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.

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.

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.

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.

Karyotype stability in the genus Phaseolus evidenced by the comparative mapping of the wild species Phaseolus microcarpus.

The genus Phaseolus L. (Fabaceae) is monophyletic and comprises approximately 75 species distributed into two principal clades. The five cultivated species, including the common bean (Phaseolus vulgaris), were placed in clade B. Clade A comprises only wild species, with more limited distribution. In the present work, bacterial artificial chromosomes (BACs) previously mapped in common bean (2n = 22) were used as probes in fluorescent in situ hybridization (FISH) in this comparative study of Phaseolus microcarpus (2n = 22), a species from clade A. We also analyzed the chromomycin A3 (CMA)/4',6-diamidino-2-phenylindole (DAPI) banding pattern and the localization of rDNA and telomeric DNA sites. The single 45S rDNA site from P. microcarpus was mapped to chromosome 6, showing conservation to the P. vulgaris homeolog. Of the two 5S rDNA sites identified in both species, only the site on chromosome 10 appeared conserved. In spite of the phylogenetic distance between the two species, all of the single-copy BACs demonstrated conservation of synteny. However, four collinearity breaks were observed, probably caused by para- and pericentric inversions. Some variation in the repetitive fraction of the genome was also observed. Thus, a broader analysis of the genus confirms that few, rare inversions seem to represent the main karyotype changes during the evolution of this genus.

Identification and characterization of functional centromeres of the common bean.

In higher eukaryotes, centromeres are typically composed of megabase-sized arrays of satellite repeats that evolve rapidly and homogenize within a species' genome. Despite the importance of centromeres, our knowledge is limited to a few model species. We conducted a comprehensive analysis of common bean (Phaseolus vulgaris) centromeric satellite DNA using genomic data, fluorescence in situ hybridization (FISH), immunofluorescence and chromatin immunoprecipitation (ChIP). Two unrelated centromere-specific satellite repeats, CentPv1 and CentPv2, and the common bean centromere-specific histone H3 (PvCENH3) were identified. FISH showed that CentPv1 and CentPv2 are predominantly located at subsets of eight and three centromeres, respectively. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of CentPv1 and CentPv2 at centromeres. Genomic analysis revealed several interesting features of CentPv1 and CentPv2: (i) CentPv1 is organized into an higher-order repeat structure, named Nazca, of 528 bp, whereas CentPv2 is composed of tandemly organized monomers; (ii) CentPv1 and CentPv2 have undergone chromosome-specific homogenization; and (iii) CentPv1 and CentPv2 are not likely to be commingled in the genome. These findings suggest that two distinct sets of centromere sequences have evolved independently within the common bean genome, and provide insight into centromere satellite evolution.

Contrasting evolution of a satellite DNA and its ancestral IGS rDNA in Phaseolus (Fabaceae).

CC4 is a satellite DNA from common bean (Phaseolus vulgaris L.) that is similar to its intergenic spacer (IGS) rDNA. CC4 was originally hypothesized to be an old, fast evolving satellite family that has invaded common bean rDNA. To test this hypothesis and contribute to the understanding of IGS-like satellite DNA evolution, we have investigated its distribution in the genus Phaseolus and related species. CC4 was cloned and used as probe for Southern blot and FISH experiments. CC4 was observed as an independent satellite in common bean, forming two to three major and a few minor pericentromeric clusters. In Phaseolus coccineus, CC4 was present in four major clusters, also not co-localized with the 45S rDNA sites. Remarkably, in the less related species of the genus, signals were detected co-localized with the 45S rDNA sites, but co-localization was not observed in the species where CC4 is present as an independent satellite. No signal was detected in species from related genera. Altogether, the data suggest that CC4 has originated from the IGS rDNA in the P. vulgaris-P. coccineus lineage and has evolved slower than the IGS rDNA from this lineage.

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.

Isolation and characterization of a new repetitive DNA family recently amplified in the Mesoamerican gene pool of the common bean (Phaseolus vulgaris L., Fabaceae).

The common bean (Phaseolus vulgaris) is one of the most important crop plants. About 50% of its genome is composed of repetitive sequences, but only a little fraction was isolated and characterized so far. In this paper, a new repetitive DNA family from the species, named PvMeso, was isolated and characterized in both gene pools of P. vulgaris (Andean and Mesoamerican) and related species. Two fragments, 1.7 and 2.3 kb long, were cloned from BAC 255F18, which has previously shown a repetitive pattern. The subclone PvMeso-31 showed a terminal block in chromosome 7. This subclone contains a 1,705 bp long, AT-rich repeat with small internal repeats and shares a 1.2 kb region with PvMeso-47, derived from the 2.3 kb fragment. The presence of this repetitive block was restricted to Mesoamerican accessions of the common bean. In P. acutifolius, P. leptostachyus and Andean P. vulgaris, only a faint, 2.3 kb fragment was visualized in Southern experiments. Moreover, in Mesoamerican accessions, two other fragments (1.7 kb and 3.4 kb) were strongly labelled as well. Taken together, our results indicate that PvMeso is a recently emerged, repeat family initially duplicated in chromosome 11, on ancestral Mesoamerican accession, and later amplified in chromosome 7, after the split of the two major gene pools of the common bean.

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.

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.

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.

A situação atual do mercado da saúde suplementar no Brasil e apontamentos para o futuro / The current situation of the private health plans and insurance market in Brazil and trends for the future

O artigo tem por objetivo descrever a situação do mercado de planos privados de assistência médica no Brasil, no período de 2000 a 2006. Analisa a situação atual no que tange aos beneficiários, às operadoras e aos planos de saúde e destaca algumas possíveis tendências sinalizadas pelo estudo. Apresenta o perfil dos beneficiários e a cobertura por planos na população.Discute a expansão dos planos coletivos, a redução dos planos individuais, bem como a acentuada concentração de beneficiários em poucas operadoras, identificando que, apesar de tratar-se de um mercado concentrado, há mais beneficiários em planos com abrangência municipal ou regional do que em planos nacionais. Por fim, aborda aspectos relacionados aos recursos financeiros, entre eles o incentivo governamental para o setor e conclui sinalizando a necessidade de estudos para melhor conhecer a dinâmica do mercado de planos privados de saúde.

This paper presents an overview of the Brazilian private health plan market over the period 2000-2006. The current situation is analyzed with respect to the profile of private insurance companies, health plans and beneficiaries and some possible trends that were identified in the study are emphasized. The increase of employer group-plans as a work-related benefit and the reduction of individual plans are discussed. Although the market is restricted to only a few companies, there are more people covered by local plans than by plans offering coverage on a national basis. Finally, the paper approaches aspects related to the financial resources, among them the governmental incentive for the health area, and points to the need of further studies for a better understanding of the supplementary healthcare market.

[The current situation of the private health plans and insurance market in Brazil and trends for the future]. / A situação atual do mercado da saúde suplementar no Brasil e apontamentos para o futuro.

This paper presents an overview of the Brazilian private health plan market over the period 2000-2006. The current situation is analyzed with respect to the profile of private insurance companies, health plans and beneficiaries and some possible trends that were identified in the study are emphasized. The increase of employer group-plans as a work-related benefit and the reduction of individual plans are discussed. Although the market is restricted to only a few companies, there are more people covered by local plans than by plans offering coverage on a national basis. Finally, the paper approaches aspects related to the financial resources, among them the governmental incentive for the health area, and points to the need of further studies for a better understanding of the supplementary healthcare market.

Portabilidade em planos de saúde no Brasil / Portability health plan in Brazil

Este estudo pretende revisar alguns fundamentos da composição do sistema de atenção à saúde no Brasil. Os benefícios advindos da regulação padronizaram e garantiram a prestação de serviços, porém levaram a aumento dos custos e diminuição da capacidade competitiva do mercado. O ônus decorrente da troca de plano de saúde expressa por perda de direitos adquiridos de cumprimento de prazos de carência e direito a atendimento de doenças preexistentes limita o poder de mercado do consumidor por tender a mantê-lo no mesmo plano de saúde mesmo quando não satisfeito com os serviços recebidos. O direito a troca de plano de saúde sem perda de direitos a portabilidade, restabeleceria o poder do consumidor e a competição do mercado ao permitir a troca de plano de saúde quando concorrentes oferecessem melhores condições de preço ou qualidade de serviços. A regulamentação da portabilidade é considerada, neste estudo, como a obrigatoriedade de cobertura de mais um procedimento, sendo, portanto geradora de custos para operadoras de planos de saúde. Estes custos estão relacionados a fatores atuariais e a fatores de difícil quantificação decorrentes de conduta não ética de beneficiários e operadoras. Algumas estratégias de contornar estas dificuldades são discutidas. A legislação em vigor também é revista procurando determinar situações onde a legislação pode ser aperfeiçoada para evitar a perda de direito de atendimento em planos de saúde após a perda de vínculo empregatício. O estudo conclui com a proposta de criação de uma taxa de portabilidade capaz de favorecer a mobilidade dos beneficiários e a competitividade do mercado sem concentrar todos os riscos sobre as operadoras.