Artur Fonsêca

Cytogenetic map of common bean (Phaseolus vulgaris L.)

A cytogenetic map of common bean was built by in situ hybridization of 35 bacterial artificial chromosomes (BACs) selected with markers mapping to eight linkage groups, plus two plasmids for 5S and 45S ribosomal DNA and one bacteriophage. Together with three previously mapped chromosomes (chromosomes 3, 4, and 7), 43 anchoring points between the genetic map and the cytogenetic map of the species are now available. Furthermore, a subset of four BAC clones was proposed to identify the 11 chromosome pairs of the standard cultivar BAT93. Three of these BACs labelled more than a single chromosome pair, indicating the presence of repetitive DNA in their inserts. A repetitive distribution pattern was observed for most of the BACs; for 38% of them, highly repetitive pericentromeric or subtelomeric signals were observed. These distribution patterns corresponded to pericentromeric and subtelomeric heterochromatin blocks observed with other staining methods. Altogether, the results indicate that around half of the common bean genome is heterochromatic and that genes and repetitive sequences are intermingled in the euchromatin and heterochromatin of the species.

Comparative cytogenetic mapping between the lima bean (Phaseolus lunatus L.) and the common bean (P. vulgaris L.)

The common bean (Phaseolus vulgaris) and lima bean (P. lunatus) are among the most important legumes in terms of direct human consumption. The present work establishes a comparative cytogenetic map of P. lunatus, using previously mapped markers from P. vulgaris, in association with analyses of heterochromatin distribution using the fluorochromes chromomycin A3 (CMA) and 4′,6-diamidino-2-phenylindole (DAPI) and localization of the 5S and 45S ribosomal DNA (rDNA) probes. Seven BACs selected from different common bean chromosomes demonstrated a repetitive pericentromeric pattern corresponding to the heterochromatic regions revealed by CMA/DAPI and could not be mapped. The subtelomeric repetitive pattern observed for BAC 63H6 in most of the chromosome ends of common bean was not detected in lima bean, indicating lack of conservation of this subtelomeric repeat. All chromosomes could be identified and 16 single-copy clones were mapped. These results showed a significant conservation of synteny between species, although change in centromere position suggested the occurrence of pericentric inversions on chromosomes 2, 9 and 10. The low number of structural rearrangements reflects the karyotypic stability of the genus.

Intra- and interchromosomal rearrangements between cowpea (Vigna unguiculata (L.) Walp.) and common bean (Phaseolus vulgaris L.) revealed by BAC-FISH

Cowpea (Vigna unguiculata) is an annual legume grown in tropical and subtropical regions, which is economically relevant due to high protein content in dried beans, green pods, and leaves. In this work, a comparative cytogenetic study between V. unguiculata and Phaseolus vulgaris (common bean) was conducted using BAC-FISH. Sequences previously mapped in P. vulgaris chromosomes (Pv) were used as probes in V. unguiculata chromosomes (Vu), contributing to the analysis of macrosynteny between both legumes. Thirty-seven clones from P. vulgaris ‘BAT93’ BAC library, corresponding to its 11 linkage groups, were hybridized in situ. Several chromosomal rearrangements were identified, such as translocations (between BACs from Pv1 and Pv8; Pv2 and Pv3; as well as Pv2 and Pv11), duplications (BAC from Pv3), as well as paracentric and pericentric inversions (BACs from Pv3, and Pv4, respectively). Two BACs (from Pv2 and Pv7), which hybridized at terminal regions in almost all P. vulgaris chromosomes, showed single-copy signal in Vu. Additionally, 17 BACs showed no signal in V. unguiculata chromosomes. The present results demonstrate the feasibility of using BAC libraries in comparative chromosomal mapping and karyotype evolution studies between Phaseolus and Vigna species, and revealed several macrosynteny and collinearity breaks among both legumes.

Speeding up chromosome evolution in Phaseolus : multiple rearrangements associated with a one-step descending dysploidy

The genus Phaseolus L. has been subject of extensive cytogenetic studies due to its global economic importance. It is considered karyotypically stable, with most of its ca. 75 species having 2n = 22 chromosomes, and only three species (Phaseolus leptostachyus, Phaseolus macvaughii, and Phaseolus micranthus), which form the Leptostachyus clade, having 2n = 20. To test whether a simple chromosomal fusion was the cause of this descending dysploidy, mitotic chromosomes of P. leptostachyus (2n = 20) were comparatively mapped by fluorescent in situ hybridization (FISH) using bacterial artificial chromosomes (BACs) and ribosomal DNA (rDNA) probes. Our results corroborated the conservation of the 5S and 45S rDNA sites on ancestral chromosomes 10 and 6, respectively. The reduction from x = 11 to x = 10 was the result of the insertion of chromosome 10 into the centromeric region of chromosome 11, supporting a nested chromosome fusion (NCF) as the main cause of this dysploidy. Additionally, the terminal region of the long arm of chromosome 6 was translocated to this larger chromosome. Surprisingly, the NCF was accompanied by several additional translocations and inversions previously unknown for the genus, suggesting that the dysploidy may have been associated to a burst of genome reorganization in this otherwise stable, diploid plant genus.

Chromosome identification in the Andean common bean accession G19833 (Phaseolus vulgaris L., Fabaceae)

Characterization of all chromosomes of the Andean G19833 bean genotype was carried out by fluorescent in situ hybridization. Eleven single-copy genomic sequences, one for each chromosome, two BACs containing subtelomeric and pericentromeric repeats and the 5S and 45S ribosomal DNA (rDNA) were used as probes. Comparison to the Mesoamerican accession BAT93 showed little divergence, except for additional 45S rDNA sites in four chromosome pairs. Altogether, the results indicated a relative karyotypic stability during the evolution of the Andean and Mesoamerican gene pools of P. vulgaris.

Epigenetic Analyses and the Distribution of Repetitive DNA and Resistance Genes Reveal the Complexity of Common Bean (Phaseolus vulgaris L., Fabaceae) Heterochromatin

The common bean (Phaseolus vulgaris L.) is the main representative of its genus and one of most important sources of proteins in African and Latin American countries. Although it is a species with a small genome, its pericentromeric and subtelomeric heterochromatin fractions are interspersed with single-copy sequences and active genes, suggesting a less compartmentalized genome organization. The present study characterized its chromatin fractions, associating the distribution of repetitive sequences and resistance genes with histone and DNA epigenetic modifications with and without biotic stress. Immunostaining with H3K4me3 and H4K5ac were generally associated with euchromatic regions, whereas H3K9me2, H3K27me1, and 5mC preferentially labeled the pericentromeric heterochromatin. The 45S rDNA and centromeric DNA sequences were hypomethylated as were most of the terminal heterochromatic blocks. The largest of them, which is associated with resistance genes, was also hypomethylated after the plants were infected with virulent and avirulent strains of the fungus Colletotrichum lindemuthianum, suggesting no correlation with control of resistance gene expression. The results highlighted the differences between subtelomeric and pericentromeric heterochromatin as well as variation within the pericentromeric heterochromatin.

Cytogenetics and Comparative Analysis of Phaseolus Species

The genus Phaseolus includes approximately 75 species, most of which have 2n = 22 small meta- or submetacentric chromosomes. This considerable karyotypic stability has been further reinforced by comparative cytogenetic mapping of single-copy sequences using Bacterial Artificial Chromosomes (BACs) and the Fluorescent in situ Hybridization (FISH) technique. These physical maps have revealed complete macrosynteny among the investigated species and only a few breaks in collinearity due to chromosomal inversions, except for Phaseolus leptostachyus, with 2n = 20 and several rearrangements. The variation in the repetitive fraction of the genome is much greater, however, as expected. Considerable variation has been found in the number of 35S rDNA sites in the representatives of the Vulgaris group, and repetitive pericentromeric and subtelomeric sequences vary among Phaseolus species. This repetitive genome fraction has also been investigated in Phaseolus vulgaris through an epigenetic approach, revealing a higher degree of complexity in the heterochromatin than previously thought. The available molecular tools for the common bean have permitted a systematic analysis of the synteny, not only between the common bean and other cultivated and wild species of the genus, but also at the intergeneric level, contributing to a more detailed understanding of the cytogenetics of the group.