Cloning and Analysis of Calmodulin Gene from Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensiss,cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exous and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca2+ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.     

Cloning and Analysis of Calmodulin Gene from Porphyra yezoensis Ueda （Bangiales, Rhodophyta）

In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensis, cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exons and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca²⁺ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.     

Use of rapd for detecting and identifyingPorphyra (Bangiales, Rhodophyta)

Randomly amplified polymorphic DNA (RAPD) technique was applied to assess the genetic variations and phylogenetic relationships in 4 species ofPorphyra. The samples were collected from the coast of Canada, Vietnam, Zhoushan, Fujian and Qingdao in China. Amplifications with 20 primers were carried out under predetermined optimal reaction conditions (samples were first heated at 94°C for 5 min. and followed by 45 cycles of 1 min at 94°C, 1 min at 36°C, and 2 min at 72°C, then held at 72°C for 10 min). The amplified products were scored as present (1) or absent (0) for each DNA sample and an index of genetic similarity (F) was calculated by using Nei & Li’s matching coefficient method (1979). The value of (1−F) was used to quantify the genetic distances between species and construct a phylogenetic tree. The relationship indicated by the UPGMA and NJ cluster analysis on the values of the genetic distance is in good overall agreement with classical taxonomy. The obvious differences between natural and cultivated population ofP. haitanensi suggest that variation or hybridization with other species occurred during the culture. Contribution No. 100 from the Experimental Marine Biology Laboratory, IOCAS. Contribution No. 3289 from the Institute of Oceanology, Chinese Academy of Sciences. The research supported by the Foundation of the Chinese Academy of Sciences.     

Development of suspended conchocelis ofPorphyra haitanensis (Bangiales, Rhodophyta)

This Mar. 1993 to Aug. 1994 study on suspended conchocelis ofPorphyra haitanensis showed that there were three patterns for development of vegetative filaments: filaments to filaments by “budding”; filaments to sporangial branchlets by “budding”, or cell swelling. There were also three patterms for sporangial branchlet development: vegetatively propagating, changing into conchospores, or dying. Each developmental stage had one or more different developmental directions between vegetative filaments and sporangial branchlets. Developments from conchosporangial branchlets to conchospores were sequential and irreversible. Although sporangial branchlets formed at 29°C could give rise to filaments, they could not propagate as healthily under the same conditions as those formed at 25°C did. Probably the crucial period of plant cell differentiation is in the late stage of sporangial branchlets. In line with the developmental directions of different stages, the authors regulated the development of conchocelis to get ideal different developmental stages materials to obtain very developmentally homogeneous stages, including filaments and sporangial branchlets. Contribution No 3283 from the Institute of Oceanology, Chinese Academy of Sciences. Study supported by the Climbing Project B PD-B6-4-2 of the State Science and Technology Commission of China.     

Identification and characterization of a Dna J gene from red alga Pyropia yezoensis(Bangiales,Rhodophyta)

Members of the Dna J family are proteins that play a pivotal role in various cellular processes, such as protein folding, protein transport and cellular responses to stress. In the present study, we identified and characterized the full-length Dna J c DNA sequence from expressed sequence tags of Pyropia yezoensis( Py Dna J) via rapid identification of c DNA ends. This c DNA encoded a protein of 429 amino acids, which shared high sequence similarity with other identified Dna J proteins, such as a heat shock protein 40/Dna J from Pyropia haitanensis. The relative m RNA expression level of Py Dna J was investigated using real-time PCR to determine its specific expression during the algal life cycle and during desiccation. The relative m RNA expression level in sporophytes was higher than that in gametophytes and significantly increased during the whole desiccation process. These results indicate that Py Dna J is an authentic member of the Dna J family in plants and red algae and might play a pivotal role in mitigating damage to P. yezoensis during desiccation.     

Complete nuclear ribosomal DNA sequence amplification and molecular analyses of B angia(Bangiales,Rhodophyta) from China

Filamentous Bangia,which are distributed extensively throughout the world,have simple and similar morphological characteristics.Scientists can classify these organisms using molecular markers in combination with morphology.We successfully sequenced the complete nuclear ribosomal DNA.approximately 13 kb in length,from a marine Bangia population.We further analyzed the small subunit ribosomal DNA gene(nrSSU) and the internal transcribed spacer(ITS) sequence regions along with nine other marine,and two freshwater Bangia samples from China.Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences(0-0.003;0-0.006,respectively) from each other,but high divergences(0.123-0.126;0.198,respectively) from freshwater samples.An exception is the marine sample collected from Weihai,which shows high divergence from both other marine samples(0.063-0.065;0.129,respectively) and the freshwater samples(0.097;0.120,respectively).A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades,with the two marine sample clades containing Bangia spp.from North America,Europe,Asia,and Australia;and one freshwater clade,containing Bangia atropurpurea from North America and China.     

Characterization of Pythium chondricola associated with red rot disease of Pyropia yezoensis (Ueda) (Bangiales,Rhodophyta) from Lianyungang,China

Pyropia yezoensis(formerly Porphyra yezoensis) is an economically important red alga that is cultured extensively in China. The red rot disease occurs commonly during Pyropia cultivation,causing serious economic losses. An incidence of red rot disease was found in a P. yezoensis farm from mid-November to mid-December 2015 at Lianyungang, Jiangsu Province, China. Histopathological examination revealed that the naturally infected thalli were infected apparently by a pathogen, leading to red rot symptoms. The causative agent was isolated and identified as the oomycete Pythium chondricola by morphological analysis and sequence analysis of the internal transcribed spacer and cytochrome oxidase subunit 1(cox1). In artificial infection experiments on the P. yezoensis blades, the P. chondricola isolate was able to cause the same characteristic histopathology seen in natural infections. P. chondricola grew well at a wide range of temperatures in the range 8-31℃, salinities at 0-45 and pH 5-9. In an orthogonal test used to determine the effects of environmental factors(temperature, salinity, and zoospore concentration)on infection, the data revealed that temperature was the most important factor to affect red rot disease development, with the optimal conditions for disease expansion being 20℃, 35 salinity, and a zoospore concentration of 10~6 zoospores/mL. The results obtained from the present study prompted us to set up a comprehensive epidemiological study on Pyropia, which will provide support to maintain the healthy development of the Pyropia industry in China.     

Phylogenetic,evolutionary, and biogeographic origin of the genus Sheathia (Batrachospermales,Rhodophyta)

Journal of Oceanology and Limnology - The genus Sheathia consists of over 20 species primarily distributed in Asia, Europe, Oceania, North America, and Africa. However, the origin and evolution of...     

Construction of high-density genetic linkage map of Pyropia yezoensis(Bangiales,Rhodophyta) and identification of red color trait QTLs in the thalli

Pyropia yezoensis is an important macroalga because of its extensive global distribution and economic importance. Color is an important trait in the thalli of P. yezoensis, it is also an effective marker to identify the hybridization in genetic breeding. In this study, a high-density genetic linkage map was constructed based on high-throughput single nucleotide polymorphism(SNP) markers, and used for analyzing the quantitative trait loci(QTLs) of red color trait in the thalli of P. yezoensis. The conchospore undergoes meiosis to develop into an ordered tetrad, and each cell has a haploid phenotype and can grow into a single individual. Based on this theory, F1 haploid population was used as the mapping population. The map included 531 SNP markers, 394.57 cM long on average distance of 0.74 cM. Collinear analysis of the genetic linkage map and the physical map indicated that the coverage between the two maps was 79.42%. Furthermore, QTL mapping identified six QTLs for the chromosomal regions associated with the red color trait of the thalli. The value of phenotypic variance explained(PVE) by an individual QTL ranged from 4.71%–63.11%. And QTL qRed-1-1, with a PVE of 63.11%, was considered the major QTL. Thus, these data may provide a platform for gene and QTL fine mapping, and marker-assisted breeding in P. yezoensis in the future.     

Development of organelle single nucleotide polymorphism(SNP) markers and their application for the identification of cytoplasmic inheritance patterns in Pyropia yezoensis(Bangiales,Rhodophyta)

The genus Pyropia contains several important cultivated species.Genetic research in nori species has mainly focused on the cell nucleus,with few studies on organelles(chloroplast and mitocho ndria).Due to the high copy numbers of organelles in cells,which influence the development and traits of algae,it is necessary to study their genetic mechanism.In this study,the marine red alga yezoensis,an important economic macroalga,was selected as the study object.To investigate organelle(chloroplast and mitochondria) inheritance in P yezoensis,the wild type RZ(maternal strain) was cro ssed with the red mutant HT(paternal strain) and 30 color-sectors from 11 F1 gametophytic blades were examined.The complete chloroplast and mitochondrial genomes of the red mutant(HT) were assembled for the first time.One reliable and stable single nucleotide polymorphism(SNP) loci filtrated by bioinformatics analysis was used as a molecular marker for chloroplast and mitochondrial DNA,respectively,in subsequent experiments.PCR amplification and sequence analysis showed that the haplotypes of color-sectors detected were consistent with those of the maternal parent,confirming that both chloroplast and mitochondrial genomes were inherited maternally in P.yezoensis.The inheritance pattern of organelles in P.yezoensis can be used to guide the hybridization and breeding of nori.Additionally,the organelle SNP markers developed in this study can be applied in subsequent genetic research.     

Effects of elevated CO2 on the photosynthesis and nitrate reductase activity of Pyropia haitanensis (Bangiales,Rhodophyta) grown at different nutrient levels

Pyropia haitanensis,a commercially important species,was cultured at two CO2 concentrations(390×10- 6 and 700×10-6(parts per million)) and at low and high nutrient levels,to explore the effect of elevated CO2 on the species under nutrient enrichment. Results show that in CO2-enriched thalli,relative growth rate(RGR) was enhanced under nutrient enrichment. Elevated CO2 decreased phycobiliprotein(PB) contents,but increased the contents of soluble carbohydrates. Nutrient enrichment increased the contents of chlorophyll a(Chl a) and PB,while soluble carbohydrate content decreased. CO2 enrichment enhanced the relative maximum electronic transport rate and light saturation point. In nutrient-enriched thalli the activity of nitrate reductase(NRA) increased under elevated CO2. An instantaneous pH change in seawater(from 8.1 to 9.6) resulted in reduction of NRA,and the thalli grown under both elevated CO2 and nutrient enrichment exhibited less pronounced reduction than in algae grown at the ambient CO2. The thermal optima of NRA under elevated CO2 and/or nutrient enrichment shifted to a lower temperature(10–15°C) compared to that in ambient conditions(20°C). We propose that accelerated photosynthesis could result in growth increment. Nassimilation remained high in acidified seawater and refl ected increased temperature sensitivity in response to elevated CO2 and eutrophication.     

Phylogeny of subclass Scuticociliatia (Protozoa, Ciliophora) using combined data inferred from genetic, morphological, and morphogenetic evidence

Gene sequence-based genealogies of scuticociliates are different from those produced by morphological analyses. For this reason, 11 representative scuticociliates and two ambiguously related genera were chosen to test the ability of combined phylogenetic analyses using both gene sequences and morphological/morphogenetic characteristics. Analyses of both the SSrRNA gene sequences and the combined datasets revealed a consistent branching pattern. While the terminal branches and the order level relationships were generally well resolved, the family level relationships remain unresolved. However, two other trees based on ITS1-5.8S-ITS2 region sequences and morphological/morphogenetic characters showed limited information, due to a lack of informative sites in these two datasets. Our data suggest, however, that the combined analysis of morphological/morphogenetic characters and gene sequences did produce some changes to the phylogenetic estimates of this group.     

Identification and characterization of a DnaJ gene from red alga <Emphasis Type="Italic">Pyropia yezoensis</Emphasis> (Bangiales,Rhodophyta)

Members of the DnaJ family are proteins that play a pivotal role in various cellular processes, such as protein folding, protein transport and cellular responses to stress. In the present study, we identified and characterized the full-length DnaJ cDNA sequence from expressed sequence tags of Pyropia yezoensis (PyDnaJ) via rapid identification of cDNA ends. This cDNA encoded a protein of 429 amino acids, which shared high sequence similarity with other identified DnaJ proteins, such as a heat shock protein 40/DnaJ from Pyropia haitanensis. The relative mRNA expression level of PyDnaJ was investigated using real-time PCR to determine its specific expression during the algal life cycle and during desiccation. The relative mRNA expression level in sporophytes was higher than that in gametophytes and significantly increased during the whole desiccation process. These results indicate that PyDnaJ is an authentic member of the DnaJ family in plants and red algae and might play a pivotal role in mitigating damage to P. yezoensis during desiccation.     

Complete nuclear ribosomal DNA sequence amplification and molecular analyses of <Emphasis Type="Italic">Bangia</Emphasis> (Bangiales,Rhodophyta) from China

Filamentous Bangia, which are distributed extensively throughout the world, have simple and similar morphological characteristics. Scientists can classify these organisms using molecular markers in combination with morphology. We successfully sequenced the complete nuclear ribosomal DNA, approximately 13 kb in length, from a marine Bangia population. We further analyzed the small subunit ribosomal DNA gene (nrSSU) and the internal transcribed spacer (ITS) sequence regions along with nine other marine, and two freshwater Bangia samples from China. Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences (00.003; 0–0.006, respectively) from each other, but high divergences (0.123–0.126; 0.198, respectively) from freshwater samples. An exception is the marine sample collected from Weihai, which shows high divergence from both other marine samples (0.063–0.065; 0.129, respectively) and the freshwater samples (0.097; 0.120, respectively). A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades, with the two marine sample clades containing Bangia spp. from North America, Europe, Asia, and Australia; and one freshwater clade, containing Bangia atropurpurea from North America and China.     

Tsengiella,a new genus of delesseriaceae—(Rhodophyta) from China

A medium-sized delesseriaceous red alga has been collected from the subtidal area of the northern Huanghai Sea coast. The characteristics of the specimens precludes their being placed in any genus defined by the currently accepted criteria.Tsengiella gen. nov., withT. spinosa sp. nov., as the type and only known species, is created and allied with theHypoglossum group of the Delesserioideae (Delesseriaceae, Ceramiales, Rhodophyta). Contribution No. 1286 Academia Sinica Institute of Oceanology, Qingdao.     

The first complete organellar genomes of an Antarctic red alga,Pyropia endiviifolia:insights into its genome architecture and phylogenetic position within genus Pyropia(Bangiales,Rhodophyta)

Pyropia species grow in the intertidal zone and are cold-water adapted. To date, most of the information about the whole plastid and mitochondrial genomes(ptDNA and mtDNA) of this genus is limited to Northern Hemisphere species. Here, we report the sequencing of the ptDNA and mtDNA of the Antarctic red alga Pyropia endiviifolia using the Illumina platform. The plastid genome(195 784 bp, 33.28% GC content) contains 210 protein-coding genes, 37 tRNA genes and 6 rRNA genes. The mitochondrial genome(34 603 bp, 30.5% GC content) contains 26 protein-coding genes, 25 tRNA genes and 2 rRNA genes. Our results suggest that the organellar genomes of Py. endiviifolia have a compact organization. Although the collinearity of these genomes is conserved compared with other Pyropia species, the genome sizes show significant differences, mainly because of the different copy numbers of rDNA operons in the pt DNA and group II introns in the mtDNA. The other Pyropia species have 2–3 distinct intronic ORFs in their cox 1 genes, but Py. endiviifolia has no introns in its cox 1 gene. This has led to a smaller mtDNA than in other Pyropia species. The phylogenetic relationships within Pyropia were examined using concatenated gene sets from most of the available organellar genomes with both the maximum likelihood and Bayesian methods. The analysis revealed a sister taxa affiliation between the Antarctic species Py. endiviifolia and the North American species Py. kanakaensis.     

Isolation and callus formation of Gracilariopsis bailiniae (Gracilariales,Rhodophyta) protoplasts

Journal of Oceanology and Limnology - This paper reports the first successful isolation of protoplasts from Gracilariopsis bailiniae and their callus formation. The base solution type,...     

Induction and cultivation of cloned filaments of Polysiphonia urceolata (Rhodomelaceae, Rhodophyta)

A filamentous clone of Polysiphonia urceolata was regenerated from segments cut from the fronds of gametophytes. Unlike wild thalli with short virgate branchlets, the clone was filamentous with few branches. Many transparent trichoblasts arose from pericentral cells during the induction culture, but these were seldom observed during normal growth. The trichoblasts were uniseriate, often colorless, and formed lobed rhizoids rapidly when they came into contact with solid substrates. In addition to morphological characteristics, the photosynthetic properties and growth conditions of the clone differed from those of the mother plant. Cross-gradient light and temperature culture experiments revealed that the most favorable conditions for culture of the filamentous clone were 22°C and 95-120 μE/(m2-s) light intensity. The photosynthetic light saturation value for filaments was approx. 100 μE/(m2-s), which is far lower than that of wild thalli. These results could be used to develop techniques for mass cultures of P. urceolata in photobioreactors for production of seed stock or bioactive products.     

Formation and early development of tetraspores of Polysiphonia urceolata (Rhodomelaceae, Rhodophyta)

Polysiphonia urceolata is one type of potential commercial red seaweeds used for breeding and cultivation, because of its significant biochemical and biomedical application. However, the information of breeding and seedling incubation for cultivation is limited, especially the early development. In this study, tetrasporohyte and gametophyte of P. urceolata were taken as the study materials in Huiquan Bay, Qingdao, China. The cleaned and sterilized tetrasporophytes and gametophytes were pre-cultured in sterilized seawater, then nurtured at 18℃, 25 μmol photons m-2 s-1 in 12:12 h (light:dark) photoperiod. Continuous observation under microscope showed that the early development consists of bipolar division stage and seedling stage. In the division stage, tetraspores germinate into bipolar sporelings that further differentiate into a colorless rhizoidal portion and a lightly pigmented upright shoot. The lightly pigmented rhizoidal cell develops to a rhizoid and the larger pigmented cell transforms to an erect axis. In the seedling stage, several quasi-protuberances appear on the erect axis and form juvenile seedlings. The results demonstrate the culture of P. urceolata from tetraspores under laboratory conditions.