Alleles Variation of Coconut Accessions (Cocos nucifera L., Arecaceae) Based on Microsatellite DNA.Procceding ICBB 2012: 235-238.
4th I NTE RNATIONAL CON FERENC E ON BIOSCI ENC ES AND BIOTECH NO LOGY
,,Advancing lde stuu'yJsfor health (anti aging development in pticalar) andfood security"
19.
FROM
COMPARING EXTRACTION METHODS TO ISOLATE ANDROGRAPHOLIDE
THE BITTER HERB {Andrographis puticulaaBurm-f Ness)
Ni Kadekffirditiani and Suwidiiyo Pramono
232
POSTER PRESEF{TATIoNS : BIoDIVERSITY AND ENVIRoNMENT
I.
ALLELESVARIATIONOFCOCONUTACCESSIONS
(Cocosnuciferal.,Arccaceae)BASEDoNMICROSATELLITEDNA
2.
3.
Eniek Krismiyanti, I Gede Rai Maya Temaja, Made Sudana
and G.N. Allt Susanta Wirya -...-..-....-G*ENETICDIVERSITY OF SOROH CELAGI (PASEK CATUR SANAK CLAN)
BASED ON Y-CHROMOSOMAL MICROSATELITES DNA
I Ketut Junitha, Made Pharmawati and Wayan Rosiana
THE STUDY OF SOCIO-ENGINEERING OF SUBAK SYSTEM DEVELOPMENT
WITH Agroecotowism ORIENTED
Sumiyati, Elik Sutiarso, Wayan Windia and Putu Sudira
4.BIOCATALYTICDESULFURIZATIONoFDIBENZOTHIOPHENE
BY Pseudmonas sP- STRAIN KWN5
Ida Bagus nhyan Gunarn, I Gusti Ayu Lani Triani, Nyoman Semadi Antara,
Agus Selamet Duniaji, Yohanes Setiyo and Dewa Adi Supata
s. INDUCE FOLIPLOIDY OF Lilium longiflorwtTHuNB. USING ORYZALIN
TREATMENT
6.
7.
8.
Gusti Ayu ilNirah Dwidaputri, Eniek Kriswiyanti and IdaAyuAstarini
COMPOSIING ACCELERATION OF KITCHEN WASTE USTNG
MICROBI-AL INOCULANT
Ni Made k*i Hapsari Arihantana, Ni Luh Yulianti """""""""'j"""""""""
HEAVY METAL BIOREMEDIATION LEAD (Pb) TO AGRICULTURAL LAND
ON THE EDGE OF LAKE BUYAN BALI WITH PLANT SANSCAiCTiA IOTCNtii
Ni Luh Sudani........
THE IDENTIFICATION AND INVESTIGATION OF PARASITIZATION RATE
oFPARASIToIDSoFHAIRYCATERPILLARLyMANTRIAMARGINATAWLK.
(LEPIDOMERA: LYMANTRIIDAE) IN BULELENG BALI
I Wayan Srsila And I Putu Sudiarta
23s
235
245
250
254
257
262
263
PRESBITAIION S: HEALTH
THE IMPIEMENTATION OF MODULE-IIGSAW COOPERATIVE LEARNING
STRATEGY, MPROVING T}{E PREVENTATIVE BEHAVIOR TOWARDS
PO STER
1.
2.
3.
DENGUE HEMORRHAGEIC FEVER
Dewa Nymran Oka.---..'.----.
CLASSIC HODGKTN'S LYMPHOMA SIMULATING ANAPLASTIC LARGE
CELL LYMPHOMA
I Gusti AYu Sri Mahendra Dewi"'
EXTENDING TIME OF KOMBUCHA TEA FERMENTATION IMPROVES THE
OF
GROWTHOF INHIBHON CAPACITY OF Escherichia coli;THE PRODUCER
EXTENDED SPECTRUM BETA LACTAMASES (ESBL) BY IN VITRO METHOD
Ni Putu A4radnyani,I Dewa Made sukrama, I wayan Putu sutirta Yasa""""'
xvi I Bali
- Indonesio | 21o - 27d September 2012
w
267
t
lth INTERNATTONALCONFERENCE ON BIOSCIENCES AND BIOTECHNOLOCY
"Advncingtife sctencesJor health (aaiagingdevelopmenl in parlicular) andJodsecarity"
&
Y9
ALLELES VARTATION OF COCONUT ACCESSIONS
(Cocos meifero L., Arecaceae) BASED ON MICROSATELLITE DNA
Eniek Krisrviyantit', I Gede Rai l\{ayeTemaia2, Made Sudana2
and G.N. Alit SusanteWirYa2
Dept Biologt of Faculty Mathemdi and Natural Science
2Faculty of Agricalture, Udapna University.
.
I
Corresponding author: [email protected]
ABSTRACT
purpose.
Coconut plays a more prominent role in the social, economic and crltural for Balinese people, especially fm ritual
Alleles
Arecaceae).
(Cocos
L.,
nucifera
accessions
oconut
variation
of
sixteen
alleles
to
determined
research
The aimed of this
variation in 16 coconut acoessions from Br. Babung, Gunaka and Pikat vitlages, Dawan, Klungkung regencywas determined
per locus, there are
using 4 microsatellite markers. A total of 28 alleles were detected by microsatellite with an average 7 alleles
7 alleles of microsatellite primer CNZ05, 8 alleles of CNZ09,7 allelesof CNZ2I, and 6 alleles of microsatelliteprimer CNZ5l.
Keyword: Alleles, coconul aeessions, microsatellite nrorkers, heteroryosity-
INTRODUCTION
There are two maintypes of coconut: 'tall', the naturally cross pollinating group with more economic
value and 'dwarf', the naturally self pollinating group with reduced size and growth habit. ft is believed
that the 'dwarfl originded from earliest 'tall' coconuts in atolls of the far east and maintained most of its
original genome because of its autogamous behaviour- Thm, dwarf coconuts are of similar stature and fruit
features irrespective ofthe geographical location. However, 'tall'genome has undergone many changes
because of bottle-neck effects of selection, though it had retained the tall stature and fruit clraracteristics
irrespective of its dispenion from far east to Indo-Atlantic regions across Africa (Bourdeix et al-,2005,
e t al, 2009').
The coconut palm (Cocos nucifera L., Arecaceae) is the most widely cultivated crop in Philippines,
Indonesia, India, Sri Lanka and China, wher€ coconut palm plays an important role in economy. It provides
food supply and industrial products, such as coconut oil, copr4 liquid endospelrn and desiccated coconutAlmost every part of the coconut tree can be used in eitlrer making commercial products ormeeting the
food requirements of rural communities (Teulet et al., 2000)gelrnplasm
Investigation of coconut genetic diversity provides sufficient scientific data
and
biochemical
traits,
palm
done
by
morphological
has been
management. Diversity analysis in coconut
Dasanayaka
fc
molecular markers. Morphological and biochemical markers have shortages as follows: Long juvenile
phase, high cost. long-term of field evaluation, environment factors and limited number of available
phenotypic markers (Manimekalai et al., 20Aq. Howeveq since molecular markers are detectable at
all stages of developmmt and can cover the entire genome, they, which detect variation ht-DNA level,
overcome most limitations of morphological and biochemical markers, (Lebrun et al, 1998;, Perera et
a1.,1996,2000, 2001; Rivera et al-,1999; Teulat et a1.,2000; Dasanayake et a1.,20A3;U@hyay et al.,
2004; Manimekalai et a1.,2006,2007,2010). Among various available molecular marker techniques,
simple sequence repeat(SSR) or microsatellite markers pmvide good signal in evaluating genetic diversity
and genetic relationship in plants. The increased number of SSR markers greatly improves tlre previously
established genetic relalionships among coconut varietieVpopulations {Liu et
al.,20ll).
MATERIALAND METHODS
plant material used: Leaf samples were obtained from l6 coconut accessions from Br. Babung
Gunaksa and Br PikatDawan Klungkung residence. Among the 16 coconut accessions. 13 were of the
'tall'category (n),trh ancak, baraldred, bingin, gadang/green, kebo, kopyor/polo/srogsoga4 manjangan.
Bali - Indonesial2l'' - 22'"t September 20121235
@
- Mr,-*nS
lifr
sctenesfu
tah
(odi agig @
mulung, penyu, rangda, salak, sangket, stdamala,
category (green, white and yellow dwarf)-
a
p*fu)
nyuh:
ad Id *z:irryBalinese coconut) and 3 were of the 'dwartr
DNA extraction and detection of microsatellite polymorphisms:
DNA was extracted from fresh coconut leaves using a CTAB based protocol modified from Doyle
and Doyle ( 1937). The primer sequences and associated information are given in Table l Polymerase Chain Reactions (PCR) assay and gel analysis
DNA was amplifled in 13 pL reactions containing 2 pL sample, 3.5 pL H,O, 6.5 pL Mastermix/
annealing
hotstart (eiagen), I pL primer The PCR programmed for 30 cycles of 60 seconds each at 94oC,
denaturation
temperature 3g-52"C, extension temperature 72oC. The first cycle was preceded by a 3 min
on 6Yo
at94"C and the last cycle ended with 5 min extension at72" C. Reaction products were separated
were
alleles
1984).The
(Tegelstorm,
polyacrylamide (denatured) and visualized with silver nitrate staining
gel.
in
a
single
scored based on the size of each PCR amplified fragment by electrophoresing all samples
(Hutchinson,
Allele size was determined by semilog plotting of distance migration of amplicon on PAGE
locus using
each
microsatellite
for
calculated
were
values based on allele frequencies
2001). Diversity
;
Nei's methods ( 1987).
Table I : Detail of microsatellites Primer tsed
Reverse primer
Forward primer (5'-3')
Primer name
(3'-5')
CNZO5
CTTATCCAAATCGTCACAGAG
AGGAGAAGCCAGGAAAGATTT
CNZO9
ATCTACCA GTGTGGTCCTCTC
ACCAGGAAAAAGAGCGCAGAA
CNZ2I
CNZ5l
ATGTTTTAGCTTCACCATGAA
TCAAGTTCAAGAAGACCTTTG
CTTTAGGGAAAAAGGACTGAG
ATCCATGAGCTGAG CTTGAAC
RESULTS AND DISCUSSION
The sixteen accessions coconut with four specific microsatellite primer pairs produced a total
28 alleles ranging from 6 to 8 alleles per locus (Fig- 1 and Table 2)'
of
Fig.l Allelograph from locus CNZ05
A.Green dwarf, B.Yellow dwarf, D.white dwarf, the tall type: c.Green tall,
E.Red tall, F. Kopyor/Polo/srogsogut,G.Salak,H. Brown, l.Rangda,J.Kebo,KMulung,L-lBingin,
M.Penlw,N.Sangket
Fig.2 Allelograph from locus CNZ2I
V: yellow dwarf; X: white dwarf and W:green dwarf; A'. Ancak.B.Baraklted,P'. Salak,M'.Mulung,Q:
Sangke t, O: Rangda, H: Green/ G adang, K: Kopyor,
R'. Sudamala,J: Kebo,N: Penyr'. "tall category"
236 | Bali - Indonesia
| 2 ln - 22'd September 201 2
'
lth INTERNAT0NALCONFERENCE ON BIOSCIENCESAND BIOTECHNOLOGY
"Advancing life scienaJor health (onti aging development in paaiular) andlood secttriry"
6
W
Table 2. Detail of microsatellite loci, alleles detected in coconut accessions
Microsatellite
CNZO5
No of alleles
7
CNZO9
8
CNZ2I
7
CNZ5I
6
Allele size (bp)
1
18, 12t, 138, l4g,
l5g, 168,
Hcterozygmity
178
lj!s, 130, 135, 140, 145, 155
224, 2X, 250, 260, 27 0, 27 6, 286
I 15,120,
160, 170, 180, 190, 200, 210
0.838il5
0.8755
0.78s092
0.80658
In the present study, the mean numberof alleles per locus (7.0) was similar to that found in other
studies of coconut palm tree populations usiag SSR markers. Rajesh et al. (2@8) the genetic diversity
in 26 coconut accessions from the Andaman and Nicobar Islands was determired using 14 microsatellite
markers- A total of 103 alleles were detected by the microsatellite markers with an average of 7 -35 alleles
per locus Dasanayaka et al. (2009) sixteen primer pairs identified 79 alleles, averaging 4.9 alleles per
locus ranging from 3 to l0 simple sequence repeat polymorphisms among tlre 43 coconut accessions
assessed. All 16 loci were polymorphic and atotal of 76 alleles were observed in tall category ranging
from 3 to l0 with an average of 4.7 alleles perlocus. Atotal of 29 alleles were obseryed in dwarf category
ranging frwr I to 3 with an average of I .8 alleles per locus, Ribeiro et al. (2010) found a total of 68 alles,
rangrng frrom 2 to l3 alleles per locus, with anaverage of 5.23, and heterorygosity were 0-459 and 0.443,
Kumar, etal-(2011) found atotal2S polymorfrric alleles produce of 8 primers were used of l4 accessions,
Liu et ol. (2011) used 26 simple sequence repeat (SSR) markers, were detected a total of 188 alleles with
an averageof7.23 alleles per locus.
On the table 3 the locus CNZ05: the highest frequency of alleles is size 128 bp, (0.281), rare
freqnency ll8 bp was found only on coconutecession'nyuh Rangda" (0.063). The Locus CNZ09 height
freqtrency on allele size 125 bp and 135 bp both with frequency 0.218, rare frequency 155 bp only on
co@nut accession 'nyuh AncaV' frgquency 0.031. Locus CNZ2I height frequerrcy on size allele 250 bp
with freqwrcy 0.344, rare frequency 286 bp only on coconut accession 'nltuh white dwarf'with frequency
0-063- Locrc CNZ5I height frequency on sizeallele 190 bp with frequency 0.344, rare frequency on I l8
bp, on co@nut accession 'nyuh ancak and gafung/green tall " frequency 0.063
ACKNOWLEDGEMENT
This research was funded by Directorate General of Higher Education, Ministry of Education and
Cuhure Republic of Indonesia by Research Dissertation Program, contract number:2 I .1/UN l4lLPPMl20l2
darcd l9 lnuary 2012.
REFERENCES
CultivarNaietyl Ecotype/Population/Variant.
In C-monut Genetic Resources. Batugal F, Ramanatha Rao V and Oliver J. (eds). Intemational Plant
Genaic Resources Institute - Regional Office for Asia, the Pacific and Oceania flPGRI-APO),
Bundeix Y&
Santos G, Labouisse JP and Baudouin L. 2005.
Ssrdarg, Selangor D.E, Malaysia.
hsana,v-ake PN, Everard EH, Karunanayake I*G and Nandadasa HG. 2003, Characterization of coconut
germplasm by microsatellite markers, Tmp Agriculture Research, I 5: 5 1 -60.
Dasana3'aka PN, Everard JMDI Karunanayaka EH and Nandadasa HG. 2009. Analysis of coconut (Cocos
mcifera L-) diversity using microsatellite markers with emphasis on management and utilisation of
Esrxticresources. J. National Sci. Found. Sri Lanka,3l:99-109.
Doyle JL. 1981. A Rapid DNA Isolation Procedure for Small Amount
Phlrm*rcm- Bull. l9: I l- 15.
Dq1te JJ
d
of Leaf
Tissue.
Bali - htdonesial2l'' - 22n September 2012lZ37
BIOTECHNOLAGY
CONFEREIff;E ON BIOKIENCES AND
4th INTERNATIONAL
':;;;;;;ii;
,ii"*irt
,
tf,iii*i
osis dr*topment
in partictttar) ondfood
sectritv"
center Education
DNA Band By Semi-Log Plot'cancer Research
Hutchinson F. 200 I . Estimating sizes of
PartnershiP.
of South
20 r l. Microsatellite Marker Based characterization
Kumar SR Manimekalai R and Kumari BDR.
lnt. J, Plant Breed. Genet. 5:34-43.
Pasific Coconut (Cocos mteiferal.) Accessions.
Genetic Diversity in coconut (cocos
Seguin M, Grivet L and Baudouin L' 1998'
Lebrun B N',Cho YP,
nuciferal.)
polymorphism (RFLP) Markers' Euphytica
Revealed by Restriction Fragment Length
101:103-108.
by Microsatellite
Diversity of Coconut Cultivars in China
Liu X, Thng H, Li DD, Hou LH. 2011. Genetic
(SSR) Markers Molecular Plant Breeding2(12):
(cocos nuciferaL')
p.2006, Ar.".riig g"netic relationships among coconut
Manimekalai R and Nagarajan
108(l): 49'54'
."**u1 markers, Scientia Horticulturae'
accessions using inter simple sequence
of genetic
Sequence Repeat markers for estimation
Manimekalai R and Nagarajan P. 2007- U."
and
Biochemistry
accessions' Journal of Plant
in coconut (cocos nuciferal.) germplasm
"iii;;le
diversity
BiotechnologY, 16(1): 29-33'
Genetic Structure of
SSR and ISSR Markers Based Population
Manimekalai R and Nagar{an P 2010:
(l):
Accssion Indian J. of Plant Genetic Resources,23
Coconut (Cocos nuciferal.) Germplasm
77-82.
columbia university Press New York'
Nei M.1gg7. Molecular Evolutionary Genetics.
of coconut (co cos nucderaL.) Biodiversity
peries
Femando wMU. 1 996. Conservation
perera L,
RRA and
106'
in Sri Lanka. Plant Genetic Resources Newletter
Distribution of Genetic Diversitybof coconut
perera L, Russel R, provan J and powell w. 2001 . Levels and
(Cocosnuciferal.,varTypicar,o*typi"4fromSriLankabyMicrosatelliteMarkers.Euphytica
122 381-389Rajesh MK, Nagarajan P, Jerard BA, Arunachalaml
of coconut accessions (cocos
nuciftral')
V and Dhanapal R' 2008' Microsatellite variability
Science 94
from Andaman and Nicobar Islands' current
ttiJ;l,i?.l
Mr and vencovskv R. 2010. Population
,ebrun L, chaves LJ, Brondani c, Zucchi
nucifera L') by microsatellite markers' Genet'Mol'
structures of Brazilian Tall coconut'7Vo',o'
*ib"ir:?I,
JHA Barker, G M Amold, G Ayad, T Hodgkin'
*,r".",,,|]'i'r('Ii*uror,
A Karp'1999' Isolation and
characterizationofpolymorphicmicrosatellitesincocosnuciferal'Genome'1999'42(4):668popuration: an Improved
r"g"rrfJ* H. 19g6. Mitochondrial DNA in Natural
Routine for Screening
of
GeneticVariationBaseonSensitivesilverStaining.ElectrophoresisT:226-229
analysis of genetic diversity of the coconut
c, Trehin R, Lebrun B Barker JHA. 200b. An
Teulat B, Aldam
geographic range using sequence-tagged microsatellites
(Cocos nucifera) populations from across the
andAFLPs' TheorAppl Genet 100:
764-771'
,r:.,-.oirl, in
iSSnr)
- L:- ^-r diversity
in Indian coconul
2004'Genetic relationship and
upadhyay A, Jayadev K, Mamimekalai R(2004): 353-362'
Horticulturae 99
accessions based on RAPD markers.Scentia
238 | Bali - Indonesia
|
2l'
- 22*t September 20I2
,,Advancing lde stuu'yJsfor health (anti aging development in pticalar) andfood security"
19.
FROM
COMPARING EXTRACTION METHODS TO ISOLATE ANDROGRAPHOLIDE
THE BITTER HERB {Andrographis puticulaaBurm-f Ness)
Ni Kadekffirditiani and Suwidiiyo Pramono
232
POSTER PRESEF{TATIoNS : BIoDIVERSITY AND ENVIRoNMENT
I.
ALLELESVARIATIONOFCOCONUTACCESSIONS
(Cocosnuciferal.,Arccaceae)BASEDoNMICROSATELLITEDNA
2.
3.
Eniek Krismiyanti, I Gede Rai Maya Temaja, Made Sudana
and G.N. Allt Susanta Wirya -...-..-....-G*ENETICDIVERSITY OF SOROH CELAGI (PASEK CATUR SANAK CLAN)
BASED ON Y-CHROMOSOMAL MICROSATELITES DNA
I Ketut Junitha, Made Pharmawati and Wayan Rosiana
THE STUDY OF SOCIO-ENGINEERING OF SUBAK SYSTEM DEVELOPMENT
WITH Agroecotowism ORIENTED
Sumiyati, Elik Sutiarso, Wayan Windia and Putu Sudira
4.BIOCATALYTICDESULFURIZATIONoFDIBENZOTHIOPHENE
BY Pseudmonas sP- STRAIN KWN5
Ida Bagus nhyan Gunarn, I Gusti Ayu Lani Triani, Nyoman Semadi Antara,
Agus Selamet Duniaji, Yohanes Setiyo and Dewa Adi Supata
s. INDUCE FOLIPLOIDY OF Lilium longiflorwtTHuNB. USING ORYZALIN
TREATMENT
6.
7.
8.
Gusti Ayu ilNirah Dwidaputri, Eniek Kriswiyanti and IdaAyuAstarini
COMPOSIING ACCELERATION OF KITCHEN WASTE USTNG
MICROBI-AL INOCULANT
Ni Made k*i Hapsari Arihantana, Ni Luh Yulianti """""""""'j"""""""""
HEAVY METAL BIOREMEDIATION LEAD (Pb) TO AGRICULTURAL LAND
ON THE EDGE OF LAKE BUYAN BALI WITH PLANT SANSCAiCTiA IOTCNtii
Ni Luh Sudani........
THE IDENTIFICATION AND INVESTIGATION OF PARASITIZATION RATE
oFPARASIToIDSoFHAIRYCATERPILLARLyMANTRIAMARGINATAWLK.
(LEPIDOMERA: LYMANTRIIDAE) IN BULELENG BALI
I Wayan Srsila And I Putu Sudiarta
23s
235
245
250
254
257
262
263
PRESBITAIION S: HEALTH
THE IMPIEMENTATION OF MODULE-IIGSAW COOPERATIVE LEARNING
STRATEGY, MPROVING T}{E PREVENTATIVE BEHAVIOR TOWARDS
PO STER
1.
2.
3.
DENGUE HEMORRHAGEIC FEVER
Dewa Nymran Oka.---..'.----.
CLASSIC HODGKTN'S LYMPHOMA SIMULATING ANAPLASTIC LARGE
CELL LYMPHOMA
I Gusti AYu Sri Mahendra Dewi"'
EXTENDING TIME OF KOMBUCHA TEA FERMENTATION IMPROVES THE
OF
GROWTHOF INHIBHON CAPACITY OF Escherichia coli;THE PRODUCER
EXTENDED SPECTRUM BETA LACTAMASES (ESBL) BY IN VITRO METHOD
Ni Putu A4radnyani,I Dewa Made sukrama, I wayan Putu sutirta Yasa""""'
xvi I Bali
- Indonesio | 21o - 27d September 2012
w
267
t
lth INTERNATTONALCONFERENCE ON BIOSCIENCES AND BIOTECHNOLOCY
"Advncingtife sctencesJor health (aaiagingdevelopmenl in parlicular) andJodsecarity"
&
Y9
ALLELES VARTATION OF COCONUT ACCESSIONS
(Cocos meifero L., Arecaceae) BASED ON MICROSATELLITE DNA
Eniek Krisrviyantit', I Gede Rai l\{ayeTemaia2, Made Sudana2
and G.N. Alit SusanteWirYa2
Dept Biologt of Faculty Mathemdi and Natural Science
2Faculty of Agricalture, Udapna University.
.
I
Corresponding author: [email protected]
ABSTRACT
purpose.
Coconut plays a more prominent role in the social, economic and crltural for Balinese people, especially fm ritual
Alleles
Arecaceae).
(Cocos
L.,
nucifera
accessions
oconut
variation
of
sixteen
alleles
to
determined
research
The aimed of this
variation in 16 coconut acoessions from Br. Babung, Gunaka and Pikat vitlages, Dawan, Klungkung regencywas determined
per locus, there are
using 4 microsatellite markers. A total of 28 alleles were detected by microsatellite with an average 7 alleles
7 alleles of microsatellite primer CNZ05, 8 alleles of CNZ09,7 allelesof CNZ2I, and 6 alleles of microsatelliteprimer CNZ5l.
Keyword: Alleles, coconul aeessions, microsatellite nrorkers, heteroryosity-
INTRODUCTION
There are two maintypes of coconut: 'tall', the naturally cross pollinating group with more economic
value and 'dwarf', the naturally self pollinating group with reduced size and growth habit. ft is believed
that the 'dwarfl originded from earliest 'tall' coconuts in atolls of the far east and maintained most of its
original genome because of its autogamous behaviour- Thm, dwarf coconuts are of similar stature and fruit
features irrespective ofthe geographical location. However, 'tall'genome has undergone many changes
because of bottle-neck effects of selection, though it had retained the tall stature and fruit clraracteristics
irrespective of its dispenion from far east to Indo-Atlantic regions across Africa (Bourdeix et al-,2005,
e t al, 2009').
The coconut palm (Cocos nucifera L., Arecaceae) is the most widely cultivated crop in Philippines,
Indonesia, India, Sri Lanka and China, wher€ coconut palm plays an important role in economy. It provides
food supply and industrial products, such as coconut oil, copr4 liquid endospelrn and desiccated coconutAlmost every part of the coconut tree can be used in eitlrer making commercial products ormeeting the
food requirements of rural communities (Teulet et al., 2000)gelrnplasm
Investigation of coconut genetic diversity provides sufficient scientific data
and
biochemical
traits,
palm
done
by
morphological
has been
management. Diversity analysis in coconut
Dasanayaka
fc
molecular markers. Morphological and biochemical markers have shortages as follows: Long juvenile
phase, high cost. long-term of field evaluation, environment factors and limited number of available
phenotypic markers (Manimekalai et al., 20Aq. Howeveq since molecular markers are detectable at
all stages of developmmt and can cover the entire genome, they, which detect variation ht-DNA level,
overcome most limitations of morphological and biochemical markers, (Lebrun et al, 1998;, Perera et
a1.,1996,2000, 2001; Rivera et al-,1999; Teulat et a1.,2000; Dasanayake et a1.,20A3;U@hyay et al.,
2004; Manimekalai et a1.,2006,2007,2010). Among various available molecular marker techniques,
simple sequence repeat(SSR) or microsatellite markers pmvide good signal in evaluating genetic diversity
and genetic relationship in plants. The increased number of SSR markers greatly improves tlre previously
established genetic relalionships among coconut varietieVpopulations {Liu et
al.,20ll).
MATERIALAND METHODS
plant material used: Leaf samples were obtained from l6 coconut accessions from Br. Babung
Gunaksa and Br PikatDawan Klungkung residence. Among the 16 coconut accessions. 13 were of the
'tall'category (n),trh ancak, baraldred, bingin, gadang/green, kebo, kopyor/polo/srogsoga4 manjangan.
Bali - Indonesial2l'' - 22'"t September 20121235
@
- Mr,-*nS
lifr
sctenesfu
tah
(odi agig @
mulung, penyu, rangda, salak, sangket, stdamala,
category (green, white and yellow dwarf)-
a
p*fu)
nyuh:
ad Id *z:irryBalinese coconut) and 3 were of the 'dwartr
DNA extraction and detection of microsatellite polymorphisms:
DNA was extracted from fresh coconut leaves using a CTAB based protocol modified from Doyle
and Doyle ( 1937). The primer sequences and associated information are given in Table l Polymerase Chain Reactions (PCR) assay and gel analysis
DNA was amplifled in 13 pL reactions containing 2 pL sample, 3.5 pL H,O, 6.5 pL Mastermix/
annealing
hotstart (eiagen), I pL primer The PCR programmed for 30 cycles of 60 seconds each at 94oC,
denaturation
temperature 3g-52"C, extension temperature 72oC. The first cycle was preceded by a 3 min
on 6Yo
at94"C and the last cycle ended with 5 min extension at72" C. Reaction products were separated
were
alleles
1984).The
(Tegelstorm,
polyacrylamide (denatured) and visualized with silver nitrate staining
gel.
in
a
single
scored based on the size of each PCR amplified fragment by electrophoresing all samples
(Hutchinson,
Allele size was determined by semilog plotting of distance migration of amplicon on PAGE
locus using
each
microsatellite
for
calculated
were
values based on allele frequencies
2001). Diversity
;
Nei's methods ( 1987).
Table I : Detail of microsatellites Primer tsed
Reverse primer
Forward primer (5'-3')
Primer name
(3'-5')
CNZO5
CTTATCCAAATCGTCACAGAG
AGGAGAAGCCAGGAAAGATTT
CNZO9
ATCTACCA GTGTGGTCCTCTC
ACCAGGAAAAAGAGCGCAGAA
CNZ2I
CNZ5l
ATGTTTTAGCTTCACCATGAA
TCAAGTTCAAGAAGACCTTTG
CTTTAGGGAAAAAGGACTGAG
ATCCATGAGCTGAG CTTGAAC
RESULTS AND DISCUSSION
The sixteen accessions coconut with four specific microsatellite primer pairs produced a total
28 alleles ranging from 6 to 8 alleles per locus (Fig- 1 and Table 2)'
of
Fig.l Allelograph from locus CNZ05
A.Green dwarf, B.Yellow dwarf, D.white dwarf, the tall type: c.Green tall,
E.Red tall, F. Kopyor/Polo/srogsogut,G.Salak,H. Brown, l.Rangda,J.Kebo,KMulung,L-lBingin,
M.Penlw,N.Sangket
Fig.2 Allelograph from locus CNZ2I
V: yellow dwarf; X: white dwarf and W:green dwarf; A'. Ancak.B.Baraklted,P'. Salak,M'.Mulung,Q:
Sangke t, O: Rangda, H: Green/ G adang, K: Kopyor,
R'. Sudamala,J: Kebo,N: Penyr'. "tall category"
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Table 2. Detail of microsatellite loci, alleles detected in coconut accessions
Microsatellite
CNZO5
No of alleles
7
CNZO9
8
CNZ2I
7
CNZ5I
6
Allele size (bp)
1
18, 12t, 138, l4g,
l5g, 168,
Hcterozygmity
178
lj!s, 130, 135, 140, 145, 155
224, 2X, 250, 260, 27 0, 27 6, 286
I 15,120,
160, 170, 180, 190, 200, 210
0.838il5
0.8755
0.78s092
0.80658
In the present study, the mean numberof alleles per locus (7.0) was similar to that found in other
studies of coconut palm tree populations usiag SSR markers. Rajesh et al. (2@8) the genetic diversity
in 26 coconut accessions from the Andaman and Nicobar Islands was determired using 14 microsatellite
markers- A total of 103 alleles were detected by the microsatellite markers with an average of 7 -35 alleles
per locus Dasanayaka et al. (2009) sixteen primer pairs identified 79 alleles, averaging 4.9 alleles per
locus ranging from 3 to l0 simple sequence repeat polymorphisms among tlre 43 coconut accessions
assessed. All 16 loci were polymorphic and atotal of 76 alleles were observed in tall category ranging
from 3 to l0 with an average of 4.7 alleles perlocus. Atotal of 29 alleles were obseryed in dwarf category
ranging frwr I to 3 with an average of I .8 alleles per locus, Ribeiro et al. (2010) found a total of 68 alles,
rangrng frrom 2 to l3 alleles per locus, with anaverage of 5.23, and heterorygosity were 0-459 and 0.443,
Kumar, etal-(2011) found atotal2S polymorfrric alleles produce of 8 primers were used of l4 accessions,
Liu et ol. (2011) used 26 simple sequence repeat (SSR) markers, were detected a total of 188 alleles with
an averageof7.23 alleles per locus.
On the table 3 the locus CNZ05: the highest frequency of alleles is size 128 bp, (0.281), rare
freqnency ll8 bp was found only on coconutecession'nyuh Rangda" (0.063). The Locus CNZ09 height
freqtrency on allele size 125 bp and 135 bp both with frequency 0.218, rare frequency 155 bp only on
co@nut accession 'nyuh AncaV' frgquency 0.031. Locus CNZ2I height frequerrcy on size allele 250 bp
with freqwrcy 0.344, rare frequency 286 bp only on coconut accession 'nltuh white dwarf'with frequency
0-063- Locrc CNZ5I height frequency on sizeallele 190 bp with frequency 0.344, rare frequency on I l8
bp, on co@nut accession 'nyuh ancak and gafung/green tall " frequency 0.063
ACKNOWLEDGEMENT
This research was funded by Directorate General of Higher Education, Ministry of Education and
Cuhure Republic of Indonesia by Research Dissertation Program, contract number:2 I .1/UN l4lLPPMl20l2
darcd l9 lnuary 2012.
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