Kein Folientitel

1
Development of Western Corn Rootworm Resistant
GEM Germplasm and its Role in Host Plant
Resistance Research
Martin Bohn Crop SciencesUniversity of Illinois
2
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
3
Background Selection
Breeders Equation
Gen 0
Density
Gen 1
Density
Genotypic variation Evaluation assay / trait
4
Background Illinois Long-term Selection
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).
5
The Insect - Adults
6
The Insect Eggs
Source J. Spencer
7
The Insect - Larvae
Source J. Spencer
8
WCR Resistance - The Challenge
Labor intensive! Resistance traits have low
heritability. Resistance to WCR larvae and adult
feeding not correlated.
9
WCR Distribution
Gray et al. 2009. Annual Rev. Entomology
10
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
11
Dekalb-Program
Germplasm Screening
Start lt 1964 N 3,800
Origin Sel Cornbelt 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)
12
NGIRLUSD-Program
Germplasm Selection
1964 Early 1980s N 57
Large, dense root systemsWCR resistance
(tolerance) Cornbelt Early midseasonSouth
D. Root rot resistantExotic West Indies,
Mexico
SDCRW1SYN
5
Traits Root lodgingVertical pull
resist. Infest Trap crop
SDCRW1C0
13
Iowa-Program
Germplasm Screening
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 ToleranceRoot traits Infes. Trap crop
(?)
14
USDA/ARS-Missouri-Program
Germplasm Screening
Start 1992 N 3,500
Corn and corn relatives
Traits RDR Infes. 600 eggs / 30cm S(tot) 0.2
(7 accessions)
15
Quintessence
12,000 corn accessions and relatives were
screened for WCR resistance. Trap crop
artificial infestation multiple traits to assess
WCR damage lt 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.
16
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.
17
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.
18
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
19
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
20
Trap crop vs. art. Infestation Results
Plant materials Inbreds 15 entries Populations
20 entries NGSDCRW1(S2)C4-15-2S2 Monsant
o Bt Monsanto Non-Bt Field experiments Locati
ons DeKalb, Monmouth, Urbana Treatments Trap
crop DeKalb, Monmouth, Urbana Artif.
Infes. Urbana (600 eggs/plant) Chemi.
prot. DeKalb, Monmouth, Urbana Experimental
design a-lattice design Replications 3 rows/p
lot 1 (I), 4 (P)
21
Germplasm Screening
Node-Injury Scale (0.00 3.00)
1.50
No. of full nodes eaten
of a node eaten
3.00
0.00
(Oleson et al. 2005. J Econ Entomol 981-8)
22
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)
23
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
24
Materials and Methods
Population 15 Entries Inbred 55 Entries
(20, 35) Location Urbana, 2003 (35), 2004
(70) Design a lattice, 4 replications Plot
size Population 4 row plots Inbreds - 2
row plots WCR eval. Trap crop
25
Root Damage Ratings(0.00 3.00)
Results Populations
2004
2003
DKXL212N11a01 UR10001N1708b UR10001N1702 CH0501
5N1204 DKB844S1612 NGSDCRW1 FS8A(T)N1804 FS8A(S
)S0907 CASHN1410 AR17056N2025 AR16026S1719 AR1
3035S11b04 AR17056S1216 UR13085N0204 AR16026N1
210
0.60
MIN
0.88
1.00
MEAN
1.40
MAX
1.74
2.00
1.80
2.28
2.55
2.81
3.00
LSD(5) 0.99
LSD(5) 0.34
26
Root Damage Ratings(0.00 3.00)
Results Inbreds
2004
CUBA117S1520-153 AR17056N2025-728 B64 CUBA117S1
520-182 AR17056N2025-5 AR17056N2025-522 CUBA117
S1520-52 CUBA117S1520-41 CUBA117S1520-156 AR170
56N2025-546 AR17056N2025-508 AR17056N2025-2 AR
17056N2025-532 B37 Mo17 AR17056N2025-4 NGSDCRW1
(S2)C4-15 AR17056N2025-1 AR17056N2025-3 B73
2003
0.50
MIN
0.56
1.08
MEAN
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
27
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.
28
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
29
Germplasm Screening
30
Germplasm Development

• 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

EU
S1 per se
SU
RU
S1 testcross
Selected
Not selected
Illinois WCR Synthetic
31
Germplasm Evaluation QTL Mapping
32
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
33
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
34
Germplasm Evaluation QTL Mapping
3.0
2.5
2.0
F23 family test cross performance Root 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 F23
family per se performance
35
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-Germpl
asm Enhancement in Maize (GEM) Test across a
large number of environments Testcross and per
se performance
36
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.
37
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.
38
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
39
Gene Expression The Model
Wound elicitorsInsect specific elicitorsAbiotic
stress
Signal cascades
ToxinsAntinutriensAntidigestionsVolatilesMetab
olic reconfiguration
40
Gene Expression
Gene Group Total Up
Down
Signal transduction 12 9 3
Metabolism 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 1
Defense 12 8 4
Transcription 30 20 10
Flavanoids 2 2 0
Misc. 5 4 1
Ntotal 141 88 53
41
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
42
Metabolic Profiling
43
Metabolic Profiling
CCC
No.
70
0
1.1
0
1.2
60
2
1.3
No.
0
1.4
CCC
No.
CCC
50
1
5.1
2
7.1
40
4
5.2
2
7.2
Number of Class Members
CCC
No.
30
No.
CCC
11
2.1
CCC
No.
2.2
2
20
0
6.1
1
3.1
2
6.2
2
3.2
10
0
1
2
3
4
5
6
7
8
Contrast Combination
Based on Discriminate Analysis (using Proc
StepDisc)
44
Metabolic Profiling
N 30 out of gt 700
45
Metabolic Profiling
Can2
10
8
6
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.
46
Molecular Breeding Gene/Metabolite networks
Control
WCR
WOU
N(Meta)150 GGMpcor gt 0.04 GeneNet R
47
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.
48
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
49
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
50
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 plants 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.
51
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
52
(No Transcript)