Abstract
Key message
The Ror1 gene was fine-mapped to the pericentric region of barley chromosome 1HL.
Abstract
Recessively inherited loss-of-function alleles of the barley (Hordeum vulgare) Mildew resistance locus o (Mlo) gene confer durable broad-spectrum disease resistance against the obligate biotrophic fungal powdery mildew pathogen Blumeria graminis f.sp. hordei. Previous genetic analyses revealed two barley genes, Ror1 and Ror2, that are Required for mlo-specified resistance and basal defence. While Ror2 was cloned and shown to encode a t-SNARE protein (syntaxin), the molecular nature or Ror1 remained elusive. Ror1 was previously mapped to the centromeric region of the long arm of barley chromosome 1H. Here, we narrowed the barley Ror1 interval to 0.18 cM and initiated a chromosome walk using barley yeast artificial chromosome (YAC) clones, next-generation DNA sequencing and fluorescence in situ hybridization. Two non-overlapping YAC contigs containing Ror1 flanking genes were identified. Despite a high degree of synteny observed between barley and the sequenced genomes of the grasses rice (Oryza sativa), Brachypodium distachyon and Sorghum bicolor across the wider chromosomal area, the genes in the YAC contigs showed extensive interspecific rearrangements in orientation and order. Consequently, the position of a Ror1 homolog in these species could not be precisely predicted, nor was a barley gene co-segregating with Ror1 identified. These factors have prevented the molecular identification of the Ror1 gene for the time being.
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Acknowledgments
R.P, J.A.G and M.B were funded by grants from the Max Planck Society. J.A.G was additionally supported by a research fellowship from the International Max Planck Research School (IMPRS). N.C.C gratefully acknowledges Syngenta for funding. Work in the P.B lab is supported by an EMBO Installation Grant. We are thankful to Margaret Corbitt for technical assistance, Takuji Sasaki and Robin Buell for sharing rice genomics information pre-publication, Pietro Spanu for performing pulsed field gel electrophoresis of YACs and Akio Kato for providing the probe pHv1112.
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Supplementary material 2 (PNG 1536 kb) Fig. S1 Micrographs of multicolor FISH on barley metaphase chromosomes of cv. Ingrid. Shown in white (in merged) is the signal of the chromosome 1H long arm-specific probe pHv-1112 (Kato 2011); the arrows indicate the position of the signals for the genic probes in green and red; the insets show a magnification of the chromosomes with the FISH signals in black and white and after pseudo-coloring (in part rotated to suit the Figure format, arrow head indicates the pHv-1112-specific signal). Scale bar = 20 µm. a Probes for Con (red) + Pol (green) + pHv-1112. b Con (red) + Unk (green) + pHv-1112. Note that only results for combinations 1 and 2 (see main text) are shown and that the different chromosome background colors in Fig. 3 (blue) and Fig. S1 (greenish) are due to the fact that results shown in Fig. S1 are multicolor overlays of three differently labeled probes, lacking a dedicated color for the chromosomes
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Supplementary material 3 (PNG 544 kb) Fig. S2 Accumulation of selected secondary metabolites in first leaves of the indicated barley genotypes at 24 and 48 hours after inoculation with Blumeria graminis f. sp. hordei spores. Compounds with retention time (RT) 10.9, 12.3, 14.1 and 16.2 minutes are shown as the ones representing the most striking induction after pathogen inoculation. Error bars indicate standard deviations of one experiment
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Supplementary material 4 (PNG 148 kb) Fig. S3 UV spectra for four UPLC-PDA metabolite peaks showing induction by Blumeria graminis f. sp. hordei inoculation in primary barley leaves. Representative UV spectra of compounds eluted at retention time (RT) 14.1 and 16.2 resemble those reported for hordatines, their precursors and derivatives (Stoessl and Unwin 1970; von Röpenack et al. 1998). AU, absorbance units
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Supplementary material 6 (XLSX 17 kb) Table S2 Detail of primer pairs used to investigate the genes from the YAC clones and the genomic DNA sequence obtained for each gene fragment in the parents Ingrid and Malteria Heda
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Supplementary material 8 (XLS 119 kb) Table S4 Details of DNA sequence polymorphism survey across three barley mapping parent genotypes
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Supplementary material 9 (XLS 52 kb) Table S5 Polymorphisms, haplotypes and marker details for the three barley mapping parent genotypes
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Supplementary material 10 (XLS 50 kb) Table S6 Genotypes of F2 recombinants for the Ror1 interval, used for fine mapping
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Supplementary material 11 (XLSX 13 kb) Table S7 Genotype in a panel of recombinants for the Ror1 interval for the genes identified in the sequenced YAC clones
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Supplementary material 12 (XLSX 16 kb) Table S8 Anchoring option, position coordinates and contigs in the barley genome for the genes present in the sequenced YAC clones close to the Ror1 locus. The analysis is based on the International Barley Sequencing Consortium (2012) genome draft release ftp://ftpmips.helmholtz-muenchen.de/plants/barley/public_data/ and http://webblast.ipk-gatersleben.de/barley
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Acevedo-Garcia, J., Collins, N.C., Ahmadinejad, N. et al. Fine mapping and chromosome walking towards the Ror1 locus in barley (Hordeum vulgare L.). Theor Appl Genet 126, 2969–2982 (2013). https://doi.org/10.1007/s00122-013-2186-6
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DOI: https://doi.org/10.1007/s00122-013-2186-6