Jurnal rssN 1410-3354

AktaAgrosiaTelah Diakreditasi

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Vol. 11 No.2 Juli - Desember 2008

DAFTAR ISIEkstrak Tumbuhan sebagai Penginduksi Ketahanan Sistemik Tanaman Cabai terhadap CucumberMosaic Virus. (Mimi Su{rawstidan Yenny Sariasih)

Sistem Tanaman Legowo dan Pemberian P-starter pada Padi Sawah Dataran Tinggi. (Azwir) ....

Identification ofDNAmarkers Linked to CMV Resistance Gene (S) in Hot Pepper. (Rustikawati,Catur Herison, Sudarsono, Eliyanti dan Dotty SuryatD ...................

PatogenitSs Sreinernema sp. Isolat Bengkulu terhadap Rayap (Coptotermes currvignathusHolnigren). @jamitah, Priyatiningsih dan-Sugeng Widi;rtoI...-........:.....................

\espon -Va1iglqs P-adi Surya pada Dosis Abu Sekam dan Umur Pindah Tanam. (Sri ViviKasmarleni, Wklodo dan Riwandi)

Resp-onBeberapa Hibrid Kakao terhadap Cekaman Kekeringan pada'Fase Bibit. (MuhammadThulikdan Hermansyah) .......................

i-:togenilitgs ls.olat Sleinerilsna dari Beberapa Ekosistem di Bengkulu terhadap SpodopteralituraF- (Priyatiningsih, Djamilah dan lVlugiyirno) ...........................:..........

Studi Perkecambahan Benih Jarak Pagar (Jatropha curcas L.). (Firdaus Sulaiman dan Andi

Changes.in Seed-Quality- of Mung Bean Genotypes with Different Seed Characteristics asAffected by Field Weatheiing Dwing Maruriry Sm[es. (Marwanto)

Potensi Cendawan Entomopatogen Metarrhizium anisopliae Sorokin Isolat Curup UntukPengendalian Spodoptera linl"a Fatrricius. (Nadrawati) ......:...............

Efektivitas Cendawan Metarrhizium onisopliae Sorokin terhadap Plutella rylostella Curtisdan Cro c i dol o m i a p wona na Zellet (Tri Su ila rdi d a n Nad rawa ti) ...............

Metode Penularan dan Uji Ketahanan Genotipe Cabai(Capsicttm spp.) terhadap Begomovirus.@wi Wahyuni Ganefianli, Sriani Sujiprihati, Sri ffenOiasfufi Hilayit, dan llftihamiO Syutur;

StabilitasCa, Mg, Ktk Tanah dan Hasil Sawit dalam Hubungannya dengan Kemiringan Lahandi Bengkulu (Mu-hammad Faiz Barchia) ....................

F-rp.yo Zygotic Rescue and Regeneration of F I Hybrid Manggo Seedling Obtained from Inter-Varieties Polycrossing. (Sya rif Hlsen and E rny tsha rta$ ......:............

Penyehatan Tanah secara Hayati di Tanah Tanaman Tomat Terkontaminasi Fusarium oxysDorumF.SP.lycopersicr. (Kusdi Haslopo,Inekas Soesanto dan Endang lVlugiastuti) ...............:..............

Lalat Pengorok Daun, Liriomyza huidobresis (Blanchard) (Diotera: Aeromvzidae) di SentraTanaman-Sayur Rejang !-efong, ,B_engkulu: Tariaman inan'g,'Paiasiotoiifs. dair kelirirpahannya.@winardiApriyanto, Mutia Farida dan Tri SunardD........-..........

Uji_Multilokqli Galur-galur Harapan Kedelai pada Lahan Rendah Fosfor. (Dotti Suryati,M oha m m ad C h ozin, Haisa n ud i n dri n lh,v ina rd i Apriya n to)

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Jurnal Alda Agrosia telah dial<reditasi melalui Keputusan DirelAur Jenderal Pendidikan TinggiDepartemen Pendidikan Nasional Republik Indonesia dengan Nomor : 26/DIKTI/Ibp/2005

JumalAktaAgrosiaVol. I I No.2 hlm 108 - ll2 Jul - Des 2008 rssN 14r0-3354

Identification of DNA Markers Linked to CMV Resistance Gene(s); , in Hot Pepper

Identifikasi Marka DNA yang Terkait den7an (ien Pengendaii Ketaha,nanterhadap CMV pada Cabui lvfe,rah

Rustikawatir, catur Herison I, sudarsonoz, .Eliyanti3, dan Dotti suryati I

3University of Jamb!, Jambirus t ikaw at i@un i b. uc. i d

cMV has caused severe damages in fl?Hf""J ,,,, intbction ,:aused major yieid reduction inIndonesia, Inheritance study on resistance to CMV showcd that resistance 1o CMV was co(rtrolled by at leastthree recessiYe genes. The objective of this study was to idenrifu i)NA rnarkers linked to CMV resistancegene(s) in hot pepper using bulk segregant analysis (BSA) sh'ategy. Molecular markers were cleveloped byRAPD analysis on the F2 generation generated from a cross between C 102,i and, C frutesscetil as rhe mappingpopulation' DNAgenomeswereisolatedfromseventytworandornlysampledpiantsofasegregatedF2popriitionand were amplified with six groups of random primers from Operon Technologies of OpA, OpC, OpE, Opt OpFland OPM each ofwhich consisted of 20 primers. Result of the experiment obtained 20 CMV resistance specificRAPD markers. These RAPD markers are grouped ^nto two linked groups and one of the nrarkers (OpH5ro) r,r.asshown to be associated with one of the three CMV resistar.ce gerres with the log-lil.:elihood (LOD) vatue of:.6+.The OPH5,* marker linked to a CMV resistance gene with a distance of 8.1 cM, and may be used to assist hotpepper breeding programs lbr CMV resistance.

Key words: DNA markers, RAPD, hot pepper, CMV resi.stqnce

INTRODUCTION :

One of the objectives of hot pepperbreeding programe in Indonesia is developing highyielding-virus resistance cultivars, especiailycucumber mosaic virus (CMV) resistance.Among 45 identified viruses infecting hot peppersin Indonesia, CMV infection has caused severedamage and resultecl in up to 75%-100% loss ofhot pepper fruit production (DEPTAN, 1999;Duriat 1996; Duriat el ql. 1993; Eliyanti, 1998;Safietal., 1997; Sulyo et a|.1996; Rustikawati,2000).

Result of the inheritance study of CMVresistance character in hot pepper hns beeninconsistence among different published reports(Singh and Thakua 1997; Rusko and Csillery I 980;Pochard et al., 1983; Lapidot et ol., 1997; I{obbs

et al., 1996). Inheritance study on resistance toCN4V showed that resistance to CllV rvasccntrolled by at least three recessiv; genes(Itustil;arvati. 2C00; Hcrison et cI.,2004)

Lre transfer of CMV resistancecharacters from resistance donor parent torecurrent recipient one can be done by backcrossbreeding. This conventional hybridizationapproach usually requires l0-15 backcrossgenerations irnd takes years to cornplete the wholecyclel. 'lb overconre this corrstraint, alternuitiveapproaclres l;u;h as molecular aided hackcrossbreeding techrrique [ras been suggested.

I(andom A.rnplifieC Polymorphic LINA(RAI')D) rs one ofthe various molecularteclmiquescomrrtr>nly appliec ln plant breeding. The RAPDhas beelr used to idcntity DNA markers linli tovarious diseases resistance genes in cuculnb{)t-,

Rustikawati, Catur Herison, Sudarsono, Eliyanti . dan Dotti Suryati: Identification of DNA 109

hot pepper, muskmelon and sweet pepper(Wechter et a1,,1995; Baoxi et al., 2C0Q: Horejsiet al., 2000; Sanjaya et al., 2002\.

The o'rjective ofth s study was to identitvDNA markers'linked to CMV resistarrce gene(s]r

in trot pepper using bulk segregant analvsis (BSrr)$:rategy and RAPD technique.

MATERIALSAND METHODS

The establishment of DNA Pool usirrg BulkSegregant Analysis (BSA) Method

Seventy two individuals of F2 mappingpcpulation generated from a cross ofa resistance

genotype (C lC24) with a' susceptible one (C

fi'utescent) were inoculated and grorrped intohighly susceptible (score 5) and resistance (score

0). D1'{A from the identified highly resistance

piants were isolated and cornhine into resistance

DNA pool (R pool). Similarly, DNA from the

iCentified highly susceptible plants rvere isolated

and eombine into susceptible DN/r oool (S pool).

The quality and purity of tNA were detenninedby cak ulating the ratio of absorbance value ofthe prepared DNA at Aruo to Arro. The value of1"8 20 indicated good quality DNA (Sambrook

et al.,1989). The R pool and S pool DNA wereused as ternplate to gerierate RAt'D markers usinga number of random DNA prinrers. The RAPDmarkers were generated through polymerasechain reaction (PCR) using PE 2400 geneAmp-DNA thermal cycler.

ITAPD lr.nalysis by BSA Strateg.vSubsequence steps were conducted to gsncrate

the desired RAPD markers:1. 120 of random primers frorn 6 groups of

Operon Primers (OPA, OPC, OPE, OPF,O['H and OPM) were useC to generateR.APD markers. These randorn printers wereused to amplifr template DNA of R pool ad S

pool in PCR and generaled F,"\PD markerswcre scparated in 0,8Yo agatose gelelectrophoresis

2. The presence or absenoe of amplified products(RAPD marrers) generate,{ by the testedprimers were scored foi R pools,ad S pools

template DNA. The nurnbers and sizes ofthe generated RAPD malkers were recorded

and compared with I kb,ladder marker

3 . Primer producing R pool specific polymorphic

marker were selected, and the R pool specificRAPD markers were identified.

4. Only the R pool specific RAPD markers were-used to genotype individual plant of the F2

segregating-population. The presence orabserrce of R pool specific RAPD and S pool

specific RAPD matkers were scored for each

F2 plant.

Liukage Analysis among RAPD Markerswith genes Controlling CMY Resistance5. The presence specific RAPD markers were

combined with scoring data for symptoms ofCMV infection. Morphological respgnses to

CMV infection were grouped into resistance,

mild, and susceptible.

6. Linkage map ofRAPD markers and resistance

score were constructed by MAPMAKER/Bxp application software. Linkage analysis

was conducted by MAPMAKER/QtI version

3.0. The program calculated thb genetic

distance of each RAPD marker to theresistance trait through the calculation oftheprop ortion of recombinants. Calc ulation of the

proportion of recombinants is a method toidentifu linkageamong genesused by Morgan

RESULTS AND DISCUSSION

Bulk segregant analysis (BSA) was used

to accelerate identification of markers associated

with the CMV resistancei.phenotype. BSA

analysis is usefulto identify linkage amongmarkers

with simple genes controlling desirable characters

(Paterson 1996).PCR amplification resulted in only 2E

primers out of 120 random primers tested produced

polymorphic markers. Out of28 primers producing

polymorphic markers, only 12 primers produced

RAPD markers specific for CM'y' resistance pool(R pool). From these selected primers, 23 RAPDmarkers were identified.

Jurnsl AldaAgrosia Vol. I I No.2 hlm 108 - I 12 Jul - Des 2008 n0

.

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RAPD marker-s identified b), bulk seg-

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Figure 2. Linkage patern 6mong markers within linkage group I-Gl and LG2 with symptom scores

Rustikuwati, Catur llcrison, Sudarcr.no, Eliyanii , dirn Dotti Suryati: Identification of DNA ill

Linkage analysis among tlrose RAPDrrrarkers showed theybelonged into two diffqrentXinkage groups w ith ths logJ ikel ihood (LOD) value

3,64. (Figure 1). Linkage group (l,G) No.lconsisted of 1l RAPD ma.rkers and covering for86,2 cM of the totalgenome. The linkage group

(LG) I{o.2 consisted of 12 RAPD markers and

covering for 221,7 cN{ of the tocal genonre. Thegenetic distance anton! markers rvere commonlyin a range of 0 - 50 cM, and oPened to be

saturated with other markers in the future.'Mhen the disepse response scores were

combined into linkage;4nalysis, ol"te out of thr.:egr:nes controlling he CMV resista.nce characters

identified in the previous genetic study was linkedto one of the CMV rq.sisiance specific RAPDmarker iderrtified using'8SA The OPII5500 and

OPtll2j0o markers was l,inked and flanked to one

of the CMV res rstance gene with the dis:tance of6,1 arrd 9,1 cM respectively. Two genes'were

considered linkage rovher,l the distance between

them'*as less thar 50 cM (Crowder, 1993).

MAPMAKEPv QTI analysis on symPtom

category data resultecl almost sirnilarly to that on

qualitative data of CI'./V resistance as a marker.'ihe closest genetic distance to CMV resistance

controlling gene was OPHSroo and OPI{12roo withthe distance of 6,1 chtf and 9,1 cM respectivell;with the log-likelihood (LOD) value 3,64 (Figure

2). The existence of OPII-(500 and OPHl2rnomarkers rvill appear coincidentally with tne CMVresistance gene at the probability of 93,9 and

90,9yo, respectively. Therefole, those marksrscan be used to assist selection on CMV resistattce.

CONCLUSION

With brrlk segregant analysis rnethod. 20

CMV resistance specifio RAPD tnarkers were

identified. These RAPD markers are grouped

into two linkage groups ancl two ofthem (OPI{S,,{,

and OPHl2roo) were shown to be associated withone of the three resistance genes. '[he'OPFISroo

marker linked to CMV resistance gerae with adistance of 6.1 cM, and may be used to assist

breeding programs of hot pepper for CMVresistanoe.

ACTC.{OWLEDGEMENT

We wish to thank RUT VnI of The

Ministry of Research and Technolory of Indonesia

for financial support to the research project. We

also wish to thank RGCI and PAU IPB forIaboratory facilities elaborated in this research-

Special thanks extended to Yudi and Dambang,

staffs of RGCI IPB, for tecirnical assistance on

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