1. Development of Western Corn Rootworm Resistant GEM Germplasm and its Role in Host Plant Resistance Research Martin Bohn Crop Sciences University 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. Density Gen 0 Gen 1 Background: Selection Genotypic variation Evaluation assay / trait “Breeder’s Equation”

4. 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

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. 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 Breeding for WCR Resistance

11. “Dekalb”-Program Germplasm Screening Start: < 1964 N:3,800 Origin% %Sel Cornbelt inbreds41 5 OPV30 7 Synth.10 10 European inbreds 1 0 Exotic inbreds 4 0 OPV14 5 Traits:Root lodging Row evaluation Anchorage 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

12. “NGIRL–USD”-Program Germplasm Selection 1964 – Early 1980s N: 57 Traits:Root lodging Vertical pull resist. Infest:Trap crop Large, dense root systems WCR resistance (tolerance) Cornbelt:Early – midseason South D.:Root rot resistant Exotic:West Indies, Mexico SDCRW1SYN5 SDCRW1C0 Germplasm Development C1:RS among S1 families C2: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 NGSDCRW1(S2)C4 registered in 1985 as source of tolerance to WCR. NGSDCRW1(S2)C4-15-2S2

13. Germplasm Screening Traits:Tolerance Root 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 lodging Root size Root re-growth Infes.:Trap crop (?) Populations with improved levels of tolerance – BS19(S)C2, BS20(S)C2

14. 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:RDR Infest:600 eggs / 30cm Germplasm Development 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 Diallel Study CRW3Syn0 -> CRW3-C8 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). Infestation Rate 0 60012001800 1 2 3 Root damage rating R 2 = 0.83

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

22. 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 00.511.522.53 RDR – (Trap) RDR – (Infes) r = 0.66 0 2 4 6 8 10 12 14 16 024681012 14 16 r = 0.64 Rank – (Infes) Rank – (Trap) -+ Trap crop vs. art. Infestation: Results Tolerant

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:α – lattice, 4 replications Plot size:Population – 4 row plots Inbreds - 2 row plots WCR eval.:Trap crop

25. Results: Populations 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 MAX MEAN 1.74 1.40 MIN0.88 1.00 1.80 0.60 LSD(5%) = 0.99 3.00 2.00 2.55 2.28 2.81 LSD(5%) = 0.34 2003 2004 Root Damage Ratings (0.00 – 3.00)

26. Results: Inbreds 1.08 LSD(5%) = 0.74 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 0.50 1.50 2.00 0.56 1.73 MAX MEAN MIN 3.00 2.00 2.47 1.83 2.80 LSD(5%) = 0.52 Root Damage Ratings (0.00 – 3.00) 2003 2004

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. -6 -4 -2 0 2 4 6 -6-4-20246 Cluster 1 Cluster 2 Cluster 3 Can 1 Can 2 Germplasm Screening

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

30. 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 = S 1 – per se S 1 – testcross Selected Not selected Illinois WCR Synthetic Germplasm Development

31. Germplasm Evaluation – QTL Mapping Mean 1.73 SD 0.50 REP0.18 0 0.1 0.2 0.3 0.4 0.5 Proportion 0 0.1 0.2 0.3 0.4 0.5 Proportion Missouri 2 South Dakota Mean 1.35 SD 0.34 REP0.38 0 0.511.522.53 Root Damage Rating [0-3 Iowa Rating Scale] 0 0.1 0.2 0.3 0.4 0.5 Proportion 0 0.1 0.2 0.3 0.4 0.5 Proportion Illinois Missouri 1 Mean 0.99 SD 0.43 REP0 Mean 2.70 SD 0.25 REP0.42 Number of F 2:3 families = 230 Number of locations = 4 (Missouri, South Dakota, Illinois) Incomplete block design, number of Reps/Loc = 3 Manual infestation, trap crop CRW3(C6)×LH51

32. Germplasm Evaluation – QTL Mapping Root Size RatingRoot Re-growth Rating Mean 4.15 SD 0.77 REP 0.25 Mean 3.95 SD 0.59 REP 0.15 Mean 4.19 SD 0.46 REP -/- Mean 3.52 SD 0.47 REP 0.21 Mean 3.74 SD 0.77 REP 0.26 Mean 5.75 SD 0.34 REP 0.09 Mean 5.38 SD 0.48 REP -/- Mean 4.60 SD 0.49 REP 0.42 Frequency [%] 123456123456

33. -15 -10 -5 0 5 15 - -10-505 15 C B A PC 1 PC 2 Missouri (2 locations ) Illinois South Dakota Germplasm Evaluation – QTL Mapping

34. 0 0.5 1.0 1.5 2.0 2.5 3.0 00.51.01.52.02.53.0 Root Damage Rating [0-3 Iowa rating scale] F 2:3 family per se performance F 2:3 family test cross performance Root Damage Rating [0-3 Iowa rating scale] Germplasm Evaluation – QTL Mapping

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 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. Wound elicitors Insect specific elicitors Abiotic stress Signal cascades Toxins Antinutriens Antidigestions Volatiles Metabolic reconfiguration Gene Expression – The Model

40. Gene Expression 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 N total 141 88 53 Gene Group Total Up Down

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. 0 10 20 30 40 50 60 70 12345678 Contrast Combination Number of Class Members 01.4 2 1.3 01.2 01.1 CCCNo. 2 2.2 112.1 CCCNo. 23.2 13.1 CCC No. 45.2 15.1 CCC No. 2 6.2 06.1 CCC No. 2 2 CCC No. 7.2 7.1 Based on Discriminate Analysis (using Proc StepDisc) Metabolic Profiling

44. N = 30 out of > 700

45. Can1 Can2 -12 -10 -8 -6 6 8 10 -20-15-10-5510 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

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

47. 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 = Gene E = Enzyme / Enzyme activity S = Substrate G1G1 G2G2 GiGi G i+1 GnGn

48. Molecular Breeding – Gene networks What information can breeders exploit? 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

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 designs intensive multi-institutional collaborations including private companies integration 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.

51. Acknowledgements Illinois Missouri Biotech Alliance University of Missouri Georgia Davis Kelly Barr USDA-ARS Bruce Hibbard Sherry Flint-Garcia Ken Dashiell D. Prischmann- Voldseth USDA-Germplasm Enhancement in Maize AgReliant Guenter Seitz Jim Uphaus Tom Koch Pioneer Andy Ross University of Illinois Mike Gray Kevin Steffey Ron Estes Indu Rupassar Silvia Bulhoes Juan Jose Marroquin Aco