Assessment Of Genetic Diversity Of Selected Laminaria (Phaeophyceae) Cultivars With RAPD And ISSR Marker

CTAB method has been selected for DNA extraction from Laminaria gametophytes in our study, and it is showed that fresh and partial dried gametophytes are lysed in CTAB buffer with grind [2 % cetyltrimethyl ammonium bromide (w/v), 1.4 M NaCl, 100 mM Tris-HCl (pH 8.0) and 20 mM ethylenediaminetetraacetate (EDTA)] could yield ideal result. About 10μg DNA were collected from 0.3 g partial dried gametophytes. The average size of the extracted genomic DNA detected is 23 kb and its purity is 1.6 (A260/A280). Our provided protocols can be applied to other future molecular application, including restriction endonuclease digestion, internal transcribed spacer ITS analysis, and random amplified polymorphism DNA (RAPD) analysis.RAPD (random amplified polymorphism DNA) analysis was applied to germplasm characterization in 33 different selected Laminaria male and female gametophytes. The positional homology of the RAPD analysis using SCAR (sequence characterized applied region) method was successfully conducted. A total of 233 polymorphic loci were obtained from 18 selected primers after screening, of which 27 stable and clear bands were selected to construct a fingerprint map for discrimination of each gametophyte. Seven RAPD markers from five primers were finally determined by a computer program to construct the fingerprint map. Three specific markers closely related with gametophytes were obtained and were converted to gametophytic SCAR markers. One SCAR marker was identified in the genome DNA by Southern blotting. This is the first SCAR marker report on Laminaria germplasm and applicable to cultivars identification. These results demonstrated the feasibility of applying RAPD markers to germplasm characterization in selected Laminaria gametophytes, and can provide a molecular basis for breeding new Laminaria strains.Inter simple sequence repeat (ISSR) analysis was used to assess genetic diversities among the ten pairs of Laminaria gametophytes, each of which including male and female gametophytes. A total of 58 amplification loci were obtained from 10 selected ISSR primers, of which, 34 revealed polymorphism among the gametophytes. Genetic distances calculated with the Dice coefficient ranged from 0.006 to 0.223. The dendrogram, based on UPMGA (unweighted pair-group method arithmetic average) in the program Phylip3.6a3, showed that most male and female gametophytes of the same species could be clustered together, ten pairs of gametophytes were divided into four groups, and were generally consistent with the taxonomic categories. The major group consisted of six pairs of gametophytes, which were selected from Laminaria japonica by intensive inbreeding and artificial hybridization. Four amplified bands from one monomorphic locus of ISSR primer 852 were cloned, and were sequenced for examining the positional homologies of ISSR markers. One specific marker was cloned but was not converted to sequence characterized amplified region (SCAR) marker successfully. These results demonstrated the feasibility of applying ISSR markers to evaluate Laminaria germplasm diversities, and can provide useful genetic information for breeding and selection.