Comparison of Different Chloroplast DNA (CPDNA) Extraction Methods for Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis of CPDNA Among the Zingiberaceae Family in Malaysia

Chow, Lee Suen (2022) Comparison of Different Chloroplast DNA (CPDNA) Extraction Methods for Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis of CPDNA Among the Zingiberaceae Family in Malaysia. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

The Zingiberaceae or ginger family is one of the most widely cultivated plants in Malaysia. However, the extraction of high concentration and purity of chloroplast DNA (cpDNA) from ginger plants is crucial due to the presence of secondary metabolites. In this study, four cpDNA extraction methods, namely LN method coupled with DNeasy Plant Mini Kit, LN method coupled with CTAB, WS method coupled with DNeasy Plant Mini Kit and WS method coupled with CTAB were compared to extract high concentration and purity of cpDNA from Bentong gingers. The result showed that a high concentration (79.2 ng/μL) and purity (A260/280, 1.89) of cpDNA were obtained using the LN method coupled with CTAB. The LN method coupled with CTAB was then used to extract cpDNA from Tanjung Sepat, Kampung, Tambunan, Bara, Karak, Thai, China, Galangal, Red, Turmeric and Red Button gingers. All of these also exhibited a high concentration of cpDNA with reliable purity. The extracted cpDNA was then used to perform the PCR-RFLP to study the genetic variations among the Zingiberaceae family in Malaysia. In the present study, the trnM-rbcL/HaeIII, rbcL1-rbcL2/HinfI and rbcL1-rbcL2/HaeIII combinations were used for PCR-RFLP analysis. From the result, a genetic variation was observed among the twelve ginger species studied. The result also showed the trnM-rbcL/HaeIII combination was found to be the most informative or potential primer/enzyme combination for determining the genetic variation among ginger species. The restriction banding patterns of Red Button ginger formed were obviously different from other ginger species, indicating it was not closely related to other genera Zingiber, Alpinia and Curcuma. Apart from that, the result also showed that Zingiber, Alpinia and Curcuma were more likely closely related to each other. Overall, the LN method coupled with CTAB was the most optimal cpDNA extraction method, capable to generate high concentration and purity of cpDNA that can use for PCR-RFLP analysis among the Zingiberaceae family. The present study also showed that PCR-RFLP was a useful technique that can use for the analysis of genetic variation