Breeding Nutritionally Enhanced Maize: The Tropical Experience

1. Breeding Nutritionally Enhanced Maize: The Tropical Experience K. Pixley, R. Babu, J. Yan, N. Palacios & colleagues GEM Cooperator Meeting, 8 December 2010, Chicago

2. QPM Normal Maize QPM: Nutritionally enhanced maize “The Pigs” QPM also “works” with chickens… • Pellagra: diarrhea, dermatitis; due to niacin deficiency. Tryptophan is a precursor of niacin • Kwashiorkor: edemas, anorexia, increased susceptibility to infections; due to low quality protein

3. Whatis QPM? • Containsone gene – opaque2 (o2) – thataffectsproteinproduction in thegrain • No change in proteinquantity • More of proteinsrich in tryptophan and lysine • o2 wasfound in maize; QPM isnottransgenic • Looks, cooks and tastes like normal maize • Mustbe, and many are, agronomicallycompetitive

4. Where is maize an important source of protein? FAO Stat WHO, 2007. Protein and amino acid requirements in human nutrition. http://whqlibdoc.who.int/trs/WHO_TRS_935_eng.pdf

6. Summary: QPM meta-analysisGunaratna et al. • 9 studies: 5 countries (Ethiopia, Ghana, India, Mexico, Nicaragua); 48-486 children; 3.5 mo – 5 yrold • ConsumingQPM instead of normal maizeresulted in: • 12% (95% CI: 7-18%) increase in weightgain • 9% (95% CI: 6-15) increase in heightgain • Theseresultswererobust; essentially un-changedby: • Variousadjustments/transformationstothe data, ormethodstocalculate CI • Discardstudieswithmost extreme results • Discardany of thestudies Gunaratna et al., FoodPolicy 2010

7. Iron deficiency affects >2 billion people • Iron deficiency anemia (IDA) • Maternal and perinatal mortality • Impaired cognitive skills and physical activity • Women and children in South Asia and Africa

8. Zinc deficiency • Zn deficiency • 800,000 child deaths per year; increased risk • Diarrhea, pneumonia, malaria • Stunting during early childhood • Equally affects males and females • South Asia and Africa Photo: N. Palacios

9. Vitamin A deficiency • Vitamin A deficiency (VAD) • Night blindness, corneal scarring & blindness • Weakened immune system: VAD associated with • 20% of measles- • 24% of diarrhea- • 20% of malaria-related mortality in children; • 20% of maternal mortality • South Asia and Africa have highest VAD prevalence • 157 million pre-school children • 30 million pregnant women * Micronutrient malnutrition affects more than half of the world’s population – United Nations SCN, 2004.

10. Dietary sources • Vitamin A • Meat (esp. liver) • Vegetables (carrot, sweet potato, spinach) • Iron • Red meat, fish, poultry • Lentils, beans, leafy vegetables • Zinc • Oysters, animal proteins, • Beans, nuts, whole grains $$$ $ $$$$ $$

11. Biofortification of staple food crops • Micronutrients available in staple foods • Sustainable, affordable • Accompanied by dietary/nutrition information • Complemented by supplementation and fortification • Acute malnutrition • Equal or better agronomic performance of biofortified crops • Yield, disease resistance, drought tolerance…

12. Cross high proA x good drought tolerance… De3, SC55, CI7

13. X X X BC1S1 X X X X BC1S4 BC1S2

14. 2nd Dose F1’s X X X BC1 2ndDose S1’s

16. 8 promising proA hybrids: 5 sites in Zambia + 2 sites in Zimbabwe ProA (ug/g) Best hybrid check Hybrid 1: 8.9 Hybrid 2: 7.1 Hybrid 3: 6.3 Hybrid 4: 6.5 Hybrid 5: 7.4 Hybrid 6: 5.7 Hybrid 7: 6.9 Hybrid 8: 5.9 Tons per hectare Harare GART Msekera Mpungwe ART, Harare SeedCo, Lus ZamSeed, Lus

17. GGPP lycopene LCYE LCYB δ-carotene γ-carotene LCYB LCYB α-carotene β-carotene HYDb HYDb1 β-cryptoxanthin zeinoxanthin HYDE HYDb zeaxanthin lutein ABA LCYE affects the ratio of carotenoids in the biosynthetic pathway PSY PDS Z-ISO ZDS/CRISTO • Alleles for LCYE identified by: • Association mapping • Linkage mapping • Expression analysis • Mutagenesis Harjes et al., Science 2008 HYDB1 has a large effect on BC • De3 • (KU1409/DE3/KU1409)S2-18-2-B Yan et al., Nature Genetics 2010

18. Total proA (ug/g) in 9 genotypic classes of 6 crosses • B104 • CML325, CML327, CML460 • Bank accessions (hets) • KUI carotenoidsyn-FS17-3-2-B

19. Seed genotyping pre-planting • Dry chipping using dog nail clippers • ≈10,000 seeds will be genotyped pre-planting this season

20. Steps to develop a hybrid cultivar (w/MAS) year UU x FF -> UF UF x UU -> UU:UF 1 Elim.50% 200UF seeds -> 50UU:100UF:50FF 50FF S1 ears -> 40FF S2 ears 2 1-2 best S6 x 3 tester -> 15 Stage3 hyb S7 -> HPLET 5 75% 40FF x tester -> 15 stage1 hybrids 15 best S3 -> 15 best S4 3 $10 per row + time 15 S4 (HPLC) x 3 tester -> 15 Stage2 hyb 5 best S5 -> 5 best S6 4-5 best x tester -> hyb Multilocation on-farm trials 4 6 $25-75/sample $5850 150+120+45 nursery + (45x6) trial rows 1-2 best -> release Multilocation on-farm demo’s 7 $2250 8 Begin marketing seed

21. What happens to provitaminA during cooking? Photo: N. Palacios Photo: H. De Groote

22. Effect of porridge preparation Shanshan Li et al., 2007 25% loss of β-carotene

23. Effect of snack preparation • 36% loss of provitamins A following nixtamalization and snack preparation by deep frying (n=13) Lozano Alejo et al., 2006

24. What happens to provitaminA after we eat them? …bioaccessibility In vitro assessment of bioaccessibilty of carotenoids from foods Mark Failla Department of Human Nutrition Parker, FASEB J, 1996

25. Combining proA and Zn …bioefficacy • Zn in the action of retinol dehydrogenase (retinol to retinal); essential pigment for vision • Zn is a probable co-factor for b-carotene mono-oxygenase (cleaves proA to vitA) • Zn deficiency depresses synthesis of the carrier protein of vitA => lower plasma retinol concentrations 2010: Stage 1 High Zn x ProA 2011: S2’s High Zn x ProA (HYDB1) to TC

26. Will farmers and consumers grow/ consume biofortified crops? • ProA sweet potatoes are orange; consumers prefer white • Will farmers choose to plant the biofortified varieties? • Will farmers choose to grow orange maize varieties? • Will seed companies market the new orange varieties?

27. No complaints from these consumers!

28. Agriculture for nutrition and health + + + + Plant breeding and agronomy Plant biochemistry Molecular biology + + + + + Education & marketing Socio-economics Nutrition Food technology Healthy families

29. Seeds of Discovery (SeeD) A Mexican initiative to contribute to global food security vis-à-vis climate change and resource scarcity by broadening the genetic base of global maize and wheat-breeding programs P. Wenzl, K. Pixley, G. Atlin, G. Edmeades, M. Banziger & many colleagues

30. Historical bottleneck 10 – 20 years Increased agricultural production Varietyadoption and improvement Breedingprograms Genetic resources SeeD: new genetic variation to raisefuturecropproduction

31. Factors limiting the use of GR • Factor 1: So many accessions, so little information! • Challenges to characterize accessions at phenotypic and molecular levels • Missing or ‘superficial’ passport data • Factor 2: Insufficient tools to mine information • Outdated/user-unfriendly data management tools • Limited query capabilities • Factor 3: How to effectively utilize exotic germplasm? • How to identify beneficial alleles in exotic germplasm? • How to capture novel, useful variation into elite backgrounds Many of the same challenges and issues of GEM!

32. Objectives of SeeD • Objective 1: To minemaize/wheat genetic resources for novel alleles and beneficial traits combining genotyping and phenotyping methods • Objective 2: To build on-line catalogues that facilitate the identification of beneficial genetic variation, and • Objective 3: To put in place practical delivery pathways that empower maize and wheat programs to broaden their genetic base by incorporating novel variation Marker-assisted introgression pipeline service facility Doubled haploid service facility? Develop and release “bridging lines”

33. SeeD: A technology intensive project! • Because of the size and complexity of the initiative, will require strategic alliances with key players. • Key partnerships: • Genotyping • Phenotyping • High-performancebioinformatics approaches for genetic analyses • Cyberinfrastructure for a SeeD web portal

34. Concept-development guidelines: • Focus on user base targeted by SeeD: maize and wheat breeding programs, especially public and SME breeding programs in developing countries • Design practical delivery paths that enable the adoption of novel and useful genetic diversity in breeding programs.

35. Draftstrategyformaize

36. General points (and workingassumptions) • The CIMMYT maize collection has about 26,000 accessions with no genotypic data, incomplete passport data, and some phenotypic data • Most accessions are heterogeneous with much more genetic variability among than within accessions. • Alleles that are rare globally, and at low frequencies in the accessions in which they occur, are unlikely to be very important or detectable. • Most traits in maize are highly polygenic. • Haplotypes with small effects likely control most variation

37. General points (cont’d) • Main diversity is to be found in the Mexican-Guatemalangermplasm, which has been in long co-existence with teosinte • Much genetic variability remains in teosinte, but there are few introgression populations available that could allow us access to this variability • Demand for direct access to landrace or teosinte accessions by breeders will be limited

38. Twomainproducts of SeeDformaize • Haplotype effect estimates for loci with small effects • Well-characterized accessions for specific traits to be used as donors for large-effect alleles

39. Haplotypes as theunit of evaluation • Because haplotypes are likely to be replicated across many accessions, it is the haplotype rather than the accession itself whose effect we want to estimate, and that is the unit of evaluation or selection • To sample haplotype frequencies and to begin estimating haplotype effects, one plant per accession will be initially genotyped at >1,000,000-plex, on the assumption that this would detect all but the rarest alleles

40. Test crosses • To estimate haplotype allele effects, the single-plant representatives of core accessions will be crossed to elite, adapted testers (testers as females) • The testcrosses will be phenotyped in screens of interest.

41. GEBVpredictionforallaccessions • Because haplotypes will usually be replicated across accessions, it not necessary to estimate the effect of each testcross with high precision via high levels of replication on individual accessions. • Based on allelic effects estimated by phenotyping the subset of core accessions, the entire collection will be examined for accessions that have high GEBVsfor traits of interest, and that are under-sampled in the existing elite germplasm. • New pre-breeding populations will be established from these accessions and improved by genomic selection.

42. “Details of this approach to delivery of new genetic variation for quantitative traits needs a lot more thought…”

43. Large-effectalleles • The objective is to identify accessions with high frequencies of unusual alleles with large effects on simple or oligogenic traits. There will likely be very few of these. • The accession is the unit of evaluation • The core and materials that have a high likelihood of having been selected for the trait (based on passport information) need to be phenotyped at high precision, either per se or in testcrosses.

44. Large-effectalleles • Map, and develop gene-based markers • Introgressthe allele of interest into elite inbreds(“bridge inbreds”), which breeding programs will be want to use • As donors for MAS-based conversion, or • As parents of pedigree starts. • SeeD probably needs to develop such inbreds as deliverables, to ensure that any genes discovered are truly accessible to smaller public and private breeding programs in the developing world

45. SeeD and GEM • Many opportunities for complementarity and partnership with GEM • Important to communicate often and learn from each other • Many questions we can work together to answer and enhance the use of genetic resources P.Wenzl@cgiar.org G.Atlin@cgiar.org K.Pixley@cgiar.org