Mitochondrial genome of Hancornia speciosa gomes: intergenic regions containing retrotransposons and predicted genes.

BACKGROUND: Plant mitochondrial genomes are characterized by high homologous recombination, extensive intergenic spacers, conservation in DNA sequences, and gene content. The Hancornia genus belongs to the Apocynaceae family, with H. speciosa Gomes being the sole species in the genus. It is an siganificant commercial fruit crop; however, only a number of studies have been conducted. In this study, we present the mitochondrial genome of H. speciosa and compare it with other mitochondrial genomes within the Apocynaceae family. METHODS AND RESULTS: A total of 2.8 Gb of Illumina paired-end reads were used to obtain the mitogenome, resulting in 22 contigs that were merged using 6.1 Gb of Illumina mate-pair reads to obtain a circular chromosome. The mitochondrial genome of H. speciosa is circular, containing 63 predicted functional genes, spanning a length of 741,811 bp, with a CG content of 44%. Within the mitogenome, 50 chloroplast DNA sequences, equivalent to 1.72% of the genome, were detected. However, intergenic spaces accounted for 703,139 bp (94.79% of the genome), and 287 genes were predicted, totaling 173,721 bp. CONCLUSION: This suggests the incorporation of nuclear DNA into the mitogenome of H. speciosa and self duplication. Comparative analysis among the mitogenomes in the Apocynaceae family revealed a diversity in the structure mediated by recombination, with similar gene content and large intergenic spaces.

Neutrophil virucidal activity against SARS-CoV-2 is mediated by NETs.

BACKGROUND: The inflammation in the lungs and other vital organs in COVID-19 are characterized by the presence of neutrophils and high concentration of neutrophil extracellular traps (NETs), which also seems to mediate host tissue damage. However, it is not known whether NETs could have virucidal activity against SARS-CoV-2. METHODS: We investigated whether NETs could prevent SARS-CoV-2 replication in neutrophils and epithelial cells, and what the consequence of NETs degradation in K18-humanized ACE2 transgenic mice infected with SARS-CoV-2. RESULTS: Here, by immunofluorescence microscopy we observed that viral particles co-localize with NETs in neutrophils isolated from COVID-19 patients or from healthy individuals and infected in vitro. The inhibition of NETs production increased virus replication in neutrophils. In parallel, we observed that NETs inhibited virus abilities to infect and replicate in epithelial cells after 24 h of infection. Degradation of NETs with DNase I prevented their virucidal effect in vitro. Using K18-humanized ACE2 transgenic mice we observed a higher viral load in animals treated with DNase I. On the other hand, the virucidal effect of NETs was not dependent on neutrophil elastase or myeloperoxidase activity. CONCLUSION: Our results provide evidence of the role of NETosis as a mechanism of SARS-CoV-2 viral capture and inhibition.

Enoxaparin improves COVID-19 by reducing Neutrophils Extracellular Traps (NETs) production.

BACKGROUND: COVID-19 causes consequences such as imbalance of the immune system and thrombotic events. During the infection process, NETs in excess induce a pro-inflammatory response and disseminated intravascular coagulation. We evaluated the role of enoxaparin as a potential inhibitor of NETs. METHODS: K18-hACE2 animals infected with the SARS-CoV-2 virus and a group of 23 individuals admitted to the hospital with COVID-19 treated with enoxaparin or without treatment and controls without the disease were included. RESULTS: Enoxaparin decreased the levels of NETs, reduced the signs of the disease and mitigated lung damage in the animals infected with SARS-CoV-2. These effects were partially associated with prevention of SARS-CoV-2 entry and NETs synthesis. Clinical data revealed that treatment with enoxaparin decreased the levels of inflammatory markers, the levels of NETs in isolated neutrophils and the organ dysfunction. CONCLUSION: This study provides evidence for the beneficial effects of enoxaparin in COVID-19 in addition to its anticoagulant role.

Targeting neutrophils extracellular traps (NETs) reduces multiple organ injury in a COVID-19 mouse model.

BACKGROUND: COVID-19 is characterized by severe acute lung injury, which is associated with neutrophil infiltration and the release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment. METHODS: Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. SARS-CoV-2-infected K18-hACE2 mice were performed for clinical sickness scores and lung pathology. Moreover, the levels of NETs were assessed and lung injuries were by histopathology and TUNEL assay. Finally, the injury in the heart and kidney was assessed by histopathology and biochemical-specific markers. RESULTS: DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potentially deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I. CONCLUSIONS: Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.

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.

Gasdermin-D activation by SARS-CoV-2 triggers NET and mediate COVID-19 immunopathology.

BACKGROUND: The release of neutrophil extracellular traps (NETs) is associated with inflammation, coagulopathy, and organ damage found in severe cases of COVID-19. However, the molecular mechanisms underlying the release of NETs in COVID-19 remain unclear. OBJECTIVES: We aim to investigate the role of the Gasdermin-D (GSDMD) pathway on NETs release and the development of organ damage during COVID-19. METHODS: We performed a single-cell transcriptome analysis in public data of bronchoalveolar lavage. Then, we enrolled 63 hospitalized patients with moderate and severe COVID-19. We analyze in blood and lung tissue samples the expression of GSDMD, presence of NETs, and signaling pathways upstreaming. Furthermore, we analyzed the treatment with disulfiram in a mouse model of SARS-CoV-2 infection. RESULTS: We found that the SARS-CoV-2 virus directly activates the pore-forming protein GSDMD that triggers NET production and organ damage in COVID-19. Single-cell transcriptome analysis revealed that the expression of GSDMD and inflammasome-related genes were increased in COVID-19 patients. High expression of active GSDMD associated with NETs structures was found in the lung tissue of COVID-19 patients. Furthermore, we showed that activation of GSDMD in neutrophils requires active caspase1/4 and live SARS-CoV-2, which infects neutrophils. In a mouse model of SARS-CoV-2 infection, the treatment with disulfiram inhibited NETs release and reduced organ damage. CONCLUSION: These results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology and suggests GSDMD as a novel potential target for improving the COVID-19 therapeutic strategy.

High-Throughput Genomic Data Reveal Complex Phylogenetic Relationships in Stylosanthes Sw (Leguminosae).

Allopolyploidy is widely present across plant lineages. Though estimating the correct phylogenetic relationships and origin of allopolyploids may sometimes become a hard task. In the genus Stylosanthes Sw. (Leguminosae), an important legume crop, allopolyploidy is a key speciation force. This makes difficult adequate species recognition and breeding efforts on the genus. Based on comparative analysis of nine high-throughput sequencing (HTS) samples, including three allopolyploids (S. capitata Vogel cv. "Campo Grande," S. capitata "RS024" and S. scabra Vogel) and six diploids (S. hamata Taub, S. viscosa (L.) Sw., S. macrocephala M. B. Ferreira and Sousa Costa, S. guianensis (Aubl.) Sw., S. pilosa M. B. Ferreira and Sousa Costa and S. seabrana B. L. Maass & 't Mannetje) we provide a working pipeline to identify organelle and nuclear genome signatures that allowed us to trace the origin and parental genome recognition of allopolyploids. First, organelle genomes were de novo assembled and used to identify maternal genome donors by alignment-based phylogenies and synteny analysis. Second, nuclear-derived reads were subjected to repetitive DNA identification with RepeatExplorer2. Identified repeats were compared based on abundance and presence on diploids in relation to allopolyploids by comparative repeat analysis. Third, reads were extracted and grouped based on the following groups: chloroplast, mitochondrial, satellite DNA, ribosomal DNA, repeat clustered- and total genomic reads. These sets of reads were then subjected to alignment and assembly free phylogenetic analyses and were compared to classical alignment-based phylogenetic methods. Comparative analysis of shared and unique satellite repeats also allowed the tracing of allopolyploid origin in Stylosanthes, especially those with high abundance such as the StyloSat1 in the Scabra complex. This satellite was in situ mapped in the proximal region of the chromosomes and made it possible to identify its previously proposed parents. Hence, with simple genome skimming data we were able to provide evidence for the recognition of parental genomes and understand genome evolution of two Stylosanthes allopolyploids.

Genome size and identification of repetitive DNA sequences using low coverage sequencing in Hancornia speciosa Gomes (Apocynaceae: Gentianales).

Repetitive DNA is an important component of eukaryotic genomes, accounting for more than 90% of the genome size of some species, including mobile elements and satellite DNA sequences. The aim of study was to characterize the genome of Hancornia speciosa Gomes using C-value genome size estimate and repetitive DNA sequences analysis. The genome size estimate was obtained by flow cytometry and the repetitive DNA sequences were accessed using graph-based clustering. Evolutionary relationships among species of Apocynaceae was obtained using reads of Catharanthus roseus L., Rhayza stricta Decne, and Asclepias syriaca L. from the NCBI and analyzed by graph-based clustering. The genome size estimates in two botanical varieties showed 2C-values ranging from 0.88 to 1.08 pg, indicating small genome size. Clusters representing repeats making up at least 0.01% of the genome revealed the proportion of repetitive DNA ranging from 19.87% (H. speciosa) to 51.674% (A. syriaca), of which the mobile elements were more abundant. Satellite DNA sequences were not found in H. speciosa and R. stricta, while at least one satellite was detected in C. roseus and A. syriaca, suggesting that the LTR retrotransposon Ty3/Gypsy/Chromovirus may have replaced the satellite DNA in H. speciosa and R. stricta.

Mitochondrial genomes of genus Atta (Formicidae: Myrmicinae) reveal high gene organization and giant intergenic spacers.

The ants of the genus Atta are considered important pests to agriculture in the Americas, although Atta species are also important contributors to ecosystem functions in the various habitats in which they occur. The aim of this study was to assemble four complete mitochondrial genomes of the genus Atta, construct the phylogenomic tree, and analyze the gene content, order, and organization. The mitogenomes of A. colombica, A. opaciceps, A. texana, and A. sexdens rubropilosa comprise 18,392, 19,257, 19,709, and 19,748 bp, respectively. The four Atta mitogenomes showed the charactistics typical of those of insects, with 13 protein-coding genes, 22 tRNAs, and 2 rRNAs, with genes displayed in the conventional order. Analysis for intergenic spacer regions showed that Atta intergenic spacers are larger than those of the outgroups. Phylogenomic analyses using partial cytochrome oxidase I gene sequences showed similar topologies to previous phylogenetic analyses, with high clade support values. We conclude that Atta mitogenomes are characterized by high conservation in gene order and have giant intergenic spacers in the genus Atta.

Complete mitochondrial genomes of the Spondias tuberosa Arr. Cam and Spondias mombin L. reveal highly repetitive DNA sequences.

Mitogenomes in plants are well-known as exhibiting high diversity in genome size architecture and repetitive DNA sequences. In this research study, we report on the complete mitochondrial genomes of S. tuberosa and S. mombin using Illumina paired-end and mate-pair end reads. These genomes were obtained by a combination of methods of de novo assembly and contig extension. The mitogenomes of S. tuberosa and S. mombin showed 779,106 bp and 685,788 bp in length, with a total of 35 genes. Genome comparisons showed many rearrangements that were mediated by repetitive DNA, and also high incorporation of DNA from chloroplast. In summary, we demonstrate: (1) first complete mitochondrial genomes for the genus Spondias; (2) the synteny between S. tuberosa and S. mombin showed rearrangements, mediated by repetitive DNA; (3) that gene content in Spondias mitogenomes is highly conserved; and (4) the high incorporation DNA from chloroplast genome, (5) the mitogenome size is due intergenic spacers and (6) the non-tandem repeats contributes for giant intergenic spacers.

Genome survey and development of 18 microsatellite markers to assess genetic diversity in Spondias tuberosa Arruda Câmara (Anacardiaceae) and cross-amplification in congeneric species.

Spondias tuberosa Arr. Cam belongs to the Anacardiaceae family, an economically important family of plants whose fruits are consumed by humans and animals. The aim of this study was to develop microsatellite markers using sequences from high-throughput sequencing and a magnetic bead enrichment method. The sequences were used to obtain contigs with a minimum of 500 nucleotides using Ray software and the mining of the simple sequence repeats (SSR) was performed with Phobos software, while the primers were designed by Primer3. We developed 18 polymorphic nuclear microsatellite markers and successfully cross-amplified them to three Spondias species. In S. tuberosa, the alleles ranged from 2 to 5 for each locus and Hardy-Weinberg equilibrium was found for 16 loci, with an expected and observed heterozygosity at 0.095-0.755 and 0.1-0.75, respectively. Cross-transferability was obtained for all loci in S. bahiensis, S. dulcis and S. purpurea. We concluded that the microsatellite markers developed in this study are useful in genetic population and conservation studies, as well as for investigating the hybrid origins of Spondias species.

The complete chloroplast genome sequences of three Spondias species reveal close relationship among the species.

This study reports the complete chloroplast sequences of three Spondias species. The genome sequences were obtained for Spondias tuberosa, Spondias bahienses, and Spondias mombin using the Illumina sequencing technology by a combination of de novo methods and a reference-guided assembly using Sapindus mukorossi as reference. The genomes of S. tuberosa, S. bahiensis, and S. mombin had 162,036, 162,218, and 162,302 bp, respectively. The coding regions exhibited 130 genes, including 34-35 tRNAs and 4 rRNAs. The results revealed synteny among the genomes, with high conservation in the gene order and content and CG content. The inverted repeat regions (IRA and IRB) and the large and small single copies were very similar among the three genomes. The phylogenomic analysis reported similar topologies as that of previous studies, which used partial chloroplast, wherein S. mombin was the first diverging lineage, while S. tuberosa and S. bahiensis were derived, indicating that the phylogenetic analysis using partial or complete genome produces similar results. In summary, (1) we presented the first complete chloroplast genome for the genus Spondias, (2) phylogenies analyzed using the complete chloroplast genomes revealed a robust phylogenetic topology for Spondias, and (3) gene order, content, and orientation in Spondias are highly conserved.

Phylogenomic and single nucleotide polymorphism analyses revealed the hybrid origin of Spondias bahiensis (family Anacardiaceae): de novo genome sequencing and comparative genomics.

The genus Spondias (family Anacardiaceae) comprises 19 taxa, ten of which occur in Neotropical regions. Spondias bahiensis has been suggested to be a hybrid, although initial evidence does not support this hypothesis. The aim of this study was to test the hypothesis of the hybrid origin of S. bahiensis using high-throughput sequencing with single nucleotide polymorphism (SNP) analysis, characterization of intragenomic nuclear ribosomal DNA (nrDNA), and nuclear and chloroplast phylogenomic analyses. The SNP analysis revealed a high number of SNPs in the S. bahiensis genome, and with respect to nrDNA, S. bahiensis shared approximately half of the SNP alleles with S. tuberosa, but not with S. mombin. Combining the SNP analysis with nrDNA phylogeny confirmed the hybrid origin of S. bahiensis and put S. tuberosa as the female genitor. Considering the phylogeny of the genus Spondias and intraspecific SNPs in S. bahiensis, the putative male genitor is S. dulcis.

Origin and parental genome characterization of the allotetraploid Stylosanthes scabra Vogel (Papilionoideae, Leguminosae), an important legume pasture crop.

Backgrounds and Aims: The genus Stylosanthes includes nitrogen-fixing and drought-tolerant species of considerable economic importance for perennial pasture, green manure and land recovery. Stylosanthes scabra is adapted to variable soil conditions, being cultivated to improve pastures and soils worldwide. Previous studies have proposed S. scabra as an allotetraploid species (2n = 40) with a putative diploid A genome progenitor S. hamata or S. seabrana (2n = 20) and the B genome progenitor S. viscosa (2n = 20). We aimed to provide conclusive evidence for the origin of S. scabra. Methods: We performed fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) experiments and Illumina paired-end sequencing of S. scabra, S. hamata and S. viscosa genomic DNA, to assemble and compare complete ribosomal DNA (rDNA) units and chloroplast genomes. Plastome- and genome-wide single nucleotide variation detection was also performed. Key Results: GISH and phylogenetic analyses of plastid DNA and rDNA sequences support that S. scabra is an allotetraploid formed from 0.63 to 0.52 million years ago (Mya), from progenitors with a similar genome structure to the maternal donor S. hamata and the paternal donor S. viscosa. FISH revealed a non-additive number of 35S rDNA sites in S. scabra compared with its progenitors, indicating the loss of one locus from A genome origin. In S. scabra, most 5S rDNA units were similar to S. viscosa, while one 5S rDNA site of reduced size most probably came from an A genome species as revealed by GISH and in silico analysis. Conclusions: Our approach combined whole-plastome and rDNA assembly with additional cytogenetic analysis to shed light successfully on the allotetraploid origin of S. scabra. We propose a Middle Pleistocene origin for S. scabra involving species with maternal A and paternal B genomes. Our data also suggest that variation found in rDNA units in S. scabra and its progenitors reveals differences that can be explained by homogenization, deletion and amplification processes that have occurred since its origin.

Validation of reference genes for accurate normalization by quantitative polymerase chain reaction in sugarcane drought stress studies using two cultivars / Validação de genes de referência para normalização de reação em cadeia de polimerase quantitativa em estudos de estresse hídricoem cana-de-açúcar

ABSTRACT: The aim of this study was to analyze the expression of putative reference genes in sugarcane under drought stress. The varieties RB72454 and RB72910 were cultivated and the treatments control and drought stress applied to 135-day-old plants grown under field conditions. The stress level of the plants was measured by rate of photosynthesis, transpiration, and stomatal conductance. For each biological replicate, expression analyses were conducted using quantitative polymerase chain reaction for the genes α-tubulin, β-tubulin, β-actin, cyclophilin, eukaryotic elongation factor 1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H3 and ubiquitin. Stability was evaluated using the geNorm, NormFinder, and BestKeeper software packages. Among the candidate genes, GAPDH was identified as the most stable in all software, indicating its suitability for gene expression studies in sugarcane undergoing drought stress; the gene β-actin was the second most stable. These findings suggest using GAPDH and β-actin for normalization in relative gene expression in sugarcane.

RESUMO: O objetivo do estudo foi analisar a expressão de genes de referência em cana-de-açúcar sob estresse hídrico. As cultivares RB72454 e RB72910 foram cultivadas em campo e os tratamentos controle e estresse foram aplicados após 135 dias. O estresse das plantas foi mensurado utilizando as variáveis fisiológicas: taxa fotossintética, transpiração e condutância estomática. Para cada repetição biológica, a expressão gênica foi conduzida usando PCR tempo real para os genes α-tubulina, β-tubulina, β-actina, ciclofilina, fator 1 de alongamento eucariótico, gliceraldeído 3-fosfato desidrogenase (GAPDH), histona H3 e ubiquitina. A estabilidade dos genes foi avaliada usando os softwares geNorm,NormFinder e BestKeeper. Entre os genes candidatos, GAPDH foi identificado como o mais estável nos três métodos de análises, indicando ser estável para estudos de expressão gênica em cana-de-açúcar sob estresse hídrico; seguindo o gene β-actina como o segundo mais estável. Os resultados concluem que os genes GAPDH e β-actina são indicados para normalização em expressão gênica relativa, associada com estresse hídrico em cana-de-açúcar.

High-level phylogeographic structuring of Neoleucinodes elegantalis Guenée (Lepidoptera, Crambridae) in Brazil: an important tomato pest

ABSTRACT Neoleucinodes elegantalis is an important tomato pest in Brazil, occurring throughout the country and resulting in economic losses in agriculture. In several species, biogeographic studies in Brazil indicate the structuring of populations, following the refuge model, with a split between the populations of the northeast and the southeast regions of Brazil. The objective of this work was to analyze the phylogeography of N. elegantalis in Brazil, understanding its population structure and the demographic patterns. Larvae were collected from eight locations throughout Brazil, and the mitochondrial cytochrome c oxidase subunit 1 gene was analyzed. A total of 628 bp in 51 individuals were obtained, showing 12 haplotypes with a haplotype diversity of 0.836. Spatial analysis of molecular variance (SAMOVA) and cluster analysis showed two populations, indicating population structuring between individuals from the northeast (population 1) and southeast (population 2) regions of Brazil. Phylogenetic analysis indicated that the clades corresponding to the groups defined by SAMOVA have a divergence time of 0.2–0.5 million years, suggesting isolation during climatic events and a separation of the two populations coinciding with the predicted refuges to the Atlantic forest.

Expression profiles of sugarcane under drought conditions: Variation in gene regulation.

Drought is a major factor in decreased sugarcane productivity because of the resulting morphophysiological effects that it causes. Gene expression studies that have examined the influence of water stress in sugarcane have yielded divergent results, indicating the absence of a fixed pattern of changes in gene expression. In this work, we investigated the expression profiles of 12 genes in the leaves of a drought-tolerant genotype (RB72910) of sugarcane and compared the results with those of other studies. The genotype was subjected to 80-100% water availability (control condition) and 0-20% water availability (simulated drought). To analyze the physiological status, the SPAD index, Fv/Fm ratio, net photosynthesis (A), stomatal conductance (gs) and stomatal transpiration (E) were measured. Total RNA was extracted from leaves and the expression of SAMDC, ZmPIP2-1 protein, ZmTIP4-2 protein, WIP protein, LTP protein, histone H3, DNAj, ferredoxin I, ß-tubulin, photosystem I, gene 1 and gene 2 was analyzed by quantitative real-time PCR (RT-PCR). Important differences in the expression profiles of these genes were observed when compared with other genotypes, suggesting that complex defense mechanisms are activated in response to water stress. However, there was no recognizable pattern for the changes in expression of the different proteins associated with tolerance to drought stress.

Differential detection of transposable elements between Saccharum species.

Cultivars of sugarcane (Saccharum) are hybrids between species S. officinarum (x = 10, 2n = 8x = 80) and S. spontaneum (x = 8, 2n = 5 - 16x = 40 - 128). These accessions have 100 to 130 chromosomes, 80-85% of which are derived from S. officinarum, 10-15% from S. spontaneum, and 5-10% are possible recombinants between the two genomes. The aim of this study was to analyze the repetition of DNA sequences in S. officinarum and S. spontaneum. For this purpose, genomic DNA from S. officinarum was digested with restriction enzymes and the fragments cloned. Sixty-eight fragments, approximately 500 bp, were cloned, sequenced and had their identity analyzed in NCBI, and in the rice, maize, and sorghum genome databases using BLAST. Twelve clones containing partial transposable elements, one single-copy control, one DNA repetitive clone control and two genome controls were analyzed by DNA hybridization on membrane, using genomic probes from S. officinarum and S. spontaneum. The hybridization experiment revealed that six TEs had a similar repetitive DNA pattern in the genomes of S. officinarum and S. spontaneum, while six TEs were more abundant in the genome of S. officinarum. We concluded that the species S. officinarum and S. spontaneum have differential accumulation LTR retrotransposon families, suggesting distinct insertion or modification patterns.

High macro-collinearity between lima bean (Phaseolus lunatus L.) and the common bean (P. vulgaris L.) as revealed by comparative cytogenetic mapping.

Common bean (P. vulgaris) and lima bean (P. lunatus) are the most important crop species from the genus Phaseolus. Both species have the same chromosome number (2n = 22) and previous cytogenetic mapping of BAC clones suggested conserved synteny. Nevertheless, karyotype differences were observed, suggesting structural rearrangements. In this study, comparative cytogenetic maps for chromosomes 3, 4 and 7 were built and the collinearity between the common bean and lima bean chromosomes was investigated. Thirty-two markers (30 BACs and 2 bacteriophages) from P. vulgaris were hybridized in situ on mitotic chromosomes from P. lunatus. Nine BACs revealed a repetitive DNA pattern with pericentromeric distribution and 23 markers showed unique signals. Nine of these markers were mapped on chromosome 3, eight on chromosome 4 and six on chromosome 7. The order and position of all analyzed BACs were similar between the two species, indicating a high level of macro-collinearity. Thus, although few inversions have probably altered centromere position in other chromosomes, the main karyotypic differences were associated with the repetitive DNA fraction.

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.