Porphyra tenera Kjellman, widely cultivated in nori farms before the development of artificial seeding, is currently listed as an endangered species in Japan. To confirm whether a wild-collected gametophytic blade was P. tenera or the closely related species P. yezoensis Ueda, morphological observations and molecular analyses were made on the pure line HGT-1 isolated from a wild blade. This pure line was identified as P. tenera based on detailed morphological features. Sequences of the nuclear internal transcribed spacer region 1 and the plastid RUBISCO spacer revealed that P. tenera HGT-1 was clearly different from P. yezoensis f. narawaensis Miura, the main species cultivated in Japan. PCR-RFLP analysis of the internal transcribed spacer region was found to be a convenient method for rapid discrimination between P. tenera and cultivated P. yezoensis. The restriction patterns generated by the endonucleases Dra I and Hae III were useful for differentiating between both gametophytic and conchocelis stages of P. tenera HGT-1 and P. yezoensis f. narawaensis strains. Thus, PCR-RFLP analysis will serve as a valuable tool for rapid species identification of cultivated Porphyra strains, culture collections of Porphyra strains for breeding material and conservation of biodiversity, and, as codominant cleaved amplified polymorphic sequence markers for interspecific hybridization products between P. tenera and P. yezoensis f. narawaensis. Under the same culture conditions, rate of blade length increase and the blade length-to-width ratio were lower in P. tenera HGT-1 than in P. yezoensis f. narawaensis HG-4. The HGT-1 became mature more rapidly than HG-4 and had thinner blades.

We investigated the genetic variations of the samples that were tentatively identified as two cultivated Porphyra species (Porphyra yezoensis Ueda and Porphyra tenera Kjellm.) from various natural populations in Japan using molecular analyses of plastid and nuclear DNA. From PCR-RFLP analyses using nuclear internal transcribed spacer (ITS) rDNA and plastid RUBISCO spacer regions and phylogenetic analyses using plastid rbcL and nuclear ITS-1 rDNA sequences, our samples from natural populations of P. yezoensis and P. tenera showed remarkably higher genetic variations than found in strains that are currently used for cultivation. In addition, it is inferred that our samples contain four wild Porphyra species, and that three of the four species, containing Porphyra kinositae, are closely related to cultivated Porphyra species. Furthermore, our PCR-RFLP and molecular phylogenetic analyses using both the nuclear and plastid DNA demonstrated the occurrence of plastid introgression from P. yezoensis to P. tenera and suggested the possibility of plastid introgression from cultivated P. yezoensis to wild P. yezoensis. These results imply the importance of collecting and establishing more strains of cultivated Porphyra species and related wild species from natural populations as genetic resources for further improvement of cultivated Porphyra strains.

We performed interspecific hybridization in the haploid blade-forming marine species (nori) of the genus Porphyra, which have a heteromorphic life cycle with a haploid gametophytic blade and a diploid microscopic sporophyte called the ''conchocelis phase.'' The green mutant HGT-6 of P. tenera var. tamatsuensis A. Miura was crossed with the wildtype HG-1 of P. yezoensis f. narawaensis A. Miura; the F 1 heterozygous conchocelis developed normally and released numerous conchospores. However, almost all the conchospore germlings did not survive past the four-cell stage or thereabouts, and only a few germlings developed into gametophytic blades. These results indicate that hybrid breakdown occurred during the meiosis, while the surviving F 1 gametophytic blades were considered a breakthrough in the interspecific hybridization of Porphyra. Organelle genomes (cpDNA and mtDNA) were found to be maternally inherited in the interspecific hybridization by molecular analyses of the organelle DNA. In particular, molecular analyses of nuclear DNA revealed that the surviving F 1 blades were allodiploids in the haploid gametophytic phase; however, there is a possibility of the occurrence of rapid chromosomal locus elimination and rearrangement in the F 1 conchocelis phase. Our findings are noteworthy to the breeding of cultivated Porphyra and will provide important information for understanding of the speciation of marine plants with high species diversity.

Detailed morphological observations were made on two strains of cultivated Porphyra : HG-1 (pure line isolated from Dai-1) and Noriken-4 (parental strain of a pure line HG-4) . The two strains were identified as P. yezoensis f. narawaensis based on their macroscopic and microscopic features, such as long linear or oblanceolate blades up to 50 cm in maximum length, division formulae of spermatangia and zygotosporangia, shape of trichogynes and carpogonia, and the second transverse divisional plane formed at the division from c/2 to c/4 in zygotosporangia. Gametophytic blades from two completely homozygous conchocelis strains isolated in this study (HG-1 and HG-4) were cultured under the same conditions and compared to confirm whether the differences in their shapes are genetically determined. The shape of blades from both of conchospores and monospores was always more slender in HG-4 than in HG-1 at the same blade age, suggesting that the difference in the blade shape between the two pure lines is due to genetic variation. To estimate the level of genetic variation the two pure lines were subjected to amplified fragment length polymorphism fingerprint analysis. A total of 230 bands were detected in HG-1 and HG-4 using eight selective primer pairs, and the number of polymorphic bands was only two in HG-1. These results indicate that the two pure lines certainly show genetic variation, which is, however, at an extremely low level. The importance of pure-line breeding and the origin of currently cultivated Porphyra are discussed. This is the first report to identify currently cultivated Porphyra strains in Japan based on combined results of detailed morphological observations and molecular analysis.

We compared the wild Porphyra strain OGATSU from northeastern Japan with cultivated Porphyra yezoensis f. narawaensis using the RuBisCO spacer, rbcL, and ITS-1 DNA sequences as well as early gametophyte development. Based on the molecular analyses and detailed morphological observations, OGATSU was identified as P. yezoensis, but also revealed important differences from the cultivated form. Under the same culture conditions, gametophytic blades of OGATSU produced more archeospores than P. yezoensis f. narawaensis strain HG-4. The length of blades and their length-to-width ratios were significantly lower in OGATSU than in HG-4, and the color of OGATSU blades was darker than that of HG-4. The first lateral cell division in conchospore germlings occurred significantly earlier in the OGATSU strain than in the HG-4 strain, resulting in the rounder shape of the OGATSU blade compared to that of P. yezoensis f. narawaensis. These results suggested that wild strains such as OGATSU can provide useful characters that could enhance cultivated varieties in a careful breeding program.