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Leveraging powdery mildew resistance in wild populations

Nick AmesSept 26th, 2014

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



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

Powdery mildew life cycle and mechanisms of defense

http://www.hgca.com/images/upload/Powdery_Mildew.gif

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: ?%

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

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

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

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

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

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

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

Gene Postulation

Distribution of Aggregate scores

Pathotyperegression

Geographical correlation

Environmental correlation

Pathotype distribution

Uniqueness of response within the collection

Phenotype data

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

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)

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

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

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

Aggregate scores shows normal distribution

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

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

Accessions carrying powdery mildew resistance can be found across the

wild barley range

Resistance can be found in geographic clusters

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

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.

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

Genetic Data

SNP (BOPA1+2)

DaRT Markers (600 set)

DaRT SEQ

PCA

STRUCTURE

LD and LD simulation



• Genotype o Postulated genetic populations are associated with broad geographic

areas

Summary of marker datasets

• DaRT (521)• SNP (2597)• DaRT-Seq (8616 markers, 3721 locations)

WBDC Population structure contains seven subpopulations

G+P

GWAS

SNP DaRT

DaRT-Seq

Geographic population structure




areas• Combined genotype and phenotype data

o Postulated genetic populations are phenotypically differento Significant markers for resistance

Sub-populations show significant separation of resistance

Aver

age

mea

n is

olat

e re

actio

n

Isolate designation

Sub-populations are geographically grouped

Distribution of markers across the genome

Legend

Found Markers

Previously known

genes

Previously

identified QTL

19 total loci

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

LD Analysis of SNP markers

MLA region

Chromosome 1H

0.39 cM 143.20 cM

D’<1 D’=1LOD<2

LOD#2




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

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

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

Advanced Backcross – Nested Association Mapping (AB-NAM) population design

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

Parental phenotypesRasmusson WBDC 016, 227, 035

BC2F4:F6 families

Phenotype distribution of families in the greenhouse

016 Family

035 Family

227 Family

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

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’.

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

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

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