Development of Western Corn Rootworm Resistant GEM Germplasm and its Role in Host Plant Resistance Research

Martin Bohn

Crop SciencesUniversity of Illinois

Outline

Breeding for WCR Resistance

Quintessence of 70 years of breeding for

WCR resistance

Tolerance vs. antibiosis

Identifying antibiosis

Trap crop vs. manual infestation

Germplasm

Alternative Approaches – Closing the

information gap

Den

sity

p p

ps

Den

sity

Gen 0

Gen 1

ppS

Sh2

gih

psR

Background: Selection

Genotypic variation

Evaluation assay / trait

“Breeder’s Equation”

0

5

10

15

20

25

0 5 10

15 20 25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

10

0

IHO

ILO

RHO

RLO

SHO

Mea

n %

Oil

of

ears

an

aly

zed

Generation

Illinois long-term high-oil/low-oil selection experiment. Plot of mean oil concentration against generation for Illinois High Oil (IHO), Reverse High Oil (RHO), Switchback High Oil (SHO), Illinois Low Oil (ILO), and Reverse Low Oil (RLO).

Background: Illinois Long-term Selection

The Insect - Adults

The Insect – Eggs

Source – J. Spencer

The Insect - Larvae

Source – J. Spencer

WCR Resistance - The Challenge

Labor intensive!

Resistance traits have low heritability.

Resistance to WCR larvae and adult feeding not correlated.

WCR Distribution

Gray et al. 2009. Annual Rev. Entomology

1930/40s Germplasm survey (Bigger, 1941)

sig. for WCR (adult, larvae) resistance resistances to WCR adult and larvae were not correlated

Resistant lines were developed large densely branched root systems quick root regeneration

2g

SD10, SD20, B69, Mo22, Oh05, B14, N38A, A251, W202

Breeding for WCR Resistance

“Dekalb”-ProgramGermplasm Screening

Start: < 1964

N: 3,800

Origin % %SelCornbelt

inbreds 41 5OPV 30 7Synth. 10 10

Europeaninbreds 1 0

Exoticinbreds 4 0OPV 14 5

Traits: Root lodgingRow evaluationAnchorage ratings

Infes.: Trap crop

S(tot): 5% (190)

Germplasm Development

RS among S1 families

Traits: RDR

Infes.: Trap crop

S: 2.2-6.9% (1964-1968)0.1-3.6% (1969-1977)

RW15, RW16, RW17

“NGIRL–USD”-ProgramGermplasm Selection

1964 – Early 1980s

N: 57

Traits: Root lodgingVertical pull resist.

Infest: Trap crop

Large, dense root systemsWCR resistance (tolerance)

Cornbelt: Early – midseasonSouth D.: Root rot resistantExotic: West Indies, Mexico

SDCRW1SYN 5

SDCRW1C0

Germplasm Development

C1: RS among S1 families C2: RS among S2 families

Traits: Root lodgingVertical pull resist.

Infest: Trap crop

C3: RS among S2 families

Traits: RDR

Infest: 600 eggs / 30cm

S: 10% (20 S2s)

C4: NGSDCRW1(S2)C4

NGSDCRW1(S2)C4 registered in 1985 as source of tolerance to WCR.

NGSDCRW1(S2)C4-15-2S2

Germplasm Screening

Traits: ToleranceRoot traits

Infes.: Trap crop (?)

“Iowa”-Program

W153R, A239, A251, A265, A297, A417, A556, A632, Msl97, Oh43, R168, SDIO

Iowa Early Rootworm Synthetic (BSER)

Iowa Late Rootworm Synthetic (BSLR)

B14A, B53, B59, B64, B67, B69, B73, N6, N28, R101, HD2286, 38-11

Germplasm Development

RS among S1 families

Traits: RDR Root lodgingRoot size Root re-growth

Infes.: Trap crop (?)

Populations with improved levels of tolerance – BS19(S)C2, BS20(S)C2

Germplasm Screening

Start: 1992

N: 3,500

Traits: RDR

Infes.: 600 eggs / 30cm

S(tot): 0.2 (7 accessions)

Corn and corn relatives

“USDA/ARS-Missouri”-Program

Traits: RDRInfest: 600 eggs / 30cm

Germplasm Development

N: 56 crossesTraits: RDR Infest: 600 eggs / 30cmS: 18% (10 crosses)

TL92A-PAR 1779 60-4 (C4)TL92A-PAR 1774 28-1 (C3)PI 340839 (Popcorn)NGSDCRW1(S2)C4-15-2S2

Diallel Study

CRW3Syn0 -> CRW3-C8

Genotypes from C3 and C5 were used in QTL studies.

Quintessence

12,000 corn accessions and relatives were screened for WCR resistance.

Trap crop – artificial infestationmultiple traits to assess WCR damage

< 1% of the screened germplasm was used in germplasm development.

large, dense root systemgood root re-growth

Tolerance (not antibiosis)

No maize cultivars with high levels of WCR host plant resistance under moderate to high insect pressure were yet released.

Tolerance vs. Antibiosis

Germplasm screening phase

Root lodgingVertical pull resistanceRow performance

Associated with root size

associated with tolerance

not associated with antibiosis

Consequences:

Genotypes with interesting antibiotic properties were not identified.

Most breeding programs improved tolerance but not antibiosis.

Tolerance vs. Antibiosis: Example 1

Rogers et al. (1977) estimated variance components in BSER and BSLR.

Root lodging * *

Root size * *

Root re-growth * *

Root damage ratings ns ns

2ˆ ge2ˆ g

Model calculations showed that the populations will respond to selection for root lodging and WCR tolerance but not for RDR.

Parental selection is crucial.

Identifying antibiosis

Associations between root size measures under insecticide protection and WCR infestation are highly correlated.

Tolerance can be improved under infestation and under protection.

Tolerance can be improved if infestation levels are variable.

Genotypes displaying antibiosis can reliably only identified if high and evenly distributed WCR larvae pressure is applied.

Example: “Dekalb”-Program

Trap crop vs. art. infestation

Significant correlation between infestation level and RDR (Branson et al. 1981).

Infestation Rate

0600 1200 1800

1

2

3

Roo

t da

mag

e ra

ting

R2 = 0.83

Trap crop vs. art. Infestation: Results

Plant materialsInbreds: 15 entriesPopulations: 20 entries

NGSDCRW1(S2)C4-15-2S2Monsanto BtMonsanto Non-Bt

Field experimentsLocations: DeKalb, Monmouth, UrbanaTreatments:

Trap crop: DeKalb, Monmouth, UrbanaArtif. Infes.: Urbana (600 eggs/plant)Chemi. prot.: DeKalb, Monmouth, Urbana

Experimental designα-lattice designReplications: 3#rows/plot: 1 (I), 4 (P)

Germplasm Screening

Node-Injury Scale (0.00 – 3.00)

1.50

No. of full nodes eaten % of a node eaten

3.000.00

(Oleson et al. 2005. J Econ Entomol 98:1-8)

GENOTYPES TRAP INFES ---------------------------- 1 B14A 2.55 1.56 2 B64 2.12 0.73 3 B67 1.45 0.68 4 B69 1.85 0.72 5 B73 2.17 1.35 6 Lo1016 1.68 0.48 7 Lo964 1.67 0.64 8 Mo12 1.47 0.83 9 Mo17 2.20 1.16 10 Mo47 2.03 1.62 11 ND251 2.70 1.15 12 NY992 2.72 1.69 13 NGSDCRW 2.35 0.68 14 NGSDCRW 1.96 0.87 15 MON_Bt 0.49 0.15 16 MON_I 2.53 1.17 ---------------------------- Mean 2.00 0.97 LSD(T) 0.19 ----------------------------

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2 2.5 3

RDR – (Trap)

RD

R –

(In

fes)

r = 0.66

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12 14 16

r = 0.64

Ran

k –

(In

fes)

Rank – (Trap) - +

Trap crop vs. art. Infestation: Results

To

lerant

Germplasm ------------------------------------ D M U Mean ------------------------------------ B14A 2.04 1.95 2.55 2.18 B64 2.25 1.60 2.12 1.99 B67 1.69 1.72 1.45 1.62 B69 2.15 1.70 1.85 1.90 B73 2.57 1.39 2.17 2.04 Lo1016 1.91 1.16 1.68 1.58 Lo964 1.57 1.64 1.67 1.63 Mo12 1.47 0.74 1.47 1.23 Mo17 2.05 0.96 2.20 1.74 Mo47 2.78 2.07 2.03 2.29 ND251 2.90 2.70 2.70 2.77 NY992 2.87 2.36 2.72 2.65 NGSDCRW 2.27 2.15 2.35 2.26 NGSDCRW 2.77 2.18 1.96 2.30 MON_Bt 0.07 0.30 0.49 0.29 MON_Iso 2.60 2.92 2.53 2.68 ------------------------------------ Mean 2.12 1.72 2.00 1.95 LSD(5%) 0.50 Rep. 0.86 ------------------------------------

Economic Threshold:

RDR = 0.3

Tolerant

Materials and Methods

“Population” 15 Entries“Inbred” 55 Entries (20, 35)

Location: Urbana, 2003 (35), 2004 (70)

Design: α – lattice, 4 replications

Plot size: Population – 4 row plotsInbreds - 2 row plots

WCR eval.: Trap crop

Results: Populations

DKXL212:N11a01UR10001:N1708bUR10001:N1702CH05015:N1204DKB844:S1612NGSDCRW1FS8A(T):N1804FS8A(S):S0907CASH:N1410AR17056:N2025AR16026:S1719AR13035:S11b04AR17056:S1216UR13085:N0204AR16026:N1210

MAX

MEAN

1.74

1.40

MIN 0.881.00

1.80

0.60

LSD(5%) = 0.99

3.00

2.00

2.552.28

2.81

LSD(5%) = 0.34

2003 2004

Root Damage Ratings(0.00 – 3.00)

Results: Inbreds

1.08

LSD(5%) = 0.74

CUBA117:S1520-153AR17056:N2025-728B64CUBA117:S1520-182AR17056:N2025-#5AR17056:N2025-522CUBA117:S1520-52CUBA117:S1520-41CUBA117:S1520-156AR17056:N2025-546AR17056:N2025-508AR17056:N2025-#2AR17056:N2025-532B37Mo17AR17056:N2025-#4NGSDCRW1(S2)C4-15AR17056:N2025-#1AR17056:N2025-#3B73

0.50

1.50

2.00

0.56

1.73MAX

MEAN

MIN

3.00

2.00

2.47

1.83

2.80

LSD(5%) = 0.52

Root Damage Ratings(0.00 – 3.00)

20032004

Conclusions

Germplasm was successfully improved for tolerance to WCR

but not for antibiosis.

Germplasm can be reliably screened for antibiosis against

WCR larvae feeding using trap crop enhanced natural

infestation.

Germplasm screening must continue! - Concentration on exotic

germplasm

Genotypic variation is present for WCR resistance /

susceptibility.

-6

-4

-2

0

2

4

6

-6 -4 -2 0 2 4 6

Cluster 1Cluster 2Cluster 3

Can 1

Can

2

Germplasm Screening

Germplasm Screening

USDA-Germplasm Enhancement in Maize (GEM) – base populations

Selfed progeny of one plant(per se and testcross evaluation).

Individual plants.

Selfed seed is used to intercross selected plants.

• Parental control

• 4 Seasons/cycle

SU =

EU =

RU =

S1

– p

er s

e S

1 –

test

cro

ss

Selected

Not selected

Illinois WCR Synthetic

Germplasm Development

Germplasm Evaluation – QTL MappingMean 1.73SD 0.50REP 0.18

0

0.1

0.2

0.3

0.4

0.5

Pro

po

rtio

n

0

0.1

0.2

0.3

0.4

0.5

Pro

po

rtio

n

Mis

so

uri

2S

ou

th D

ak

ota

Mean 1.35SD 0.34REP 0.38

0 0.5 1 1.5 2 2.5 3

Root Damage Rating [0-3 Iowa Rating Scale]

0

0.1

0.2

0.3

0.4

0.5

Pro

po

rtio

n

0

0.1

0.2

0.3

0.4

0.5

Pro

po

rtio

n

Illi

no

isM

iss

ou

ri 1 Mean 0.99

SD 0.43REP 0

Mean 2.70SD 0.25REP 0.42

Number of F2:3 families = 230 Number of locations = 4 (Missouri, South Dakota, Illinois)Incomplete block design, number of Reps/Loc = 3Manual infestation, trap crop

CRW3(C6)×LH51

Germplasm Evaluation – QTL Mapping

Root Size Rating Root Re-growth Rating

Mean 4.15SD 0.77REP 0.25

Mean 3.95SD 0.59REP 0.15

Mean 4.19SD 0.46REP -/-

Mean 3.52SD 0.47REP 0.21

Mean 3.74SD 0.77REP 0.26

Mean 5.75SD 0.34REP 0.09

Mean 5.38SD 0.48REP -/-

Mean 4.60SD 0.49REP 0.42

Fre

quen

cy [

%]

1 2 3 4 5 61 2 3 4 5 6

-15

-10

-5

0

5

10

15

-15 -10 -5 0 5 10 15

**ˆ

20.0ˆ

2,

2

RDRg

RDRh

C

B

A

ns

h

RDRg

RDR

2,

2

ˆ

0ˆ

ns

h

RDRg

RDR

2,

2

ˆ

0ˆ

PC 1

PC

2

Missouri (2 locations )

Illinois

South Dakota

Germplasm Evaluation – QTL Mapping

0

0.5

1.0

1.5

2.0

2.5

3.0

0 0.5 1.0 1.5 2.0 2.5 3.0

Root Damage Rating [0-3 Iowa rating scale]F2:3 family per se performance

F2:

3 fa

mily

test

cro

ss p

erfo

rman

ce R

oot

Dam

age

Rat

ing

[0-3

Iow

a ra

ting

scal

e]

28.0

45.0%5

14.1

2

h

LSD

x

20.0

78.0%5

22.2

2

h

LSD

x

nsr 11.0

Germplasm Evaluation – QTL Mapping

Conclusions – Germplasm Evaluation

Traits used to determine WCR resistance show low to moderate heritabilities due to

lack of genotypic variance

presence of G × E interactions

large error variances

USDA-Germplasm Enhancement in Maize (GEM)

Test across a large number of environments

Testcross and per se performance

gihR

We need to learn more!

Genomic evaluation of defense response of maize (Zea mays L.) against herbivory by the western corn rootworm (Diabrotica vigifera

virgifera LeConte)

Gene expression patterns in the presence and absence of WCR larvae.

Root ‘metabolome’ of maize cultivars and relatives with different levels of WCR resistance in the presence and absence of WCR larvae.

QTL involved in the inheritance of WCR resistance in maize using multiple mapping populations derived from a maize diallel experiment and relate these to gene expression pattern and metabolite profiles.

Material and Methods

Plant Material:

CRW-C6 (USDA - Missouri)

14d in growth chamber 14h photoperiod - 28C, 60% rel. humidity10h scotoperiod – 22C, 80% rel. humidity

Treatments:

Plant stage V3

Mechanical wounding

50 neonate WCR larvae

Tissue Collection:

1d after treatment

First cm of all seminal root tips

Collection in the dark / green florescent light.

Material and Methods: Gene Expression

Experimental design

Contrasts:WCR vs. mechanical wounding, WCR vs. control, mechanical wounding vs. control.

Biological replicates R = 3

Microarray - 50,000+ element maize oligoarray from the University of Arizona.

Mixed Linear Model - SAS

Wound elicitorsInsect specific elicitors

Abiotic stress

Signal cascades

ToxinsAntinutriens

AntidigestionsVolatiles

Metabolic reconfiguration

Gene Expression – The Model

Gene Expression

Signal transduction 12 9 3Metabolism 51 28 23

Hormone 5 0 5

Translation 13 7 6

Post translational control 3 3 0

Silencing 3 3 0

Chromatin remodeling 5 4 1Defense 12 8 4

Transcription 30 20 10

Flavanoids 2 2 0

Misc. 5 4 1

Ntotal 141 88 53

Gene Group Total Up Down

Metabolic Profiling: Experimental Design

The same plant material as in gene expression study.

Contrasts:WCR vs. mechanical wounding, WCR vs. control, mechanical wounding vs. control.

Biological replicates R = 3

Six different extraction method, only water-soluble face, GC/MS

Mixed Linear Model - SAS

Metabolic Profiling

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8

Contrast Combination

Num

ber

of

Cla

ss M

embe

rs

01.421.301.201.1

CC

C

No.

22.2112.1

CC

C

No.

23.213.1

CC

C

No.

45.215.1

CC

C

No.

26.206.1

CC

C

No.

22

CC

C

No.

7.27.1

Based on Discriminate Analysis (using Proc StepDisc)

Metabolic Profiling

Metabolic Profiling

N = 30 out of > 700

Can1

Can2

-12

-10

-8

-6

6

8

10

-20 -15 -10 -5 5 10

CONTROL

WOUND

WCR

CONTROL_ANOVA

WOUND_ANOVA

WCR_ANOVA

Wilks’s Λ

< 0.0004**

< 0.0402**

Plot of three groups on two discriminant functions derived from two different sets of metabolites selected by a stepwise procedure (SAS Proc STEPDISC) or a single metabolite analysis of variance (SAS Proc GLM), respectively.

Metabolic Profiling

Control

WCR

WOU

Molecular Breeding – Gene/Metabolite networks

N(Meta)=150

GGM|pcor| > 0.04

GeneNet – R

Molecular Breeding – Gene networks

Pathway analysis

Information about gene/metabolic networks is so far limited.

Tools are still under development

Statistical issues are open.

G = GeneE = Enzyme / Enzyme activity S = Substrate

i

ii E

dEFdF

Z

11

n

iiZ

1121121 ......

nnii SESESESESES nii

G1 G2 Gi Gi+1 Gn

Molecular Breeding – Gene networks

What information can breeders exploit?

43

32

21

1 SESESES 1Z 2Z 3Z

Screen germplasm for variation in gene expression level or activity at these loci

Incorporate this information in selection index or BLUPs together with other information

Goal - Maximum output of S4

Summary and Conclusion

Recently, progress was made improving host plant resistance in maize against WCR feeding on roots. This progress was possible due to

However, conventional methods employed for improving WCR resistance are labor intensive. Progress is still slow and mostly hampered by lack of detailed knowledge about the genetic basis of the resistance.

New inbreds with improved WCR resistance provide the means for genetic research. Using these sources, we developed segregating populations of double haploids for mapping quantitative loci involved in WCR resistance.

improved high throughput screening methods and experimental designsintensive multi-institutional collaborations including private companiesintegration of exotic materials to broaden the genetic base for WCR resistance

Summary and Conclusion

Genes responding to wounding and WCR feeding are part of central metabolism, transcription, signal transduction, and defense pathways. Genes involved in gene silencing and chromatin remodeling were also identified – This is interesting!

No “magic” key compound involved in the plant’s response to WCR root feeding was found.

The metabolic response is complex as suggested by the metabolic response networks.

Integration of gene expression and metabolic profiles is of key importance.

Diverse sets of maize need to be screened in order to link expression patterns and metabolic signatures with WCR resistance. QTL population development is underway. eQTL and mQTL mapping will follow.

Gene and metabolite information has the potential to greatly enhance selection efficiency and will allow effective screening of germplasm banks for new resistance sources.

Acknowledgements

Illinois Missouri Biotech Alliance

University of Missouri

Georgia Davis Kelly Barr

USDA-ARS

Bruce HibbardSherry Flint-GarciaKen DashiellD. Prischmann-Voldseth

USDA-Germplasm Enhancement in Maize

AgReliant

Guenter SeitzJim UphausTom Koch

Pioneer

Andy Ross

University of Illinois

Mike GrayKevin SteffeyRon Estes

Indu RupassarSilvia BulhoesJuan Jose Marroquin Aco