Towards identification of molecular sex markers for Gracilaria changii, (Rhodophyta) a red sea weed

Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia (1963) is one of the most abundant agarophytic seaweeds in Malaysia. G. changii grows abundantly on intertidal mudflats, and as attached epiphytes to mangrove roots, shells, pebbles and fish cages. Gracilaria changii produces high yields of good quality agar with high gel strength. The agar can be used to produce good food grade agar and agarose. Malaysian G. changii has potential commercial application in the agar industry due to its good quality agar and adaptibility to the harsh mangrove conditions. The random amplified polymorphic DNA (RAPD) assay has become a widely used method to identify DNA polymorphisms for genetic analysis in plants and animals as well. RAPD is also used to identify molecular sex markers in plants. Immature Gracilaria is difficult to separate as to sex and life-stage based morphology. Therefore, the objective of this study was to use RAPD techniques to identify genetic or molecular sex markers for sex and life stages. G. changii specimens were collected from two different locations, namely Morib, Selangor and Sungai Pulai, Johor. Intraspecies relationships among G. changii collected from Morib and Sungai Pulai were studied where Caulerpa racemosa and C. lentillifera were used as out groups. Two methods were carried out for DNA extraction: The CTAB and Phenol-Chloroform combination method and the DNA easy Plant Mini Kit. The CTAB and Phenol-Chloroform method gave the best yield of DNA both in quality and quantity and was suitable for larger-sized specimens like G. changii. The RAPD approach was successful in generating individual fingerprints for individual specimens. In the present study, 69 random primers were studied to identify sex in G. changii, and one sex-linked marker was identified using OPA 18 which is a male-specific marker. Of the 69 primers that were screened, only seven primers - OPA7, OPA18, S14, S61, S64, S75 and S76 were selected for interpopulation RAPD analysis for DNA isolated from Morib and Sungai Pulai. In the present study the combination of all data generated by the seven primers was able to develop a cluster which grouped the specimen, into different clades according to their sexes and life stages using the UPGMA method. It showed that RAPD was able to differentiate tetra-sporophytes, females and males.