| In the present work, we analyzed large ribosomal subunit hypervariable region, inter transcribed spacer (ITS) and intergenic spacer (IGS) sequences in Porphyra species, comparing the sequences distinctions and genetic variations, to provide the molecular proof for phylogeny study, germplasm resources investigation, genetic diversity protecting, and strain breeding and improving.The sequences of 5.8S rDNA and ITS region of the wild thalli and the cultivated thalli of Porphyra yezoensis, were amplified, sequenced and analyzed. The template DNA was extracted by alkaline-heating method for PCR amplification. The results were as follows: the 5.8S rDNA and ITS sequences were essentially identical among different resources of P. yezoensis. The length of ITS1 sequences was 337bp to 338bp; the length of 5.8S rDNA was 152 bp. The ITS1 sequences of the wild and the cultivated had the high level homology (99.7%) with the sequences of P. yezoensis (DQ813581) retrieved from GenBank. Meanwhile, the ITS region of three cultivated strains of Porphyra haitanensis thallus, were also amplified, sequenced and analyzed. The results were as follows: the sequences of ITS and 5.8S rDNA were essentially identical among the three strains. The sequences of ITS1 were 331bp to 334bp; the sequenced of ITS2 were 673bp to 681bp. The sequences of ITS had a high level homology with the P. haitanensis (DQ662228) retrieved from GenBank, and the homology was up to 99.5%, while compared with other species of Porphyra, it was lower, only about 50%.The intergenic spacer region (IGS) was first time applied to analyze the genetic variability of Porphyra haitanensis from different populations. The partial IGS sequences of cultivated strains of P. haitanensis thallus, were amplified, sequenced and analyzed. The results of sequence analysis indicated that the partial IGS sequences from the three populations were the external transcribed spacers (ETS) of 3'end of the IGS gene. In the three populations, the length range of IGS sequence was 1085bp to 1100bp and the content of G+C varied from 50.88% to 51.27%. There were 55 variable sites which occupied 5% of ETS sequences approximately. The results of the similarity analysis and the multisequencing alignment of sequences concluded that the partial IGS sequences of the three populations in P. haitanensis existed notable variabilities.The 28S rDNA sequences were first time applied to analyze the phyletic evolution of interspeicies genetic relationship in different Porphyra species. The partial 28S rDNA sequences of three Porphyra species (Porphyra haitanensis, Porphyra yezoensis and Porphyra katadai), were amplified, sequenced and analyzed. The results of sequence analysis indicated that the length range of the obtained sequence in three species was 932bp to 952bp and the content of G+C varied from 54.08% to 55.25%. There were 808 conservative sites and 127 variable sites. It concluded that the partial 28S rDNA sequences of the different populations in Porphyra species existed notable variabilities, which could be distinguished them effectively.The study suggested that 28S rDNA sequences and IGS sequences in different Porphyra species presented conspicuous variabilities, which could be judged the relationship of Porphyra interspecies phyletic evolution, providing the proof for Porphyra identification study. While the ITS sequences and 5.8S rDNA in Porphyra intraspecies had little genetic variation so that they were not available for as the classification markers in intraspecies of Porphyra. The IGS sequences in the different strains of P. haitanensis presented conspicuous variabilities, which could effectively discriminate the different populations of P. haitanensis. Therefore, the IGS sequence could be used as the critical genetic marker in intraspecies of P. haitanensis. Furthermore, it will be a powerful tool for genetic diversity and classification in intraspecies of other Porphyra species. |
