Characterization of qEt8.06 using near-isogenic line (NIL) pairsnear-isogenic line (NIL) pairsTo be able to analyze qEt8.06 in detail, NIL pairs contrasting for the 8.06 region were developed using heterogeneous inbred family (HIF) strategy (2). In HIF analysis, intermediate materials from breeding programs are used to develop NIL pairs that are isogenic at the majority of loci, but differ at a specific QTL. In order to capture alleles contributing broad-spectrum resistance in NIL pairs, we chose to start from F6 families derived from DK888 x S11. DK888 is a tropical genotype with superior resistance to multiple diseases.

Conclusions

1. Consistent detection of qEt8.06 in diverse mapping populations indicates that it accounts for a large proportion of NLB resistance in maize germplasm.

2. High-resolution nested association mapping and break-point analysis using NIL pairs has localized qEt8.06 to an overlapping region of < 4 Mb (142.9 – 146.5 Mb on physical map). The tightly linked marker umc2210 can be applied for marker-assisted selection in maize breeding.

3. Race-specificity, map position and gene action of resistance suggested that qEt8.06 can be Ht2, Htn1 or a novel resistance locus. Concurrent work of fine-mapping Htn1 locus using F2 populations derived from B68Htn1 x B68 will

resolve this question.

4. The enrichment of disease QTL in the 8.06 region and its genetic complexity implies the possibility that instead of a single major gene, qEt8.06 may consist of a cluster of resistance genes. Different levels and phenotypes of resistance can be due to various combinations of alleles for multiple genes, and their expression modified by genetic backgrounds and environmental conditions. The hypothesis will be further tested through map-based positional cloning.

AcknowledgementsStephen Kresovich Institute for Genomic Diversity, Cornell UniversityMargaret Smith Dept. of Plant Breeding and Genetics, Cornell UniversityFunding from Ministry of Education, Taiwan; the Generation Challenge Program; and The McKnight Founda

tion.

Genetic Dissection of Loci Conditioning Disease Resistance in Maize Bin 8.06Chia-Lin Chung 1*; Jesse Poland 1*; Randall Wisser 2; Judith Kolkman 1; The Maize Diversity Project 1,2,3,4,5,6,7; Rebecca Nelson 1

1 Cornell University, Ithaca, NY; 2 USDA-Agricultural Research Service; 3 Cold Spring Harbor Laboratory, NY; 4 University of California-Irvine; 5 North Carolina State University, Raleigh, NC; 6 University of Missouri, Columbia, MO; 7 University of Wisconsin, Madison, WI; * Joint first authors

Student's t-testDK888 S11 (P-value)

Northern Incubation period days after inoculation 17.4 ± 1.7 10.0 ± 0.3 < 0.0001 ***

leaf blight (NLB) Primary diseased leaf area % 9.0 ± 4.1 65.0 ± 6.2 < 0.0001 ***

Southern Lesion length mm 1.2 ± 0.05 1.2 ± 0.06 0.719

leaf blight (SLB) Primary diseased leaf area % 29.5 ± 1.0 30.0 ± 1.5 0.585

Anthracnose Incubation period days after inoculation 7.7 ± 0.2 7.8 ± 0.4 0.698

leaf blight (ALB) Latent period days after inoculation 10.4 ± 0.7 10.4 ± 0.7 1.000

Primary diseased leaf area % 39.1 ± 12.1 38.6 ± 14.7 0.963

Anthracnose stalk rot (ASR)

Discolored internode tissueTotal % of internode 1 to 8

121.7 ± 12.9 120.0 ± 23.0 0.901

Rust First postule appearance days after inoculation 7.5 ± 0 7.5 ± 0 1.000

Number of pustules # pustules 96.0 ± 64.0 149.5 ± 37.7 0.706

Primary diseased leaf area % 14.4 ± 3.1 15.0 ± 2.7 0.790

Smut Volume of gall cm3 273.8 ± 157.4 167.5 ± 99.1 0.258

Weight of gall grams 127.4 ± 68.5 78.9 ± 46.1 0.247

Stewart's wilt Primary diseased leaf area % 72.5 72.5 –

Disease Parameter UnitAllele(s) at qEt8.06

The sixth segment of maize chromosome 8 (bin 8.06) is known to be associated with resistance to NLB and several other diseases (4). Two qualitative resistance loci (Ht2 and Htn1) and several QTLs for NLB resistance have been localized to this region. In response to a recurrent selection program for NLB resistance, significant changes in allele frequencies provided evidence of selection acting at several loci in bin 8.06. One of the putatively selected allele has been validated in F2 families derived from the selection mapping population (5). To dissect the complex region, and to understand the relationship between qualitative and quantitative disease resistance in maize, a set of genetic stocks capturing a range of resistance alleles at bin 8.06 has been used for QTL mapping and characterization.

Maize disease QTL consensus map

(Wisser et al., 2006)Chromosome

8

Erwinia wilt

Viral diseases

Aspergillus flavus

Ear rot and stalk rot

Common smut

Downy mildew

Common rust

Southern rust

Gray leaf spot

Southern leaf blight

Northern leaf blight

Disease QTLFlowering time

QTL

Ht2

Htn1

Fig. 1. Chromosomal regions associated with multiple disease resistance

qEt8.06 is the largest-effect NLB-QTL identified in the nested association mapping (NAM) populationnested association mapping (NAM) population

The nested association mapping (NAM) population is a large-scale mapping resource in maize, consisting of 5,000 recombinant inbred lines (RILs) developed from 25 diverse inbred lines crossed with a common inbred line B73. This resource is designed to combine the advantages of linkage mapping and association mapping, for high resolution QTL mapping with genome-wide coverage (7). Evaluating a subset of the NAM population for NLB for a first year led to mapping of 6 QTLs conditioning increased incubation period (IP) and 15 QTLs conditioning decreased disease severity (AUDPC) (Fig. 2). Of the 21 QTL detected, qEt8.06 (qEt for quantitative resistance to Exserohilum turcicum) was identified as the largest-effect QTL across all populations, and one of the two QTLs significantly contributing to both resistance parameters, IP and AUDPC (relative allele effects for decreasing AUDPC shown in Fig. 3). Most of the QTLs identified in this study co-localized with previously reported disease resistance QTLs for NLB, but novel QTLs were also detected.

Fig. 2. Position and relative effect of QTL for resistance to Northern Leaf Blight referenced against previously reported QTL.

IP

AUDPC

0

5

10

15

20

DK888 S11

DK888 S11

EtNY001 race 0 race 1 race 23N

DK888 S11

DK888 S11

10

20

15

5

0

Incu

ba

tion

pe

rio

d

Resistance spectrum of qEt8.06Although DK888 harbors multiple disease resistance, the DK888 allele at 8.06 (qEt8.06DK888 ) is effective only for NLB resistance. Resistance spectra and effectiveness of diverse alleles at this locus will be characterized in NIL pairs being developed from the NAM population.

Race specificity of qEt8.06qEt8.06DK888 conditions resistance to race 0, race 1, but not race23N of E. turcicum. Race specificity suggests that it may encompass the major genes Ht2 and/orHtn1.

Gene action at qEt8.06

qEt8.06 identified in DK888 HIF showed partially dominant resistance, differing from the completely dominance of Ht2 documented in previous reports (6).

11

12

13

14

15

16

17

18

DK888 Het S11

Allele(s) at umc2210

Inc

ub

ati

on

pe

rio

d(d

ay

s a

fte

r in

oc

.)

Genotype - Genotype IP difference P-value

DK888/DK888 S11/S11 3.8 days < 0.0001 ***

DK888/DK888 Heterozygote 3.2 days < 0.0001 ***

Heterozygote S11/S11 0.6 days 0.0119 *

References1. Carson and van Dyke (1994) Plant Dis. 78: 519-522.2. Tuinstra et al. (1997) Theor. Appl. Genet. 95: 1005-

1011.3. Simcox and Bennetzen (1993) Phytopathology 83: 1326-1330.4. Wisser et al. (2006) Phytopathology 96: 120-129.

5. Wisser et al. (2008) Genetics (in press).6. Yin et al. (2003) Chinese Science Bulletin 48(2): 165-

169.7. Yu et al. (2008) Genetics 178: 539-551.

Genetic dissection of qEt8.06The QTL interval for qEt8.06DK888 in F7 was ~20 Mb. Trait-marker association with ~2,800 individuals (F9 or F10) segregating for bin 8.06 has delimited the resistance locus to a region of < 4 Mb tightly linked to the marker umc2210. High marker density in the NAM population also allowed mapping of qEt8.06 to an overlapping region. Since all available SSR markers have been exhausted in the region, we have started to develop single nucleotide polymorphism markers (SNPs) surrounding umc2210. We are working to further saturate the resistance locus with SNPs to identify further recombinants for positional cloning.

Previously reported NLB-QTL

Chr. 1 Chr. 2 Chr. 3 Chr. 4 Chr. 5 Chr. 6 Chr. 7 Chr. 8 Chr. 9 Chr. 10

-0.9

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

Ki1

1

CM

L5

2

CM

L2

47

CM

L3

33

NC

35

8

MS

71

Oh

43

CM

L2

28

CM

L3

22

B9

7

P3

9

M3

7W

M1

62

W

CM

L6

9

Tx

30

3

CM

L2

77

Ky

21

Mo

18

W

Tzi

8

NC

35

0

Il14

H

B7

3

CM

L1

03

Ki3

Hp

30

1

Oh

7B

Maize genotype

Re

lativ

e a

llele

effe

ct(L

Sm

ean

of

AU

DP

C s

tan

dar

diz

ed t

o B

73)

Fig. 3. Relative allele effects for qEt8.06

from 25 NAM parents

Negative values:lower disease severity relative to the common parental line B73.

qEt8.06 explains the most genetic variance of NLB resistance in NAM.

0

10

20

30

40

50

60

70

80

90

- L

og

P

GH (Dec 07; n = 576)

GH (May 08; n = 1191)

Aurora NY (Jul 08; n = 1043)

QTL region identified in F7

um

c18

28

um

c19

97

um

c23

95

um

c23

56

um

c11

49

bnlg

24 0

um

c23

61

um

c21

99

um

c17

77

um

c13

16

bnlg

17

24

um

c17

28

um

c12

87

um

c22

10

Evidence for NLB-QTLs in maize bin 8.05-8.06

umc1

828

umc1

997

umc2

395

umc2

356

umc1

149

bnlg

240

umc2

361

umc2

199

umc1

777

umc1

316

bnlg

1724

umc1

728

umc1

287

umc2

210

130 135 140 145 150 155

8.05 8.06

115 120 125um

c237

8

umc1

712

umc1

846

110

umc2

367

bnl2

.369

qEt8.06 in NAM

qEt8.06DK888

Ht2 (6) Htn1 (3)NLB-QTL

qEt8.06 in recurrent selection population (5)

** * * ** * **

* Putatively selected loci in recurrent selection population (5).

Htn1• Susceptible lesion type• Delay of lesion

development• Partially dominant,

genetic background dependent

Ht2• Chlorotic lesion type• Fewer lesions, prolonged

incubation period• Dominant, resistance breaks

down at low light intensities

umc1

121

Ht2 (3)

Physical map of bin 8.05-8.06 in maize

BackgroundNorthern Leaf Blight (NLB), caused by Exserohilum turcicum, is one of the most important diseases affecting maize production worldwide. Several qualitative loci (Ht genes) and a large number of quantitative trait loci (QTL) for NLB resistance have been identified and widely used in breeding programs for disease control. Qualitative race-specific resistance of Ht genes is characterized as inducing hypersensitive response and/or delaying lesion development, in a monogenic manner. However, the expression of Ht genes can be quantitative in certain environments and genetic backgrounds (1). Co-localization of major R genes and disease QTLs in some chromosomal regions of the maize genome (4) also suggests that the distinction between qualitative and quantitative resistance is ambiguous. Isolating and characterizing gene(s) underlying resistance loci is needed for resolving the question.