linked-in defense
TRANSCRIPT
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Leveraging powdery mildew resistance in wild populations
Nick AmesSept 26th, 2014
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Background of Barley• Hordeum vulgare and the wild progenitor Hordeum vulgare subsp.
spontaneum
• Fourth in both quantity produced and area devoted to cultivation, as of 2007
• Center of domestication: Fertile Crescent
• In the US, 51% is used for animal feed, 44% for malt production, 3% for seed, and 2% for food http://www.barleyfoods.org/BarleyFacts-Industry.pdf
http://www.nap.edu/books/12692/xhtml/images/p20019ad4g94001.jpg
10,000
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Powdery Mildew• Blumeria graminis f. sp. hordei • Tends to thrive in high humidity
environments• Yield losses in temperate
climates range from 5-40%• Obligate biotroph
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Powdery mildew life cycle and mechanisms of defense
http://www.hgca.com/images/upload/Powdery_Mildew.gif
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Worldwide losses Average Losses: 10%Epidemic losses: 20%
Average Losses: 10%Epidemic losses: 30%
Average losses: 10%Epidemic losses: 25%
Winter and facultative barley losses: ?%
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Powdery mildew resistance genes
• Mla:Cluster of genes• Chromosome 1H• Numerous alleles due to intensive selection pressure
• mlo: is a defective repressor gene that was discovered in Ethiopian barley• Not used in winter barley
• Other genes include: Mlk, Mlh, Ml(Ru2), Mlg, and much, much more
• These genes are insufficient to protect barley against the full range of Bgh pathothypes
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Previous screening efforts• NSGC wild barley screening:
o 22 isolates screened against 1,383 accessions • Double haploid wild barley screening:
o 6 isolates against two wild/Harrington barley populations found 9 QTL• Evaluation of 116 Jordan and Israel based lines:
o 1 isolate against 116 accessions• Evaluation of a wild barley RIL population
o 1 isolate tested, found 2 QTL
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Wild Barley Diversity Collection
• Assembled by Brian Steffenson here in Minnesotao 316 accessions collected from ICARDA in the Middle Easto Expanded from a previous, smaller seto Designed to cover the breadth of wild barley growing areao Covers a diverse set of geographic areas
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Hypotheses• There are novel powdery mildew resistance genes
in the Wild Barley Diversity Collection (WBDC) • We can identify loci that confer resistance to
powdery mildew• There are resistant accessions within the WBDC
that would be useful for resistance breeding
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Objectives for the current work
• Our objectives in this research project• Characterize 316 lines of wild barley in response to 40
different isolates of powdery mildew • Identify loci that are associated with powdery mildew
resistance in wild barley
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Resources• 40 isolates of powdery mildew, diverse virulence
ratings• 316 Accessions of Wild barley
o Geographic locationso Environmental data
• Genetic datao DaRT markerso DaRT-Seq markerso SNP markers
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Experimental set up40 Seeds from each of the WBDC accessions were each planted in individual pots
3 leaf sections are cut for each isolate tested and placed in a petri dish containing benzomidazole to prevent senescence.
2 weeks
Spores were blown from a super susceptible infected leaf onto the petri dishes 10-11 days later, leaves
were evaluated.
10-11 days
12,064
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Gene Postulation
Distribution of Aggregate scores
Pathotyperegression
Geographical correlation
Environmental correlation
Pathotype distribution
Uniqueness of response within the collection
Phenotype data
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Major conclusions• Phenotype
o Environmental correlations are not strong enough to use predictiono There is a geographic hotspot of resistanceo The collection’s response to powdery mildew indicates diversity
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Evaluation Scale
Infection type
Mycelium growth Sporulation Development of
Chlorosis/Necrosis General reaction
0 None None - Resistant0-1 None None + Resistant1 Weak None + Resistant
1-2 Weak Weak + Resistant2 Moderate Weak + Intermediate
2-3 Moderate Moderate + Intermediate3 Strong Moderate + Susceptible
3-4 Strong Strong + Susceptible4 Strong Strong - Susceptible
1Table taken from Torp et al (1978)
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Near Isogenic Lines• To evaluate isolates, a series of lines were
developed that contain a single resistance gene• These ‘Near Isogenic Lines’ allow for precise
designation of isolates into pathotypes• The NIL lines also allow for the assessment of
virulence
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Overview of the 40 isolates of powdery mildew
Each isolate is defined by it’s ability to overcome a combination of 12 resistance genes
We have a range of isolates, ranging from very avirulenttovery virulent
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The WBDC is a rich source of powdery mildew resistance
-52 accessions show resistance to at least 20 isolates
-67 show no resistance to any isolate
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Aggregate scores shows normal distribution
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1044
0 0.5 1 1.5 2 2.5 3 3.5 4
7377
0 0.5 1 1.5 2 2.5 3 3.5 4
0 0.5 1 1.5 2 2.5 3 3.5 4
H-148
Isolate distributions show distinctive patterns
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Environment is weakly correlated with resistance
Isolate Precipitation per year
Yearly Max Temperature
Yearly Min Temperature Aridity
0004 0.09 **-0.21 *-0.16 0.10020 0.05 *-0.17 *-0.15 0.080023 0.06 *-0.16 *-0.13 0.080061 0 -0.1 -0.07 0.010235 0.03 *-0.18 *-0.14 0.040323 0 **-0.19 *-0.18 0.030331 0.06 **-0.22 *-0.16 0.090574 -0.02 *-0.18 *-0.17 0.021002 0.06 *-0.17 *-0.14 0.081044 -0.01 **-0.32 **-0.28 0.051377 -0.01 **-0.19 **-0.19 0.021541 0.05 *-0.16 -0.09 0.062567 0.03 **-0.2 *-0.18 0.073707 0.02 **-0.2 *-0.17 0.063777 -0.01 -0.11 -0.09 0.024404 0.02 *-0.18 *-0.18 0.034523 -0.09 -0.1 -0.07 -0.074611 0.03 *-0.13 -0.08 0.044761 -0.04 *-0.16 *-0.17 -0.024776 -0.11 *-0.13 *-0.16 -0.095137 -0.04 *-0.17 *-0.16 05425 0.02 *-0.17 *-0.14 0.035511 -0.01 *-0.15 -0.12 -0.015735 -0.05 *-0.16 *-0.14 -0.025765 -0.07 *-0.14 *-0.12 -0.055715 -0.02 -0.05 -0.02 -0.036000 -0.08 0.01 -0.01 -0.16040 -0.06 *-0.14 *-0.15 -0.046045 0 **-0.24 **-0.2 0.046737 -0.05 **-0.24 **-0.22 07377 *-0.13 -0.11 -0.11 -0.17557 0.04 -0.11 -0.07 0.047737 -0.07 *-0.14 *-0.15 -0.047777 -0.1 *-0.18 **-0.21 -0.06H-148 *0.19 0.03 *0.13 *0.18J-462 0.09 -0.1 -0.02 *0.15Q-301 *0.16 -0.01 0.1 *0.15S-016 *0.12 -0.04 -0.02 *0.12Y-035 *0.19 -0.03 0.09 **0.19Y-069 *0.13 -0.07 -0.01 *0.13
Aggregate -0.01 *-0.18 *-0.15 0.01
Correlations for each of the univariate tests. * indicates .001<p<.05 and ** indicates p<.001
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Accessions carrying powdery mildew resistance can be found across the
wild barley range
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Resistance can be found in geographic clusters
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WBDC contains diverse resistance patterns
• 249/316 lines were resistant to at least one isolate
• 204/249 showed a unique resistance spectrum• A large number of resistant accessions are not
redundant• The resistant accessions are useful
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Gene postulation logic• We can also logically eliminate isoline genes used
to type the isolates.• If a line is susceptible to an isolate, the line
cannot contain any of the isoline genes that would confer resistance to that isolate.
• After all isolates are examined, a line may have genes that are not eliminated. These are postulated genes. A line may contain any of the postulated genes, but does not necessarily contain all of the postulated genes.
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0 2 4 6 8 10 12 140
20
40
60
80
100
120
140
160
180
Aggregate scorePredicted Aggregate score
Number of genes eliminated
Aggr
egat
e sc
ore
P-value 6.62657E-45
The number of postulated genes in a line is correlated with aggregate score overall
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Genetic Data
SNP (BOPA1+2)
DaRT Markers (600 set)
DaRT SEQ
PCA
STRUCTURE
LD and LD simulation
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Major conclusions• Phenotype
o Environmental correlations are not strong enough to use predictiono There is a geographic hotspot of resistanceo The collection’s response to powdery mildew indicates diversity
• Genotype o Postulated genetic populations are associated with broad geographic
areas
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Summary of marker datasets
• DaRT (521)• SNP (2597)• DaRT-Seq (8616 markers, 3721 locations)
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WBDC Population structure contains seven subpopulations
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G+P
GWAS
SNP DaRT
DaRT-Seq
Geographic population structure
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Major conclusions• Phenotype
o Environmental correlations are not strong enough to use predictiono There is a geographic hotspot of resistanceo The collection’s response to powdery mildew indicates diversity
• Genotype o Postulated genetic populations are associated with broad geographic
areas• Combined genotype and phenotype data
o Postulated genetic populations are phenotypically differento Significant markers for resistance
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Sub-populations show significant separation of resistance
Aver
age
mea
n is
olat
e re
actio
n
Isolate designation
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Sub-populations are geographically grouped
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Distribution of markers across the genome
Legend
Found Markers
Previously known
genes
Previously
identified QTL
19 total loci
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LD Analysis• We examined the linkage disequilibrium across
the chromosomes with TASSEL and Haploview using SNP markers. o We also examined LD decay on chromosome 1H, where Mla is foundo All chromosomes were examined, and show similar patterns of low LD,
as reported in previous publications about the WBDCo Analysis on SNP markers is shown here, but DaRT-Seq markers, despite
the higher number of markers shows just as little LD
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LD Analysis of SNP markers
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MLA region
Chromosome 1H
0.39 cM 143.20 cM
D’<1 D’=1LOD<2
LOD#2
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Major conclusions• Phenotype
o Environmental correlations are not strong enough to use predictiono There is a geographic hotspot of resistanceo The collection’s response to powdery mildew indicates diversity
• Genotype o Postulated genetic populations are associated with broad geographic
areas• Combined genotype and phenotype data
o Postulated genetic populations are phenotypically differento Significant markers for resistance
• Efficacy of Nested Association Mapping (NAM) familieso Resistant NAM families showed more than expected resistance
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Further testing of a multi-parent advanced backcross
population• To introgress resistance from the WBDC into elite
lines a multi-parent advanced backcross population was developed by Liana Nice
• Parents and families of the multi-parent advanced backcross population were evaluated for powdery mildew resistance
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Infection phenotypes of the multi-parent advanced backcross
population• (G:Resistant, Y:Intermediate, R:susceptible)
Race/Pathotype 0004
0020
0023
0061
0235
0323
0331
0574
1002
1044
1377
1541
2567
3707
3777
4404
4523
4611
4761
4776
5137
5425
5511
5735
5765
5715
6000
6040
6045
6737
7377
7557
7737
7777
H-14
8J-
462
Q-3
01S-
016
Y-03
5Y-
069
WBDC020WBDC350WBDC103WBDC302WBDC092WBDC255WBDC172 (Not Wild)WBDC336WBDC142WBDC227WBDC028WBDC016WBDC173WBDC355 (OUH602)WBDC150WBDC061WBDC035WBDC182WBDC234WBDC292WBDC032WBDC340WBDC042WBDC348WBDC082WBDC115
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Advanced Backcross – Nested Association Mapping (AB-NAM) population design
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Isolate testing in Minnesota• An isolate of powdery mildew was typed on a
differential panel similar to the ones used to type the original set
• This isolate shows similarity to a previously tested isolate
• Three families were chosen for testing that showed high resistance in testing for this isolate
• We tested it on the wild parents as well as Rasmusson, the cultivated parent
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Parental phenotypesRasmusson WBDC 016, 227, 035
BC2F4:F6 families
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Phenotype distribution of families in the greenhouse
016 Family
035 Family
227 Family
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Evaluation results• Three families contained lines (8) that were highly
resistant• Overall resistance in the 3 BC2F4:F6s was higher
than might be expected with a susceptible parent, despite the only 12.5% wild composition of the lines
• Many of the lines showed intermediate phenotypes
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Conclusions• None of the plants in any of the families showed
as much susceptibility as Rasmusson.• These plants will be useful as sources for powdery
mildew resistance, as they have already been ‘domesticated’.
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Summary• Rich source of resistance• There are geographic hotspots containing
aggregate resistance for the collection of isolates• 19 markers associated with resistance were
identified , of which six are novel.• Mla was not found• Multi-parent advanced backcross populations may
provide a bridge between wild barley and elite cultivars
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Acknowledgements• Advisor
o Gary Muehlbauer• Expert on Powdery Mildew and the WBDC
o Brian Steffensono Antonin Dreiseitl
• Readers for the Thesiso Gary, Brian, and Kevin Smith
• Assistance with STRUCTUREo María Muñoz-Amatriaín
• Assistance with the WBDCo Liana Nice