Andrea Pedrosa-Harand

Extensive ribosomal DNA amplification during Andean common bean (Phaseolus vulgaris L.) evolution

The extent of 5S and 45S ribosomal DNA (rDNA) variation was investigated in wild and domesticated common beans (Phaseolus vulgaris) chosen to represent the known genetic diversity of the species. 5S and 45S rDNA probes were localized on mitotic chromosomes of 37 accessions by fluorescent in situ hybridization (FISH). The two 5S rDNA loci were largely conserved within the species, whereas a high variation in the number of 45S rDNA loci and changes in position of loci and number of repeats per locus were observed. Domesticated accessions from the Mesoamerican gene pool frequently had three 45S rDNA loci per haploid genome, and rarely four. Domesticated accessions from Andean gene pool, particularly from the race Peru, showed six, seven, eight or nine loci, but seven loci were found in all three races of this gene pool. Between three and eight loci were observed in accessions resulting from crosses between Andean and Mesoamerican genotypes. The presence of two to eight 45S rDNA loci in wild common beans from different geographic locations indicates that the 45S rDNA amplification observed in the Andean lineage took place before domestication. Our data suggest that ectopic recombination between terminal chromosomal regions might be the mechanism responsible for this variation.

Genomics of Phaseolus Beans, a Major Source of Dietary Protein and Micronutrients in the Tropics

Common bean is grown and consumed principally in developing countries in Latin America, Africa, and Asia. It is largely a subsistence crop eaten by its producers and, hence, is underestimated in production and commerce statistics. Common bean is a major source of dietary protein, which complements carbohydrate-rich sources such as rice, maize, and cassava. It is also a rich source of minerals, such as iron and zinc, and certain vitamins. Several large germplasm collections have been established, which contain large amounts of genetic diversity, including the five domesticated Phaseolus species and wild species, as well as an incipient stock collection. The genealogy and genetic diversity of P. vulgaris are among the best known in crop species through the systematic use of molecular markers, from seed proteins and isozymes to simple sequence repeats, and DNA sequences. Common bean exhibits a high level of genetic diversity, compared with other selfing species. A hierarchical organization into gene pools and ecogeographic races has been established. There are over 15 mapping populations that have been established to study the inheritance of agronomic traits in different locations. Most linkage maps have been correlated with the core map established in the BAT93 x Jalo EEP558 cross, which includes several hundreds of markers, including Restriction Fragment Length Polymorphisms, Random Amplified Polymorphic DNA, Amplified Fragment Length Polymorphisms, Short Sequence Repeats, Sequence Tagged Sites, and Target Region Amplification Polymorphisms. Over 30 individual genes for disease resistance and some 30 Quantitative Trait Loci for a broad range of agronomic traits have been tagged. Eleven BAC libraries have been developed in genotypes that represent key steps in the evolution before and after domestication of common bean, a unique resource among crops. Fluorescence in situ hybridization provides the first links between chromosomal and genetic maps. A gene index based on some P. vulgaris 21,000 expressed sequence tags (ESTs) has been developed. ESTs were developed from different genotypes, organs, and physiological conditions. They resolve currently in some 6,500–6,800 singletons and 2,900 contigs. An additional 20,000 embryonic P. coccineus ESTs provides an additional resource. Some 1,500 M2 Targeting Local Lesions In Genomes populations exist currently. Finally, transformation methods by biolistics and Agrobacterium have been developed, which can be applied for genetic engineering. Root transformation via A. rhizogenes is also possible. Thus, the Phaseomics community has laid a solid foundation towards its ultimate goal, namely the sequencing of the Phaseolus genome. These genomic resources are a much-needed source of additional markers of known map location for marker-assisted selection and the accelerated improvement of common bean cultivars.

A Nomadic Subtelomeric Disease Resistance Gene Cluster in Common Bean

The B4 resistance (R) gene cluster is one of the largest clusters known in common bean (Phaseolus vulgaris [Pv]). It is located in a peculiar genomic environment in the subtelomeric region of the short arm of chromosome 4, adjacent to two heterochromatic blocks (knobs). We sequenced 650 kb spanning this locus and annotated 97 genes, 26 of which correspond to Coiled-Coil-Nucleotide-Binding-Site-Leucine-Rich-Repeat (CNL). Conserved microsynteny was observed between the Pv B4 locus and corresponding regions of Medicago truncatula and Lotus japonicus in chromosomes Mt6 and Lj2, respectively. The notable exception was the CNL sequences, which were completely absent in these regions. The origin of the Pv B4-CNL sequences was investigated through phylogenetic analysis, which reveals that, in the Pv genome, paralogous CNL genes are shared among nonhomologous chromosomes (4 and 11). Together, our results suggest that Pv B4-CNL was derived from CNL sequences from another cluster, the Co-2 cluster, through an ectopic recombination event. Integration of the soybean (Glycine max) genome data enables us to date more precisely this event and also to infer that a single CNL moved from the Co-2 to the B4 cluster. Moreover, we identified a new 528-bp satellite repeat, referred to as khipu, specific to the Phaseolus genus, present both between B4-CNL sequences and in the two knobs identified at the B4 R gene cluster. The khipu repeat is present on most chromosomal termini, indicating the existence of frequent ectopic recombination events in Pv subtelomeric regions. Our results highlight the importance of ectopic recombination in R gene evolution.

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.

Molecular Analysis of a Large Subtelomeric Nucleotide-Binding-Site–Leucine-Rich-Repeat Family in Two Representative Genotypes of the Major Gene Pools of Phaseolus vulgaris

In common bean, the B4 disease resistance (R) gene cluster is a complex cluster localized at the end of linkage group (LG) B4, containing at least three R specificities to the fungus Colletotrichum lindemuthianum. To investigate the evolution of this R cluster since the divergence of Andean and Mesoamerican gene pools, DNA sequences were characterized from two representative genotypes of the two major gene pools of common bean (BAT93: Mesoamerican; JaloEEP558: Andean). Sequences encoding 29 B4-CC nucleotide-binding-site–leucine-rich-repeat (B4-CNL) genes were determined—12 from JaloEEP558 and 17 from BAT93. Although sequence exchange events were identified, phylogenetic analyses revealed that they were not frequent enough to lead to homogenization of B4-CNL sequences within a haplotype. Genetic mapping based on pulsed-field gel electrophoresis separation confirmed that the B4-CNL family is a large family specific to one end of LG B4 and is present at two distinct blocks separated by 26 cM. Fluorescent in situ hybridization on meiotic pachytene chromosomes revealed that two B4-CNL blocks are located in the subtelomeric region of the short arm of chromosome 4 on both sides of a heterochromatic block (knob), suggesting that this peculiar genomic environment may favor the proliferation of a large R gene cluster.

The Arabidopsis thaliana MND1 homologue plays a key role in meiotic homologous pairing, synapsis and recombination

Mnd1 has recently been identified in yeast as a key player in meiotic recombination. Here we describe the identification and functional characterisation of the Arabidopsis homologue, AtMND1, which is essential for male and female meiosis and thus for plant fertility. Although axial elements are formed normally, sister chromatid cohesion is established and recombination initiation appears to be unaffected in mutant plants, chromosomes do not synapse. During meiotic progression, a mass of entangled chromosomes, interconnected by chromatin bridges, and severe chromosome fragmentation are observed. These defects depend on the presence of SPO11-1, a protein that initiates recombination by catalysing DNA double-strand break (DSB) formation. Furthermore, we demonstrate that the AtMND1 protein interacts with AHP2, the Arabidopsis protein closely related to budding yeast Hop2. These data demonstrate that AtMND1 plays a key role in homologous synapsis and in DSB repair during meiotic recombination.

Cytogenetic mapping of common bean chromosomes reveals a less compartmentalized small-genome plant species

Cytogenetic maps of common bean chromosomes 3, 4 and 7 were constructed by fluorescence in-situ hybridization (FISH) of BAC and a few other genomic clones. Although all clones were selected with genetically mapped markers, mostly with single-copy RFLPs, a large subset of BACs, from 13 different genomic regions, contained repetitive sequences, as concluded from the regional distribution patterns of multiple FISH signals on chromosomes: pericentromeric, subtelomeric and dispersed. Pericentromeric repeats were present in all 11 chromosome pairs with different intensities, whereas subtelomeric repeats were present in several chromosome ends, but with different signal intensities depending on the BAC, suggesting that the terminal heterochromatin fraction of this species may be composed of different repeats. The correlation of genetic and physical distances along the three studied chromosomes was obtained for 23 clones. This correlation suggests suppression of recombination around extended pericentromeric regions in a similar way to that previously reported for plant species with larger genomes. These results indicate that a relatively small plant genome may also possess a large proportion of repeats interspersed with single-copy sequences in regions other than the pericentromeric heterochromatin and, nevertheless, exhibit lower recombination around the pericentromeric fraction of the genome.

Chiasmatic and achiasmatic inverted meiosis of plants with holocentric chromosomes.

Meiosis is a specialized cell division in sexually reproducing organisms before gamete formation. Following DNA replication, the canonical sequence in species with monocentric chromosomes is characterized by reductional segregation of homologous chromosomes during the first and equational segregation of sister chromatids during the second meiotic division. Species with holocentric chromosomes employ specific adaptations to ensure regular disjunction during meiosis. Here we present the analysis of two closely related plant species with holocentric chromosomes that display an inversion of the canonical meiotic sequence, with the equational division preceding the reductional. In-depth analysis of the meiotic divisions of Rhynchospora pubera and R. tenuis reveals that during meiosis I sister chromatids are bi-oriented, display amphitelic attachment to the spindle and are subsequently separated. During prophase II, chromatids are connected by thin chromatin threads that appear instrumental for the regular disjunction of homologous non-sister chromatids in meiosis II.

Complex rearrangements are involved in Cephalanthera (Orchidaceae) chromosome evolution.

The genus Cephalanthera is an excellent plant group for karyotype evolution studies because it exhibits a dysploid series and bimodal karyotypes. With the aim of understanding their chromosomal and phylogenetic relationships, rRNA genes and the Arabidopsis-type telomeric sequence were mapped by fluorescence in-situ hybridization (FISH), and the rDNA intergenic spacer (ITS) was sequenced for the first time in three European species: C. longifolia (2n = 4x = 32), C. damasonium (2n = 4x = 36) and C. rubra (2n = 4x = 44). One 45S and three 5S rDNA sites are observed in C. longifolia, one 45S and two 5S sites in C. damasonium, and two 45S and one 5S site in C. rubra. Telomeric signals were observed at every chromosome end in all three species and C. damasonium also displays interstitial signals on three chromosome pairs. In agreement with chromosome data, molecular analyses support C. longifolia and C. damasonium as closely related taxa, while C. rubra stands apart. Possible pathways of karyotype evolution are discussed in reference to a previous hypothesis. The results indicate that complex chromosomal rearrangements, possibly involving Robertsonian fusions and fissions, loss of telomeric repeats, gain or loss of rDNA sites and other heterochromatic sequences and inversions, may have contributed to generating the present-day karyotypes.

Holocentromeres in Rhynchospora are associated with genome-wide centromere-specific repeat arrays interspersed among euchromatin

Significance Holocentric chromosomes are characterized by kinetochore activity along each sister chromatid. Although the kinetochore structure seems to be well conserved, as in monocentric organisms, the organization of holocentromeres is still elusive, and no centromeric repeat has been found associated with centromeric histone H3 variant-positive centromeric nucleosomes for any holocentric organism studied hitherto. We demonstrate that holocentrics of the sedge (Cyperaceae) Rhynchospora pubera possess different classes of centromere-specific repeats. Holocentromeres are composed of multiple centromeric units interspersing the gene-containing chromatin, and, as a functional adaption, a cell-cycle–dependent shuffling of centromeric units results in the formation of functional (poly)centromeres during cell division. The genome-wide distribution of centromeric repeat arrays interspersing the euchromatin provides a previously unidentified type of centromere organization. Holocentric chromosomes lack a primary constriction, in contrast to monocentrics. They form kinetochores distributed along almost the entire poleward surface of the chromatids, to which spindle fibers attach. No centromere-specific DNA sequence has been found for any holocentric organism studied so far. It was proposed that centromeric repeats, typical for many monocentric species, could not occur in holocentrics, most likely because of differences in the centromere organization. Here we show that the holokinetic centromeres of the Cyperaceae Rhynchospora pubera are highly enriched by a centromeric histone H3 variant-interacting centromere-specific satellite family designated “Tyba” and by centromeric retrotransposons (i.e., CRRh) occurring as genome-wide interspersed arrays. Centromeric arrays vary in length from 3 to 16 kb and are intermingled with gene-coding sequences and transposable elements. We show that holocentromeres of metaphase chromosomes are composed of multiple centromeric units rather than possessing a diffuse organization, thus favoring the polycentric model. A cell-cycle–dependent shuffling of multiple centromeric units results in the formation of functional (poly)centromeres during mitosis. The genome-wide distribution of centromeric repeat arrays interspersing the euchromatin provides a previously unidentified type of centromeric chromatin organization among eukaryotes. Thus, different types of holocentromeres exist in different species, namely with and without centromeric repetitive sequences.