Molecular Taxonomic Studies Of Gracilaria Changii From Various Locations Using The Random Amplified Polymorphic DNA (Rapid) Technique

Author

Gan Sook Yee

Date

1999

Keyword

Gracilaria changii , seaweeds, molecular taxonomoc studies, random amplified polymorphic DNA(RAPD) technique

Abstract

Malaysia which has abundant Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia has the potential to become a producer of good food  grade agar.   However, for effective utilisation of this agarophytic seaweed, Bird and Van der Meer (1993) emphasised the importance of taxonomic studies in ensuring the correct identification and recognition of this seaweed. Molecular biology has been applied in the approach to study the phylogenetic relationship of the Gracilaria species. In this study, Gracilaria changii samples were collected from six geographical locations, namely, Phuket & Takbai (Thailand) and Ban Merbok, Morib, Carey Island & Sungai Pulai (Malaysia). Although these samples were identified as Gracilaria changii, some morphological differences were observed such as thallus appearance, diameter of thallus and the reproductive stage. The objective of this study is to assess the use of the Random Amplified Polymorphic DNA (RAPD) in characterising these samples. Interspecies relationship between selected Gracilaria species collected from Morib such as Gracilaria changii, Gracilaria edulis, and Gracilaria salicornia were also studied while Sargassum oligocystum was used as an outgroup. In this study, two DNA extraction protocols were carried out: the cetyltrimethylammonium (CTAB) and phenol combination adapted from Sinnappah (1994) and a modification of this protocol (with additional steps of isolating from the residue and a reextraction step). The modified protocol was found to produce higher purity of DNA with better yield. Of the 20 primers screened, four primers: OP A3 (AGTCAGCCAC), OPA10 (GTGATCGCAG), OPA11 (CAATCGCCGT) and OPA13 (CAGCACCCAC) were selected to generate polymorphic band patterns for all the samples under an optimised temperature. The RAPD data analysis was carried out using the Gelcompar Software 4.1 which calculated the matrix of similarities based on the Dice coefficient (So) and dendrograms were generated using the unweighted pair group method using arithmetic averages (UPGMA). All the four primers showed that there were great intrapopulational variations among the Gracilaria changii as well as interpopulational variations. Overall, these primers were unable to form a relationship at the geographical scales studied as there is high intrapopulational variation. There is a possibility of developing individual fingerprints with these primers. However, for larger geographical distance, primer OPA13 can separate the Malaysian Gracilaria changii from the Gracilaria changii collected from Thailand. In the interspecies study, OPA13 can separate Gracilaria changii from both Gracilaria salicornia and Gracilaria edulis while OP All showed that there is a possibility of hybridisation between Gracilaria changii and Gracilaria salicornia or between Gracilaria changii and Gracilaria edulis. Both OP All and OPA13 separate Gracilaria samples from Sargasssum samples.