Transcript non-toxic

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
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
BIOLOGY TO BENEFIT SOCIETY
Jatropha biodiesel & energy balance
Energy outputs
•Biodiesel
•Glycerol
•Seedcake
-fertiliser
-biogasification
-animal feed
0
Main data - Fulton et al., (2006); Jatropha - ICRISAT Working Paper (2007)
BIOLOGY TO BENEFIT SOCIETY
Oil-palm
Jc- intensive
1
Castor
2
Rapeseed
3
Soybean
4
Sunflower
5
Jc- wasteland
6
thousand litres of oil per hectare
Energy inputs
•Cultivation
-marginal sites
-intensive agriculture
•Seed harvesting
•Oil extraction
-mechanical
-solvent
•Transesterification
•Transport of fuel
•Disposal of wastes
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)
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.
BIOLOGY TO BENEFIT SOCIETY
Phorbol esters
6 Jatropha PEs described to date:
• Analogues of diacylglycerol activate protein kinase C (PKC)
• Acutely toxic
Phorbol
nucleus
Diester 3 & 4
Diester 1
Diester 2
Diester 5
Diester 6
All thought to be derived from single
parent molecule, therefore same MW
Haas et al., 2002. J. Nat. Prod. 65, 14341440.
BIOLOGY TO BENEFIT SOCIETY
• 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
Hirota et al., 1998. Cancer Res. 48, 58005804.
Phorbol ester analysis- LC-MS
[M-diester-H2O+]
O
310.3
100
43.77
Relative Abundance
Mass detector:
-382.2
H
O
H
39.03
44.59
45.38
45.79
38.46
38.13
54.93
Relative Abundance
OH
m/z 727-728.5
0
O
O
H
Exact mass
710.4
HO
OH
[M+NH4+]
-18
693.0
727.7
709.9
56.85
UV detector:
300
250
39.02
0.93
mAU
44.68
100
0
0
300
58.92
41.30
150
50
346.0
367.1
399.0
657.3
200
0
O
100
5
10
51.8355.77
17.75 23.1027.32
7.8810.58
15
20
25
30
35
40
Time (min)
BIOLOGY TO BENEFIT SOCIETY
45
50
55
400
450
500
550
m/z
600
650
700
Mass spectrum of
phorbol ester
15.97
2.87
350
60
750
800
PE analysis of non-toxic seeds
2200
2000
2000
2000
1800
1800
1800
1800
1600
1600
1600
1600
1400
1400
1400
1400
1200
1200
1200
1200
ISTD
800
600
800
600
600
400
200
200
0
0
600
400
400
200
200
35
40
Relative Abundance
100
90
80
70
60
50
40
30
20
10
100
90
80
70
60
50
40
30
20
10
0
43.8
Single toxic seed
39.0
44.6
45.4
54.9
54.2
12.7
20 non-toxic
seeds
13.2
13.4
20.6
36.9
56.5
13.8
48.3 53.4
48.9
43.0
58.2
0
0
Minutes
30
800
mAU
1000
1000
800
400
Non-toxic
Relative Abundance
mAU
1000
1000
2200
ISTD
Toxic
2000
mAU
mAU
2200
2200
Minutes
45
50
30
35
40
45
HPLC: UV detector trace
BIOLOGY TO BENEFIT SOCIETY
50
HPLC: Mass spectrometer trace
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
BIOLOGY TO BENEFIT SOCIETY
Madagascar project
Seeds & soil collected from 23 field sites across Madagascar in 2007
Average seed mass (mg)
750
700
650
600
AM3
VF3
LA3
MO2 (rpt)
SO1
MO2
AM2
AN1
SA2
TO2
LA1
MO1
SA1
TO1
MO4
AM1
MO3
BO2 (rpt)
BO2
LA2
AM2
VF1
VF2
LA3
BO1
BO1
SO2
MO5
TO2
550
BIOLOGY TO BENEFIT SOCIETY
55%
50%
45%
VF1
AM1
MO1
MO5
VF2
MO2
SA2
MO4
AN1
LA2
TO1
AM3-2
VF3
AM3-2
BO2
AM3-1
SO2
LA1
SA1
MO3
40%
SO1
Analysis as follows:
• Soil nutrients
• Seed & kernel mass
• Oil content
• Phorbol ester content
• AFLP
oil content of kernal (%)
60%
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
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
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
Markers
include SNPs
and AFLPs
Var1
Var1
Var2
Var2
h1
h2
h1
h2
ATGTTTGAACGACTTCAA
ATGTTTGAACGACTTCAA
ATGTTTGTACGACTTCAA
ATGTTTGTACGACTTCAA
*
BIOLOGY TO BENEFIT SOCIETY
1
1
2
2
•Plants with correct genotype
can then be subjected to
phenotypic verification
Developmental stage selection
Oil production
35.00%
100%
90%
30.00%
others
80%
% oil
25.00%
C20-24:0
70%
18:3n3
20.00%
60%
18:2n6c
15.00%
50%
18:1n7c
40%
18:1n9c
10.00%
18:0
30%
16:0
44
56
58
BIOLOGY TO BENEFIT SOCIETY
se
ed
*
AP
M
at
ur
e
D
AP
Develomental stage
77
D
AP
70
D
AP
63
D
AP
58
D
AP
56
D
at
ur
e
M
16:1n7
0%
44
se
ed
*
AP
77
D
AP
70
D
AP
D
63
D
58
D
56
D
44
AP
0.00%
10%
AP
20%
AP
5.00%
63
70
77
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
>200,000 reads each from toxic and nontoxic seeds, av. 235 bp per read
Total = 98 Mbp
•Assembled sequences
Toxic: 10,995 contigs, 25,381 singletons
Non-toxic: 11,341 contigs, 25,301 singletons
•Gene expression levels
•SNP/SSR marker detection
BIOLOGY TO BENEFIT SOCIETY
‘Digital northern’
Marker assisted breeding (>400 SNPs)
Sufficient for a dense map
Gene expression & candidate genes
PE biosynthesis:
A number of terpene cyclases, including one expressed only in the
‘toxic’ variety
Numerous CYP450 oxygenases
GGPP
1
Tigliane
diterpene
2
Phorbol
3
+
Acyl-CoA
1.
Phorbol 2.
ester 3.
Terpene cyclase
P450 oxygenases
Acyltransferases
Other trait for which molecular markers could be developed:
Oil content/yield
Seed phytate levels
Plant architecture
Disease resistance
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
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
BIOLOGY TO BENEFIT SOCIETY
Acknowledgements
University of York
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
BIOLOGY TO BENEFIT SOCIETY
Universidad Autónoma de Chapingo
Jesús Axayacatl Cuevas-Sanchez
Edgardo Bautista Ramírez
SNP markers
Var1
Var1
Var2
Var2
A1
A2
A1
A2
ATGTTTGAACGACTTCAA
ATGTTTGAACGACTTCAA
ATGTTTGTACGACTTCAA
ATGTTTGTACGACTTCAA
*
782 polymorphisms in 370 contigs
1
1
2
2
Var1
Var1
Var2
Var2
A1
A2
A1
A2
ATGTTTGAACGACTTCAA
ATGTTTGTACGACTTCAA
ATGTTTGTACGACTTCAA
ATGTTTGTACGACTTCAA
*
95 polymorphisms in 118 contigs
1
2
2
2
Var1
Var2
Var1
Var2
A1
A2
A1
A2
ATGTTTGAACGACTTCAA
ATGTTTGTACGACTTCAA
ATGTTTGAACGACTTCAA
ATGTTTGTACGACTTCAA
*
13 polymorphisms in 37 contigs
1
2
1
2
Var1
Var1
Var2
Var2
A1
A2
A1
A2
ATGTTTGAACGACTTCAA
ATGTTTGTACGACTTCAA
ATGTTTGAACGACTTCAA
ATGTTTGCACGACTTCAA
*
3 polymorphisms in 3 contigs
1
2
1
3
Var1
Var1
Var2
Var2
A1
A2
A1
A2
1
1
2
3
ATGTTTGAACGACTTCAA
ATGTTTGAACGACTTCAA
ATGTTTGTACGACTTCAA
ATGTTTGCACGACTTCAA
*
1 polymorphisms in 1 contig
BIOLOGY TO BENEFIT SOCIETY
Example:
India
India
India
India
India
India
India
India
India
India
Mexico
Mexico
Mexico
Mexico
Mexico
Mexico
Mexico
Mexico
Mexico
Mexico
01
02
03
04
05
06
07
08
09
10
01
02
03
04
05
06
07
08
09
10
390
400
410
|
|
|
ATGTTNGAACGACTTCAATTCGTTACCTN
ANGTTNGAACGACTTCAATTCGTTACCTN
ATGTTNGAACGACTTCAATTCGTTACCTT
ATGTTNGAACGACTTCAATTCGTTACCTN
ATGTTNGANCGACTTCAATTCGTTACCTG
ATGNTTGAACNACTTCAATTCGTTACCTT
ATGTTNGANCGACTTCAATTCGTTACCTT
ATGTTNGAACGACTTCAATTCGTTACCTT
ATGTTNGAACGACTTCAATTCGTTACCTT
ATGTTNGAACGACTTCAATTCGTTACCTT
ATGTTNGTACGACTTCAATTCGCTACCTT
ATGTTNGTACGACTTCAATTCGCTACCTT
ANGTTNGTACGACTTCAATTCGCTACCTT
ATGTTNGNACGACTTCAATTCGCTACCTT
ATGTTNGTACGACTTCAATTCGCTACCTT
ATGTTNGTACGACTTCAATTCGCTACCTT
ATGTTNGTACGACTTCAATTCGCTACCTT
ATGTTNGTACGACTTCAATTCGCTACCTT
ATGTTNGTACGACCTCAATTCGCTACCTT
ATGTTNGTACGACTTCANTTCGC-----*
*
• 11 chromosomes (1n)
• Genome (1c) = approx 400 Mbp (unpublished)
• SNP & AFLP markers should therefore produce
a fairly dense map