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

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

Molecular mapping in the highlyresistant CIMMYT line CM-82036

Fhb1 confers deoxynivalenol (DON) resistance and detoxifies DON into DON-3-glucoside

Transcriptomic and metabolomiccharacterization

Introgressing in European winter wheat, durum wheat and triticale

Buerstmayr et al. 2002, 2003

Lemmens et al. 2005, 2008

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. 2017

Salameh et al. 2011Prat et al. 2016 , Oiller et al.

Fhb1 – milestones from BOKU University

0

5

10

1

5

20

2

5

30

Xbarc0750.0

Xgwm3895.1

Xgwm5338.5Xbarc13311.0Xbarc14711.8

Xgwm49315.4

Xbarc10222.7

Xs18m18_943.7cM

LOD

Establishment of the genomic region spanning Fhb1 and fine-mapping

DON severity*

* mean number of FHB/DON-bleached spikelets/head

FHB severity*

Localisation of Fhb1 in an 860 kb region harboring 28 genesGene(s) confer FHB and DON resistance

R = resistant S = susceptible

Schweiger and Steiner et al. (2016) TAG

resistant alleles

susceptible alleles

resistant alleles

Control lines:

Identification of the DON detoxification geneMutant populations of the highly resistant line CM-82036:EMS (ethyl methane sulfonate)-treated: 6,000 linesGamma-irradiated: 1,200 lines

Forward geneticslines were inoculated with Fusariumor infiltrated with DON-> susceptible mutants were genotyped

Reverse genetics - TILLINGScreening for mutations in candidates

Reverse genetics excludes 9 candidate genes

EMS population was screened for mutations in 9 genes~ 10 lines/gene with truncation/deleterious missense mutations phenotyped

control near isogenic lines differing in Fhb1 mutant lines

Fhb1

F. gram. point inoculation

Fhb1 fhb1 fhb1

DON infiltration DON infiltration

few mutants showed susceptible phenotypes, but no conclusive results for one candidate

all tested genes excluded as Fhb1

Nu

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f in

fect

edsp

ikel

ts/s

pik

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Fhb1/Fhb1

fhb1/fhb1

Fhb1/fhb1

Fhb1 acts dominant resistant

Forward genetics: Fhb1 is a dominat resistance gene

1,200 radiated lines inoculated with Fusarium80 lines FHB susceptible in the field12 lines confirmed in the greenhouse4 of these also DON susceptible

genes for FHB and DON resistance are located in this genomic region of CM-82036

controls lacking Fhb1

controls with Fhb1

Nu

mb

er o

f lin

es

Mean number of infected spikelets per head

Genotypic characteristation:3 lines have deletions covering the whole Fhb1 region, these are FHB and DON susceptible

Forward genetics for DON susceptibility

1,300 EMS-treated lines infiltrated with DON8 lines DON susceptible in the field and greenhouse7 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 line5 amplicons insufficient coverage

60K

50K

40K

30K

20k

10K

0

AmpliconsAve

rage

nu

mb

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f re

ads/

amp

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Zero to 5 mutations/line Stop codon and putative deleterious

missense mutations were found in several genes

Current work confirmation of mutations identified by NGS

re-sequencing of missing coding regions

co-segregation analysis: mutation - phenotype

Outlook and conclusions

Fhb1 – achievements

Complete contig with all annotated genes

Fhb1 gene-specific primers developed

Dominant resistance gene(s) present in CM-82036

Confer resistance against FHB and DON

DON susceptibility associated with FHB susceptibility

Acknowledgments

BOKU UniversityMatthias Fidesser – fieldTheresia Köstlbauer – greenhouseEvelyn Weissbacher – greenhouse

Simon AllerstorferMolla Fentie Mengist Simon MühlRoman Polzer Bernhard Seidl

Marc Lemmens – DON, Fusarium inoculum

Austrian Science Fund SFB F3711: Functional genomics of Fusarium resistance in wheat. Project coordinator: Gerhard Adam

External cooperation:Bradley Till, IAEA Laboratory, A – NGS, irradiationHélène Berges, CNRGV-INRA, F – BAC library

Greenhouse or fieldF. gram point inoculation or DON infiltration at anthesis

Evaluation of FHB/DON severity at several time-points

SNP detection in genes: TILLING8-fold 2D pool screening, heteroduplex analysis on Fragment AnalyzerTM System

Sequencing of the ‘Fhb1 genic region‘Fhb1 gene-specific primers, sequencing of amplicons on MiSeq System

Phenotyping for FHB and DON susceptibility

Genotyping of the Fhb1 region

RNA-seq data mapped to the Fhb1 contig

-> selection of promising Fhb1 candidate genes

F. gram inoculation and DON infiltration2 spikelet (4 florets) / head

10 µl spore suspension (500 conidia) / floret20 μl DON solution (12 g/L DON) / floret

26 days after inoculation/infiltration: number of diseased /DON bleached spikelets= FHB / DON severity

Materials and Methods –Phenotyping of mutant lines for FHB and DON resistance

Genotypes 2CM-82036 NILs differing in Fhb1:NIL38 (Fhb1)NIL51

Treatments 2 Fusarium inoculatedMock inoculated

Sampling time points

6 3 6 12 24 36 48 hai

Replications 3 1 2 3

Total number of samples

72

Transcriptomic characterisation of the Fhb1 region using RNA sequencing

spikelet 2spikelet 1

spikelet 3

spikelet 5spikelet 4

spikelet 6

F. gram spore conc. 50.000 conidia/mL5 heads/sample with 6 inoculated spikelets/head

Greenhouse experiment:

Plant Material

CM82036 Remusx

Duplication of the genom of the haploid plants (n) to doubled haploid plants (2n)

Fhb1region

CM82036DH line x5 times backcrossed with CM82036

chr. 3B chr. 3B

BC5F2

CM Nil 38 CM Nil 51

Fhb1region

Qfhs.ifa-5A region

Fhb1region

Qfhs.ifa-5A region

chr. 3B 5A chr. 3B 5A

BC5F1 x 98.5 % CM82036 alleles

F1

Regeneration of haploid plants (n) from F1 plants

Fhb1region

Fhb1region