Evaluation of Three Extraction Methods for DNA Quantification and PCR Detection in Cocoa-Derived Products. Assessment of Genetic Diversity of the Main Fourteen Cocoa Varieties in Vietnam using Non-coding c/d sequences Lam Thi Viet Ha ab, Tran Nhan Dung b, Ha Thanh Toan b, Koen Dewettinck a and Kathy Messens a a Ghent University, b Cantho University INTRODUCTION METHODS MATERIALS: Cocoa-derived products: cocoa leaves, cocoa beans, cocoa powder, cocoa butter, cocoa mass, and dark chocolate. - CTAB-SDS ( Dung et al., 2011) - DNeasy Plant kit with and without polyvinyl polypyrrolidone (PVPP) - The primer pair: plant c (5’-CGA AAT CGG TAG ACG CTA CG-3’) and plant d (5’-GGG GAT AGA GGG ACT TGA AC-3’) (Taberlet et al., 1991). -The primers: 5S I (5’-TTT AGT GCT GGT ATG ATC GC-3’) and 5S II (5’-TGG GAA GTC CTC GTG TTG CA-3’); SSP III (5’-GGC AAT TTA CTT CGT GAC  AAA CG-3’) and SSP IV (5’-CTC ATA TTT GCC AGG AGA ATT AC-3’) (Petiard and Crouzillat, 2004) -The non-coding regions c/d trnL (UAA) exon (Taberlet et al., 1991) -PCR reaction/ polyacrylamide gel/ encode bands by into binary matrix / the dendrogram is detected using the program NTSYS-PC 2.1/ clusteranalysis by unweighted pair group method for arithmetic mean (UPGMA). Faculty of Bioscience Engineering – Laboratory of Food Technology and Engineering Contact person: Lam Thi Viet Ha ( ) Coupure links 653, 9000 Ghent, Belgium, tel , fax CONCLUSIONS 1. DNeasy Plant kit and the CTAB-SDS methods seemed appropriate for DNA purification from cocoa leaves, cocoa powder and cocoa mass. The CTAB-SDS procedure is mostly effective for DNA extraction from cocoa beans and dark chocolate However, in all 3 examined procedures, the lowest DNA yield was obtained from the cocoa butter matrix. The genetic clustering of 14 cocoa varieties showed genetic differences ranging from 0 to 41% At around 59% similarity, fourteen TD clones could be divided into three groups: A, B, and C. Group A (TD1, TD2, TD3, TD5, TD6, TD7, TD8, TD9, TD11, TD12), in group B (TD10 and TD13), and group C (TD14 and TD15). Regarding homologous relationship within members of a group, B adopted 80%, while A and C had approximately 76.4% and 73% respectively RESULTS 1. OD measurem ent 2. PCR reaction CTAB- SDS procedure DNeasy Plant kit DNeasy Plant kit with PVPP +, positive amplification -, negative amplification Table 2. Comparison of DNA yield, purity and PCR results for cocoa leaves and cocoa beans isolated with different methods SampleExtraction method DNA concentration (ng/µL) A260/280 rangePCR amplification Plant c/d5S I/IISSP III/IV Cocoa leaves Cocoa beans Cocoa powder Cocoa butter Cocoa mass Dark chocolate DNeasy Plant kit DNeasy Plant kit with PVPP CTAB-SDS DNeasy Plant kit DNeasy Plant kit with PVPP CTAB-SDS DNeasy Plant kit DNeasy Plant kit with PVPP CTAB-SDS DNeasy Plant kit DNeasy Plant kit with PVPP CTAB-SDS DNeasy Plant kit DNeasy Plant kit with PVPP CTAB-SDS DNeasy Plant kit DNeasy Plant kit with PVPP CTAB-SDS ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± < < < Several studies have reported the efficiency of using cocoa leaf material for the extraction of DNA. These different authors proved the efficiency of DNA extraction methods for cocoa studies and most of these methods were used for cocoa leaf tissues. Thus, to the best of our knowledge, alternative methods for extracting DNA from different cocoa-derived products have yet to be published. In this research, the DNA of cocoa-derived products was extracted with three different DNA extraction procedures, such as the commercial kits DNeasy Plant kit and DNeasy Plant kit with polyvinyl polypyrrolidone (PVPP), and CTAB (cetyltrimethyl- ammonium bromide) based protocol CTAB-SDS. Three PCR primer pairs were selected to detect the presence of specific genes in cocoa derived products and to target a fragment of the chloroplast DNA, the 5S rDNA and the Seed Storage Protein (SSP) (Taberlet et al., 1992; Petilliard & Crouzillat, 2004). The efficiency of these methods was evaluated by the purity, quantity, and amplification of the extracted DNA. The 2 nd part focused on clarifying the genetic diversity of 14 cocoa clones in Vietnam. Primers non coding c/ d were used to analyze the genetic relationship. This result contributed important signification for Vietnamese Cocoa breeding and selection cultivar activities. Table 1. Overview of the collected samples Fig. 1. Dendrogram of the main fourteen cocoa clones in southern Vietnam using non-coding c/d regions