Physiological effects of nitrogen deficiency and recovery on the macroalga Gracilariopsis lemaneiformis (Rhodophyta)

Algal metabolites are the most promising feedstocks for bio‐energy production. Gracilariopsis lemaneiformis seems to be a good candidate red alga for polysaccharide production, especially relating to the agar production industry. Nitrogen deficiency is an efficient environmental pressure used to increase the accumulation of metabolites in algae. However, there are no studies on the physiological effects of G. lemaneiformis in response to nitrogen deficiency and its subsequent recovery. Here we integrated physiological data with molecular studies to explore the response strategy of G. lemaneiformis under nitrogen deficiency and recovery. Physiological measurements indicated that amino acids and protein biosynthesis were decreased, while endogenous NH₄⁺ and soluble polysaccharides levels were increased under nitrogen stress. The expression of key genes involved in these pathways further suggested that G. lemaneiformis responded to nitrogen stress through up‐regulation or down‐regulation of genes related to nitrogen metabolism, and increased levels of endogenous NH₄⁺ to complement the deficiency of exogenous nitrogen. Consistent with the highest accumulation of soluble polysaccharides, the gene encoding UDP‐glucose pyrophosphorylase, a molecular marker used to evaluate agar content, was dramatically up‐regulated more than 4‐fold compared to the relative expression of actin after 4 d of nitrogen recovery. The present data provide important information on the mechanisms of nutrient balance in macroalgae.     

Structure of reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis (Gracilariaceae,Rhodophyta)

Reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis collected from Qingdao city were studied with a light and a transmission electron microscope. The special superficial arrangement of spermatangium for this species was clearly observed, and the ultrastructure of spermatangial devel-opment revealed the similar cytodynamic pattern followed by all the Gracilariaceae members devel-oped from spermatangial mother cells to spermatangium. The female reproductive apparatus before fertilization was also observed and trichogyne was found protruding above the cortex, contrary to the earlier reports. Tetrasporangium was formed by an outer cortical cell and the tetraspores became spherical and expended after being released.     

Structure of reproductive apparatus of Gracilaria/ Gracilariopsis lemaneiformis (Gracilariaceae, Rhodophyta)

Reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis collected from Qingdao city were studied with a light and a transmission electron microscope. The special superficial arrangement of spermatangium for this species was clearly observed, and the ultrastructure of spermatangial development revealed the similar cytodynamic pattern followed by all the Gracilariaceae members developed from spermatangial mother cells to spermatangium. The female reproductive apparatus before fertilization was also observed and trichogyne was found protruding above the cortex, contrary to the earlier reports. Tetrasporangium was formed by an outer cortical cell and the tetraspores became spherical and expended after being released. Supported by the National Natural Sciences Foundation of China (Grant No. 40606034) and the National High-Tech Research and Development Program of China (Grant No. 2006AA10A413)     

Relationship between gene expression of UDP-glucose pyrophosphorylase and agar yield in Gracilariopsis lemaneiformis (Rhodophyta)

UDP-glucose pyrophosphorylase (UGPase) is an enzyme involved in the biosynthesis of UDP-D-galactose, a subunit of agar in red seaweeds. The relationship between agar content and expression levels of the UGPase encoding gene (glugp) was studied in thalli under different treatment conditions using a quantitative real-time PCR-based method (qPCR). Moreover, this qPCR method for the measurement of glugp expression was also applied to commercial varieties of Gracilariopsis lemaneiformis, a red macroalga, in order to examine its reliability on material obtained from field cultivation. Both the agar content and glugp gene expression in G. lemaneiformis grown under low salinity (17?‰) conditions for 1 week showed a slight increase in comparison with the control group (33?‰ salinity, natural salinity of seawater), but the difference was not statistically significant (P?>?0.05). However, when the culture time was extended to 2 weeks, the increase in both the agar yield and glugp expression became significant (P?glugp expression (P?>?0.05). Our results suggest that glugp gene expression and agar content are highly positively correlated and that the measurement of glugp expression, using only a small amount of thalli material, may be an efficient approach to evaluate agar content. In addition, both the agar content and glugp expression in cultivars 981, 07-2, and ZC differed significantly from those of MT-18. The findings of this study suggest that UGPase may be involved in agar biosynthesis and indicate that glugp gene expression could be a fairly reliable molecular marker to reflect the agar content of strains during breeding and selection of G. lemaneiformis.     

Cloning and characterization of two types of ubiquitin genes from Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta)

Two types of ubiquitin genes were isolated from the marine red alga Gracilaria lemaneiformis: a ubiquitin-52 amino acid fusion protein gene, and a 6-unit polyubiquitin gene. Alignment of polyubiquitins among three red algae (Gracilaria lemaneiformis, Gracilaria verrucosa, Aglaothamnion neglectum) and other species revealed that there were six ubiquitin repeats in all three red algae polyubiquitins, and that glutamine was the final amino acid residue in the terminal repeat of the polyprotein in the two Gracilaria sequences. Southern blot analysis revealed that both genes were encoded by low-copy number genes. Semi-quantitative RT-PCR was performed to investigate the expression of these two genes in two phases of G. lemaneiformis. The result revealed that the monoubiquitin was phase-relative, and upregulated in tetrasporophytes compared with female gametophytes. The polyubiquitin gene was expressed at similar levels in both phases.     

Genome Survey Sequencing for the Characterization of the Genetic Background of Rosa roxburghii Tratt and Leaf Ascorbate Metabolism Genes

Rosa roxburghii Tratt is an important commercial horticultural crop in China that is recognized for its nutritional and medicinal values. In spite of the economic significance, genomic information on this rose species is currently unavailable. In the present research, a genome survey of R. roxburghii was carried out using next-generation sequencing (NGS) technologies. Total 30.29 Gb sequence data was obtained by HiSeq 2500 sequencing and an estimated genome size of R. roxburghii was 480.97 Mb, in which the guanine plus cytosine (GC) content was calculated to be 38.63%. All of these reads were technically assembled and a total of 627,554 contigs with a N50 length of 1.484 kb and furthermore 335,902 scaffolds with a total length of 409.36 Mb were obtained. Transposable elements (TE) sequence of 90.84 Mb which comprised 29.20% of the genome, and 167,859 simple sequence repeats (SSRs) were identified from the scaffolds. Among these, the mono-(66.30%), di-(25.67%), and tri-(6.64%) nucleotide repeats contributed to nearly 99% of the SSRs, and sequence motifs AG/CT (28.81%) and GAA/TTC (14.76%) were the most abundant among the dinucleotide and trinucleotide repeat motifs, respectively. Genome analysis predicted a total of 22,721 genes which have an average length of 2311.52 bp, an average exon length of 228.15 bp, and average intron length of 401.18 bp. Eleven genes putatively involved in ascorbate metabolism were identified and its expression in R. roxburghii leaves was validated by quantitative real-time PCR (qRT-PCR). This is the first report of genome-wide characterization of this rose species.     

Glutamine Synthetase (GS): A Key Enzyme for Nitrogen Assimilation in The Macroalga Gracilariopsis lemaneiformis (Rhodophyta)

This study aimed to address the importance of glutamine synthetase II (GSII) during nitrogen assimilation in macroalga Gracilariopsis lemaneiformis. The cDNA full‐length sequence of the three glGSII genes was revealed to have the 5′ m⁷G cap, 5′‐untranslated region, open reading frame (ORF), 3′‐untranslated region, and a 3′ poly (A) tail. The three glGSIIs were classified into plastid glGS2 and cytosolic glGS1‐1 and glGS1‐2, having conserved GSII domains but different cDNA sequences. The complicated 5′ end flanking region indicates complex function of glGS genes. glGS1 genes were significantly up‐regulated under the different NH₄⁺: NO₃^? ratio (i.e., 40:10, 25:25, 10:40, and 0:50) except glGS2 which dramatically up‐regulated under the low NH₄⁺: NO₃^? ratio (i.e., 10:40 and 0:50) during different cultivation times. These different expression patterns perhaps are due to the different biological roles of GS1 and GS2 in the gene family. Furthermore, hypothetical working model of nitrogen assimilation pathway exhibiting the role of glGS1 and glGS2 is proposed. Finally, glGS2 was expressed in Escherichia coli BL21 (DE3), and the optimal conditions for culture (15°C, overnight), purification (500 mM imidazole washing), and activity (pH 7.4, 37°C) were established. This study lays a very important foundation for exploring the role of GS in nitrogen assimilation in algae and plants.     

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).     

Isolation and identification of two strains of pathogenic bacteria and their effects on the volatile metabolites of Gracilariopsis lemaneiformis (Rhodophyta)

Apical necrosis is a widely distributed disease in the culture of the marine agar-producing alga, Gracilariopsis lemaneiformis. In this study, 16 strains of epiphytic bacteria were isolated from the tip bleaching parts of G. lemaneiformis; of the 16 strains, two strains could induce healthy algal tips to become necrotic. They were identified as Thalassospira sp. and Vibrio parahaemolyticus by 16S rDNA sequence analysis and biochemical characterization. Using solid phase microextraction and gas chromatography–mass spectrometry, the variation of volatile metabolites of G. lemaneiformis infected by these two strains of pathogenic bacteria was analyzed. The results showed that E-2-nonenal and 1-octen-3-one differed very significantly (P < 0.01) between the Thalassospira infected group and the control group, while trichloromethane, 3-methyl butanal, benzaldehyde, and E-2-decenal differed significantly (P < 0.05). The difference of 1-octen-3-one, benzaldehyde, and E-2-nonenal between the V. parahaemolyticus infected group and the control group was very significant (P < 0.01), while 3-methyl butanal and octanal were significantly different (P < 0.05). In conclusion, 1-octen-3-one, E-2-nonenal, and benzaldehyde might be the characteristic metabolites for the pathogenic infection and could be used as biomarkers for the disease prevention of G. lemaneiformis.     

Effect of dark treatment on the starch degradation and the agar quality of cultivated Gracilariopsis lemaneiformis (Rhodophyta,Gracilariales) from Venezuela

The effect of darkness and ammonia on Gracilariopsis lemaneiformis agar yield and quality was studied. Plants were cultivated under controlled conditions in the laboratory and fertilized with ammonia prior to a month storage in the dark. Starch inclusions are known to interfere with the mechanical properties of the isolated agar. The starch content of algal tissue and the activities of the floridean starch phosphorylase and -glucosidase were measured during this storage period as a way to follow the enhancement of starch degradation in the absence of light, and to measure the effect of darkness on the content and quality of the synthesized agar. The agar yield, gel strength, 3,6-anhydrogalactose, sulphate and starch content were considerably affected by this dark treatment. The values obtained reflect a pattern of optimization of agar quality when Gracilariopsis lemaneiformis was stored in the dark. The isolated agar following dark treatment is less contaminated by starch inclusions. This is shown by the improvement in the agar yield and quality, the nutrient status of the plant and the reduction of the starch content.Abbreviations DW: Dry weight - FW: Fresh weight - S: Sulphate - 3,6-AG: 3,6-anhydrogalactose - GS: Gel strenght at 1.5% concentration - CX: Cold extract at 25 °C - A: Autoclaved extract at 121 °C - MW: Molecular weight - PFD: Photon flux density     

Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.     

Temperature response of photosynthetic light‐ and carbon‐use characteristics in the red seaweed Gracilariopsis lemaneiformis (Gracilariales,Rhodophyta)

The red seaweed Gracilariopsis is an important crop extensively cultivated in China for high‐quality raw agar. In the cultivation site at Nanao Island, Shantou, China, G. lemaneiformis experiences high variability in environmental conditions like seawater temperature. In this study, G. lemaneiformis was cultured at 12, 19, or 26°C for 3 weeks, to examine its photosynthetic acclimation to changing temperature. Growth rates were highest in G. lemaneiformis thalli grown at 19°C, and were reduced with either decreased or increased temperature. The irradiance‐saturated rate of photosynthesis (P_max) decreased with decreasing temperature, but increased significantly with prolonged cultivation at lower temperatures, indicating the potential for photosynthesis acclimation to lower temperature. Moreover, P_max increased with increasing temperature (~30 μmol O₂ · g^?1FW · h^?1 at 12°C to 70 μmol O₂ · g^?1FW · h^?1 at 26°C). The irradiance compensation point for photosynthesis (I_c) decreased significantly with increasing temperature (28 μmol photons · m^?2 · s^?1 at high temperature vs. 38 μmol photons · m^?2 · s^?1 at low temperature). Both the photosynthetic light‐ and carbon‐use efficiencies increased with increasing growth or temperatures (from 12°C to 26°C). The results suggested that the thermal acclimation of photosynthetic performance of G. lemaneiformis would have important ecophysiological implications in sea cultivation for improving photosynthesis at low temperature and maintaining high standing biomass during summer. Ongoing climate change (increasing atmospheric CO₂ and global warming) may enhance biomass production in G. lemaneiformis mariculture through the improved photosynthetic performances in response to increasing temperature.     

The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation Genetic Diagnosis of Chromosomal Abnormalities

Reliable and accurate pre-implantation genetic diagnosis(PGD) of patient’s embryos by next-generation sequencing(NGS) is dependent on efficient whole genome amplification(WGA) of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA(15 pg) and single cells, we showed that the two PCR-based WGA systems Sure Plex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification(MDA) system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing(CNV-Seq) was applied to single cell WGA products derived by either Sure Plex or MALBAC amplification, we showed that known disease CNVs in the range of 3e15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either Sure Plex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient’s cohort for uterine transplantation.     

Comprehensive Draft Genome Analyses of Three Rockfishes (Scorpaeniformes,Sebastiscus) via Genome Survey Sequencing

Sebastiscus species, marine rockfishes, are of essential economic value. However, the genomic data of this genus is lacking and incomplete. Here, whole genome sequencing of all species of Sebastiscus was conducted to provide fundamental genomic information. The genome sizes were estimated to be 802.49 Mb (S. albofasciatus), 786.79 Mb (S. tertius), and 776.00 Mb (S. marmoratus) by using k-mer analyses. The draft genome sequences were initially assembled, and genome-wide microsatellite motifs were identified. The heterozygosity, repeat ratios, and numbers of microsatellite motifs all suggested possibly that S. tertius is more closely related to S. albofasciatus than S. marmoratus at the genetic level. Moreover, the complete mitochondrial genome sequences were assembled from the whole genome data and the phylogenetic analyses genetically supported the validation of Sebastiscus species. This study provides an important genome resource for further studies of Sebastiscus species.     

基于全基因组测序的法医单核苷酸多态性系谱推断研究

目的 通过全基因组测序（whole genome sequencing，WGS）获得高密度单核苷酸多态性（single nucleotide polymorphism，SNP）分型数据，评估分型准确性，研究建立WGS数据用于法医SNP系谱推断的方法。方法 通过华大MGISEQ-200RS测序平台对样本进行深度为30×的WGS，从测序数据中提取Wegene GSA芯片中的645 199个常染色体SNP位点，质控过滤后运用IBS/IBD算法计算预测亲缘关系，并对样本的族群来源进行分析。结果 从测序数据中提取的SNP分型与Wegene GSA芯片分型的一致率大于99.62%。测序获得的SNP数据使用IBS算法可预测1~4级亲缘关系，4级亲缘预测置信区间准确性达100%，使用IBD算法可预测1~7级亲缘关系，7级亲缘预测为有亲缘关系的准确性达100%，通过高深度WGS数据获取的SNP系谱推断能力与芯片预测结果无显著差异。同时，WGS数据用于族群推断与调查结果一致。结论 WGS技术可应用于法医SNP系谱推断，为案件侦破提供线索。