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Streptococcus pyogenes Cas9 (SpCas9) is the most widely used genome editing tool in plants. The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif (PAM), significantly limiting its scope of application. Recently, five SpCas9 variants, SpCas9-NRRH, SpCas9-NRCH, SpCas9-NRTH, SpG, and SpRY, were developed to recognize non-canonical PAMs in human cells. In this study, these variants were engineered for plant genome editing, and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice (Oryza sativa) by stable transformation. Moreover, both cytosine base editors using a rat APOBEC1 or a human APOBEC3a and adenine base editors using a directly evolved highly compatible TadA∗-8e deaminase were developed from these SpCas9 variants. Our results demonstrated that the developed SpCas9 variants-based base editors readily generated conversions between C∙G and T∙A in the target sites with non-canonical PAMs in transgenic rice lines. Collectively, the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.

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.

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 mitochondria). 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 Pyropia 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 crossed 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.

Pyropia haitanensis (Bangiales, Rhodophyta), a major economically important marine crop, is also considered as an ideal research model of Rhodophyta to address several major biological questions such as sexual reproduction and adaptation to intertidal abiotic stresses. However, comparative genomic analysis to decipher the underlying molecular mechanisms is hindered by the lack of high‐quality genome information. Therefore, we integrated sequencing data from Illumina short‐read sequencing, PacBio single‐molecule sequencing and BioNano optical genome mapping. The assembled genome was approximately 53.3 Mb with an average GC% of 67.9%. The contig N50 and scaffold N50 were 510.3 kb and 5.8 Mb, respectively. Additionally, 10 superscaffolds representing 80.9% of the total assembly (42.7 Mb) were anchored and orientated to the 5 linkage groups based on markers and genetic distance; this outcome is consistent with the karyotype of five chromosomes (n = 5) based on cytological observation in P. haitanensis. Approximately 9.6% and 14.6% of the genomic region were interspersed repeat and tandem repeat elements, respectively. Based on full‐length transcriptome data generated by PacBio, 10,903 protein‐coding genes were identified. The construction of a genome‐wide phylogenetic tree demonstrated that the divergence time of P. haitanensis and Porphyra umbilicalis was ~204.4 Ma. Interspecies comparison revealed that 493 gene families were expanded and that 449 were contracted in the P. haitanensis genome compared with those in the Po. umbilicalis genome. The genome identified is of great value for further research on the genome evolution of red algae and genetic adaptation to intertidal stresses.

Pyropia yezoensis is an economically important marine macroalgae, naturally distributed in the upper intertidal zone. Owing to the nature of its habitat, the thallus will periodically be exposed to seawater or atmosphere, and can lose up to 95% of its cellular water content. This makes the alga an ideal research model to investigate the mechanisms of desiccation tolerance. In this study, we investigated the response mechanisms to dehydration and rehydration stresses at the transcription level in Pyropia yezoensis. The differently expressed genes were analyzed based on the different functions of encoding proteins: effector proteins (chloroplast proteins, macromolecular protective substances, and toxicity degradation enzymes) and regulatory proteins (protein kinases and phosphatases). Under osmotic stress, the unigenes related to photosynthesis were down-regulated significantly while those encoding glutathione transferase, superoxide dismutase and heat shock proteins were up-regulated significantly. We inferred that the photosynthetic activity was reduced to prevent damage caused by photosynthetic by-products and that the expression of antioxidant enzyme was increased to prevent the damage associated with reactive oxygen species. Additionally, unigenes encoding serine/threonine kinases and phospholipases were up-regulated in response to osmotic stress, indicating that these kinases play an important role in osmotolerance. Our work will serve as an essential foundation for the understanding of desiccation tolerance mechanisms in Pyropia yezoensis in the upper intertidal zones of rocky coasts.

An eCDAL was established from Japanese lamprey CDA1-like 4 to achieve a high editing frequency in a broad region as a C-terminal cytosine base editors (CT-CBE). Then, a novel plant dual-base editors DuBE (pDuBE1) was developed by integrating TadA-8e into eCDAL. The editing efficiency of pDuBE1 could reach to 87.6%, with frequencies of concurrent A-to-G and C-to-T conversions as high as 49.7% in stably transformed plant cells. Our results showed that pDuBE1 could mediate robust dual editing in plant genome, providing a powerful manipulation tool for precise crop breeding and screening platforms for in planta direct evolution. This article is protected by copyright. All rights reserved.

Botryococcus braunii is widely studied due to its high hydrocarbon content. In this study, B. braunii Abt02 was subjected to several analyses, including cytological observation, hydrocarbon composition analysis by gas chromatography mass spectrometry (GC-MS), phylogenetic identification using known races (A, B and L) of B. braunii strains based on their 18S rDNA sequences, and qPCR-based investigation of transcript accumulation levels of hydrocarbon biosynthesis-related enzymes (DXS, MCS, DLS, SQS) during different growth phases (lag phase, log phase, early stationary growth phase, late stationary growth phase) under nitrogen-replete and nitrogen-depleted growth conditions, respectively. Based on cytological observation and on the 18S rDNA phylogenetic analysis, strain Abt02 was assigned to race B. Analysis of the strain’s chemical composition showed that the B. braunii Abt02 contained high levels of hydrocarbons, which accounted for 43.75% of the cell’s dry weight. Of these hydrocarbons, squalene and its derivatives accounted for up to 87.54%. In addition, all four enzymes investigated were expressed at higher levels during the log growth phase under nitrogen depleted conditions than under nitrogen replete conditions.

Changes in atmospheric CO2 concentration have played a central role in algal and plant adaptation and evolution. The commercially important red algal genus, Pyropia (Bangiales) appears to have responded to inorganic carbon (Ci) availability by evolving alternating heteromorphic generations that occupy distinct habitats. The leafy gametophyte inhabits the intertidal zone that undergoes frequent emersion, whereas the sporophyte conchocelis bores into mollusk shells. Here, we analyze a high-quality genome assembly of Pyropia yezoensis to elucidate the interplay between Ci availability and life cycle evolution. We find horizontal gene transfers from bacteria and expansion of gene families (e.g. carbonic anhydrase, anti-oxidative related genes), many of which show gametophyte-specific expression or significant up-regulation in gametophyte in response to dehydration. In conchocelis, the release of HCO3- from shell promoted by carbonic anhydrase provides a source of Ci. This hypothesis is supported by the incorporation of 13C isotope by conchocelis when co-cultured with 13C-labeled CaCO3., The nori producing seaweed Pyropia yezoensis has heteromorphic generations that occupy distinct habitats. Here, via genome assembly, transcriptome analysis, and 13 C isotope labeling, the authors show the interplay between inorganic carbon availability and life cycle evolution in the intertidal environment.

Background Pyropia yezoensis, a marine red alga, is an ideal research model for studying the mechanisms of abiotic stress tolerance in intertidal seaweed. Real-time quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method to analyze gene expression levels. To accurately quantify gene expression, selection and validation of stable reference genes is required. Results We used transcriptome profiling data from different abiotic stress treatments to identify six genes with relatively stable expression levels: MAP, ATPase, CGS1, PPK, DPE2, and FHP. These six genes and three conventional reference genes, UBC, EF1-α, and eif4A, were chosen as candidates for optimal reference gene selection. Five common statistical approaches (geNorm, ΔCt method, NormFinder, BestKeeper, and ReFinder) were used to identify the stability of each reference gene. Our results show that: MAP, UBC, and FHP are stably expressed in all analyzed conditions; CGS1 and UBC are stably expressed under conditions of dehydration stress; and MAP, UBC, and CGS1 are stably expressed under conditions of temperature stress. Conclusion We have identified appropriate reference genes for RT-qPCR in P. yezoensis under different abiotic stress conditions which will facilitate studies of gene expression under these conditions. Electronic supplementary material The online version of this article (10.1186/s12864-018-4643-8) contains supplementary material, which is available to authorized users.

The mitogen-activated protein kinase (MAPK) cascade is composed of three hierarchically organized protein kinases (MAP kinase kinase kinases, MAP kinase kinases, and MAPKs) and plays a vital role in regulating developmental processes and stress responses in plants. Pyropia yezoensis belongs to the old Rhodophyta and grows in intertidal zones. Although MAPKs have been well studied in some model plants, there is little information available concerning the P. yezoensis MAPK gene family and their roles in adaptation to intertidal environments. In this study, we identified 15 MAPKs from P. yezoensis (PyMAPKs) based on genome-wide analysis of the omics database. Conserved motif analysis indicated that all PyMAPKs had typical protein kinase domains; however, in addition to TEY/TDY activation loop motifs, some new types of activation loop motifs were identified: TTY, TAY, TQY, TSH, TAH, and TQA. Phylogenetic analysis showed that, with other red algae, PyMAPKs formed three groups independent from green plants, indicating that red algae retained older MAPK types than green plants. Transcriptomic analysis suggested that members of the PyMAPK gene family were involved in response to dehydration/rehydration and temperature stress and showed differential expression patterns. This work represents the first complete inventory of MAPKs from red algae and could help elucidate the mechanisms involving these proteins in P. yezoensis responses to abiotic stress.

Mitogen-activated protein kinase (MAPK) signaling cascades are ubiquitous in eukaryotes and play a pivotal role in a range of biotic and abiotic stress responses. Phosphorylated mitogen-activated protein kinase is the last component of the cascade pathway and activates cellular responses. Pyropia yezoensis (Rhodophyta), a red alga, grows in intertidal zones. Here, we systematically analyzed PyMAPK2 (Group D) expression patterns at the mRNA and protein levels in response to different abiotic stresses. The results showed that PyMAPK2 responded to various stresses with different patterns. Yeast two-hybrid assays indicated that 17 candidate proteins putatively interacted with PyMAPK2. Bimolecular fluorescence complementation analysis confirmed that two cytoskeleton-related proteins, PyActin3 and PyFasciclin, interacted with PyMAPK2 in onion epidermal cells. PyActin3 and PyFasiclin mRNA expression also increased significantly in response to desiccation/rehydration stress. This study helps to clarify the molecular mechanisms and signal pathways associated with PyMAPK2 that regulate the response of P. yeozensis to environmental stresses.

Pyropia yezoensis, belonging to the Rhodophyta, is an economically important seaweed. In this study, we developed a high-efficiency plastid transformation platform for P. yezoensis. In the plastid transformation vector, psbA UTR of P. yezoensis, including the promoter and 3' UTR, was used to express foreign genes. The integration site was a transcriptionally active intergenic region between the rrsB and trnI genes, located in the inverted repeat regions of the plastid genome. The CAT and eGFP genes were integrated into the plastid genome at this site. The expression of CAT in the transformants confers resistance to chloramphenicol through the action of chloramphenicol acetyltransferase, which inactivates the drug, thereby allowing the plant to grow well under selective pressure. The eGFP fluorescence signal was also observed in transformed cells and the transformants. The average survival rate of treated cells was estimated to be approximately 4.2‰ (4 transplastomic colonies per 1000 gametophyte cells). Multiple-PCR analyses confirmed that the CAT and eGFP genes were successfully integrated in the site between rrsB and trnI. Western blot also showed eGFP expression in the cells of transformants. Thus, this study presents the first convenient plastid gene expression system for P. yezoensis and provides an important platform for studying gene function in P. yezoensis.

Aquaporins are a class of ancient channel proteins. The role of aquaporin proteins (AQPs) has been extensively studied in land plants. However, the information on AQPs in seaweeds is unclear. Pyropia yezoensis (Rhodophyta) grows in the intertidal zone and is believed to be a model research organism for seaweeds in the intertidal zone. In this study, we isolated two AQPs from P. yezoensis (PyAQPs). They contained two conserved Asn-Pro-Ala (NPA) motifs and six transmembrane α-helices (H1–H6). Phylogenetic analysis showed that they are classified into two subfamilies, with much greater evolutionary distance with green plants. PyAQP1 belonged to the Glp group, whereas PyAQP2 belonged to a single small group. Subcellular localization showed that PyAQP1 was distributed on the plasma membrane of the cells, and PyAQP2 was mainly distributed in the plasma membrane and cytoplasm of the cells. The two genes displayed differential expression profiles for different abiotic stresses (desiccation, salinity, and temperature) at the mRNA and protein levels. Over-expression of PyAQPs in yeast cells indicated that PyAQPs improved the growth of yeast under osmotic stress. These results provide an insight towards understanding the molecular mechanisms of P. yezoensis responding to stress.

During photosynthesis, photosynthetic electron transport generates a proton motive force (pmf) across the thylakoid membrane, which is used for ATP biosynthesis via ATP synthase in the chloroplast. The pmf is composed of an electric potential (ΔΨ) and an osmotic component (ΔpH). Partitioning between these components in chloroplasts is strictly regulated in response to fluctuating environments. However, our knowledge of the molecular mechanisms that regulate pmf partitioning is limited. Here, we report a bestrophin-like protein (AtBest), which is critical for pmf partitioning. While the ΔpH component was slightly reduced in atbest, the ΔΨ component was much greater in this mutant than in the wild type, resulting in less efficient activation of nonphotochemical quenching (NPQ) upon both illumination and a shift from low light to high light. Although no visible phenotype was observed in the atbest mutant in the greenhouse, this mutant exhibited stronger photoinhibition than the wild type when grown in the field. AtBest belongs to the bestrophin family proteins, which are believed to function as chloride (Cl- ) channels. Thus, our findings reveal an important Cl- channel required for ion transport and homeostasis across the thylakoid membrane in higher plants. These processes are essential for fine-tuning photosynthesis under fluctuating environmental conditions.

Photosynthesis includes the collection of light and the transfer of solar energy using light-harvesting chlorophyll a/b-binding (LHC) proteins. In high plants, the LHC gene family includes LHCA and LHCB sub-families, which encode proteins constituting the light-harvesting complex of photosystems I and II. Zostera marina L. is a monocotyledonous angiosperm and inhabits submerged marine environments rather than land environments. We characterized the Lhca and Lhcb gene families of Z. marina from the expressed sequence tags (EST) database. In total, 13 unigenes were annotated as ZmLhc, 6 in Lhca family and 7 in ZmLhcb family. ZmLHCA and ZmLHCB contained the conservative LHC motifs and amino acid residues binding chlorophyll. The average similarity among mature ZmLHCA and ZmLHCB was 48.91% and 48.66%, respectively, which indicated a high degree of divergence within ZmLHChc gene family. The reconstructed phylogenetic tree showed that the tree topology and phylogenetic relationship were similar to those reported in other high plants, suggesting that the Lhc genes were highly conservative and the classification of ZmLhc genes was consistent with the evolutionary position of Z. marina. Real-time reverse transcription (RT) PCR analysis showed that different members of ZmLhca and ZmLhcb responded to a stress in different expression patterns. Salinity, temperature, light intensity and light quality may affect the expression of most ZmLhca and ZmLhcb genes. Inorganic carbon concentration and acidity had no obvious effect on ZmLhca and ZmLhcb gene expression, except for ZmLhca6.

The reaction center (RC) of photosystem II (PSII), which is composed of D1, D2, PsbI, and cytochrome b559 subunits, forms at an early stage of PSII biogenesis. However, it is largely unclear how these components assemble to form a functional unit. In this work, we show that synthesis of the PSII core proteins D1/D2 and formation of the PSII RC is blocked specifically in the absence of ONE-HELIX PROTEIN1 (OHP1) and OHP2 proteins in Arabidopsis (Arabidopsis thaliana), indicating that OHP1 and OHP2 are essential for the formation of the PSII RC. Mutagenesis of the chlorophyll-binding residues in OHP proteins impairs their function and/or stability, suggesting that they may function in the binding of chlorophyll in vivo. We further show that OHP1, OHP2, and HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244), together with D1, D2, PsbI, and cytochrome b559, form a complex. We designated this complex the PSII RC-like complex to distinguish it from the RC subcomplex in the intact PSII complex. Our data imply that OHP1, OHP2, and HCF244 are present in this PSII RC-like complex for a limited time at an early stage of PSII de novo assembly and of PSII repair under high-light conditions. In a subsequent stage of PSII biogenesis, OHP1, OHP2, and HCF244 are released from the PSII RC-like complex and replaced by the other PSII subunits. Together with previous reports on the cyanobacterium Synechocystis, our results demonstrate that the process of PSII RC assembly is highly conserved among photosynthetic species.

Red algae are important primary photosynthetic organisms. The Bangiales comprise a morphologically diverse order of red algae. Until now, complete plastid genomes of the Bangiales were only mapped for foliose species. To date, no filamentous plastomes have been published. The aim of this study was to determine and analyze the complete plastid genome of the filamentous marine species ‘Bangia’ sp. OUCPT-01. It is a circular molecule, 196,913 bps in length with a guanine-cytosine (GC) content of 33.5%. It has a quadripartite structure with two single copy regions separated by two direct non-identical repeats. It has 205 protein-coding genes, 37 tRNAs, and 6 rRNAs. Therefore, it has a high coding capacity and is highly similar to other Bangiales species in terms of content and structure. In particular, it reveals that the genera in the Bangiales have highly conserved gene content and plastome synteny. This plastome and existing data provide insights into the phylogenetic relationships among the Bangiales genera of the Rhodophyta. According to its plastid- and mitochondrial genomes, ‘Bangia 2′ is a sister group to Porphyra. However, the position of Wildemania schizophylla in the Bangiales is still controversial. Our results show that the Bangiales divergence time was ~225 million years ago.

Highlight A novel CRY1-interacting protein, TCP2, regulated by blue light and involved in the CRY1-mediated blue light signal pathway to regulate photomorphogenesis, is described., Light regulates plant growth and development via multiple photoreceptors including phytochromes and cryptochromes. Although the functions of photoreceptors have been studied extensively, questions remain regarding the involvement of cryptochromes in photomorphogenesis. In this study, we identified a protein, TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR 2 (TCP2), which interacts with the cryptochrome 1 (CRY1) protein in yeast and plant cells via the N-terminal domains of both proteins. Transgenic plants overexpressing TCP2 displayed a light-dependent short hypocotyl phenotype, especially in response to blue light. Moreover, light affected TCP2 expression in a wavelength-dependent manner and TCP2 positively regulates mRNA expression of HYH and HY5. These results support the hypothesis that TCP2 is a transcription activator which acts downstream of multiple photoreceptors, including CRY1.

This study investigated a new fungal disease that attacks Pyropia yezoensis, an economically important red alga that is extensively cultured in China. An incidence was found in a P. yezoensis farm during mid to end November 2012 at Haizhou Bay, Jiangshu Province, China. Histopathology revealed that the naturally infected thalli were overwhelmed by a fungus, leading to progressive red rot symptoms. The causative agent was isolated, grown in pure culture, and identified as a member of the genus Alternaria by morphology and sequence analysis of the nuclear ribosomal DNA containing the internal transcribed spacer (ITS) region of ITS1 and ITS2. In artificial infection experiments on P. yezoensis blades, the fungal isolate was able to cause the same characteristic histopathology seen in natural infections. This fungal isolate grew well at a wide range of temperatures (8–36 °C) and at low salinities (5–50 ‰). In an orthogonal test used to determine the effects of environmental factors (temperature, salinity, and conidia concentration) on disease expansion, it was found that higher temperatures and lower salinities easily caused red rot disease, with the optimal conditions for disease development being 23 °C, 24 ‰ salinity, and a conidia concentration of 105 mL−1. This is the first report to show that Alternaria causes red rot disease in P. yezoensis.

Bangia fuscopurpurea is a traditional mariculture crop having high nutritional value, eicosapntemacnioc acid (EPA) production, and protein content. As an intertidal species, it can tolerate drastic changes in abiotic factors such as temperature, hydration, and light radiation; however, genomic information on the evolutionary aspect and mechanism of EPA enrichment in B. fuscopurpurea and the role of EPA in cold tolerance in this species remain elusive. We conducted transcriptome profile analysis in B. fuscopurpurea to investigate the biological functions of genes associated with resistance to various environment factors. We identified 41,935 unigenes that were assembled and applied to public databases to define their functional annotation (NR, GO, KEGG, KOG, and SwissProt). We further identified genes that encoded key enzymes in EPA biosynthesis; five paralogous genes encoding delta5 desaturase were detected in B. fuscopurpurea. Fatty acid profiling and gene expression analysis of B. fuscopurpurea grown under cold stress were simultaneously performed. The EPA content was increased by 29.8% in the samples grown at 4°C, while the total amount of fatty acids remained unchanged. Moreover, all the EPA biosynthesis-related desaturase and elongase genes were upregulated under cold stress. Thus, we hypothesized that diverse EPA biosynthesis pathways and significant increase in gene copy numbers of fatty acid desaturases, together with the concomitant elevation in the transcriptional level of genes associated with fatty acid metabolism, lead to EPA accumulation and subsequently affect membrane fluidity, contributing to cold stress resistance in B. fuscopurpurea. Our findings not only provide a fundamental genetic background for further research in B. fuscopurpurea, but also have important implications for screening and genetic engineering of algae and plants for EPA production.

Quantitative real-time PCR (qRT-PCR) is frequently used for gene expression analysis. The selection of reference genes is required for normalization of the variation to avoid misinterpretation of experimental results and erroneous analyses. Pyropia yezoensis, growing in the intertidal zone, is an economically important seaweed. Although this intertidal seaweed has been an experimental system for understanding stress tolerance and developmental mechanisms, reference genes suitable for the normalization of qRT-PCR data have not previously been identified. In this study, expression stability of six candidate reference genes, including ACT, eIF4A, EF1α, GAPDH, TUA, and UBQ (traditional housekeeping genes), has been validated in a diverse set of samples representing different developmental stages and stress conditions of P. yezoensis. Three qRT-PCR analysis methods, geNorm, NormFinder, and BestKeeper, were evaluated systematically. The results indicated that ACT3, eIF4A, and EF1α were the optimal reference genes for P. yezoensis under stress conditions; UBQ, EF1α, and eIF4A were suitable for studying the expression of genes related to P. yezoensis development. TUA showed the lowest expression stability both under stress conditions and over developmental stages. Our results have provided a reference gene application guideline for P. yezoensis gene expression characterization using the qRT-PCR system.

In this article, high-throughput de novo transcriptomic sequencing was performed in Alexandrium catenella , which provided the first view of the gene repertoire in this dinoflagellate based on next-generation sequencing (NGS) technologies. A total of 118,304 unigenes were identified with an average length of 673 bp (base pair). Of these unigenes, 77,936 (65.9%) were annotated with known proteins based on sequence similarities, among which 24,149 and 22,956 unigenes were assigned to gene ontology categories (GO) and clusters of orthologous groups (COGs), respectively. Furthermore, 16,467 unigenes were mapped onto 322 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). We also detected 1143 simple sequence repeats (SSRs), in which the tri-nucleotide repeat motif (69.3%) was the most abundant. The genetic facts and significance derived from the transcriptome dataset were suggested and discussed. All four core nucleosomal histones and linker histones were detected, in addition to the unigenes involved in histone modifications.190 unigenes were identified as being involved in the endocytosis pathway, and clathrin-dependent endocytosis was suggested to play a role in the heterotrophy of A . catenella . A conserved 22-nt spliced leader (SL) was identified in 21 unigenes which suggested the existence of trans-splicing processing of mRNA in A . catenella .

Zostera marina, a monocotyledonous angiosperm, is one of the most important seagrass species. To investigate the salt-tolerance mechanism and discover salt-tolerant genes in Z. marina, a cDNA library was constructed. Single-pass sequencing of the 5′ ends of 4 081 clones yielded 4 002 high quality expressed sequence tags (ESTs), which were assembled into 241 contigs and 1 673 singletons, representing 1 914 unigenes. The average length of the ESTs was 582 bp, with sizes ranging from 100–1 500 bp. Basic Local Alignment Search Tool (BLASTX) analysis revealed that 1 664 unigenes had significant homology to known genes in the National Center for Biotechnology Information (NCBI) non-redundant (nr) database (E-value⩽5–10). Among them, the two most abundant genes encoded metallothionein (157 ESTs) and chlorophyll a/b-binding protein (38 ESTs), accounting for 7.1% and 1.7% of the total ESTs, respectively. Using Kyoto Encyclopedia of Genes and Genomes (KEGG), 1 462 unigenes were assigned to 1 161 pathways (E-value⩽5–10). A total of 938 unigenes were assigned Gene Ontology (GO) terms based on the GO hierarchy analysis, and InterProScan searches recognized 1 003 InterPro families. Three genes for metallothionein in Z. marina that belonged to Class II was identified. Results of this study will improve understanding of the molecular mechanisms of saline tolerance in Z. marina.

High light-inducible proteins are critical for photosynthetic organisms when responding to high light stress. We cloned and characterized the HLIP gene from Porphyra yezoensis, which was designated as PyHLIP. Sequence analysis revealed that the open reading frame of PyHLIP was 387 bp in length that encoded a 128 amino acid-polypeptide containing a plastid transit peptide and an obvious membrane-spanning α-helix region. No intron existed in the genomic DNA of PyHLIP. The putative promoter of PyHLIP was TATA-less, and the initiator element (Inr) existed upstream of ATG. Analysis of genomic organization indicated there were two copies of PyHLIP gene in the P. yezoensis nuclear genome. The expression of PyHLIP can be induced by various stress conditions and is especially sensitive to high light.

Green tides are caused by the proliferation of chlorophytes under suitable hydrographic conditions. These blooms lead to environmental degradation and negatively impact the waters and seagrass beds, as well as fishing and other recreational activities in the bay. A comprehensive ecological understanding of the bloom dynamics, including the origin and persistence, is needed to foster management decisions. The algae in the great majority of green tide blooms usually belong to two genera of Ulvophyceae, Ulva and Enteromorpha. Ulva has been observed more often in recent years. In China, green tides occurred for the first time in the middle area of the Yellow Sea in 2007, and a large-scale algae blooming broke out in the middle and southern areas of the Yellow Sea in late May 2008. We identified them as Ulva prolifera by comparative analysis of the rDNA internal transcribed spacer 1 (ITS1), 5.8S and ITS2 sequences in combination with microscopic observation. Morphological differences were found between the free-floating algae and the attached thalli. Various reproduction patterns of the free-floating algae include sexual, asexual and vegetative propagations, which played important roles in the long-term green tide persistence in China. The ITS sequences of the blooming algae were identical to those of the samples from the Lianyungang sea area but were different from the attached samples from the Qingdao sea area. The results infer that the blooms are originated from other sea areas rather than from the local attached populations.

The agarophyte Gracilariopsis lemaneiformis is both important for biological research and of significant economic value. However, the genetic diversity of wild populations of the alga has not been studied. We used amplified fragment length polymorphism (AFLP) PCR and simple sequence repeat (SSR) analysis to investigate diversity in four field populations, three from the coast of Qingdao and one from Weihai, China. Forty G. lemaneiformis isolates collected from the four different geographical groups were analyzed using 16 pairs of SSR primers for PCR amplification. However, no polymorphisms were detected, indicative of a degree of genetic homogeneity. A total of 347 reproducible bands were then amplified using eight AFLP primer pairs, and genetic indices of diversity within and between populations were calculated. This analysis revealed only low levels of genetic diversity both within and between the four geographical groups of G. lemaneiformis. The Weihai population showed a higher within-population genetic diversity than any of the Qingdao populations. The data suggest that there is only limited gene flow between populations. An UPGMA dendrogram revealed two main clusters, and one of these included all the Qingdao isolates. The order of clustering was in accordance with their geographical distribution. These results suggest that the wild G. lemaneiformis populations are closely related and that there is little genetic diversity within wild germplasm in the regions sampled.

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.

Microsatellites are repetitive genomic elements that show high levels of variation and therefore are useful tools for studying genetic polymorphism and constructing genetic linkage maps of eukaryotic organisms. Porphyra yezoensis Ueda is an economically important seaweed that is being targeted for genetic improvement using marker-assisted breeding. Hence, in an attempt to develop microsatellite markers for P. yezoensis, a microsatellite (or simple sequence repeat)-enriched library was constructed to identify (GA)n and (CA)n motifs. A total of 71 perfect microsatellite clones were identified, of which 30 simple sequence repeat primer pairs were developed. Of these, 24 (80%) amplified polymerase chain reaction products of expected sizes. Twelve primer pairs amplified two to four bands, whereas another 12 primer pairs produced monomorphic banding patterns. Data for 12 loci were analyzed using POPGENE software version 1.32. A total of 29 alleles were produced at 12 loci, with an average of 2.42 alleles (Na) and 1.81 effective alleles (Ne) per locus. These markers were used to analyze the genetic diversity within 11 geographically different lines of P. yezoensis. Overall, these lines were clustered into two divisions with those from close geographic locations clustering together. Further cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of motif repeats in different alleles were major sources of polymorphisms.

Dihydrodipicolinate synthase (DHDPS) is the main enzyme of a specific branch of the aspartate pathway leading to lysine biosynthesis in higher plants. We have cloned and characterized the DHDPS gene from Zizania latifolia Griseb, which was named ZlDHDPS. Sequence analysis indicates that it contains an ORF of 954 bp interrupted by two exons and one intron encoding a polypeptide of 317 amino acids lacking a plastid transit peptide and a stop codon. The sequence of ZlDHDPS has high identity with known plant DHDPS in GenBank. Southern blotting analysis indicates that there are two copies of Z. latifolia DHDPS (ZlDHDPS) gene in the Z. latifolia nuclear genome. RT-PCR analysis shows the expression of ZlDHDPS is tissue specific and high level expression is present in fast-growing tissue and reproductive tissue. The 5′-regulatory sequence of ZlDHDPS contains a GT-1 box and a (CA)n element, which may play a role in regulating the expression of ZlDHDPS. The fusion construct of the ZlDHDPS sequence with the reporter gene GUS was transiently expressed in the onion epidermal cells by particle gun-mediated bombardment suggesting that ZlDHDPS was located in the cytoplasm, different from DHDPS gene of other species. Functional complementary analysis showed that ZlDHDPS can recover the DHDPS-deleted mutant of Escherichia coli.

Background Pyropia haitanensis is an economically important marine crop grown in harsh intertidal habitats of southern China; it is also an excellent model system for studying mechanisms of stress tolerance. To understand the molecular mechanisms underlying osmotic tolerance and adaptation to intertidal environments, a comprehensive analysis of genome-wide gene expression profiles in response to dehydration and rehydration in Py. haitanensis was undertaken using digital gene expression profile (DGE) approaches combined with de novo transcriptome sequencing. Results RNA-sequencing of the pooled RNA samples from different developmental phases and stress treatments was performed, which generated a total of 47.7 million clean reads. These reads were de novo assembled into 28,536 unigenes (≥200 bp), of which 18,217 unigenes (63.83 %) were annotated in at least one reference database. DGE analysis was performed on four treatments (two biological replicates per treatment), which included moderate dehydration, severe dehydration, rehydration, and normal conditions. The number of raw reads per sample ranged from 12.47 to 15.79 million, with an average of 14.69 million reads per sample. After quality filtering, the number of clean reads per sample ranged from 11.83 to 15.04 million. All distinct sequencing reads were annotated using the transcriptome of Py. haitanensis as reference. A total of 1,681 unigenes showed significant differential expression between moderate dehydration and normal conditions, in which 977 genes were upregulated, and 704 genes were downregulated. Between severe dehydration and normal conditions, 1,993 unigenes showed significantly altered expression, which included both upregulated (1,219) and downregulated genes (774). In addition, 1,086 differentially expressed genes were detected between rehydration and normal conditions, of which 720 genes were upregulated and 366 unigenes were downregulated. Most gene expression patterns in response to dehydration differed from that of rehydration, except for the synthesis of unsaturated fatty acids, several transcription factor families, and molecular chaperones that have been collectively implicated in the processes of dehydration and rehydration in Py. haitanensis. Conclusions Taken together, these data provide a global high-resolution analysis of gene expression changes during osmotic stress that could potentially serve as a key resource for understanding the biology of osmotic acclimation in intertidal red seaweed. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2226-5) contains supplementary material, which is available to authorized users.