Malays. Appl.ECOLOGY
Biol. (2007)AND
36(2):
21–31
DNA
FINGERPRINTING OF CRYPTOCORYNE PALLIDINERVIA ENGLER
21
ECOLOGY AND DNA FINGERPRINTING OF CRYPTOCORYNE
PALLIDINERVIA ENGLER (ARACEAE) ACCESSIONS USING
POLYMERASE CHAIN REACTION M13 UNIVERSAL PRIMER
I.B. IPOR1, W.S. HO, C.S. TAWAN, M.S. SALMIZANA and M. NORYATIMAH
Faculty of Resource Science & Technology
University Malaysia Sarawak
94300 Kota Samarahan, Sarawak
Malaysia
1E-mail: [email protected]
ABSTRACT
The ecological study of Cryptocoryne pallidinervia Engler was conducted at three different locations vis. Sungai Keranji
(Lundu), Sungai Batu (Triso, Sri Aman) and Lingga Peat Swamp Forest Reserve (Lingga, Sri Aman).The study
comprised of morphological characteristics, growth pattern and biomass allocation of C. pallidinervia, forest structure
and edaphic conditions of the location of C. pallidinervia were carried out. The total dry weight (g/m2), plant density,
total leaves per m2, total leaf area (cm2/m2), leaf area ratio (cm2/g), specific leaf area (cm2/g), leaf weight ratio (g/g),
petiole weight ratio (g/g) and root weight ratio (g/g) varied between localities. Genomic DNA of 18 accessions was
amplified with the M13 universal primer (5’- TTATGAAACGACGGCCAGT-3’). A total of 18 distinctive PCR
patterns were obtained which composed of 3 to 20 bands with the size ranging from 500bp to 3kb. The PCR profile
was further analyzed to establish genetic diversity between through the construction of dendrogram. Cluster analysis
of genetic relatedness had divided the C. pallidinervia accessions into four different major clusters. All big leaf
accessions were grouped together whereas the small leaf accessions can be found in all clusters. Besides that, Bintulu
accessions were highly differentiated among the others since they grouped together in different clusters. This indicates
that the PCR method with M13 universal primer is a rapid and reliable method to study genetic relatedness of C.
pallidinervia accessions from different locations.
Key words: Araceae, morphological characters, DNA fingerprinting
INTRODUCTION
Cryptocoryne (Araceae) has for many years been
given attention because of their value as aquarium
plants (Rataj and Horeman, 1977). They are
common fresh water aquatic plant. According to
Mansor (1991), Bastmeijer (2005) and de Wit
(1990), most of the species of Cryptocoryne are
exploited for the aquarium industry in the
international aquarium market. Fourteen species
of Cryptocoryne have been recognized from
Borneo and one of them being Cryptocoryne
pallidinervia Engler. It is a plant of lowland
forests where it grows in slow running rivers and
streams and seasonally inundated forest pools
under extremely acid conditions with around pH4
or so. This species is endemic to Borneo that is
in the peat swamp forests of Sarawak and West
Kalimantan. To date, in Sarawak, it can be found
in Kampung Keranji in Lundu, Lingga Peat
Swamp Forest Reserve and Sungai Batu,
Kampung Teriso in Sri Aman, Sungai Ayang,
Dalat in Mukah, and near Kemena Waterfall in
Bintulu (Figure 1). C. pallidinervia is characterized
by the cordate, more or less bullate leaves as
illustrated in Figure 2. The spathe has a long tube
and the limb is red with protuberances. Its collar
zone is yellowish with small red spots that become
smaller towards the throat. The spadix has the
male and female flowers with very short naked
part of spadix, situated adjacent to each other.
The kettle is black purple inside. The present
study is to determine the ecology and genetic
relatedness among accessions of C. palidinervia
from five different locations in Sarawak.
* To whom correspondence should be addressed.
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ECOLOGY AND DNA FINGERPRINTING OF CRYPTOCORYNE PALLIDINERVIA ENGLER
Fig. 1. Five areas of C. pallidinervia natural habitat in Sarawak.
(a)
(b)
(c)
(d)
Fig. 2. Morphological characteristics of spathe, leaves and habitats of C. Pallidinervia collected in the
study area. (a) Kampung Keranji, Lundu; (b) Lingga, Sri Aman; (c) Kampung Teriso, Sri Aman; (d)
Sungai Ayang, Dalat, Mukah.
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ECOLOGY AND DNA FINGERPRINTING OF CRYPTOCORYNE PALLIDINERVIA ENGLER
MATERIALS AND METHODS
Biomass allocation patterns of C. pallidinervia
Sampling was conducted at three sites vis. Sg.
Keranji, Lundu, Kuching (thereafter Keranji), Sg.
Batu, Triso, Sri Aman (thereafter Triso) and
Lingga Peat Swamp forest reserve, Lingga, Sri
Aman (thereafter Lingga) was conducted (Figure
1). All plants within 1m x 1m quadrate were
collected to determine the total number of plants
and leaves. Dry weight of the rhizomes, leaves and
petioles was also determined by drying at 60°C for
7 days. The leaf areas were taken by using AT
Delta–T Scan before dried up in the oven. The
biomass pattern was mathematically analyzed by
using the method of Peterson and Flint (1983).
Floristic composition and above ground biomass
estimation
Four plots (10m x 20m) were established
randomly where C. pallidinervia occurred along
Sg. Keranji. All trees with the diameter at breast
height (DBH) > 5cm were enumerated by taking
their height and DBH. The important value (IV),
relative basal density (RD), relative density (Rd)
and relative frequency (Rf) were calculated by
using the method developed by Brower et al.
(1990). Biomass estimation and leaf area index
(LAI) were calculated based on the formula
developed by Yamakura et al. (1986).
Soil analysis
Soil samples were taken at the depth of
between 0 to 25cm from Keranji, Triso and
Lingga. The soil samples were dried at room
temperature for two weeks and sieved with the 2
mm filter. The soil samples were analyses for the
pH (Hesse, 1979; Mc Lean, 1986), soil organic
carbon (Dewis and Freites, 1970), nitrogen (Beitz,
1974), cation exchange changes (CEC), calsium,
magnesium, potassium, sodium and basic
saturation (BS) (Anon, 1980).
Genetic relatedness of C. pallidinervia accessions
Eighteen accessions of C. pallidinervia from
five different locations were studied, i.e. Keranji,
Lingga and Triso, Sungai Ayang, Dalat in Mukah
(thereafter Ayang) and Kemena Waterfall in
Bintulu (thereafter Kemena) (Table 1 & Figure 2).
Total genomic DNA was isolated from fresh
leaves of C. pallidinervia using a modified CTAB
method (Doyle and Doyle, 1987). The genomic
DNA purification was carried out using Wizard®
Genomic DNA Purification Kit (Promega, USA).
PCR amplification reaction was performed in a
total volume of 25 μl containing 1X PCR buffer
(200 mM Tris – HCl pH 8.4, 500 mM KCl), 3 mM
MgCl2, 200 μM each of dNTPs which consist of
dATP, dCTP, dTTP and dGTP, 0.2 μM M13
universal primer (5’-TTATGAAACGACGGC
CAGT-3’) (Welsh et al. 1991; Chong et al. 1995),
1.0 U Taq DNA polymerase and 15 ng C.
pallidinervia genomic DNA.
Photograph from GelStar-stained agarose gel
was used to score the data for PCR analysis. The
PCR bands were named after the primer and a
hyphenated number corresponding to the order of
their migrations. Starting from the slowest to the
Table 1. Details of C. pallidinervia accessions
21 - 31 I B Ipor.pmd
Place of collection
Leaves
Kampung Keranji, Lundu
Small
Flat land, dry river, rubber plantation
KRJ
a)
b)
c)
KRJ2
KRJ3
KRJ5
Lingga, Sri Aman
Large
Logged over peat swamp forest
LNG
a)
b)
c)
d)
LNG2
LNG3
LNG4
LNG5
Sungai Batu, Kampung Teriso, Sri Aman
Large
Peat swamp riverine forest
TRS
a)
b)
c)
TRS3
TRS4
TRS5
Sungai Ayang, Dalat, Mukah
Small
Sago farm, intertidal
DLT
a)
b)
c)
d)
e)
DLT2
DLT3
DLT4
DLT5
DLT6
Kemena Waterfall, Bintulu
Small
Secondary forest
BTL
a)
b)
c)
BTL1
BTL3
BTL4
23
Habitat
Abbreviation
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Accession
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ECOLOGY AND DNA FINGERPRINTING OF CRYPTOCORYNE PALLIDINERVIA ENGLER
fastest migrating fragment, the PCR amplified
bands were designated as M13-01, M13-02, M1303 and so on. The presence of a band was scored
as 1 and absence was scored as 0, based on the
several criteria which are each locus was assumed
as independent or non–allelic, there is no bias in
scoring monomorphic fragments versus polymorphic fragments and only fragments in the
range of 500bp to 3kb are considered in order to
increase the data reliability. The data was
analyzed using the NTSYpc (Version 2.2)
software (Rohif, 1998). The data was quantified
by the similarity index, Jij = Cij / (ni + nj – Cij)
(Jaccard, 1908), where Jij is the number of bands
common to individuals i and j, ni is the number
of bands in individual i and nj is the number of
bands in individual j. A dendrogram was
generated using the Unweighted Pair–Group
Method with Arithmetical Averaging (UPGMA)
as described by Sneath and Sokal (1973).
RESULTS AND DISCUSSION
Biomass allocation of C. pallidinervia
Total dry weight of C. pallidinervia in 1m x
1m quadrate at Keranji was 69.9g while 60.0g for
Triso and Lingga with 88.26 g (Figure 3a). These
locations had 260 plants, 150 plants and 343
plants respectively (Figure 3b). Lingga peat
swamp forest reserve is presently utilized as
catchment area for water intake to supply treated
water to the major part of Lingga. The established
logged over forest has no significant ecological
disturbance with regards to the pristine condition.
The luxuriant growth of C. pallidinervia is
probably influenced by the sufficient penetration
of available light through wide openings of forest
gaps. The total number of leaves was the highest
at Triso (734 total leaf/m2), followed by Lingga
(521 total leaf/m2) and Keranji (379 total leaf/m2)
(Figure 3c). The plants at Triso were generally
comprised of smaller leaves probably as a result
of regular exposure of plants to direct sunlight
that mainly caused by severe deforestation of the
study site. Total leaf area of C. pallidinervia in 1m
x 1m quadrate at Keranji was 38502cm2 (leaf area
index of 3.85 cm2/cm2) while at Triso was 9954cm2
(leaf area index of 0.99 cm2/cm2) and at Lingga
was 10707cm2 (leaf area index of 1.07 cm2/cm2)
(Figure 3d). The plants at Triso had smaller leaves
and the plants were more condensed due to their
occurrence was within freshwater intertidal zone.
The habitat in Triso is considerably disturbed due
to illegal logging activities and indiscriminate
clearing of riverine forest. The immediate opening
of the canopy and strong water current
particularly during high tide and raining season
21 - 31 I B Ipor.pmd
24
probably influenced the growth performance of C.
pallidinervia. The leaves of C. pallidinervia were
longer in deep water. The same trend was
observed by Ipor et al. (2005) for C. striolata in
Sungai Stuum, Lundu. This phenomenon may be
due to the direct interaction to availability of
sunlight. The leaf dry weight was 20.15g, 17.3g
and 22.6g at Keranji, Triso and Lingga
respectively. The weight of leaf petioles at Keranji
was 11.94g while at Sg.Triso was 11.77g and
Lingga with 19.91g.The dry weight of roots and
rhizomes at Keranji was 37.84g, 30.98g at Triso
and 45.75g at Lingga (Figure 3e). The result
revealed that the highest dry weight of leaves,
leaves petioles and root and rhizomes was at
Lingga.
The leaf area ratio (LAR) of C. pallidinervia
for Sg.Keranji was significantly higher than those
in Triso and Lingga. There was no significant
difference of the leaf area ratio for Triso and
Lingga (Figure 4a). It was obvious that the
occurrence of C. pallidinervia was at the deeper
level from the surface water. The water flow at
Triso was swifted and the water level will rise
when it is rain. According to Tootil (1984), a lot
of aquatic and semi aquatic plant had heterophilic
characteristic whereby, they have more than one
type of morphological and functions. Leaves in
deeper water adapted to current resistance while
those on water surface have broad lamina as to
maintain it floating. The specific leaf area (SLA)
was highest at Keranji with 1910 followed by
Triso with 575 and Lingga with 474. The specific
leaf area for Keranji was significantly higher than
those in Triso and Lingga. Hence there was no
significant difference of specific leaf area in Triso
and Lingga (Figure 4b). The leaf weight ratio
(LWR) between Keranji, Triso and Lingga was
significantly differed (Figure 4c). The leaf weight
ratio at Keranji was 0.35, Triso 0.41 and Lingga
0.07 (Figure 4c).
The petiole weight ratio (PWR) was highest
at Lingga with 0.25 followed by Triso and Keranji
(Figure 4d). The petiole weight ratio between these
locations had no significant difference. Hence,
there are no differences among all of the locations.
The root/rhizome weight ratio (RWR) was highest
at Keranji (0.45) followed by Lingga (0.40) and
Triso with 0.39 (Figure 4c). The root/rhizome
weight ratio in these locations had no significant
difference.
Floristic composition of secondary forest at Kg.
Keranji, Lundu
The surrounding structure of Keranji was a
secondary forest after the shifting cultivation
activities that had been carried out by the
villagers. The forest was mainly comprised of
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ECOLOGY AND DNA FINGERPRINTING OF CRYPTOCORYNE PALLIDINERVIA ENGLER
25
Fig. 3. Comparison of vegetative parts of C. pallidinervia from: Keranji = Keranji, Lundu, Triso = Triso, Sri Aman
and Lingga = Lingga, Sri Aman (3a=Total dry weight (g/m2); 3b= Total plants/m2; 3c= Total leaves/m2; 3d= Total
leaf area cm2/m2; 2e= Dry weight (g/m2). Figure 3a, 3b, 3c, 3d and 3e (within cluster), values sharing the same letter
have no significant difference at the 5% level.
rubber farm intercropped with local fruit trees and
nuts such as Durio zibethinus (durian) and Shorea
macrophylla (engkabang). According to Mansor
(1994) rivers that are situated along or in rubber
plantations can be the best medium for
Cryptocoryne growing when we are dealing with
cultivated areas. Hence, the environment in this
area can support the growth of C. pallidinervia
very well. The canopy from the surroundings trees
21 - 31 I B Ipor.pmd
25
also gives a great protection for the growth of C.
pallidinervia. Jacobsen (1985) reported that
Cryptocoryne could grow well under a thick
canopy.
A total of 101 individuals comprising of 23
tree species were recorded at Keranji in 4 plots of
10 m x 20 m in size. Five dominant species in this
forest were Hevea brasiliensis, Shorea macrophylla,
Sandoricum borneense, Kibessia gracilis and
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ECOLOGY AND DNA FINGERPRINTING OF CRYPTOCORYNE PALLIDINERVIA ENGLER
Fig. 4. Comparison of biomass allocations of C. pallidinervia from: Keranji = Sg Keranji (Lundu), Triso = Triso
(Sri Aman) and Lingga = Lingga (Sri Aman), (4a= Leaf area ratio (cm2/g); 4b= Specific leaf area (cm2); 4c= Leaf
weight ratio (g/g); 4d= Petiole weight ratio (g/g); 3e=Root/rhizome weight ratio (g/g). Vertical bars are values of
standard error.
Shorea myrionerva. H. brasiliensis contributed to
the highest value of important value (IV= 75.45)
(Table 2). It was followed by S. macrophylla (IV=
63.81), S. borneense (IV=18.42), K. gracilis
(IV=17.72) and S. myrionerva (IV= 15.50) (Table
2). The relative density (Rd= 24.75), relative
frequency (Rf= 8.89) and relative dominance
(RD= 41.81) of H. brasiliensis also contributed to
the highest important value (IV). The occurrence
of rubber farm hardly influenced the existence of
21 - 31 I B Ipor.pmd
26
the aquatic plant as it provides thick shade
condition. The least dominant species were
Baccaurea bracteata with the important value,
IV= 3.23, Timonius havescens (IV= 3.23) and
Elaocarpus griffithii (IV= 3.22). The relative
density (Rd) of B. bracteate, T. havescens and E.
griffithii was 0.99 each. The relative frequency
(Rf) of each of them was also the same or 2.2.
The total above ground biomass (TAGB)
from 4 plots of 20 m x 10 m was 5129.68 kg or
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ECOLOGY AND DNA FINGERPRINTING OF CRYPTOCORYNE PALLIDINERVIA ENGLER
Table 2. Relative density (Rd), relative frequency (Rf), relative dominance (RD), important value (IV) and estimated total
above ground biomass (TAGB) of trees with DBH > 5cm from the secondary forest at Keranji
SPECIES
Hevea brasiliensis (Willd. ex A. Juss.) Muell. Arg.
Shorea macrophylla (de Vr.) Ashton
Sandoricum borneense Miq.
Kibessia gracilis Cogn.
Shorea myrionerva Symington ex P.S. Ashton
Callophyllum soulattri Burm.
Macaranga triloba (Bl.) M. A.
Eugenia arcuatinerva Merr.
Eugenia subsessilifolia Merr.
Baccaurea macrocarpa (Miq.) Mull. Arg.
Melastoma imbricatum Wall. Ex C. B. Clarke
Artocarpus rigidus Blume
Artocarpus integer (Thunb.) Merr.
Durio zibethinus Murray
Vitex pubescen Vahl
Eugenia palembanica (Miq.) Merr.
Dillenia eximia Miq.
Blumeodendron tokbraii (Bl.) J. J. Smith
Artocarpus anisophyllus Miq.
Mangifera maingayi Hk. f.
Baccaurea bracteata M. A.
Timonius flavescens (Jack) Baker
Elaeocarpus griffithii (Wight) A. Gray
Rd
Rf
RD
IV
24.75
13.86
6.93
6.93
3.96
5.94
2.97
3.96
3.96
4.95
2.97
2.97
1.98
3.96
1.98
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
8.89
8.89
8.89
8.89
6.67
4.44
6.67
4.44
4.44
2.22
4.44
2.22
4.44
2.22
4.44
2.22
2.22
2.22
2.22
2.22
2.22
2.22
2.22
41.81
41.06
2.60
1.91
4.88
1.49
0.62
1.53
0.34
0.47
0.14
1.79
0.53
0.32
0.08
0.18
0.08
0.07
0.05
0.03
0.02
0.02
0.01
75.45
63.81
18.42
17.72
15.50
11.87
10.25
9.93
8.74
7.64
7.56
6.98
6.95
6.50
6.50
3.39
3.30
3.28
3.26
3.24
3.23
3.23
3.22
TAGB (kg)
164.11
672.20
120.61
81.80
1129.54
101.34
158.29
256.06
25.81
20.49
22.86
629.37
316.98
32.31
137.90
650.94
324.47
142.72
53.82
34.75
21.34
21.34
10.63
5129.68 or
64.12 ton/ha
Table 3. The soil characteristics from the study site of Keranji, Triso and Lingga
Location
pH
N (%)
Exchange
(+cmol/kg)
Total
organic
carbon
Ca
Mg
K
CEC
Na
Mechanical analysis
hyrometer (%)
Clay
Silt
Fine
BS
Coarse
Keranji
4.4
0.33
06.63
1.04
0.31
0.12
0.11
07.88
04.03
05.76 53.63
36.58
20.05
Batu Triso
3.3
1.63
45.06
1.83
5.42
0.62
0.80
22.83
10.73
03.15 77.29
08.83
37.98
Lingga
4.1
0.28
04.58
2.08
5.36
0.28
0.51
13.33
36.80
50.45 08.25
04.51
61.74
N = Nitrogen, CEC = Cation exchange capacity, BS = Base saturation.
64.12 ton/ha (Table 2). S. myrionerva (1129.54kg)
contributed to the highest above ground biomass
in the location. It was followed by S. macrophylla
with 672.20kg and Eugenia palembanica with
650.94 kg. E. griffithii with 10.63 kg contributed
the least above ground biomass.
Soil characteristics
The soil test revealed that Keranji had a pH
of 4.4 with 0.33% of nitrogen (N) content (Table
3). The total organic carbon was 6.63% while the
exchange of calcium (Ca), magnesium (Mg),
potassium (K) and sodium (Na) was 1.04 +cmol/
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27
kg, 0.31 +cmol/kg, 0.12 +cmol/kg and 0.11 +cmol/
kg respectively. The cation exchange capacity
(CEC) in the study site was 7.88 +cmol/kg while
the percentage of clay was 4.03% with 5.20% of
silt. The finest soil was 53.63% whilst the coarse
component was 36.58%. Base saturation (BS)
value was 20.05.
The pH of soil at Triso was 3.3 with a
nitrogen (N) content of 1.63% (Table 3). The total
organic carbon was 45.06% and the highest in this
study. The content of Ca, Mg, K, and Na was
1.83 +cmol/kg, 5.42 +cmol/kg, 0.62 +cmol/kg and
0.80 +cmol/kg respectively. The cation exchange
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ECOLOGY AND DNA FINGERPRINTING OF CRYPTOCORYNE PALLIDINERVIA ENGLER
capacity (CEC) in the location was 22.83 +cmol/
kg. The percentage of clay was 10.73% with 3.15%
of silt. The finest soil was 77.29% and the course
soil was 8.83% with base saturation (BS) of 37.98.
Lingga soil had pH of 4.1 with nitrogen content
of 0.28%. The percentage of the total organic
carbon was 4.58%, Ca, Mg, K and Na were 2.08
+cmol/kg, 5.36 +cmol/kg, 1.28 +cmol/kg, and 0.51
+cmol/kg respectively (Table 3). The cation
exchange capacity (CEC) was 13.33 +cmol/kg as
there was 36.80% of clay, 50.45 % of silt, 8.25%
of finest soil and 4.51% coarse soil in the study
site. The base saturation value was 61.74.
Genetic relatedness of C. pallidinervia accessions
Total genomic DNA of C. pallidinervia
accessions collected from five different locations
in Sarawak, namely Keranji, Lingga, Teriso,
Ayang and Kemena was extracted using the
modified CTAB miniprep DNA extraction
method (Doyle and Doyle, 1987) together with
Wizard® Genomic DNA Purification Kit method.
Ahigh degree of polymorphism was detected
among the was used to extract the genomic DNA
of 18 C. pallidinervia accessions collected from five
different locations in Sarawak namely Keranji,
Lingga, Teriso, Ayang and Kemena. The result
showed that a high degree of polymorphism
among the C. pallidinervia using the M13
universal primer. A total of 18 distinctive PCR
profiles were observed in this study which
composed of molecular weight ranging from
500bp to 3kb. Although most of the C.
pallidinervia accessions showed a high degree of
polymorphisms, but two accessions, DLT4 and
DLT6 produced identical banding profiles.
There were several accessions with one or
more unique band(s) and these bands were useful
in differentiating the C. pallidinervia accessions in
the present study (Figure 5). For example, locus
M13–07 was specific to accession TRS4, locus
M13–14 was specific to accession TRS5, locus
Fig. 5. DNA profiles of 18 C. pallidinervia accessions collected from 5 different locations in Sarawak, namely Keranji
(KRJ), Lingga (LNG), Teriso (TRS), Ayang (DLT) and Kemena (BTL) generated by using M13 universal primer.
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ECOLOGY AND DNA FINGERPRINTING OF CRYPTOCORYNE PALLIDINERVIA ENGLER
M13-21 was specific to accession LNG4, locus
M13–24 was specific to accession DLT3, and
locus M13–27 was specific to accession BTL4
(Figure 5). Three bands (locus M13–25, locus
M13–29 and locus M13–30) were found in all
accessions except accession BTL1. besides that,
different presence and absence behaviors of the
remaingin bands were also useful in differentiating
the samples within accessions (Chong et al. 1995).
For example, accessions KRJ2 and KRJ5 could
be discriminated from the other accessions at
locus M13–33 as well as accessions TRS4 and
DLT2, which differed at locus M13–06.
The constructed dendogram based on Jaccard
similarity matrix had grouped C. pallidinervia
accessions into 4 major clusters (Figure 6). Cluster
1 contained three accessions from Keranji and
Lingga with average value of J = 0.82. The
greatest value was observed between accession
KRJ5 and LNG2 with J = 0.70. The smallest
value of J = 0.52 within this cluster was observed
between accessions KRJ2 and LNG2. The genetic
Fig. 6. Dendrogram representing the genetic relatedness among the C. pallidinervia
Accessions. KRJ = Keranji, LNG = Lingga, TRS = Teriso, DLT = Dalat, BLT = Bintulu.
21 - 31 I B Ipor.pmd
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ECOLOGY AND DNA FINGERPRINTING OF CRYPTOCORYNE PALLIDINERVIA ENGLER
Table 4. Geographical distance between five different locations of C. pallidinervia accessions in Sarawak
KERANJI
LINGGA
KERANJI
LINGGA
TRISO
AYANG
KEMENA
variation of the accessions was greatly differed
even from the same location. Cluster 2 was the
largest cluster with 12 accessions derived from
Keranji, Lingga, Triso and Ayang. The average
Jaccard coefficient value among the entire
accessions was J = 0.76. The greatest value of J =
0.83 within this cluster was observed between
accessions DLT4 and DLT5 as well as accessions
DLT5 and DLT6. While the smallest value of J =
0.07 within this cluster was observed between
accessions DLT5 and LNG3. In this cluster,
accessions DLT4 and DLT6 showed identical
banding profiles. Since the differences in the PCR
banding profiles were high among accessions in
this study and therefore, one plausible explanation
is that the accessions DLT4 and DLT6 are derived
from the same source. Chong et al. (1995) showed
that the banding profiles generated by M13
universal primer not only verified the Salix species
identity, but they also differentiated among 14 of
the 15 Salix clones. Two clones of S. eriocephala,
ERIO21 and ERIO23 showed identical banding
profile and therefore, suggested that these two
clones were two ramets of a clone. Thus, in this
regard, the M13 universal primer was indeed
valuable.
The Kemena accessions were grouped
together in Cluster 3 that comprised of two
accessions, i.e. BTL3 and BTL4. Meanwhile
Cluster 4 composed only accession BTL1. The
geographical distance between Kemena and
Ayang, Triso, Lingga and Keranji may influence
the Kemena accessions. The distance between
Kemena and other locations was larger compared
to others (Table 4). For instance, the distance
between Kemena and Keranji is 400 km and they
were not closely related based on the dendrogram.
In fact, their genetic similarity value was 0.22.
Therefore, it suggests that C. pallidinervia
accessions from Kemena can be considered as the
subvarieties of C. pallidinervia. Besides the
geographical distance factor, the differences
between Kemena accessions and the other
accessions may also be due to habitat. The
21 - 31 I B Ipor.pmd
TERISO
AYANG
KEMENA
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142 Km
272 Km
400 Km
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177 Km
290 Km
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172 Km
285 Km
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130 Km
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30
habitat for all of the accessions is peat
swampland, lowland area along the slow running
rivers, except Kemena accessions, which is in
secondary forest. The Kemena accessions were
raised up and collected only at mineral soil as
compare to other accessions that were originated
from peat swamp habitat. Hence, the different
geographic or environmental origins of these
samples also have significant effects on the
clustering of DNA profiles obtained from PCR
(Lai, 2003).
Based on the dendrogram, it was observed
that accessions from different locations tended to
group together in a cluster, as shown in Cluster
2. This suggests that extensive gene flow has been
occurred within the C. pallidinervia accessions.
The gene flow occurs due to the pollination, which
is one of the mating systems besides vegetative
reproduction. The pollen is possible to be
transferred from one location to another through
insects especially between adjacent locations such
as Lingga and Triso with the distance of 26 km.
However, further research need to be conducted
due to the because there is lack of evidence
regarding the pollen viability in C. pallidinervia.
Leaf character also shows the main differences
among the accessions. All large leaf accessions
except for LNG2, KRJ2, KRJ5, BLT3, BLT4 and
BLT1 were grouped together in Cluster 2, whereas
small leaf accessions can be found in all of the
clusters. A similar result was also reported by
Upadhyay et al. (2002) after analyzing the
phylogenetic relationships among coconut
accessions. They found that all dwarf coconut
accessions were grouped together while tall
accessions formed three groups.
In conclusion, the present study has
established the ability of using the PCR method
with M13 universal primer to distinguish C.
pallidinervia accessions with high efficiency. The
results also indicate that the PCR method with
M13 universal primer is a rapid and reliable
method to study genetic relatedness of C.
pallidinervia accessions from different locations.
3/27/2008, 3:39 PM
ECOLOGY AND DNA FINGERPRINTING OF CRYPTOCORYNE PALLIDINERVIA ENGLER
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