), oligopaints of chromosomes 2 and 3 of Phaseolus vulgaris L. were applied to Macroptilium atropurpureum (Sessé & Moc.ex DC.) Urb., confirming a reciprocal translocation between these chromosomes in P. vulgaris and the conservation of the APK pattern in Macroptilium and Vigna (Montenegro et al. 2022). ...
... Due to the large variation observed in rDNA sites among species in many groups, identification of orthologous chromosomes relies on chromosome-specific markers to identify chromosomes individually, such as BAC clones from genomic libraries (Peterson et al. 2000;Fonsêca et al. 2010;Feng et al. 2013;Sader et al. 2021;Ferraz et al. 2020;Oliveira et al. 2020;de Oliveira Bustamante et al. 2021;). These markers have been used in phylogenetically related groups, showing, for example, the chromosome conservation of one 35S and one 5S rDNA site in orthologous chromosomes 6 and 10, respectively, in Phaseolus and Vigna (Fonsêca and Pedrosa-Harand 2013;Vasconcelos et al. 2015;Ferraz et al. 2020;Oliveira et al. 2020). ...
... Due to the large variation observed in rDNA sites among species in many groups, identification of orthologous chromosomes relies on chromosome-specific markers to identify chromosomes individually, such as BAC clones from genomic libraries (Peterson et al. 2000;Fonsêca et al. 2010;Feng et al. 2013;Sader et al. 2021;Ferraz et al. 2020;Oliveira et al. 2020;de Oliveira Bustamante et al. 2021;). These markers have been used in phylogenetically related groups, showing, for example, the chromosome conservation of one 35S and one 5S rDNA site in orthologous chromosomes 6 and 10, respectively, in Phaseolus and Vigna (Fonsêca and Pedrosa-Harand 2013;Vasconcelos et al. 2015;Ferraz et al. 2020;Oliveira et al. 2020). Considering the reported karyotype variability observed in the genus despite the low sampling of cytogenetic data, as well as the presence of chromosome rearrangements in Phaseolus and related genera, the aim of this work was to understand the main events involved in the karyotype evolution of Macroptilium by analysing its heterochromatin and rDNA distribution, as well as the conservation of orthologous chromosomes using chromosome-specific markers. ...
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 (M. atropurpureum, M. bracteatum, M. erythroloma, M. gracile, M. lathyroides and M. martii) from the two main clades that form the genus. Heterochromatin distribution was investigated with CMA/DAPI staining and fluorescent in situ hybridization (FISH) was used to localise the 5S and 35S ribosomal DNA (rDNA) sites. Single copy BACs (Bacterial Artificial Chromosomes) 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.
... and P. macvaughii Delgado (Leptostachyus clade), on the other hand, evidenced extensive genome reshuffling associated with descending dysploidy involving a nested chromosome fusion between chromosomes 10 and 11 (Fonsêca et al. 2016;Ferraz et al. 2020). Furthermore, even though comparative cytogenetics and sequence alignments between Vigna Savi species and P. vulgaris revealed a high degree of macrosynteny between the genera, at least five chromosomes were found to be involved in synteny breaks (Vasconcelos et al. 2015;Lonardi et al. 2019;Oliveira et al. 2020;do Vale Martins et al. 2021). A detailed analysis of chromosomes 2 and 3 of V. angularis, V. unguiculata, and P. vulgaris, based on integrative sequence alignment and oligo-FISH painting approaches, identified additional macro-and micro-inversions, translocations, and intergeneric centromere repositioning among the species analysed (do Vale Martins et al. 2021). ...
... Few translocations and many inversions were identified within Phaseolinae. Some of those rearrangements were previously identified by BAC-FISH, oligo-FISH, and comparative genomics in P. vulgaris, P. lunatus, and V. unguiculata (Bonifácio et al. 2012;Vasconcelos et al. 2015;Lonardi et al. 2019;Oliveira et al. 2020;do Vale Martins et al. 2021;Garcia et al. 2021;de Oliveira Bustamante et al. 2021). Based on our APK and oligo-FISH approaches, we are now able to propose the direction of those rearrangements within a phylogenetic context. ...
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.
... Ohwi and Ohashi (do Vale Martins et al. 2021) and Vigna aconitifolia (Jacq.) Maréchal (Oliveira et al. 2020). These studies enabled the expansion of the comparative cytogenetic analyses between Vigna and Phaseolus species. ...
... The positions of each barcode marker were measured in 20 chromatids from five metaphases per species, following Fonsêca et al. (2010), except for using the DRAWID 0.26 software (Kirov et al. 2017). The new markers were integrated into the already established maps of V. unguiculata (Vasconcelos et al. 2015;Oliveira et al. 2020) and P. vulgaris (Fonsêca et al. 2010). ...
... Additionally, the sequences available for BAC markers used in previous studies for both species were included. BACs from V. unguiculata were previously used by Oliveira et al. (2020) and do Vale Martins et al. (2021) and are available at HarvEST:cowpea (harvest.ucr.edu). The P. vulgaris BAC sequence accessions used for the integrated map are provided in Table S2 and were obtained by BLAST using the sequences of corresponding genetic markers used to select each BAC and provided by the studies of Vallejos et al. (1992), Murray et al. (2002), Hougaard et al. (2008), and Geffroy et al. (2009). ...
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.
Abstract
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.
... Macrosynteny breaks were observed between these two species, by identifying rearrangements involving five V. unguiculata chromosomes, as translocations, inversions, and duplication, with major rearrangements involving chromosomes 2 and 3. Later, Iwata-Otsubo et al. (2016a) integrated the genetic and physical cowpea maps on V. unguiculata pachytene chromosomes by BAC-FISH. Oliveira et al. (2020) generated the first BAC-FISH map to V. aconitifolia (Jacq.) Maréchal (Ceratotropis subgenus) and compared this map to those of V. unguiculata and P. vulgaris chromosome maps, corroborating chromosomes 2 and 3 as hotspots for chromosomal changes. ...
... In Vigna, the physical BAC-FISH mapping has been performed only in V. unguiculata (Vasconcelos et al. 2015) and V. aconitifolia (Oliveira et al. 2020). This limited number of species has prevented an analysis of chromosome evolution in the whole genus. ...
... Phaseolus vulgaris was chosen as reference genome because of the Pv2/Pv3 BAC complex translocation described previously for chromosomes 2 and 3 of Vigna species (Vasconcelos et al. 2015;Oliveira et al. 2020). Briefly, the genomic sequences were divided into 45 bp oligos in a step size of 5 bp. ...
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.
... On the other hand, similar comparative cytogenetic mapping of two species of the Leptostachyus clade from the same genus showed extensive genome reshuffling associated with descending dysploidy involving a nested chromosome fusion between chromosomes 10 and 11 (Fonsêca et al., 2016;Ferraz et al., 2020). Furthermore, comparative cytogenetics and sequence alignment between Vigna species and P. vulgaris revealed a high degree of synteny with five chromosomes involved in synteny breaks (Vasconcelos et al., 2015;Lonardi et al., 2019;Oliveira et al., 2020;do Vale Martins et al., 2021). A detailed analysis of chromosomes 2 and 3 of V. angularis, V. unguiculata and P. vulgaris based on sequence alignment and oligo-FISH painting integrative approaches, identified additional macro-and micro inversions, translocations, and intergeneric centromere repositioning (do Vale Martins et al., 2021). ...
... lunatus and V. unguiculata (Bonifácio et al., 2012;Vasconcelos et al., 2015;Lonardi et al., 2019;Oliveira et al., 2020;do Vale Martins et al., 2021;Garcia et al., 2021;Bustamante et al., 2021). Based on our APK and oligo-FISH approaches, we can propose the direction of these rearrangements in a (which was not certified by peer review) is the author/funder. ...
The tribe Phaseoleae (Leguminosae; Papilionoideae) includes several legume crops with assembled genomes. Comparative genomic studies indicate the preservation of large genomic blocks among legumes, however, the chromosome dynamics during Phaseoleae evolution has not been investigated yet. We conducted a comparative genomic analysis to define an informative genomic block (GB) system and to reconstruct the ancestral Phaseoleae karyotype (APK). We defined the GBs based on the orthologous genes between Phaseolus vulgaris and Vigna unguiculata genomes. We searched for these GBs in different genome species belonging to the Phaseolinae ( P. lunatus ) and Glycininae subtribes ( Amphicarpaea edgeworthii and Spatholobus suberectus ), and in the Medicago truncaluta outgroup. To support our in silico analysis, we used oligo-FISH probes of P. vulgaris chromosomes 2 and 3 to paint the orthologous chromosomes of two non-sequenced Phaseolinae species ( Macroptilium atropurpureum and Lablab purpureus ). We inferred the APK with n = 11 and 19 GBs (A to S). We hypothesized five chromosome fusions that reduced the ancestral legume karyotype with n = 16 to n = 11 in APK. Furthermore, we identified the main rearrangements within Phaseolinae and observed an extensive centromere repositioning resulting from evolutionary new centromeres (ENC) in the Phaseolus lineage. Additionally, we demonstrated that the A. edgeworthii genome is more reshuffled than the dysploid S. suberectus genome, in which we could reconstruct the main events that lead the chromosome number reduction. The development of the GB system and the proposed APK provide useful approaches for future comparative genomic analyses of legume species.
... and V. unguiculata (L.) Walp. without detected changes in collinearity and may be related to variation in the 35S rDNA block size or other repetitive sequences (Oliveira et al. 2020). ...
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.
Macroptilium (Benth.) Urb. is a forage genus native to regions from North to South America. Phylogenetic analyses place it close to Phaseolus L., but its infrageneric division into two sections is not well supported. Despite its chromosomal number stability (2 n = 22), interspecific rDNA loci variation enabled species differentiation, suggesting that a cytogenomic approach might be valuable for inferring species relationships and genome evolution. Here, we (1) characterized nine Macroptilium species through oligonucleotide-based chromosome painting and barcoding (Oligo-FISH); (2) generated genome skimming data for six species and used it to investigate their repeatome dynamics, and (3) performed phylogenomic reconstruction using complete plastomes. Oligo-FISH data unveiled de novo translocations between chromosomes 2 and 6, and 3 and 11 in species from proposed groups II and III, respectively, in disagreement with the currently proposed phylogenetic hypothesis. Our phylogenomic (plastid) and repeatome (nuclear) analyses supported groups II and III as clades, with shared satDNA families. Group I was paraphyletic and resembled the Ancestral Phaseolinae Karyotype. We demonstrated the efficiency of different cytogenomic approaches to characterize Macroptilium species, providing insights into its genomic evolution and indicating the need for a systematic re-evaluation of the genus. These findings also support the power of these approaches to solve phylogenetic relationships even in groups with chromosome number stability and recent diversification.
