Jurnal
rssN
1410-3354
AktaAgrosia Telah Diakreditasi
Vol.
11
No.2 Juli - Desember 2008
DAFTAR ISI Ekstrak Tumbuhan sebagai Penginduksi Ketahanan Sistemik Tanaman Cabai terhadap Cucumber Mosaic Virus. (Mimi Su{rawstidan Yenny Sariasih)
tr
Sistem Tanaman Legowo dan Pemberian P-starter pada Padi Sawah Dataran Tinggi. (Azwir) .... ;
\J
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 currvignathus Holnigren). @jamitah, Priyatiningsih dan-Sugeng Widi;rtoI...-........:.....................
\espon -Va1iglqs P-adi Surya pada Dosis Abu Sekam dan Umur Pindah Tanam. (Sri Vivi Kasmarleni, Wklodo dan Riwandi)
tvz
i
lo8i 113
119
Resp-onBeberapa Hibrid Kakao terhadap Cekaman Kekeringan pada'Fase Bibit. (Muhammad
Thulikdan
Hermansyah)
.......................
t?5
i-:togenilitgs ls.olat Sleinerilsna dari Beberapa Ekosistem di Bengkulu terhadap Spodoptera
lituraF- (Priyatiningsih, Djamilah dan lVlugiyirno) ...........................:..........
t32
Studi Perkecambahan Benih Jarak Pagar (Jatropha curcas L.). (Firdaus Sulaiman dan Andi 139
Changes.in Seed-Quality- of Mung Bean Genotypes with Different Seed Characteristics as Affected by Field Weatheiing Dwing Maruriry Sm[es. (Marwanto)
Potensi Cendawan Entomopatogen Metarrhizium anisopliae Sorokin Isolat Curup Untuk Pengendalian Spodoptera linl"a Fatrricius. (Nadrawati) ......:...............
15l
Efektivitas Cendawan Metarrhizium onisopliae Sorokin terhadap Plutella rylostella Curtis dan 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 Lahan di Bengkulu (Mu-hammad Faiz Barchia) ....................
r70
F-rp.yo Zygotic
Rescue and Regeneration of F I Hybrid Manggo Seedling Obtained from InterVarieties Polycrossing. (Sya rif Hlsen and E rny tsha rta$ ......:............
t75
Penyehatan Tanah secara Hayati di Tanah Tanaman Tomat Terkontaminasi Fusarium oxysDorum F.SP.lycopersicr. (Kusdi Haslopo,Inekas Soesanto dan Endang lVlugiastuti) ...............:..............
180
Lalat Pengorok Daun, Liriomyza huidobresis (Blanchard) (Diotera: Aeromvzidae) di Sentra Tanaman-Sayur Rejang !-efong, ,B_engkulu: Tariaman inan'g,'Paiasiotoiifs. dair kelirirpahannya. @winardiApriyanto, Mutia Farida dan Tri SunardD........-..........
188
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)
Jurnal Alda Agrosia telah dial
JumalAktaAgrosiaVol. I I No.2 hlm 108 -
ll2
rssN 14r0-3354
Jul - Des 2008
Identification of DNA Markers Linked to CMV Resistance Gene(s) ; , in Hot Pepper Identifikasi Marka DNA yang Terkait den7an (ien Pengendaii Ketaha,nan terhadap CMV pada Cabui lvfe,rah Rustikawatir, catur Herison I, sudarsonoz, .Eliyanti3, dan Dotti suryati
I
3University of Jamb!, Jambi rus t ikaw at i@un i b. uc.
i
d
fl?Hf""J
cMV has caused severe damages in ,,,, intbction ,:aused major yieid reduction in Indonesia, Inheritance study on resistance to CMV showcd that resistance 1o CMV was co(rtrolled by at least three recessiYe genes. The objective of this study was to idenrifu i)NA rnarkers linked to CMV resistance gene(s) in hot pepper using bulk segregant analysis (BSA) sh'ategy. Molecular markers were cleveloped by RAPD analysis on the F2 generation generated from a cross between C 102,i and, C frutesscetil as rhe mapping population' DNAgenomeswereisolatedfromseventytworandornlysampledpiantsofasegregatedF2popriition and were amplified with six groups of random primers from Operon Technologies of OpA, OpC, OpE, Opt OpFl and OPM each ofwhich consisted of 20 primers. Result of the experiment obtained 20 CMV resistance specific
RAPD markers. These RAPD markers are grouped ^nto two linked groups and one of the nrarkers (OpH5ro) r,r.as shown 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 hot pepper breeding programs lbr CMV resistance. Key words: DNA markers, RAPD, hot pepper, CMV resi.stqnce
INTRODUCTION
:
One of the objectives of hot pepper breeding programe in Indonesia is developing high
yielding-virus resistance cultivars, especiaily cucumber mosaic virus (CMV) resistance. Among 45 identified viruses infecting hot peppers in Indonesia, CMV infection has caused severe damage and resultecl in up to 75%-100% loss of
hot 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 CMV
resistance character in hot pepper hns been inconsistence among different published reports (Singh and Thakua 1997; Rusko and Csillery I 980; Pochard et al., 1983; Lapidot et ol., 1997; I{obbs
al., 1996). Inheritance study on resistance to CN4V showed that resistance to CllV rvas ccntrolled by at least three recessiv; genes et
(Itustil;arvati. 2C00; Hcrison et cI.,2004) Lre transfer of CMV resistance characters from resistance donor parent to recurrent recipient one can be done by backcross
breeding. This conventional hybridization approach usually requires l0-15 backcross generations irnd takes years to cornplete the whole alternuitive approaclres l;u;h as molecular aided hackcross breeding techrrique [ras been suggested.
cyclel. 'lb overconre this corrstraint,
I(andom A.rnplifieC Polymorphic LINA (RAI')D) rs one ofthe various molecularteclmiques comrrtr>nly appliec ln plant breeding. The RAPD has beelr used to idcntity DNA markers linli to various diseases resistance genes in cuculnb{)t-,
Rustikawati, Catur Herison, Sudarsono, Eliyanti . dan Dotti Suryati: Identification of DNA
hot pepper, muskmelon and sweet pepper (Wechter et a1,,1995; Baoxi et al., 2C0Q: Horejsi et al., 2000; Sanjaya et al., 2002\. The o'rjective ofth s study was to identitv DNA 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 Bulk Segregant Analysis (BSA) Method Seventy two individuals of F2 mapping pcpulation generated from a cross ofa resistance genotype (C lC24) with a' susceptible one (C fi'utescent) were inoculated and grorrped into
highly 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 detennined by cak ulating the ratio of absorbance value of the prepared DNA at Aruo to Arro. The value of 1"8 20 indicated good quality DNA (Sambrook et al.,1989). The R pool and S pool DNA were used as ternplate to gerierate RAt'D markers using a number of random DNA prinrers. The RAPD markers were generated through polymerase chain reaction (PCR) using PE 2400 geneAmpDNA thermal cycler.
109
template DNA. The nurnbers and sizes of the 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 specific RAPD markers were identified. 4. Only the R pool specific RAPD markers wereused to genotype individual plant of the F2 segregating-population. The presence or abserrce of R pool specific RAPD and S pool specific RAPD matkers were scored for each F2 plant.
Liukage Analysis among RAPD Markers with genes Controlling CMY Resistance
5.
The presence specific RAPD markers were combined with scoring data for symptoms of CMV 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 the resistance trait through the calculation ofthe prop ortion of recombinants. Calc ulation of the
proportion of recombinants is a method to identifu linkageamong genesused by Morgan
RESULTS AND DISCUSSION ITAPD lr.nalysis by BSA Strateg.v Subsequence 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 generate R.APD markers. These randorn printers were used to amplifr template DNA of R pool ad S pool in PCR and generaled F,"\PD markers wcre scparated in 0,8Yo agatose gel electrophoresis 2. The presence or absenoe of amplified products (RAPD marrers) generate,{ by the tested primers were scored foi R pools,ad S pools
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 RAPD markers were identified.
Jurnsl AldaAgrosia Vol. I I No.2 hlm 108
- I 12
n0
Jul - Des 2008
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l" Linkage groups among CMV resistance specific RAPD marker-s identified
b), bulk seg-
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Figure 2. Linkage patern 6mong markers within linkage group
L
I-Gl
and LG2 with symptom scores
ill
Rustikuwati, Catur llcrison, Sudarcr.no, Eliyanii , dirn Dotti Suryati: Identification of DNA
Linkage analysis among tlrose RAPD rrrarkers showed theybelonged into two diffqrent Xinkage groups w ith ths logJ ikel ihood (LOD) value
3,64. (Figure 1). Linkage group (l,G) No.l consisted of 1l RAPD ma.rkers and covering for 86,2 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. The genetic distance anton! markers rvere commonly in 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.:e gr:nes controlling he CMV resista.nce characters identified in the previous genetic study was linked to one of the CMV rq.sisiance specific RAPD marker 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 of 6,1 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 with the 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 OPHl2rno markers rvill appear coincidentally with tne CMV resistance gene at the probability of 93,9 and 90,9yo, respectively. Therefole, those marksrs can be used to assist selection on CMV resistattce.
CONCLUSION With brrlk segregant analysis rnethod. 20
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 for Iaboratory facilities elaborated in this researchSpecial thanks extended to Yudi and Dambang, staffs of RGCI IPB, for tecirnical assistance on
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