Alleles Variation of Coconut Accessions (Cocos nucifera L., Arecaceae) Based on Microsatellite DNA.Procceding ICBB 2012: 235-238.

(1)

(2)

19.

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"

COMPARING EXTRACTION

METHODS

TO ISOLATE

ANDROGRAPHOLIDE FROM

THE

BITTER HERB {Andrographis

puticulaaBurm-f

Ness)

Ni Kadekffirditiani

and

Suwidiiyo

Pramono

232

POSTER PRESEF{TATIoNS

:

BIoDIVERSITY

AND

ENVIRoNMENT

I.

ALLELESVARIATIONOFCOCONUTACCESSIONS

(Cocosnuciferal.,Arccaceae)BASEDoNMICROSATELLITEDNA

Eniek Krismiyanti, I Gede Rai Maya Temaja, Made Sudana and G.N.

Allt

Susanta Wirya

-...-..-....--2.

G*ENETICDIVERSITY OF SOROH

CELAGI

(PASEK CATUR

SANAK CLAN)

BASED ON Y-CHROMOSOMAL MICROSATELITES

DNA

I

Ketut Junitha, Made Pharmawati and Wayan Rosiana

3.

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

Gusti Ayu ilNirah Dwidaputri, Eniek

Kriswiyanti

and

IdaAyuAstarini

6.

COMPOSIING

ACCELERATION OF

KITCHEN

WASTE USTNG

MICROBI-AL

INOCULANT

Ni

Made

k*i

Hapsari Arihantana,

Ni

Luh

Yulianti

"""""""""'j"""""""""

7.

HEAVY METAL BIOREMEDIATION LEAD

(Pb)

TO AGRICULTURAL

LAND

ON THE

EDGE OF

LAKE BUYAN BALI WITH PLANT

SANSCAiCTiA IOTCNtii

Ni

Luh

Sudani...

8.

THE IDENTIFICATION

AND

INVESTIGATION

OF

PARASITIZATION RATE

oFPARASIToIDSoFHAIRYCATERPILLARLyMANTRIAMARGINATAWLK.

(LEPIDOMERA: LYMANTRIIDAE)

IN

BULELENG

BALI

23s

w

235

245

250

254

t

257

262

263

267

I Wayan Srsila And I Putu Sudiarta

PO STER

PRESBITAIION

S:

HEALTH

1.

THE IMPIEMENTATION

OF

MODULE-IIGSAW COOPERATIVE

LEARNING

STRATEGY,

MPROVING

T}{E PREVENTATIVE BEHAVIOR TOWARDS

DENGUE HEMORRHAGEIC FEVER

Dewa Nymran

Oka.---..'.----.

2.

CLASSIC

HODGKTN'S

LYMPHOMA

SIMULATING ANAPLASTIC LARGE

CELL LYMPHOMA

I Gusti

AYu Sri

Mahendra

Dewi"'

3.

EXTENDING TIME

OF

KOMBUCHA TEA FERMENTATION IMPROVES THE

GROWTHOF

INHIBHON

CAPACITY

OF Escherichia

coli;THE PRODUCER

OF

EXTENDED

SPECTRUM

BETA LACTAMASES

(ESBL)

BY

IN

VITRO

METHOD

Ni

Putu A4radnyani,I Dewa Made sukrama, I wayan Putu sutirta

Yasa""""'

(3)

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

I

Dept Biologt of Faculty Mathemdi and Natural Science

.

2Faculty of Agricalture, Udapna University. Corresponding author: [email protected]

ABSTRACT

Coconut plays a more prominent role in the social, economic and crltural for Balinese people, especially fm ritual purpose. The aimed of this research to determined alleles variation of sixteen oconut accessions (Cocos nucifera L., Arecaceae). Alleles variation in 16 coconut acoessions from Br. Babung, Gunaka and Pikat vitlages, Dawan, Klungkung regencywas determined using 4 microsatellite markers. A total of 28 alleles were detected by microsatellite with an average 7 alleles per locus, there are 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,

Dasanayaka 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

coconut-Almost

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)-Investigation

of

coconut

genetic diversity

provides

sufficient scientific data

fc

gelrnplasm management. Diversity analysis in coconut palm has been done by morphological traits, biochemical and 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.

(4)

@

- _Mr,-*nS

lifr

_sctenes

fu

tah

(odi agig

@

a

p*fu)

ad

Id

*z:irry-mulung, penyu, rangda, salak, sangket, stdamala,

nyuh:

Balinese coconut) and 3 were

of the 'dwartr

category (green, white and

yellow

dwarf)-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/ hotstart

(eiagen),

I

pL

primer

The PCR programmed for 30 cycles of 60 seconds each at 94oC, annealing temperature 3g-52"C, extension temperature 72oC. The

first

cycle was preceded by a 3

min

denaturation at94"C and the last cycle ended

with

5 min extension

at72"

C. Reaction products were separated on 6Yo polyacrylamide (denatured) and visualized with silver nitrate staining (Tegelstorm, 1984).The alleles were scored based on the size of each PCR amplified fragment by electrophoresing

all

samples

in

a single gel.

Allele

size was determined by semilog plotting of distance migration

of

amplicon on PAGE (Hutchinson, 2001).

Diversity

values based on allele frequencies were calculated

for

each microsatellite locus using

Nei's

methods ( 1987).

Table I : Detail

of

microsatellites Primer tsed

;

Primer

name

Forward primer (5'-3')

Reverse

primer

(3'-5')

CNZO5

CNZO9

CNZ2I

CNZ5l

CTTATCCAAATCGTCACAGAG

ATCTACCA GTGTGGTCCTCTC

ATGTTTTAGCTTCACCATGAA

CTTTAGGGAAAAAGGACTGAG

AGGAGAAGCCAGGAAAGATTT

ACCAGGAAAAAGAGCGCAGAA

TCAAGTTCAAGAAGACCTTTG

ATCCATGAGCTGAG

CTTGAAC

RESULTS AND DISCUSSION

The sixteen accessions coconut with four specific microsatellite primer pairs produced a total

of

28 alleles ranging

from

6 to 8 alleles per locus (Fig- 1 and Table

2)'

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,

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

(5)

'

lth INTERNAT0NALCONFERENCE ON BIOSCIENCESAND BIOTECHNOLOGY

6

"Advancing life scienaJor health (onti aging development in paaiular) andlood secttriry"

W

Table 2. Detail of microsatellite loci, alleles detected in coconut accessions

Microsatellite

No of alleles Allele size

(bp)

Hcterozygmity

CNZO5 CNZO9

CNZ2I

CNZ5I

7 8 7 6

1 18, 12t, 138,

l4g,

l5g,

168, 178

I 15,120,

lj!s,

130, 135, 140, 145, 155 224,

2X,

250, 260, 27 0, 27 6, 286

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

Bundeix Y&

Santos G, Labouisse JP and Baudouin L. 2005.

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),

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.

Dq1te JJ

d

Doyle

JL.

1981.

A

Rapid DNA

Isolation Procedure

for

Small

Amount

of

Leaf

Tissue. Phlrm*rcm-

Bull.

l9:

I

l-

15.

(6)

4th INTERNATIONAL CONFEREIff;E ON BIOKIENCES AND BIOTECHNOLAGY

':;;;;;;ii;

,ii"*irt

,

tf,iii*i

osis dr*topment in partictttar) ondfood sectritv"

Hutchinson F. 200 I . Estimating sizes of

DNA

Band

By

Semi-Log Plot'cancer Research center Education PartnershiP.

Kumar SR Manimekalai R and Kumari BDR. 20 r

l.

Microsatellite Marker Based characterization of South

Pasific Coconut (Cocos

mteiferal.)

Accessions. lnt. J, Plant Breed. Genet.

5:34-43.

Lebrun

B

N',Cho YP, Seguin

M, Grivet L

and Baudouin

L'

1998' Genetic

Diversity in

coconut

(cocos

nuciferal.)

Revealed by Restriction Fragment Length polymorphism

(RFLP)

Markers' Euphytica

101:103-108.

Liu X,

Thng H,

Li

DD, Hou LH. 2011. Genetic Diversity of Coconut Cultivars in China by Microsatellite (SSR) Markers Molecular Plant Breeding2(12):

Manimekalai R and Nagarajan p.2006,

Ar.".riig

g"netic relationships among coconut

(cocos

nuciferaL')

accessions using inter simple sequence ."**u1 markers, Scientia

Horticulturae' 108(l): 49'54'

Manimekalai R and Nagarajan P. 2007-

U."

"iii;;le

Sequence Repeat markers

for

estimation of genetic

diversity

in coconut (cocos

nuciferal.)

germplasm accessions' Journal

of

Plant Biochemistry and BiotechnologY, 16(1): 29-33'

Manimekalai

R

and

Nagar{an

P 2010: SSR and ISSR Markers Based Population Genetic Structure

of

Coconut (Cocos

nuciferal.)

Germplasm Accssion Indian J.

of

Plant Genetic Resources,23

(l):

77-82.

Nei M.1gg7. Molecular Evolutionary Genetics.

columbia university

Press New

York'

perera _{L, peries RRA}and Femando

wMU.

1 996. Conservation of coconut

(co

cos nucderaL.) Biodiversity

in

Sri Lanka. Plant Genetic Resources

Newletter

106'

perera _L,_RusselR, provan J and powell

w.

2001 . Levels and Distribution of Genetic Diversitybof

coconut

(Cocosnuciferal.,varTypicar,o*typi"4fromSriLankabyMicrosatelliteMarkers.Euphytica

122

381-389-Rajesh

MK,

Nagarajan P, Jerard

BA, Arunachalaml

V

and Dhanapal

R'

2008' Microsatellite

variability

of coconut accessions (cocos

nuciftral')

from Andaman and Nicobar Islands' current

Science 94

*ib"ir:?I,

ttiJ;l,i?.l

,ebrun

L, chaves LJ, Brondani

c,

Zucchi

Mr

and vencovskv R. 2010. Population structures

of

Brazilian Tall coconut'7Vo',o' nucifera

L')

by

microsatellite markers' Genet'Mol'

*,r".",,,|]'i'r('Ii*uror,

JHA

Barker,

G

M

Amold, G

Ayad,

T

Hodgkin'

A

Karp'1999' Isolation

and

characterizationofpolymorphicmicrosatellitesincocosnuciferal'Genome'1999'42(4):668-r"g"rrfJ* H.

19g6. Mitochondrial

DNA in Natural

popuration: an Improved Routine

for

Screening

of

GeneticVariationBaseonSensitivesilverStaining.ElectrophoresisT:226-229

Teulat B, Aldam

c,

Trehin R, Lebrun B Barker JHA. 200b.

An

analysis of genetic diversity of the coconut

(Cocos nucifera) populations from across the geographic range using sequence-tagged microsatellites

iSSnr)

andAFLPs'

TheorAppl

Genet 100:

764-771'

-

L:-

^-r

,r:.,-.oirl,

in

upadhyay

A,

Jayadev

K,

Mamimekalai

R-

2004'Genetic relationship and

diversity

in

Indian

coconul

accessions based on RAPD markers.Scentia Horticulturae 99 (2004): 353-362'