Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker

8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker

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Genetic Diversity of Jatropha curcasL. detected on AFLP markers

Wahyu PurbowasitoBIOTECH CENTER- BPPT


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INTRODUCTION

Increasing the prices ofworld fuel at the end of 2005,

has affected the production

cost of national and home

industrial sectors in

Indonesia

This also influenced the availability anddistribution of national energy.

$$ $


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Development of bio-fuel

became an alternative that hasto be considered which fit to

Indonesian condition, since

we have many bio-resources

with biodiversity for plant

producing oil.

One of the potential plant isJatropha curcas. The seed

contains about 25-35% oil.


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The new green fuel extracted from the

seeds of the Jatrophaplant has an idealcharacteristic as future alternative

biodiesel


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The cultivation ofJatropha curcas inIndonesia is short ofhigh quality clones.

There is hugevariation in seed yieldcapacity and oilconcentration in theseeds among theseedling used for

cultivation or plantation.

Problems Formulation


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Uniformity ofmaturity willreduce harvestingcost.

Fruits of Jatrophain one brunch ripe atdifferent time. It causes difficulty duringharvesting, that farmer has to select ripeningfruits only. This makes harvesting time slow

and high cost.

Problems Formulation


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As a result, plantvarieties may beproduced with

combination of traitspreviously too difficultor time- consuming toproduce otherwise.

Since some economical traits are quantitativelyinherited, using appropriate methods such asquantitative trait loci analysis, combined with marker-assisted selection, however would enhance thebreeding methods for Jatropha.

Problem Solving


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APPROACHES

Develop the F2 orbackcross populationbased on the selectedmother plants

Construct the linkagemapping using DNAmarkers

Mother plants collection with specific characters

In vitro propagation

DNA finger printing method to characterize themother plants


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Genetic Marker

Represent variation at a particular site onthe genome which is heritable, easy to

assay and can be followed over generations Type of genetic markers:

Morphological

Biochemical

Molecular


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Molecular Markers

Reflect heritable differences inhomologous DNA sequences amongindividuals

They may be due to:

Base pair changes

Rearrangements (translocation or inversion)

Insertions or deletions


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A wide array of different moleculartechniques are used to detect

polymorphism at the DNA level

Most Molecular markers fall into 3 basic

categories:Hybridization-based (non-PCR)

techniques

Sequence targeted and single locus PCR

Arbitrarily primed PCR techniques


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Amplified FragmentLenght Polymorphism

An arbitrarily amplified DNA techniquesthat uses ligation of adaptors to the ends

of restricted DNA and amplification withhemispecific primers

Amplification products are generaly

visualized by polyacrilamide gelelectrophoresis, and silverstaining


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Strengths of AFLP

PCR-based

Requires minimal amounts of DNA

Automatable Robust, Reliable and reproducible

No prior sequence knowledge

High marker density


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OBJECTIVES

To identify, characterize some of

potential plants/clones which werealready collected from 8 places, andgenetics variation based on AFLP

techniques


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Padang

Tangerang

Purwakarta G. Kidul Kupang

Kendari Jayapura

Merauke


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16 pairs of Primer used

Primer MseI

MCTA MCAC MCTG MCAG

Pr

imerEcoRI

EACA EACA

MCTA

EACA

MCAC

EACA

MCTG

EACA

MCAG

EACT EACT

MCTA

EACT

MCAC

EACT

MCTG

EACT

MCAG

EAAG EAAGMCTA

EAAGMCAC

EAAGMCTG

EAAGMCAG

EACG EACG

MCTA

EACG

MCAC

EACG

MCTG

EACG

MCAG


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RESULT


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Polyacryl amid gel

eletrophoresis of primer E-

ACA and M-CTG

1 2 3 4 5 6 7 8 9 M1M2

1000 pb

750 pb

500 pb

400 pb

300 pb

legend:M1= 1 kb ladderM2= 50 bp ladder

1=Padang2= Kupang

3= Merauke4= Jayapura

5= Kendari

6= Tangerang7= Gunung Kidul

8= Purwakarta9= natural rubberPB 260.

T l b f AFLP b d


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Primer Padang Kupang Merauke Jayapura Kendari Tangerang G.kidul Purwakarta Total

E-AAG M-CAC 57 51 61 40 33 59 59 47 407

E-AAG M-CAG 74 66 75 54 42 72 72 63 518

E-AAG M-CTA 97 110 108 101 80 116 113 110 835

E-AAG M-CTG 31 34 36 31 30 26 36 41 265

E-ACA M-CAC 95 109 101 100 87 107 105 109 813

E-ACA M-CAG 72 95 73 88 81 91 92 90 682

E-ACA M-CTA 78 78 79 66 57 85 80 82 605

E-ACA M-CTG 76 81 60 78 71 83 84 77 610

E-ACG M-CAC 27 39 27 28 38 33 36 32 260

E-ACG M-CAG 24 42 28 42 34 31 36 24 261

E-ACG M-CTA 53 72 76 75 68 76 71 72 563

E-ACG M-CTG 27 29 32 24 23 31 34 36 236

E-ACT M-CAC 87 60 86 56 43 82 90 90 594

E-ACT M-CAG 70 68 61 72 67 75 76 71 560

E-ACT M-CTA 108 94 104 91 81 99 104 100 781

E-ACT M-CTG 57 59 63 64 65 67 70 59 504

Total 1.033 1.087 1.070 1.010 900 1.133 1.158 1.103 8.494

Total number of AFLP bands

1 2 3 4 5 6 7 8 9


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500 pb

1 2 3 4 5 6 7 8 9

Legend:

1=Padang; 2= Kupang; 3= Merauke; 4= Jayapura; 5= Kendari; 6= Tangerang;7= Gunung Kidul; 8= Purwakarta; 9= natural rubber PB 260.

Primers Total Common Polimorphic Polimorphics bands


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Primers Total

bands

Common

bands

Polimorphic

bands

Polimorphics bands

(%)

E-AAG M-CAC 74 17 57 77,03

E-AAG M-CAG 95 29 66 69,47

E-AAG M-CTA 126 61 65 51,59

E-AAG M-CTG 46 14 32 69,57

E-ACA M-CAC 118 70 48 40,68

E-ACA M-CAG 126 32 94 74,60

E-ACA M-CTA 97 41 56 57,73

E-ACA M-CTG 108 30 78 72,22

E-ACG M-CAC 62 10 52 83,87

E-ACG M-CAG 64 7 57 89,06

E-ACG M-CTA 104 26 78 75,00

E-ACG M-CTG 41 16 25 60,98

E-ACT M-CAC 102 39 63 61,76

E-ACT M-CAG 97 35 62 63,92

E-ACT M-CTA 126 68 58 46,03

E-ACT M-CTG 82 37 45 54,88

Total 1.468 532 936

Rata-rata 91,75 33,25 58,5 63,76

1 2 3 4 5 6 7 8 9 M1M2


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1.000 pb

750 pb

500 pb

300 pb

250 pb

200 pb

legend:M1= 1 kb ladderM2= 50 bp ladder

1=Padang2= Kupang

3= Merauke4= Jayapura

5= Kendari


8= Purwakarta9= natural rubber PB 260.

Selective band from primer E-

ACG and M-CAC

1 2 3 4 5 6 7 8M1/29


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1.000 pb

750 pb

500 pb

300 pb

250 pb

200 pb

150 b

legend:M1= 1 kb ladder

M2= 50 bp ladder

1=Padang

2= Kupang

3= Merauke4= Jayapura5= Kendari


8= Purwakarta9= nat. rubber PB 260.

Polyacryl amid gel

eletrophoresis of primer E-

AAG and M-CTG


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Primer Padang Kupang Merauke Jayapura Kendari Tangerang G.Kidul Purwakarta

E-AAG M-CAC 1 3E-AAG M-CAG 7 6

E-AAG M-CTA 2 1

E-AAG M-CTG

E-ACA M-CAC 3 1 1

E-ACA M-CAG 5 12

E-ACA M-CTA 3 2

E-ACA M-CTG 9 2

E-ACG M-CAC 3 1 3 4 1 1

E-ACG M-CAG 1 4 1 1

E-ACG M-CTA 1 3 2 2

E-ACG M-CTG 2 1 1

E-ACT M-CAC 3

E-ACT M-CAG 10 1

E-ACT M-CTA 5 2 1

E-ACT M-CTG 3 3 1 1

Total 54 2 37 1 14 2 3 7

Total number of Clones specific DNA band

B d th d t l t


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AFLP bands

Binary data

Dendogram

Software

PAUP*

Based on these data, clusteranalysis done used PAUP Software


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Padang

Merauke

Kupang

Jayapura

Kendari

Purwakarta

Tangerang

Gunung Kidul

Rubber PB 260

100

87

86

100

99

100

Dendogram

Cluster A

Cluster B

Cluster B1

Cluster B2

Dendogram analysis compare with oil contents


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Padang

Merauke

Kupang

Jayapura

Kendari

Purwakarta

Tangerang

Gunung Kidul

Karet PB 260

100

87

86

100

99

100

Oil contents

30--35%

30--35%

30--35%

30--35%

35--40%

19--20%

19--20%

19--20%

Dendogram analysis compare with oil contents


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Based on AFLP marker analysis, we found genetic variationon Jatropha curcas.

From 16 primer pairs, total AFLP band 8.494 bands andwith mean of polimorphisme has 63,76%.

120 specific AFLP bands have detected from 8 clones.

From dendogram analysis: there are 2 major groups andPadang and Merauke groups have clearly differ from theother groups: Kupang, Jayapura, Kendari, Tangerang,Gunung Kidul, dan Purwakarta.

Tangerang, Gunung Kidul, and Purwakarta have found tobe one group and shows lower oil contents (19--20%)


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Faculty of Science, Universtty of Indonesia

Andreas Agustian

Retno Lestari

Lab of Gene Technology/Genetic Engineering

Biotech Center -BPPT Dudi Hardianto

Nila Kusumawaty

Indra Rachmawati

Anna Safarrida Teuku Tajuddin

Irvan Faizal

Nadirman Haska


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Documents

Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker