Identification, characterization and expression profiles of E2 and E3 gene superfamilies during the development of tetrasporophytes in Gracilariopsis lemaneiformis (Rhodophyta).

E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases play important roles in the growth and development of plants and animals. To date, the systematic analysis of E2 and E3 genes in Rhodophyta is limited. In this study, 14 E2 genes and 51 E3 genes were identified in Gracilariopsis lemaneiformis, an economically important red alga. E2 genes were classified into four classes according to the structure of the conserved domain, UBC. E3 genes were classified into 12 subfamilies according to individual conserved domains. A phylogenetic tree of seven algae species showed that functional differentiation of RING-type E3s was the highest, and the similarity between orthologous genes was high except in Chlamydomonas reinhardtii and Chara braunii. RNA-seq data analysis showed significant differential expression levels of E2 and E3 genes under the life stages of tetraspore formation and release, especially GlUBCN and GlAPC3. According to GO and KEGG analysis of two transcriptomes, GlUBCN and GlAPC3 were involved in ubiquitin-mediated proteolysis, and other subunits of the anaphase promoting complex or cyclosome (APC/C) and its activators GlCDC20 and GlCDH1 were also enriched into this process. The CDH1 and CDC20 in 981 were down-regulated during tetraspores formation and release, with the down-regulation of CDH1 being particularly significant; CDH1 and CDC20 in WLP-1, ZC, and WT were up-regulated during tetraspores formation and release, with CDC20 being more significantly up-regulated. Therefore, GlCDH1, rather than GlCDC20, in '981' might play the leading role in the activation of the APC/C, and GlCDC20 might play the leading role rather than GlCDH1 in strains WLP-1, ZC and wild type. The low fertility of cultivar 981 might be highly correlated with the inactivity of activators CDH1 and CDC20. This study provided a basic and comprehensive understanding of characteristic of E2 and E3 genes in Gp. lemaneiformis and set a foundation for further understanding of E2 ubiquitin conjugating enzymes and E3 ubiquitin ligase in regulating tetrasporophytes development of Gp. lemaneiformis.

Calmodulin and Its Interactive Proteins Participate in Regulating the Explosive Growth of Alexandrium pacificum (Dinoflagellate).

Alexandrium pacificum is a typical dinoflagellate that can cause harmful algal blooms, resulting in negative impacts on ecology and human health. The calcium (Ca2+) signal transduction pathway plays an important role in cell proliferation. Calmodulin (CaM) and CaM-related proteins are the main cellular Ca2+ sensors, and can act as an intermediate in the Ca2+ signal transduction pathway. In this study, the proteins that interacted with CaM of A. pacificum were screened by two-dimensional electrophoresis analysis and far western blots under different growth conditions including lag phase and high phosphorus and manganese induced log phase (HPM). The interactive proteins were then identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Four proteins were identified, including Ca2+/CaM-dependent protein kinase, serine/threonine kinase, annexin, and inositol-3-phosphate synthase, which all showed high expression levels under HPM. The gene expression levels encoding these four proteins were also up-regulated under HPM, as revealed by quantitative polymerase chain reaction, suggesting that the identified proteins participate in the Ca2+ transport channel and cell cycle regulation to promote cell division. A network of proteins interacting with CaM and their target proteins involved in the regulation of cell proliferation was raised, which provided new insights into the mechanisms behind the explosive growth of A. pacificum.

Estimating the ploidy of Gracilariopsis lemaneiformis at both the cellular and genomic level1.

The determination of the ploidy level of an organism is a prerequisite for studies of evolution, cellular function, and genomic construction. Identification of the ploidy of the economically important red alga Gracilariopsis lemaneiformis has been hindered by its small genome and large number of chromosomes. Therefore, in the current study, PloidyNGS, a tool that calculates the number of reads supporting different alleles at each position along the genome sequence, and fluorescence in situ hybridization coupled with tyramide signal amplification (TSA-FISH) were used to clarify the ploidy of G. lemaneiformis. In addition, flow cytometry (FCM) was used to estimate the ploidy of different somatic cells. The PloidyNGS results showed that most of the alleles in the gametophyte were monomorphic, whereas the TSA-FISH results showed that one hybridization signal was observed in gametophytic nuclei and two in tetrasporophytic nuclei when the nuclei were hybridized by single copy gene probes. These results confirmed that G. lemaneiformis is a haploid in the gametophytic generation and diploid in the sporophytic generation. Moreover, the FCM result suggested that G. lemaneiformis was not an endopolyploid. Based on previous studies, we hypothesize that the nuclear number is important for the cellular differentiation and function of this species. We also suggest that G. lemaneiformis evolved from a paleopolyploid, the genome of which has been diploidized, and that traces of genomic doubling are no longer apparent. Thus, this study provides important evidence for further studies on the evolution and genomes of red algae.

Antihypertensive Effects of Two Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from Gracilariopsis lemaneiformis (Rhodophyta) in Spontaneously Hypertensive Rats (SHRs).

A variety of biologically active products have been isolated from Gracilariopsis lemaneiformis. In the present study, two novel angiotensin-converting enzyme (ACE) inhibitory peptides, FQIN [M(O)] CILR, and TGAPCR, were screened and identified from G. lemaneiformis protein hydrolysates by LC-MS/MS. The IC50 values of FQIN [M(O)] CILR and TGAPCR were 9.64 ± 0.36 µM and 23.94 ± 0.82 µM, respectively. In the stability study, both peptides showed stabilities of pH, temperature, simulated gastrointestinal digestion, and ACE hydrolysis. The Lineweaver⁻Burk plot showed that the two peptides were noncompetitive inhibitors of ACE. Molecular docking simulated the intermolecular interactions of two peptides and ACE, and the two peptides formed hydrogen bonds with the active pockets of ACE. However, FQIN [M(O)] CILR was more closely linked to the active pockets of ACE, thereby exerting better ACE inhibition. Spontaneously hypertensive rats (SHRs) were studied with an oral dose of 10 mg/kg body weight. Both peptides reduced systolic blood pressure (SBP) and diastolic blood pressure (DBP) in SHRs, of which FQIN [M(O)] CILR was able to reduce the systolic blood pressure by 34 mmHg (SBP) (p < 0.05). Therefore, FQIN [M(O)] CILR was an excellent ACE inhibitory peptide.

Effects of sodium bicarbonate concentration on growth, photosynthesis, and carbonic anhydrase activity of macroalgae Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae (Gracilariales, Rhodophyta).

There is potential for bicarbonate to improve crop yields and economic efficiency of marine algae. However, few studies have focused on the effect of bicarbonate on the growth, photosynthesis, and enzyme activity associated with carbon utilization, especially in commercial macroalgae. Here, the addition of bicarbonate (up to 420 mg L(-1)) to macroalgal cultures has been evaluated for Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae with respect to growth rate, photosynthetic activity, carbonic anhydrase activity, and biochemical composition. The results showed that the effects of NaHCO3 on growth, chlorophyll a, phycoerythrin, photosynthetic oxygen evolution, photochemical parameters of PSI and PSII, carbonic anhydrase activity, and nitrogen content were significant (P < 0.05) and followed the same pattern in the three species. The parameter values were promoted in lower NaHCO3 concentrations (up to 252 or 336 mg L(-1)) and inhibited in higher NaHCO3 concentrations (>336 mg L(-1) for Gp. lemaneiformis and >420 mg L(-1) for the other two species). Moreover, species-specific differences induced by supplementation with bicarbonate were discovered during culture. Optimal concentrations of NaHCO3 used in this study were 252 mg L(-1) for Gp. lemaneiformis and 336 mg L(-1) for G. vermiculophylla and G. chouae. These results suggest that an adequate supplementation of sodium bicarbonate is a viable strategy for promoting growth and photosynthetic activity in some macroalgae as well as for improving biochemical composition. The study will help to accelerate the growth rate of algae and improve the quality of thalli, and will also be useful for enhancing the understanding of carbon utilization in macroalgae.

The complete chloroplast genome of Gracilariopsis lemaneiformis (Rhodophyta) gives new insight into the evolution of family Gracilariaceae.

The complete chloroplast genome of Gracilariopsis lemaneiformis was recovered from a Next Generation Sequencing data set. Without quadripartite structure, this chloroplast genome (183,013 bp, 27.40% GC content) contains 202 protein-coding genes, 34 tRNA genes, 3 rRNA genes, and 1 tmRNA gene. Synteny analysis showed plasmid incorporation regions in chloroplast genomes of three species of family Gracilariaceae and in Grateloupia taiwanensis of family Halymeniaceae. Combined with reported red algal plasmid sequences in nuclear and mitochondrial genomes, we postulated that red algal plasmids may have played an important role in ancient horizontal gene transfer among nuclear, chloroplast, and mitochondrial genomes. Substitution rate analysis showed that purifying selective forces maintaining stability of protein-coding genes of nine red algal chloroplast genomes over long periods must be strong and that the forces acting on gene groups and single genes of nine red algal chloroplast genomes were similar and consistent. The divergence of Gp. lemaneiformis occurred ~447.98 million years ago (Mya), close to the divergence time of genus Pyropia and Porphyra (443.62 Mya).

Comparison of potential diatom 'barcode' genes (the 18S rRNA gene and ITS, COI, rbcL) and their effectiveness in discriminating and determining species taxonomy in the Bacillariophyta.

Diatoms form an enormous group of photoautotrophic micro-eukaryotes and play a crucial role in marine ecology. In this study, we evaluated typical genes to determine whether they were effective at different levels of diatom clustering analysis to assess the potential of these regions for barcoding taxa. Our test genes included nuclear rRNA genes (the nuclear small-subunit rRNA gene and the 5.8S rRNA gene+ITS-2), a mitochondrial gene (cytochrome c-oxidase subunit 1, COI), a chloroplast gene [ribulose-1,5-biphosphate carboxylase/oxygenase large subunit (rbcL)] and the universal plastid amplicon (UPA). Calculated genetic divergence was highest for the internal transcribed spacer (ITS; 5.8S+ITS-2) (p-distance of 1.569, 85.84% parsimony-informative sites) and COI (6.084, 82.14%), followed by the 18S rRNA gene (0.139, 57.69%), rbcL (0.120, 42.01%) and UPA (0.050, 14.97%), which indicated that ITS and COI were highly divergent compared with the other tested genes, and that their nucleotide compositions were variable within the whole group of diatoms. Bayesian inference (BI) analysis showed that the phylogenetic trees generated from each gene clustered diatoms at different phylogenetic levels. The 18S rRNA gene was better than the other genes in clustering higher diatom taxa, and both the 18S rRNA gene and rbcL performed well in clustering some lower taxa. The COI region was able to barcode species of some genera within the Bacillariophyceae. ITS was a potential marker for DNA based-taxonomy and DNA barcoding of Thalassiosirales, while species of Cyclotella, Skeletonema and Stephanodiscus gathered in separate clades, and were paraphyletic with those of Thalassiosira. Finally, UPA was too conserved to serve as a diatom barcode.

Subclinical highly pathogenic avian influenza virus infection among vaccinated chickens, China.

Subclinical infection of vaccinated chickens with a highly pathogenic avian influenza A(H5N2) virus was identified through routine surveillance in China. Investigation suggested that the virus has evolved into multiple genotypes. To better control transmission of the virus, we recommend a strengthened program of education, biosecurity, rapid diagnostics, surveillance, and elimination of infected poultry.

Analysis of Hyperosmotic Tolerance Mechanisms in Gracilariopsis lemaneiformis Based on Weighted Co-Expression Network Analysis.

We conducted transcriptome sequencing on salt-tolerant mutants X5 and X3, and a control (Ctr) strain of Gracilariopsis lemaneiformis after treatment with artificial seawater at varying salinities (30‱, 45‱, and 60‱) for 3 weeks. Differentially expressed genes were identified and a weighted co-expression network analysis was conducted. The blue, red, and tan modules were most closely associated with salinity, while the black, cyan, light cyan, and yellow modules showed a close correlation with strain attributes. KEGG enrichment of genes from the aforementioned modules revealed that the key enrichment pathways for salinity attributes included the proteasome and carbon fixation in photosynthesis, whereas the key pathways for strain attributes consisted of lipid metabolism, oxidative phosphorylation, soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE) interactions in vesicular transport, and porphyrin and chlorophyll metabolism. Gene expression for the proteasome and carbon fixation in photosynthesis was higher in all strains at 60‱. In addition, gene expression in the proteasome pathway was higher in the X5-60 than Ctr-60 and X3-60. Based on the above data and relevant literature, we speculated that mutant X5 likely copes with high salt stress by upregulating genes related to lysosome and carbon fixation in photosynthesis. The proteasome may be reset to adjust the organism's proteome composition to adapt to high-salt environments, while carbon fixation may aid in maintaining material and energy metabolism for normal life activities by enhancing carbon dioxide uptake via photosynthesis. The differences between the X5-30 and Ctr-30 expression of genes involved in the synthesis of secondary metabolites, oxidative phosphorylation, and SNARE interactions in vesicular transport suggested that the X5-30 may differ from Ctr-30 in lipid metabolism, energy metabolism, and vesicular transport. Finally, among the key pathways with good correlation with salinity and strain traits, the key genes with significant correlation with salinity and strain traits were identified by correlation analysis.

Breeding of New Strains of Gracilariopsis lemaneiformis with High Agar Content by ARTP Mutagenesis and High Osmotic Pressure Screening.

ARTP (atmospheric and room temperature plasma mutagenesis) mutagenesis was tried on G. lemaneiformis, and mutagenesis conditions were confirmed. An osmotic pressure screening program was established. Mutants were identified and characterized of relevant physiological traits. The aim of the study is to try to use ARTP mutagenesis and osmotic pressure screening for the breeding of high-agar G. lemaneiformis. Treatment time of 46 s was found to be an optimal mutagenesis time. The mutagenized spores were initially screened with 58‰ salinity artificial seawater, and then, the surviving spores were screened twice with 60‰ salinity artificial seawater in their vertical growth phase and branch growth phase, respectively. Four fast-growing and hypertonic resistance gametophytes were selected. The actual photosynthetic efficiency [Y(PSII)], photochemical quenching (qL), and non-photochemical quenching (NPQ) of four mutants were measured. The values of Y(PSII) and qL of HAGL-X3 and HAGL-X5 were higher than those of the control in the early stage of salt stress. NPQs of HAGL-X3 and HAGL-X5 were higher than control in most of the times. The growth rates of the four mutants were higher than that of the control. HAGL-X4 was the highest. The agar content was measured; HAGL-X5 displayed the highest agar content among the tested strains. HAGL-X5 was more in line with expectations, because of its high agar content and good hypertonic resistance. In this study, the mutant of G. lemaneiformis with high agar content was obtained by the procedure, which provided a certain reference for the selection of G. lemaneiformis strains with high agar content.

H3K79 methylation promotes rapid growth of Alexandrium pacificum under high light intensity via increased photosynthesis.

Alexandrium pacificum is one of the main species responsible for harmful algal blooms, posing serious threats to coastal ecosystems, economies, and public health. Light intensity is an important abiotic factor affecting the occurrence of red tides. In a certain range, increasing light intensity can promote the rapid growth of A. pacificum. This study aimed to elucidate the molecular mechanisms of H3K79 methylation (H3K79me) in response to high light intensity during the rapid growth of A. pacificum and the formation of toxic red tides. The research found that the abundance of H3K79me increased 2.1-fold under high light (HL, 60 µmol photon m-2 s-l) compared to control light conditions (CT, 30 µmol photon m-2 s-l), which was consistent with the trend of rapid growth under HL, and both can be inhibited by EPZ5676. Then effector genes of H3K79me under HL were identified using ChIP-seq and a virtual genome constructed based on transcriptome data of A. pacificum for the first time. The results showed that the differential modification-associated genes were primarily enriched in the pathways of "energy metabolism", "carbon metabolism", and "amino acid metabolism". These findings were confirmed through ChIP-qPCR. Subsequently, H3K79me-associated genes CP43 and GOGAT were identified by combined analysis of ChIP-seq and differentially expressed genes. Finally, pharmacological experiments using the H3K79me inhibitor EPZ5676 showed that the expression of the photosynthesis-related gene CP43 was significantly reduced by 2.5-fold and the maximum photochemical quantum efficiency of A. pacificum was reduced by 1.2 to 1.8-fold in HL compared with CT, leading to inhibited growth of A. pacificum. These results suggest that H3K79me plays a role in regulating the rapid growth of A. pacificum and photosynthesis is likely an important regulatory pathway, which is the first to provide epigenetic evidence underlying the formation of toxic red tides from the perspective of H3K79me.

The H3K79 methylase DOT1, unreported in photosynthetic plants, exists in Alexandrium pacificum and participates in its growth regulation.

Alexandrium pacificum is one of the typical toxic dinoflagellate species leading to harmful algal blooms (HABs). Histone modifications play key roles in many cellular events, but little is known about the mechanism of regulating A. pacificum growth. In this study, a total of 30 proteins containing the DOT1 domain were identified and analyzed. Some ApDOT1 gene expression levels were significantly influenced by light intensity and nitrogen by expression analysis and RT-qPCR validation. The enrichment of H3K79 methylation also showed a similar trend. In addition, ApDOT1.9 protein was proved to have the function of catalyzing the methylation of H3K79 by homology analysis and in vitro methylation. The results suggested that ApDOT1 proteins and H3K79 methylation were involved in responding to harmful algal blooms-inducing conditions (high light intensity, and high nitrogen), which provided basic information for further exploration of the regulatory mechanism of histone methylation in A. pacificum rapid growth.

Macropinocytosis in Gracilariopsis lemaneiformis (Rhodophyta).

Macropinocytosis is an endocytic process that plays an important role in animal development and disease occurrence but until now has been rarely reported in organisms with cell walls. We investigated the properties of endocytosis in a red alga, Gracilariopsis lemaneiformis. The cells non-selectively internalized extracellular fluid into large-scale endocytic vesicles (1.94 ± 0.51 µm), and this process could be inhibited by 5-(N-ethyl-N-isopropyl) amiloride, an macropinocytosis inhibitor. Moreover, endocytosis was driven by F-actin, which promotes formation of ruffles and cups from the cell surface and facilitates formation of endocytotic vesicles. After vesicle formation, endocytic vesicles could be acidified and acquire digestive function. These results indicated macropinocytosis in G. lemaneiformis. Abundant phosphatidylinositol kinase and small GTPase encoding genes were found in the genome of this alga, while PI3K, Ras, and Rab5, the important participators of traditional macropinocytosis, seem to be lacked. Such findings provide a new insight into endocytosis in organisms with cell walls and facilitate further research into the core regulatory mechanisms and evolution of macropinocytosis.

Identification of epigenetic histone modifications and analysis of histone lysine methyltransferases in Alexandrium pacificum.

Alexandrium pacificum is a toxic dinoflagellate that can cause harmful algal blooms (HABs). The molecular mechanisms of HABs are still poorly understood, especially at the epigenetics level. Organism growth and metabolic processes are affected by histone modifications, an important mode of epigenetic regulation. In this study, various types of modifications, including methylation, acetylation, ubiquitination, and phosphorylation in A. pacificum cells were identified by using pan-antibodies, mass spectrometry, and an H3 modification multiplex assay kit. The modification abundance of H3K4me2 and H3K27me3 of A. pacificum varied under different growth conditions detected by Western blots. A class of SET domain genes (SDGs) encoding histone lysine methyltransferase was analyzed. A total of 179 SDG members were identified in A. pacificum, of which 53 sequences encoding complete proteins were classified into three categories by phylogenetic analysis, conserved domains and motifs analysis. Expression analysis and real-time polymerase chain reaction validation showed that the expressions of some SDGs were significantly influenced by light, nitrogen, phosphorus and manganese supplements. The results revealed that histone lysine methylation played an important role in responding to HABs inducing conditions. This study provided useful information for the further exploration of the role and regulatory mechanism of SDGs in the rapid growth of A. pacificum.

Single-cell transcriptome sequencing revealing the difference in photosynthesis and carbohydrate metabolism between epidermal cells and non-epidermal cells of Gracilariopsis lemaneiformis (Rhodophyta).

The allocation of photoassimilates is considered as a key factor for determining plant productivity. The difference in photosynthesis and carbohydrate metabolism between source and sink cells provide the driven force for photoassimilates' allocation. However, photosynthesis and carbohydrate metabolism of different cells and the carbon allocation between these cells have not been elucidated in Gracilariopsis lemaneiformis. In the present study, transcriptome analysis of epidermal cells (EC) and non-epidermal cells (NEC) of G. lemaneiformis under normal light conditions was carried out. There were 3436 differentially expressed genes (DEGs) identified, and most of these DEGs were related to photosynthesis and metabolism. Based on a comprehensive analysis both at physiological and transcriptional level, the activity of photosynthesis and carbohydrate metabolism of EC and NEC were revealed. Photosynthesis activity and the synthesis activity of many low molecular weight carbohydrates (floridoside, sucrose, and others) in EC were significantly higher than those in NEC. However, the main carbon sink, floridean starch and agar, had higher levels in NEC. Moreover, the DEGs related to transportation of photoassimilates were found in this study. These results suggested that photoassimilates of EC could be transported to NEC. This study will contribute to our understanding of the source and sink relationship between the cells in G. lemaneiformis.

Ploidy Identification by Flow Cytometry and Application of the Method to Characterize Seasonal Ploidy Variation of Wild Populations of the Red Alga Gracilariopsis lemaneiformis.

Gracilariopsis lemaneiformis (Gp. lemaneiformis) is an economically important alga. At present, there is no way to quickly and easily determine its ploidy and life cycle dynamics in wild populations, which affects the process of genetic breeding. In this study, we developed and verified a ploidy identification method using flow cytometry and then used it to explore the seasonal fluctuation of the ploidy ratio and the environmental factors that influence it in wild populations of this species. Of the three methods we tested for nucleus extraction, quick chopping was the best because of its high extraction efficiency, low debris background, obvious subcellular scatter plot, and clear typical histogram. Samples from the tip of the alga were more suitable for preparing the nuclear suspension than samples from the base. Generalized linear model analysis based on diagnosis of multicollinearity revealed a negative correlation between temperature and ploidy ratio. Among the environmental factors tested, temperature had the greatest influence on the ploidy ratio, whereas precipitation and sunshine duration had no effect on the ploidy ratio fluctuation. Our study will be useful for material collection and studies of utilization and life cycle dynamics. Moreover, understanding of ploidy dynamics may provide a theoretical basis for improving variety and breeding of Gp. lemaneiformis in the future.

Detection, characterization and expression dynamics of histone proteins in the dinoflagellate Alexandrium pacificum during growth regulation.

Histones are the most abundant proteins associated with eukaryotic nuclear DNA. The exception is dinoflagellates, which have histone protein expression that is mostly reported to be below detectable levels. In this study, we investigated the presence of histone proteins and their functions in the dinoflagellate, Alexandrium pacificum. Histone protein sequences were analyzed, focusing on phylogenetic analysis and histone code. Histone expression was analyzed during the cell cycle and under nutritionally enhanced conditions using quantitative-PCR and western blots. Acid-soluble proteins were subjected to mass spectrometry analysis. To our knowledge, this is the first report of immunological detection of histone proteins (H2B and H4) in any dinoflagellate species. Absolute quantification of histone transcript in activily dividing cells revealed significant transcription in cells. The stable expression of histones during the cell cycle suggested that the histone genes in A. pacificum belonged to a replication-independent class and appeared to have a limited role in DNA packaging. The conservation of numerous post-translationally modified residues of multiple histone variants and differential expression of histones under nutritionally enhanced conditions suggested their functional significance in dinoflagellates. However, we detected histone H2B protein only via mass spectrometry. Histone-like protein was identified as most abundant acid-soluble protein of the cells.

Heterozygous Single Nucleotide Polymorphic Loci in Haploid Gametophytes of Gracilariopsis lemaneiformis (Rhodophyta).

Gracilariopsis lemaneiformis is an important commercial macroalga. Whole-genome resequencing was conducted to identify single nucleotide polymorphisms (SNPs) in parental gametophytes and 60 F1 gametophytes of Gp. lemaneiformis in this study, and 9,989 SNPs located in nonrepetitive sequences were obtained. Among these SNPs, 92.02% of loci were identified as having a heterozygous genotype in at least one gametophyte, and 48.07% of loci had identical heterozygous genotypes in the 62 gametophytes. For each gametophyte, the proportions of homozygous and heterozygous loci ranged between 13.74 and 21.61% (mean of 17.04%) and between 66.36 and 83.59% (mean of 77.12%), respectively. The remainder were missing loci, representing an average 5.84%. Sources of heterozygous SNPs were free of exogenous DNA contamination, cross contamination among individuals, plastid and mitochondrial sequences, chimeras of different thallus parts or different cells, and repetitive sequences. Genotypes of heterozygous SNPs were verified by Sanger sequencing of PCR products and monoclones. Duplications of chromosomal rearrangements in the genome of Gp. lemaneiformis might explain the presence of heterozygous SNPs in haploid gametophytes.

Inhibition of pds gene expression via the RNA interference approach in Dunaliella salina (Chlorophyta).

To investigate the potential of double-stranded RNA interferencing with gene expression in Dunaliella salina, a plasmid pBIRNAI-Dsa was constructed to express hairpin RNA (hpRNA) containing sequences homologous to phytoene desaturase gene (pds), a key gene in carotenoid biosynthesis, and transformed into D. salina by electroporation. The relative transcription level of pds in pBIRNAI-Dsa-treated cells to nontreated cells was quantitated and the gene silencing efficiency by RNAi was evaluated via real-time polymerase chain reaction (PCR). The transcriptions of pds of the pBIRNAI-Dsa-treated group changed compared to those of the control group, and the 2(-delta deltaC)(T) was lowest on the 7th day, corresponding to 0.281265-fold of the relative pds control transcript; a relatively strong gene inhibition effect was therefore deduced. The transcript of pds may be modulated in a wide range, and a reduced transcription even to 28% of the normal level may be tolerated for its survival. This study shows that dsRNA-mediated genetic interference can induce sequence-specific inhibition of gene expression and pBIRNAI-Dsa can be used for transient suppression of gene expression in D. salina. The aim of this study was to exploit dsRNA-mediated gene silencing and to provide a foundation for gene function research in D. salina.