RESEARCH ARTICLE

SABRAO Journal
of Breeding 129-144, 2018

GENETIC VARIABILITY AND CLASSIFICATION OF GANDARIA (Bouea)

IN INDONESIA BASED ON INTER SIMPLE SEQUENCE REPEAT (ISSR)
MARKERS

T. HARSONO1*, N. PASARIBU2, SOBIR3, FITMAWATI4 and

E. PRASETYA1
1
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Medan,
Indonesia
2
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara,
Indonesia
3
Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University,
Indonesia
4
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Riau, Indonesia
*Corresponding author’s email: [email protected]
Email addresses of coauthors: [email protected], [email protected],
[email protected], [email protected]

SUMMARY

The genus Bouea is a member of the Anacardiaceae family whichis widespread in

the Malesian Region. This genus consists of two species, namely Bouea oppositifolia
(Roxb.) Adelb. and Bouea macrophylla Griffit. This study aims to analyze the
genetic diversity Bouea in Indonesia. A total of 75 accessions of B. macrophylla and
30 accessions of B. oppositifolia were analyzed using inter simple sequence repeat
(ISSR) markers. The results on B. macrophylla showed that the species were
divided into three clusters with a similarity coefficient of 0.35. Group I consists of
53 accessions from Ambon, South Kalimantan, West Kalimantan, Banten, Bogor
(Loji, Pandeglag, Leuwisadeng and Jasinga), Cibinong, and Bogor Botanical Garden,
whereas Group II consists of 17 accessions from Riau (Batu Sangkar), West
Sumatra, Cibinong, Aceh, Medan, Jambi, Palembang, Lampung, and Bangka
Belitung. Group III consists of 5 accessions from west Sumatra Province (Batu
Sangkar), Bogor Botanical Garden, Jambi, and South Kalimantan. B. oppositifolia is
divided into four groups with a similarity coefficient of 0.84. Group I consists of one
accession from Bogor Botanical Garden, Group II consists of 25 accessions from
North Sumatera, Bogor Botanical Garden and Bangka Belitung, Group III consists of
two accessions from North Sumatra, whereas group IV consists of two accessions
from the Bogor Botanical Garden. The ISSR marker could separate B. macrophylla
and B. oppositifolia with a similarity coefficient of 0.34. It was determined that B.
macrophylla and B. oppositifolia were ancestors based on ISSR markers.

Key words: Bouea, Indonesia, intraspecies, ISSR

129
SABRAO J. Breed. Genet. 50 (2) 129-144

Key findings: Genetic variability and classification of gandaria (Bouea) based on

inter simple sequence repeat (ISSR) markers provide useful information to analyze
genetic diversity and can be used for the conservation of gandaria in Indonesia.

Manuscript received: January 9, 2018; Decision on manuscript: March 17, 2018; Accepted: April 7, 2018.
© Society for the Advancement of Breeding Research in Asia and Oceania (SABRAO) 2018

Communicating Editor: Prof. Dr. Naqib Ullah Khan

INTRODUCTION of ISSR indicates a considerable

variation between accessions that are
Bouea is a genus of Anacardiaceae morphologically indistinguishable
widely distributed in the Malesian (Ghazali et al., 2015).
region (Ghazali and Mohammad, Linking characteristics between
2014). Malesian region is an area with the varieties and the magnitude of the
distinctive flora and fauna and have plasticity of morphological features
the highest levels of vegetation has made it difficult to determine the
diversity in the world (Bass et al., limits of existing cultivars. Thus, it
2012). Bouea distribution covers the needs to be supported by data sources
territory of West Malesian, including which are obtained through other
the islands of Sumatra, Java, approaches (Fitmawati and Hartana,
Kalimantan, Brunei Darussalam, 2010). Morphological markers often
Singapore, and Malaysian Peninsula cause different perceptions among
(Rifai, 1992). Bouea consists of two researchers because of their high level
species: Bouea oppositifolia (Roxb.) of plasticity and sensitivity to
Adelb. and Bouea macrophylla Griffith environmental factors (Tanksley and
(Hou, 1974). Another species of Bouea Bematzy, 1989). Identification of
is reported to originate from Trang familial relationship of a plant can be
Bom, Vietnam under scientific name of carried out by combining
Bouea poilanei Evr. (“Xoai Mu” and morphological with molecular markers
“Xoai Muc”) with the distinct (Waugh, 1997). The researchers used
characteristics of having red-colored molecular markers to support
fruits (Le and Hancock, 1999). identification with morphological
The classification of Bouea into markers because they are more stable
two species, namely Bouea (Yunus, 2007) and less sensitive to
macrophylla and Bouea oppositifolia, changes in the environment and aging
Hou (1974) is the only grouping, process, rendering the data obtained
which becomes the main reference in relatively more accurate (Julisaniah et
discussing this genus. Classification of al., 2008). One of the methods that
Bouea was performed using can used to minimize environmental
morphological data. Harsono et al. influence on species or cultivars is the
(2016) reported that Bouea showed use of molecular markers (Finkeldey
high morphological variations. et al., 2010).
Morphological variation of B. The inter simple sequence
oppositifolia is higher compared to repeat (ISSR) marker is based on PCR
those of B. macrophylla. Variation of amplification products with a size of
the genus Bouea in Peninsular about 100-3000 bp near microsatellite
Malaysia using the molecular marker area that forms the basis of some

130
 Harsono et al. (2018)

simple sequence repeat (SSR) motifs. MATERIALS AND METHODS

The ISSR technique is very useful in
determining genetic instability in the Plant Material
early stages of in vitro culture, genetic
diversity evaluation, cultivar Two different species of Gandaria
identification, and monitoring of (Bouea) i.e., B. oppositifolia and B.
somaclonal variation (Trojanowska macrophylla were used to study the
and Bolibok, 2004). ISSR is more genetic variability and classification
informative than RAPD in wheat, fruit among different accessions based on
crops (strawberries and apples) and inter simple sequence repeat (ISSR)
pea (Trojanowska and Bolibok, 2004; markers. Fresh samples of B.
Korbin et al., 2002). This marker oppositifolia were obtained from North
system is quite reproducible and has Sumatra, Riau, Bangka Belitung, and
been used for rapid characterization Bogor Botanical Garden, which still
on many cultivars such as poplars have B. oppositifolia collection from
(Gao et al., 2006), common beans Lampung, Bangka Belitung, and West
(Gonzales et al., 2005), Cycad (Xiao Sumatra. Fresh samples of B.
et al. 2005), study of kinship between macrophylla were obtained from
ginger relatives (Wahyuni et al., Ambon, Banten, West Sumatra,
2004), and Fusarium culmorum isolate Bogor, Jambi, West Kalimantan, South
(Mishra et al., 2003). This study aims Kalimantan, Bogor Botanical Gardens,
to analyze the genetic variation of Palembang, Lampung, Bangka
Bouea using ISSR markers. Belitung, Medan, and Aceh (Figure 1).
Fresh samples used in observations
using the ISSR molecular markers
include 75 accessions of B.
macrophylla and 30 accessions of B.
oppositifolia (Table 1).

Figure 1. Bouea sampling area based on field studies.

131
SABRAO J. Breed. Genet. 50 (2) 129-144

Table 1. Fresh samples of Bouea obtained from the territory of Indonesia.

No. Type Province Total
1 B. oppositifolia (Roxb.) Adelb. North Sumatra 5
2 B. oppositifolia (Roxb.) Adelb. Riau 1
3 B. oppositifolia (Roxb.) Adelb. Isles of Bangka Belitung 19
4 B. oppositifolia (Roxb.) Adelb. Bogor Botanical Gardens 5
5 B. macrophylla Griffit. Ambon 14
6 B. macrophylla Griffit. Banten 8
7 B. macrophylla Griffit. West Sumatra 5
8 B. macrophylla Griffit. Bogor 13
9 B. macrophylla Griffit. Jambi 2
10 B. macrophylla Griffit. West Kalimantan 6
11 B. macrophylla Griffit. South Kalimantan 18
12 B. macrophylla Griffit. Palembang 2
13 B. macrophylla Griffit. Lampung 1
14 B. macrophylla Griffit. Bangka Belitung 1
15 B. macrophylla Griffit. Medan 1
16 B. macrophylla Griffit. Aceh 1
17 B. macrophylla Griffit. Bogor Botanical Gardens 3
Total 105

DNA Extraction The amplified PCR product was

visualized through electrophoresis
Total DNA was isolated from fresh using 1% agarose gel in TBE buffer
leaves using CTAB method of (Doyle and stained with 4 μl of SYBR® Safe
and Doyle, 1987) with modification. DNA Gel Stain (Invitrogen). 7 µl of
DNAs were suspended in TE buffer. PCR product was added with 1 µl of
loading dye when running along with
ISSR Amplification 100 bp DNA Ladder marker using
electrophoresis machine at 100 Volts
DNA amplification was performed for 45 minutes. Visualization of
using seven ISSRs primers (Table 2) marker bands was carried out using
which had been selected from eleven gel documentation equipped with UV
ISSRs primers with high polymorphic illumination.
band rates. PCR reaction volume was
25 μl, which consists of 2 μl of DNA Data recording and analysis
genome, 1 μl of each reverse and
forward primers (10 pmol), 12,5 μl Scoring of DNA band polymorphism
Taq polymerase (KAPA2GTM Fast was done using Gel Pro Analyzer
ReadyMix (2x) with Loading Dye) and program and the creation of a
9.5 μl of ddH2O (aquabidest). dendogram and genetic distance
PCR Program for ISSR were as analyses were done using NTSys PC
follows: (1) initial denaturation at 97 (version 2.02). Individual grouping
o
C for 4 minutes (1 cycle); (2) PCR patterns based on genetic similarity
which consists of denaturation at 97 matrices were reflected in the shape
o
C for one minute, annealing at 55 oC of dendrogram with a genetic
for one minute and at 72 oC for 2 similarity range of 0.00 (0%) to 1.00
minutes (35 cycles); and (3) final (100%). The average number of allele
extension at 72 oC for 4 minutes (1 counts, the average number of
cycle), followed by (4) cooling at 4 oC. effective alleles, genetic diversity, the

132
 Harsono et al. (2018)

Table 2. Primer to be used in ISSR analysis.

No Name of Primer Sequence Sequence Tm (oC)

1 PKBT3 (AG)8T AGAGAGAGAGAGAGAGT 55
2 PKBT4 (AG)8AA AGAGAGAGAGAGAGAGAA 55
3 PKBT5 (AG)8TA AGAGAGAGAGAGAGAGTA 55
4 PKBT7 (GA)9A GAGAGAGAGAGAGAGAGAA 55
5 PKBT9 (GA)9T GAGAGAGAGAGAGAGAGAT 55
6 PKBT10 (GA)9A GTGTGTGTGTGTGTGTGTA 55
7 PKBT12 (GT)9T GTGTGTGTGTGTGTGTGTT 55
(Source: Tomar, et.al 2011)

Table 3. Analysis of B. macrophylla’s genetic diversity using ISSR markers.

Polymorphic Shannon’s
Number of
Primers Heterozygosity Information Information
Effective Alleles
Content (PIC) Index
PKBT3 1.4342 0.2625 0.915 0.4075
PKBT4 1.3970 0.2392 0.885 0.3723
PKBT5 1.2878 0.1952 0.835 0.3223
PKBT7 1.4761 0.2916 0.925 0.4485
PKBT9 1.3663 0.2365 0.883 0.3804
PKBT10 1.3894 0.2431 0.880 0.3803
PKBT12 1.6092 0.3602 0.933 0.5391
Average 1.4228 0.2611 0.893 0.4072

Shannon information index, the observations is capable of producing

number of polymorphic loci, and the high polymorphic alleles. Polymorphic
percentage of polymorphic loci were information content (PIC) is used to
analyzed using the program POPGENE determine the level of polymorphism
(version 1.32). Analysis of molecular of a molecular marker. According to
variance (AMOVA) was used to Botstein et al. (1980), PIC value is the
measure genetic diversity in index used to measure the value of
populations and outside populations polymorphism value. According to Gou
and was analyzed using GenAlex 6.5. and Elston (1999), the PIC is defined
as the probability of genotype markers
of a given offspring that allows
RESULTS AND DISCUSSION detection in the absence of crossing-
over from the two-marker alleles of
Allelic variation among B. the affected parents it received. PIC
macrophylla Griffit accessions for dominant markers have a
maximum value of 1.0 (De Riek et al.,
Polymorphic ISSR markers from seven 2001; Bolaric et al., 2005).
primers ISSR are listed in Table 3, Polymorphism is considered high if the
where primer of PKBT10 produced the PIC value is ≥ 0.5, medium if PIC =
highest polymorphic information 0.25 < PIC < 0.5, and low if the PIC
content (PIC). PIC values obtained value is ≤ 0.25 (Botstein et al., 1980).
ranged from 0.880 to 0.933 with an The number of alleles for each
average value of 0.893. This indicates ISSR primer ranges from 14 (PKBT5)
that the primary ISSR marker used on to 21 (PKBT3) with an average of 18

133
SABRAO J. Breed. Genet. 50 (2) 129-144

alleles per primer. The highest number greater geographical isolation, the
of effective alleles (1.6092) went to lower the flow of genes.
PKBT12 primer and the lowest
effective alleles detected went to Cluster analysis of B. macrophylla
PKBT5 primer (1.2878). PKBT12 Griffit.
primer (0.3602) shows the highest
heterozygosity value and the highest Cluster analysis based on ISSR marker
Shannon information index is shown data in B. macrophylla and the
by PKBT12 primer (0.5391). outgroup (Mangifera indica and
Shannon’s information index is a Anacardium occidentale) has similarity
measure of gene diversity (Lewontin, coefficients ranging from 0.18 to 0.83
1974). The heterozygosity value is and is classified into four main groups
one of the parameters to measure the at a coefficient of 0.35 (Figure 2).
level of genetic diversity in a Group I is the largest group consisting
population. Heterozygosity is the of 53 accessions with a similarity
result of calculation of the frequency coefficient of 0.36 consisting of
of genes in each locus (Nei, 1978). accessions from Ambon, South
The higher the heterozygous Kalimantan, Banten, West Kalimantan,
frequency in a population, the higher Bogor (Loji, Pandeglag, Leuwisadeng,
the level of diversity (Vilas et al., and Jasinga), Cibinong, and Botanical
2015) Gardens. Group II consists of 10
Heterozygosity value of the accessions with a similarity coefficient
total population (HT) is 0.2219, which of 0.37 consisting of accessions from
indicates that there is considerable Batu Sangkar, West Sumatra,
genetic variation among individuals Cibinong, Aceh, Medan, Jambi,
within the B. macrophylla population. Palembang, Lampung, and Bangka
The coefficient of genetic Belitung. Group III with a similarity
differentiation (GST) in B. macrophylla coefficient of 0.35 consists of five
has a value of 0.5750, which indicates accessions from accessions derived
that this figure has a higher value from Batu Sangkar (West Sumatra),
than the amount of standard genetic Kebun Raya (Bogor) (KR7 origin Jambi
differentiation proposed by Nybom and KR8 origin Peninsula Malaysia),
and Bartish (2000) with a value of and South Kalimantan). Group IV
0.23 (for cross-cultivated crops) and consists of the following outgroup
0.19 for endemic plants. Geographical Anacardium occidentale and Mangifera
isolation is the main factor of the high indica.
value of genetic differentiation Group I is divided into seven
coefficient of B. macrophylla because subgroups again, group I has 11
the geographical isolation has accessions from Ambon with a
inhibited the gene flow. This condition similarity coefficient of 0.51. Group II
is evidenced by the low value of gene has eight accessions originating from
flow (0.3681). Gene flow is a South Kalimantan and Ambon with a
collective term encompassing all similarity coefficient of 0.56. Group III
mechanisms, which cause movement has 11 accessions originating from
of genes from one population to South Kalimantan and Ambon with a
another (Slatkin, 1995). Fischer and similarity coefficient of 0.54. Group IV
Matthies (1998) stated that the has nine accessions from Banten and
West Kalimantan with a similarity

134
 Harsono et al. (2018)

Figure 2. Dendogram of B. macrophylla using ISSR data with Unweighted Pair Group
Method with Arithmetic Average (UPGMA).

index of 0.49, group V has five the population of Lampung and Medan
accessions from West Kalimantan and and the nearest genetic distance
Banten with a similarity index of 0.43. exists between the Ambon and South
Group VI has four accessions from Kalimantan populations.
Bogor (Loji, Pandeglang, Leuwisadeng,
and Jasinga) with a similarity Analysis of molecular variance
coefficient of 0.64, and group VII has (AMOVA) B. macrophylla
five accessions from Cibinong and four
from Botanic Garden with a similarity The results of AMOVA analysis shows
coefficient of 0.47. that genetic variation in the population
The similarity index within 75 (86%) is greater than the genetic
accessions of B. macrophylla ranged variation between populations (14%).
between 0.6429 - 0.994 with the This suggests that the variations
highest observable similarity between present in B. macrophylla are largely
Ambon and South Kalimantan due to the influence of variation in the
populations, whereas the lowest population compared to the variations
observable similarity is found among caused by differences in geographical
the population of Medan and conditions. This is probably due to the
Lampung. Similarity index matrix high level of distribution of these
between populations using ISSR plants in a population, which caused
markers in 14 populations of B. high level of variation. Despite its vast
macrophylla is presented in Table 4. geographical distribution, it is likely
The observable genetic distance that B. macrophylla can only live in
ranged from 0.0509 - 0.4418, where the same environmental conditions
the furthest distance is found between between one population and another.

135
 SABRAO J. Breed. Genet. 50 (2) 129-144

Table 4. Matrix of similarity index and genetic distance among B. macrophylla populations using ISSR marker.
Pop AM BA BL BS EP JB KB KR KS PLB LP MDN SN BO
AM ***** 0,9134 0,8204 0,9035 0,9090 0,8695 0,9171 0,8880 0,9504 0,8474 0,7928 0,7665 0,8016 0,8858
BA 0,0905 ***** 0,7925 0,8823 0,8829 0,8347 0,9319 0,8543 0,9268 0,8139 0,7486 0,7507 0,8166 0,8442
BL 0,1979 0,2326 ***** 0,8340 0,8234 0,8502 0,7762 0,7679 0,8195 0,8456 0,8254 0,6905 0,7540 0,7927
BS 0,1014 0,1253 0,1815 ***** 0,8821 0,9123 0,8759 0,8705 0,9028 0,8700 0,8166 0,7626 0,8079 0,8666
EP 0,0954 0,1245 0,1943 0,1255 ***** 0,8626 0,8712 0,8859 0,8976 0,8189 0,8072 0,7717 0,8041 0,8835
JB 0,1398 0,1807 0,1623 0,0918 0,1478 ***** 0,8233 0,8629 0,8519 0,8475 0,8165 0,7576 0,7913 0,8594
KB 0,0865 0,0706 0,2533 0,1325 0,1378 0,1944 ***** 0,8640 0,9140 0,8317 0,7381 0,7498 0,8026 0,8696
KR 0,1188 0,1575 0,2641 0,1387 0,1211 0,1475 0,1462 ***** 0,8729 0,8394 0,7679 0,7136 0,7707 0,8980
KS 0,0509 0,0760 0,1991 0,1022 0,1080 0,1603 0,0899 0,1360 ***** 0,8315 0,7957 0,7413 0,8096 0,8697
PLB 0,1656 0,2059 0,1677 0,1392 0,1997 0,1654 0,1843 0,1751 0,1846 ***** 0,8202 0,7187 0,7864 0,8820
LP 0,2321 0,2896 0,1919 0,2026 0,2142 0,2027 0,3037 0,2641 0,2285 0,1982 ***** 0,6429 0,6587 0,8322
MDN 0,2659 0,2867 0,3704 0,2710 0,2592 0,2776 0,2879 0,3374 0,2993 0,3303 0,4418 ***** 0,7302 0,7532
SN 0,2211 0,2026 0,2824 0,2133 0,2180 0,2341 0,2198 0,2604 0,2112 0,2403 0,4174 0,3145 ***** 0,7663
BO 0,1213 0,1693 0,2323 0,1431 0,1239 0,1515 0,1397 0,1076 0,1396 0,1256 0,1837 0,2835 0,2661 *****
AM = Ambon, BA = Banten, BL = Bangka Belitung, BS = Batu Sangkar, West Sumatra, EP = Cibinong, KB = West Kalimantan, KR = Botanical Gardens,
KS = South Kalimantan, PLB = Palembang, LP = Lampung, MDN = Medan, SN = Lhoksukon, Aceh, BO = Bogor
Above diagonal Similarity Index
Below diagonal Genetic Distance

Table 5. Results of AMOVA on 75 accessions of B. macrophylla in 14 populations using ISSR markers.

Source of Variation df SS SS Est. Var. % Fst P-value
Inter Population 10 325.519 29.59 2.423 14% 0.140 0.001
In Population 64 965.884 14.86 14.86 86%
Total 74 1291.403 17.283 100%

136
 Harsono et al. (2018)

Table 6. Analysis of B. oppositifolia's genetic diversity using ISSR markers.

Polymorphic Shannon’s
Number of
Primers Heterozygosity Information Information
Effective Alleles
Content (PIC) Index
PKBT3 1.0711 0.0622 0.6799 0.1225
PKBT4 1.1165 0.0207 0.7596 0.0408
PKBT5 1.0345 0.0322 0.4987 0.0731
PKBT7 1.2168 0.2056 0.6254 0.3001
PKBT8 1.3369 0.2286 0.7559 0.3599
PKBT9 1.1330 0.1590 0.7789 0.2815
PKBT10 1.1126 0.0861 0.6684 0.1521
PKBT12 1.2484 0.2147 0.8405 0.3524
Average 1.1587 0.1261 0.7009 0.2103

High level of variation in obtained from PKBT8 (0.2286) primer.

populations indicates genetic diversity The Shannon information index varies
in the population. The genetic between 0.0408-0.3524, with the
diversity in high populations indicates highest value indicated by the PKBT8
considerable population differentiation. primer (0.3599). Shannon information
Results of AMOVA also show that index measures the level of diversity,
variation in population and between the appropriateness of markers, and
populations are significant (P-value < the emergence of genetic
0.01). AMOVA can be used to separate polymorphisms (Kesari et al., 2010).
the variation when there is adequate The observed heterozygosity is
genetic distance to describe the expected to be able to define the
difference of an allele with another probability that certain randomly
(Holsinger et al. 1996). Results of selected individuals from the
AMOVA within 75 accessions of B. population will be heterozygous at a
macrophylla in 14 populations using particular loci and observed
ISSR markers are presented in Table heterozygosity will be lower than
5. expected (Mishra, 2013). This
information and heterozygosity index
Allelic variations among the also support the diversity between
accessions of B. oppositifolia populations, in comparison with the
population (Sirkar, 2017)
The results of diversity analysis on 30 The number of effective alleles
accessions of B. oppositifolia, using in Table 6 shows the frequently similar
seven ISSR primers are presented in size of alleles taken to achieve a
Table 6. PIC values ranged from certain level of gene diversity. This
0.4987 – 0.8405 with an average means that it is possible to compare
value of 0.7009, which indicates that when the number and distribution of
the primary ISSR used in this study is alleles differ significantly (Hartl and
capable of producing high polymorphic Clark, 1989). The concept of
data, except for the PKBT five primers polymorphism used to define genetic
with PIC value below 0.5, specifically variation in the population. PIC has
0.4987. The highest number of become the most commonly used
effective allelic observed is found in formula for genetic studies in
PKBT12 (1.2484) primer and the measuring the information content of
highest heterozygosity value is a molecular marker (Botstein et al.,

137
SABRAO J. Breed. Genet. 50 (2) 129-144

1980). A high PIC value indicates the that greater geographical isolation,
high informative value of a primer. the flow of genes diminishes, whereas
The results shows that the value of adjacent geography will cause high
PIC from the observations ranged gene flow.
from 0.4987 to 0.8405 with an
average of 0.7009, which means that Cluster analysis of B. oppositifolia
the informative value of data
generated by the molecular marker Cluster analysis based on ISSR marker
used in this study is quite high. data in B. macrophylla and its
The heterozygosity value of the outgroup (Mangifera indica and
total population (HT) is 0.1528, which Anacardium occidentale) has similarity
indicates that there are considerable coefficients ranging from 0.56 to 0.99
genetic variations among individuals and is classified into 5 main groups at
within the B. oppositifolia population. a coefficient of 0.84 (Figure 3). Group
The genetic differentiation coefficient 1 is an outgroup which consists of M.
(GST) in B. oppositifolia has a value of indica and A. occidentale. Group 2 is a
0.4316, which indicates that this group with 1 accession, namely the
figure has a high value based on the Botanical Gardens (KR1). Group 3 is
standards determined by (Nybom and the largest group with 23 accessions,
Bartsih, 2000). Geographical isolation namely accession from North Sumatra
is the main factor of low value of (GT, HA, SO), Riau, Botanical Gardens
genetic differentiation coefficient of B. (KR2 and KR3), and Bangka Belitung.
oppositifolia because geographical Group 4 is a group with 2 accessions
isolation has determined the gene from North Sumatra (SP and LG).
flow. This condition has evidenced by Group 5 is a group with 2 accessions
the high value of gene flow (0.6584). from the Botanical Gardens (KR5 and
Fischer and Matthies (1998) stated KR6).

Figure 3. Dendogram of B. oppositifolia using ISSR data with Unweighted Pair

Group Method with Arithmetic Average (UPGMA).

138
 Harsono et al. (2018)

Table 7. Matrix of similarity index and genetic distance among populations using
ISSR marker.
Populations Botanical Gardens North Sumatra Riau Bangka Belitung
Botanical Gardens ****** 0.9164 0.8946 0.9428
North Sumatra 0.0873 ****** 0.9288 0.9317
Riau 0.1113 0.0739 ****** 0.9186
Bangka Belitung 0.0589 0.0707 0.0849 ******
Above diagonal Similarity Index
Below diagonal Genetic Distance

Table 8. AMOVA on 30 accessions of B. oppositifolia in 4 populations using ISSR

markers.
Source of Variation df SS SS Est. Var. % Fst P-value
Inter Population 2 47.629 15.876 2.054 40% 0.400 0.001
In Population 27 86.246 3.080 3.080 60%
Total 29 133.875 5.134 100%

The similarity index on 30 Analysis of molecular variance

accessions of B. oppositifolia ranged (AMOVA) of B. oppoositifolia
between 0.8946 and 0.9428 with the
highest observable similarity between The results of molecular variance
Botanical Gardens and Bangka analysis indicate that the genetic
Belitung populations, whereas the variation in the population is 60%,
lowest observable similarity is found whereas genetic variation among
among the populations of Botanical populations is 40%. This suggests that
Gardens and Riau. Similarity index the variations present in B.
matrix between populations using oppositifolia are largely due to the
ISSR markers in four populations of B. influence of variation in the population
oppositifolia is presented in Table 7. compared to the variations caused by
The observable genetic distance differences in geographical conditions.
ranges from 0.0589-0.1113, where This is probably due to the low level of
the furthest distance is found between distribution of these plants in a
the populations of Botanical Gardens population, which caused low level of
and Riau and the nearest genetic variation. The variation index between
distance exists between Botanical populations and in nearby populations
Gardens and Bangka Belitung appears to be quite comparable
populations. Genetic distance matrix between geographic influences and
between populations using ISSR genetic influences from within the
markers in four populations of B. population. Results of AMOVA also
oppositifolia is presented in Table 7. show that variation in population and
between populations are very
significant (P < 0.01). Results of
AMOVA on 30 accessions of B.
oppositifolia in four populations using
ISSR markers are presented in Table
8.

139
SABRAO J. Breed. Genet. 50 (2) 129-144

Cluster analysis among accessions According to Harsono et al.

of B. macrophylla and B. (2016), based on the diversity
Oppositifolia analysis of Bouea based on
morphological characters, B.
The results of cluster analysis of 30 oppositifolia has a similarity coefficient
accessions of B. oppositifolia, 75 between 0.49-1.00, whereas B.
accessions of B. macrophylla, and two macrophylla has a similarity coefficient
outgroups (M. indica and A. between 0.77-1.00. This indicates that
occidentale) are presented in Figure 4. the morphological variation of B.
Thirty accessions of B. oppositifolia oppositifolia is higher compared to
and 75 accessions of B. macrophylla that of B. macrophylla. Morphological
added with 2 outgroups (Mangifera variation is influenced by the level of
indica and Anacardium occidentale) individual plasticity. Phenotypic
have similarity coefficients ranging plasticity is influenced by the
from 0.17 to 1.00 and are classified interaction between the individual and
into 3 main groups at a coefficient of his environment (Mboumba and Ward,
0.34 (Figure 4). The results of the 2008). Phenotypic plasticity and local
analysis show that the results data adaptation are considered important
using ISSR markers are able to mechanisms in the adaptation of
distinguish both Bouea genus and its plants to new environments (Sexton
outgroups. et al., 2002). This is inversely
Studies using molecular proportional to the results obtained
markers related to the genus of Bouea using ISSR markers. This difference
are still very rare, and therefore the may be due to the differences in
search for comparative data for geographical distribution. The
molecular analysis in this study proves geographical distribution of B.
to be very difficult. The results in this macrophylla is more vast than that of
study are quite different from those of B. oppositifolia. The vast distribution
Ghazali et al. (2015) who examined of B. macrophylla causes low gene
the relationship between species in flow due to the long distances
the genus Bouea in the Malaysian between populations. According to
peninsula based on ISSR markers, Harsono et al. (2017), based on the
which indicated that the similarity genetic analysis using cpDNA
coefficient of B. macrophylla ranged sequence of trnL-F intergenic space,
from 0.659-0.955 and similarity B. oppositifolia is considered as the
coefficient of B. oppositifolia ranged ancestor of B. macrophylla. This
from 0.591-0.977. This is due to the research can be used by breeders to
differences in sample split distance. identify the diverse genotypes of
Among the Bouea genus originating different groups and use them in
from Indonesia, most samples were future breeding programs. Based on
far in between the provinces and all information obtained in this study,
islands, whereas Bouea originating the existence of Bouea as an endemic
from the Malaysian peninsula has low plant in western Indonesia should
level of distribution. This geographic always be preserved. This study
position may affect the genetic provides a baseline data for Bouea
variations, which emerged from each conservation programs in Indonesia.
member of the Bouea genus.

140
 Harsono et al. (2018)

Figure 4. Dendogram of Joint ISSR data of B. macrophylla and B. oppositifilia using

ISSR data with Unweighted Pair Group Method with Arithmetic Average (UPGMA).
Red (B. macrophylla), Blue (B. oppositifolia), and Green (M. indica and A.
occidentale) backgrounds show cluster differences.

141
SABRAO J. Breed. Genet. 50 (2) 129-144

CONCLUSION Botstein D, White RL, Skolnick M (1980).

Construction of a genetic linkage
ISSR markers can be used to map in man using restriction
distinguish species from the Bouea fragment length polymorphisms.
Am. J. Hum. Genet. 32: 314–331.
genus originating from Indonesia. B.
De Riek J, Calsyn E, Everaert I, Van
macrophylla (0.18-0.83) has a greater Bockstaele E, & De Loose M
genetic variation compared to B. (2001). AFLP based alternatives for
oppositifolia (0.56-0.99). The value of the assessment of distinctness,
gene flow (NM) in B. oppositifolia uniformity and stability of sugar
(0.6584) is higher compared to that of beet varieties. Theor. Appl. Genet.
B. macrophylla (0.3691). The primers 103: 1254-1265.
used in this study resulted in greater Doyle JJ. Doyle JL (1987). A rapid DNA
polymorphic information content value isolation procedure for small
in B. macrophylla (0.893) compared to quantities of fresh leaf tissue.
Phytochem Bull. 19: 11–15.
B. oppositifolia (0.7009). B.
Finkeldey R, Leinemann L, Gailing O
macrophylla is grouped into 3 clusters (2010). Molecular genetic tools to
with a similarity coefficient of 0.35 infer the origin of forest plants and
whereas B. oppositifolia is grouped wood. Appl. Microbiol. Biotechnol.
into 4 clusters with similarity 85: 1251–1258.
coefficient of 0.84. The ISSR marker is Fitmawati, Hartana A (2010). Phylogenetic
capable of separating B. macrophylla Study of Mangifera laurina and its
and B. oppositifolia with a similarity Related Species Using cpDNA trnL-
coefficient of 0.34. This indicates that F Spacer Markers. Hayati J. Biosci.
B. macrophylla and B. oppositifolia 17: 9–14.
Fischer M, Matthies, D (1998). RAPD
came from the same ancestor. The
variation in relation to population
ISSR marker can used to distinguish size and plant performance in the
members of the Bouea genus. rare Gentianella germanica. Am. J.
Bot. 85: 811–819.
Ghazali MN, Mohammad AL (2014).
ACKNOWLEDGEMENT Comparative Leaves Anatomical
Studies of Bouea, Mangifera, and
This research was supported by KEMENRISTEK Spondias (Anacardiaceae) in
DIKTI through HIBAH FUNDAMENTAL 2017. Malaysia. J. Life Sci. 8: 758–767.
Thanks to all parties involved in this study. Ghazali MN, Yunus FM, Mohammad AL
(2015). Assessment of genetic
relationships within Bouea
REFERENCES (Anacardiaceae) accessions in
Peninsular Malaysia using inter
Bass P, Kalkman K, Geesink R (2012). The simple sequence repeats (ISSR)
Plant Diversity of Malesia, in marker. Afr. J. Biotechnol. 14: 76–
Proceeding of the Flora Malesiana 85.
Symposium Commemorating Gao J, S. Zhang, L. Zhang QIY, Wang C,
Professor Dr. CGGJ Van Steenis Song W, Han S (2006). Application
Leiden, August 1989. of ISSR markers to fingerprinting
Bolaric S, Barth S, Melchinger AE, Posselt of elite cultivars (varieties/clones)
UK (1974). Genetic diversity in from different sections of the
European perennial ryegrass Genus Populus L. Silvae. Genet.
cultivars investigated with RAPD 55: 1–6.
markers. Plant Breed. 124: 161– Gou X, Elston R (1999). Linkage
166. information content of polymorphic

142
 Harsono et al. (2018)

genetic markers. Hum. Hered. 49: Le HT, Hancock JF (1999). Germplasm

112–118. Resources in Vietnam: Major
Gonzales A, Wong A, Delgado-Salinas A, horticultural and industrial crops.
Papa R, Gepts P (2005). Hort Sci. 34: 175–180.
Assessment of Inter simple Lewontin RC (1974). The genetic basis of
sequence repeat markers to evolutionary change. Columbia
differentiate sympatric wild and University Press. New York. 560
domesticated Populations of Mishra P, Ali AS, Kuralkar SV, Dixit SP,
common bean. Crop Sci. 45: 606– Anggarwal RAK, Dangi PS, Verma
615. NK (2013). Analysis of genetic
Harsono T, Pasaribu N, Sobir, Fitmawati diversity in berari goat population
(2017). Phylogenetic analysis of of Maharashtra State. Iran J. Appl.
Indonesian gandaria (Bouea) using Anim. Sci. 3: 553–559.
molecular markers of cpDNA trnL-F Mishra PK, Fox RT, Culham A (2003).
intergenic spacer. Biodiversitas J. Inter simple sequence repeat and
Biol. Divers. 18: 51–57. aggressiveness analyses revealed
Harsono T, Pasaribu N, Sobir, Fitmawati high genetic diversity,
(2016). Diversity of Gandaria recombination and long range
(Bouea) based on morphological dispersal in Fusarium culmorum.
characters in Indonesia. SABRAO J. Ann. Appl. Biol. 143: 291–301
Breed. Genet. 48: 504–517. Mboumba G, Ward D (2008). Phenotypic
Hartl DL, Clark AG (1989). Effective allele plasticity and local adaptation in
number in Principle of population two extreme populations of Acacia
genetics, 2nd ed., Sinaure karroo. Afri. J. Range and Forage
Associates: 125. Sci. 25: 121-130.
Holsinger HE, Mason-Gamer RJ (1999). Nei M (1978). Estimation of average
Hierarchical analysis of nucleotide heterozygosity and genetic
diversity in geographically distance from a small number of
structured populations. Genet. individuals. Genet. 89: 583–590.
142: 629–639. Nybom H, Bartish (2000). Effects of life
Hou D (1974). Anacardiaceae, Flora history traits and sampling
Malesiana-Series 1, strategies on genetic diversity
Spermatophyta. 8: 395–548. estimates obtained with RAPD
Julisaniah NI, Sulistyowati L, Sugiharto AN markers in plants. Perspect. Plant
(2008). Analisis Kekerabatan Ecol. Evol. Syst. 3: 93–114.
Mentimun (Cucumis sativus L.) Rifai MA (1992) Bouea macrophylla
menggunakan Metode RAPD-PCR Griffith, in plant resources of
dan Isozim. J. Biodiversitas 9: 99– South-East Asia. No. 2: Edible
102. fruits and nuts, R. E. Coronel and
Kesari V, Madurai Sathyanarayana V, E. W. M. Verheij, Eds. Bogor:
Parida A, Rangan L (2010). Porsea Foundation, 104–105.
Molecular marker-based Sexton JP, McKay JK, Sala A (2002)
characterization in candidate plus Plasticity and genetic diversity may
trees of Pongamia pinnata, a allow saltcedar to invade cold
potential biodiesel legume. AoB climates in North America. Ecol
Plants 20: 1–12. Appl. 12: 1652–1660.
Korbin M, Kuras A, Zurawicz E (2002). Slatkin M (1995). A measure of population
Fruit plant germplasm subdivision based on microsatellite
characterization using molecular allele frequencies. Genet. 139:
markers generated in RAPD and 457-462.
ISSR-PCR. Cell Molec Biol Lett. Tanksley SD, Bernatzky R (1989).
7:785–794. Restriction fragments as molecular
markers for germplasm evaluation

143
SABRAO J. Breed. Genet. 50 (2) 129-144

and utilisation, in In the Use of

Plant Genetic Resources, A. H. D.
Brown, O. H. Frankel, D. R.
Marshall, and J. T. Williams, Eds.
Cambridge: Cambridge University
Press: 353–362.
Trojanowska MR, Bolibok H (2004).
Characteristics and comparison of
three classes of microsatellite-
based markers and their
application in plants. Cell Mol. Biol.
Lett. 9: 221–238.
Vilas A, Pérez Figueroa A, Quesada H,
Caballero A (2015). Allelic diversity
for neutral markers retains a
higher adaptive potential for
quantitative traits than expected
heterozygosity. Mol. Ecol. 24:
4419–4432.
Wahyuni S, Xu DH, Bermawiel N,
Tsunematsu H, Ban T (2004).
Skrining ISSR Primer Studi
Pendahuluan Kekerabatan antar
Jahe Merah, Jahe Emprit, dan Jahe
Besar. Bul. Penelit. Tanam.
Rempah dan Obat. 15: 33–42.
Waugh R (1997). RAPD analysis: use for
genome characterization, tagging
traits and mapping, in Plant
Molecular Biology-A Laboratory
Manual, Berlin: Springer Berlin
Heidelberg: 305–333.
Xiao L, Gong L, Hao G, Ge X, Tian B,
Zheng S (2005). Comparison of the
genetic diversity in two species of
cycads. Aust. J. Bot. 53: 219–223.
Yunus A (2007). Identifikasi Keragaman
Genetik Jarak Pagar (Jatropha
curcas L.) Berdasarkan Penanda
Isozim. J. Biodiversitas. 8: 249–
252.

144