Abstract
Isolating genetic markers is often costly and time-consuming for non-model fungal species. However, these markers are of primary importance to identify the origin of invasive species and to infer their reproductive mode and dispersal ability. We slightly modified a recent molecular method to quickly isolate and validate single-nucleotide polymorphism (SNP) markers, from a first Erysiphe alphitoides draft genome, one of the main causal agent of oak powdery mildew in Europe. Although the draft assembly was strongly fragmented (555,289 contigs), we successfully isolated 1700 SNPs from 75 single-copy genes conserved in most fungal genomes. Ninety percent of them allowed to clearly distinguish the two main Erysiphe species reported on European oaks: E. alphitoides and E. quercicola. Thirty-six SNPs, located in distinct genes, were then validated using a strategy of MassArray genotyping on 95 E. alphitoides isolates sampled in Europe. This genotyping showed that only monospore isolates had the expected haploid signature, whereas direct genotyping from field leaves showed signature of mixed infection. Considering haploid isolates, these markers led to the first results of population genetic diversity, and suggested that E. quercicola may have a more asexual reproduction than its sister species, E. alphitoides.
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Raw sequencing data of the draft genome are available on the Bioproject PRJNA593204. The first assembly, contigs > 10 kb homologs to Blumeria graminis, and predicted genes are available on arachne.pierroton.inra.fr/AlphiGeno. Erysiphe alphitoides Jbrowse is available on https://urgi.versailles.inra.fr/jbrowse/gmod_jbrowse/?data=myData/AlphiGeno. Raw Ion Torrent sequencing data, scripts for detecting SNP, reference sequences for each amplicon from the E. alphitoides draft genome, MassArray genotyping data, and supplementary tables of this paper are available on https://doi.org/10.15454/UGMTBK.
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Acknowledgments
We thank Olivier Fabreguettes, Gilles Saint-Jean, and Martine Martin-Clotté for their help in the laboratory, especially for Erysiphe alphitoides sub-cultures, and DNA extraction; Nacer Mohellibi for helping in the genome browser; and Françoise Alfama-Depauw for blast banks, using the facilities of the URGI platform (https://urgi.versailles.inra.fr/). MassArray, Access, and Ion Torrent experiments were performed at the Genome Transcriptome Facility of Bordeaux (Grants from Investissements d’Avenir, Convention attributive d’aide EquipEx Xyloforest ANR-10-EQPX-16-01), with the help of Christophe Boury. Illumina sequencing was performed in collaboration with the GeT core facility, Toulouse, France (http://get.genotoul.fr) and the help of Céline Jeziorski, and was supported by France Génomique National infrastructure, funded as part of “Investissement d’avenir” program managed by Agence SNPs from an Erysiphe alphitoides draft genome - 8Nationale pour la Recherche (contract ANR-10-INBS-09). Andrin Gross benefited from the IdEx Bordeaux post-doctoral fellowship program. This work was also supported by the ANR-12-ADAP-0009 (Gandalf project), and an innovative project INRA department EFPA. Part of the sampling was issued from a European BiodivERsA project (RESIPATH: Responses of European Forests and Society to Invasive Pathogens – BIODIVERSA/0002/2012) with a national grant ANR-13-EBID-0005-01. We are also grateful to the Genotoul bioinformatics platform Toulouse Midi-Pyrénées (Bioinfo Genotoul) for providing computing and storage resources. We finally thank colleagues from RESIPATH and the herbaria curators D. Triebel (Bot. Staatssammlungen Munich, Germany), C. Lange (Natural history museum of Denmark), A. D. Bond (Royal Botanical Gardens Fungarium, UK), R. Berndt (Fungarium Z + ZT, Switzerland), V. Queloz and V. Dubach (forest protection service herbarium, WSL, Switzerland), and Nathalie Séjalon-Delmas et Paul Seimandi (Paul Sabatier Toulouse university, France) for Erysiphe sp. samples used in the genotyping test.
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Dutech, C., Feau, N., Lesur, I. et al. An easy and robust method for isolation and validation of single-nucleotide polymorphic markers from a first Erysiphe alphitoides draft genome. Mycol Progress 19, 615–628 (2020). https://doi.org/10.1007/s11557-020-01580-w
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DOI: https://doi.org/10.1007/s11557-020-01580-w