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Development of Western Corn Rootworm Resistant GEM Germplasm and its Role in Host Plant Resistance Research. Martin Bohn Crop Sciences University of Illinois. Outline. Breeding for WCR Resistance Quintessence of 70 years of breeding for WCR resistance Tolerance vs. antibiosis

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Martin bohn crop sciences university of illinois

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

Martin Bohn

Crop SciencesUniversity of Illinois


Martin bohn crop sciences university 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


Martin bohn crop sciences university of illinois

Background: Selection

“Breeder’s Equation”

Gen 0

Density

Gen 1

Density

Genotypic variation

Evaluation assay / trait


Martin bohn crop sciences university of illinois

Background: Illinois Long-term Selection

25

IHO

ILO

20

RHO

RLO

SHO

15

Mean % Oil of ears analyzed

10

5

0

5

0

30

40

50

65

70

25

35

45

55

60

75

80

20

85

95

10

15

90

100

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).


Martin bohn crop sciences university of illinois

The Insect - Adults


Martin bohn crop sciences university of illinois

The Insect – Eggs

Source – J. Spencer


Martin bohn crop sciences university of illinois

The Insect - Larvae

Source – J. Spencer


Martin bohn crop sciences university of illinois

WCR Resistance - The Challenge

Labor intensive!

Resistance traits have low heritability.

Resistance to WCR larvae and adult feeding not correlated.


Martin bohn crop sciences university of illinois

WCR Distribution

Gray et al. 2009. Annual Rev. Entomology


Martin bohn crop sciences university of illinois

Breeding for WCR Resistance

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

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


Martin bohn crop sciences university of illinois

“Dekalb”-Program

Germplasm Screening

Germplasm Development

RS among S1 families

Start: < 1964

N:3,800

Origin% %Sel

Cornbeltinbreds41 5OPV30 7Synth.10 10

Europeaninbreds 1 0

Exoticinbreds 4 0OPV14 5

Traits:RDR

Infes.:Trap crop

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

Traits:Root lodgingRow evaluationAnchorage ratings

Infes.:Trap crop

S(tot):5% (190)

RW15, RW16, RW17


Martin bohn crop sciences university of illinois

“NGIRL–USD”-Program

Germplasm Selection

Germplasm Development

C1:RS among S1 families

C2:RS among S2 families

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

C3:RS among S2 families

Traits:RDR

Infest:600 eggs / 30cm

S:10% (20 S2s)

SDCRW1SYN

5

Traits:Root lodgingVertical pull resist.

Infest:Trap crop

C4:NGSDCRW1(S2)C4

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

NGSDCRW1(S2)C4-15-2S2

SDCRW1C0


Martin bohn crop sciences university of illinois

“Iowa”-Program

Germplasm Screening

Germplasm Development

RS among S1 families

Iowa Early Rootworm Synthetic (BSER)

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

Iowa Late Rootworm Synthetic (BSLR)

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

Traits:RDR

Root lodging

Root size

Root re-growth

Infes.:Trap crop (?)

Traits:ToleranceRoot traits

Infes.:Trap crop (?)

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


Martin bohn crop sciences university of illinois

“USDA/ARS-Missouri”-Program

Germplasm Screening

Germplasm Development

Diallel Study

Start:1992

N:3,500

N: 56 crosses

Traits:RDR

Infest:600 eggs / 30cm

S: 18% (10 crosses)

TL92A-PAR 1779 60-4 (C4)

TL92A-PAR 1774 28-1 (C3)

PI 340839 (Popcorn)

NGSDCRW1(S2)C4-15-2S2

Corn and corn relatives

CRW3Syn0 -> CRW3-C8

Traits:RDR

Infes.:600 eggs / 30cm

S(tot):0.2 (7 accessions)

Traits:RDR

Infest:600 eggs / 30cm

Genotypes from C3 and C5 were used in QTL studies.


Martin bohn crop sciences university of illinois

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.


Martin bohn crop sciences university of illinois

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.


Martin bohn crop sciences university of illinois

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

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

Parental selection is crucial.


Martin bohn crop sciences university of illinois

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


Martin bohn crop sciences university of illinois

Trap crop vs. art. infestation

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

3

2

Root damage rating

1

R2 = 0.83

0

600

1200

1800

Infestation Rate


Martin bohn crop sciences university of illinois

Trap crop vs. art. Infestation: Results

Plant materials

Inbreds:15 entries

Populations:20 entries

NGSDCRW1(S2)C4-15-2S2

Monsanto Bt

Monsanto Non-Bt

Field experiments

Locations:DeKalb, Monmouth, Urbana

Treatments:

Trap crop:DeKalb, Monmouth, Urbana

Artif. Infes.:Urbana (600 eggs/plant)

Chemi. prot.:DeKalb, Monmouth, Urbana

Experimental design

α-lattice design

Replications:3

#rows/plot:1 (I), 4 (P)


Martin bohn crop sciences university of illinois

Germplasm Screening

Node-Injury Scale (0.00 – 3.00)

1.50

No. of full nodes eaten

% of a node eaten

0.00

3.00

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


Martin bohn crop sciences university of illinois

Trap crop vs. art. Infestation: Results

3

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

----------------------------

r = 0.66

2.5

2

RDR – (Infes)

1.5

1

0.5

0

0

0.5

1

1.5

2

2.5

3

RDR – (Trap)

16

r = 0.64

14

12

Tolerant

Rank – (Infes)

10

8

6

4

2

0

0

2

4

6

8

10

12

16

14

-

+

Rank – (Trap)


Martin bohn crop sciences university of illinois

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

Lo10161.91 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

NGSDCRW2.27 2.15 2.35 2.26

NGSDCRW 2.77 2.18 1.96 2.30

MON_Bt0.07 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


Martin bohn crop sciences university of illinois

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


Martin bohn crop sciences university of illinois

Root Damage Ratings(0.00 – 3.00)

Results: Populations

2004

2003

DKXL212:N11a01

UR10001:N1708b

UR10001:N1702

CH05015:N1204

DKB844:S1612

NGSDCRW1

FS8A(T):N1804

FS8A(S):S0907

CASH:N1410

AR17056:N2025

AR16026:S1719

AR13035:S11b04

AR17056:S1216

UR13085:N0204

AR16026:N1210

0.60

MIN

0.88

1.00

1.40

MEAN

MAX

1.74

2.00

1.80

2.28

2.55

2.81

3.00

LSD(5%) = 0.99

LSD(5%) = 0.34


Martin bohn crop sciences university of illinois

Root Damage Ratings(0.00 – 3.00)

Results: Inbreds

2004

CUBA117:S1520-153

AR17056:N2025-728

B64

CUBA117:S1520-182

AR17056:N2025-#5

AR17056:N2025-522

CUBA117:S1520-52

CUBA117:S1520-41

CUBA117:S1520-156

AR17056:N2025-546

AR17056:N2025-508

AR17056:N2025-#2

AR17056:N2025-532

B37

Mo17

AR17056:N2025-#4

NGSDCRW1(S2)C4-15

AR17056:N2025-#1

AR17056:N2025-#3

B73

2003

0.50

MIN

0.56

MEAN

1.08

1.83

1.50

2.00

1.73

MAX

2.47

2.80

2.00

3.00

LSD(5%) = 0.52

LSD(5%) = 0.74


Martin bohn crop sciences university of illinois

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.


Martin bohn crop sciences university of illinois

Germplasm Screening

6

Cluster 1

4

Cluster 2

Cluster 3

2

Can 2

0

-2

-4

-6

Can 1

-6

-4

-2

0

2

4

6


Martin bohn crop sciences university of illinois

Germplasm Screening

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


Martin bohn crop sciences university of illinois

Germplasm Development

EU =

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

  • Individual plants.

  • Selfedseed is used to intercross selected plants.

  • Parental control

  • 4 Seasons/cycle

S1 – per se

SU =

RU =

S1 – testcross

Selected

Not selected

Illinois WCR Synthetic


Martin bohn crop sciences university of illinois

Germplasm Evaluation – QTL Mapping

CRW3(C6)×LH51

Mean 1.73

SD 0.50

REP0.18

South Dakota

Proportion

Mean 1.35

SD 0.34

REP0.38

Proportion

Missouri 2

0.5

0.5

0.5

0.5

0.4

0.4

0.4

0.4

0.3

0.3

0.3

0.3

Mean 0.99

SD 0.43

REP0

0.2

0.2

0.2

0.2

Proportion

Missouri 1

0.1

0.1

0.1

0.1

0

0

0

0

Mean 2.70

SD 0.25

REP0.42

Proportion

Illinois

0

0.5

1

1.5

2

2.5

3

Root Damage Rating [0-3 Iowa Rating Scale]

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


Martin bohn crop sciences university of illinois

Germplasm Evaluation – QTL Mapping

Mean 4.15SD 0.77REP 0.25

Mean 3.74SD 0.77REP 0.26

Mean 3.95SD 0.59REP 0.15

Mean 5.75SD 0.34REP 0.09

Frequency [%]

Mean 4.19SD 0.46REP -/-

Mean 5.38SD 0.48REP -/-

Mean 3.52SD 0.47REP 0.21

Mean 4.60SD 0.49REP 0.42

1

2

3

4

5

6

1

2

3

4

5

6

Root Size Rating

Root Re-growth Rating


Martin bohn crop sciences university of illinois

Germplasm Evaluation – QTL Mapping

15

A

10

C

5

B

PC 2

0

-

5

-

10

Missouri (2 locations )

Illinois

South Dakota

-

15

-

15

-

10

-

5

0

5

10

15

PC 1


Martin bohn crop sciences university of illinois

Germplasm Evaluation – QTL Mapping

3.0

2.5

2.0

F2:3 family test crossperformanceRoot Damage Rating [0-3 Iowa rating scale]

1.5

1.0

0.5

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


Martin bohn crop sciences university of illinois

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


Martin bohn crop sciences university of illinois

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 togene expression pattern and metabolite profiles.


Martin bohn crop sciences university of illinois

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.


Martin bohn crop sciences university of illinois

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


Martin bohn crop sciences university of illinois

Gene Expression – The Model

Wound elicitorsInsect specific elicitorsAbiotic stress

Signal cascades

ToxinsAntinutriensAntidigestionsVolatilesMetabolic reconfiguration


Martin bohn crop sciences university of illinois

Gene Expression

Gene Group Total Up Down

Signal transduction 12 9 3

Metabolism 512823

Hormone 5 0 5

Translation 13 7 6

Post translational control 3 3 0

Silencing 3 3 0

Chromatin remodeling 5 4 1

Defense 12 8 4

Transcription 302010

Flavanoids 2 2 0

Misc. 5 4 1

Ntotal 141 88 53


Martin bohn crop sciences university of illinois

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


Martin bohn crop sciences university of illinois

Metabolic Profiling


Martin bohn crop sciences university of illinois

Metabolic Profiling

CCC

No.

70

1.1

0

1.2

0

60

1.3

2

No.

CCC

1.4

0

No.

CCC

50

5.1

1

7.1

2

40

5.2

4

7.2

2

Number of Class Members

No.

CCC

30

No.

CCC

2.1

11

CCC

No.

2.2

2

20

6.1

0

3.1

1

6.2

2

3.2

2

10

0

1

2

3

4

5

6

7

8

Contrast Combination

Based on Discriminate Analysis (using Proc StepDisc)


Martin bohn crop sciences university of illinois

Metabolic Profiling

N = 30 out of > 700


Martin bohn crop sciences university of illinois

Metabolic Profiling

Wilks’s Λ

Can2

CONTROL

10

< 0.0004**

WOUND

8

WCR

CONTROL_ANOVA

6

< 0.0402**

WOUND_ANOVA

WCR_ANOVA

Can1

-20

-15

-10

-5

5

10

-6

-8

-10

-12

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.


Martin bohn crop sciences university of illinois

Molecular Breeding – Gene/Metabolite networks

Control

WCR

WOU

N(Meta)=150

GGM|pcor| > 0.04

GeneNet – R


Martin bohn crop sciences university of illinois

G1

G2

Gi

Gi+1

Gn

Molecular Breeding – Gene networks

G = GeneE = Enzyme / Enzyme activity S = Substrate

Pathway analysis

Information about gene/metabolic networks is so far limited.

Tools are still under development

Statistical issues are open.


Martin bohn crop sciences university of illinois

Molecular Breeding – Gene networks

What information can breeders exploit?

Goal - Maximum output of S4

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


Martin bohn crop sciences university of illinois

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


Martin bohn crop sciences university of illinois

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.


Martin bohn crop sciences university of illinois

Acknowledgements

University of Missouri

Georgia Davis Kelly Barr

USDA-ARS

Bruce HibbardSherry Flint-GarciaKen DashiellD. Prischmann-Voldseth

University of Illinois

Mike GrayKevin SteffeyRon Estes

Indu RupassarSilvia BulhoesJuan Jose Marroquin Aco

AgReliant

Guenter SeitzJim UphausTom Koch

Pioneer

Andy Ross

USDA-Germplasm Enhancement in Maize

Illinois Missouri Biotech Alliance


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