Pemetaan tersebut telah menghasilkan peta genetik yang lebih padat dan meliputi genom yang berukuran 1, 714cM dan 1,225cM bagi pisifera dan dura.
PertanikaJ. Trop. Agric. Sci. 29 (l & 2): 35-45 (2006)
ISS : 1511-3701 ©Universiti Putra Malaysia Press
Statistical Mapping of Quantitative Trait Loci Controlling the Time to First Callusing in Oil Palm (Elaeis guineensis Jacq.) Tissue Culture lTING NGOOT CHIN, 'CHEAH SUAN CHOO, lZAMZURI ISHAK, 2'fAN SOON GUAN, 2FARIDAH QAMARUZ ZAMAN, lMAIZURAITHNIN & 'RAJINDERSI GH ] Advanced Biotechnology and Breeding Centre, Biological Research Division, Malaysian Palm Oil Board, Malaysia 2 Department ofBiology, Faculty ofScience, Universtii Putra Malaysia 43400, UPM Serdang, Selangor Malaysia
Keywords: Quantitative Trait Loci (QTL), Time to First Callusing (TFC), oil palm, genomic loci
ABSTRAK Sebanyak 400 penanda genetik (126 RFLP, 274 AFLP) telah berjaya dipetakan daripada 87 progeni F] dari kacukan Deli dura X Yangambi pisifera. Pemetaan tersebut telah menghasilkan peta genetik yang lebih padat dan meliputi genom yang berukuran 1, 714cM dan 1,225cM bagi pisifera dan dura. Menggunakan Kosambi interval mapping, perisian MapQTL Versi 4.0, kajian selanjutnya telah dijalankan untuk mencari 'quantitative trait loci' (QTL) yang mempengarnhi tempoh pembentukan kalus (TFC) bagi kultur tisu sawit. Data kultur tisu menunjukkan taburan yang berterusan. Dalam kajian ini, tiga QTLs telah dikesan pada pisifera manakala dua untuk dura pada nilai signifikan 99% dan 95 %. Ini menunjukkan lokasi QTL tersebut adalah signifikan secara statistik dalam mempengarnhi variasi bagi TFG. aleh yang demikian, maklumat ini menunjukkan bahawa loci genomik memberi kesan terhadap kemampuan sawit untuk dikulturkan. ABSTRACT An additional 400 genetic markers (126 RFLPs, 274 AFLPs) were successfully mapped on the earlier developed linkage maps using 87 F] progenies derived from Deli dura X Yangambi pisifera cross. This resulted in a denser map with coverage length of 1, 714cM and 1, 225cMfor pisifera and dura, respectively. Further exploration to searchfor quantitative trait loci (QTL) associated with time to first callusing (TFC) was carried out by Kosambi Interval Mapping using the computer program MapQTL Version 4. O. The tissue culture trait data showed a continuous distribution. In this paper; three likelihood QTLs were detected in pisifera and two QTLs in dura at 99% and 95% significant thresholds. These QTL locations can be designated as statistically significant for contributing to the variation of TFG. Therefore, the information points to a genomic loci affecting tissue culturability in oil palm.
INTRODUCTION In the current tissue culture production of oil palm planting material, some genotypes are observed to be more amenable to tissue culture than others. The rate of callus formation has been reported to be variable between clones (Ginting and Fatmawati, 1995; Rival et aL, 1997; Wooi, 1995). This variability also extends to time to first callusing (TFC). This was clearly
observed in TFC data collected in the current study where, some of the palms easily formed callus as early as 3 weeks and others took up to 33 weeks to form callus. The development of molecular markers has made it possible to generate a linkage map for individual palm and consequently allows identification ofQTLs based on the framework map. QTL mapping for tissue culture traits
TING NGOOT CHIN ET AL.
have been reported in other crops such as rice (Taguchi-Shiobara et al., 1997) and barley (Mano and Kounatsuda, 2002). This has clearly showed that tissue culture traits can be mapped as QTLs in order to find out significant regions within the genome affecting the variation. Detection of QTLs opens up a fine view of quantitative genetic architecture and provides potential tools in marker-assisted selection. The methodology of QTL detection is somehow laborious and involves complex mathematical calculations. However, computer software packages such as JoinMap and MapQTL have made mapping of QTLs possible for almost every species. Several QTL mapping approaches have been developed such as, Interval Mapping (Lander and Botstein, 1989) and MQM mapping (Jansen, 1993, 1994) which can be used together to improve the mapping resolution. A simple calculation for determining the significant LOD threshold for declaring a QTL was developed by Van Ooijen (1999) based on the result of stochastic simulation of a diploid species with a map density of one marker every lcM. This helps to provide confidence in detection of QTLs and avoid false positives. The objective of this study was to generate additional RFLP and AFLP markers to improve the dura (E L48) and the pisifera (MLl61) linkage maps developed by Chua et al. (2006). The dura linkage map constructed by Chua et al. (2006) consisted of 42 RFLPs and 36 AFLPs while the pisifera linkage map, had 65 RFLPs and 68 AFLPs. The QTL analysis was then carried out by using the updated framework maps to identifY QTLs associated with TFC with the eventual aim to develop diagnostic tools for selection of ortets that are amenable to tissue culture. MATERIALS AND METHODS Plant Materials The mapping material used in this study consisted of 87 F} palms produced by a cross between Ulu Remis Deli dura (E L48) and Yangambi pisifera (MLl61). The oil palm
36
material was supplied by FELDA Agricultural Services Sdn. Bhd. Tissue Culture Response: Time to First Callusing (1FC)
The tissue culture of the F} palms was carried out by the collaboration of seven major laboratories of the oil palm industry namely: Guthrie Biotech Laboratory Sdn. Bhd., 101 Corporation Bhd, United Plantation Bhd, Golden Hope Plantation Bhd., Ebor Laboratories, Applied Agriculture Research Sdn. Bhd. and Malaysian Palm Oil Board (MPOB). The cultures were observed closely for the first callus formation via TFC. The TFC, which is a phenotypic data, was transformed to arctan'" (x+1) for improving the normality of variance prior to QTL analysis. RFLP Analysis RFLP procedures which included D A extraction (Doyle and Doyle, 1990), digestion using various restriction enzymes (BamBI, Bell, BgIlI, DraI, EcoRI, HindIII, HindI, Bst I, Rsal, HaeIII, TaqI and XbaI) , electrophoresis, Southern blot and Southern hybridization were performed as described by Cheah et al. (1993). The oil palm cDNA probes used in the RFLP analysis were generated from different tissues and stages of development such as, young etiolated seedling, mesocarp, kernel, inflorescence, callus and embryoids which were kindly supplied by the MPOB Biological Research Centre (MBRC). AFLP Analysis Two restriction enzyme-combinations: EcoRI/ MseI and PstI/ MseI were used for generating AFLP markers. The experimental assay for EcoRI/ MseI was carried out as described by the manufacturer in the manual (cat no. 10717015 and 10719-011, Invitrogen™ Life Technologies) with some minor modifications as described by Rajinder et al. (1998). For PstI/ MseI enzyme-combination, adapters and primers were synthesized by Invitrogen™ Life Technologies.
PERTANIKAJ. TRap. AGRIC. SCI. VOL. 29 NOS. I & 2 (2006)
MAPPING OF QTLS CO TROLLING THE TIME TO FIRST CALLUSI G I
OIL PALM
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ranging from 7cM to 270cM (Fig. 2). For dura, a map comprising of 23 linkage groups was constructed ranging from12cM to 161cM (Fig.
RFLP and AFLP Linkage Mapping
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A total of 299 and 173 segregating bands were scored from RFLP and AFLP analysis for the pisifera and dura parent, respectively. Linkage maps were constructed with density 395 loci/ 1,714cM for pisifera and 214loci/l,225cM for dura. Correspondingly, the map distances between adjacent markers were 4.34cM and 5.72cM for the pisifera and dura maps, respectively. For pisifera, all the mapped loci were assigned into 16 linkage groups with size
QTLs Associated to
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Using the Interval mapping analysis, three associated QTLs were identified in pisifera (MLl61) and two in dura (ENL48) at the genome-wide significant threshold level calculated according to Van Ooijen (1999) (Table 1). Two highly significant QTLs, FDBI12+EACG/MCTA>330+FDBI20 and CB6F+TAGG/HCTA-550 were identified at
PERTANIKAJ. TROP. AGRIC. SCI. VOL. 29 NOS. 1 & 2 (2006)
39
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