Cytogenetic Mapping of Common Bean Chromosomes Reveals a Less Compartmentalized Small-Genome Plant Species

Chromosome Research (2009) 17:405–417 DOI 10.1007/s10577-009-9031-4 Cytogenetic mapping of common bean chromosomes reveals a less compartmentalized small-genome plant species Andrea Pedrosa-Harand & James Kami & Paul Gepts & Valérie Geffroy & Dieter Schweizer Received: 13 November 2008 /Revised and Accepted: 21 January 2009 / Published online: 28 March 2009 # The Author(s) 2009. This article is published with open access at Springerlink.com Abstract Cytogenetic maps of common bean chro- with genetically mapped markers, mostly with single- mosomes 3, 4 and 7 were constructed by fluorescence copy RFLPs, a large subset of BACs, from 13 in-situ hybridization (FISH) of BAC and a few other different genomic regions, contained repetitive genomic clones. Although all clones were selected sequences, as concluded from the regional distribu- tion patterns of multiple FISH signals on chromosomes: pericentromeric, subtelomeric and dispersed. Electronic supplementary material The online version of this article (doi:10.1007/s10577-009-9031-4) contains Pericentromeric repeats were present in all 11 chro- supplementary material, which is available to authorized users. mosome pairs with different intensities, whereas subtelomeric repeats were present in several chromo- Responsible Editor: Jiming Jiang. some ends, but with different signal intensities : A. Pedrosa-Harand D. Schweizer depending on the BAC, suggesting that the terminal Department of Chromosome Biology, University of Vienna, heterochromatin fraction of this species may be Vienna, Austria composed of different repeats. The correlation of J. Kami : P. Gepts genetic and physical distances along the three studied Department of Plant Sciences/MS1, chromosomes was obtained for 23 clones. This Section of Crop and Ecosystem Sciences, correlation suggests suppression of recombination University of California, around extended pericentromeric regions in a similar Davis, CA, USA way to that previously reported for plant species with V. Geffroy larger genomes. These results indicate that a relatively Institut de Biotechnologie des Plantes, UMR-CNRS 8618, small plant genome may also possess a large INRA, Université Paris Sud, proportion of repeats interspersed with single-copy Orsay, France sequences in regions other than the pericentromeric D. Schweizer heterochromatin and, nevertheless, exhibit lower GMI - Gregor Mendel Institute of Molecular Plant Biology, recombination around the pericentromeric fraction of Austrian Academy of Sciences, the genome. Vienna, Austria Present Address: A. Pedrosa-Harand (*) Laboratory of Plant Cytogenetics, Department of Botany, Keywords fluorescence in-situ hybridization (FISH) . Federal University of Pernambuco, Recife, PE, Brazil 50670-420 bacterial artificial chromosome (BAC) . cytogenetic e-mail: [email protected] map . recombination . Fabaceae 406 A. Pedrosa-Harand et al. Abbreviations of this consortium is to develop genomic resources for 32P radioactive isotope of phosphorus the species (Broughton et al. 2003). A physical map APA loci family of seed proteins for the common bean was also considered to be a consisting of α-amylase inhibitors priority among researchers from the legume commu- (α-AI), phytohaemagglutinins (PHA) nity and should be established by 2011 (Gepts et al. and arcelins (ARL) 2005). Recently, a draft physical map of P. vulgaris BAC bacterial artificial chromosome cv. G19833 was developed, with 34 264 clones Bng (common) bean genomic plasmid clone assembled into 1183 contigs and the remaining 6385 C0tC0 is the initial concentration clones existing as singletons (Schlueter et al. 2008). of single stranded DNA in mol/l; To date, several genetic maps are available for the t is the reannealing time in seconds common bean (Gepts et al. 2008). This is a diploid CCD charge-coupled device species with 22 chromosomes and a haploid genome CIAT International Center for Tropical size estimated to be around 600 Mb (Arumuganathan Agriculture and Earle 1991; Bennett and Leitch 1995). Linkage cM centimorgan groups from previously developed genetic maps Cy3-dUTP 5-aminopropargyl-2′-deoxyuridine (Vallejos et al. 1992; Freyre et al. 1998) have been 5′-triphosphate coupled to red assigned to the corresponding chromosome pairs by cyanine fluorescent dye pooling RFLP markers from each linkage group and DAPI 4′,6-diamidino-2-phenylindole hybridizing them in situ to chromosomes (Pedrosa et DNA deoxyribonucleic acid al. 2003). This study provided a set of markers that FISH fluorescence in-situ hybridization allowed the identification of each chromosome of HindIII restriction enzyme from the species. It also revealed a lack of correlation Haemophilus influenzae between chromosome and linkage group sizes. PCR polymerase chain reaction However, the differences in recombination frequen- RFLP restriction fragment length cies among and along chromosomes could not be polymorphism investigated because linkage groups and chromo- SD standard deviation somes were not connected by multiple shared SDS sodium dodecyl sulfate sequences: only one probe, composed of several SSC saline-sodium citrate buffer RFLP markers of the same linkage group, was (20× SSC: 3 M NaCl, 0.3 M available for most chromosomes. sodium citrate, pH 7.0) Large-insert BAC clones have frequently been SSPE saline-sodium phosphate-EDTA used in fluorescence in-situ hybridization (FISH) buffer (20× SSPE: 3 M sodium experiments to physically map single-copy, genomic chloride, 0.2 M sodium sequences to chromosomes of plants with a small hydrogenphosphate, 0.02 M genome size, such as Arabidopsis (Fransz et al. 2000), EDTA, pH 7.4) rice (Cheng et al. 2001a,b), sorghum (Kim et al. TAC transformation-competent 2005a,b), Medicago truncatula (Kulikova et al. 2001) artificial chromosome and Lotus japonicus (Pedrosa et al. 2002). Since several BAC clones have been selected using genetically mapped markers and mapped by FISH along Introduction chromosomes, genetic and physical distances could be compared and associated with chromosome land- The common bean (Phaseolus vulgaris L.) is the marks such as centromeres, telomeres and hetero- major legume crop for direct consumption, and is an chromatin. In the present work, we established important source of dietary proteins for populations in cytogenetic maps based on fluorescence in-situ Africa and Latin America. In an effort to accelerate hybridization of BACs and a few other genomic improvement of the common bean, the Phaseomics clones for the first three common bean chromosomes. consortium has been established, bringing together These maps enabled the evaluation of recombination bean researchers from different fields. One of the aims frequencies along these chromosomes, gave an Mapping common bean chromosomes 407 overview of the common bean genome organization was purified in a Sephadex G-50 column (Sambrook and provided anchoring points for future contig maps. et al. 1989). Filters were hybridized for 16 h at 65°C in 7.5% SDS, 5× SSPE and washed with 2× SSC- 0.1% SDS, 1× SSC-0.1% SDS, 0.5× SSC-0.1% SDS Materials and methods and 0.1× SSC-0.1% SDS at 65°C. Radioactivity signals were detected using a FX Molecular Imager Plant material (BioRad, Hercules, CA, USA). Each probe was individually hybridized to one filter and was used Seeds from the P. vulgaris Mesoamerican breeding for a second hybridization only if no positive clone line BAT93 were obtained from the germplasm bank was identified in the first trial. Bacteriophages co- of the International Center for Tropical Agriculture - localized at the end of linkage group B4 with a CIAT, Cali, Colombia. complex disease resistance gene cluster (Geffroy et al. 2008, 2009) were selected as described by Ferrier- Chromosome preparation Cana et al. (2003). BAC and bacteriophage DNA were isolated using the Plasmid Mini Kit (Qiagen, Root tips obtained from germinating seeds were Hilden, Germany) and Nucleobond AX columns pre-treated in 2 mM 8-hydroxyquinoline for 5 h at (Macherey-Nagel, Düren, Germany), respectively, 16°C, fixed in ethanol-acetic acid 3:1 (v/v) and following the manufacturer’s instructions. All selected stored in fixative at −20°C for up to several weeks. genomic clones were labelled by nick translation Mitotic chromosome preparation was performed as (Roche Diagnostics, Indianapolis, IN, USA) with described in Pedrosa-Harand et al. (2006), with Cy3-dUTP (GE Healthcare, Bucks, UK) or maceration in 3% (w/v) cellulase ‘Onozuka R-10’ SpectrumGreen-dUTP (Vysis, Des Plaines, IL, USA). (Serva, Heidelberg, Germany) plus 30% (v/v) pecti- nase (Sigma-Aldrich, St. Louis, MO, USA) in DNA dot blot analysis 0.01 M citric acid-sodium citrate buffer pH 4.8 for 1.5 h at 37°C. In order to confirm the orientation of DNA (10 ng) from BAC clones that showed strong clones mapped to the short arm of chromosome 7, positive signals after library screening was spotted young flower buds were fixed as described above for onto an N+ Hybond membrane. Hybridization of the pachytene chromosome preparation. Pachytene chro- C0t-1 fraction, isolated according to Zwick et al. mosome spreads were prepared according to Armstrong (1997), was performed with the ECF random priming et al. (2001), except that whole flower buds were labelling and signal amplification system (GE Health- digested in 0.33% (w/v) cellulase ‘Onozuka R-10’ care) according to the manufacturer’s instructions. (Serva) and 0.33% (w/v) pectolyase (Sigma-Aldrich) Fluorescent signals were detected using an FX for 3 h at 37°C, and meiocytes were left in 60%

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