Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
Transcript of Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
1/33
Genetic Diversity of Jatropha curcasL. detected on AFLP markers
Wahyu PurbowasitoBIOTECH CENTER- BPPT
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
2/33
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.
$$ $
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
3/33
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.
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
4/33
The new green fuel extracted from the
seeds of the Jatrophaplant has an idealcharacteristic as future alternative
biodiesel
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
5/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
6/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
7/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
8/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
9/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
10/33
Molecular Markers
Reflect heritable differences inhomologous DNA sequences amongindividuals
They may be due to:
Base pair changes
Rearrangements (translocation or inversion)
Insertions or deletions
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
11/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
12/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
13/33
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
14/33
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
15/33
Strengths of AFLP
PCR-based
Requires minimal amounts of DNA
Automatable Robust, Reliable and reproducible
No prior sequence knowledge
High marker density
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
16/33
OBJECTIVES
To identify, characterize some of
potential plants/clones which werealready collected from 8 places, andgenetics variation based on AFLP
techniques
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
17/33
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
18/33
Padang
Tangerang
Purwakarta G. Kidul Kupang
Kendari Jayapura
Merauke
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
19/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
20/33
RESULT
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
21/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
22/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
23/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
24/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
25/33
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
6= Tangerang7= Gunung Kidul
8= Purwakarta9= natural rubber PB 260.
Selective band from primer E-
ACG and M-CAC
1 2 3 4 5 6 7 8M1/29
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
26/33
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
6= Tangerang7= Gunung Kidul
8= Purwakarta9= nat. rubber PB 260.
Polyacryl amid gel
eletrophoresis of primer E-
AAG and M-CTG
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
27/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
28/33
AFLP bands
Binary data
Dendogram
Software
PAUP*
Based on these data, clusteranalysis done used PAUP Software
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
29/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
30/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
31/33
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%)
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
32/33
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
-
8/14/2019 Genetic Diversity of Jathropha curcas L. Detected on AFLP Marker
33/33