Study On The Genetic Diversity Of Four Species Free-Living Conchocelis Of Porphyra With Isozyme And ISSR-PCR

P. oligospermatangia and 5 cultivars of P. yezoensis free-living conchocelis were done by vertical flat electrophoresis of polyacrylamide gel electrophoresis. Six isozyme patterns were obtained, namely MDH, ME, LDH, GDH, IDH, G-6-PDH. The six isozymes patterns showed the number of isozyme bands was 58. The average portion polymorphic loci of these four species porphyra was 0.655, and which of the 5 cultiv-ars of P. yezoensis was 0.586; every isozyme pattern can dicriminate the eighe samples. Comparability coefficient was calculated by PHILIP sofeware: the comparability coefficient of 5 cultivars of P. yezoensis was 0.7528, and which between P. yezoensis and P. katadai Miura var. hemiphylla, P. oligospermatangia, P. haitanensis was 0.7199, 0.7681 and 0.7598. Cluster analysis based on the isozyme results was performed, divided the eight simples into two group. Four samples of 5 cultivars of P. yezoensis and P. katadai Miura var. hemiphylla were together, Y-9308 and P. oligospermatangia, P. haitanensis were in one group. The experiment on isozymes of P. haitanensis, P. katadai Miura var. hemiphylla,The DNA of the free-living conchocelis of Porphyra was extracted with CTAB (cetyltrimethyl ammonium bromide), which was used to ISSR-PCR research. The yield DNA is approximately 20μg DNA/free-living conchocelis weight . g^-1, and the average size of genomic DNA was about 23Kb estimated by agrose gel electrophoresis. The optimized reactions was composed of 2.5μl PCRbuffer, 2.5 mmol/L MgCl₂ 1.5μl, dNTP (2.5mmol/L) 2μl, 0.2μmol/L ISSR primer, 20ng template DNA, 1.5U Taq DNA polymerase. The optimized programs were predenaturation at 94℃ for 5 min; then 45 cycles containing denaturation 45s at 94℃, annealing 45s at 52℃, and enlongation 2 minat 72 °C; and at last holding 10 min at 72 °C.One hundred ISSR primers were screened, of which 23 polymorphic and informative primers were selected. Total 217 DNA bands were amplified, 201 of which were polymorphic (92.6%). The mean of each primer amplified was 9.34 bands. The amplified bands size were mostly between 150bp-2500bp. Cluster analysis based on the ISSR results was performed, which ordinarily accord with the analysis by isozyme. Among the 5 cultivars of P. yezoensis the genetic distance between Y-9965 and Y-9505 was 0.3897; and the distance between the two samples from Qingdao (Y-9308,Y-2035) was 0.4299; further more, the cultivar come from Japan (Y-0008) had the average genetic distance 0.4469 compared with other four samples. At the same time, DNA profiles based on the ISSR markers had revealed potential fingerprints for various individual cultivars.