The document discusses genomic research for improving stress tolerance in cereals like wheat and barley. It summarizes: 1) Breeding cereals for resistance to biotic and abiotic stresses is important to avoid yield losses and ensure sustainable production. 2) The document outlines various approaches the research group uses including marker-assisted breeding, gene isolation, and allele editing to improve resistances. 3) Preliminary results are presented on identifying QTL and genes for pre-haustorial resistance to rust in wheat and resistance to viruses in barley.

1. www.jki.bund.de
Genomics based breeding research for
improving resistance to biotic and abiotic
stress in cereals
Dragan Perovic, Albrecht Serfling, Katja Perner, Sandra Färber, Cristina Silvar,
Ilona Krämer, Antje Habekuß, Doris Kopahnke, Heike Lehnert, Thomas Vatter,
Gwendolin Wehner, Esther Mitterbauer, Andreas Graner, Nils Stein
and Frank Ordon

2. Institute for Resistance Research and Stress Tolerance
Acreage of cereals 2012
Wheat and barley growing area (ha)
and average yield (t/ha) in 2012
ha (Mio) t/ha
Wheat
World 215.49 3.11
India 29.86 3.18
Germany 3.06 7.33
Barley
World 49.52 2.68
India 0.77 2.10
Germany 1.68 6.19
http://faostat.fao.org
home.arcor.de http://www.nurbier.de/category/biergeschichte/http://www.abzonline.de/praxis/kasten-weizenbrot,707243491.html

3. Institute for Resistance Research and Stress Tolerance
Challenges for plant production
Food security
Growing population
Bioenergy
Change in dietary habits
Climate change
Anstieg der weltweiten Mitteltemperatur für die
Zeitspanne 071 - 2100 relativ zu der Zeitspanne 1961 -
1990. © MPI Met

4. Institute for Resistance Research and Stress Tolerance
Climatechange
http://www.umweltdaten.de/publikationen/fpdf-l/GGTSPU-styx2.bba.de-6248-7152625-DAT/3133.pdf
www.digiklix.de
Beschreibende Sortenliste 2010
+1°C = 10% yield reduction in wheat
-27% predicted for 2050 compared to 2000 in some regions
Wheat Initiative

5. Institute for Resistance Research and Stress Tolerance
http://www.transgen.de/pflanzenforschung/pflanzengesundheit/
Insects
Diseases
Weeds
Totalharvest
Rice
Sorghum
Maize
Oats
Wheat
Barley
Rye
Potato
Sugarcane
Average yield losses
Breeding for resistance to biotic and abiotic stress in cereals is of prime
importance to:
•avoid yield losses
•to ensure a consumer and environmental friendly production
Wheat (2012)
~140 million t
~$ 35 billion
FAOSTAT 2014

6. Institute for Resistance Research and Stress Tolerance
Mildew Leaf rust
No. Cultivars Yield
Year resistant susceptible resistant susceptible
1986 6 37 4.3* 5.6
1995 24 41 6.5 6.3
2005 52** 23 6.7 6.1
2011 55 9 6.9 6.4
Success of breeding for resistance in barley
BaMMV/BaYMV
Ahlemeyer pers. comm.
1=minimum, 9=maximum

7. Institute for Resistance Research and Stress Tolerance
Asfaw Adugna , 2004. Alternate Approaches in Deploying Genes for Disease Resistance in Crop
Plants. Asian Journal of Plant Sciences, 3: 618-623.
The never ending story
P. hordei P. striiformis B. graminis
P. teres R. commune U. nuda
BaMMV/BaYMV BYDV
Barley Wheat

8. Institute for Resistance Research and Stress Tolerance
Marker type RFLPs Genomic SSRs AFLPs
EST
SNPs/SSRs DArTs BOPAs/OPAs iSelect
Genotyping by
sequencing
Throughput
single marker
application
single marker
application
few marker
application
single marker
application 6K 1,5K 9K 50K
Multiplexing no mutiplexing
few markers
multiplexing
low
multiplexing
few markers
multiplexing
platform/
simultaneous
analysis
platform/
simultaneous
analysis
platform/
simultaneous
analysis
platform/
simultaneous
analysis
simultaneous
multiplexing
NGS/GBS
Amount of D N A Large amount low amount low amount low amount low amount low amount low amount low amount low amount
Quality of D N A very good average average average very good very good very good very good very good
Plant breeders toolbox

9. Institute for Resistance Research and Stress Tolerance
Marker based harnessing of genetic resources: B. graminis
7HS: 12.1 cM 5.3 cM 1.5cM
7HL: 41.9 cM 2.8 cM 1.3cM

10. Institute for Resistance Research and Stress Tolerance
Nested association mapping
NAM population HEB-25
• 25 wild accessions (H. spontaneum)
• 1 elite recipient (Barke)
• 1420 BC1S3 lines
TASSEL 4 (Q + K)
Significant differences (p <.0001; tukey-test) between and within families
Barke
incl. wildtype
Marker based harnessing of genetic resources: P. teres
Vatter et al. (unpublished)

11. Institute for Resistance Research and Stress Tolerance
Marker based harnessing of genetic resources: P. triticina
0
0,25
0,5
0,75
1
1996 1998 2000 2002 2004 2005 2006 2007 2008
Sorten ohne Resistenzgen Sorten mit LR37
Thatcher NIL-
Lr37
Thatcher without
resistance
Isolates Lr10 Lr11 Lr17 Lr18 Lr20 Lr28 Lr37 Lr49 T. monococcum T. boeticum
77WxR s s s s s s s s r s
167/176WxR s s s s s s s s r ps
Tommi 1 s s s s s s s s r s
13/20WxR s s s s s s s s r s
4136 ps s s s s r s s r s
s
ps
r
Analyzed isolates
virulent against all
known Lr-genes
located on the A
genome
The prehaustorial resistance of T. monococcum
0
20
40
60
80
12 24 48 72 96
HMC/Infection
Time after inoculation (h)
Borenos wxr77
Pi272560 wxr77
Susceptible
accession
Resistant
accession
Susceptible
accession
Resistant
accession
24 hai 96 hai 168 hai
24 hai 96 hai 168 hai
HMC
Lr37: 2004 2013
Serfling et al. (in preparation)

12. Institute for Resistance Research and Stress Tolerance
Molecular characterization of the prehaustorial resistance by Massive
Analysis of cDNA (MACE)
Number of RNA samples: 12 Time after inoculation
0 to 8 hai 8 to 16 hai 16-24 hai
Resistant accession rust inoculated 1 1 1
Resistant accession mock inoculated 1 1 1
Susceptible accession rust inoculated 1 1 1
Susceptible accession mock inoculated 1 1 1
Number of differentially expressed tags after comparison of the inoculated resistant and susceptible accession 0-24 hai
Quantitativelly differentially expressed 6810 6780 4832 1648
Qualitativelly differentially expressed 4413 3592 3592 340
In silico map on the basis of SNP detection of annotated
tags
1A 2A 3A 4A 5A 6A 7A
Comprises 1136 genes in which
4358 SNPs were detected
Serfling et al. (in preparation)

13. Institute for Resistance Research and Stress Tolerance
Detailed analysis of peroxidases and chitinases
-6
-4
-2
0
2
4
6
0-8hai
8-16hai
16-24hai
0-8hai
8-16hai
16-24hai
0-8hai
8-16hai
16-24hai
0-8hai
8-16hai
16-24hai
0-8hai
8-16hai
16-24hai
Pox6 Pox1 Prx113 Pox 54 Pox prec.
Log2ofexpressionInoculated/
noninoculated
Resistant accession Susceptible accession
By Go terms identified Peroxidases
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
0-8
hai
8-16
hai
16-24
hai
0-8
hai
8-16
hai
16-24
hai
Chitinase1 Chitinase 2
By Go terms identified Chitinases
Serfling et al. (in preparation)

14. Institute for Resistance Research and Stress Tolerance
Resistant
accesssion
Susceptible
accesssion
Resistant
accession
Susceptible
accession
0 6 12 24 48 72 96 168 hai
*
*
*
*
µMolH2O2
Diaminobenzidine stain
Peroxidase activity
Resistant
accesssion
Susceptible
accesssion
0
15
30
45
60
Peroxidaseactivity
(Unitsmin-1)
0 6 12 24 48 72 96 168 hai
*
Chitinaseactivity
(Unitsmin-1)
0
6
12
18
30
24
*
*
*
* *
Chitinase activity
Characterization of prehaustorial resistance
Calcofluor stain
? ?
0
0.05
0.1
0.15
0.2
0.25
0 50 100 150 200 250 300
Calibration curve
µMol H2O2 l-1
Absorption
0 min ai
15 min ai
30 min ai
Serfling et al. (in preparation)

15. Institute for Resistance Research and Stress Tolerance
Chr cM Number of markers
1A 217.7 588
2A 260.1 771
3A 171.2 503
4A 111.5 339
5A 236.7 570
6A 232.6 620
7A 258.4 727
Total 1488.3 4118
LOD
3.15
24.4%
LOD
3.32
16.5%
LOD
3.84
13.0%
LOD
3.62
13.3%
Phenotyping:
Number of haustorial mother
cells 72 hai (F2/F3)
Identification of QTL for pre-haustorial resistance
Serfling et al. unpublished
Localization of
candidate
genes in QTLs
is ongoing

16. Institute for Resistance Research and Stress Tolerance
Gene isolation: BaYMV/BaYMV-2 resistance
distance marker5H
‘HOR4224‘ (r) x ‘HOR10714‘ (s)
Based on 3369 F2 - plants,
Resolution 0.015% rec.
Exome capture
Perner et al. (in preparation)

17. Institute for Resistance Research and Stress Tolerance
Gene isolation: BaMMV resistance
GBS
Färber et al. (in preparation)

18. Institute for Resistance Research and Stress Tolerance
rym4/rym5
Isolation of resistance genes - allele mining
Hofinger et al. 2011. Molecular Ecology 20, 3653-3668
A. Graner
1000 accessions selected
27 resistant haplotypes
40 novel exon haplotypes
known haplotypes
13 susceptible
non allelic genes
identification of
8 novel eIF4E alleles
resequencing
resistance tests
test crosses,
resistance tests
year1
year2/3
year3
eIF4E allele mining
Hv-eIF4E
HvPDIL5-1
rym11
Yang et al. 2014. www.pnas.org/cgi/doi/10.1073/pnas.1320362111
Yang et al. 2014. Theor. Appl. Genet
Kanyuka et al.

19. Institute for Resistance Research and Stress Tolerance
Allele Editing: Directed mutagenesis using endonucleases
Puchta and Fauser (2014) The Plant Journal
ZFNs Zinc-Finger Nucleases
TALENs Transcription Activator-Like Effector Nucleases
CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
Cas CRISPR-associated, RNA-guided endonuclase
Meganucleases
A. Graner

20. Institute for Resistance Research and Stress Tolerance
Wheat – powdery mildew resistance
A. Graner
Allele Editing: Directed mutagenesis using endonucleases

21. Institute for Resistance Research and Stress Tolerance
Drought stress in the juvenile stage
EQTL
156 BARLEY GENOTYPES
PHENOTYPING
Biomass yield
Chlorophyll content
Chlorophyll fluorescence
Osmotic adjustment
Content of free proline
Total content of soluble sugars
DROUGHT STRESS
Stress application starts 7das
BBCH11 – BBCH33
4 weeks stress period
Stress 20% water capacity of soil
3 replicates per genotype
3 years trials
CONTROL STRESS
CONTROL STRESS
GWAS
QTL
GENE EXPRESSION
PROTEIN DETECTION
Illumina 9k iSelect SNP Chip
Consensus Map of markers
SNP Scoring
LD and Population structure
Significant SNPs
Chomosome 5H + 2H
NCBI BlastX
Protein function UniProt
Genetic map
GENOTYPING
DROUGHTSTRESS
LEAFSENESCENCE
qPCR Fluidigm Chip array
Drought stress genes
Genes for leaf senescence
Genes out of GWAS
Tassel 3.0
Detection of QTL
Localisation of QTL

22. Institute for Resistance Research and Stress Tolerance
Correlations (Pearson) and Heritability:
Treatment BY SPAD ETR OA CFP CSS
h² Control 0.80 0.64 0.08 0.00 0.13 0.13
Stress 0.58 0.61 0.50 0.27 0.29 0.30
BY Control 0.395 *** 0.091 -0.127 -0.328 *** -0.220 **
Stress 0.361 *** -0.087 -0.124 0.307 *** 0.367 ***
SPAD Control 0.160 * -0.185 * -0.239 ** -0.192 *
Stress -0.105 0.034 0.425 *** 0.418 ***
ANOVA: significant effects (p <0.001) of genotype and treatment; significant GxT effect for BY, CFP and CSS
Significance level: P≤0.05 *. P≤0.01 **. P≤0.001 ***
Wehner et al. (submitted)
Drought stress in the juvenile stage

23. Institute for Resistance Research and Stress Tolerance
Trait
Number of genomic regions associated with the traits on the seven linkage groups (barley chromosomes)
*highest R²
1H 2H 3H 4H 5H 6H 7H Total QTL
BY 81.7 cM (3 SNP) 2 cM (3 SNP) 76.2 cM (1 SNP) 99.1 cM (1 SNP) 46.7 cM (8 SNP) 48.3 cM (1 SNP) 19 (32 SNPs) 0.20%
92.2 cM (1 SNP) 5.5 cM (1 SNP) 135.5 cM (1 SNP)* 59.7 cM (1 SNP) 70.2 cM (1 SNP)
12.1 cM (1 SNP) 80.3 cM (1 SNP) 133.9 cM (1 SNP)
90.2 cM (3 SNP) 110.1 cM (1 SNP)
139.1 cM (1 SNP)
152.4 cM (1 SNP)
167.7 cM (1 SNP)
SPAD 49.2 cM (1 SNP)* 44.2 cM (4 SNP) 128.3 cM (1 SNP) 3 (6 SNPs) 3.80%
ETR 59.4 cM (1 SNP) 2.1 cM (1 SNP)* 2 (2 SNPs) 5.50%
OA 116.8 cM (1 SNP) 51.8 cM (1 SNP) 2.4 cM (1 SNP) 52.3 cM (1 SNP) 46.5 cM (1 SNP) 10.3 cM (1 SNP) 106.5 cM (1 SNP) 22 (29 SNPs) 3.50%
60.8 cM (2 SNP) 36.8 cM (2 SNP) 110.2 cM (1 SNP) 55.7 cM (1 SNP) 47.5 cM (1 SNP)
81.5 cM (4 SNP)* 51.8 cM (1 SNP) 95 cM (1 SNP) 51 cM (2 SNP)
135.8 cM (1 SNP) 61.9 cM (1 SNP) 137.9 cM (1 SNP)
146.5 cM (1 SNP) 89.4 cM (1 SNP)
100.7 cM (2 SNP)
CSS 95.8 cM (1 SNP)* 1 (1 SNP) 1.60%
Total QTL 4 (6 SNPs) 10 (18 SNPs) 8 (10 SNPs) 3 (3 SNPs) 12 (22 SNPs) 4 (5 SNPs) 6 (6 SNPs) 47 (70 SNPs)
Wehner et al. (submitted)
Drought stress in the juvenile stage

24. Institute for Resistance Research and Stress Tolerance
FTSH3_BY_0.2%
PME49_CSS_1.6%
1H
SUS4_SPAD_3.8%
YSL2_OA_0.7%
YSL15_OA_0.7%
GDH2_OA_1.4%
AMP1_OA_2.3%
GPX1_BY_0.2%
2H
FBL21_OA_2.8%
ACO1_OA_2.3%
3H
PYL5_OA_2.5%
4H
AVP1_SPAD_BY_0.2%
ATM_SPAD_BY_2.6%
TRIUR3_SPAD_BY_3.1%
SAPK9_SPAD_BY_3.1%
DREB1A_SPAD_OA_2.4%
EGY1_OA_1.4%
5H 6H
CHX_ETR_5.5%
ERF062_BY_0.2%
7H
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
Genetic map of QTLs including the significant associated SNP marker positions for
significant blasted proteins (BlastX) linked to drought stress or leaf senescence,
related traits for drought stress treatment and percentage of phenotypic variance
(explained R² in %) of the SNPs for all linkage groups (barley chromosomes).
cM
Wehner et al. (submitted)
Drought stress in the juvenile stage

25. Institute for Resistance Research and Stress Tolerance
New breeding goals: Mycorrhization and drought stress
Experimental design:
• 94 Genotypes, 2 Years, 3 Replications
• Treatments
– Mycorrhization (Myco, N-Myco)
– Irrigation (25% and 75% maximal water capacity, MWC)
Quantification root colonization:
 Ink vinegar staining (Vierheilig et al., 1998)
 Magnified intersection method (McGonigle et al., 1990)
N-myco Myco Myco Myco Myco
Myco 25% MWC Myco 75% MWC
Lehnert et al. (in preparation)

26. Institute for Resistance Research and Stress Tolerance
Trait: Root colonization (%)
Myco 25% MWC Myco 75% MWC
-log10(p)
-log10(p)
Chromosome Chromosome
Lehnert et al. (in preparation)
New breeding goals: Mycorrhization and drought stress

27. Institute for Resistance Research and Stress Tolerance
• Trait: Biomass (g), Yield (g), Ears per plant, 1000 grain weight (g),
Grains per ear
•
Lehnert et al. (in preparation)
New breeding goals: Mycorrhization and drought stress

28. Institute for Resistance Research and Stress Tolerance
amb: 400 ppm
eCO2: 700 ppm
New breeding goals: CO2

29. Institute for Resistance Research and Stress Tolerance
Mitterbauer et al. (in preparation)
YieldEars/plantTKW
Kernel/Ear(2-rowed)Kernel/Ear(6-rowed)Protein
New breeding goals: CO2

30. Institute for Resistance Research and Stress Tolerance
• Genome wide association
analyses
• (QK mixed model
approach;
• minor allele frequency
>5%)
• 3886 marker
Yield response (E/A)
Biomass response (E/A)
Kernel #/ear(E/A)
Stem weight (E/A)
Mitterbauer et al. (in preparation)
New breeding goals: CO2

31. Institute for Resistance Research and Stress Tolerance
Summary and future prospects
•Genomic tools facilitate an enhanced marker development and isolation of major
genes and QTL for resistance to biotoc and abiotic stress leading to a deeper
understanding of trait development and the transfer of marker based selection to the
allele level.
•This will lead to a more directed and faster use of genetic variation.
•High throughput marker systems also offer the opportunity to implement new
breeding goals efficiently into applied breeding procedures.
•Knowledge on gene sequences will facilitate the targeted editing of respective alleles
in the future by endonucleases.
•Genomic tools will speed up breeding for resistance to biotic
and abiotic stress
http://www.nature.com/mtna/journal/v1/n1/full/mtna20115a.html
Fernie, A.R., N. Schauer, 2008: Trends in Genetics 25, 39-48

32. Institute for Resistance Research and Stress Tolerance
Thanks
Dr. Dragan Perovic
Dr. Ilona Krämer
Dr. Antje Habekuß
Dr. Christiane Balko
Dr. Esther Mitterbauer
Dr. Nadine Knöchel
Gwendolin Wehner
Katja Perner
Sandra Färber
Dr. Doris Kopahnke
Thomas Vatter
Dr. Albrecht Serfling
Prof. Dr. Wolfgang Friedt
Prof. Dr. Andreas Graner
Dr. Nils Stein
Dr. Ping Yang
Martin Mascher
Prof. Dr. Klaus Pillen
Dr. Ernesto Igartua
Dr. Ana Casas
Dr. Cristina Silvar
Dr. Brian Steffenson
Dr. Kostya Kanyuka
Prof. Dr. Olga Afanasenko