Towards the development of new Jatropha varieties:

1
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
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
Jatropha biodiesel energy balance
Energy inputs
Energy outputs

• Cultivation
• -marginal sites
• -intensive agriculture
• Seed harvesting
• Oil extraction
• -mechanical
• -solvent
• Transesterification
• Transport of fuel
• Disposal of wastes

• Biodiesel
• Glycerol
• Seedcake
• -fertiliser
• -biogasification
• -animal feed

4
Priorities for Jatropha RD

• 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
Research collaboration
Centre for Novel Agricultural Products Graham
Lab Oilseed Research Metabolomics Facility
Method development Gene discovery/bioinformatics/p
lant 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
Phorbol esters
6 Jatropha PEs described to date

• 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

All thought to be derived from single parent
molecule, therefore same MW
Hirota et al., 1998. Cancer Res. 48, 5800-5804.
Haas et al., 2002. J. Nat. Prod. 65, 1434-1440.
7
Phorbol ester analysis- LC-MS
8
PE analysis of non-toxic seeds
Toxic
Non-toxic
ISTD
ISTD
HPLC UV detector trace
9
Location of phorbol esters within the seed
Testa 0.33 0.11 U mg-1
Inner skin 25.23 1.45 U mg-1
Endosperm 4.71 0.71 U mg-1
Embryo 0.55 0.03 U mg-1
Mature seed
10
Madagascar project
Seeds soil collected from 23 field sites
across Madagascar in 2007

• Analysis as follows
• Soil nutrients
• Seed kernel mass
• Oil content
• Phorbol ester content
• AFLP

11
Jatropha genotyping
In Gh Pu QR
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
Conventional plant breeding
Cross plants, e.g., high oil cultivated with wild
disease resistant
x
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
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
Developmental stage selection
Oil production
44
63
70
56
58
77
15
454 sequencing project
Toxic variety
Non-toxic variety
10,000
Conventional sequencing 2000 x 500 bp 1
Mbp 454 sequencing 400,000 x 235 bp 94 Mbp
cDNA from developing seeds
454 sequencing
gt200,000 reads each from toxic and non-toxic
seeds, av. 235 bp per read Total 98 Mbp
Toxic 10,995 contigs, 25,381 singletons Non-toxi
c 11,341 contigs, 25,301 singletons

• Assembled sequences

Digital northern

• Gene expression levels

• SNP/SSR marker detection

Marker assisted breeding (gt400 SNPs) Sufficient
for a dense map
16
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

• Terpene cyclase
• P450 oxygenases
• Acyltransferases

• 2

• 1

• 3

Tigliane diterpene
Phorbol ester
GGPP
Phorbol
Acyl-CoA
17
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
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
Acknowledgements
University of York
Universidad Autónoma de Chapingo
Jesús Axayacatl Cuevas-Sanchez Edgardo Bautista
Ramírez
Andy King Wei He Yi Li (Bioinformatics) Beate
Reinhardt Tony Larson Valeria Gazda
FOFIFA, Madagascar
Daniele Ramiaramanana
UNAM Morelos
Patricia León
Ecoregional Initiatives, Madagascar
Mark Freudenberger
Yara Phosyn (Soil analysis)
Funding Garfield Western Foundation
20
SNP markers
Example

• 11 chromosomes (1n)
• Genome (1c) approx 400 Mbp (unpublished)
• SNP AFLP markers should therefore produce a
  fairly dense map