1. ASSESSMENT OF GENETIC VARIATION IN CACAO CLONES COLLECTION OF
MCB HILIR PERAK USING SIMPLE SEQUENCE REPEAT (SSR) MARKERS
NURAZIAWATI, M.Y.1, ALIAS, A.D.2 , LEA, J.2 and KHAIRUL BARIAH, S.1
1Cocoa Research and Development Center, Malaysian Cocoa Board, P.O.Box 30, Jln. Sg. Dulang, Sg. Sumun, Perak (
2Cocoa Biotechnology Research Centre, Malaysian Cocoa Board, Zon Komersial 1, KKIP Selatan Jalan Norowot, Taman Perindustrian Kota Kinabalu, Sabah
INTRODUCTION
Cocoa, Theobroma cacao (Linnaeus) is a perennial species and belongs to the genus of Theobroma which consists of twenty-two species. The consumption of cacao is worldwide and high on demand. Thus, there is a need to collect many cacao varieties in developing and improving new superior cacao materials. Germplasm collections help conserve the genetic and phenotypic diversity of cacao as a sources in various field of studies. A rational and efficient use of available accessions collection depends largely on the knowledge of the nature and relationships of the genetic diversity present in the collections. There are more than 2000 accessions of local and international through MCB research stations. However, there is inadequate information on their genetic diversity. Such the information need to be identified especially for breeding purpose. The objective of this study was to access the genetic variability in cacao clones collection of MCB Hilir Perak using microsatellite markers.
MATERIALS AND METHOD
DNA extraction, SSR Marker and Polymerase Chain Reaction (PCR) Amplification
Two hundred and eighty-seven (287) accessions were used in the study. Total genomic DNA was extracted from 100mg of fresh leaf sample using DNeasy Plant Mini Kit (Qiagen Ltd, UK) according to manufacturer’s instruction. DNA was then quantified either by fluorimetry or by comparing band intensities with DNA standards after agarose gel electrophoresis.
Five fluorescently labeled SSR primers were tested across all the plant materials. The PCR reaction mixtures consisted of 2µL of 2mM dNTP mix, 2µL of 10x Taq buffer, 1.6µL of 25mM MgCl2, 0.8µL of a stock solution containing both forward and reverse primer (10mM), 1µL containing ng of genomic DNA template, 1µL of 0.5unit/µl Taq DNA polymerase and sterile distilled water for a total volume of 20µL. PCR amplification was performed in a Perkin Elmer Cetus DNA Thermocycler model 2400 (Perkin Elmer, USA) with following profile: 94oC for 4 minutes (initial cycle), followed by 35 cycles of 94oC for 30 seconds, 51oC (annealing temperature of the primer pair) for 1 minute, 72oC for 1 minute; followed by a hold at 72oC for 5 minutes and stored at 4oC indefinitely. The PCR products were then outsourced for fragment analysis.
Data analysis
Data on fragment analysis were prepared by CMDV/MARDI using GeneMapper 5 Software. Genotype data were then used to generate dendogram cluster.
RESULTS AND DISSCUSSION
The cluster analysis result showed that the 287 cacao accessions could be clustered into two main groups with multiple subgroups and the genetic distance is The total of 178 numbers of polymorphic bands were detected from 5 loci. This data indicating that 5 loci can be used in discriminating the genetic variability in cacao and exploited for breeding plans. However, more microsatellite markers need to be used for precise result.
Table 1: List of primer used in fragment analysis
Figure 1: Dendogram of 287 cacao accessions shows the genetic relatedness among the accessions
ACKNOWLEDGEMENT
This work was supported by eScience Fund project No SF0062. The author would like to thank Datin Norhaini Udin, acting of Director General of MCB, for the permission to present this paper. Thanks are also extended to Mr. Haya Ramba, Director of Cocoa Upstream Technology Department for his valuable constructive suggestions and comments. Our sincere appreciation to Dr. Khairun Hisam bin Nasir and his teams of CMDV and also to the first base for their assistance and contribution on this research project.