Vegetative Amplification And Genetic Diversity Analysis Of Laminaria Gametophyte Clones

Laminaria is a group of macroalgae, which are very important. As the development of the cloning technique of Laminaria gametophytes, gametophyte clones can be widely used in germplasm preservation, genetic breeding and seedling raising and in genetic variation analysis.In this study, the effects of the amount of nutrient salts, illumination intensity and time and temperature on the vegetative amplification of Laminaria gametophytes were determined. In addition, the genetic diversity of the gametophyte clones isolated from L. japonica and L. lonngissima was analyzed with RAPD markers and compared each other.The major findings were listed as the following:Instantaneous growth rate of Laminaria gametophyte clones at static cultivation was obviously lower than that at suspension cultivation. Therefore, static cultivation was not appropriate for the large scale vegetative amplification of Laminaria gametophyte clones. However, at a low initial density, the medium containing 6.0ppm N and 1.0ppm P and the replacing frequency of one time each week, static cultivation met the need of laboratory use.At static cultivation, temperature affected the vegetative amplification of Laminaria gametophyte clones obviously. The optimum temperature for the amplification of Laminaria gametophyte clones ranged from 10 to 15℃. It was found that 25℃was lethal to Laminaria gametophyte clones.Laminaria gametophyte clones showed the highest growth rate under an illumination intensity of 4500lx. However, the decrease of cytochrome was observed at such intensity, indicating that Laminaria gametophyte clones were not tolerant to high illumination intensity. It was suggested that the illumination intensity of 2500lx should be used in large scale amplification of Laminaria gametophyte clones, the optimal illumination time is 24h/d.Nineteen random primers were obtained after primary selection. Among 252 loci, 228 were polymorphic. The ratio of polymorphic loci was 90.5%. Some loci showed obvious differentiation between clones with different species origins and genders. The phylogenetic tree constructed based on the genetic distances depicted that Laminaria gametophyte clones isolated from different species and those with different genders can be easily discriminated. RAPD marker was proved to be effective for determining the genetic diversity of Laminaria gametophyte clones.Nei's gene diversity of L .longissima gametophyte clones and Laminaria japonica gametophyte clones were relatively high. There was obvious genetic differentiation between the clones isolated from two species. The variation levels between male and female gametophyte clones within a species and among the whole collection of clones were different obviously. The differentiation between different genders of the gametophyte clones from L. longissima was obviously higher than that of L. japonica. For Laminaria of the same species, female gametophytes have significantly higher genetic diversity than their male analogs.