1. BIOLOGY TO BENEFIT SOCIETY Towards the development of new Jatropha varieties: Molecular and biochemical analysis of toxic and non-toxic lines Ian Graham, Centre for Novel Agricultural Products, University of York

2. BIOLOGY TO BENEFIT SOCIETY Jatropha & biofuel sustainability Environmental: GHG & energy balance – depends on land use, cultivation intensity and downstream processing Social: Non-displacement of food production – dependent on land use Rural income generation – need more reliable data Economic: Reliable income generation – dependent on oil price and political factors Jatropha has been promoted for its ability to grow on marginal lands Current Jatropha plantations use wild varieties More information needed on energy inputs v outputs to allow more sustainable practice

3. BIOLOGY TO BENEFIT SOCIETY Jatropha biodiesel & energy balance Energy inputs Cultivation -marginal sites -intensive agriculture Seed harvesting Oil extraction -mechanical -solvent Transesterification Transport of fuel Disposal of wastes Energy outputs Biodiesel Glycerol Seedcake -fertiliser -biogasification -animal feed Soybean Sunflower Rapeseed Castor Jc- wasteland Jc- intensive Oil-palm Main data - Fulton et al., (2006); Jatropha - ICRISAT Working Paper (2007)

4. BIOLOGY TO BENEFIT SOCIETY Priorities for Jatropha R&D Identify the available varieties using robust genotyping techniques Assess performance of different varieties under different field conditions Monitor crop performance in relation to agricultural inputs Develop varieties with improved agronomic value through plant breeding Develop ‘non-toxic’ varieties as a dual purpose crop (oil and animal feed)

5. BIOLOGY TO BENEFIT SOCIETY Research collaboration Centre for Novel Agricultural Products Graham Lab: Oilseed Research Metabolomics Facility: Method development Gene discovery/bioinformatics/plant breeding Dr Cuevas: Ethnobotanist with extensive experience of use of Jatropha in Mexico. Includes local non-toxic varieties. Mark Freudenberger - Ecoregional Initiative, Madagascar FOFIFA: ‘Le Centre National de la Recherché Appliqué de Développement Rural’: Jatropha trials across diverse climatic environments.

6. BIOLOGY TO BENEFIT SOCIETY Phorbol esters Analogues of diacylglycerol - activate protein kinase C (PKC) Acutely toxic Not destroyed by heat treatment Jatropha meal from ‘toxic’ varieties therefore cannot be used as animal feed Tumour promoting activity i.e., Increase incidence of tumour formation in the presence of carcinogens Phorbol nucleus Diester 1 Diester 2 Diester 3 & 4 Diester 5 Diester 6 6 Jatropha PEs described to date: All thought to be derived from single parent molecule, therefore same MW Haas et al., 2002. J. Nat. Prod. 65, 1434- 1440. Hirota et al., 1998. Cancer Res. 48, 5800- 5804.

7. BIOLOGY TO BENEFIT SOCIETY Phorbol ester analysis- LC-MS 051015202530354045505560 Time (min) 0 50 100 150 200 250 300 mAU 0 100 Relative Abundance 43.77 39.03 44.59 45.38 38.46 54.93 38.13 45.79 56.85 39.02 0.93 41.30 58.92 44.68 15.97 2.87 55.77 51.83 17.75 27.32 23.10 10.58 7.88 Mass detector: m/z 727-728.5 UV detector: 300350400450500550600650700750800 m/z 0 100 Relative Abundance 310.3 693.0 727.7 709.9 367.1 346.0399.0 657.3 [M-diester-H 2 O + ] [M+NH 4 + ] Exact mass 710.4 -382.2 -18 OH H HO H H OH O O O O O Mass spectrum of phorbol ester

8. BIOLOGY TO BENEFIT SOCIETY Toxic ISTD Non-toxic ISTD PE analysis of non-toxic seeds HPLC: UV detector trace 10 20 30 40 50 60 70 80 90 100 Relative Abundance 43.8 39.0 44.6 45.4 54.9 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 54.2 12.7 13.2 13.4 36.9 20.6 56.5 13.8 53.4 48.3 48.9 58.2 43.0 Single toxic seed 20 non-toxic seeds HPLC: Mass spectrometer trace

9. BIOLOGY TO BENEFIT SOCIETY Location of phorbol esters within the seed Mature seed Testa: 0.33 ± 0.11 U mg -1 Endosperm: 4.71 ± 0.71 U mg -1 Embryo: 0.55 ± 0.03 U mg -1 Inner ‘skin’: 25.23 ± 1.45 U mg -1

10. BIOLOGY TO BENEFIT SOCIETY Analysis as follows: Soil nutrients Seed & kernel mass Oil content Phorbol ester content AFLP Madagascar project Seeds & soil collected from 23 field sites across Madagascar in 2007

11. BIOLOGY TO BENEFIT SOCIETY Jatropha genotyping In Gh Pu QR 13 primer pairs selected for use in further studies These reveal 69/453 polymorphic bands (15.2%) Results indicate very little variation between accessions from India, Ghana, Tanzania & Madagascar

12. BIOLOGY TO BENEFIT SOCIETY Conventional plant breeding x Cross plants, e.g., high oil cultivated with wild disease resistant Phenotypic screen of all progeny – usually requires mature plants Limited by number of plants than can be brought to maturity and screened. Selected progeny then backcrossed with cultivated variety to remove undesirable traits

13. BIOLOGY TO BENEFIT SOCIETY Marker assisted breeding Involves creation of a genetic map using ‘Markers’ Co-inheritance of phenotype and ‘genotype’ reveals linked markers These can then be used in fast-track breeding programmes Genotype analysis performed at seedling stage More rapid, and higher throughput than phenotypic selection Plants with correct genotype can then be subjected to phenotypic verification Markers include SNPs and AFLPs

14. BIOLOGY TO BENEFIT SOCIETY Developmental stage selection 44 56 58 6370 77 Oil production

15. BIOLOGY TO BENEFIT SOCIETY 454 sequencing project 454 sequencing >200,000 reads each from toxic and non- toxic seeds, av. 235 bp per read Total = 98 Mbp Toxic varietyNon-toxic variety cDNA from developing seeds Assembled sequences ‘Digital northern’ Marker assisted breeding (>400 SNPs) Sufficient for a dense map Toxic: 10,995 contigs, 25,381 singletons Non-toxic: 11,341 contigs, 25,301 singletons SNP/SSR marker detection Gene expression levels £10,000 Conventional sequencing: 2000 x 500 bp = 1 Mbp 454 sequencing: 400,000 x 235 bp = 94 Mbp

16. BIOLOGY TO BENEFIT SOCIETY Gene expression & candidate genes PE biosynthesis: A number of terpene cyclases, including one expressed only in the ‘toxic’ variety Numerous CYP450 oxygenases Other trait for which molecular markers could be developed: Oil content/yield Seed phytate levels Plant architecture Disease resistance GGPP 2 Tigliane diterpene 1 Phorbol ester + Acyl-CoA 3 1.Terpene cyclase 2.P450 oxygenases 3.Acyltransferases

17. BIOLOGY TO BENEFIT SOCIETY Summary Jatropha varieties used in plantations are currently wild; crop improvement can increase yields CNAP has set up a research collaboration (Chapingo/Madagascar) to conduct research in priority areas Preliminary genotyping analysis reveals little difference in accessions collected in India, Ghana, Tanzania & Madagascar but significant variation with Mexican accessions CNAP have developed robust techniques for oil & phorbol ester analysis, and identified varieties lacking phorbol esters 454 sequencing projects has produced 97 Mbp of data from toxic and non-toxic varieties SNP markers will be used in mapping population and breeding programmes

18. BIOLOGY TO BENEFIT SOCIETY Perspectives The future is very promising for Jatropha breeding - there is substantial variation and we can benefit from new technologies and ‘piggy-back’ on knowledge gained from other crops to go after specific traits such as yield, architecture and disease resistance We need robust standards for describing genetic variation and ‘new’ elite lines We should set ourselves challenging targets for ‘rapid domestication’ of Jatropha and work together to achieve these for the benefit of all

19. BIOLOGY TO BENEFIT SOCIETY Acknowledgements University of York Andy King Wei He Yi Li (Bioinformatics) Beate Reinhardt Tony Larson Valeria Gazda Funding: Garfield Western Foundation UNAM Morelos Patricia León FOFIFA, Madagascar Daniele Ramiaramanana Jesús Axayacatl Cuevas-Sanchez Edgardo Bautista Ramírez Universidad Autónoma de Chapingo Yara Phosyn (Soil analysis) Ecoregional Initiatives, Madagascar Mark Freudenberger

20. BIOLOGY TO BENEFIT SOCIETY SNP markers Example: 11 chromosomes (1n) Genome (1c) = approx 400 Mbp (unpublished) SNP & AFLP markers should therefore produce a fairly dense map