1. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Cassava Biofortification: An Overview of IITA Harvest Plus Cassava Breeding Activities PA Kulakow, EY Parkes, NG Maroya, B Maziya-Dixon, H Ceballos, SK Friedrich, E Alamu, P Iluebbey, M Bakare, P Ilona, C Egesi, AGO Dixon 19 June 2012

2. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Biofortification Our goal is increase the nutritional quality of cassava to help reduce micronutrient deficiencies especially for women and young children Biofortification should a an economic and sustainable way to address hidden hunger Harvest Plus Phase 1 and Phase 2 has supported this work since 2004 Target countries – Nigeria and DR Congo

3. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Outline of presentation Stages of the cassava breeding program First release of ProVitamin A cassava varieties in Nigeria Next genotypes in the pipeline Early breeding stages High throughput screening tools - iCheck™ Carotene to complement other methods The future – new breeding methods

4. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Goal of IITA Biofortified Cassava Breeding Yellow cassava with a target concentration of 15 μg/g fresh wt. of β-carotene Roots with high dry matter to produce high quality biofortified foods from yellow cassava – garri, fufu, boiled roots Disease and pest resistance High yield Plant types preferred by farmers

5. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Typical Cassava Breeding Cycle Seedling nursery (SN) (no replication) Multi-location Uniform yield trial (UYT) (4 replications) Clonal evaluation (CE) (no replication) Preliminary yield trial (PYT) (2 replications) Advanced yield trial (AYT) (4 replications) Crossing Blocks – Source Populations National Variety Testing and On-Farm Trials Year 1 Year 2 Year 3 Year 4 Years 5 & 6 Multiplication and Release NARS Years 7 & 8 Rapid Multiplication One-cycle of recombination in 8 years.

6. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Cassava Agroecosystems in West Africa

7. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Performance of released ProVitamin A Varieties 2009-2010 Clone Virus Disease Severity Fresh Tuber YieldDry matterTotal carotene scoret/ha%μg/g frwt 01/13682.921.428.06.1 01/13711.222.126.58.1 01/14121.029.629.06.4 30572 (chk)2.821.038.00.6 Performance of 3 First Release Candidate Varieties in Ibadan Nigeria during 2009/10. Clone Virus Disease Severity Fresh Tuber Yield Dry matter Total carotene scoret/ha%μg/g frwt TMS I0113682.921.428.06.1 TMS I0113711.222.126.58.1 TMS I0114121.029.629.06.4 TMS I30572 (chk)2.821.038.00.6.

8. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org ProVitamin A Cassava Variety IITA TMS I011371

9. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org CRP 4: Biofortification Released pVAC varieties Parents Clonefemale x male IITA TMS 01/136894/0561 X 94/0263 IITA TMS 01/137194/0561 X 94/0263 IITA TMS 01/141295/0971 X 94/0561

10. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Historical Development of 01/1368, 01/1371, and 01/1412 YearActivity 1992-1993Parent’s seed produced 1994-1995Parent’s cloned 1999Parent clones hybridized 2000Seedling nursery in Ibadan 2001Genotypes cloned in Ibadan 2002Preliminary Yield Trial 2003Advanced Yield Trial 2004-2008Multi-location Yield Trials 2008-2010National Variety Release Trials – NRCRI 2010-2011On-farm trials 2011Variety Release Decisions and release

11. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Effect of Planting Date and Time of Harvest on Pro Vitamin A content in 3 yellow Cassava Varieties Locations : Ibadan, Umudike, Ubiaja Varieties: 01/1368, 01/1371 and 01/1412 Design: Split-split plot in Randomized Complete Block Design Main plot: Planting date Sub-plot: Harvesting age Sub-sub plot: Varieties

12. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Breeder's Seed Multiplication Established 2010 and cut in 2011 Ratooned in July-Aug 2011 Candidate VarietyArea (m 2 )Plant # IITA TMS I011368772921728 IITA TMS I011371626313066 IITA TMS I01141218644118 Combined 1368 and 13712080041600 Total3665680512 Multiplication ratio Foundation Seed ha established6516.25

13. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Breeder's Seed Multiplication available for planting in 2012 VarietyArea (m2)ha First Wave IITA TMS I011368 17504017.5 IITA TMS I011371 24544024.5 IITA TMS I011412 19747019.7 61.7 Second Wave Candidates IITA TMS I070593 11209011.2 IITA TMS I061635 504805.0 IITA TMS I070539 372003.7 19.3

14. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Outline of presentation Stages of the cassava breeding program First release of ProVitamin A cassava varieties in Nigeria Next genotypes in the pipeline Early breeding stages High throughput screening tools - iCheck™ Carotene to complement other methods The future – new breeding methods

15. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Mean CMD Severity Fresh yield Dry matter Total carotene Genotype t/ha%ug/g frwt TMS I0705931.110.624.311.7 TMS I0709521.93.518.79.2 TMS I0616351.110.122.18.8 TMS I0708243.310.319.58.8 TMS I070539111.820.18.7 TMS I0113682.717.922.78 TMS I0113711.712.620.96.9 Second Wave Candidate Varieties: Top genotypes from UYT Yellow Root 6 grown in ten environments

16. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org TMS I070593 shows stable high TC across ten environments TMS I070593

17. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Promising Clone IITA TMS I070593 Highest in TC over 5 locations (11.4 ug/g frwt) CMD Resistant Poundable Relatively high dry matter Good plant architecture Above average yield

18. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org National Performance Testing for Second Wave ProVitamin A Cassava Varieties National Coordinated Research Program 2011-2012 – 9 locations Yellow Roots White roots IITA TMS I070593 IITA TMS I010046 IITA TMS I070539 IITA TMS I010085 IITA TMS I061635 IITA TMS I011097 NR 07/0167CIAT CR 36-2 NR 07/0220NR 04/0053 NR 04/0071 NR 04/0328

19. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Outline of presentation Stages of the cassava breeding program First release of ProVitamin A cassava varieties in Nigeria Next genotypes in the pipeline Early breeding stages High throughput screening tools - iCheck™ Carotene to complement other methods The future – new breeding methods

20. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org 2011-12 Cassava Breeding Cycle Seedling nursery (SN) 43,845 Multi-location Uniform yield trial (UYT) 46 + 81 multilocational Clonal evaluation (CE) 1,625 Preliminary yield trial (PYT) 735 + 477 mapping popn Advanced yield trial (AYT) 473 + 522 multilocational Crossing Blocks – 652,192 HS 50,470 S1, FS National Variety Testing 14 Year 1 Year 2 Year 3 Year 4 Years 5 & 6 Multiplication 9.5 ha and Release NARS Years 7 & 8 Rapid Multiplication Additional Trials: Local Germplasm Genetic Gain Mapping Populations Special Studies

21. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org New sources of variation for total carotenoids Production of elite x elite crosses Need to increase the genetic base of yellow root germplasm to avoid crossing only closely related parents Sources –Latin America – CIAT Botanic seeds Tissue culture plantlets –Yellow root African landraces –Potential new sources from inbreeding

22. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org CIAT Seedling Nursery 2010-2011 CIAT HS Pedigree Genotype Number Total Carotene (µg/g fr. wt.) GM 36640029-4613.9 SM 3442 0043-1713.4 0043-2610.7 GM 3653 0027-613.3 0027-6512.3 0027-6611.3 0027-5610.7 GM 36580026-110.8 GM 3594 0014-110.5 0014-1210.5 SM 34270039-3010.3 SM 34430044-6810.3 Parameter Total Carotene Means7.25 N81 Min3.2 Max13.9

23. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Botanic Seed Groups and Seedling nurseries 2011-2012 Location Family No. No. surviving Ibadan (planting 1)* 112014,250 Ibadan (planting 2)4361494 Ikenne* 52110,551 Ubiaja33515,310 Zaria2233734 Total 43,845 Hybridization Group Elite Yellow Root X Elite Yellow Root Elite Yellow Root X Multiple Pest Resistance Elite Yellow Root X Poundable Elite Yellow Root X Yellow Root Other YR and WR X Poundable Yellow Root and CMD Resistance X CIAT Seedlings Elite Yellow Root Selfed Lines Other Selfed Lines Yellow Root and White Roots X CBSD Tolerant Lines Polyploid Parents Elite and other Yellow Root Half Sib Families *harvested Botanic Seed is available for distribution to partners throughout Africa

24. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Promising Parents: Ikenne Seedling Nursery – 2011-2012 obsparentN iCheck (n) % samples mean iCheckSEminmax 1I011610805613.80.811.516.5 2I070539485471013.20.48.018.6 3I06163528419712.70.66.118.1 4I07059398955612.60.48.821.1 5 GM 3653-66 (11.3) 955612.21.18.614.8 6I011646545911.90.99.714.8 7I011368155990611.50.35.918.9 8I07082460128511.30.46.915.0 9I07096210042 11.10.47.117.9 10GM 3569-1225124810.90.86.615.4 11I0113711469128910.90.25.918.1 12I07037415712810.90.96.117.8 58124481711.9

25. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Outline of presentation Stages of the cassava breeding program First release of ProVitamin A cassava varieties in Nigeria Next genotypes in the pipeline Early breeding stages High throughput screening tools - iCheck™ Carotene to complement other methods The future – new breeding methods

26. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org iCheck TM Carotene Portable Device to Estimate ProVitamin A in Cassava Samples Validated in comparison to spectrophotometer and also HPLc Rapid processing produces results in one day Field durable

27. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Comparison of HPLC, Spectrophotometer, NIRS and iCheck™ Carotene – Chopped root samples Trait Reference Values Range a Mean a SD a b-cryptoxanthin0.04 – 0.550.240.09 13-cis BC0.11 – 2.040.900.41 trans BC0.20 – 8.983.261.79 9-cis BC0.10 – 2.721.090.55 Total BC0.41 – 11.735.262.40 Total Carotenoids (Spectrophotometer) 0.46 – 10.555.922.42 Total Carotenoids (iCheck™ Carotene) 0.79 – 13.186.153.14

28. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Comparison of HPLC, Spectrophotometer, NIRS and iCheck™ Carotene – Mashed or blended root samples Trait Reference Values Range a Mean a SD a b-cryptoxanthin0.04 – 0.550.240.09 13-cis BC0.11 – 2.040.900.41 trans BC0.20 – 8.983.261.79 9-cis BC0.10 – 2.721.090.55 Total BC0.41 – 11.735.262.40 Total Carotenoids (spectrophotometer)0.46 – 10.555.922.42 Total Carotenoids (iCheck™ Carotene)0.79 – 13.186.153.14

29. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org 109 genotypes sampled in four replications (from 4 trials) correlation 0.83 R2 0.69

30. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Outline of presentation Stages of the cassava breeding program First release of ProVitamin A cassava varieties in Nigeria Next genotypes in the pipeline Early breeding stages High throughput screening tools - iCheck™ Carotene to complement other methods The future – new breeding methods

31. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Next Generation Cassava Breeding: Genomic Selection Modify the breeding process using selection based on genotype information 1.Reduce the cassava breeding cycle to one year 2.Enhance early flowering and recombination 3.Share information – Cassavabase.org 4.Efficient use of genomic tools

32. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Typical Cassava Breeding Cycle Seedling nursery (SN) (no replication) Multi-location Uniform yield trial (UYT) (4 replications) Clonal evaluation (CE) (no replication) Preliminary yield trial (PYT) (2 replications) Advanced yield trial (AYT) (4 replications) Crossing Blocks – Source Populations National Variety Testing and On-Farm Trials Year 1 Year 2 Year 3 Year 4 Years 5 & 6 Multiplication and Release NARS Years 7 & 8 Rapid Multiplication One-cycle of recombination in 8 years.

33. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Cassava Breeding on a One-Year Cycle Potential to increase gain from selection per unit time. Faster multiplication and phenotyping of selected genotypes to evaluate proof of concept for genomic selection Rapid production of potential varieties for farmer evaluation Rapid variety release and impact Complements two-year and longer approaches

34. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Genomic Selection on an Annual Cycle MonthsObjective Activity 1Activity 2Activity 3Activity 4 Jun-DecFirst cycle Recombination Crossing block June Hybridization Aug-Oct Harvest early seeds Oct-Dec. Harvest late seeds Jan-MaySeedling nurserySeed germination CMD - screening Multiplication Mar- May GenotypingDNAGBSGS model Jun-JunField - Phenotyping GBS – genotype by sequencing, GS – apply genomic selection statistical model Jun-DecSecond cycle Recombination Crossing block June Hybridization Aug-Oct Harvest early seeds Oct-Dec. Harvest late seeds Jan-MaySeedling nurserySeed germination CMD - screening Multiplication Mar- May GenotypingDNAGBSGS model Jun-JunField - Phenotyping

35. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org CRP RTB: Next Generation Cassava Breeding –Genotype by Sequencing Elite Cassava Breeding lines –Assembly of historical phenotypic data in genetic gain trials and uniform yield trials –Association mapping –Assessement of genetic diversity to facilitate exploitation of heterosis 641 Elite Cassava Breeding Lines Relationships between yellow root genotypes based on 4000 SNP markers

36. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Actions to Achieve a One Year Breeding Cycle Efficient organization of all field activities –Planting, hybridization, seed harvest, DNA processing and data analysis Fast cassava technology –Reduce time to onset of flowering and increase prolificacy of flowers –Can 6-month buds be grafted to high flowering root stocks? Optimal nursery management –Irrigation and fertilization Selection for reduced seed dormancy, high flowering and high fertility

37. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Cassavabase.org Database for genomic and phenotypic data to support Next Generation Cassava Breeding Source of information on genotypes including images, fingerprints, characterization Genomic selection tools

38. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org CRP-RTB: Cassava Trait Ontology 120 traits defined for ICASS database with partners 60 default traits identified for IBP fieldbook – trans. to Spanish, French, Portuguese Utilized by Cassavabase.org and Agtrial.org Root Neck Length CO_334:0000022 Root Shape CO_334:0000020 Stem Color CO_334:0000062 Root Number CO_334:0000011 Outer Skin Color CO_334:0000064

39. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Acknowledgement Project Support from Harvest Plus through IFPRI and CIAT with funding from the Bill and Melinda Gates Foundation Harvest Plus I (2004-2008) Harvest Plus II (2009-2013)

40. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org A Bright Future for Cassava Breeding