BREEDING.AID POPI.JLATION  GEITETICSSTIJDIESON                   COCON!{ (Cocosnuciferu L.) COIfTOSITE VARIEIY            ...
The dissenationattachedhereto. entitled "BREEDING AND POPIJI-ATIONGENETICSSTUDIESON COCONUT(Cocostwcifera L ) COMPOSITE   ...
BIOGRAPHICAL SKETCH     The authorwasbom in Manado,                               Indonesia August25, 1959. is the eldest ...
He marriedZubaidaKanini Pomantoin 1986;they are blessed                                                           with one...
ACKNOWLtrDGEMENT                 In the nameof Allah, Most Gracious,Most Merciful             Praisebe to Allah, The Cheri...
my      appreciation the membem my Ph.D advisory     I alsowishto express sincere          to         ofcommrttee,        ...
generations M&pangetTall populationsand providing valuableinformation on those           ofvariety;k Miftahorrahiuq Ir Ism...
Kak Ari, Imrar! JunaidandNur, Iskandarand Vonny, Syaifirl and Aam, Arifin, Syamsi,      Mut Godeh ard Ali Utina, for conti...
TABLE OT CONTENTS                                                        PageGENERAL INTRODUCTION                         ...
Paqe   RESULTS                                                       31      Eva.luation the Assumptions the Mixed-MatingM...
MATERIALS AND METEODS                                              76                                                     ...
PagePOPULATION GENtrTIC STRUCTURE OF COCONUT (Cacasnaciferu L.) COMPOSITE VARIETIf,S                   138   INTRODUCTION ...
PaqeGENERAL DISCUSSION                 187GENERAL SIJMMARY AID CONCLUSION   zo1RECOMMENDATION                     209LITER...
LIST OF TABLESTable                                                                    Page        Single locus linkage di...
Table                                                                  Paqe l1     Mean Auft components inbreedingdepressi...
Table                                                                  Page        Corelation betwe€ngeneticdistancesofpa....
LIST OF FIGURISFiqu.e                                                                   Pase  I      Inheritanceofthree po...
Figure                                                                       Pese ll      Genetic divergenc€of parentalpop...
Figure 2l      Dendrogram CCV I derivedfrom Fl hybrids                      of                              (CCV 0) based ...
LIST OF APPEI{DD( TA}LESABpeodix Tables   I       List of samplepalmsof CCV 0 andmotherpalmsof CCV 1
LIST OF APPENDD( FIGI]RESAopendix                                                              Page Figure   I       Tiang...
ABSTRACTAKUBA, RUSTHAMRIN HARIS. University of the Philippines Los Baios April2002. Breedins and Population GeneticsStudie...
multilocuslevels. Hence,balancedheterozygositywas not attainedafter one generationofraodom mating                         ...
fixation index. Wrights Fsr and Rho va.luesof unbiasedestimator of Slatkins RSTdistancewas computedas an indicator of popu...
GENERALINTRODUCTION     Coconut plays an imponant role in most coconut producing countries. In thePhilippines,it contribut...
2      Hybrid)yielded to 3.8 t coprape. hectare yearat 10yea.sold compa.edCoconut             3.5                      per...
3Tall, and selected                 elite open-pollinated       namely:                                     varieties     ...
4perfomance of the advancedgenerationof compositevarieties(Busbice, 1970).Heterosis,the superiority in performanceof hybri...
5it meansthat the genotypefrequencies, this case,the heterozygous                                    in                   ...
bpolymorphism (MLP) (Teulat et al, 2000), random amplified polymorphic DNA orRAPD (Ashbumer,1999), simplesequence         ...
7     The main goal of this researchwork is to €valuatethe me.its of breedingcoconutcomposite        varietyin order to en...
8chapter. Chapter is focused population               IV          on        geneticstructure CCV 0 and CCV 1              ...
ANALYSIS OF THE MATING SYSTEM AND Gf,NETIC EQUILIBRIUM IN         COCONUT COMPOSITE VARIETIES (Cocos nuciftra L.)         ...
10      Estimation of sellpollination rate and/or outcrossing rate using conventionalmethods      suchasfruit colorinherit...
t1     The knowledge matingsystem necessary choosing                 of            is       in        suitableand elicient...
ItrA.TERIALS AND METEODS                       Plant Materialsand SamplinsStrategvCoconutCompositevariety 0 (CCV 0)     Co...
27145palns witlt a total of 1,805palns. Therewere 1,650palmsremaining the field in                                        ...
28hybridsand palrn number,and germinatedseparately th€ nursery.Alter 6 months,leal                                        ...
2965 C for t hr. The tubeswere invertedgentlyat 1i min interval. The soliddebriswasremoved filteringthe slurryin several  ...
30AmplificationReaction                     was cardedout in a 96-well microtiterplate in a PTC-100     PCR amplificationP...
31(IPB Genetics Lab protocol, unpublished).Once dried, the gel was photographed,scanned scored.      and                  ...
32     The estimation the parameters conducted                  of             was                   software             ...
33                                RISULTS           Evaluationof the Assumotions the Mixed-Matins Model                   ...
LocusCN2A4       M3Pl        5t1.    511     4 P l_5      P]_.-__.j     r r_.__.__j r]_-_._.1                             ...
35     One ofthe assumptioiN this model is that the loci usedare indep€ndent are not                          in          ...
36Tablel. Singlelocuslinkagedisequilibria SSRloci in CCV I with unknown                                      of           ...
37SSRGeneFrequencies Pollen                  inand Ovuleof CCV I     The sevenSSR loci were 100 % polymoryhicso that all l...
38Table3. SSRgene&equencies pollenandolule ofCoconutComposite                          in                              Var...
39Table4. SSRgen€liequencies pollenandowle ofCoconutComposite                         in                               Var...
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Disertation of rustham0001

  1. 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. 2. The dissenationattachedhereto. entitled "BREEDING AND POPIJI-ATIONGENETICSSTUDIESON COCONUT(Cocostwcifera L ) COMPOSITE VARIETYUSING MORPHOLOGICAI AND MICROSATELLITEMARKERS-. prepared and ofthe requiremenlssurnrtted RUSTHAMRIHARISAKuBA. in panialtulfrllnrent by olDoctor ofPhilosophyforthe degree Breeding) hereby (Piaot is accepted r ?-, al--1 JOSE€ I#RNINDEZ CALIXTO M. PROTACIOMembet Advisoq/Committee Advisory Member, Committee furr(s,aoz ,forl+ t a- Date signed Datesigned /, | . t /i//J"4 Xv4t.*tC DESIREE HAUTEA N4. AdvisoryCon1miiteeCo-Chai., Chair,Advisory Committee 4V-y o, ,o, 7 1 Datesigoed Accepted as panial fulfillment of the requirementsfor the degreeof Doctor ofPhilosophy(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. 3. BIOGRAPHICAL SKETCH The authorwasbom in Manado, Indonesia August25, 1959. is the eldest on He ol children Alm. MusaAkubaandSartinseven of Wahibu. He accomplished primary the school SDNPineleng, at Mnahasa, North Sulawesi, in1971,the secondary schoolat SMPNegeriI Manadoin 1973-andhigh school SMA atNegeriI Manado 1976. He took up theBachelor Science in of in degree Statistics Sam atRatulangi University Manadoin 1983. He rorks at Research Institutefor Coconut and (RICP)Manado,Palmae Indonesia an agronomist as since1984 He obtained Masterof aScience degree Agroclimate in from Bogor Agriculture Bogor, lndonesia University, in1988undera scholarship the Agrjculture Aom Resea.ch Management Project, AgencyforAgricultural Researchand Development(ARMP), Governmentof Indonesia.He got the grantedby (ARMP II), AARD Government Indonesia pursuea PhD,,scholarship of toprogramin Plant Breedingand minor in Plant GeneticResources, Management andConservation the University at ofthe Philippines Baflos 1998. Los in He was elected President Inte.national as of Students (ISA), University Associationof the Philippines Los Baiios @PLB) in 2000 For his active participation the inorganization,the Chancellorof IIPLB awardedhim the Most Outstandingand DynamicLeadership awardfbr foreignstudents 2000. He is a member in ofthe Honor Society of GAMltrA SIGMA DELTAAgriculture University ofthe Philippiness Chapter
  4. 4. He marriedZubaidaKanini Pomantoin 1986;they are blessed with one son,AdityaAkuba, andone daughter, Citra Arini Ahrba. c->45 RUSTHAMRIN HARIS AKIBA
  5. 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 followingagencies institutions: and the Department of Agriculturg Bureau of Agricultural Research (DA-BAR),Republic of the Philippines,for financing this studies through a researchgrant to DrDesireeM. Ifuutea; the Agency for Agricuhural Researchand Development(AARD), DepartmentofAgricullure, Republicoflndonesia,for the Ph.D. scholanhipgrant andpartly financingtheresearch underAgricultureResearch ProjectII (ARMP II); Management the PhilippineCoconutAuthodty (PCA) through PCA-Zamboanga Research Cente.(PCA-ZRC), the pemission access coconutsynthetic for to the varietyandother.elatedcoconutva.ietiesasmaterialsofthe reseaxch providingthe rel€vantdata, and the Institute of Plant Breeding,Collegeof Agriculture,Universityof the PhilippinesLos Bafros,for the use of laboratoryfacilitiesand other .esources the Geo€tics ofLaboratory, and the Research Institute for Coconut and Palmae, Manado, Indonesia, for thepermission access selfinggeneratioNofN{apangetTall coconut. to the
  6. 6. my appreciation the membem my Ph.D advisory I alsowishto express sincere to ofcommrttee, nalnelyl (Chairman)andDr DesireeM. Hautea(Co-Chair)for their Dr JuanitoB. Sangalangenomous interestto the research, invaluabl€ cofftructive comments lriendly guidance, andencouragement dudngthethesis indebted Dr Desiree Hautea work. I amparticularly to M.for teachingme molecularbiology and moleculargeneticsand enhancing knowledge myby giving me opportunities to participate in intemational and nationsl slanposiaandtrainingin the Philippines; and Dr JoseE. Hemandez Dr Calixto M. Protaciofor their significantcontributionto andthis 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, hisPCA-ZRC,Zamboanga; GerardoA. Santos, Mr lorbrilliant ideasin developingcoconut syntheticvariety that hasinspiredthe thesisworkand facilitating the researchwork at PCA-ZRC; Mr RamonRivera, Researcher PCA- in providing relevantinfomationZRC, for his assistance settingup the field experiment, in vadety, and developing SSR primersfor coconut;MJon thecoconut synthetic theErnesto Efirnanuel, Lea Kingco,Ms Joyce E. Ms Desoacido, Mr Lucilo Kingcofor andfieldassistance PCA-ZRC; at Dr David Allorerun& Director of Research Institute for Coconut and PaLmae, foradministrativeand financial support in the fieldwork at RICP; Ir Tine Rompasand DrNovarianto Hengky, Breeders at RICP, for their efforts in developing the selfing
  7. 7. generations M&pangetTall populationsand providing valuableinformation on those ofvariety;k Miftahorrahiuq Ir IsmailMaskromo,Ir ElsjeT. Tenda,Drs DjunaidALubaand RICP stafs, for field assistance RICP Manado,Indonesi4Drs JohnWurangian, at Dr Pons A. Batugal, COGENT CoordinatorIPGRI, for his great concernson thisresearchand providing relevant literaturc; Dr Roger Ashburnerfor providing valuablepaper on matilg systemof coconut; Dr llasnanLthe director of Center ResearchforIndustrial Crcps (CRIC); Bogor for administrativesupport,Dr Pasril Wa.hid,the fomerdirectors of CRIC, Dr Zainal Mahmudfor continuouslyencouraging to continuethe mestudy andDr CesarMadamba "my Philipino parents",for their and Mam Lilia Madamba, lhat my wasveryconvenienlhospitaljty made stayat Los Banos I also wish to acknowledge valuableassistance the following staff of the IPB the ofGenetics Lab: Dr Conrado Balaterc, University Researcher the Institute of Plant atBreeding, for fruitful discussions, sharing knowledge on molecular biology, andconstructivecommentsduring the lab and thesis trork; Ms Hayde Galvez, Ms ShirleySegovia,Ms Carol Padlan,Ms Nancy B. Coronado,Ms Alma O. Canama, Joy Marie MsBartolo andMs Mailln A. Latiza for sharing and their knowledgeon moleculartechniquescreatingthe friendly atmosphere the lab that madethe lab work exciting; Tita Rowena in especiallyin providing laboratory administrativeFrankie has extendedgreat assistancessuppot andKuya CesarCaspillofor technicalassistarce the lab, in and appreaiationis extendedto my wonderfirl big The geat acknowledgementfamily: Ma SartirLMa Masitah,Ma Imun, Kak Sophianand Sri, Ana and Imeng, Syari!
  8. 8. Kak Ari, Imrar! JunaidandNur, Iskandarand Vonny, Syaifirl and Aam, Arifin, Syamsi, Mut Godeh ard Ali Utina, for continuousSyamsu, prayersandencouagement; and A heartfelt gratitude finally is extended to my beloved wife, Zubaida KartiniPomanto, tlrc Pogi sol! Aditya, and the smart daughter, Citra for their patience, encouragement prayersduringthe diffcult time ofthe reseaichunderstanding, and work. May Allah Blessandgivesrewardsto all the generous peoplementionedabove-
  9. 9. TABLE OT CONTENTS PageGENERAL INTRODUCTION IANALYSIS OF MATING SYSTEM AI{D GENETIC EQUILIBRIUMIN COCONUT (Cacasnuciferu L) COMPOSITE VARIETmS USINGMICROSATELLTTEMARKERS 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. 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 63INBREEDING DEPRI,SSION AND HETEROSIS IN COCONUT(Cocosnucifera L.) COMPOSITE VARIf,TIES 67 INTRODUCTION 67 71 RXVIEW OF LITERATURX Effectsof Inbreeding 7l Heterosis Composite in Varieties
  11. 11. MATERIALS AND METEODS 76 76 Plart Materialsand SamplingStrategies Morphological Observation 19 MicrosatelliteAnalysis 80 Data Analysis 81RESULTS 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 109DISCUSSION t26 InbreedingDepression t26 Homozygosity 129 Heterosis t32SUMMARY AND CONCLUSION 134
  12. 12. PagePOPULATION GENtrTIC STRUCTURE OF COCONUT (Cacasnaciferu 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 SamplingStrategy 151 Moryhological Evaluation 152 MolecularEvaluation 153 Data Analysis 153 RESULTS Allelicrichness l55 Allelic Evenness 158 Heterozygosity 160 PopulationDifferentiation 164 GeneticDistance 170 DISCUSSION 179 SUMMARY AND CONCLUSTON t84
  13. 13. PaqeGENERAL DISCUSSION 187GENERAL SIJMMARY AID CONCLUSION zo1RECOMMENDATION 209LITERATURES CITf,D 211.APPENDICES 228
  14. 14. LIST OF TABLESTable 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 Wrights fixatlon index (F,.) and 1r test for 51 equilibriumofCCV 1 with known (CCV lb) andunloown (CCV la) mate.nalparentage
  15. 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 Wrights fixation index @;") as a measue of heterozygous 104 deficiency or excessin selfing generationsof Mapanget Tall (IvrTT) 19 Neis 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 Slatkins 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. 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$ity, and 124 heteozygosity SSRloci andliuit components a1 ofCCV 0 27 Number a.nd size of alleles detected i4 CCV 0 and CCV I 157 populations 28 Allele richnessof the populationsthat make up of CCV 0 and 159 ccv 1 29 Heterozygosity ard fixation index of CCV 0 and CCV I 163 populations Fsr andgeneflow 0Im) ofCCV 0 and CCV 1 populations 165 31 Matrix ofRho valueaveraging varjance ove. components oICCV 167 0 population Matrix ofRho valueaveragingovervadancecomponents CCV of 169 I population 33 Matrix of(delta-p)ofCCV 0 population 171 Matrix of (delta-p)? CCV 1 population of 1,75 Meanofcopraweightofparental populations, hybrids, Fl CCV 0 196 andCCV 1
  17. 17. LIST OF FIGURISFiqu.e Pase I Inheritanceofthree polymorphicSSRloci in CoconutComposite 34 Variety 1 with known matemalpalm parentage(CCV lb). Lanes: M8 : MarkerVlll (Promega), = Matemal P parents (CCV 0); l-5 : individualpalmofeachp.ogeny (CCV 1) Estimates ofave.age multilocusoutcrossing (t.), single rate locus 49 outcrossing late (t.), correlation of outcrossing rutes (r), coeficient correlation of outcrossed patemity (rp) based on differentnumberof SSRloci Polynorphism of SSR loci CNZ21 and CN2A4 in selfing 93 generationsof Laguna Tall and Bago-OshiroTall in the multi- loadingpolyacrylamyde electrophoresis gel Allele evenness ofselfing generations oflaguna Tall 96 Allele evenness selfinggenerations of ofBago-Oshirc Tall Level of homozygosity selfinggelerationsLagunaTall (S0 and of 98 S1LAGT) andopen-pollinated Bago-Oshiro Tall(S0BAOT) and SI BAOTar ieven microsalellile loci Polyrnorphismsof four representativesSSR loci in selfng 100 generations Mapanget of Tall (M8 = MarkerVIII; 1,2,3,4,5,6 : individualpalm) Allele evenness selfng generatio$ 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. 18. Figure Pese ll Genetic divergenc€of parentalpopulationsof CCV 0 basedon 111 unbiasedestirnator Slatkins 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 t7 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. 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. 20. LIST OF APPEI{DD( TA}LESABpeodix Tables I List of samplepalmsof CCV 0 andmotherpalmsof CCV 1
  21. 21. LIST OF APPENDD( FIGI]RESAopendix 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. 22. ABSTRACTAKUBA, RUSTHAMRIN HARIS. University of the Philippines Los Baios April2002. 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 Cederas a supplementary coconutbreeding. breedingstrategyto the hybdd and open-pollinatedThe objectives creatingcoconutcomposite of varietieswer€ to obtaincoconutvarietywithhigher yield potential than the open-pollinated coconut variety because heterosis tall ofefect, greater genetic variability, ald balanced heterozygosity. Morphological andmicrosatellite inbreeding markerswere usedin the evaluationofmating system, depression geneticvariability andgenaic equilibriumin the ooconutcompositeandheterosis, variety. Analysis of the mating system and genetic equilibdum in Coconut CompositeVarieties{CCV 0 and CCV 1) were conducted. Resultsirdicate that CCv 0 waspredominantly ratesof 91.1 Yolo 91.4o/o, cross-pollinated outcrossing with and selfingates8.6 yoto 8.9 yo. A largeproportionofthe cross-pollination eventswere dueto full-sibmating. The biparentalinbreeding crossing or betweencloselyrelatedindMdual occurredat tie rate of 6.9 yo to 10.3 %. CCV 1 did not attain equilibrium at the single and
  23. 23. multilocuslevels. Hence,balancedheterozygositywas not attainedafter one generationofraodom mating wasalsoperformed. Th€ effec1 ofinbreedingdepression heterosis Assessment and ofiobreeding depressionon the economic yield was indirectly studied by using selfinggenerations tlree tall coconutvarieties(LagunaTall and Bago-OshiroTall, Mapanget ofTall). Inbreedingdepression CCV 1 was also assessed molecularapproaclr, which in by in was measured reducedlevel of heterozygosity. Selfingin Tallinbreedingdepression ascoconut populationsresulted in inbreedingdepressionand increasedhomozygosityinLAGT, BAOT, and MTT populations. Inbreeding depr€ssionthat was measutedasreducedfitnessbased the changes fixation index at SSRloci washigh in 53 MTT and on in54 MTT. Selfing,fiil sib mating and biparentalinbrcedingin CCV 0 resultedin CCv 1with lower level of heterozygosity. Md-parent heterosiswas presentin the intervarietalFl Tall x Tall coconuthybridsthat constitutedthe CCV 0 for whole nut weight, meat weight and copra weight. Thepredictedcopraweight per nut ofCCV 1 washigherthan the copraweight per nut oftheparentalTall populations,however,it was lower than that ofFl tall x tall hybrids. Therelationship baits andg€n€ticdistances betweenheterosis fruit component in ofthe Fl Tallx Tall hybridswas also determined.Data indicatedthal or y mid-parentheterosis copra inweight couldbe correlatedwith geneticdistance. The assessment population genetic structure was studied in terms of genetic ofvariability and populationdifferentiationof CCV 0 and CCV l. The geneticdiversitywascharacterized terms of numberof alleles,observedand expgctedheterozygosity, in and
  24. 24. fixation index. Wrights Fsr and Rho va.luesof unbiasedestimator of Slatkins RSTdistancewas computedas an indicator of population diferentiation. Genetic distanc€sbetreencomponents weremadeup ofCCV 0 andCCV I were computed that on basedmorphological in vigor in CCV 1, and SSR tni1s, i.e. fruit components CCV 0 andseedlingmarkem. The rcsu1ts showedthat CCV 1 waslessdiversecompared CCV 0 population. toThe CCV I had less number of alleles and was deficient in heterozygousgenot)?es.Geneticdifferentiationwithin populationwas higher in CCV 1 than in CCV 0 indicatingthat CCV 1 was more fragmented. The imptcations of the results of this researchtococonutbrcedingandgermplasm were discussed management extensively.
  25. 25. GENERALINTRODUCTION Coconut plays an imponant role in most coconut producing countries. In thePhilippines,it contributesUS$ 900M yearly for the country, benefiting1.5M farmersandlarm workers, and 24 million people directly or indirectly. It supportsthe sustainablemanagement envircnment,as the pa]ms prctect the soil fiom erosion and nutdent oflosses(PhilippineCouncil for Agriculture, Forestry and Natural Resources Research and 1999).Development, programsin the Philippines.It Replantingis one of the main coconut dev€lopmentis aimed at rcplacing the senile coconut trees and replanting of fams damagedbytyphoonsand other natural calamitieswith promisinglocal cultivars and/or hybrids. Thetargetsfor 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 is240,000seed-nuts year, which is ody enoughfor 1,000ha per year (Santos per and 1995).Rivera, To overcomethe problem of insumcienl seed-nutproduction, the development ofcoconuthybrids, Dwarf x Tall hybrids, beenconducted. i.e. has Hyb.id breeding the hasadvantages 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 theTall va.ieties.Coconut hybrid developmentrelease nine coconuthybrids(PCA 15-1 to PCA 15-9)by the Philippines of Coconut Nut productionAuthority. ofthesehybrids ftom 117to 155nutsper palmperyear ranges(Santos,1998).Luntungan(1997) reportedthat the ten-yearold KHINA (Indonesian
  26. 26. 2 Hybrid)yielded to 3.8 t coprape. hectare yearat 10yea.sold compa.edCoconut 3.5 perto 1.3 1.6 t copraper hectare yeai ofthe Tall coconutvarieties aboutthe same per atage. However, hybrid breeding in coconut faces the problems of narow geneticvariabiliry, moresusceptibility pests, to diseases droughtstress, high costofseed and andproduction.It takes time to multiply a suitablevariety or hybrid for commercialplanting(Baudouin, rate,the final costof planting 1999).Due to the low multiplication materialswould be unaffordable resource-poor to fa.mersunlessgovemments providea subsidythat many count.iescannotafford (Batugal, 1999). In the Philippines, current theestimat€d of coconut cost is per (CIF nurser]site, hybridseednut aboutPhP33.00 seednutunpubl.).In contrast, seed-nuts open-pollinated the of varieties costaround per PhP6.00 (Santos dl,2O1O).The hybridizationseed-nut el technique not as simpleas in annual iscrops sincecoconuthas giant featu.es.This biological constrainthasresultedin the highcostbfhybrid seed production lessnumber and produced palm. ofhybrid seed-nuts per ResearchCenter (PCA-ZRC) The Philippine Coconut Authority-Zamboangainitiated the development of coconut s]rthetic variety way back in 1979 as asupplem€ntary breeding strategy to the hybrid and open-pollinatedcoconut breedingprograms (Sartos, Bahala Cano,1989;Santos a/, 2000). To oblainhigher and e/ yieldingvariety with greater genetic variability, the slartheticvariety should be developedfromrandommating of inbredlineswith differentgeneticbackground, then testedfor their andcombiningability (Agrawal, 1998).Theserequirements difficult to implement are forcoconutsinceit is a perennial crop. The development inbred lines is also time- ofconsuming costly.Therefore, and Santos a/ (1989)modified method e/ the ofgeneratingsyntheticva.iety by using fust generationselfing (Sl) of Laguna Tall and Bago-Oshiro
  27. 27. 3Tall, and selected elite open-pollinated namely: varieties Bay-bayTall, Tagnanan Tall, IslandTall, andWestAfricanTall, asthe parentsRennel ofsyntheticvarietyzero(Syn0), instead usinginbredlines(S4 to 56 populations). Syn 0 is composed 15 of The ofintervarietal Fl hybrids among parentalpopulations. Randommating the Syn 0 hasresulted inSlal 1. Theoretically, coconutsyrthetic the varietydeveloped Santos a/ (1989)is a by elcompositeva.rietysincethe base populationsconsistsof inteNa.rietal hybrids of a fixed varietiesor populations.set of heterogeneous Nonetheless, syntheticand compositevarieties very similarin structure are (Busbice, 1970;Hallauer and Miranda,1981).Inthis study,the term compositevariety is usedinsteadofthe term slnthetic variety basedon theoretical considerations. The intervarietal hybrid population (or Syn 0) is calledCoconut CompositeVariety 0 (CCV 0). Meanwhile,the progenies resultingfromranddmmating the CCV 0 is called Coconut CompositeVariety 1 (CCV 1), which waslaunchedas GeneticallyMulti-Ancestors (Gtr4-A)coconut farmersvariety jn August2001. It is expectedthat the development coconut compositevariety would resuit in a ofvarietythat hashigheryield potential because h€terosis of effectandwider adaptabiiitysinceit is composedof various lines with differcnt genetic constitutions(Santose/ a/,1989). Fu.the.more,this variety (CCV 1) is also er?ected to havebaiancedhete.ozygosityin which the proportion of heterozygotegenotypes will not changeovergeneration randommating. of The mating system of the populations a.ffectsthe genetic variability (Clay ardLevin, 1989;Ritland,1989),the attainment genetic of equilibrium(Liu, 1998),and the
  28. 28. 4perfomance 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 theint€rvarietal hybrids as the base populations of composite va.riety will affect theperformance generations ofsucceeding (Eberhart dl, 1967). ofcomposites et geneaction and degree The level of heterosisis affectedby the level of dominanceof geneticrelatedness between (Falconer Mackay,1996). The presence parents and ofdominancegene action can be detected indirectly by the occurrence of inbreedingdepression a consequence selfing. However, inbreedingdepression economic as of inyield cannotbe detectedin CCV 1 sinceit is not in the fiuit-bea.ringstage. ln this aasethe presenceof inbreedingdepressiolin Tall populationsof coconut would beinvestigatedby 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 composileof populations with different genetic background. Genetic variability of compositevarietiesdependson the geneticvariability of the parents,inbreedinglevel, outcrossingrate, and genefrequencies the population.Hallauerand Miranda(1981)mentioned inthal greater genetic variability expected be available is to ifpopulations ofdiverseoriginsare combined. Heterosisamongintervarietalhybrids hasalso beenlbund to be relativelyhigh. The meanyield of new populationis expected be greaterthan the average to oftheparental varieties. The other objective in generatingthe coconut compositevariety is to obtain avarietywith balanc€d (Santos a/, 1989).In population heterozygosity el genetics tenrL
  29. 29. 5it meansthat the genotypefrequencies, this case,the heterozygous in fequ€flcy doesnotchange from generation to generation. lt implies that the population is in genetjcequilibrium.If nndom mating occurs in a very la.rgepopulation of compositevarietieswith the assumptions no selection,mutatiorLmigratiorLand randomdrift then genetic ofequilibrium a locusis expected be attained one generation randommating in to in of(Falconer and Maakay,19961 Weir, 1996). Sincenot all of the assumptions be oanfuifilled in coconutcompositevariety populatio4 then the balancedheterozygosity mightnol bereached onegeneralion in ofrandommating. Agro-morphological markers have been used in tie mating system, inbreedingdepressionand hete.osis, and genetic diversity studies of coconut. Ashbumer el a/(2001)usedliuit color inheritance methodto assess nating system Gazelle the on Tallpopulation. The studiesof inbreedingdepression were carriedout on the onsetflowering(Santbsand Sangar€,1992); and height of seedling, girth aircumference, numberof andgreenleaves(Rompas a/, 1988). Heterosis coconut el on hybrids wasreported copra onweight and oil content in PBl21 hybrid (Vanialingan,Khoo, and Chew, 1978);onset offlowering 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 andevaiuationof genetictraits of many crops, in additionto morphologicalcharacterization.DNA maikels are not dependent the stageof plant development.They are distributed onin th€ whole genome,and are highly pollmorphic Several molecular techniquesareavailable for genetic study of coconut. These include restriction lragment length
  30. 30. bpolymorphism (MLP) (Teulat et al, 2000), random amplified polymorphic DNA orRAPD (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 ofshould meet severalrequircments.The results must be highly reproduciblein differentlaboratoriesand analyzedusing standardized scoring and aml)tical methods.Data mustbe easily incorporatedinto databases. Assaysshould be applicabiefor high throughputand necessarymaterialsshould be exchangeable. Co-dominantmarkers are preferableover dominantmarker systems, high polyrnorphism and levels are requiredto distinguishcloselyrelatedgenotypes. SSRor microsatellites these fit precisely are requirements andconsideredto provide the most informative method of evaluatinggenetic diversity incocodut(Karp, 1999;Ashbumer, 1999).ln this study,SSRor microsatellites used werein coljunctjon with morphological traits to evaluateor assess allele and genotypefrequencies,mating system param€te.s,genetic equilibrium, and population geneticstructureof the populations. In rcsponse the development to ofcoconut synthetic vadetiesby Santos a/ e/(1989), Baudouin(1999) mentioned that thecoconut slnthetic varietyhas severaldisadvantages such as: (1) the Fl hybridswill alwaysbe better than this variety, (2) theresultingvariety vrill be rather heterogeneous, the geneticvalue could vary with time (3)due to seasonal va.riations flowering and production, and (4) selfingis not prevented. ofHis doubts about the vadety were finally expressed salng: "Only experience by will sayifthis type ofmaterial reliable economically is and interesting".
  31. 31. 7 The main goal of this researchwork is to €valuatethe me.its of breedingcoconutcomposite varietyin order to ensure release the and distribution improvedcoconut ofvarieties to farmels. To ensurethe accomplishment the objectivesand answerany ofdoubts about the coconut compositevariety, characterization evaluationhave to b€ andcarded out for the facto$ that affect its performance. The mating systerL heterosis,geneticva.riabilityand geneticequilibdumare the main factors involved in the evaluationof coconut composite varieties. Threestudieswer€ conducted answerthe following specificobjectives: to1. 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 reportedinChapter Thischapter ll. alsodiscusses status the ofgeneticequilibrium ofCCV I at SSRloci. ChapterIII presents inbreeding the depression selfinggenerations Laguna in ofTall, Bago-OshiroTall, MapangetTall and Coconut CompositeVadety I (CCV l) thatobs€rved iiuit comporents SSRloci. Heterosis on and ofCCV 0 is alsodescribed this in
  32. 32. 8chapter. Chapter is focused population IV on geneticstructure CCV 0 and CCV 1 ofpopulations. This research lvas conducted from April 2000 to February2002. The nurseryexperiments and agro-morphologiaal evaluationlere conductedat the PhilippineCoconut Authority-Zamboanga ResearchCenter (PCA-ZRC), San Ramon, ZarllboangaCity from April to November2001. All molecular markq experimentswere done atGeneticlaboratory of the Institute of Plant Breeding,College of Agriculture, Universityofthe Philippines Bafros Los (LTPLB), College Laguna liom Julyto Decembe. 2001.
  33. 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 hasirflorescences that exhibit herkogamy (dicliny) and protandry, which promote inflorescences is predominantly Dwarf varietyexhibits dichogamousoutc.ossing. no andself-pollinated.The Tall variety is consideredas allogamousand the Dwarf variety as varieties(Fremondet dl, 1966). Tall varietieshave a mixed mating system,autogamous since pistillatei.e. cross-and self-pollination, the phase phase with staminate overlaps inthe sameinilorcscence well as in subsequent as (Rognon, inflorescences 1976;Santos ela|,2000). The rate of self-pollinalionin Tall and Dwarf vadetieshasbeenestimated using by model (Ashburner dl, 2001; Bourdeix, 1988). The self-f.uit color inheritance etpollination rate of Gazelle PenimulaTall of PapuaNew Guineawas27.89/o(Ashbumer 2001)whilein Dwarfvarieties, self-pollination rangede1a1, the rates from 88.3% to 100% (Bourdeix, 1988). The coconut compositevadety developed PCA-ZRC is bycomposed l5 F1 Ta[ x Tall coconuthybridswith differentg.owth and flowering ofhabits(Santos al, 2O0O). It is expected the outcrossjng et that ratesof the Tall x Tallhybrids will differfrom the outcrossing ofthe parental populations seashas rate Tall per reporled Ashburner o/ (200l)been by et
  34. 34. 10 Estimation of sellpollination rate and/or outcrossing rate using conventionalmethods suchasfruit colorinheritance doesnot providedirectmeasures ofthe success ofmating in the populations. The information they provide is often iladequale for theanalysisof genetic transmission the population level. In additiorl the morphological at the genotypeof the progeny.markersmethod has also a problemin determiningMorphological charactersare highly affected by the enviroment, dominanceof thecharacters, sometimes expressed the seedlingstage(Shaw,Kahler, aad Allard, or not inr 9 8) . r The use of DNA markers is consideredas a powerfirl method in determiningoutcrossing rates DNA markers are not affected by the environment, highlypolymorphic, and non-growthstagedependent. this method,the genotypeof the Inprogenies as well as the pzrents can be precisely dete.mined so that the estimatedoutcfossingratesarc more accurate. Among the DNA markersavailable,simplesequencerepeat or SSRmarkershave severaladvantages estimatingoutcrossingrates. The co indominant characteristicpermits the identiflcation of the genot)?es of each indMdualwithoutprogeny testing(ShawandAllard, 1982). Breeding systemshave important consequences the geletic structur€ of plant inpopulations. They dete.mine the amount as well as the distribution of the geneticvariationsuTithinand amongpopulations (Wright, 1921; Stebbins, 1957;Brolvn andAlard, 1970;Hamrick, Linhan,andMitton, 1979). Breeding govemthe amount systemsof assortativeor disassortativemating that takes place during the formation of open-pollinated progeniesand thus, the degreeof relatedness among offspringswithin suclrprogenies (ShawandAllard, 1982).
  35. 35. t1 The knowledge matingsystem necessary choosing of is in suitableand elicient g€n€tic strategy in optimizingbreeding ard conservation utilization. It is resources andimpo.tantin giving sampling recommendations sampling since variesaccording methodsto the mating systems the population.It can also assistin the maintenance of ofgermplasm in the multiplication selected and of genotypes. Data on matingsystemcanaiso 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 forFi.stly, it is usefulin choosing parentalpopulations. the Secondly, can be used in itassessing yieid performance the advanced the ol generations composite of varieties.Thirdly, it is important in predictingthe level of inbreedingas well as geneticequilibriumof colnpositevariety. Thisstudywasconducted thefollowingobjectives: wjth1. 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 03. Assess genetic the equilibrium status coconut of varieties (CCV 1) based composite 1 on microsatellite loci.
  36. 36. ItrA.TERIALS AND METEODS Plant Materialsand SamplinsStrategvCoconutCompositevariety 0 (CCV 0) Coconut compositevarietieswas developedand plantedby PcA-zamboanga Center (?CA-ZRC), Zamboang4 Philippines. The detail of the method ofResea-rchcreatingcoconutcomposite was described Santos a/ (1989).CCV 0 is varieties by etcomposed 15 F1 Tall x Tall coconuthybrids.Thesehybridswere generated of bycrossing Tall coconutvadeties 6 namelySl LagunaTall (Sl LAGT), Sl Bago-OshiroTall (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 xWAT; (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 (3.03ha) and Block 21 (10.62ha) with a total of 13.65hectares. The plantingsystemwasdesigned ensure to randommatingamongpalms. Eachindividual hybridwas sunoundedby other six differenl hybrids. The lay out of the palms followed a triangular plantingsystem(Appendix Figure 1). The plantingdistance was 8.5 m x 8.5 m with an avengedensity 132palmsper hectare.Thenumber of ofpalmsof eachhybridranges from 96 to
  37. 37. 27145palns witlt a total of 1,805palns. Therewere 1,650palmsremaining the field in in1998.The layout of coconutcomposite varieties the field is presented Appendix in inFigure2. For this study,the sample palrns CCV 0 were drawnrandonlyfrom Block 11 ofand Block 21, which contain204 and 310 palms,respectively. tota] of 30 mate.nal Apaknsweresarnpled, 15 palmsfrom eachblock;each hybridwasrepresented 2 i.e. Fl bypalms, 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, CCVla, refersto the l.5-yearold seedljngs unknown with palmparentage, matemal grown inpolybagsat PCA-ZRC, Zamboanga. The seedlings were derivedfrom the seed-nuts 15 ofFl Tall x Tall hybrids(CCV 0) haftested October-November, in 1999 The seed-nuts ofeach hybrid were planted in bulk in different ro{s. HeIrce,the maternalpalm of eachseediing not knorn. Therewere 75 seedlings the nursery 2001in which each was in inhybrid has of three to six seedlings. From the 75 seedlings the bulk CCV 1a ofpopulation,45 samples were dmwn randomly. Each ofthe 15 Fl hybridscomprisingCCV 0 wasrepresented three seedlings by takenat random. The secondpopulation, paln parentage.This was CCV lb, hasknownmatemalobtainedby harvestingnuts fiom 30-selected palms of CCV 0. Each Fl hybridcomprising CCV 0 u?as represented onepalm selected random eachblock. The by at innuts werc harvested May 2001. The nuts werc labeled in properlyaccording the to
  38. 38. 28hybridsand palrn number,and germinatedseparately th€ nursery.Alter 6 months,leal insamples seedling. total of 150sample were takenfrom eachsample A seedlings ofCCV drawn at random(5lb were used,whereineachhybrid was represented 10 seedlings by hybrid/block). The nurserymanagement the CCV la and CCV lbseedlings/Fl ofpopulations followed the Manual on StandardizedResearchTechniquesin Coconut (Santos a/, 1992).Brceding el Simple SequenceRepeat (SSRI AnalysisSsmplePreparationand GenomicDNA Isolation Leaf samples palmsof CCV 0 were collectedftom frond number2 or 3 of selectedpopulation (adultpalms)and leaf number1 or 2 of CCV 1 seedlings. composite A ieaf of (four leafletssample eightleaflets from eachsideof larnina CCV 0) andoneto two ofopened leaves CCV I seedlings of weretakenfrom the middleofthe lamina. Eachleafletsamplewas tainmed to 10 cm length, the midrib was removed, and flnally sealedinplastic bags.The leaf samples were lyophilizedfor 72 hours and then ground in a Wileymill. Eachgroundsample plasticbag and storedat -20C until was placed a sealed inuse. Exlraction of DNA was performedusing 0 2 g of dry-groundleaf samplefollowingDoyle and Doyles method@oyle ard Doyle, 1990),with slight modification(IPB Lab, unpublished). groundleafwas placedGenetics The into a 15 ml tube,andthen l0rL of preheatedCTAB buffer was added. The slurry was mixed well and incubatedat
  39. 39. 2965 C for t hr. The tubeswere invertedgentlyat 1i min interval. The soliddebriswasremoved filteringthe slurryin several b), layersof gauze. Al equalvolume(7.5 mL) isoamylalcohol(24i1 v/v) was addedinto the filtrate. Theof chloroformcontainingmixture were mixed slowly for 5 min and centrifugedin a refrigeratedHeraeusMegafugewith swing out rotor at 2500 rpm for 30 minutesat 22 "C. The aqueous layer waspipetted a new i5 r tubeandprecipitated an equal into with volumeofcoid isopropanol.The solutionuTas inverteduntil DNA strands visible.The precipitated became DNA was with a glassspooled hook,transfe.red a 1.5 ml tubeandwashed into with 1 nri of70 %ethanol. The tube was spunbrieflyin a microfugeand the ethanol was decanted.TheDNA pelletwas air-dried remove residual to the ethanol thenresuspended 500 !L TE inbuffer (10 mM Tris-Cl,pH 8 0, 1 mM EDTAepH 8.0) with RNAse (0.1 prg/prlfinalconcentration). The DNA was further purified following the phenol puriicationprocedure (CIMMYT, 1998). The purifiedDNA wastransferred a new 1.5ni tube intoandstored 20 C at DNA quantification doneusinga gel quantification was method(CIMMYT, 1998)Horizontal gel electrophoresis the DNA samples!as performedwith known ofconcentrations lambda phage of (1") DNA asstandard. The gelwasstained with ethidiumbromideand photographed a IJV trans-illuminator. concentration genomic on The ofDNA of eachsample estimated comparing intensity was by the ofthe DNA sample bandswith kno&nconcentrctions phage ofi, DNA.
  40. 40. 30AmplificationReaction was cardedout in a 96-well microtiterplate in a PTC-100 PCR amplificationProgrammableThermal Controller (MJ ResearchInc., Wateitown, ltrA). The reactionmixture (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 mMpdmer,200 FN{ dNTPs@romega), 0.75MgCl:, lx PCR buffer (10 mM Tris-HCl,pH 8.3, 50 nM KCI), and stedlenanopureHrO. The PTC-I0oProgrammable was programmed 35 cycles: ThermalController for94 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 powerfinal exlension 72 "C for 10 min. SevenSSRprimerpairswith discriminating of 2001) were used. The SSR primer peirs arei CN2A4,greaterthar 0.99 (Carcallas, primersCNZ51,CNZl8, CNZ21,CNZ09,CNlG4, andCN1C6.These wereisolated andcharacterized Riveraet dl (1999). byGel Electrophoresis Staining and EachPCR productwasmixedwith 12.5pL 3x STRloadingdye(98 % formamideaontaining rnM EDTA" 0.01 % [w/v] xylenecyanoland 0.01 % [w/v] Bromphenol l0Blue). A 3.5 Fl aliquotof the mixturewas loadedin eachlane of a polyacrylamide (PAGE) gel. The PCR productswere sepamted a DNA sequencingelectrophorcsis in gelcontaining % 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 wasstainedfollowing the silver stainingprotocol of Promega(1996) with slight modification
  41. 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 foindependent 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) seedwhere s is an estimateof the efective selfing.ate, which includesreal selfing as well asbiparentalinbreeding;(2) the populationhasto displayMerdelian segregation marker ofgenes; (3) loci are unlinked or therc are no linkage disequilibrium; and (4) genefrequencieswere equally distributedin pollen and oMrle pool. To test fbr assocnhons loci, Burowss compositebetween of (A66)wascalculated measure lintagedisequilibriafor all possiblepairs of loci within family with 12 tests for significance(Weir, 1996), packageusingthe 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 corelationofoutcrossingprogeny measured the conelationrate within progeny arrays as a measureof the vaaiation of outcrossingrates amongprogenyanays (r.), and (e) fixation index of maternalparents(F). The differencebetweent- and t, (t- - t"l) provides a measureof the amountof biparentalinbreeding(Ritland,1984).
  42. 42. 32 The estimation the parameters conducted of was software usingcomputer MLTRver 2.2 (Ritland, (EM) 2001) Expectation-Marirnization methodtas usedfor estimatingthe matingsystempalameters. errorsfor these Slandard weregenemted parameters using500 bootstrap estimates, resampling with Genefrequencies the pollen amongfamilies. in separately. methodofBrown andAllard (1970)wasand olule pool were estimated Theappliedto infer maternalgenolT,es for each family of CCV 1 with unknown matemalpalmpar€ntage. GeneticEquilibrium Test Wrights fixation index (F;.), the within-population inbreedingcoeflcient, wasestimated an indicator deviation as for fiom Hardy-Weinberg (Weir, 1996). proportionsExpegted 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 totest for geneticequilibriumat singlelocus andmulti loci. The expectedinbreedingequilibrium coefficientif the mating systemwas the onlyfactorcausing deviation equiiibrium wasestimated Fe: (1- from Hardy-Weinberg (F") ast",)/(l+tJ. The Fi"-F.valueis the indicator ofthe effectof factorsotherthanselfing thatcause departure fiom Hardy-Weinberg equilibrium.
  43. 43. 33 RISULTS Evaluationof the Assumotions the Mixed-Matins Model ofMendellianInheritanceof SSRMarkersin CCV 1 Population Resultsof this studyindicatethat indMdualsamplepalm of CCV 1 popuiations palmparentagewith known(CCV lb) andunknom (CCV la) maternal possesses or one representjngtwo alleles, homozygous haerozygous and suchasthe natureof individual,a diploid species. As shownin Figure 1, eachpaim of CCV lb cafied one of thematernal alleles the ofthe alleies.lt inherita[ce from CCV 0. This confinned Mendelian that oneofthe alleles the progeny inheritedmeans in is parentandthe from the maternalothel allele derived is from the paternal parent. CCV lb populations Therefore, meetoneof the assumptions mixed mating model, whereinthe populationhas to display of inheritance the markergenesMendelian of (RitlandandJain, 1981). In additionto the inheritance the SSRloci usedwere establishedresultsof this study,the Mendelian ofpreviously ftom a coconutlinkagemappingstudy(IPB Genetics Lab, unpublished data)andhybridity testingstudy(Hautea 4/, 2001). e/LinkageDisequilibriaamongSSRMarkersin 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. 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. 45. 35 One ofthe assumptioiN this model is that the loci usedare indep€ndent are not in orlinked with eachother. Resultsof this study $howthat amongthe 54 allelesofthe sevenloci used, linlage disequilibria occurredonly among 12 allelesof the five SSR locinamely: CNZ21,CNZ51,CNZI8, CN2A4,andCNIG4 in the CCV la (Table1). ln theCCV lb, 28 alleles six SSRloci namely of CN2A4,CNZ21,CNZ18,CN1G4,CNZ09,and CN1C6exhibited linkage(Table2). Therewasan inconsistency in the number of oflinted allelesbelween CCV la (unknown)and CCV lb (known). Weir (1996)mentioned the statistical mightshowthe presence that test oflinkagedisequilibna amongloci; however,the loai are not linked physically. The diJfer€nce the numberoflinked- inloci observed betweenCCV 1a and CCV lb populations may alsobe attributed the todifferent number samples of usedin the 1wopopulations. Therewere45 samples ofCCVla and 149samples ofCCV lb. All the ninealleles CN2A4werelinkedto the seven ofalleles ofCNZlS. However, otherpairsofloci werelinkedonly in oneo. two alleles. theTheseresultsindicalethat loci CN2A4 and CNZ18 occur mosl likely in the samechromosome.Thus, CN2A4 and CNZ18 are the only loci considered linkedloci since asall ofthe alleles bothloci arelinked. in The analysis of mi{ed mating model assumesthat the loci used have to beindependent. Sincelocus CN2A4 and CNZ18 were considered linked, then in the matingsystemanaiysis ofCCV la" oneof thesetwo lid<edloci(eitherCN2A4or CNZ18)haveto be removedliom the analysis. However, all loci (including linkedJoci) were used tostudy the effects of lirkage disequilibriaon the estimatedmating systemparameters. Inthe CCV lb population,all loci were includedin the anaiysis.
  46. 46. 36Tablel. 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 0.00tThe 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 4.37 0.037 CNZ2l/F CNZO9AI 0.006 0.202 6.08 0.01:l CNZ2l/D CN1C6/G 0.005 0.216 6.96 0.008The numberofsignifica (P < 0.050) lintage drsequilibria (LD) = 1.1.
  47. 47. 37SSRGeneFrequencies Pollen inand Ovuleof CCV I The sevenSSR loci were 100 % polymoryhicso that all loci we.e used inestimatingmating systemparameters. polymorphic the The locus was considered iffiequency ofthe mostcommon allelewasiessthan0.95.In CCV la (unknown), total of a weregenerated the seven andthe number54 alleles by loci ofallelesranged from 4 to 1lper locus(Table3). In knownmaternal palmparentage CCV lb, a total of53 alleles weredetected seven whereeach in loci (Table locuscontained to 9 alleles 7 4). Estimates alleleliequencies pollenand owle in CCV la and CCVIb for of forsevenSSRloci are presented Table3 andTable4. The mix€d-mating in modelanalysis that the allelefrequencies po11en or,uleare homogenous. pollenassumes in and Thefrequencyis uniform over materna]genotlTes(Brown and Allard, 1970). Thehomogeneity allelefrequencies pollen and ovule were examined using1?test of in bydescribed Weir (1996). In CCV la, SSRloci CNZ5l andCNZ09showed by signjficantdifferences < 0.05)in geneliequencies (P pollenand orule pool. It implies between thatpollenpool did not contribute plants thosetwo loci. On the other equally maternal to forhand,there were no significantdifferences genefrequencies of betweenpollen and oulepools in all loci in CCV lb. It meansthat genefrequencies CCV lb were equally indistributed pollenandolule pool asassumed mixedmating in in model(Ritiand, 1989).
  48. 48. 38Table3. 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 0.3i7 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.150 0 231 0.200 00110 0.369 0 2 0 0 0 . 4 0 0 0 3 1 3 0 . 0 3 3 0.033CN1C6 Polla 0030 0.092 0.294 0.514 2.626* O$le 0.031 0.233 0.167 0.567N- non{i8nillcdt (P<005)r = sie ficanl(P > 0.05)*" = highlv siemficant(P < 0.01)
  49. 49. 39Table4. SSRgen€liequencies pollenandowle ofCoconutComposite in VarietyI lvith palmparentage knownmaternal (CCV 1b) LOCI SOLIRCE ALLELES tCN2A4 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 0.0t7CNZ51 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"" Oule 0.050 0.183 0 . 1 3 3 0 . 2 1 7 0 . 1 5 0 0.183 0.067 0.017CNZ09 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 0.017CNZ18 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 0.275 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)

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