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