Not included the Literature Study. The full file is under copyright of UPLB

1. BREEDING.AID POPI.JLATION GEITETICSSTIJDIESON
' COCON!{ (Cocosnuciferu L.) COIfTOSITE VARIEIY
. ".USING MORPEOLOGICAL ANI)
''" I,flCROSATELLITE MARIGRS
-
nni , ,],t:
'i.:
F+
'! r'. i'
ri
4.
,:'
,1
AroBA
Rus[ii+uRtr{ EARrs
,sl
TO TEE FACULTY Otr TEE GRADUATESCHOOL
.IE{TYERSITYOF TEE PEILIPPINESLOS BANOS
IN PARTIAL FIJLFIIIMENT.OF THE
REQUREMEITT FOR
.THE DEGREN OF
DOCTOR OF PHILOSOPHY
Clant Breeding)
April, 2002

2. The dissenationattachedhereto. entitled "BREEDING AND POPIJ'I-ATION
GENETICSSTUDIESON COCONUT(Cocostwcifera L ) COMPOSITE VARIETY
USING MORPHOLOGICAI AND MICROSATELLITEMARKERS-. prepared and
ofthe requiremenls
surnrtted RUSTHAMRIHARISAKuBA. in panialtulfrllnrent
by
olDoctor ofPhilosophy
forthe degree Breeding) hereby
(Piaot is accepted
r ?-',
al--1
JOSE€ I#RNINDEZ CALIXTO M. PROTACIO
Membet Advisoq/Committee Advisory
Member, Committee
furr(s,aoz ,forl+ t a-
Date signed Datesigned
/, | . t
/i//'J"4 Xv4t.*'t'C
DESIREE HAUTEA
N4.
AdvisoryCon1miitee
Co-Chai., Chair,Advisory
Committee
4V-y o, ,o, 7
1
Datesigoed
Accepted as panial fulfillment of the requirementsfor the degreeof Doctor of
Philosophy(Plant Breeding)
h*r1,0"--"-
,/ BE€zuNOE CL,EVAS
DepartmentHorticulture
Chair. of
t/frr/ t , o.u,
Date sigoed
-
?a^.q_
RITA P LAUDE
Dean,GraduateSchool
ofthePhilippines Baios
Universi(y Los

3. BIOGRAPHICAL SKETCH
The authorwasbom in Manado,
Indonesia August25, 1959. is the eldest
on He ol
children Alm. MusaAkubaandSartin
seven of Wahibu.
He accomplished primary
the school SDNPineleng,
at Mnahasa,
North Sulawesi,
in
1971,the secondary
schoolat SMPNegeriI Manadoin 1973-andhigh school SMA
at
NegeriI Manado 1976. He took up theBachelor Science
in of in
degree Statistics Sam
at
Ratulangi
University
Manadoin 1983. He rorks at Research
Institutefor Coconut
and
(RICP)Manado,
Palmae Indonesia an agronomist
as since1984 He obtained Masterof
a
Science
degree Agroclimate
in from Bogor Agriculture Bogor, lndonesia
University, in
1988undera scholarship the Agrjculture
Aom Resea.ch
Management
Project,
Agencyfor
Agricultural Researchand Development(ARMP), Governmentof Indonesia.He got the
grantedby (ARMP II), AARD Government Indonesia pursuea PhD
,,scholarship of to
programin Plant Breedingand minor in Plant GeneticResources,
Management
and
Conservation the University
at ofthe Philippines Baflos 1998.
Los in
He was elected President Inte.national
as of Students (ISA), University
Association
of the Philippines
Los Baiios @PLB) in 2000 For his active participation the
in
organization,the Chancellorof IIPLB awardedhim the Most Outstandingand Dynamic
Leadership
awardfbr foreignstudents 2000. He is a member
in ofthe Honor Society
of
'GAMltrA SIGMA DELTA'
Agriculture University
ofthe Philippines's
Chapter

4. He marriedZubaidaKanini Pomantoin 1986;they are blessed
with one son,Aditya
Akuba, andone daughter,
Citra Arini Ahrba.
c->45
RUSTHAMRIN HARIS AKI'BA

5. ACKNOWLtrDGEMENT
In the nameof Allah, Most Gracious,Most Merciful
Praisebe to Allah, The Cherisher Sustainer
and ofthe Worlds
I would like to expressmy grateful, appreciationand glatitude to the following
agencies institutions:
and
the Department of Agriculturg Bureau of Agricultural Research (DA-BAR),
Republic of the Philippines,for financing this studies through a researchgrant to Dr
DesireeM. Ifuutea;
the Agency for Agricuhural Researchand Development(AARD), Departmentof
Agricullure, Republicoflndonesia,for the Ph.D. scholanhipgrant andpartly financingthe
research
underAgricultureResearch ProjectII (ARMP II);
Management
the PhilippineCoconutAuthodty (PCA) through PCA-Zamboanga
Research
Cente.
(PCA-ZRC), the pemission access 'coconutsynthetic
for to the variety'andother.elated
coconutva.ietiesasmaterialsofthe reseaxch providingthe rel€vantdata,
and
the Institute of Plant Breeding,Collegeof Agriculture,Universityof the Philippines
Los Bafros,for the use of laboratoryfacilitiesand other .esources the Geo€tics
of
Laboratory,
and
the Research Institute for Coconut and Palmae, Manado, Indonesia, for the
permission access selfinggeneratioNofN{apangetTall coconut.
to the

6. my appreciation the membem my Ph.D advisory
I alsowishto express sincere to of
commrttee,
nalnelyl
(Chairman)andDr DesireeM. Hautea(Co-Chair)for their
Dr JuanitoB. Sangalang
enomous interestto the research,
invaluabl€ cofftructive comments lriendly
guidance, and
encouragement
dudngthethesis indebted Dr Desiree Hautea
work. I amparticularly to M.
for teachingme molecularbiology and moleculargeneticsand enhancing knowledge
my
by giving me opportunities to participate in intemational and nationsl slanposiaand
trainingin the Philippines;
and
Dr JoseE. Hemandez Dr Calixto M. Protaciofor their significantcontributionto
and
this work, guidance, constructive
and ofthe maruscript.
comments the imprcvement
for
I have many more people to thank: Mr Carlos B. Carpio, Deputy Administrator,
PhilippineCoconut Authority (PCA), for the facilitating accessto planting matenalsat
ManagerofPCA-ZRC Zamboanga, his
PCA-ZRC,Zamboanga; GerardoA. Santos,
Mr lor
brilliant ideasin developing'coconut syntheticvariety' that hasinspiredthe thesiswork
and facilitating the researchwork at PCA-ZRC; Mr RamonRivera, Researcher PCA-
in
providing relevantinfomation
ZRC, for his assistance settingup the field experiment,
in
vadety', and developing SSR primersfor coconut;MJ
on the'coconut synthetic the
Ernesto Efirnanuel, Lea Kingco,Ms Joyce
E. Ms Desoacido, Mr Lucilo Kingcofor
and
fieldassistance PCA-ZRC;
at
Dr David Allorerun& Director of Research
Institute for Coconut and PaLmae,
for
administrativeand financial support in the fieldwork at RICP; Ir Tine Rompasand Dr
Novarianto Hengky, Breeders at RICP, for their efforts in developing the selfing

7. generations M&pangetTall populationsand providing valuableinformation on those
of
variety;k Miftahorrahiuq Ir IsmailMaskromo,Ir ElsjeT. Tenda,Drs DjunaidALubaand
RICP stafs, for field assistance RICP Manado,Indonesi4
Drs JohnWurangian, at
Dr Pons A. Batugal, COGENT CoordinatorIPGRI, for his great concernson this
researchand providing relevant literaturc; Dr Roger Ashburnerfor providing valuable
paper on matilg systemof coconut; Dr llasnan'Lthe director of Center Researchfor
Industrial Crcps (CRIC); Bogor for administrativesupport,Dr Pasril Wa.hid,the fomer
directors of CRIC, Dr Zainal Mahmudfor continuouslyencouraging to continuethe
me
study andDr CesarMadamba "my Philipino parents",for their
and Mam Lilia Madamba,
lhat my wasveryconvenienl
hospitaljty made stayat Los Banos
I also wish to acknowledge valuableassistance the following staff of the IPB
the of
Genetics Lab: Dr Conrado Balaterc, University Researcher the Institute of Plant
at
Breeding, for fruitful discussions, sharing knowledge on molecular biology, and
constructivecommentsduring the lab and thesis tr'ork; Ms Hayde Galvez, Ms Shirley
Segovia,Ms Carol Padlan,Ms Nancy B. Coronado,Ms Alma O. Canama, Joy Marie
Ms
Bartolo andMs Mailln A. Latiza for sharing and
their knowledgeon moleculartechniques
creatingthe friendly atmosphere the lab that madethe lab work exciting; Tita Rowena
in
especiallyin providing laboratory administrative
Frankie has extendedgreat assistances
suppot andKuya CesarCaspillofor technicalassistarce the lab,
in
and appreaiationis extendedto my wonderfirl big
The geat acknowledgement
family: Ma SartirLMa Masitah,Ma Imun, Kak Sophianand Sri, Ana and Imeng, Syari!

8. Kak Ari, Imrar! JunaidandNur, Iskandarand Vonny, Syaifirl and A'am, Arifin, Syamsi,
Mut Godeh ard Ali Utina, for continuous
Syamsu, prayersandencouagement;
and
A heartfelt gratitude finally is extended to my beloved wife, Zubaida Kartini
Pomanto, tlrc Pogi sol! Aditya, and the smart daughter, Citra for their patience,
encouragement prayersduringthe diffcult time ofthe reseaich
understanding, and work.
May Allah Blessandgivesrewardsto all the generous
peoplementionedabove-

9. TABLE OT CONTENTS
Page
GENERAL INTRODUCTION I
ANALYSIS OF MATING SYSTEM AI{D GENETIC EQUILIBRIUM
IN COCONUT (Cacasnuciferu L) COMPOSITE VARIETmS USING
MICROSATELLTTEMARKERS
INTRODUCTION
R.E!'ITW OF LTTf,RATURE t2
The Role of Mating Systems GenetioStudy
in t2
Matiflg System Coconut
of t3
DNA Markers 15
Method of EstimationOutqossingRate 2l
GeneticEquilibrium
MATERIALS AND METHODS
PlantMaterialsand SamplingStrat€gy 26
SimpleSequence (SSR)Analysis
Repeat
Estimationof Outcrossing
Rates 31
GeneticEquilibriumTest

10. Paqe
RESULTS 31
Eva.luation the Assumptions the Mixed-MatingModel
of of
Mating System 40
GeneticEquilibrium 50
DISCUSSION 53
Evaluationof the Assumptions 53
Outcrossing
RateofCoconut CompositeVariety 0
Correlationof Outcrossed
Patemity 5',|
Mating SystemParameters FI Hybdds
of 59
Efects of Number of Loci on the Estimation of Mating System
Pa.ameters 60
GeneticEquilibriumofCoconut CompositeVarieties(CCV) 61
SUMMARY AND CONCLUSION 63
INBREEDING DEPRI,SSION AND HETEROSIS IN COCONUT
(Cocosnucifera L.) COMPOSITE VARIf,TIES 67
INTRODUCTION 6'7
'71
RXVIEW OF LITERATURX
Effectsof Inbreeding 7l
Heterosis Composite
in Varieties

11. MATERIALS AND METEODS 76
'76
Plart Materialsand SamplingStrategies
Morphological
Observation 19
MicrosatelliteAnalysis 80
Data Analysis 81
RESULTS 85
Estimates of Inbreeding Depressionin Tall Coconut Populations
Based Fruit Component
on Traits i5)
Estimates of Inbreeding Depressionin Tall Coconut Populations
based MolecularMarkers
on 88
EstimatesofHomozygosityLevelsin Ta.llCoconutPopulations
Using
Microsalellit€sMarkers
Heterosisin CoconutCompositeVariety 109
DISCUSSION t26
InbreedingDepression t26
Homozygosity 129
Heterosis t32
SUMMARY AND CONCLUSION 134

12. Page
POPULATION GENtrTIC STRUCTURE OF COCONUT (Cacas
naciferu L.) COMPOSITE VARIETIf,S 138
INTRODUCTION 138
RETEW OF LITERATURE 141
GeneticDiversity in Coconut t4l
Population ofcomposileVa eties
Slructure
Genetic 149
MATERIALS AND METEODS 151
PlantMaterialsand SamplingStrateg'y 151
Moryhological
Evaluation 152
MolecularEvaluation 153
Data Analysis 153
RESULTS
Allelicrichness l55
Allelic Evenness 158
Heterozygosity 160
PopulationDifferentiation 164
GeneticDistance 170
DISCUSSION 1'79
SUMMARY AND CONCLUSTON t84

13. Paqe
GENERAL DISCUSSION 187
GENERAL SIJMMARY AID CONCLUSION zo1
RECOMMENDATION 209
LITERATURES CITf,D 211.
APPENDICES 228

14. LIST OF TABLES
Table Page
Single locus linkage disequilibriaof SSR loci in CCV 1 with 36
unknownmatemalpalm parentage (CCV 1a)
Singlelocus linkagedisequilibria SSR loci in CCV I with
of 36
known matemalpa.lm parentage(CCV lb)
SSRgenefrequencies pollenandoule ofCoconutComposite
in 38
(CCV la)
Variety I with unknownmatemalparentage
SSRgene &equencies pollenandonrle ofCoconutComposite
in 39
Variety parentage
I with known maternal (CCV 1b)
Mating system parameters
ofcoconut CompositeVariety 0 (CCV 4l
0) estimatedfrom CoconutComposite Variety1 (CCV lb) with
known matemalpdn parentage
Mating system pararnete$ of Coconut Composite Vari€ty 0
estimatedftom CCV 1 with known (CCV 1b) and unknolvn
(CCV la) matemalpalm parcntage
Estimales of multilocus outcrossing rate ( ), single locus
outcrossingmte (t,), biparentalinbreeding(t- tJ, corelation of
outcrossing rat€s (rr), coefficient corelation of outqossed
p&temity ofthe 15Fl Tallx Tall hybrids
(rp)
Estimates of multilocus outcrossirg rate (tt, single locus 47
outcrossing rate (t"), correlation of outuossing rates (rt),
coefficient conelation of outcrossedpatemity (rp) of CCV 0 in
differentnumberofloci
Estimate of outcrossingrate (t), observedfixation index (F1"), 51
equilibriumflxation index (FJ andequilibriumseltug rate (SJ of
CCV 0 population
l0 Eslimalesof Wright's fixatlon index (F,.) and 1r test for 51
equilibriumofCCV 1 with known (CCV lb) andunloown (CCV
la) mate.nalparentage

15. Table Paqe
l1 Mean Auft components inbreedingdepressions Sl Laguna
and in 86
Tall andSl Bago-Oshiro (1994-2000)
Tall
t2 Inbreedingdepressions fruit components selfinggenerations
in of a7
ofMapanget Tall
13 Estimalesof fixation indicesand inbreedingdepression selfing
of 89
generations
ofMapanget Tall based microsatellite
on markerdata
14 The inbreedingcoefficientof CCV 0 andCCV 1 populations 91
15 Allele richness (fla*) and ShannonInformation lndex (I) of 94
selfing generations Laguna Tall (I-AGT) and Bago-OshiroTall
(BAor)
16 diversityindex (I) of selting
Numberof alleles(Na) and Shannon 102
generations MapangetTall
of
t7 Level of homozygosity selfinggenerations
in ofMapanget Tall at 104
SSRloci
18 Wright's fixation index @;") as a measue of heterozygous 104
deficiency or excessin selfing generationsof Mapanget Tall
(IvrTT)
19 Nei's unbiasedestimates geneticidentity and geneticdistance
of 106
of selfinggenerations
ofMapangetTall
20 Level of homozygosity of Coconut Composite Varieties al 108
microsatellite
loci
2l Averagetaxonomicdistances the parentalpopulations
of of ll0
CoconutComposite Variety 0 (CCV 0) based fruit components
on
datayears1994to 2000
22 Rho matrix of unbiased€stimator of Slatkin's microsatellite 113
ofthe parental
dista:rce populations
ofthe Fl hybrids
23 Fruit componentdata of parentalpopulationsand Fl Tall x Tall 1t7
hybrids composed CCV 0 population
the
Heterosisin 15 Fl Tall x Tall hybridscomprisirgCoconut 119
Composite Variety 0 (CCV 0)

16. Table Page
Corelation betwe€ngeneticdistancesofpa.ental populationsand 121
mid-pa.ent heterosisof iluit aomponents 15 Fl Tall x Tall
of
hybridscomprisingCCV 0
26 Correlation between number of alleles, genetic dive$ity, and 124
heteozygosity SSRloci andliuit components
a1 ofCCV 0
27 Number a.nd size of alleles detected i4 CCV 0 and CCV I 157
populations
28 Allele richnessof the populationsthat make up of CCV 0 and 159
ccv 1
29 Heterozygosity ard fixation index of CCV 0 and CCV I 163
populations
Fsr andgeneflow 0'Im) ofCCV 0 and CCV 1 populations 165
31 Matrix ofRho valueaveraging varjance
ove. components
oICCV 16'7
0 population
Matrix ofRho valueaveragingovervadancecomponents CCV
of 169
I population
33 Matrix of(delta-p)'ofCCV 0 population 171
Matrix of (delta-p)'? CCV 1 population
of 1',75
Meanofcopraweightofparental
populations, hybrids,
Fl CCV 0 196
andCCV 1

17. LIST OF FIGURIS
Fiqu.e Pase
I Inheritanceofthree polymorphicSSRloci in CoconutComposite 34
Variety 1 with known matemalpalm parentage(CCV lb). Lanes:
M8 : MarkerVlll (Promega), = Matemal
P parents
(CCV 0); l-5
: individualpalmofeachp.ogeny (CCV 1)
Estimates ofave.age multilocusoutcrossing (t.), single
rate locus 49
outcrossing late (t.), correlation of outcrossing rutes (r),
coeficient correlation of outcrossed patemity (rp) based on
differentnumberof SSRloci
Polynorphism of SSR loci CNZ21 and CN2A4 in selfing 93
generationsof Laguna Tall and Bago-OshiroTall in the multi-
loadingpolyacrylamyde electrophoresis
gel
Allele evenness
ofselfing generations
oflaguna Tall 96
Allele evenness selfinggenerations
of ofBago-Oshirc Tall
Level of homozygosity selfinggelerationsLagunaTall (S0 and
of 98
S1LAGT) andopen-pollinated Bago-Oshiro Tall(S0BAOT) and
SI BAOTar ieven microsalellile
loci
Poly'rnorphismsof four representativesSSR loci in selfng 100
generations Mapanget
of Tall (M8 = MarkerVIII; 1,2,3,4,5,6
:
individualpalm)
Allele evenness selfng generatio$ of MapangetTall
of l0l
Dendrogramof seling generationsof Mapanget Tall basedon 107
sevenmicrosatelliteloci
10 Dendrogram of pa.ental populations of Coconut Composit€ 111
Va.iety 0 (CCV 0) basedon fiuit componentdatayeam 1994to
2000. S0LAGT:SOLaguna Tall, S1LAGT=SI LagunaTall, S0
BAOT=S0Bago-Oshiro Tall, 51 BAOT=S1Bago-Oshiro Tall,
BAYT=Bay-bay Tall, TAGT = TagnananTall, RIT = Rennell
Island Ta1l,andWAT :West Afticaa Tall. The dendrogram was
c.eated using MEGA ver 2.0 (Molecular Evolutionary Genetic
Aralysis) with distancedatat]?e.

18. Figure Pese
ll Genetic divergenc€of parentalpopulationsof CCV 0 basedon 111
unbiasedestirnator Slatkin's genetic distance(Rho) using five
microsatelliteloci
12 Variation of fiuit components of F1 Tall x Tall hybrids 116
comprising CCV 0 (courtesy of Ramon Rivera, PCA-ZRC,
Philippines)
t3 Linear relationship betreen genetic distances of parental t22
populationsandheterosis copraweight of CCV 0 (A : genetic
in
distancebasedon fruit componentsdata, B : genelic distance
based SSRmarkerdata)
on
The relation between mid-parent heterosisof nut weight and 125
heterozygosity SSRloci in CCV 0
at
t) Polyrnorphism SSR locus CN2A4 in CCV 0 (A) and CCV 1
of
(B) run asmulti-loading (M8- l : markerVIII firstloading,
M8-2
= marLer.III second loading,M8-3 = markerVlIl third loading,
I, II, III : first, second third loading)
and
16 Distribution allele
of fiequencies CCV 0 population
in 161
t'7 ofallelefrequencies CCV 0 population
Distribution in 162
18 Dendrogram of Fl hybrids comprising CCV 0 based on 172
microsatellitedata. LAG : Laguna Tall, BAO = Bago-Oshiro
Tall, BAY = Bay-bayTall, TAG = Tagnanan Tall, RIT : Rennel
Island Tall, WAT = West African Tall. BAOBAY rcpresents
BAOT x BAYT hybrids,and so on.
19 Dendrogram Fl hybridscomprising
of CCV 0 basedon Auit 173
component (LAG = Laguna
data Tall, BAO = Bago-Oshiro Tall,
BAY : Bay-bayTall, TAG = Tagnanan Tall, RIT : Rennel
Island
Tall, WAT : West Aflican Tall. BAOBAY represents BAOT x
BAYT hybrids,and so on)
20 Dendrograinof CCV 1 based delta p2distance
on derivedfrom Fl 176
hybrids(CCV 0). LAG = Laguna Tall,BAO = Bago-Oshiro Tall,
BAY : Bay-bayTall, TAG: Tagnanan Tall, RIT : RennelIsland
Tall, WAT = West African Ta.ll. BAOBAY represenls BAOT x
BAYT hybrids,and so on.

19. Figure
2l Dendrogram CCV I derivedfrom Fl hybrids
of (CCV 0) based 178
on vigor of seedling.
LAG = LagunaTall, BAO : Bago-Oshiro
Tall, BAY = Bay-bayTall, TAG : Tagnanan Tall, RIT : Renn€l
Island Tall, WAT - West African Tall. BAOBAY represents
BAOT x BAYT hybrids,and so on

20. LIST OF APPEI{DD( TA}LES
ABpeodix
Tables
I List of samplepalmsof CCV 0 andmotherpalmsof CCV 1

21. LIST OF APPENDD( FIGI]RES
Aopendix Page
Figure
I Tiangular plantingsystem CoconutComposite
of Variety 0 (Hi =
rrlf r r ujulrus, I r, L. .... rJt
Map offield plantingofCoconut Compositevariety 0 230

22. ABSTRACT
AKUBA, RUSTHAMRIN HARIS. University of the Philippines Los Baios' April
2002. Breedins and Population GeneticsStudieson Coconut (C.rcas
tt&cifelaL.)
ComoositeVarietv UsinsMorpholosicaland MicrosrtelliteMarher{.
Major Advisers:Dr. JuanitoB. Sangalang
Dr. Desiree Hautee
M.
CoconutComposit€Varieties1 was developed PCA-Zamboanga
by Research
Ceder
as a supplementary coconutbreeding.
breedingstrategyto the hybdd and open-pollinated
The objectives creatingcoconutcomposite
of varietieswer€ to obtaincoconutvarietywith
higher yield potential than the open-pollinated coconut variety because heterosis
tall of
efect, greater genetic variability, ald 'balanced heterozygosity'. Morphological and
microsatellite inbreeding
markerswere usedin the evaluationofmating system, depression
geneticvariability andgenaic equilibriumin the ooconutcomposite
andheterosis, variety.
Analysis of the mating system and genetic equilibdum in Coconut Composite
Varieties{CCV 0 and CCV 1) were conducted. Resultsirdicate that CCv 0 was
predominantly ratesof 91.1 Yolo 91.4o/o,
cross-pollinated outcrossing
with and selfing
'ates8.6 yoto 8.9 yo. A largeproportionofthe cross-pollination
eventswere dueto full-sib
mating. The biparentalinbreeding crossing
or betweencloselyrelatedindMdual occurred
at tie rate of 6.9 yo to 10.3 %. CCV 1 did not attain equilibrium at the single and

23. multilocuslevels. Hence,'balancedheterozygosity'was not attainedafter one generation
ofraodom mating
wasalsoperformed. Th€ effec1
ofinbreedingdepression heterosis
Assessment and of
iobreeding depressionon the economic yield was indirectly studied by using selfing
generations tlree tall coconutvarieties(LagunaTall and Bago-OshiroTall, Mapanget
of
Tall). Inbreedingdepression CCV 1 was also assessed molecularapproaclr, which
in by in
was measured reducedlevel of heterozygosity. Selfingin Tall
inbreedingdepression as
coconut populationsresulted in inbreedingdepressionand increasedhomozygosityin
LAGT, BAOT, and MTT populations. Inbreeding depr€ssionthat was measutedas
reducedfitnessbased the changes fixation index at SSRloci washigh in 53 MTT and
on in
54 MTT. Selfing,fiil sib mating and biparentalinbrcedingin CCV 0 resultedin CCv 1
with lower level of heterozygosity.
Md-parent heterosiswas presentin the intervarietalFl Tall x Tall coconuthybrids
that constitutedthe CCV 0 for whole nut weight, meat weight and copra weight. The
predictedcopraweight per nut ofCCV 1 washigherthan the copraweight per nut ofthe
parentalTall populations,however,it was lower than that ofFl tall x tall hybrids. The
relationship baits andg€n€ticdistances
betweenheterosis fruit component
in ofthe Fl Tall
x Tall hybridswas also determined.Data indicatedthal or y mid-parentheterosis copra
in
weight couldbe correlatedwith geneticdistance.
The assessment population genetic structure was studied in terms of genetic
of
variability and populationdifferentiationof CCV 0 and CCV l. The geneticdiversitywas
characterized terms of numberof alleles,observedand expgctedheterozygosity,
in and

24. fixation index. Wright's Fsr and Rho va.luesof unbiasedestimator of Slatkin's RST
distancewas computedas an indicator of population diferentiation. Genetic distanc€s
betreencomponents weremadeup ofCCV 0 andCCV I were computed
that on
based
morphological in vigor in CCV 1, and SSR
tni1s, i.e. fruit components CCV 0 andseedling
markem. The rcsu1ts
showedthat CCV 1 waslessdiversecompared CCV 0 population.
to
The CCV I had less number of alleles and was deficient in heterozygousgenot)?es.
Geneticdifferentiationwithin populationwas higher in CCV 1 than in CCV 0 indicating
that CCV 1 was more fragmented. The imptcations of the results of this researchto
coconutbrcedingandgermplasm were discussed
management extensively.

25. GENERALINTRODUCTION
Coconut plays an imponant role in most coconut producing countries. In the
Philippines,it contributesUS$ 900M yearly for the country, benefiting1.5M farmersand
larm workers, and 24 million people directly or indirectly. It supportsthe sustainable
management envircnment,as the pa]ms prctect the soil fiom erosion and nutdent
of
losses(PhilippineCouncil for Agriculture, Forestry and Natural Resources
Research
and
1999).
Development,
programsin the Philippines.It
Replantingis one of the main coconut dev€lopment
is aimed at rcplacing the senile coconut trees and replanting of fams damagedby
typhoonsand other natural calamitieswith promisinglocal cultivars and/or hybrids. The
targetsfor the replantingprogram are 5,000 ha per year for the first five yeals (starting
However, seedproduction
years.
1990)and20,000ha for the succeeding the capacity
is
240,000seed-nuts year, which is ody enoughfor 1,000ha per year (Santos
per and
1995).
Rivera,
To overcomethe problem of insumcienl seed-nutproduction, the development
of
coconuthybrids, Dwarf x Tall hybrids, beenconducted.
i.e. has Hyb.id breeding the
has
advantages heterosis,
of and the economics seedproduction
uniformity, of (Lee, 1995)
Coconut h-vbrids,
under favorable conditions, can yield three times morc than the best
programin the Philippineshasresultedin the
Tall va.ieties.Coconut hybrid development
release nine coconuthybrids(PCA 15-1 to PCA 15-9)by the Philippines
of Coconut
Nut production
Authority. ofthesehybrids ftom 117to 155nutsper palmperyear
ranges
(Santos,1998).Luntungan(1997) reportedthat the ten-yearold KHINA (Indonesian

26. 2
Hybrid)yielded to 3.8 t coprape. hectare yearat 10yea.sold compa.ed
Coconut 3.5 per
to 1.3 1.6 t copraper hectare yeai ofthe Tall coconutvarieties aboutthe same
per at
age. However, hybrid breeding in coconut faces the problems of narow genetic
variabiliry,
moresusceptibility pests,
to diseases droughtstress, high costofseed
and and
production.It takes time to multiply a suitablevariety or hybrid for commercialplanting
(Baudouin, rate,the final costof planting
1999).Due to the low multiplication materials
would be unaffordable resource-poor
to fa.mersunlessgovemments
providea subsidy
that many count.iescannotafford (Batugal, 1999). In the Philippines, current
the
estimat€d of coconut
cost is per (CIF nurser]site,
hybridseednut aboutPhP33.00 seednut
unpubl.).In contrast, seed-nuts open-pollinated
the of varieties
costaround per
PhP6.00
(Santos dl,2O1O).The hybridization
seed-nut el technique not as simpleas in annual
is
crops sincecoconuthas giant featu.es.This biological constrainthasresultedin the high
costbfhybrid seed
production lessnumber
and produced palm.
ofhybrid seed-nuts per
ResearchCenter (PCA-ZRC)
The Philippine Coconut Authority-Zamboanga
initiated the development of 'coconut s]'rthetic variety' way back in 19'79 as a
supplem€ntary
breeding strategy to the hybrid and open-pollinatedcoconut breeding
programs
(Sartos,
Bahala Cano,1989;Santos a/, 2000). To oblainhigher
and e/ yielding
variety with greater genetic variability, the slartheticvariety should be developedfrom
randommating of inbredlineswith differentgeneticbackground, then testedfor their
and
combiningability (Agrawal, 1998).Theserequirements difficult to implement
are for
coconutsinceit is a perennial
crop. The development inbred lines is also time-
of
consuming costly.Therefore,
and Santos a/ (1989)modified method
e/ the ofgenerating
syntheticva.iety by using fust generationselfing (Sl) of Laguna Tall and Bago-Oshiro

27. 3
Tall, and selected
elite open-pollinated namely:
varieties Bay-bayTall, Tagnanan
Tall,
IslandTall, andWestAfricanTall, asthe parents
Rennel of'syntheticvariety'zero(Syn
0), instead usinginbredlines(S4 to 56 populations). Syn 0 is composed 15
of The of
intervarietal Fl hybrids among parentalpopulations. Randommating the 'Syn 0' has
resulted
in'Slal 1'.
Theoretically, 'coconutsyrthetic
the variety'developed Santos a/ (1989)is a
by el
compositeva.rietysincethe base populationsconsistsof inteNa.rietal
hybrids of a fixed
varietiesor populations.
set of heterogeneous Nonetheless,
syntheticand composite
varieties very similarin structure
are (Busbice,
1970;Hallauer
and Miranda,1981).In
this study,the term 'compositevariety' is usedinsteadofthe term slnthetic variety based
on theoretical considerations. The intervarietal hybrid population (or Syn 0) is called
Coconut CompositeVariety 0 (CCV 0). Meanwhile,the progenies
resultingfrom
randdmmating the CCV 0 is called Coconut CompositeVariety 1 (CCV 1), which was
launchedas GeneticallyMulti-Ancestors (Gtr4-A)coconut farmers'variety jn August
2001.
It is expectedthat the development coconut compositevariety would resuit in a
of
varietythat hashigheryield potential
because h€terosis
of effectandwider adaptabiiity
sinceit is composedof various lines with differcnt genetic constitutions(Santose/ a/,
1989). Fu.the.more,this variety (CCV 1) is also er?ected to have'baianced
hete.ozygosity'in which the proportion of heterozygotegenotypes
will not changeover
generation randommating.
of
The mating system of the populations a.ffectsthe genetic variability (Clay ard
Levin, 1989;Ritland,1989),the attainment genetic
of equilibrium(Liu, 1998),and the

28. 4
perfomance of the advancedgenerationof compositevarieties(Busbice, 1970).
Heterosis,the superiority in performanceof hybrids comparedto their parenls Gehr,
1987), determinesthe yield potential of composite varieties. The heterosis in the
int€rvarietal hybrids as the base populations of composite va.riety will affect the
performance generations
ofsucceeding (Eberhart dl, 196'7).
ofcomposites et
geneaction and degree
The level of heterosisis affectedby the level of dominance
of geneticrelatedness
between (Falconer Mackay,1996). The presence
parents and of
dominancegene action can be detected indirectly by the occurrence of inbreeding
depression a consequence selfing. However, inbreedingdepression economic
as of in
yield cannotbe detectedin CCV 1 sinceit is not in the fiuit-bea.ringstage. ln this aase
the presenceof inbreedingdepressiolin Tall populationsof coconut would be
investigatedby using the fiIst selfing generationof Laguna Tall and Bago-OshiroTall,
andthe 52, 53, and54 generations
ofMapanget
Tall.
Greatergenetic is in sinceit is composed
varieties
variability expected composile
of populations with different genetic background. Genetic variability of composite
varietiesdependson the geneticvariability of the parents,inbreedinglevel, outcrossing
rate, and genefrequencies the population.Hallauerand Miranda(1981)mentioned
in
thal greater
genetic
variability expected be available
is to ifpopulations
ofdiverseorigins
are combined.
Heterosisamongintervarietalhybrids hasalso beenlbund to be relatively
high. The meanyield of new populationis expected be greaterthan the average
to ofthe
parental
varieties.
The other objective in generatingthe coconut compositevariety is to obtain a
varietywith 'balanc€d (Santos a/, 1989).In population
heterozygosity' el genetics
tenrL

29. 5
it meansthat the genotypefrequencies, this case,the heterozygous
in fequ€flcy doesnot
change from generation to generation. lt implies that the population is in genetjc
equilibrium.If nndom mating occurs in a very la.rgepopulation of compositevarieties
with the assumptions no selection,mutatiorLmigratiorLand randomdrift then genetic
of
equilibrium a locusis expected be attained one generation randommating
in to in of
(Falconer
and Maakay,19961
Weir, 1996). Sincenot all of the assumptions be
oan
fuifilled in coconutcompositevariety populatio4 then the balancedheterozygosity
might
nol bereached onegeneralion
in ofrandommating.
Agro-morphological markers have been used in tie mating system, inbreeding
depressionand hete.osis, and genetic diversity studies of coconut. Ashbumer el a/
(2001)usedliuit color inheritance
methodto assess nating system Gazelle
the on Tall
population. The studiesof inbreedingdepression
were carriedout on the onsetflowering
(Santbsand Sangar€,1992); and height of seedling,
girth aircumference, numberof
and
greenleaves(Rompas a/, 1988). Heterosis coconut
el on hybrids
wasreported copra
on
weight and oil content in PBl21 hybrid (Vanialingan,Khoo, and Chew, 1978);onset of
flowering ard number of nut of Indonesian hybrids CNovarianto, 1987)- Agro-
moryhologicalmarkerswere also intensivelyusedin geneticdiversity study (Sugimurae/
41,1997,
Vargas Blanco,
and 2000;Zizumbo-Villareal Arellano-Morin,
and 1991,.,.
DNA markertechnologyis considered an importanttool for characterization
as and
evaiuationof genetictraits of many crops, in additionto morphologicalcharacterization.
DNA maikels are not dependent the stageof plant development.They are distributed
on
in th€ whole genome,and are highly pollmorphic Several molecular techniquesare
available for genetic study of coconut. These include restriction lragment length

30. b
polymorphism (MLP) (Teulat et al, 2000), random amplified polymorphic DNA or
RAPD (Ashbumer,1999), simplesequence
repeats(SSR) or microsatellites
(Rivera,
1999; Perera" 1999), and the ampliflcation fiagment length polymoryhism or AFLP
(Perer41998).
The jdeal molecular
technique the characterization evaluation crops
for and of
should meet severalrequircments.The results must be highly reproduciblein different
laboratoriesand analyzedusing standardized
scoring and aml)'tical methods.Data must
be easily incorporatedinto databases.
Assaysshould be applicabiefor high throughput
and necessarymaterialsshould be exchangeable.
Co-dominantmarkers are preferable
over dominantmarker systems, high polyrnorphism
and levels are requiredto distinguish
closelyrelatedgenotypes.
SSRor microsatellites these
fit precisely are
requirements and
consideredto provide the most informative method of evaluatinggenetic diversity in
cocodut(Karp, 1999;Ashbumer,
1999).ln this study,SSRor microsatellites used
were
in coljunctjon with morphological
traits to evaluateor assess
allele and genotype
frequencies,mating system param€te.s,genetic equilibrium, and population genetic
structureof the populations.
In rcsponse the development
to of'coconut synthetic
vadeties'by Santos a/
e/
(1989), Baudouin(1999) mentioned
that the'coconut slnthetic variety'has several
disadvantages
such as: (1) the Fl hybridswill alwaysbe better than this variety, (2) the
resultingvariety vr'ill be rather heterogeneous, the geneticvalue could vary with time
(3)
due to seasonal
va.riations flowering and production, and (4) selfingis not prevented.
of
His doubts about the vadety were finally expressed salng: "Only experience
by will say
ifthis type ofmaterial reliable economically
is and interesting".

31. 7
The main goal of this researchwork is to €valuatethe me.its of breedingcoconut
composite
varietyin order to ensure release
the and distribution improvedcoconut
of
varieties to farmels. To ensurethe accomplishment the objectivesand answerany
of
doubts about the coconut compositevariety, characterization evaluationhave to b€
and
carded out for the facto$ that affect its performance. The mating systerL heterosis,
geneticva.riabilityand geneticequilibdumare the main factors involved in the evaluation
of coconut
composite
varieties.
Threestudieswer€ conducted answerthe following specificobjectives:
to
1. To chalacterizethe mating system ard study the status of genetic equilibrium in
coconut
composite
va.ietyusingSimple Repeats
Sequence (SSR)markers.
2. To estimate level of inbreeding
the depression the selfinggenerations Laguna
in of
Tall, Bago-OshiroTall andMapangetTall coconutvarieties.
3. Td assess level of inbreeding
the depression heterosis coconutcompositg
and in
variety.
4. To studythe populationgeneticstructureof coconutcomposite
varietybasedon
morphologicalandmolecularcharacteristics.
Results thesestudies presented ChapterII, Chapter and Chapter
of are in III IV.
The matingsystem CCV 0 population
of and 15 Fl Tall x Tall hybridsare reportedin
Chapter Thischapter
ll. alsodiscusses status
the ofgeneticequilibrium
ofCCV I at SSR
loci. ChapterIII presents inbreeding
the depression selfinggenerations Laguna
in of
Tall, Bago-OshiroTall, MapangetTall and Coconut CompositeVadety I (CCV l) that
obs€rved iiuit comporents SSRloci. Heterosis
on and ofCCV 0 is alsodescribed this
in

32. 8
chapter. Chapter is focused population
IV on geneticstructure CCV 0 and CCV 1
of
populations.
This research
lvas conducted
from April 2000 to February2002. The nursery
experiments
and agro-morphologiaal
evaluationlere conductedat the Philippine
Coconut Authority-Zamboanga
ResearchCenter (PCA-ZRC), San Ramon, Zarllboanga
City from April to November2001. All molecular markq experimentswere done at
Geneticlaboratory of the Institute of Plant Breeding,College of Agriculture, University
ofthe Philippines Bafros
Los (LTPLB),
College
Laguna
liom Julyto Decembe.
2001.

33. ANALYSIS OF THE MATING SYSTEM AND Gf,NETIC EQUILIBRIUM IN
COCONUT COMPOSITE VARIETIES (Cocos nuciftra L.)
USING MICROSATTLLIT E MARKERS
INTRODUCTION
Coconut is classifiedinto Tall and Dwarf varieties.The Tall variety has
irflorescences that exhibit herkogamy (dicliny) and protandry, which promote
inflorescences is predominantly
Dwarf varietyexhibits dichogamous
outc.ossing. no and
self-pollinated.The Tall variety is consideredas allogamousand the Dwarf variety as
varieties(Fremondet dl, 1966). Tall varietieshave a mixed mating system,
autogamous
since pistillate
i.e. cross-and self-pollination, the phase phase
with staminate
overlaps in
the sameinilorcscence well as in subsequent
as (Rognon,
inflorescences 1976;Santos
el
a|,2000).
The rate of self-pollinalionin Tall and Dwarf vadetieshasbeenestimated using
by
model (Ashburner dl, 2001; Bourdeix, 1988). The self-
f.uit color inheritance et
pollination
rate of Gazelle
PenimulaTall of PapuaNew Guineawas27.89/o(Ashbumer
2001)whilein Dwarfvarieties, self-pollination ranged
e1a1, the rates from 88.3% to 100
% (Bourdeix, 1988). The coconut compositevadety developed PCA-ZRC is
by
composed l5 F1 Ta[ x Tall coconuthybridswith differentg.owth and flowering
of
habits(Santos al, 2O0O). It is expected the outcrossjng
et that ratesof the Tall x Tall
hybrids
will differfrom the outcrossing ofthe parental populations seashas
rate Tall per
reporled Ashburner o/ (200l)
been by et

34. 10
Estimation of sellpollination rate and/or outcrossing rate using conventional
methods
suchasfruit colorinheritance
doesnot providedirectmeasures
ofthe success
of
mating in the populations. The information they provide is often iladequale for the
analysisof genetic transmission the population level. In additiorl the morphological
at
the genotypeof the progeny.
markersmethod has also a problemin determining
Morphological charactersare highly affected by the enviroment, dominanceof the
characters, sometimes expressed the seedlingstage(Shaw,Kahler, aad Allard,
or not in
r 9 8) .
r
The use of DNA markers is consideredas a powerfirl method in determining
outcrossing rates DNA markers are not affected by the environment, highly
polymorphic,
and non-growthstagedependent. this method,the genotypeof the
In
progenies as well as the pzrents can be precisely dete.mined so that the estimated
outcfossingratesarc more accurate.
Among the DNA markersavailable,simplesequence
repeat or SSRmarkershave severaladvantages estimatingoutcrossingrates. The co'
in
dominant characteristicpermits the identiflcation of the genot)?es of each indMdual
withoutprogeny
testing(ShawandAllard, 1982).
Breeding systemshave important consequences the geletic structur€ of plant
in
populations. They dete.mine the amount as well as the distribution of the genetic
variationsuTithinand amongpopulations
(Wright, 1921; Stebbins,
1957;Brolvn and
Alard, 1970;Hamrick,
Linhan,andMitton, 1979).
Breeding govemthe amount
systems
of assortativeor disassortativemating that takes place during the formation of open-
pollinated progeniesand thus, the degreeof relatedness
among offspringswithin suclr
progenies
(ShawandAllard, 1982).

35. t1
The knowledge matingsystem necessary choosing
of is in suitableand elicient
g€n€tic
strategy in optimizing
breeding ard conservation utilization. It is
resources and
impo.tantin giving sampling
recommendations sampling
since variesaccording
methods
to the mating systems the population.It can also assistin the maintenance
of of
germplasm in the multiplication selected
and of genotypes.
Data on matingsystemcan
aiso be used to analyzegene flow within a populationor gemplasm coliection
(Ashbumer,
1999).
Since compositevarietiesare developed random mating of the parental
by
knowledge the mating system necessary the following reasons.
populations, of is for
Fi.stly, it is usefulin choosing parentalpopulations.
the Secondly, can be used in
it
assessing yieid performance the advanced
the ol generations composite
of varieties.
Thirdly, it is important in predictingthe level of inbreedingas well as geneticequilibrium
of colnpositevariety.
Thisstudywasconducted thefollowingobjectives:
wjth
1. Characterize matingsystem coconut
the of varieties usingmiqosatellite
composite by
markers;
2. Esiimatethe mating systemparameters 15 Fl Tall x Tall coconuthybridsthat
of
constitute coconut
the varieties (CCV 0); ard
composite 0
3. Assess genetic
the equilibrium
status coconut
of varieties (CCV 1) based
composite 1
on microsatellite
loci.

36. ItrA.TERIALS AND METEODS
Plant Materialsand SamplinsStrategv
CoconutCompositevariety 0 (CCV 0)
Coconut compositevarietieswas developedand plantedby PcA-zamboanga
Center (?CA-ZRC), Zamboang4 Philippines. The detail of the method of
Resea-rch
creatingcoconutcomposite was described Santos a/ (1989).CCV 0 is
varieties by et
composed 15 F1 Tall x Tall coconuthybrids.Thesehybridswere generated
of by
crossing Tall coconutvadeties
6 namelySl LagunaTall (Sl LAGT), Sl Bago-Oshiro
Tall (S1 BAOT), Bay-Bay Tall (BAYT), R€nnelIsland Tall (RIT), Tagnanan
Tall
(TAG-T)8rd West African Tall 0IVAT). The hybridizationhasresultedin 15 Fl hybrjdsi
(1) 51 BAOTxBAYT;(2)S1 BA0T xRIT; (3) Sl BAOT x rAGT, (a) S1BAOT x
WAT; (5) BAYT x TAGT; (6) LAGT x BAOT; (7) LAGT x BAYT; (8) LAGT x RIT;
(9) LAGT x TAGT; (10) LAGT x WAT; (11) RIT x BAYT; (12) RIT x TAGT; (13)
WAT x BAYT; (14)WAT x RIT; ard (15)WAT x TAGT.
The hybridswere planted August 1992in two Blocksnamely
in Block I lG (3.03
ha) and Block 21 (10.62ha) with a total of 13.65hectares.
The plantingsystemwas
designed ensure
to randommatingamongpalms.
Eachindividual
hybridwas sunounded
by other six differenl hybrids. The lay out of the palms followed a triangular planting
system(Appendix
Figure 1). The plantingdistance
was 8.5 m x 8.5 m with an avenge
density 132palmsper hectare.Thenumber
of ofpalmsof eachhybridranges
from 96 to

37. 2'7
145palns witlt a total of 1,805palns. Therewere 1,650palmsremaining the field in
in
1998.The layout of coconutcomposite
varieties the field is presented Appendix
in in
Figure2.
For this study,the sample
palrns CCV 0 were drawnrandonlyfrom Block 11
of
and Block 21, which contain204 and 310 palms,respectively. tota] of 30 mate.nal
A
paknsweresarnpled, 15 palmsfrom eachblock;each hybridwasrepresented 2
i.e. Fl by
palms, from each
one palms presented Appendix
block.Thelist ofmaternal is in Table1.
Variety 1 (CCV I ,
CoconutComposite
CCV I was the progenythat resulted
liom randommatingCCV 0. In this study,
CCV I consisted two populations,
of CCV la andCCV lb. The first population,
CCV
la, refersto the l.5-yearold seedljngs unknown
with palmparentage,
matemal grown in
polybagsat PCA-ZRC, Zamboanga.
The seedlings
were derivedfrom the seed-nuts 15
of
Fl Tall x Tall hybrids(CCV 0) haftested October-November,
in 1999 The seed-nuts
of
each hybrid were planted in bulk in different ro{s. HeIrce,the maternalpalm of each
seediing not knorn. Therewere 75 seedlings the nursery 2001in which each
was in in
hybrid has of three to six seedlings. From the 75 seedlings the bulk CCV 1a
of
population,45 samples
were dmwn randomly.
Each ofthe 15 Fl hybridscomprising
CCV 0 wasrepresented three seedlings
by takenat random.
The secondpopulation, paln parentage.This was
CCV lb, hasknownmatemal
obtainedby harvestingnuts fiom 30-selected
palms of CCV 0. Each Fl hybrid
comprising
CCV 0 u?as
represented onepalm selected random eachblock. The
by at in
nuts werc harvested May 2001. The nuts werc labeled
in properlyaccording the
to

38. 28
hybridsand palrn number,and germinatedseparately th€ nursery.Alter 6 months,leal'
in
samples seedling. total of 150sample
were takenfrom eachsample A seedlings
ofCCV
drawn at random(5
lb were used,whereineachhybrid was represented 10 seedlings
by
hybrid/block). The nurserymanagement the CCV la and CCV lb
seedlings/Fl of
populations followed the Manual on StandardizedResearchTechniquesin Coconut
(Santos a/, 1992).
Brceding el
Simple SequenceRepeat (SSRI Analysis
SsmplePreparationand GenomicDNA Isolation
Leaf samples palmsof CCV 0
were collectedftom frond number2 or 3 of selected
population
(adultpalms)and leaf number1 or 2 of CCV 1 seedlings. composite
A ieaf
of (four leaflets
sample eightleaflets from eachsideof larnina CCV 0) andoneto two
of
opened
leaves CCV I seedlings
of weretakenfrom the middleofthe lamina.
Eachleaflet
samplewas tainmed to 10 cm length, the midrib was removed, and flnally sealedin
plastic bags.The leaf samples
were lyophilizedfor 72 hours and then ground in a Wiley
mill. Eachgroundsample plasticbag and storedat -20'C until
was placed a sealed
in
use.
Exlraction of DNA was performedusing 0 2 g of dry-groundleaf samplefollowing
Doyle and Doyle's method@oyle ard Doyle, 1990),with slight modification(IPB
Lab, unpublished). groundleafwas placed
Genetics The into a 15 ml tube,andthen l0
rL of preheatedCTAB buffer was added. The slurry was mixed well and incubatedat

39. 29
65 'C for t hr. The tubeswere invertedgentlyat 1i min interval. The soliddebriswas
removed filteringthe slurryin several
b), layersof gauze. Al equalvolume('7.5 mL)
isoamylalcohol(24i1 v/v) was addedinto the filtrate. The
of chloroformcontaining
mixture were mixed slowly for 5 min and centrifugedin a refrigeratedHeraeusMegafuge
with swing out rotor at 2500 rpm for 30 minutesat 22 "C. The aqueous
layer was
pipetted a new i5 r tubeandprecipitated an equal
into with volumeofcoid isopropanol.
The solutionuTas
inverteduntil DNA strands visible.The precipitated
became DNA was
with a glass
spooled hook,transfe.red a 1.5 ml tubeandwashed
into with 1 nri of70 %
ethanol. The tube was spunbrieflyin a microfugeand the ethanol
was decanted.The
DNA pelletwas air-dried remove residual
to the ethanol
thenresuspended 500 !L TE
in
buffer (10 mM Tris-Cl,pH 8 0, 1 mM EDTAepH 8.0) with RNAse (0.1 prg/prlfinal
concentration).
The DNA was further purified following the phenol puriication
proce'dure
(CIMMYT, 1998). The purifiedDNA wastransferred a new 1.5ni tube
into
andstored 20 'C
at
DNA quantification doneusinga gel quantification
was method(CIMMYT, 1998)
Horizontal gel electrophoresis the DNA samples!as performedwith known
of
concentrations lambda phage
of (1") DNA asstandard.
The gelwasstained
with ethidium
bromideand photographed a IJV trans-illuminator. concentration genomic
on The of
DNA of eachsample estimated comparing intensity
was by the ofthe DNA sample
bands
with kno&nconcentrctions phage
ofi, DNA.

40. 30
AmplificationReaction
was cardedout in a 96-well microtiterplate in a PTC-100
PCR amplification
ProgrammableThermal Controller (MJ ResearchInc., Wateitown, ltrA). The reaction
mixture (25 pL) consistsof 10 ng genomicDNA5 0.2 pM of forward primer and reve$e
0.75 to I U 744 DNA pol}'rnemse, to 1 mM
pdmer,200 FN{ dNTPs@romega), 0.75
MgCl:, lx PCR buffer (10 mM Tris-HCl,pH 8.3, 50 nM KCI), and stedlenanopure
HrO. The PTC-'I0oProgrammable was programmed 35 cycles:
ThermalController for
94 'C for 40 sec (denaturation),
54-55 "C (depending the primerused)for I min
on
(annealing),ard, 72 ''C fot 2 min, with an initial denaturatiorof 94 "C for 2 min and a
power
final exlension 72 "C for 10 min. SevenSSRprimerpairswith discriminating
of
2001) were used. The SSR primer peirs arei CN2A4,
greaterthar 0.99 (Carcallas,
primers
CNZ51,CNZl8, CNZ21,CNZ09,CNlG4, andCN1C6.These wereisolated
and
characterized Riveraet dl (1999).
by
Gel Electrophoresis Staining
and
EachPCR productwasmixedwith 12.5pL 3x STRloadingdye(98 % formamide
aontaining rnM EDTA" 0.01 % [w/v] xylenecyanoland 0.01 % [w/v] Bromphenol
l0
Blue). A 3.5 Fl aliquotof the mixturewas loadedin eachlane of a polyacrylamide
(PAGE) gel. The PCR productswere sepamted a DNA sequencing
electrophorcsis in gel
containing % polyacrylamidefois-acrylamide 7 M Urea and 1x TBE (90 InM
5 [19:1],
Tris-borate, mM EDTA) at 75 W constant
2 powerfor 45 min to 1.5hrs. The gel was
stainedfollowing the silver stainingprotocol of Promega(1996) with slight modification

41. 31
(IPB Genetics Lab protocol, unpublished).Once dried, the gel was photographed,
scanned scored.
and
Estimationof OutcrossinsRates
Estimation ol outcrossingrates was performed using mixed mating model fo
independent proposed Ritlandand Jain (1981) and further modifiedby Ritland
loci by
(1989).The matingpajameters
werc estimated on (1)
based the followingassumptions:
the seed made ofa proportion
is up seed andself-fertilized (s = l-t),
ofoutcrossed (t) seed
where s is an estimateof the efective selfing.ate, which includesreal selfing as well as
biparentalinbreeding;(2) the populationhasto displayMerdelian segregation marker
of
genes; (3) loci are unlinked or therc are no linkage disequilibrium; and (4) gene
frequencieswere equally distributedin pollen and oMrle pool. To test fbr assocnhons
loci, Burows's composite
between of (A66)wascalculated
measure lintagedisequilibria
for all possiblepairs of loci within family with 12 tests for significance(Weir, 1996),
package
usingthe computer POPGENE1.32(Yeah,
YangandBoyle,2001).
The mating systemparameters
estimatedwere: (a) multilocusoutcrossingrate (t-),
(b) single locus outcrossingrate (t), (c) the proportion of full-sibs among outcrossed
as by ofpatemity(h), (d) the corelationofoutcrossing
progeny measured the conelation
rate within progeny arrays as a measureof the vaaiation of outcrossingrates among
progenyanays (r.), and (e) fixation index of maternalparents(F). The differencebetween
t- and t, (t- - t"l) provides a measureof the amountof biparentalinbreeding(Ritland,
1984).

42. 32
The estimation the parameters conducted
of was software
usingcomputer MLTR
ver 2.2 (Ritland, (EM)
2001) Expectation-Marirnization methodtas usedfor estimating
the matingsystempalameters. errorsfor these
Slandard weregenemted
parameters using
500 bootstrap
estimates, resampling
with Genefrequencies the pollen
amongfamilies. in
separately. methodofBrown andAllard (1970)was
and olule pool were estimated The
appliedto infer maternalgenolT,es for each family of CCV 1 with unknown matemal
palmpar€ntage.
GeneticEquilibrium Test
Wright's fixation index (F;.), the within-population
inbreedingcoeflcient, was
estimated an indicator deviation
as for fiom Hardy-Weinberg (Weir, 1996).
proportions
Expegted
selfingrate (s.) at equilibrium
was computed fixationindex
liom the observed
(F^) by equation = s,l(2-s").Asideliom inbreeding
Fi. X2-test applied
coefficient, was to
test for geneticequilibriumat singlelocus andmulti loci.
The expectedinbreedingequilibrium coefficientif the mating systemwas the only
factorcausing
deviation equiiibrium wasestimated Fe: (1-
from Hardy-Weinberg (F") as
t",)/(l+tJ. The Fi"-F.valueis the indicator
ofthe effectof factorsotherthanselfing
that
cause
departure
fiom Hardy-Weinberg
equilibrium.

43. 33
RISULTS
Evaluationof the Assumotions the Mixed-Matins Model
of
MendellianInheritanceof SSRMarkers
in CCV 1 Population
Resultsof this studyindicatethat indMdualsamplepalm of CCV 1 popuiations
palmparentage
with known(CCV lb) andunknom (CCV la) maternal possesses or
one
representjng
two alleles, homozygous haerozygous
and suchasthe natureof
individual,
a diploid species. As shownin Figure 1, eachpaim of CCV lb cafied one of the
maternal
alleles the ofthe alleies.lt
inherita[ce
from CCV 0. This confinned Mendelian
that oneofthe alleles the progeny inherited
means in is parentandthe
from the maternal
othel allele derived
is from the paternal
parent. CCV lb populations
Therefore, meetone
of the assumptions mixed mating model, whereinthe populationhas to display
of
inheritance the markergenes
Mendelian of (RitlandandJain, 1981). In additionto the
inheritance the SSRloci usedwere established
resultsof this study,the Mendelian of
previously
ftom a coconutlinkagemappingstudy(IPB Genetics
Lab, unpublished
data)
andhybridity
testingstudy(Hautea 4/, 2001).
e/
LinkageDisequilibriaamongSSRMarkers
in CCV 1 Populations
were reportedto havemixed matingsystem, self and
Tall coconutvarieties i.e.
cross-pollination. this reason, mixedmatingmodeiof Ritlandand Jain(1981)
For the
{asusedin estimating
matingsystemparameters the CCV 0 population.
in

44. LocusCN2A4
M3Pl 5t1. 511 4 P l_5 P]_.-__.j r r_.__.__j r]_-_._.1
LocusCNZ18
l3P ]_l? I 5ll 5Pl 5tI 5 P l , - , - _ _ _ l I t - * _ _ _. J
-
Figure L lnheritanceof three polymorphic SSR loci in Coconut CompositeVa.riety 1
with known maternal palm parentage
(CCV 1b). Lanes: M8 : Marker rlII
(Promega): P : Maternalparents(CCV 0); 1-5 = individualpalm of each
progeny (CCV 1)

45. 35
One ofthe assumptioiN this model is that the loci usedare indep€ndent are not
in or
linked with eachother. Resultsof this study $howthat amongthe 54 allelesofthe seven
loci used, linlage disequilibria
occurredonly among 12 allelesof the five SSR loci
namely:
CNZ21,CNZ51,CNZI8, CN2A4,andCNIG4 in the CCV la (Table1). ln the
CCV lb, 28 alleles six SSRloci namely
of CN2A4,CNZ21,CNZ18,CN1G4,CNZ09,
and CN1C6exhibited
linkage(Table2). Therewasan inconsistency in the number
of of
linted allelesbelween CCV la (unknown)and CCV lb (known). Weir (1996)
mentioned the statistical mightshowthe presence
that test oflinkagedisequilibna
among
loci; however,the loai are not linked physically. The diJfer€nce the numberoflinked-
in
loci observed
betweenCCV 1a and CCV lb populations
may alsobe attributed the
to
different
number samples
of usedin the 1wopopulations.
Therewere45 samples
ofCCV
la and 149samples
ofCCV lb. All the ninealleles CN2A4werelinkedto the seven
of
alleles
ofCNZlS. However, otherpairsofloci werelinkedonly in oneo. two alleles.
the
Theseresultsindicalethat loci CN2A4 and CNZ18 occur mosl likely in the same
chromosome.Thus, CN2A4 and CNZ18 are the only loci considered linkedloci since
as
all ofthe alleles bothloci arelinked.
in
The analysis of mi{ed mating model assumesthat the loci used have to be
independent.
Sincelocus CN2A4 and CNZ18 were considered
linked, then in the mating
systemanaiysis
ofCCV la" oneof thesetwo lid<edloci(eitherCN2A4or CNZ18)have
to be removedliom the analysis. However, all loci (including linkedJoci) were used to
study the effects of lirkage disequilibriaon the estimatedmating systemparameters.
In
the CCV lb population,all loci were includedin the anaiysis.

46. 36
Tablel. Singlelocuslinkagedisequilibria SSRloci in CCV I with unknown
of maternal
palmparentage (CCV la)
LOCUS/ALLELE-LOCUS/ALLELEBI]RROWS CORRELATION T, PROBABtr-ITY
-
cNz21^{ CNZsI/A 0.011 0.309 0.040
CNZ2l,D - CNZI8,D 0.011 0.352 5.,14 00 1 0
CNZ2I/G CNZIS/H 0.021 0.359 5.65 t).0t7
CNZ51/A CN2A,I/A 0.021 0.359 0.016
_
CNZT8/B CNIG4D 0.011 0.503 1l.13 0.001
CNZ18/A CN2Al?ts 0.011 0.500 I1.00 0.00t
The numberof significant (P < 0.05) linl€ge disequilibria (LD) = 6.
Table 2. Singlelocus linkage disequilibriaof SSR loci in CCV I with known matemal
palmparentage (CCV 1b)
LOCUS/AILELE-LOCUS/ALLELE BIJRROWS CORRELATION t' PRoBABILITY
(rre)
CN2A4/A CNZl8/A 0.008 0..t55 30.83 0.000
cN2A,l/B CNZt8,ts 0.0t2 0.198 5.85 0.016
CN2A4/C - CNZI8,ts 0.036 0.2,t8 9.20 0.002
CN2A4,D CNZl8/C 0.094 0.:t91 22.82 0 000
CN2A4/E CNZ18?D 0.006 0.163 3.97 0.046
CN2A4/F CNZ18/E 0.0.t4 0.328 16.04 0.000
CN2A4/G CNZT8/G -0.034 -0.162 3.89 0.049
CN2A4/G . CNZI 8/G 0.030 0.175 4.56 0.033
CN2A4A{ _ CNZI8,tr 0.048 0.240 8.56 0.003
CN2A4?D CNZ2]/D 0.02t 0.170 4.28 0.039
CN2A4/G CN1G4/G 0.029 0.205 6.29 0.012
CN2A4/I{ CN1G4/C 0.035 0.171 4.3'7 0.03'7
CNZ2l/F CNZO9AI 0.006 0.202 6.08 0.01:l
CNZ2l/D CN1C6/G 0.005 0.216 6.96 0.008
The numberofsignifica (P < 0.050) lintage drsequilibria (LD) = 1.1.

47. 37
SSRGeneFrequencies Pollen
in
and Ovuleof CCV I
The sevenSSR loci were 100 % polymoryhicso that all loci we.e used in
estimatingmating systemparameters. polymorphic the
The locus was considered if
fiequency
ofthe mostcommon
allelewasiessthan0.95.In CCV la (unknown), total of
a
weregenerated the seven andthe number
54 alleles by loci ofallelesranged
from 4 to 1l
per locus(Table3). In knownmaternal
palmparentage
CCV lb, a total of53 alleles
were
detected seven whereeach
in loci (Table
locuscontained to 9 alleles
7 4).
Estimates alleleliequencies pollenand owle in CCV la and CCVIb for
of for
sevenSSRloci are presented Table3 andTable4. The mix€d-mating
in modelanalysis
that the allelefrequencies po11en or,uleare homogenous. pollen
assumes in and The
frequencyis uniform over materna]genotlTes(Brown and Allard, 1970). The
homogeneity allelefrequencies pollen and ovule were examined using1'?test
of in by
described Weir (1996). In CCV la, SSRloci CNZ5l andCNZ09showed
by signjficant
differences < 0.05)in geneliequencies
(P pollenand orule pool. It implies
between that
pollenpool did not contribute plants thosetwo loci. On the other
equally maternal
to for
hand,there were no significantdifferences genefrequencies
of betweenpollen and o'ule
pools in all loci in CCV lb. It meansthat genefrequencies CCV lb were equally
in
distributed pollenandolule pool asassumed mixedmating
in in model(Ritiand,
1989).

48. 38
Table3. SSRgene&equencies pollenandolule ofCoconutComposite
in VarietyI with
(CCV la)
unknolvnmatemaipalm pareotage
AILEL,IS x'
t0 tl
0042 0.234 0.141 0.167 0.000 0.2i0 00c0 0.000 0.125 0.000 0.042 11.01?*
0.033 0.100 0.233 0033 0.000 0000 0031 0.031 0067 0.100 0.3'i7
0.000 0.042 0.250 0.000 0.000 00r0 0.090
0.000 0.000 0.250 0000 0 1 2 5 0 0 4 2 0 . t 2 5 0 0 0 0 0.203 0.250
0.0.10 0.000 0.240 0137 0 . 0 4 0 0 2 0 2 0 1 3 0 20.15-
0 0 3 1 0 . 0 0 0 0 . 2 1 9 0 . 1 3 3 0 . 0 6 3 0 . 3 1 2 0 . 1 2 5 0.063 0.000
0 000 0 046 0 l:1,1 0 000
0.031 i:).375 i1.063 0.r88 0 . 2 5 0 0 . 0 3 1 0 . 0 6 1 0.000
0.320 0.040 0.040 0.267 0 . 0 4 0 0 . 1 3 3 0 . 1 6 0
0.031 0094 0.156 0 031 0l2i 0 . 1 3 8o t 2 i
(r.1'50 0 231 0.200 00110 0.369
0 2 0 0 0 . 4 0 0 0 3 1 3 0 . 0 3 3 0.033
CN1C6 Polla 0030 0.092 0.294 0.514 2.626*
O$le 0.031 0.233 0.167 0.567
N- non{i8nillcdt (P<005)
r = sie ficanl(P > 0.05)
*" = highlv siemficant(P < 0.01)

49. 39
Table4. SSRgen€liequencies pollenandowle ofCoconutComposite
in VarietyI lvith
palmparentage
knownmaternal (CCV 1b)
LOCI SOLIRCE ALLELES t
CN2A4 Pollen 0.029 0.029 0.158 0.301 0.029 0.086 0.213 0.l.ll 0.010 7.672""
Oule 0.017 0.017 0.117 0.200 0.033 0.150 0.167 0.283 0.0t7
CNZ51 Pollen 0.020 0.22r 0 . 1 5 1 0 . 1 r 5 0 . 1 0 8 0.22,10.078 0 084 - 7.834""
O''ule 0.050 0.183 0 . 1 3 3 0 . 2 1 7 0 . 1 5 0 0.183 0.067 0.017
CNZ09 Pollen 0.t40 0.052 0.21.10.242 0.148 0.116 0.078 0.010 -- 9.266-
orule 0.250 0.083 0 . 1 1 7 0 . 1 5 0 0 . 1 6 7 0.183 0.033 0.017
CNZ18 Pollen 0.039 0.t29 0.398 0.0 0.105 0 1 9 0 0 . 1 2 9 - - 9 250^
(}ule 0.017 0.100 0.233 0.050 0.t83 0.200 0.217 -
CNZ21 Pollen 0.163 0.23,1 0.299 0.08{ 0.180 0.030 0.010 - - 2.951^
Omle 0.183 0.200 0.300 0.050 0.167 0.083 0.017 -
CN 1G4 .Pollen 0.047 0.097 0.258 0.t01 0.078 0.068 0.t,18 -
Grne 0.067 0.117 0.267 0.283 0.050 0 . l l 7 0 . 1 0 0 -
CNIC6 Pollen 0.039 0.059 0.2'75 0.111 0.326 0.t,+7 0.0t0 "
Oule 0.017 0.033 0.250 0.150 0.283 0.267 0.000 -
' = nor-sigrrficant
(P>0.05)

50. 40
Thereare two possible
reasons the differences
for observed CCV la and
between
CCV lb. The CCV la population
r/asthe progeny CCV 0 harvested October
of in to
1999. On the otherhand,the CCV 1b population
November consisted the seed-nuts
of
fiom CCV 0 in May 2001. The two populations
harvested from diferentperiods
resulted
differences pollenandowle gene
ofpollination. The periodolpollinationcouldcause in
also affectsthe genefrequencies.
fiequencies.The numberof samples The CCV la
consisted palms
of45 sample whiJe of palms
CCV lb wascomposed 149sample
Matins SYstem
Mating SystemofCoconut Composite
Variety
rutes (t- and tJ, corelation of
The multilocusand single locus outcrossing
outcrossing among
rate progeny
arays (rJ andcomelation patemjty
oloutcrossed wjthin
progenyarays (ro) of CCV 0 estimated
usinglinkedandudinked (or independent)
loci
from CCV lb (knorm matemal)
popuiation presented Table 5. The multilocus
are in
mtes(t-) ofCCV 0 estimated
outcrossing from CCV 1b rangedfrom 0.911to 0.914so
that the multjlocusselfirg rates were 0.086 to 0.089 (8.6-8.9%).The singlelocus
outqossingrates(t") rangedfrom 0.790to 0.804. lt implies
that the singlelocusselfing
rates.angedfiom 0.196to 0.210(19.6-21 %). This findingconinns the predominantly
0
nature
outcrossing ofTall coconut
variety