biology to benefit society towards the development of new jatropha varieties: molecular and...

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


Jatropha & biofuel sustainabilityEnvironmental:GHG & energy balance – depends on land use, cultivation intensity and downstream processing

Social:Non-displacement of food production – dependent on land useRural 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


Jatropha biodiesel & energy balanceEnergy 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

0

1

2

3

4

5

6

thousand litres of oil per hectare

So

ybea

n

Su

nfl

ow

er

Rap

esee

d

Cas

tor

Jc-

was

tela

nd

Jc-

inte

nsi

ve

Oil-

pal

m

Main data - Fulton et al., (2006); Jatropha - ICRISAT Working Paper (2007)


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)


Research collaborationCentre for Novel Agricultural ProductsGraham Lab: Oilseed ResearchMetabolomics Facility: Method developmentGene discovery/bioinformatics/plant breedingDr 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.


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 activityi.e., Increase incidence of tumour formation in the presence of carcinogens

Phorbol nucleus

Diester 1Diester 2

Diester 3 & 4Diester 5

Diester 6

6 Jatropha PEs described to date:

All thought to be derived from single parent molecule, therefore same MWHaas et al., 2002. J. Nat. Prod. 65, 1434-

1440.

Hirota et al., 1998. Cancer Res. 48,

5800-5804.


Phorbol ester analysis- LC-MS

0 5 10 15 20 25 30 35 40 45 50 55 60Time (min)

0

50

100

150

200

250

300

mA

U

0

100

Rel

ativ

e A

bu

nd

ance

43.77

39.0344.59

45.3838.46 54.9338.13 45.79

56.85

39.020.93

41.30 58.9244.68

15.972.87 55.7751.8317.75 27.3223.1010.587.88

Mass detector:

m/z 727-728.5

UV detector:

300 350 400 450 500 550 600 650 700 750 800m/z

0

100

Rel

ativ

e A

bu

nd

ance

310.3

693.0727.7

709.9

367.1346.0 399.0

657.3

[M-diester-H2O+]

[M+NH4+]

Exact mass 710.4

-382.2

-18

OH

H

HO

HH

OH

O

O

O

O

O

Mass spectrum of phorbol ester


Min

utes

30 35 40 45 50

mA

U

020

040

060

080

010

0012

0014

0016

0018

0020

0022

00

mA

U

020

040

060

080

010

0012

0014

0016

0018

0020

0022

00

ToxicIS

TD

Min

utes

30 35 40 45 50

mA

U

020

040

060

080

010

0012

0014

0016

0018

0020

0022

00

mA

U

020

040

060

080

010

0012

0014

0016

0018

0020

0022

00

Non-toxic

ISTD

PE analysis of non-toxic seeds

HPLC: UV detector trace

102030405060708090

100

Rel

ativ

e A

bu

nd

ance

43.8

39.044.6

45.454.9

010

20

3040

506070

8090

100

Rel

ativ

e A

bu

nd

ance

54.212.7

13.2

13.4

36.920.6

56.513.8 53.448.3

48.958.243.0

Single toxic seed

20 non-toxic seeds

HPLC: Mass spectrometer trace


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


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

40%

45%

50%

55%

60%

TO2 BO1SO1 LA3 AM2MO3SA1 LA1 SO2AM3-1

BO2AM3-2

VF3AM3-2

TO1 LA2 AN1MO4SA2 MO2VF2 MO5MO1AM1VF1

oil content of kernal (%)

550

600

650

700

750

MO5 BO1 SO2 VF2 LA2 VF1 BO2

BO2 (rpt)

MO3AM1 MO4 TO1 SA1 MO1 LA1 TO2 SA2 AN1 AM2 MO2 SO1

MO2 (rpt)

LA3 VF3 AM3

Average seed mass (mg)


Jatropha genotypingIn 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


Conventional plant breeding

xCross 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


Marker assisted breedingInvolves 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

Var1 h1 ATGTTTGAACGACTTCAA 1 Var1 h2 ATGTTTGAACGACTTCAA 1 Var2 h1 ATGTTTGTACGACTTCAA 2 Var2 h2 ATGTTTGTACGACTTCAA 2 *

Markers include SNPs and AFLPs


Developmental stage selection

44

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

44 DAP56 DAP58 DAP63 DAP70 DAP77 DAP

Mature seed*

others

C20-24:0

18:3n3

18:2n6c

18:1n7c

18:1n9c

18:0

16:1n7

16:0

56 58 63 70 77

0.00%

5.00%

10.00%

15.00%

20.00%

25.00%

30.00%

35.00%

44 DAP56 DAP58 DAP63 DAP70 DAP77 DAP

Mature seed*

Develomental stage

% oil

Oil production


454 sequencing project

454 sequencing

>200,000 reads each from toxic and non-toxic seeds, av. 235 bp per read

Total = 98 Mbp

Toxic variety

Non-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 singletonsNon-toxic: 11,341 contigs, 25,301 singletons

•SNP/SSR marker detection

•Gene expression levels

£10,000Conventional sequencing:

2000 x 500 bp = 1 Mbp454 sequencing:

400,000 x 235 bp = 94 Mbp


Gene expression & candidate genesPE biosynthesis:A number of terpene cyclases, including one expressed only in the ‘toxic’ varietyNumerous CYP450 oxygenases

Other trait for which molecular markers could be developed:

Oil content/yieldSeed phytate levels Plant architectureDisease resistance

GGPP2Tigliane

diterpene1

Phorbol Phorbolester +

Acyl-CoA

31. Terpene cyclase2. P450 oxygenases3. Acyltransferases


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


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


AcknowledgementsUniversity of York

Andy KingWei HeYi Li (Bioinformatics)Beate ReinhardtTony LarsonValeria Gazda

Funding:Garfield Western Foundation

UNAM Morelos

Patricia León

FOFIFA, MadagascarDaniele Ramiaramanana

Jesús Axayacatl Cuevas-SanchezEdgardo Bautista Ramírez

Universidad Autónoma de Chapingo

Yara Phosyn (Soil analysis)

Ecoregional Initiatives, MadagascarMark Freudenberger


SNP markers 390 400 410 | | | India 01 ATGTTNGAACGACTTCAATTCGTTACCTN India 02 ANGTTNGAACGACTTCAATTCGTTACCTN India 03 ATGTTNGAACGACTTCAATTCGTTACCTT India 04 ATGTTNGAACGACTTCAATTCGTTACCTN India 05 ATGTTNGANCGACTTCAATTCGTTACCTG India 06 ATGNTTGAACNACTTCAATTCGTTACCTT India 07 ATGTTNGANCGACTTCAATTCGTTACCTT India 08 ATGTTNGAACGACTTCAATTCGTTACCTT India 09 ATGTTNGAACGACTTCAATTCGTTACCTT India 10 ATGTTNGAACGACTTCAATTCGTTACCTT Mexico 01 ATGTTNGTACGACTTCAATTCGCTACCTT Mexico 02 ATGTTNGTACGACTTCAATTCGCTACCTT Mexico 03 ANGTTNGTACGACTTCAATTCGCTACCTT Mexico 04 ATGTTNGNACGACTTCAATTCGCTACCTT Mexico 05 ATGTTNGTACGACTTCAATTCGCTACCTT Mexico 06 ATGTTNGTACGACTTCAATTCGCTACCTT Mexico 07 ATGTTNGTACGACTTCAATTCGCTACCTT Mexico 08 ATGTTNGTACGACTTCAATTCGCTACCTT Mexico 09 ATGTTNGTACGACCTCAATTCGCTACCTT Mexico 10 ATGTTNGTACGACTTCANTTCGC------ * *

Var1 A1 ATGTTTGAACGACTTCAA 1 Var1 A2 ATGTTTGAACGACTTCAA 1 Var2 A1 ATGTTTGTACGACTTCAA 2 Var2 A2 ATGTTTGTACGACTTCAA 2 * 782 polymorphisms in 370 contigs Var1 A1 ATGTTTGAACGACTTCAA 1 Var1 A2 ATGTTTGTACGACTTCAA 2 Var2 A1 ATGTTTGTACGACTTCAA 2 Var2 A2 ATGTTTGTACGACTTCAA 2 * 95 polymorphisms in 118 contigs Var1 A1 ATGTTTGAACGACTTCAA 1 Var2 A2 ATGTTTGTACGACTTCAA 2 Var1 A1 ATGTTTGAACGACTTCAA 1 Var2 A2 ATGTTTGTACGACTTCAA 2 * 13 polymorphisms in 37 contigs Var1 A1 ATGTTTGAACGACTTCAA 1 Var1 A2 ATGTTTGTACGACTTCAA 2 Var2 A1 ATGTTTGAACGACTTCAA 1 Var2 A2 ATGTTTGCACGACTTCAA 3 * 3 polymorphisms in 3 contigs Var1 A1 ATGTTTGAACGACTTCAA 1 Var1 A2 ATGTTTGAACGACTTCAA 1 Var2 A1 ATGTTTGTACGACTTCAA 2 Var2 A2 ATGTTTGCACGACTTCAA 3 * 1 polymorphisms in 1 contig

Example:

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

biology to benefit society towards the development of new jatropha varieties: molecular and...

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