COVER Buku Studi on the Level

ISBN 978-602-8964-17-3
STUDY ON THE LEVEL OF GENETIC DIVERSITY OF
Diospyros celebica, Eusideroxylon zwageri AND
Michelia spp. USING RAPD MARKERS
Anthonius YPBC Widyatmoko
ILG Nurtjahjaningsih
Prastyono
CENTER FOR CONSERVATION AND REHABILITATION
RESEARCH AND DEVELOPMENT,
FORESTRY RESEARCH AND DEVELOPMENT AGENCY
MINISTRY OF FORESTRY
BOGOR – INDONESIA
2011
STUDY ON THE LEVEL OF GENETIC DIVERSITY OF
Diospyros celebica, Eusideroxylon zwageri AND Michelia spp.
USING RAPD MARKERS
Anthonius YPBC Widyatmoko
ILG Nurtjahjaningsih
Prastyono
ITTO PROJECT PD 539/09 REV.1 (F)
IN COOPERATION WITH
CENTER FOR CONSERVATION AND REHABILITATION
RESEARCH AND DEVELOPMENT,
FORESTRY RESEARCH AND DEVELOPMENT AGENCY
MINISTRY OF FORESTRY
BOGOR – INDONESIA
February 2011
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri
and Michelia spp. Using RAPD Markers
Copyright©2011
By: Anthonius YPBC Widyatmoko, ILG Nurtjahjaningsih and Prastyono
This report is a part of program ITTO Project PD 539/09 Rev.1 (F) “Promoting
Conservation of Selected Tree Species Currently Threatened by Habitat Disturbance and
Population Depletion” in cooperation with Center for Conservation and Rehabilitation
Research and Development, Forestry Research and Development Agency, Ministry of
Forestry.
ISBN 978-602-8964-17-3
Published by
Center for Conservation and Rehabilitation Research and Development,
Forestry Research and Development Agency, Ministry of Forestry, Indonesia
Jl. Gunung Batu No. 5 Bogor, Indonesia 16610
Phone : 62-251-8315222, 7520067
Facs.
: 62-251-8638111
e-mail : [email protected]
Printed by
CV. Biografika, Bogor
PREFACE
This Technical Report is result of Activity 1.2.1, “to observe the level of genetic
diversity and vulnerability of selected species to determine the conservation
strategy of the selected species”. The activity is part of ITTO Project PD 539/09
Rev.1 (F), “Promoting Conservation of Selected Tree Species Currently Threatened
by Habitat Disturbance and Population Depletion”.
Sincere thanks and appreciation go to the Project Coordinator, Dr. Ir. Murniati,
M.Si for support and invaluable advice. I would like also to express my gratitude
thanks to all staff of ITTO Project PD 539/09 Rev.1 (F) for administration support.
Colleagues at Molecular Genetic Laboratory, Center for Forest Biotechnology and
Tree Improvement Research, are gratefully thanks for their support on laboratory
works.
Finally, we hope the result of this activity can support genetic conservation
activities and strategy of the selected species in Indonesia.
Bogor, February 2011
Authors
iii
iv
TABLE OF CONTENT
PREFACE
…………………………...………………………………….
TABLE OF CONTENT
iii
………………………………………………...
v
LIST OF TABLES ……..…………………………………………………
vi
LIST OF FIGURES ………………………………………………………
vii
ABSTRACT ……………………………………………………………...
viii
INTRODUCTION ………………………………………………………..
1
APPLIED METHODOLOGY …………………………………………...
DNA extraction ………………………………………………………
RAPD analysis ……………………………………………………….
Screening of RAPD primers …………………………………………
2
2
2
3
PRESENTATION OF THE DATA ……………………………………….
4
ANALYSIS AND INTERPRETATION OF THE DATA AND RESULTS
Screening of RAPD Primers …………………………………………
Genetic Diversity and Population Relationship of Diospyros celebica
Genetic Diversity and Provenances Relationship of Eusideroxylon
zwageri ……………………………………………………………….
Genetic Diversity and Provenances Relationship of Michelia spp …..
5
5
5
8
10
CONCLUSIONS …………………………………………………………
13
RECOMMENDATIONS …………………………………………………
14
IMPLICATION FOR PRACTICE ……………………………………….
15
BIBLIOGRAPHY ………………………………………………………..
17
v
LIST OF TABLES
Table 1.
Table 2.
Table 3.
Table 4.
Table 5.
Table 6.
vi
List of used samples …………………………………………...
Number of screened RAPD primer, selected primers, total
number of loci and means of loci per primer for the three
species …………………………………………………………
Genetic diversity within (diagonal) and between (below
diagonal) provenances for three provenances of Ebony from
South Sulawesi and Central Sulawesi …………………………
Genetic diversity within (diagonal) and between (below
diagonal) group for six sample groups of Ebony from South
Sulawesi and Centre Sulawesi …………………………………
Genetic diversity within (diagonal) and between (below
diagonal) groups for six sample groups of Ulin from Jambi and
South Sumatera ………………………………………………...
Genetic diversity within (diagonal) and between (below
diagonal) groups for seven sample groups of Michelia spp.
from South Sumatera and East Java …………………………...
4
5
6
7
9
10
LIST OF FIGURES
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Dendrogram of genetic relationship between three
provenances of Ebony from South Sulawesi (SS) and Centre
Sulawesi (CS) based on UPGMA (Nei, 1978) ..........................
Dendrogram of genetic relationship between six sample
groups of Ebony from South Sulawesi (SS) and Centre
Sulawesi (CS) based on UPGMA (Nei, 1978) ..........................
Dendrogram of genetic relationship between six sample
groups of Ulin from Jambi (Jb) and South Sumatera (SS)
based on UPGMA (Nei, 1978) ………………………………..
Dendrogram of genetic relationship between seven groups of
Michelia spp. from South Sumatera (SS) and East Java (EJ)
based on UPGMA (Nei, 1978) ..................................................
6
8
10
11
vii
ABSTRACT
Diospyros celebica, Eusideroxylon zwageri and Michelia spp are involed in
threatened species according to their potential in the natural forest. Thus
conservation of these species becomes a very crucial activity to be carried out. In
order to conserve the species effective and efficient, information of genetic
diversity, its distribution and genetic relationship between populations is very
important. Information on genetic erosion in within population is also important to
decide conservation strategy of each species. Analysis of genetic diversity and
genetic erosion of each species were carried out using 5 selected RAPD primers.
Total number of loci obtained from the primers has variation between 22 to 25.
Mean genetic diversity of three provenances of Diospyros celebica (Ebony) was
0.2886. Mean genetic distance between provenances of D. celebica was 0.3029.
Mean genetic diversity of two provenances of Eusideroxylon zwageri (Ulin) was
0.3678. Mean genetic distance between provenances of E. zwageri was 0.2572.
Mean genetic diversity of two provenances of Michelia spp was 0.1878. Mean
genetic distance between provenances of Michelia spp. was 0.6648. Based on
cluster analysis, provenances of the three species were divided into two clusters.
The clusters were correlated with geographic originality of the samples. Basically,
different province will be clustered into different group. Genetic erosion from trees
into poles and or wildling was not revealed in this study for D. celebica and
E. zwageri. However, for M. champaca, genetic diversity was tend to decrease
from trees to poles and wildlings.
Key words:
viii
Diospyros celebica, Eusideroxylon zwageri, Michelia, RAPD,
genetic diversity, genetic erosion.
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
INTRODUCTION
In Activity 2.1, study on the level of genetic diversity of three species
(Diospyros celebica, Eusideroxylon zwageri and Michelia spp) was carried out in
order to provide genetic information of the species for a purpose of establishing ex
situ conservation plots. This was achieved by employing DNA markers.
The level of genetic diversity of the species is limited. Ideally, any genetic
conservation works should have information on genetic diversity of the species
before hand. However, the genetic diversity information for most tropical rain
forest species is still limited. Several studies on genetic diversity have been
reported for Alstonia scholaris (Hartati et al., 2007), Instia bijuga (Rimbawanto
and Widyatmoko, 2006), Santalum album (Rimbawanto et al., 2006) and Gyrinops
verstigii (Widyatmoko et al., 2009). Genetic diversity of E. zwageri has been
reported by Sulistyowati et al. (2005) and Rimbawanto et al. (2006a).
The availability of information on genetic diversity of these species is
essential for designing an appropriate sampling strategy for genetic conservation
purposes. Genetic markers are needed to study many aspects of forest trees such as
reproduction system, genetic diversity and gene flow.
RAPD (Random Amplified Polymorphic DNA) analysis (Wiliams et al.,
1990; Welsh and McClelland, 1991) is one of the most effective tools of DNA
based fingerprinting techniques applied to analyze genetic diversity. RAPD
analysis that is based on PCR (polymerase chain reaction) with 10-mer random
oligonucleotide primer is relative easier than any DNA markers and could be
carried out in a simple instrument.
The aim of this study is to investigate genetic diversity, its distribution and
genetic relationship between populations of Diospyros celebica (ebony),
Eusideroxylon zwageri (ulin) and Michelia spp (cempaka) using RAPD markers.
1
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
APPLIED METHODOLOGY
DNA extraction
Total genomic DNA was extracted using a modified Cetyl Trimethyl
Ammonium Bromide (CTAB) protocol reported by Shiraishi and Watanabe (1995).
RAPD analysis
RAPD analysis was performed in a reaction containing 10 mM Tris-HCl (pH
8.3), 10 mM KCl, 3.0 mM MgCl2, 0.2 mM each of dNTPs, 0.5 unit/10µl AmpliTaq
DNA polymerase, a Stoffel Fragment (Applied Biosystem), 0.25 µM each of
primers (Operon Technologies), and 10 ng/10µl template DNA. The condition of
amplification was 94°C for 1 min., 45 cycles of 30 s at 94°C, 30 s at 37°C, and 90 s
at 72°C, followed by 7 min. at 72°C. The amplification products were separated by
electrophoresis in 1% agarose gel with ethidium bromide and detected with a 302nm UV transilluminator.
Twenty RAPD primers were tested for screening polymorphic RAPD
primers which will be used in the study of genetic diversity for the three species.
All the RAPD primers were supplied by Operon Technologies. Some criteria have
been used to select polymorphic RAPD primers in the screening. Those criteria
were: number of polymorphic loci, clear bands between 200 - 800 bps and
reproducibility of the locus.
The presence (1) or absence (0) of the polymorphic fragments attained from
electrophoresis was noted as 1/0 data. Based on this data, the genetic similarity (S)
and genetic distance (D = 1 - S) among all individuals were calculated using a
simple matching coefficient (Sokal and Michener, 1958). Parameter genetic i.e.
level of genetic diversity (h) was analyzed within populations. A dendrogram was
constructed using the UPGMA method from the matrix of genetic distance among
individuals. These parameters genetic were calculated using Popgene 1.32
computer program (Yeh et al., 1999).
2
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
Screening of RAPD primers
Three criteria were used to select polymorphic RAPD primers in the
screening of RAPD primers. The first criterion was number of polymorphic loci.
This criterion is important to obtain as many information as possible with less
number of primers. This method is used not only because of its time efficiency, but
also the relatively low cost for analysis. The second criterion was clearance of
RAPD band. RAPD analysis normally produces a lot of bands/loci, however
because of competition among the bands some of them are weak band. The weak
band can also be caused by mis-annealing of RAPD primers. Thus, the weak band
normally has low reproducibility. Only the clear RAPD bands were selected since
the clear bands are always high reproducibility. Some loci have clear and weak
bands, in which clear band maybe homozygote and the weak band is heterozygote.
Another reason is the loci contain two different loci. Thus, this type of loci was not
selected in order to avoid mislabeling. The clear band is all bands which appear in
one locus. Thus, reproducibility of the locus should be checked for all bands which
appear in the locus. Reproducibility of the locus was the third criterion for
screening.
3
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
PRESENTATION OF THE DATA
Plant Materials collection
Leaf sample of individual trees was collected from the tree species.
Diospyros celebica leaf samples were collected from South Sulawesi and Central
Sulawesi. Eusideroxylon zwageri leaf samples were also collected from two
provenances, Jambi and South Sumatera. Michelia spp leaf samples were collected
from South Sumatera and East Java. Details of collected samples of the three
species are shown in Table 1.
Table 1. List of used samples
No.
Provenance
Materials
Diospyros celebica
Wasupoda District
1
Luwu Timur, South Sulawesi
S 2o29.191’, E 121o06.474’, 401 m asl
Mangkutana District
2
Luwu Timur, South Sulawesi
S 2o27.087’, E 120o48.891’, 66 m asl
Parigi Moutong, Central Sulawesi
3
S 01o03’45’’ - 01o04’25’’
E 121o31’12” - 121o31’55”, 150 m asl
Eusideroxylon zwageri
1
Batanghari, Jambi
2
Musi Rawas, South Sumatera
Michelia champaca
Arjuno Mountain, Purwodadi SubDistrict, Pasuruan District
1
Wilis Mountain, Ngawi
2
Lahat1, South Sumatera
Lahat2, South Sumatera
Michelia alba
Arjuno Mountain, Purwodadi
District, Pasuruan District
Bumiaji Arboretum, Malang
4
Sub-
No.
DBH (cm)
of samples
Trees
Wildlings
8
8
35 – 45
Trees
Wildlings
8
8
35 – 45
Trees
Wildlings
8
8
35 – 45
Trees
Poles
Wildlings
Trees
Poles
Wildlings
8
8
8
8
8
8
35 – 120
15 – 30
Trees
Poles
Poles
Trees
Poles
Wildlings
Trees
8
2
6
6
6
8
4
15 – 32.5
4
1 – 7.6
75 – 90
15 – 40
Trees
4
16 – 37,7
Trees
2
21.3 – 56.3
83 – 95
5 – 15
50 – 70
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
ANALYSIS AND INTERPRETATION OF THE DATA
AND RESULTS
Screening of RAPD Primers
Total of twenty Operon RAPD primers were screened for each species to find
polymorphic RAPD primers for a genetic diversity analysis. Screening of RAPD
primers were carried out using three criteria as mentioned above. By using the
three criteria, number of selected RAPD primers, total loci and mean of loci per
primer are presented in Table 2.
Table 2. Number of screened RAPD primer, selected primers, total number of loci
and mean of loci per primer for the three species
Criteria
Number of screened primer
Selected RAPD primers
Total number of loci
Mean of loci per primer
D. celebica
20
5
23
4,6
E. zwageri
20
5
25
5
Michelia spp
20
5
22
4,4
Genetic Diversity and Population Relationship of Diospyros celebica
Based on selected RAPD markers, genetic variation of populations of ebony
ranged from 0.2476 to 0.3217 (Table 3). The highest genetic diversity was shown
by Parigi population (0.3217) and followed by Mangkutana population (0.2966).
The lowest genetic diversity was shown by Wasupoda population (0.2476). Details
of genetic diversity of each population are presented in Table 3. Mean of genetic
diversity of the three populations of ebony was 0.2886, which is higher than
coniferous species i.e. Pinus attenuata (0,011), P. radiata (0.08), P. sylvestris
(0.022), P. menziesii (0.050), even another broadleaf species i.e. Alstonia scholaris
(0.247).
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Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
Table 3. Genetic diversity within (diagonal) and between (below diagonal)
provenances for three provenances of ebony from South Sulawesi and
Central Sulawesi
Provenance
Parigi
Wasupoda
Mangkutana
Parigi
0.3217
0.4542
0.4191
Wasupoda
Mangkutana
0.2476
0.0354
0.2966
Eventhough number of individual trees of the species in several provenances
have been decreased, the populations still have high genetic diversity. Reasons of
this condition are due to some following posibilities: genetic diversity base of the
provenances was initially high, there is high cross pollination between individual
trees within provenance, and the exploitation of the trees in the provenance was
carried out in the recent year.
In order to clarify the relationship of the species among the provenances, a
UPGMA dendrogram based on Nei’s standard genetic distance was constructed
from the genetic distances (Fig. 1). The highest mean genetic distance between
provenances was recognized between Parigi and Wasupoda (0.4542), and the close
relation between two provenances was shown by Wasupoda and Mangkutana
(0.0354).
Parigi (CS)
Wasupoda (SS)
Mangkutana (SS)
Fig 1. Dendrogram of genetic relationship between three provenances of ebony
from South Sulawesi (SS) and Centre Sulawesi (CS) based on UPGMA
(Nei, 1978).
The UPGMA cluster analysis reflected two main clusters. The first cluster
comprised population from Central Sulawesi (Parigi), and the second cluster
consisted of populations from South Sulawesi (Wasupoda and Mangkutana). The
6
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
genetic distance between the two clusters was 0.4366. These results indicate that
the two clusters are genetically different according to geographic position.
The proportion of ± 70% of genetic diversity was distributed within
provenance, while the remaining 30% was distributed between provenances. This
condition is likely due to evolution and adaptation process of the species and its
provenances. The high genetic distance between the two clusters (province) might
be influenced by high exploitation of the species.
In order to reveal genetic degradation within population, two sample groups
were collected from each provenance i.e. trees and wildlings. Genetic diversity
within and between samples groups is shown in Table 4.
Table 4. Genetic diversity within (diagonal) and between (below diagonal) group
for six sample groups of ebony from South Sulawesi and Centre
Sulawesi
Groups
P-t
P-w
W-t
W-w
M-t
Parigi-trees (P-t)
0.2527
Parigi-wildlings (P-w)
0.1062 0.3048
Wasupoda-trees (W-t)
0.5186 0.3664 0.1848
Wasupoda-wildlings (W-w)
0.4029 0.3648 0.1499 0.3533
Mangkutana-trees (M-t)
0.5729 0.5536 0.1397 0.1110 0.2080
M-w
Mangkutana-wildlings (M-w) 0.5270 0.4862 0.0886 0.0762 0.0508 0.2290
The highest genetic diversity was shown by Wasupoda-widlings (0.3533),
yet Wasupoda-trees had the lowest genetic diversity. Whilst, there was no
significant different of genetic diversity between trees and wildings groups of the
other two provenances (Parigi and Mangkutana). Random mating system in these
two provenances and/or small samples using in this study may be reasonable for
this condition. If the number of samples is increased, genetic diversity between
trees and wildlings may be similar.
7
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
Wildlings of Parigi and Mangkutana showed slightly higher genetic
diversity than their parent trees. Random mating system in the provenance and
small number of sample used in this study may account for this condition. If the
number of samples is increased, genetic diversity between trees and wildlings may
be similar. Significant difference of genetic diversity between Wasupoda-trees and
Wasupoda-wildling may be due to related to following factors, such as random
mating system, small number of trees and gene flow from other provenances.
Parigi-trees
Parigi-widlings
Wasupoda-trees
Wasupoda-wildlings
Mangkutana-trees
Mangkutana-wildlings
Fig 2. Dendrogram of genetic relationship between six sample groups of ebony
from South Sulawesi (SS) and Centre Sulawesi (CS) based on UPGMA
(Nei, 1978).
Fig. 2 shows a UPGMA dendrogram of the ebony when each provenance
was divided into two groups (trees and wildings). There are distinct groups of
ebony taken from Central Sulawesi and South Sulawesi. Generally, wildling of
each provenance has a close relationship with their parent trees, however in this
case, wildling of Wasupoda group was in a group of Mangkutana population. This
condition is most likely due to the high genetic diversity of Wasupoda-wildlings
and low genetic diversity of Wasupoda-trees.
Genetic Diversity and Provenances Relationship of Eusideroxylon zwageri
Genetic diversity of provenances of ulin based on selected RAPD markers
ranged from 0.2461 to 0.4258 (Table 5). The highest genetic diversity was shown
by Musi Rawas-wildlings (0.4258) and followed by Musi Rawas-poles (0.4102).
8
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
The lowest genetic diversity was shown by Batanghari-big trees (0.2461). Detail of
genetic diversity of each provenance is presented in Table 5. Mean genetic
diversity of two populations of ulin was 0.3678, which is higher than ebony’s mean
genetic diversity obtained in this study (0.2886).
Table 5. Genetic diversity within (diagonal) and between (below diagonal) groups
for six sample groups of ulin from Jambi and South Sumatera
Groups
B-t
B-p
B-w
M-t
M-p
Batanghari- trees (B-t)
0.2461
Batanghari-poles (B-p)
0.0226
Batanghari-wildlings (B-w)
0.0276 0.0060
0.3672
Musi Rawas-trees (M-t)
0.3897 0.2396
0.2195
0.4062
Musi Rawas-poles (M-p)
0.3609 0.2204
0.1926
0.0234
0.4102
Musi Rawas-wildlings (M-w) 0.3144 0.1992
0.1792
0.0167
0.0202
M-w
0.3516
0.4258
In order to reveal the relationship among six groups of Eusideroxylon
zwageri (ulin), a UPGMA dendrogram was constructed from the genetic distances
(Fig. 3). The highest mean genetic distance between groups was 0.3897 which was
between Batanghari-trees and Musi Rawas-trees , and the closest relation between
two groups was shown by Batanghari-poles and Batanghari-wildlings (0.0060).
There were two distinct clusters identified. The first cluster comprised
groups from Jambi (Batanghari-trees, Batanghari-poles and Batanghari-wildlings),
and the second cluster consisted of groups from South Sumatera (Musi Rawastrees, Musi Rawas-poles and Musi Rawas-widlings). A very close relationship was
shown among three groups in each cluster.
Genetic degradation from trees to poles, and from poles to wildlings almost
did not reveal in both provenances of ulin, otherwise, it tends to increase. Trees
group of Batanghari population in particular has the lowest genetic diversity to
compare with poles and wildlings. There was a clear evidence that number of trees
in the provenances was decreased significantly caused by over exploitation.
9
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
Batanghari- trees (Jb)
Batanghari- poles (Jb)
Batanghari- wildlings (Jb)
Musi Rawas-poles (SS)
Musi Rawas-trees (SS)
Musi Rawas-wildlings (SS)
Fig 3. Dendrogram of genetic relationship between six sample groups of ulin
from Jambi (Jb) and South Sumatera (SS) based on UPGMA (Nei, 1978).
Genetic Diversity and Provenances Relationship of Michelia spp
Based on selected RAPD markers, genetic variation of provenances of
Michelia spp ranged from 0.1000 to 0.2560 (Table 6). The highest genetic diversity
was shown by Lahat1-poles (0.2560) and followed by Pasuruan-trees (0.2966). The
lowest genetic diversity was shown by Lahat1-seedling (0.1000). Detail of genetic
diversity of each provenance is presented in Table 6. Mean genetic diversity of six
groups of Michelia champaca was 0.1878, which is lower than ebony (0.2886) and
ulin (0.3678) obtained in this study.
Table 6. Genetic diversity within (diagonal) and between (below diagonal) groups
for seven sample groups of Michelia spp. from South Sumatera and East
Java
Groups
L1-t
L1-p
L1-s
Lahat1-trees (L1-t)
Lahat1-poles (L1-p)
Lahat1-seedlings (L1-s)
Lahat2-trees (L2-t)
Pasuruan-trees (P-t)
Pasuruan-poles (P-p)
M. alba
0.1731
0.0256
0.0473
0.1565
0.5847
0.7282
0.7399
0.2560
0.0428
0.1573
0.5141
0.6145
0.6458
0.1000
0.2292
0.6470
0.8405
0.8623
10
L2-t
P-t
P-p
M.alba
0.1512
0.6790 0.2419
0.7103 0.0888 0.2050
0.8116 0.2382 0.0754 0.1368
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
In order to clarify the relationship among six groups of Michelia champaca
and one population of M. alba, a UPGMA dendrogram was constructed from these
genetic distances (Fig. 4). The highest mean genetic distance between groups was
0.8405 which was between Lahat1-seedling and Pasuruan poles (0.8405), and the
closest relation between two groups was shown by Lahat1-trees and Lahat1-poles
(0.0256).
Lahat1-trees (SS)
Lahat1-poles (SS)
Lahat1-seedlings (SS)
Lahat2-trees (SS)
Pasuruan-trees
Pasuruan-poles
M. alba (EJ)
Fig 4. Dendrogram of genetic relationship between seven groups of Michelia spp.
from South Sumatera (SS) and East Java (EJ) based on UPGMA (Nei,
1978).
Two distinct clusters were clearly identified. The first cluster comprised
groups from South Sumatera (Lahat1-trees, Lahat1-poles, Lahat1-seedlings and
Lahat2-trees), and the second cluster consisted of groups from East Java (Pasuruantrees, Pasuruan-poles, M. alba). There were four groups included in the first cluster
which were clustered into two groups, namely Lahat 1 samples and Lahat 2
samples. The grouping revealed the origin of the groups. Lahat 1 was owned by
one farmer, and Lahat 2 was belonged to another farmer. Each farmer established
the plantation/seed orchard from different population, provenance or mother trees.
Group of samples from East Java was also divided into two small clusters.
However, Pasuruan-poles close to M. Alba rather than their parents. This condition
is most likely due to two factors, namely inter-species hybrid between
M. champaca and M. alba occurred in the same area and small number of samples
of M. alba used in this study.
11
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
Genetic diversity of seedling in Lahat1 (South Sumatera) and poles in
Pasuruan (East Java) tended to decrease comparing to their parents trees which is
different to the fact in ebony as described above. High inbreeding caused by a nonrandom mating system of mother trees may account for the genetic degradation of
Michelia spp.
12
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
CONCLUSIONS
1.
Mean genetic diversity of three provenances of Diospyros celebica (ebony)
was 0.2886. Mean genetic distance between the provenances of D. celebica
was 0.3029. Genetic distance between provenances within province was
0.0354.
2.
Mean genetic diversity of two provenances of Eusideroxylon zwageri (ulin)
was 0.3678. Mean genetic distance between the provenances of E. zwageri
was 0.2572.
3.
Mean genetic diversity of two provenances of Michelia champaca was
0.1878. Mean genetic distance between the provenances of Michelia spp.
was 0.6648.
4.
Based on cluster analysis, provenances of the three species were divided into
two clusters. The clusters were related to geographic originality of the
samples. Basically, different province will be clustered into different group.
5.
Genetic degradation from trees to poles and or wildling was not revealed in
this study for D. celebica and E. zwageri. On the other hand genetic
diversity of M. champaca tended to decrease from trees to poles and
wildlings.
13
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
RECOMMENDATIONS
Study on the level of genetic diversity of Diospyros celebica, Eusideroxylon
zwageri and Michelia spp. has been carried out in this project. Based on the results
obtained in this study, three recommendations are proposed as follows:
1.
Diospyros celebica and Eusideroxylon zwageri have been recognized as
threatened species due to their remaining populations or provenances and
number of remaining individual in their natural distribution. According to
analysis of genetic diversity of the two species in this project, however, they
still have high genetic diversities indicating that both species will be survived
and exist providing the genetic diversity of both species are well maintained..
It is therefore, in-situ and ex-situ conservation program are required.
2.
Michelia spp has different condition to compare with the other two species
(Diospyros celebica and Eusideroxylon zwageri). Although natural
distributions of Michelia spp are as limited as the others, many plantations
have been established, especially for M. champaca. Therefore, natural
distribution survey and mapping for Michelia spp are of critically important
to decide in-situ conservation plan of the species. Ex-situ conservations of M.
champaca can be carried in parallel with tree breeding programs or
plantations establishment.
3.
A number of information on genetic diversity of the three species (Diospyros
celebica, Eusideroxylon zwageri and Michelia spp) have been obtained.
However, information of mating system of natural population, include
inbreeding coefficient and gene flow of D. celebica and E. zwageri is still
required to design a conservation plan for both species.
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Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
IMPLICATION FOR PRACTICE
The potential applications of molecular markers to facilitate gene
conservation in the tropics could be divided in to two steps. Firstly, they can be
used to evaluate the status of genetic background of ex situ plantations and in situ
sites of any forest tree species which are already established based on conventional
silviculture practice whether they contain correct clones and ramets and have
sufficient genetic diversity for the conservation as the representative of the species
gene pool.
Secondly, they can be applied to evaluate the status of genetic resources of
those which have never been established but planned to establish conservation
programs by determining genetic variation within and among population or
provenance and mating system as well as gene flow. Generally speaking, they can
be used as a guideline of how and where to collect samples for ex-situ gene
conservation and which sites are suitable as in-situ gene conservation. To
maximize the latter application, it should be combined with an eco-geographic
survey and adaptive traits measurement.
In this project, the second step is appropriate to the three species because exsitu and in-situ conservations of the species have never been established in
Indonesia but planned to be established. Information on genetic variation within
and between populations or provenances, and genetic relationship between
populations or provenances has been obtained in this study. Genetic diversity of
ebony and ulin is still high. Genetic variation within provenance is higher than that
between populations. However, genetic distance between clusters was also high.
Based on this information, collection of genetic materials for an ex-situ
conservation should be focused both on individual trees within populations and
each cluster. Each province should be represented by one population.
Genetic degradation was not revealed for both D. celebica and E. zwageri. It
is indicating that the genetic diversity of both species can be kept in this recent
level when the remaining populations are well maintained and conserved. Genetic
materials for establishing an ex-situ conservation can be treated as individual
mother tree where information of every single tree is recorded or can be bulked
during the collection. It is critically important, however, to ensure that the genetic
materials were collected from several individual mother trees.
15
Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
In case of M. champaca, genetic diversity tended to decrease from trees to
poles, and from poles to wildlings/seedlings. Therefore, in order to establish an exsitu conservation plot of the species, there are two important factors to be
concerned during genetic materials collection. The first is that genetic materials
should be collected from as many as individual trees (collected separately). The
second, the individual mother trees should represent all populations in the area.
For example in Lahat area, mother trees should be selected from several individual
trees from each farmer area.
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Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp.
Using RAPD Markers
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Using RAPD Markers
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