Genetic Dissection of Quantitative Disease Resistance in Maize

Southern Leaf BlightCochliobolus heterostrophus

Gray Leaf SpotCercospora zeae-maydis

Aspergillus Ear RotAspergillus flavus

Common RustPuccinia sorghi

Jesse A. Poland1, Chialin Chung1, Randall J. Wisser2, Peter J. Balint-Kurti2,Kristen L. Kump2, Jacqueline M. Benson1, Judith M. Kolkman1, Erik L. Stromberg3

The Maize Diversity Project 1,2,4,5,6,7,8, Rebecca J. Nelson1

1Cornell University, Ithaca, NY2North Carolina State University, Raleigh, NC3Virginia Polytechnic Institute and State University, Blacksburg, VA4Cold Spring Harbor Laboratory, NY5University of California-Irvine 6USDA-Agricultural Research Service7University of Missouri, Columbia, MO 8University of Wisconsin, Madison, WI

Anthracnose Stalk RotColletotrichumgraminicola

Northern Leaf BlightExheriohilum turcicum

Presented at Generation Challenge Program Workshop, Plant and Animal Genome, Jan. 12, 2009

Resistant, Susceptible, and shades of gray…

Ph

en

oty

pe

Interaction

R

S

Specific General

Non-hostGene-

for-gene

Ph

en

oty

pe

Interaction

R

S

Specific General

Non-hostGene-

for-gene

Nested Association

Mapping

Near-Isogenic Lines

Selection Mapping

Research Approach: Genetic Platforms

Maize Diversity

Panel

Ph

en

oty

pe

Interaction

R

S

Specific General

Non-hostGene-

for-gene

Nested Association

Mapping

Near-Isogenic Lines

Selection Mapping

Alle

lic D

ive

rsit

y

Phenotyping Power

Maize Diversity

Panel

Utility of Genetic Platforms

Nested Association

Mapping

Alle

lic D

ive

rsit

y

Phenotyping Power

Maize Diversity

Panel

Near-Isogenic Lines

Selection Mapping

uniform backgrounddetailed phenotyping

diversity high resolutionenrichment for

resistance allelesbreeding material

power!resolution

Utility of Genetic Platforms

1 2 3 4 5 6 7 8

12

34

56

78

Gray Leaf Spot

So

uth

ern

L

ea

f B

lig

ht

0

0.4

0.8

1.2

1.6

2

2.4

2.8

3.2

3.6

4

No

rth

ern

L

ea

f B

lig

ht

So

uth

ern

Le

af B

lig

ht

No

rth

ern

Le

af B

lig

ht

Gray Leaf Spot

disease GLS NLB

SLB ***0.61 ***0.62

GLS - ***0.45

Evidence for Multiple Disease Resistance in the Maize Diversity Panel

Genetic component of multiple disease

resistance

Phenotypic Evaluation: P.Balint-Kurti, J.Kolkman, R.Wisser Genotyping: The Maize Diversity Project, E.Bucker et.al. Analysis: R.Wisser

Phenotypic EvaluationsSLB: 4 environmentsNLB: 3 environmentsGLS: 3 environments

Analysis:Linear Mixed Model

Covariates:•Population Structure (K)•Relatedness (Q)•Relative maturity (days to anthesis)

Candidate genes for quantitative disease resistance

tasselseed2 (TS2)

Genotypes:~850 genome-wide SNPs

-0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0

SLB

GLS

NLB{

{

{

allele effect

a

b

c

maize EST of unknown function

Glutathione S-transferase

Analysis:Linear Mixed ModelCovariates: Population Structure (K), Relatedness (Q), and relative maturity (days to anthesis)

Sequence candidate gene from diversity panel (includes 26 NAM parents) and test for association

Analysis: R.Wisser

NAM

1 2 200. . . .

B73

F1s SSD25 DL

B97

CML103

CML228

CML247

CML277

CML322

CML333

CML52

CML69

Hp301

Il14H

Ki11

Ki3

Ky21

M162W

M37W

Mo18W

MS71

NC350

NC358

Oh43

Oh7B

P39

Tx303

Tzi8

NAM is a QTL mapping strategy that simultaneously exploits the advantages of linkage analysis and association mapping for high

resolution genome scans.

NESTED ASSOCIATION MAPPING (NAM)

Use sequence information from parents for assocation analysis

Parental sequence imputed to RILs

CORRELATION

1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

Multiple disease resistance in NAM

COVARIATES NLB GLS

NONE SLB 0.5 0.49

NLB 0.44

NLB GLS

w/Population SLB 0.19 0.18

NLB 0.12

NLB GLS

w/ flowering time

SLB 0.37 0.28

NLB 0.24

NLB GLS

w/ Population & flowering time

SLB 0.18 0.17

NLB 0.11

NLB GLS

SLB 0.62 0.61

NLB 0.45

Diversity Panel NAM

NAM Phenotypic Evaluation: SLB - Kristen Kump & P.Balint-Kurti; NLB – J.Poland; GLS – J. Benson; Data Analysis – J.Poland

Physical break-up of population structure(recombinant inbred lines)

mixed model correction for population

structure, flowering time

B97

CML103

CML228

CML247

CML277

CML322

CML333

CML52

CML69

Hp301

Il14H

Ki11

Ki3

Ky21

M162W

M37W

Mo17

Mo18W

MS71

NC350

NC358

Oh43

Oh7B

P39

Tx303

Tzi8

Position and relative effect of QRL identified in NAM

1 2 3 4 5 6 7 8 9 10

SLBNLB

GLS

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

-0.6 -0.4 -0.2 0 0.2 0.4

SLB

NLB

0

10

20

30

40

50

60

70

Physical map of

bin 8.05/06 (Mb)-L

og

P

GH (May 08; IP)

Aurora 08 (IP)

Aurora 08 (DLA)

ctg359-0

1

ctg359-0

2

ctg358-0

1

ctg360-0

2

ctg360-0

4

QTL region identified in F7 u

mc1

828

um

c1997

um

c2395

um

c2356

um

c1149

bnlg

240

um

c2361

um

c2199

um

c17

77

um

c1316

bnlg

1724

um

c1728

um

c1287

um

c2210

ctg358-

05ctg358-0

3

ctg358-0

7

ctg358-

08

ctg358-

09

ctg358-

13

ctg358-

14

ctg358-

16, 18

ctg358-2

0

ctg358-

37

ctg358-

44 ctg358-

32, 33

Characterizing and Fine-mapping a Quantitative Resistance Loci on Chr. 8

NIL development, evaluation, fine-mapping – C.Chung; NAM evaluation - J.Poland;

Near Isogenic Lines: DK888/S11 F7 lines from HIF (heterozygous inbred family)

Further detailed phenotyping

possible in NILs(race-testing)

Conidium

Appressorium

Infection hyphae

Successful infection

Initial infection

NIL development, evaluation – C. Chung

Infection efficiency of E. turcicum (Northern Leaf Blight)

+ QTL @ 1.02

Near Isogenic Lines from Tx303 in B73 background

Characterizing Resistance Effect in Near Isogenic Lines

Number of hyphe growing into the xylem(E. turcicum)

+ QTL @ 1.06Reduce infection efficiency

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

-QTL @ 1.02 +QTL @ 1.02

nu

mb

er o

f h

yph

e in

xyl

em

per

infe

ctio

n s

ite

+ QTL @ 1.02Reduce fungal growth in xylem

(no effect on infection)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

- QTL +QTL @1.02 +QTL @ 1.06

Infe

ctio

n e

ffic

ien

cy (

2 d

ays

po

st

ino

c.)

- QTL @ 1.02

Nested Association

Mapping

Maize Diversity

Panel

Near-Isogenic Lines

Selection Mapping

Moving forward

race-testingmicroscopic phenotypesfine mapping map based cloning

sequencing candidate genestest for association

Evaluation of selected alleles in RIL populationFollow-up in BC lines

3rd season for NLB2nd & 3rd seasons for GLSTest SNP associations

Rebecca Nelson LabThe Generation Challenge ProgramThe McKnight FoundationCornell University

Peter Balint-Kurti LabThe Generation Challenge ProgramUSDA-ARSNorth Carolina Corn Growers

Erik L. Stromberg Lab

The Maize Diversity ProjectNSF (DBI-0321467: Molecular and Functional Diversity in the Maize Genome)

USDA-ARS

Thank you

0

1

2

3

RILs NILs

Ru

st S

eve

rity

Common Rust

B73

CML52

0

20

40

60

80

100

120

140

160

RILs NILs

Dis

eas

e S

eve

rity

Northern Leaf Blight

B73

CML52

0

50

100

150

200

250

RILs NILs

Stal

k R

ot

Seve

rity

Anthracnose Stalk Rot

B73

CML52

Allele effects for QRL in bin 6.05: A region conferring resistance to multiple pathogens

No effect on SLB severity

Dissection of a QRL for multiple diseases

NIL development, evaluation – C.Chung; RIL evaluation - J.Poland;

RILs: CML52/B73 Population (S5 inbred lines)

NILs: CML52/B73 S6 lines derived from HIF (heterozygous inbred family)

Trypan blue staining

• Targeted stages: penetration, initial intracellular hyphal growth

• Microscopic parameter: infection efficiency

Conidium

Appressorium

Infection hyphae

Successful infection

Initial infection

0

0.2

0.4

0.6

0.8

1

Infe

cti

on

eff

icie

nc

y

(su

cce

ssfu

l in

fectio

n/

ge

rmin

ate

d c

on

idia

)

Maize genotype

2 dpi

4 dpi

7 dpi

Infection efficiency of E. turcicum

**

Recurrent Selection and Selection Mapping

Recurrent Selection: Improving the mean performance of a population

through iterative cycles of selection by

1) Identifying the superior individuals/families

2) Intercrossing superior individuals to form the next generation

Phenotype

Cycle 0Cycle 0 Cycle 1Cycle 1 Cycle 2Cycle 2 Cycle 3Cycle 3 Cycle 4

Nu

mb

er

of

ind

ivid

ual

s

S R

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

0 1 2 3 4

Dis

ea

se

se

ve

rity

(A

UD

PC

)

mean AUDPC decreased by an

average of 17% cycle-1

Significant improvement for NLB resistance

Recurrent Selection Population:

CIMMYT; Ceballos et al., 1991 Crop Sci 31: 964-971

Cycle of selection

MDR in recurrent selection population

0.5

0.55

0.6

0.65

0.7

0.75

0.8

Cycle 0 Cycle 4 B73 Mo17

AU

DP

C-1

SLB

0.1

0.11

0.12

0.13

0.14

0.15

0.16

0.17

0.18

Cycle 0 Cycle 4 B73 Mo17

AU

DP

C-1

GLS

- Significant improvement for unselected diseases (SLB and GLS)

- Correlated response = genetic correlation = MDR

*evaluated at NCSU 2007

0

50

100

150

200

RILs NILs

Dis

eas

e S

eve

rity

NLB

B73

CML52

0

50

100

150

200

250

RILs NILs

Stal

k R

ot

Seve

rity

ASR B73

Northern Leaf Blight

P < 0.0001***

qEt6.05B73

qEt6.05CML52

0

20

40

60

80

100

120

140

160

RILs NILs

Dis

eas

e S

eve

rity

NLB

B73

CML52

Anthracnose Stalk Rot

qEt6.05B73 qEt6.05CML52

0

50

100

150

200

250

RILs NILs

Stal

k R

ot

Seve

rity

ASR B73

CML52

P = 0.0001***

qEt6.05B73

qEt6.05CML52

Stewart’s wilt

0

20

40

60

80

B73 CML52

Pri

ma

ry

dis

ea

se

d l

eaf

are

a (

%)

Allele(s) at bin 6.05

Resistance to Stewart's wilt in CML52 NILs differing for bin 6.05

P < 0.0001***

0

1

2

3

RILs NILs

Ru

st S

eve

rity

Rust

B73

CML52

Common Rust

qET6.05 – Multiple disease resistance effect

-NLB-ASR-Stewart’s wilt-Common rust (?)

Nelson Lab Activities

Nested Association

Mapping

Alle

lic D

ive

rsit

y

Phenotyping Power

Maize Diversity

Panel

Near-Isogenic Lines

Selection Mapping

Near-Isogenic Lines

Survey allele series

Fine-mapping QTL

Test effect of selection on

candidate genes

Evaluate for GLS resistance

sqrtAUDPC

sqrtAUDPC