Transcript Development of Western Corn Rootworm Resistant GEM Germplasm and its Role in Host Plant Resistance Research 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 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
Background: Selection
“Breeder’s Equation”
Gen 0
S    p
Density

p
R  s   p
p
 p
Gen 1
 h2 S
Density
 ih g
Genotypic variation
p
s
Evaluation assay / trait
Background: Illinois Long-term Selection
Mean % Oil of ears analyzed
25
IHO
ILO
RHO
RLO
SHO
20
15
10
5
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
0
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).
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
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
2
g
Resistant lines were developed
large densely branched root systems
quick root regeneration
SD10, SD20, B69, Mo22, Oh05, B14, N38A, A251, W202
“Dekalb”-Program
Germplasm Screening
Germplasm Development
Start: < 1964
RS among S1 families
N:
3,800
Origin
Cornbelt
inbreds
OPV
Synth.
European
inbreds
Exotic
inbreds
OPV
Traits:
%
41
30
10
5
7
10
1
0
4
14
0
5
Root lodging
Row evaluation
Anchorage ratings
Infes.:
Trap crop
S(tot):
5% (190)
%Sel
Traits:
RDR
Infes.:
Trap crop
S:
2.2-6.9% (1964-1968)
0.1-3.6% (1969-1977)
RW15, RW16, RW17
“NGIRL–USD”-Program
Germplasm Selection
Germplasm Development
1964 – Early 1980s
C1:
C2:
N: 57
Large, dense root systems
WCR resistance (tolerance)
Cornbelt:
South D.:
Exotic:
Early – midseason
Root rot resistant
West Indies, Mexico
SDCRW1SYN 5
Traits:
Infest:
Root lodging
Vertical pull resist.
RS among S1 families
RS among S2 families
Traits:
Root lodging
Vertical pull resist.
Infest:
Trap crop
C3:
RS among S2 families
Traits:
RDR
Infest:
600 eggs / 30cm
S:
10% (20 S2s)
C4:
NGSDCRW1(S2)C4
Trap crop
NGSDCRW1(S2)C4 registered in 1985
as source of tolerance to WCR.
SDCRW1C0
NGSDCRW1(S2)C4-15-2S2
“Iowa”-Program
Germplasm Screening
Iowa Early Rootworm Synthetic
(BSER)
Germplasm Development
RS among S1 families
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:
Tolerance
Root traits
Infes.:
Trap crop (?)
Traits:
RDR
Root lodging
Root size
Root re-growth
Infes.:
Trap crop (?)
Populations with improved levels of
tolerance – BS19(S)C2, BS20(S)C2
“USDA/ARS-Missouri”-Program
Germplasm Screening
Start:
1992
N:
3,500
Corn and corn relatives
Germplasm Development
Diallel Study
N:
Traits:
Infest:
S:
56 crosses
RDR
600 eggs / 30cm
18% (10 crosses)
TL92A-PAR 1779 60-4 (C4)
TL92A-PAR 1774 28-1 (C3)
PI 340839 (Popcorn)
NGSDCRW1(S2)C4-15-2S2
CRW3Syn0 -> CRW3-C8
Traits:
RDR
Infes.:
600 eggs / 30cm
S(tot):
0.2 (7 accessions)
Traits:
Infest:
RDR
600 eggs / 30cm
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 infestation
multiple traits to assess WCR damage
< 1% of the screened germplasm was used in
germplasm development.
large, dense root system
good 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 lodging
Vertical pull resistance
Row 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.
ˆ g2
ˆ ge2
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.
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).
Root damage rating
3
2
1
R2 = 0.83
0
600
1200
1800
Infestation Rate
Trap crop vs. art. Infestation: Results
Plant materials
Inbreds:
Populations:
15 entries
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:
#rows/plot:
3
1 (I), 4 (P)
Germplasm Screening
Node-Injury Scale (0.00 – 3.00)
1.50
No. of full nodes eaten
% of a node eaten
0.00
(Oleson et al. 2005. J Econ Entomol 98:1-8)
3.00
Trap crop vs. art. Infestation: Results
r = 0.66
2.5
RDR – (Infes)
2
1.5
1
0.5
0
0
0.5
1
1.5
2
2.5
3
RDR – (Trap)
16
r = 0.64
14
12
Rank – (Infes)
Tolerant
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
----------------------------
3
10
8
6
4
2
0
0
-
2
4
6
8
10
Rank – (Trap)
12
14
16
+
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”
“Inbred”
15 Entries
55 Entries (20, 35)
Location:
Urbana, 2003 (35), 2004 (70)
Design:
α – lattice, 4 replications
Plot size:
Population – 4 row plots
Inbreds - 2 row plots
WCR eval.:
Trap crop
Results: Populations
2004
2003
0.60
MIN
0.88
1.00
MEAN
1.40
MAX
1.74
1.80
Root Damage Ratings
(0.00 – 3.00)
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
2.00
2.28
2.55
2.81
3.00
LSD(5%) = 0.99
LSD(5%) = 0.34
Results: Inbreds
2003
MIN
0.50
0.56
MEAN
1.08
1.50
MAX
1.73
2.00
LSD(5%) = 0.74
Root Damage Ratings
(0.00 – 3.00)
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
2004
1.83
2.00
2.47
2.80
3.00
LSD(5%) = 0.52
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.
Germplasm Screening
6
Cluster 1
Cluster 2
Cluster 3
Can 2
4
2
0
-2
-4
-6
-6
-4
-2
0
Can 1
2
4
6
Germplasm Screening
USDA-Germplasm Enhancement in Maize (GEM) – base populations
Germplasm Development
S1 – per se
EU = Selfed progeny of one plant
(per se and testcross evaluation).
SU = Individual plants.
S1 – testcross
RU = Selfed seed is used to intercross
selected plants.
• Parental control
• 4 Seasons/cycle
Selected
Not selected
Illinois WCR Synthetic
Proportion
Proportion
0.5
Proportion
Missouri 1
Missouri 2
South Dakota
Germplasm Evaluation – QTL Mapping
0.4
0.3
Mean 1.73
SD
0.50
REP 0.18
CRW3(C6)×LH51
0.2
0.1
0
0.5
0.4
0.3
Mean 1.35
SD
0.34
REP 0.38
0.2
0.1
0
0.5
0.4
0.3
Mean 0.99
SD
0.43
REP 0
0.2
0.1
Proportion
Illinois
0
0.5
0.4
0.3
Mean 2.70
SD
0.25
REP 0.42
0.2
0.1
0
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 = 3
Manual infestation, trap crop
Germplasm Evaluation – QTL Mapping
Mean 3.74
SD
0.77
REP 0.26
Mean 3.95
SD
0.59
REP 0.15
Mean 5.75
SD
0.34
REP 0.09
Mean 4.19
SD
0.46
REP -/-
Mean 5.38
SD
0.48
REP -/-
Mean 3.52
SD
0.47
REP 0.21
Mean 4.60
SD
0.49
REP 0.42
Frequency [%]
Mean 4.15
SD
0.77
REP 0.25
1
2
3
4
5
Root Size Rating
6
1
2
3
4
5
Root Re-growth Rating
6
Germplasm Evaluation – QTL Mapping
15
A
10
ˆ g2,RDR ns
2
hˆRDR
0
5
PC 2
2
hˆRDR
0
C
B
ˆ g2,RDR ns
0
2
hˆRDR
 0.20
ˆ g2,RDR * *
-5
Missouri (2 locations )
-10
Illinois
South Dakota
-15
-15
-10
-5
0
PC 1
5
10
15
Germplasm Evaluation – QTL Mapping
F2:3 family test cross performance Root
Damage Rating [0-3 Iowa rating scale]
3.0
x  1.14
LSD5%  0.45
2.5
h 2  0.28
2.0
1.5
1.0
x  2.22
LSD5%  0.78
0.5
r  0.11ns
h 2  0.20
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
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
R  ih g
large error variances
USDA-Germplasm Enhancement in Maize (GEM)
Test across a large number of environments
Testcross and per se performance
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. humidity
10h 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
Gene Expression – The Model
Wound elicitors
Insect specific elicitors
Abiotic stress
Signal cascades
Toxins
Antinutriens
Antidigestions
Volatiles
Metabolic reconfiguration
Gene Expression
Gene Group
Total
Up
Down
Signal transduction
12
9
3
Metabolism
51
28
23
5
0
5
13
7
6
Post translational control
3
3
0
Silencing
3
3
0
Chromatin remodeling
5
4
1
Defense
12
8
4
Transcription
30
20
10
Flavanoids
2
2
0
Misc.
5
4
1
141
88
53
Hormone
Translation
Ntotal
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
20
2.1 11
2.2 2
3.1
3.2
10
1
2
No.
CCC
1
4
No.
CCC
No.
30
5.1
5.2
7.1
7.2
No.
40
CCC
0
0
2
0
No.
1.1
1.2
1.3
1.4
CCC
50
No.
60
CCC
Number of Class Members
70
CCC
Metabolic Profiling
6.1
6.2
0
2
2
2
0
1
2
3
4
5
Contrast Combination
Based on Discriminate Analysis (using Proc StepDisc)
6
7
8
Metabolic Profiling
N = 30 out of > 700
Metabolic Profiling
Wilks’s Λ
CONTROL
Can2
< 0.0004**
WOUND
10
WCR
8
CONTROL_ANOVA
WOUND_ANOVA
< 0.0402**
6
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.
Molecular Breeding – Gene/Metabolite
networks
Control
WCR
WOU
N(Meta)=150
GGM
|pcor| > 0.04
GeneNet – R
Molecular Breeding – Gene networks
G1
G2
Gi
Gi+1
Gn
E1 S 
E2 ...  S 
Ei S ... 
E
En S
i 1
S1 

S


2
i
i 1
n
n 1
G = Gene
E = Enzyme / Enzyme activity
S = Substrate
dF
Zi 
F
n
Z
i
dEi
Ei
1
i 1
Pathway analysis
Information about gene/metabolic networks is so far limited.
Tools are still under development
Statistical issues are open.
Molecular Breeding – Gene networks
What information can breeders exploit?
E3  S
E1 S 
E2  S 
S1 
2
3
4
Z1
Z2
Z3
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
Summary and Conclusion
Recently, progress was made improving host plant resistance in maize
against WCR feeding on roots. This progress was possible due to
improved high throughput screening methods and experimental
designs
intensive multi-institutional collaborations including private
companies
integration of exotic materials to broaden the genetic base for WCR
resistance
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.
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
University of Illinois
Mike Gray
Kevin Steffey
Ron Estes
Indu Rupassar
Silvia Bulhoes
Juan Jose Marroquin Aco
University of Missouri
Georgia Davis
Kelly Barr
USDA-ARS
Bruce Hibbard
Sherry Flint-Garcia
Ken Dashiell
D. PrischmannVoldseth
USDA-Germplasm Enhancement in Maize
Illinois Missouri Biotech Alliance
AgReliant
Guenter Seitz
Jim Uphaus
Tom Koch
Pioneer
Andy Ross