1. Using rice science to developrice varieties for better human nutrition B. P. Mallikarjuna Swamy November 14, 2012
2. Outline• Importance of rice in human nutrition• Micronutrient deficiencies (MD)• Resources, efforts and approaches to address MD• Biofortification of Provitamin A• Biofortification of Fe and Zn• QTLs and genes for Fe and Zn enrichment in rice• Environmental effect on Fe and Zn biofortification• Proof of the concept and the status of biofortification• Benefits of Fe and Zn biofortification in rice• Plans and time line
3. Rice is Life Energy • Rice is the major staple food crop for more than half of the worlds population • Rice supplies 30-50% to the daily caloric intake • Rice is the major source of employment • Rice plays an important role in food security • Time has come to play role in nutritional security Rice and Food security Nutrition Compositon Brown rice White rice Units800 8.0 Calories 111 123 Kcal Production Population700 7.0 Moisture 12.9 8.9 g Employment600 6.0 Total fat 0.9 0.37 g500 5.0 Protein 2.6 2.9 g Total carbo 23 26 g400 4.0 Dietary fibre 1.8 0.9 g300 3.0 Sugars 0.35 0.11 g200 2.0 Calcium 10 19 mg100 1.0 Magnesium 43 9 mg 0 0.0 Iron 0.42 0.24 mg 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2009 Zinc 0.63 0.5 mg Stuttgart, 1991
4. Nutritional improvement is a priority area of rice research Vitamin AYield potential/Stability Biotic stresses eye sight problem reduced immunity night blindness retarded growth, dry skin, Iron (Fe) Anemia Reduced immunity, Dizziness, headache,Abiotic stresses chest pain, weakness, glossitis Healthy and Quality rice Frequent worm infestation Zinc(Zn) Reduced growth, immunity Vitamin A, Iron and Zinc Loss of appetite , weight loss diarrhea, skin dryness, rashes, reduced fertility, allergy, hair loss, bowel inflammation
5. Major causes of illness and disease in low income countries Developed: 10% Developing: 61% Worldwide: 49% > 3 billion people afflicted Zn deficiency
6. Approaches to relieve Vitamin A, Iron and Zinc deficiencies1. Change of diet2. Supplementation Fe and Zn in modern rice varieties3. Biofortification Fe (mg/100g Zn (mg/100g of Varieties of brown rice) brown rice) Swarna 0.78 2.28 - Increase in concentrations MTU1010 0.73 2.54 IR64 1.05 1.05 Suraksha 1.06 2.53 - Increase in promoter compounds BR 2655 1.05 2.37 Improved BPT 1.07 2.2 - decrease in anti-nutrients Three billion live on less than 2 $ per day, 1.5 billion on less than 1 $ per daysaving one healthy life year cost as little as and cannot afford a diversified diet or$US 0.73–7.31 if both wheat and rice biofortified industrially produced supplements Biofortification of rice varieties is the most sustainable, targeted and cost-effective approach to alleviate the problems of Vit A and micronutrient deficiency!!
7. Target crops and alliance for Bio-fortificationTarget crops in Africa Target crops in Asia Rice alliance for enhancing micronutrients Country/Region Institute Australia Adelaide University School of Agriculture, Waite Campus Bangladesh Bangladesh Rice Research Institute Germany University of Freiburg Indonesia Indonesian Institute for Rice Research, Plant Breeding Division + Japan Agriculture University of Indonesia University of Tokyo Philippines International Rice Research Institute Philippine Rice Research Institute Maligaya, Muñoz Switzerland Syngenta United States International Food Policy Research Institute (IFPRI) Michigan State University Childrens Nutrition Research Center, Baylor College of Medicine
8. Frame of work for Biofortified food crops by Harvest Plus Biofortfied crop must be: • High yielding and profitable • Effective and efficient in reducing malnutrition • Acceptable to both farmers and consumers in the target region
9. Variability for Pro Vitamin A, Iron and Zinc in rice germplasmProvitamin A: No variability Rice germplasm at IRRI gene bank O. sativa 111,123Iron: Variability exists - upto 35ppm O. glaberrima 1,657 Wild Oryza spp. 4,465Zinc: Variability exists - upto 45ppm Related genera 15 Total 117,263 Gene modification where necessary Breeding where possible Genetic engineering – Vitamin A Breeding/GE - Fe and Zn How to use this in a faster and efficient way?
10. “I challenge the next generation to use newscientific tools and techniques to addressthe problems that plague the world’s poor.” Dr. Norman Borlaug Combination of Breeding and Biotechnology hastens the process of development of Commercial ProductsGenomics based technologies have been successfully used in plant breeding to address the complex problems
11. Advances in rice genomics and modern breeding programs Nipponbare (Japonica) 93-11 (Indica) SNP-detection assays for rice Description # SNPs Platform Reference O. sativa Diversity array 1M Affymetrix McCouch, per. Comm. O. sativa Diversity Array 44,100 Affymetrix Tung et al. (2010) Global Diversity Primer Set 4,357 Sanger sequencing Ebana et al. (2010) O. sativa Diversity OPA 1,536 Illumina GoldenGate Zhao et al. (2010) Japanese Core Set 768 Illumina GoldenGate Yamamoto et al. (2010) O. sativa_indica × japonica 384 Illumina BeadXpress Thomson et al. (2011) O. sativa_indica/aus 384 Illumina BeadXpress Thomson et al. (2011) O. sativa_tropical japonica 384 Illumina BeadXpress Thomson et al. (2011) O. sativa_indica × O. rufipogon 384 Illumina BeadXpress Thomson et al. (2011) O. sativa_japonica × O. rufipogon 384 Illumina BeadXpress Thomson et al. (2011) Mc Couch et al. 2011 1000 and 3000 rice genome sequencing projects Next generation sequencing technologies (NGS) Further hasten the Genotype by sequencing technology (GBS) breeding process!
13. Biofortification of Pro Vitamin A Lycopene cyclases (LCYs), alpha and beta carotene hydroxylases (HYD) are produced in the wild type endosperm But phytoene synthase, and one or both carotene desaturases – Phytoene desaturase and Z-Carotene desaturase are not produced - Schaub et al. 2005
14. Development of efficient construct for Pro Vitamin A GR II – Best construct so far •Endosperm specific promoter Glutelin – Gt1p • Nos terminator •Carotene desaturase from E. uredovora catalysing multiple steps in carotenoid synthesis • Maize phytoene synthase • Maize ubiquitine promoter • Phosphomannose –isomerase marker system • T-DNA right and left border sequence
15. Development of ß-Carotene rice in the background of popular rice mega varieties by MABC IRRI
16. Versions of Golden riceGolden rice ß-CarotenePrototype 1.6ug/gGolden rice 1 (SGR1) - change of promoter 5- 7ug/gGolden rice 2 – replacement of daffodil Pys with maize gene 31ug/gGR2 GR1 Wild-Type Paine et al. 2005
17. Bioavailability tests and expected release of Golden riceBioconversion of ß –Carotene to Vit A inGolden rice is better than conventional foods• Excellent in both in Adults and children• Effect of fat is minimal
18. Biofortification of Iron and Zinc in rice
19. Genotypic variation for Iron and Zinc content in rice grain Fe and Zn content in land races and wild rice species Fe content Zn content Genotype ug/g ug/g WR-1 (O. officinalis) 17.5 39.3 WR-2 (O. latifolia) 18.6 36.9 WR-3(O. nivara) 12.5 32.4 WR-8 (O. nivara) 21.2 41.7 WR-171 (O. longistaminata) 21.1 38.1 WR-175(O. officinalis) 22.6 39.1 Anandan et al.2011 Chandel et al.2011
20. Effect of polishing on Iron and Zinc content in rice grain Longhva et al. 2011
21. Iron and Zinc in rice grains , antinutrients and promoters Antinutrients Phytic acid, tannins, lectins, fibre, oxalic acid, phenols heavy metals, Promoters Organic acids, fatty acids, aminoacids, Vit A,Low phytic acid rice mutant (lpa) Prebiotics Furctans, Oligosaccharides Chr 3 Phenotyping phytate in rice grains Fine mapped region of lpa
22. Iron and Zinc phenotyping methods More accurate methods - ICP-OES, AAS, XRF
23. Genetic basis of high Iron and Zinc content in rice grainsHeritability Genetic basis• Moderate narrow sense heritability • Additive genetic effects• Moderate to high broad sense heritability • Dominance genetic effects • Epistatic effects • Maternal effects • Significant genotype x environment Are there any major effect QTLs worthy of MAS? Yes!
24. Heterosis and Correlation for Iron and Zinc content in rice Correlation Fe and Zn References 0.346* Anandan et al. 2011 0.691*** Stangoulis et al. 2007 0.215* Oliviera et al. ??? 0.700*** Swamy et al. 2009
25. Molecular diverity analysis and mapping QTLs for Iron and Zinc from wild progenitor species Swamy et al. 2009
26. Development of mapping population BC1Parents F1 Swarna Trans segregant BC2 BC2F2 Swamy et al. 2009
27. Phenotyping of mapping population Fe concentrations in Swarna X O.nivara F2 population (S) Zn concentrations in Swarna X O.nivara F2 population (s) 140 60 55 54 123 120 50 100 No. of linesNo. of lines 40 80 26 28 30 60 20 40 24 10 16 4 20 5 0 0 1-3.9 4-6.9 7-9.0 10.0 - 13.9 14.0 - above 5.0 - 10.0 10.0 - 15.0 15.0 - 20.0 60.0 - 65.0 Iron in ppm Zn in ppm ….normal distribution
31. Co-location of Iron and Zinc QTLs with resistance genes (H. Biradar et al. 2007)
32. Genes involved Iron and Zinc uptake and accumulation Ferritin – Storage Phytase – Antinutrient reduction NAS – Uptake and transport
33. Location of genes involved Iron and Zinc homeostasis Giraldo et al. at CIAT have developed and validated functional SNP markers for 23 genes involved in Iron and Zinc Gross et al. 2003 accumulation in rice
34. Environmental influence on Fe and Zn content in rice grains Effect of water management Effect of Nitrogen application Effect of Nitrogen application Effect of various soil parameters on Fe and Zn uptake
35. Iron and Zinc content of rice genotypes across the locations soil properties influence phyto availability of micronutrients, yet genetic makeup of a plant determines its response to varied soil conditions Chandel et al.2010
36. Proof of the concept of Iron and Zinc Biofortified riceMS13 – Maligaya Special – with high Fe and Zinc released in PhilippinesImproved line IR68144-3B-2-2-3 – Fe (21ppm) and Zn (34 ppm) in Brown rice, 80% improvement over IR 72 after polishingThree improved IR lines - with high Fe and Zn are in advanced stage of evaluation in Bangaldesh - IRRI Nicaragua - CT15679-1-1-1-4 Fe (5-6 ppm) and zinc (21 ppm) Panama - released four varieties Colombia - FA336-1-1-V5-MA Dominican Republic - CT18245-11-6-2-3-4-3-M Fe(4.62ppm) and Zn (14.17 ppm) CT18247-11-5-2-3-1-1 Fe (5.60ppm) and Zn (15.93 ppm ) - Martinez et al.2010Improved Nipponbare - OsNAS2 – Fe ( 14 -19ppm) and Zn( two fold increase) - Jhonson et al.2011
37. Current status of Biofortification projects Countries: Philippines, Bangladesh, Indonesia, and India Higher beta carotene (Golden Rice)• Current level: ~ 0 ppm Product Development and• Target level: 6-8 ppm (+50 % EAR) Deployment; future improvements• Level reached: 14 ppm Higher Zinc Rice Product Validation and Development;• Current level: 16 ppm research• Target level : 24 ppm (+40 % EAR)• Level reached: 25/51 ppm• Higher Iron Rice• Current level: 2 ppm Product Validation; research• Target level: 12-14 ppm (+30 % EAR)• Level reached: 8/12 ppmEAR = estimated average requirement (USA) G. Barry, IRRI
38. Biofortification of Vitamin A, high Iron and Zinc – Gist• Golden rice 1 (6ppm) and GR2 (31ppm) developed, introgression to popular varieties by MAS in progress.• GR- MAS products tested for bio availability, efforts are going on to release in partner countries.• GR I expected to supply up to 40-50% of RDA and GR II expected to meet 100% of RDA.• Variability exists within the rice gene pool for Fe and Zn and can be improved by breeding approaches.• Fe and Zn accumulates in different compartments within rice, absorption and accumulation positively correlated, moderate to high heritability, so simultaneous improvement is possible.• Environmental factors influence the Fe and Zn content, genetic make up of the rice genotypes play very important role in stable performance.• Germplasm with high Fe and Zn identified , used in developing improved lines with high Fe and Zn.• Lines with high Fe and Zn developed in the I phase of the project are being released.• Bioavailability tests with high Fe rice showed 20% improvement.• The improved lines expected to supply 40-50% of RDA of Fe and Zn.• Major effect QTLs for Fe and Zn identified , needs to be validated and introgressed to develop high Fe and Zn MAS products.
39. Benefits of Iron and Zinc Biofortified rice Higher yield under Fe and Zn deficiency Seed enriched with Fe and Zn
40. Iron and Zinc Biofortification – Time line • New high Fe and Zn donors with low phytic acid identified • Development of improved lines with high Fe and Zn 2013- 2015 • Mapping populations developed and large effect QTLs identified • QTLs validated for consistency - genetic backgrounds and environment Bio efficacy tests • MAB –QTL pyramiding for Fe and Zn initiated • Identification of novel QTLs from wild progenitor species of rice • QTL pyramiding in new varieties, combined with Fe and Zn transgenic, with Vitamin A 2016 - 2018 • Fine mapping of selected large effect QTLs • Candidate gene analysis and development of functional markers Bioavailability , • MAS products successfully developed and tested in TPE and released • Product development, testing, release, candidate gene identification continued • Candidate genes validated by transgenic approaches in collaboration2018 beyond • Genetic stocks developed for best combination of QTLs • Understanding the mechanisms for high Fe and Zn accumulation in rice grains • QTLs pyramided and tested in hybrid parental backgrounds • MARS and GWS initiated for stable and better genetic gain
41. “ Food is the moral right of all who are born into this world.” - Borlaug“Nutritious food is the moral right of all who are born into this world.”
45. Pyramiding of major effect drought grain yield QTLs DTY1.1, DTY2.1 and DTY3.1 in SwarnaSub1 Background recovery of Swarna ILsBC4F3 Swarna lLs (Two QTLs + Sub1) Submergence screening 1 day after draining BC4F3 Swarna lLs (Three QTLs + Sub1) 6 days after draining Swarna
46. Introgression of major effect drought grain yield QTLs DTY3.1 and DTY12.1 Anjali IR81896-B-195 X Anjali (DS2010) Fore ground selection Major effect drought grain yield QTLs ( DTY3.1) Additive QTLs Chr Interval R2 Donor F1 X Anjali (WS2010) Fore ground selection effect DTY3.1 3 RM520-RM16030 30 25 Apo BC1 X Sub1Swarna (DS2011) Fore ground selection DTY 12.1 12 RM28048-RM28166 36 47 Way Rarem BC2F1 (WS2011) Fore ground selection Selection of BC2F2 (DS2012) homozygote for DTY3.1 IR 84984-83-15-18-B-B-93 X Anjali (DS2010) Fore ground selection ( DTY12.1) F1 X Anjali (WS2010) Fore ground selection BC1 X Sub1Swarna (DS2011) Fore ground selection Anjali lLs with DTY12.1 , 12DAS BC2F1 (DTY3.1) X BC2F1 (DTY12.1) (WS2011) Fore ground selectionFore ground selection Selection of BC3F1 BC2F2 (DS2012) homozygote for DTY 12.1 Selection of homozygote BC3F2 (WS2012) for DTY3.1 and DTY12.1 Selected homozygotes with DTY3.1, DTY12.1 and their combinations will be tested under drought DS2013 Anjali lLs with DTY3.1 12DAS
48. Intogression of DTY QTLs in Korean parents (RDA) Back ground Stage QTLs Hanareumbyeo BC1 DTY1.1 and DTY2.2 Jinmybyeo BC1 DTY1.1 and DTY2.2 Gayabyeo BC1 DTY1.1 and DTY2.2 Sagnambatbyeo BC1 DTY1.1 and DTY3.1BC2 – confirmed for foreground markers and will be backcrossed Introgression of QTLs in Smbha Mahsuri• QTLs – DTY2.2 and DTY4.1• Generation - BC2F2• Foreground selection and selection of homozygotes• Background selection
49. Wild species derived mapping population development Rice varieties SL No Female Parentage Male Parent Diversity 1 MTU 1010/IRGC 81994 MTU1010 based onSSR markers 2 MTU 1010/IRGC 105757 MTU1010 3 MTU 1010/IRGC 106109 MTU1010 4 MTU 1010/IRGC 106283 MTU1010 5 MTU 1010/IRGC 106285 MTU1010 6 Saro 5/IRGC 81994 Saro 5 7 Saro 5/IRGC 105757 Saro 5 Wild accessions 8 Saro 5/IRGC 106109 Saro 5 9 Saro 5/IRGC 106283 Saro 5 10 Saro 5/IRGC 106285 Saro 5 11 NericaL-14/IRGC 105757 NericaL-14 12 Nerica-L-14/IRGC 106277 NericaL-14 13 Nerica-L-14/IRGC 106285 NericaL-14 14 Nerica-L-31/IRGC 104639 Nerica-L-31 15 Nerica-L-31/IRGC 106277 Nerica-L-31 16 Nerica-L-5/IRGC 106109 Nerica-L-5 17 Nerica-L-7/IRGC 106283 Nerica-L-7 18 Nerica-L-8/IRGC 106285 Nerica-L-8 Can be used for mapping Fe and Zn content in rice grains!!
50. qDTY3.1 : Major effect and consistent QTL in Swarna and BR11 QTLs Chr Interval R2 Additive effect Donor Recipient • QTL validation DTY3.1 3 RM520-RM16030 30 25 Apo Swarna DTY3.1 3 RM15935-RM520 20-25 22 Apo BR11 • Fine mapping • Physiogical characterization • Insilico candidate gene identification Apo x Swarna Apo x BR11 •Transciptome analysis to identify differentially expressed genes • Validation of genes by RT and QRT PCR