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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 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

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  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 “Breeder’s Equation” Gen 0 Density Gen 1 Density Genotypic variation Evaluation assay / trait

  4. 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).

  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 Germplasm Development RS among S1 families Start: < 1964 N: 3,800 Origin % %Sel Cornbelt inbreds 41 5OPV 30 7Synth. 10 10 Europeaninbreds 1 0 Exoticinbreds 4 0OPV 14 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

  12. “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

  13. “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

  14. “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.

  15. 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.

  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 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)

  21. 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)

  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 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

  24. 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 plots Inbreds - 2 row plots WCR eval.: Trap crop

  25. 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

  26. 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

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

  30. 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

  31. Germplasm Evaluation – QTL Mapping CRW3(C6)×LH51 Mean 1.73 SD 0.50 REP 0.18 South Dakota Proportion Mean 1.35 SD 0.34 REP 0.38 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 REP 0 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 REP 0.42 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

  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 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

  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-Germplasm 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 togene 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 ToxinsAntinutriensAntidigestionsVolatilesMetabolic 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 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)

  44. Metabolic Profiling N = 30 out of > 700

  45. 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.

  46. Molecular Breeding – Gene/Metabolite networks Control WCR WOU N(Meta)=150 GGM|pcor| > 0.04 GeneNet – R

  47. 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.

  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 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.

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