| Porphyra is one of the most important marine algae, which has a global distribution and an important economic value. The output value of Porphyra has occupied the first rank of marine algae in our country. At present, the most Porphyra lines used in produce are collected from natural world and the main taxonomy is based on the morphological characteristics. The classification of Porphyra lines still behinds the times, it can not meet the needs of the production and research of Porphyra. Therefore, the issue of germplasm identification has been a serious limited factor in developing and exploiting the Porphyra resources over the years, In order to reduce the losses caused by using inferior Porphyra lines in practice, it is necessary and urgent to establish a rapid and accurate way for the identification of Porphyra lines. In this study, genetic diversity of 15 filaments of Porphyra representing four groups, Porphyra yezoensis, P Haitanensis, P Katadai var. Hemiphylla and P Oligosperrnatangia, was studied with RAPD (random amplified polymorphic DNA). After screening 8 reproducible amplification patterns were obtained by 8 primers. Among the total of these amplified fragments, 70-97% were polymorphic. Cluster analysis based on the RAPD results was performed. The 15 Porphyra lines were divided into 3 groups. The results were consistent with taxonomy analysis. A DNA fingerprint based on the 8 stable, clear and repeatable bands amplified by RAPD from two primers, OPN-02 and OPJ-18, was constructed. It could be used in Porphyra variety identification. In the fingerprint the RAPD amplification results were scored and converted to computer language expressed with two digits, 1 and 0, which representing the presence(numbered as 1) or absence (numbered as 0) of each band, respectively. Each Porphyra line has its unique fingerprinting pattern and can be easily distinguished from the others by the constructed DNA fingerprint, 10 Porphyra lines from Seed Multiplication Farm of Poiphyra of Jiangsu were identified by this new method, The identified Porphyra lines have been accepted by the related government organization and then become the first group of Porphyra lines to be stored in Porphyra Germplasm Resources Storehouse of China. Later, a software named as PhGI was designed according to this DNA fingerprinting. Eight specific RAPD products were obtained from 8 individual Porphyra lines and all of them have been recovered and cloned. In this study, 8 specific and valuable RAPD markers were obtained, five of them were sequenced, then 5 pairs of SCAR-PCR primers were designed and synthesized according to the 5?terminal nucleotide sequences of the 5 RAPD markers. Through successful optimization of the SCAR-PCR reaction conditions, the RAPD marker of Porphyra line K9401 was converted into a SCAR marker and the RAPD marker of Poq.ihyra line Y9502 was converted into a STS marker. Both specific SCAR marker and STS marker can be directly used for Porphyra identification in practice. It is the first time to apply the method of current molecular marker to alga research, which was used to apply only to plant study. The development of these specific molecular markers is very important for rational and continued utilization and selection of elite Porphyra varieties, and for property protection of Porphyra germplasin. Eight specific RAPD products were obtained from 8 individual Porphyra lines and all of them have been recovered and cloned. In this study, 8 specific and valuable RAPD markers were obtain... |
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