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Isolation of Resistance Gene Candidates (RGCs) and characterization of an RGC cluster in cassava

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Abstract

Plant disease resistance genes (R genes) show significant similarity amongst themselves in terms of both their DNA sequences and structural motifs present in their protein products. Oligonucleotide primers designed from NBS (Nucleotide Binding Site) domains encoded by several R-genes have been used to amplify NBS sequences from the genomic DNA of various plant species, which have been called Resistance Gene Analogues (RGAs) or Resistance Gene Candidates (RGCs). Using specific primers from the NBS and TIR (Toll/Interleukin-1 Receptor) regions, we identified twelve classes of RGCs in cassava ( Manihot esculenta Crantz). Two classes were obtained from the PCR-amplification of the TIR domain. The other 10 classes correspond to the NBS sequences and were grouped into two subfamilies. Classes RCa1 to RCa5 are part of the first subfamily and were linked to a TIR domain in the N terminus. Classes RCa6 to RCa10 corresponded to non-TIR NBS-LRR encoding sequences. BAC library screening with the 12 RGC classes as probes allowed the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons. Members of the two TIR and non-TIR NBS-LRR subfamilies occurred together within individual BAC clones. The BAC screening and Southern hybridization analyses showed that all RGCs were single copy sequences except RCa6 that represented a large and diverse gene family. One BAC contained five NBS sequences and sequence analysis allowed the identification of two complete RGCs encoding two highly similar proteins. This BAC was located on linkage group J with three other RGC-containing BACs. At least one of these genes, RGC2, is expressed constitutively in cassava tissues.

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Acknowledgements

We are grateful to Ivan Acosta and Gerardo Gallego (CIAT) for helping in the analysis of RGCs and for fruitful discussions. Thanks to Christel Llauro-Berger and Michèle Laudié for sequencing, and to Céline Grasset for her technical support. We thank Jérôme Salse for fruitful discussions and technical advice during this work. Thanks to Véronique Jorge for analyzing the mapping data and to Benoit Piégu for advice on bioinformatics. We are grateful to Chikelu Mba for critically reading the manuscript. We thank Martin Fregene for fruitful discussions and suggestions during this work and for providing us with the BAC library. We thank the anonymous reviewers for helpful comments and suggestions on the manuscript. This research was supported by grants from the Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnología "Francisco José de Caldas" (Colciencias) (Contract No. 344-98, code 2236-12-051-98), IRD and CIAT. Camilo López was supported by a doctoral fellowship awarded by IRD

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  1. Biotechnology Research Unit, Centro Internacional de Agricultura Tropical (CIAT), AA 6713, Cali, Colombia

    C. E. López, A. P. Zuluaga & J. Tohme

  2. UMR5096 CNRS, Institut de Recherche pour le Développement (IRD), Université de Perpignan, 52 Avenue de Villeneuve, 66860, Perpignan, France

    C. E. López, R. Cooke, M. Delseny & V. Verdier

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  1. C. E. López
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  2. A. P. Zuluaga
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Correspondence to V. Verdier.

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Communicated by M.-A. Grandbastien

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López, C.E., Zuluaga, A.P., Cooke, R. et al. Isolation of Resistance Gene Candidates (RGCs) and characterization of an RGC cluster in cassava. Mol Gen Genomics 269, 658–671 (2003). https://doi.org/10.1007/s00438-003-0868-5

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