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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<- phenotyping of lines with mutations
in candidate genes
Ph
eno
typ
e
Gen
oty
pe
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
mb
er o
f in
fect
edsp
ikel
ts/s
pik
e
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
er o
f re
ads/
amp
lico
n
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 x98.5 % CM82036 alleles
F1
Regeneration of haploid plants (n) from F1 plants
Fhb1region
Fhb1region
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