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Haplotype variability and identification of new functional alleles at the Rdg2a leaf stripe resistance gene locus

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Abstract

The barley Rdg2a locus confers resistance to the leaf stripe pathogen Pyrenophora graminea and, in the barley genotype Thibaut, it is composed of a gene family with three highly similar paralogs. Only one member of the gene family (called as Rdg2a) encoding for a CC-NB-LRR protein is able to confer resistance to the leaf stripe isolate Dg2. To study the genome evolution and diversity at the Rdg2a locus, sequences spanning the Rdg2a gene were compared in two barley cultivars, Thibaut and Morex, respectively, resistant and susceptible to leaf stripe. An overall high level of sequence conservation interrupted by several rearrangements that included three main deletions was observed in the Morex contig. The main deletion of 13,692 bp was most likely derived from unequal crossing over between Rdg2a paralogs leading to the generation of a chimeric Morex rdg2a gene which was not associated to detectable level of resistance toward leaf stripe. PCR-based analyses of genic and intergenic regions at the Rdg2a locus in 29 H. vulgare lines and one H. vulgare ssp. spontaneum accession indicated large haplotype variability in the cultivated barley gene pool suggesting rapid and recent divergence at this locus. Barley genotypes showing the same haplotype as Thibaut at the Rdg2a locus were selected for a Rdg2a allele mining through allele re-sequencing and two lines with polymorphic nucleotides leading to amino acid changes in the CC-NB and LRR encoding domains, respectively, were identified. Analysis of nucleotide diversity of the Rdg2a alleles revealed that the polymorphic sites were subjected to positive selection. Moreover, strong positively selected sites were located in the LRR encoding domain suggesting that both positive selection and divergence at homologous loci are possibly representing the molecular mechanism for the generation of high diversity at the Rdg2a locus in the barley gene pool.

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Acknowledgments

We thank Donata Pagani and Nadia Faccini for excellent technical assistance. This work was supported by the Italian national projects ‘Proteine e geni per la protezione delle piante’ (‘PROTEO-STRESS’) and ‘Nuove tecnologie molecolari per l’analisi del genoma di organismi di interesse agrario’ (‘AGRO-NANOTECH’) funded by MiPAAF. The work was also supported by a grant ‘Barlex’ of the Ministry of Education and Research (BMBF, FKZ 0314000) to N.S.

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Author notes

  1. Burkhard Steuernagel

    Present address: The Sainsbury Laboratory, John Innes Centre, Colney Lane, NR4 7UH, Norwich, UK

  2. Daniela Schulte

    Present address: KWS SAAT AG, Grimsehlstraße 31, 37555, Einbeck, Germany

Authors and Affiliations

  1. Genomics Research Centre, CRA-Consiglio per la ricerca e la sperimentazione in agricoltura, Via S Protaso 302, 29017, Fiorenzuola d’Arda, Piacenza, Italy

    Chiara Biselli, Simona Urso, Gianni Tacconi, Alberto Gianinetti, Paolo Bagnaresi, Luigi Cattivelli & Giampiero Valè

  2. Rice Research Unit, CRA-Consiglio per la ricerca e la sperimentazione in agricoltura, S.S. 11 to Torino, Km 2,5, 13100, Vercelli, Italy

    Chiara Biselli & Giampiero Valè

  3. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466, Seeland OT Gatersleben, Germany

    Burkhard Steuernagel, Daniela Schulte & Nils Stein

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  1. Chiara Biselli
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Correspondence to Giampiero Valè.

Additional information

Communicated by T. Komatsuda.

Electronic supplementary material

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122_2013_2075_MOESM1_ESM.pdf

Figure S1 Alignment between Morex rdg2a (Mrdg2a) and Thibaut Rdg2a and Nbs2-Rdg2a CDSs. In the consensus sequence, for each polymorphism, lower-case indicates conservation in only two genes, while a missing space represents no conservation among the three sequences. Colour legend: white background = conservation; grey background = conservation between only two sequences; black background = absence of conservation among the three genes. (PDF 265 kb)

122_2013_2075_MOESM2_ESM.pdf

Figure S2 Alignment between Morex RDG2A (MRDG2A) and Thibaut RDG2A and NB2-RDG2A proteins. For each polymorphism, in the consensus sequence, lower-case represents conservation in only two proteins, numbers indicate that all the residues at that position have the same biochemical properties (1 = acid; 2 = negative charged; 3 = hydrophilic; 4 = positive charged; 5 = aromatic; 6 = hydrophobic). Colour legend: white background = conservation; light grey background = conservation between only two sequences; white letter on dark grey background = different residue but with the same biochemical property; white letter on black background = different residue among the other sequences. (PDF 95 kb)

122_2013_2075_MOESM3_ESM.pdf

Figure S3 Alignment between the genomic sequences of Optic, Rebelle, Galleon, Acuario, Haruna Nijo and Thibaut Rdg2a alleles. The colour and consensus sequence legends were as in Fig. S1. (PDF 769 kb)

122_2013_2075_MOESM4_ESM.pdf

Figure S4 Alignment between Optic, Rebelle, Galleon, Acuario, Haruna Nijo and Thibaut RDG2A proteins. The colour and consensus sequence legends were as in Fig. S2. (PDF 125 kb)

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Biselli, C., Urso, S., Tacconi, G. et al. Haplotype variability and identification of new functional alleles at the Rdg2a leaf stripe resistance gene locus. Theor Appl Genet 126, 1575–1586 (2013). https://doi.org/10.1007/s00122-013-2075-z

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