Functional identification of the wheat gene enhancing mycotoxin detoxification of the major Fusarium resistance QTL Fhb1

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Schweiger, Hermann Buerstmayr BOKU-University of Natural Resources and Life Sciences Vienna, Department

1 Functional identification of the wheat gene enhancing mycotoxin detoxification of the major Fusarium resistance QTL Fhb1 Barbara Steiner, Simone Zimmerl, Marc Lemmens, Gerhard Adam, Bradley Till, Wolfgang Schweiger, Hermann Buerstmayr BOKU-University of Natural Resources and Life Sciences Vienna, Department IFA-Tulln, Institute for Biotechnology in Plant Production, Konrad Lorenz Str. 20, A-3430 Tulln, Austria

Fhb1 milestones from BOKU University Molecular mapping in the highly resistant CIMMYT line CM-82036 Buerstmayr et al.

0 Xbarc133 11.8 Xbarc147 15.4 Xgwm493 22.

2 Fhb1 milestones from BOKU University Molecular mapping in the highly resistant CIMMYT line CM Buerstmayr et al. 2002, 2003 Fhb1 confers deoxynivalenol (DON) resistance and detoxifies DON into DON-3-glucoside Lemmens et al. 2005, Xbarc075 Xgwm Xgwm Xbarc Xbarc Xgwm Xbarc102 LOD Transcriptomic and metabolomic characterization Introgressing in European winter wheat, durum wheat and triticale Steiner et al. 2009, Schweiger et al. 2013, Kugler et al. 2013, Warth et al. 2014, Nussbaumer et al. 2015, Kluger et al. 2015, Samad-Zamini et al Salameh et al Prat et al. 2016, Oiller et al cm Xs18m18_9

alleles susceptible alleles Localisation of Fhb1 in an 860 kb region harboring 28 genes Gene(s)

3 Establishment of the genomic region spanning Fhb1 and fine-mapping FHB severity* DON severity* * mean number of FHB/DONbleached spikelets/head Control lines: resistant alleles resistant alleles susceptible alleles Localisation of Fhb1 in an 860 kb region harboring 28 genes Gene(s) confer FHB and DON resistance R = resistant S = susceptible Schweiger and Steiner et al. (2016) TAG

4 Phenotype Genotype Identification of the DON detoxification gene Mutant populations of the highly resistant line CM-82036: EMS (ethyl methane sulfonate)-treated: 6,000 lines Gamma-irradiated: 1,200 lines Forward genetics lines were inoculated with Fusarium or infiltrated with DON -> susceptible mutants were genotyped Reverse genetics - TILLING Screening for mutations in candidates <- phenotyping of lines with mutations in candidate genes

5 Reverse genetics excludes 9 candidate genes EMS population was screened for mutations in 9 genes ~ 10 lines/gene with truncation/deleterious missense mutations phenotyped few mutants showed susceptible phenotypes, but no conclusive results for one candidate all tested genes excluded as Fhb1 control near isogenic lines differing in Fhb1 mutant lines Fhb1 fhb1 Fhb1 fhb1 F. gram. point inoculation DON infiltration DON infiltration

6 Number of infected spikelts/spike Number of lines Forward genetics: Fhb1 is a dominat resistance gene 1,200 radiated lines inoculated with Fusarium 80 lines FHB susceptible in the field 12 lines confirmed in the greenhouse 4 of these also DON susceptible controls with Fhb1 Genotypic characteristation: 3 lines have deletions covering the whole Fhb1 region, these are FHB and DON susceptible controls lacking Fhb1 genes for FHB and DON resistance are located in this genomic region of CM Mean number of infected spikelets per head Fhb1 acts dominant resistant fhb1/ fhb1 Fhb1/ fhb1 Fhb1/ Fhb1

Average number of reads/amplicon Forward genetics for DON susceptibility 1,300 EMS-treated lines infiltrated with DON 8 lines DON susceptible in the field and greenhouse 7 of these lines also FHB

7 Average number of reads/amplicon Forward genetics for DON susceptibility 1,300 EMS-treated lines infiltrated with DON 8 lines DON susceptible in the field and greenhouse 7 of these lines also FHB susceptible Sequencing of the Fhb1 genic region 80 kb analysed (70 amplicons cover 34 genes) of 32 lines (susceptible mutants and controls) MiSeq sequencing: 500k to 1500k reads per line 5 amplicons insufficient coverage Zero to 5 mutations/line Stop codon and putative deleterious missense mutations were found in several genes 60K 50K 40K 30K 20k 10K 0 Amplicons

8 Outlook and conclusions Current work confirmation of mutations identified by NGS re-sequencing of missing coding regions co-segregation analysis: mutation - phenotype Fhb1 achievements Complete contig with all annotated genes Fhb1 gene-specific primers developed Dominant resistance gene(s) present in CM Confer resistance against FHB and DON DON susceptibility associated with FHB susceptibility

Fusarium inoculum External cooperation: Bradley Till, IAEA Laboratory, A NGS, irradiation Hélène Berges,

9 Acknowledgments BOKU University Matthias Fidesser field Theresia Köstlbauer greenhouse Evelyn Weissbacher greenhouse Simon Allerstorfer Molla Fentie Mengist Simon Mühl Roman Polzer Bernhard Seidl Marc Lemmens DON, Fusarium inoculum External cooperation: Bradley Till, IAEA Laboratory, A NGS, irradiation Hélène Berges, CNRGV-INRA, F BAC library Austrian Science Fund SFB F3711: Functional genomics of Fusarium resistance in wheat. Project coordinator: Gerhard Adam

time-points Genotyping of the Fhb1 region SNP detection in genes: TILLING 8-fold 2D pool screening,

11 Phenotyping for FHB and DON susceptibility Greenhouse or field F. gram point inoculation or DON infiltration at anthesis Evaluation of FHB/DON severity at several time-points Genotyping of the Fhb1 region SNP detection in genes: TILLING 8-fold 2D pool screening, heteroduplex analysis on Fragment Analyzer TM System Sequencing of the Fhb1 genic region Fhb1 gene-specific primers, sequencing of amplicons on MiSeq System

12 RNA-seq data mapped to the Fhb1 contig -> selection of promising Fhb1 candidate genes

13 Materials and Methods Phenotyping of mutant lines for FHB and DON resistance F. gram inoculation and DON infiltration 2 spikelet (4 florets) / head 10 µl spore suspension (500 conidia) / floret 20 μl DON solution (12 g/l DON) / floret 26 days after inoculation/infiltration: number of diseased /DON bleached spikelets = FHB / DON severity

time points Replications 3 1 2 3 Total number of samples 6 3 6 12 24 36 48 hai 72 F. gram spore conc. 50.

14 Transcriptomic characterisation of the Fhb1 region using RNA sequencing Greenhouse experiment: Genotypes 2 CM NILs differing in Fhb1: NIL38 (Fhb1) NIL51 Treatments 2 Fusarium inoculated Mock inoculated Sampling time points Replications Total number of samples hai 72 F. gram spore conc conidia/ml 5 heads/sample with 6 inoculated spikelets/head spikelet 6 spikelet 4 spikelet 5 spikelet 3 spikelet 2 spikelet 1

15 Plant Material CM82036 chr. 3B Fhb1 region x chr. 3B Fhb1 region Remus F1 Regeneration of haploid plants (n) from F1 plants Duplication of the genom of the haploid plants (n) to doubled haploid plants (2n) DH line Fhb1 region x CM times backcrossed with CM82036 BC5F1 x 98.5 % CM82036 alleles BC5F2 chr. 3B 5A chr. 3B 5A Fhb1 Qfhs.ifa-5A Fhb1 region region region Qfhs.ifa-5A region CM Nil 38 CM Nil 51

Current Knowledge on the Genetics of Fusarium Head Blight Resistance in Wheat - Implications for Resistance Breeding

Current Knowledge on the Genetics of Fusarium Head Blight Resistance in Wheat - Implications for Resistance Breeding Hermann Buerstmayr, Barbara Steiner, Marc Lemmens BOKU-University of Natural Resources