Confirmation of cultivated Porphyra tenera (Bangiales, Rhodophyta) by polymerase chain reaction restriction fragment length polymorphism analyses of the plastid and nuclear DNA

Polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) analysis of the plastid ribulose‐1,5‐bisphosphate carboxylase (RuBisCo) spacer region was developed for a more reliable and rapid species identification of cultivated Porphyra in combination with PCR‐RFLP analysis of the nuclear internal transcribed spacer (ITS) region. From the PCR‐RFLP analyses of the plastid and nuclear DNA, we examined seven strains of conchocelis that were used for cultivation as Porphyra tenera Kjellman but without strict species identification. The PCR‐RFLP analyses suggested that two strains, C‐32 and 90‐02, were cultivated P. tenera and that the other five strains, C‐24, C‐28, C‐29, C‐30 and M‐1, were Porphyra yezoensis f. narawaensis Miura. To identify species more accurately and to reveal additional genetic variation, the two strains C‐32 and 90‐02 were further studied by sequencing their RuBisCo spacer and ITS‐1 regions. Although RuBisCo spacer sequences of the two strains were identical to each other, each of their ITS‐1 sequences showed a single substitution. The sequence data again confirmed that the two strains (C‐32 and 90‐02) were cultivated P. tenera, and suggested that the two strains showed some genetic variation. We concluded that PCR‐RFLP analysis of the plastid and nuclear DNA is a powerful tool for reliable and rapid species identification of many strains of cultivated Porphyra in Japan and for the collection of genetically variable breeding material of Porphyra.     

MORPHOLOGICAL AND MOLECULAR ANALYSIS OF THE ENDANGERED SPECIES PORPHYRA TENERA (BANGIALES,RHODOPHYTA)1

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.     

GENETIC DIVERSITY AND INTROGRESSION IN TWO CULTIVATED SPECIES (PORPHYRA YEZOENSIS AND PORPHYRA TENERA) AND CLOSELY RELATED WILD SPECIES OF PORPHYRA (BANGIALES,RHODOPHYTA)1

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.     

Research Note: Simple molecular discrimination of cultivated Porphyra species (Porphyra yezoensis and Porphyra tenera) and related wild species (Bangiales,Rhodophyta)

To discriminate between cultivated Porphyra species (Porphyra yezoensis and Porphyra tenera) and closely related wild Porphyra species, we developed a polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) analysis of the rbcL gene using five restriction enzymes. Although our previous PCR‐RFLP analyses of internal transcribed spacer (ITS) rDNA and plastid RuBisCO spacer regions could not always discriminate wild P. yezoensis, wild P. tenera, and closely related wild species, the PCR‐RFLP profiles of the rbcL gene were useful in discriminating samples collected from natural habitats. Therefore, PCR‐RFLP analysis of the rbcL gene will help in the simple identification of a large number of samples, not only for the establishment of reliable cultures as breeding material, but also for the taxonomic investigations of species that are closely related to cultivated Porphyra.     

Rapid DNA extraction from conchocelis and ITS-1 rDNA sequences of seven strains of cultivated Porphyra yezoensis (Bangiales, Rhodophyta)

In order to extract DNA rapidly from cultivated Porphyra, we extracted total DNA from conchocelis using the ISOPLANT II kit (Nippon Gene) without liquid nitrogen treatment or CsCl-gradient ultracentrifugation. By confirming the reproducibility of RAPD patterns, it is concluded that the quality of the extracted DNA is sufficient to use as a template for molecular investigation. Using this rapid method, the nuclear ribosomal DNA of the internal transcribed spacer (ITS) regions was amplified from seven strains of cultivated Porphyra, which had been maintained as free-living conchocelis by subculturing in the laboratory. From the amplified DNAs, the ITS-1 sequences were determined in order to identify the species and genetic relationship of the strains. The sequences were identical in the seven strains, and all the strains were identified as P. yezoensis. Furthermore, the gametophytic blades of these strains showed long linear or oblanceolate shapes in the laboratory culture. It was concluded that these strains are P. yezoensis form. narawaensis. This rapid DNA extraction method from conchocelis will be a powerful tool for phylogenetic analysis and for genetic improvement of cultivated Porphyra.     

INTERSPECIFIC HYBRIDIZATION IN THE HAPLOID BLADE‐FORMING MARINE CROP PORPHYRA (BANGIALES,RHODOPHYTA): OCCURRENCE OF ALLODIPLOIDY IN SURVIVING F1 GAMETOPHYTIC BLADES1

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 F₁ 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 F₁ 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 F₁ 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 F₁ 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.     

Molecular features of a defined genetic marker for the determination of the Porphyra tenera lineage

Nucleotide sequences of the nuclear SSU rDNA and ITS1 are presented as a defined genetic marker for Porphyra tenera as a species. Exon nucleotide sequences were identical within all the P. tenera specimens. Intron nucleotide sequences varied between populations. The introns and ITS1 variations are presented as defined genetic markers to establish the Porphyra tenera strains. Wild-collected thalli identified by morphological systematics, from five populations of Porphyra tenera throughout Japan, were discriminated by comparing sequences of the various regions utilizing the results of this and previous studies.     

Comparative study of wild and cultivated <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Bangiales,Rhodophyta) based on molecular and morphological data

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.     

Development of an expression system using the heat shock protein 70 promoter in the red macroalga, Porphyra tenera

Porphyra is a commercially valuable source of food and drugs and an important model organism for algal research. However, genetic research on Porphyra tenera has been limited by a lack of a heterologous gene expression system. In this study, we isolated native promoter PtHSP70 for the efficient expression of foreign genes in this organism. This promoter lies approximately 1 kb upstream of the heat shock protein 70 coding sequence and was isolated using adapter ligation-mediated genomic polymerase chain reaction. Promoter activity was evaluated using the synthetic GUS gene (PyGUS) with optimized codons for Porphyra yezoensis. Interestingly, the PtHSP70 promoter allowed the efficient expression of PyGUS in P. tenera and P. yezoensis, whereas the PyGAPDH promoter from P. yezoensis was not fully functional in P. tenera. The PtHSP70 promoter may have a more conserved regulatory mechanism than the PyGAPDH promoter between these species, suggesting that PtHSP70 could serve as a universal promoter for Porphyra species. We also established an efficient transient transformation system for P. tenera by evaluating transformation parameters including gold particle quantity, helium and vacuum pressure, developmental stages of leafy gametophytes, and target distance. Under optimal conditions of transient transformation, the frequency of GUS expression was determined by histochemical staining as 30–50 cells per bombardment. In addition, PyGUS expression was detected during the regeneration of monospores in P. tenera, indicating successful genetic transformation. Therefore, the new transient transformation system using the PtHSP70 promoter can be used for foreign gene expression in P. tenera, which may advance the development of P. tenera as a model organism.     

Rapid discrimination of <Emphasis Type="Italic">Porphyra tenera</Emphasis> Kjellman var. <Emphasis Type="Italic">tamatsuensis</Emphasis> Miura by PCR-RFLP

To allow to discriminate rapidly the strains of Porphyra tenera var. tamatsuensis, cultivars of which grow more vigorously than strains of P. tenera var. tenera, strains of both varieties were examined by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis using mitochondrial DNA related to the ATP synthase F0 subunit 6 (ATP6) gene. The lengths of all sequences in this region of three strains of each variety were 670 bp and had just a single nucleotide substitution. Digestion with the restriction enzyme of TaaI yielded three visible bands which appeared in the three strains of P. tenera var. tamatsuensis, whereas two bands appeared in the three strains of P. tenera var. tenera. We therefore conclude that PCR-RFLP analysis is a valuable tool for discrimination of P. tenera var. tamatsuensis among the stock of P. tenera strains used for mariculture.     

C55 bacteriocin produced by ETB‐plasmid positive Staphylococcus aureus strains is a key factor for competition with S. aureus strains

Exfoliative toxin (ET) produced by Staphylococcus aureus is closely associated with the onset of bullous impetigo. To date, three ETs (ETA, ETB and ETD) have been identified. The gene encoding ETB is located in a plasmid designated pETB. Bacteriocin synthesis genes are also located in this plasmid and pETB‐positive strains reportedly produce the C55 bacteriocin. In this study, the antibacterial activity against S. aureus strains of the bacteriocin produced by the pETB‐positive strain TY4 was investigated. This bacteriocin demonstrated antibacterial activity against all pETB‐negative but not pETB‐positive strains, including TY4. Additionally, a TY4? strain from which the pETB plasmid had been deleted exhibited susceptibility to the bacteriocin. Further experiments revealed that two immunity factors (orf 46‐47 and orf 48) downstream of the bacteriocin synthesis genes in the pETB plasmid are associated with immunity against the bacteriocin produced by TY4. The TY4? with orf46‐47 strain exhibited complete resistance to bacteriocin, whereas the TY4? with orf48 strain exhibited partial resistance. Whether bacteriocin affects the proportion of each strain when co‐cultured with S. aureus strains was also investigated. When TY4 or TY4? was co‐cultured with 209P strain, which is susceptible to the bacteriocin, the proportion of 209P co‐cultured with TY4 was significantly less than when 209P was co‐cultured with TY4?, whereas the proportion of TY4? with orf46‐48 co‐cultured with TY4 was greater than with TY4?. These results suggest that the C55 bacteriocin produced by pETB‐positive strains affects the proportion of each strain when pETB‐positive and ‐negative strains co‐exist.

     

Red rot resistance in interspecific protoplast fusion product progeny of Porphyra yezoensis and P. tenuipedalis (Bangiales, Rhodophyta)

Nine primary regenerants were recovered by interspecific protoplast fusion of Porphyra yezoensis Ueda T‐14 (Py) (cultivated Porphyra) and Porphyra tenuipedalis Miura (Pt). This combination is difficult to achieve with conventional sexual hybridization, yet is important in that non‐cultivated P. tenuipedalis is partially resistant (PR) to red rot disease, caused by the microbial pathogen, Pythium porphyrae Takahashi et Sasaki. Out of the nine primary regenerants, two strains (Py‐Pt‐4 and Py‐Pt‐7) were like the parent, P. tenuipedalis, while the rest were like the other cultivated parent P. yezoensis T‐14 in their life cycle. Red rot resistance was assessed in parents and interspecific fusion product progeny (FPP) by exposing the foliose thalli to equivalent infection and measuring two parameters of the host‐pathogen interactions: supported fungal biomass and amount of disease produced. Intermediate resistance between P. yezoensis T‐14 (1.00) and P. tenuipedalis (0.13) was observed in two of the Py‐type FPP, Py‐Pt‐2F₂ (0.25) and Py‐Pt‐5F₂ (0.23). Stable inheritance of resistance was observed through two subsequent generations. The morphologic and reproductive characteristics of the regenerated foliose thalli, and nature of host‐pathogen interactions were used to further verify the hybrid origin of the FPP. Host‐pathogen interactions were followed using epi‐fluorescence and scanning electron microscopy (SEM). The zoospores encysted at higher rates on the susceptible cultivated parent (P. yezoensis T‐14) germinated immediately and the short germ tubes formed appres‐soria and penetrated the algal cells near the site of encystment. While on the PR parental (P. tenuipedalis) and partially resistant FPP (PRFPP) progeny (Py‐Pt‐2F₂ and Py‐Pt‐5F₂) the low rate of zoospore encystment was followed by cyst germination, but only a few of the germ tubes formed appressoria and penetrated the thallus surface. Long germ tubes (with no appressoria) were seen growing on the thallus surface without host penetration. The minimal rate of encystment concomitant with low rate of appressorium formation on the PR parent and PRFPP was observed as the major factor responsible for the partial resistance in these thalli.     

Cryptic species in the Pyropia yezoensis complex (Bangiales,Rhodophyta): Sympatric occurrence of two cryptic species even on same rocks

In a previous study on wild populations of Pyropia, the occurrence of two possible new species (Pyropia sp. 2 and Pyropia sp. 3) which are closely related to the two commercially important Pyropia species, P. yezoensis and P. tenera, was confirmed as the result of molecular phylogenetic analyses. To characterize the morphological features of the two wild Pyropia species, we collected Pyropia blades in a natural population in which Pyropia sp. 3 was known to occur, and carried out molecular identification before detailed morphological observations. Through the molecular identification we found, unexpectedly, that Pyropia sp. 2 blades grew sympatrically in the same site. Therefore, after molecular identification, we examined in detail the external morphology and anatomy of the two wild Pyropia species using more than 10 blades each. As a result, it is concluded that all of the blades of the two species are morphologically identical to P. yezoensis, but distinct from P. tenera. It is therefore considered that both of the two wild Pyropia species are cryptic species within the P. yezoensis complex. Furthermore, this study revealed that the two cryptic species grew sympatrically, even on the same rocks within the natural habitat.     

Podosphaera pannosa (syn. Sphaerotheca pannosa) on Rosa and Prunus spp.: Characterization of Pathotypes by Differential Plant Reactions and ITS Sequences

The powdery mildew fungus Podosphaera pannosa (Wallr.: Fr.) de Bary (syn. Sphaerotheca pannosa) is a major problem on roses worldwide. Twenty‐six monoconidial isolates of Podosphaera collected on roses and Prunus spp. in Belgium, Germany, France, Denmark, Israel and The Netherlands were characterized on the basis of differential reactions on in vitro rose genotypes and Prunus avium L. and by DNA sequence analysis of the rDNA ITS (internal transcribed spacer) region. Twenty‐four isolates were determined as P. pannosa. Amongst these, different groups could be distinguished. A first group of 18 isolates was highly virulent on rose and avirulent or very weakly virulent on P. avium. A second group of four isolates was highly virulent on both rose and P. avium. Analysis of the ITS sequence could discriminate these two groups of P. pannosa strains by a one base pair difference. Finally, two isolates of powdery mildew collected on Prunus sp. could be classified as P. pannosa based on their ITS sequence, which was identical to the ITS sequence of the isolates only highly virulent on roses. However, these two isolates were not able to infect roses. These results indicate that different strains of P. pannosa exist with varying host specificity. We demonstrated by ITS sequencing and plant reactions that the host range of P. pannosa comprises roses and Prunus spp.     

The determination of multiple microsatellite genotypes and DNA sequences from a single pollen grain

Pollen plays important roles in the reproduction and gene flow of flowering plants, and its haploid DNA sequence provides useful information for studies of plant evolution and genealogy. We describe a new method for multiple microsatellite genotyping and DNA sequencing from a single pollen grain. The haploid DNA was extracted from a single pollen grain by using a simple DNA extraction method, and multiple microsatellite genotypes and DNA sequences of multiple chloroplast loci were determined. Using nine pairs of microsatellite primers, more than 90% of genotypes were successfully determined, and 71% and 100% of DNA sequences were determined at two chloroplast DNA loci, the trnL intron region and the trnL/trnF intergenic spacer region, respectively. This simple method of genetic analysis for a single pollen grain will facilitate detailed study of pollination, evolution and genealogy.     

Morphological and AFLP variation of Porphyra yezoensis Ueda form, narawaensis Miura (Bangiales, Rhodophyta)

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.     

INTER‐ AND INTRASPECIFIC COMMUNITY STRUCTURE WITHIN THE DIATOM GENUS PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE)1

Pseudo‐nitzschia‐specific PCR primers (PnAll F/R) were designed to amplify a polymorphic region of the internal transcribed spacer 1 (ITS1) from at least 11 Pseudo‐nitzschia species. The primers were used to generate environmental clone libraries from Puget Sound, Washington, and Vancouver Island, British Columbia, to confirm that the primers were specific for Pseudo‐nitzschia and to determine the extent of ITS1 sequence diversity within individual species. All environmental ITS1 sequences generated with PnAll primers displayed the greatest similarity to known Pseudo‐nitzschia ITS1 sequences. The length of cloned ITS1 fragments differed among species but was conserved within a species. Intraspecific genotypes exhibited <3% sequence divergence for seven of the 10 species detected in clone libraries. Several ITS1 genotypes unique to the Pacific Northwest were identified in environmental samples, and other genotypes were more broadly distributed. The Pseudo‐nitzschia primers were also used to develop an automated ribosomal intergenic spacer analysis (ARISA) to rapidly identify Pseudo‐nitzschia species in environmental samples based on species‐specific variation in the length of the targeted ITS1 region. The ARISA peaks were then associated with the environmental clone sequences for Pseudo‐nitzschia species. Surveying the genetic composition of communities at both the inter‐ and intraspecific levels will enhance our understanding of Pseudo‐nitzschia bloom dynamics.