Construction of a dense genetic linkage map and mapping quantitative trait loci for economic traits of a doubled haploid population of Pyropia haitanensis (Bangiales, Rhodophyta)

The genotypes of 4550 LP markers that were mapped onto the genetic map. (XLSX 1645 kb

Genetic analyses of six quantitative traits of a doubled haploid population of Porphyra haitanensis Chang et Zheng (Bangiales, Rhodophyta)

National High Technology Research & Development Program of China ("863" Program) [2006AA10A413]; National Natural Science Foundation of China [40806065]; Construction Program of Science and Technological Platform of Fujian Province, China [2007 N2011]The first doubled haploid (DH) population of Porphyra haitanensis was created by crossing a wild-type line with a red type, artificial pigmentation, mutant line, by means of single somatic cell clone cultivation. Six quantitative traits (frond length (FL), frond width (FW), frond thickness (FT), fresh weight (W), growth rate of frond length (LGR), and growth rate of fresh weight (WGR)) from the DH population were analyzed. The frequency of each quantitative trait is in accordance with a normal distribution. Variable coefficients were between 20.43% and 57.35%, and while the mean of each quantitative trait was between the parents, it was closer to the paternal for six traits. Correlation analysis among the six traits showed significant positive correlations between FL and W, FW, and W, LGR and W, and WGR and W. There appears to be no correlation between FT and W. Heritability, number of genes controlling each quantitative trait and gene interactions, were also analyzed. Heritability of the six traits was greater than 70%, and heritability was not correlated with the number of genes controlling the corresponding quantitative trait. Frond thickness was the most heritable trait (95.30%), but this had the fewest control genes (7.52). According to the estimated coefficients of skewness and kurtosis, gene interaction was absent for LGR, but complementary gene interaction was observed in FW and W. In FL, FT, and WGR it is possible that complementary or duplicate gene interaction is involved. Our results enrich our understanding of Porphyra genetics and will help determine selection and breeding procedures for P. haitanensis

Cells tile a flat plane by controlling geometries during morphogenesis of Pyropia thalli

Background Pyropia haitanensis thalli, which are made of a single layer of polygonal cells, are a perfect model for studying the morphogenesis of multi-celled organisms because their cell proliferation process is an excellent example of the manner in which cells control their geometry to create a two-dimensional plane. Methods Cellular geometries of thalli at different stages of growth revealed by light microscope analysis. Results This study showed the cell division transect the middle of the selected paired-sides to divide the cell into two equal portions, thus resulting in cell sides ≥4 and keeping the average number of cell sides at approximately six even as the thallus continued to grow, such that more than 90% of the cells in thalli longer than 0.08 cm had 5–7 sides. However, cell division could not fully explain the distributions of intracellular angles. Results showed that cell-division-associated fast reorientation of cell sides and cell divisions together caused 60% of the inner angles of cells from longer thalli to range from 100–140°. These results indicate that cells prefer to form regular polygons. Conclusions This study suggests that appropriate cell-packing geometries maintained by cell division and reorientation of cell walls can keep the cells bordering each other closely, without gaps

Mutagenesis and Blade Phenotypic Traits of Neoporphyra haitanensis F1 Lines

Neoporphyra haitanensis is a traditional coastal aquaculture species in China; it is rich in essential amino acids, minerals, and vitamins. It is not only beneficial economically, but also adjusts the ecosystem. However, fine varieties of N. haitanensis are lacking even with the enlargement and extension of the N. haitanensis growing area. Since humans began breeding N. haitanensis, most produced strains have been domesticated from wild N. haitanensis collected from rocks. The germplasm collection and usage is considered the bottleneck issue that often causes N. haitanensis quality degradation. Therefore, it is important to breed new varieties. Selective breeding, cross-breeding, and mutation breeding are among the most common methods used to breed new N. haitanensis varieties. Mutation breeding is a quick, simple, and convenient genetic tools. For several decades, natural means to induce genetic diversity have been exploited to breed new varieties, as the naturally occurring mutations are insufficient. One of the most important genetic breakthroughs was the invention of artificial methods to induce mutations. Physical mutagenesis is a safer and more efficient procedure than chemical mutagenesis. To obtain artificial mutants, N. haitanensis (NSD35) gametophytes were treated with different γ-ray irradiation intensities (700 Gy, 900 Gy, 1100 Gy, 1300 Gy, and 1500 Gy). After culture recovery, the results showed that gametophytic cell death increased with the irradiation escalation, with more than 90% cell viability after low radiation dose treatment (700 Gy and 900 Gy). With doses more than 1100 Gy, the cell viability decreased, in which the NSD35 cellular space became larger and a few middle cells died, although no obvious change was observed in tip cells. After induction, the cell morphology changed with higher radiation doses (1300 Gy and 1500 Gy). Some cells exhibited microscopic features, such as enlarged size and alternation from irregular cell shape to circular for some cells. Notably, the cell death rate decreased systematically from the blade base to its tip and from that to its center. Microscopic examination revealed that the number of mutant cells increased and then decreased with the radiation dose rise. The pigmented mutant cells frequency was higher in low radiation doses, further increasing with the rise in irradiation from 700 Gy to 1300 Gy. Nonetheless, the pigmented mutant cells frequency decreased with the highest dose of 1500 Gy. The best mutagenic effect was observed in gametophytes treated with 1300 Gy as they had the largest number of pigmented mutant cells. Cells cultured in vitro were obtained by enzymatic hydrolysis and individual regeneration. Preliminary screening of 67 mutants was performed to construct the mutant library, and 45 pigmented mutant strains, 51 morphological mutants, and 11 other mutants were obtained. Most of the mutants showed two or more mutations of phenotypic character. Twenty-one F1 generation cell lines were screened, and 14 traits (length, width, fresh weight, daily length growth rate, daily width growth rate, daily weight growth rate, length-breadth ratio, base thickness, center thickness, tip thickness, color, base section form, leaf form, saw tooth size, twist, and maturity) were analyzed by correlation analysis and system analysis. The coefficients of variation results showed significant phenotypic differences between F1 and control cells. The F1 coefficients of variation ranged from 8.74% to 59.49%, revealing a moderate variability. Correlation analysis also showed that most of the traits had significant correlations. There was no significant correlation between the length and width or fresh weight, while a significant positive correlation was observed between the width and leaf fresh weight (P < 0.01). Significant positive correlations were also found between quantitative traits, while there was no correlation between qualitative and quantitative traits. These allow indirect selection to improve breeding efficiency. Cluster analysis showed that at the Euclidean distance of 20, all 21 individuals analyzed were clustered into four groups. Overall, the phenotypic traits of different groups were significantly different. Mutants are critical materials for studying the genetic regulation of mechanisms involved in the control of economically important N. haitanensis traits. Pigmented mutants are a dominant-marker trait used for breeding that have high value for theoretical research and practical applications. Our study identified many pigmented mutants that can be used to study relevant biological mechanisms. Nevertheless, the variation coefficients of four quantitative characters (length, width, fresh weight, and thickness) of the F1 N. haitanensis generation were less than those of the control group and were biased towards negative variation. For example, more thin leaf mutants were obtained after the gamma rays´ treatment. It is interesting as a thin N. haitanensis is more suitable for automatic processing, and its primary products can be reprocessed with higher added value. In conclusion, after being treated with γ-ray, the N. haitanensis blades were guided to variation enabling the selection and cultivation of new strains, from which strains with improved traits strains were selected. This study provides interesting materials for accelerating the N. haitanensis breeding research and the selection of excellent varieties

cDNA Cloning and Functional Verification of Transcription Factor NhbZIP1 from Neoporphyra haitanensis

Neoporphyra haitanensis is a macroalgae available in the south coast of China, and it is one of the most widely cultivated seaweeds in China. In recent years, due to global warming, the continuously high temperatures following the White Dew solar term, has led to the decomposition of seedlings of N. haitanensis in Fujian, Zhejiang and other provinces. This has had a huge impact on the coastal N. haitanensis cultivation industry in terms of production and development. Therefore, investigation of the molecular mechanism of high temperature stress response of N. haitanensis and the high-temperature resistance related genes is essential, and the results can also lay a foundation for the breeding of high-temperature resistant varieties of N. haitanensis. A previous study revealed that the basic region Leucine Zipper (bZIP) family transcription factors are one of the largest and most conserved transcription factor families in plants. The family plays an important role in plant response to abiotic stresses, such as high temperature, drought, and osmosis. bZIP transcription factors regulate plant response to abiotic stress by binding to functional genes or regulatory gene promoter cis-elements to activate and induce downstream gene expression. So far, 127, 89, and 216 bZIP transcription factors have been found in Arabidopsis, rice, and maize, respectively. However, previous studies on bZIP have mainly focused on model plants and only some field crops, and the functions of bZIP in macroalgae have not been reported. To this end, NhbZIP1 was screened based on the whole genome and transcriptomic data of N. haitanensis, and the NhbZIP1 gene was cloned and functionally analyzed by molecular biology and bioinformatics techniques. Its structure and expression pattern were also analyzed. Finally, the NhbZIP1 gene was transformed into Chlamydomonas reinhardtii by the "glass bead transformation" method for gene function verification. In this study, a gene product with a length of approximately 1000 bp was obtained by PCR amplification. After sequencing and BLAST analysis, the gene was identified as the bZIP gene of N. haitanensis and named NhbZIP1. Studies have shown that the open reading frame of NhbZIP1 gene is 825 bp in length and encodes 274 amino acids. There are five low-complexity domains and one BRLZ (115~179 aa) structure. BRLZ is a conserved domain of bZIP family and contains a α-coiled helix structure (121~171 aa). The molecular formula of NhbZIP1 is C1193H1935N339O375S7, and the predicted molecular weight of NhbZIP1 is 27 251.95 Da; its theoretical isoelectric point is 5.03, and it contains 32 negative charge residues and 26 positive charge residues in total. Ala (A) content of the protein was 27%, and Arg (R) content was 4.7%. The total average hydrophilic coefficient was 0.089, which indicates that the protein is hydrophilic, and the instability coefficient was 42.68, which indicates that the protein is unstable. There were 15 potential phosphorylation sites and 12 potential O-linkage glycosylation sites in this protein, which had no signal peptide or transmembrane structure. The protein was located in the nucleus and its characteristics were consistent with those of the genes encoding transcription factors, indicating that NhbZIP1 was a bZIP family transcription factor. Phylogenetic analysis showed that NhbZIP1 gene was isolated from Porphyra umbilicalis and was different from that higher plants, indicating that NhbZIP1 gene was relatively conserved in Porphyra and was genetically distant from higher plants. It is speculated that the NhbZIP1 gene of N. haitanensis has a different evolutionary mode from that of other species. Real-time fluorescence quantitative PCR (qRT-PCR) showed that NhbZIP1 gene was significantly induced by high temperature stress, and the expression level of NhbZIP1 gene was about 3.4 times that of the initial level after 6 h of stress. Under long-term high temperature stress, the heat tolerance of N. haitanensis could be enhanced by enhancing the expression of the resistance gene NhbZIP1. To further clarify the molecular function of NhbZIP1 gene, we transformed it into C. reinhardtii for functional verification. The results showed that the expression level of NhbZIP1 gene was relatively stable before 30 min of high temperature treatment, while the gene expression level was significantly increased after 60 min of the treatment, which was 1.8 times that of the initial level. Under subsequent high temperature stress, the gene expression level remained high, which was about 2.6 times that of the initial level after 180 min of high temperature treatment. The biomass of transgenic lines under heat stress was always higher than that of the wild type, and the difference became more significant with the increase of treatment time. The expression levels of heat shock protein family and genes related to antioxidant system in transgenic lines were significantly higher than that of the wild type. The results showed that NhbZIP1 gene plays an important role in activating the expression of downstream stress-resistant genes in the response to heat stress in N. haitanensis, suggesting that NhbZIP1 gene may enhance the heat tolerance of algae by regulating the expression of HSPs and activating the expression of genes encoding antioxidant enzymes. This study helps to clarify the molecular mechanism of bZIP transcription factor in regulating the response of N. haitanensis to high temperature stress and provides theoretical basis for guiding the breeding of new varieties with high temperature tolerance

Investigating the Mechanisms Underlying the Low Irradiance-Tolerance of the Economically Important Seaweed Species <i>Pyropia haitanensis</i>

Pyropia haitanensis, one of the most economically and ecologically important seaweed species, is often exposed to persistent or transient low irradiance (LI), resulting in limited yield and quality. However, the mechanisms mediating P. haitanensis responses to LI are largely unknown. In this study, LI-tolerant (LIT) and LI-sensitive (LIS) P. haitanensis strains were compared regarding their physiological and transcriptomic changes induced by 1 and 4 days of LI (5 μmol photons/m2·s). The results indicated that the inhibition of photomorphogenesis and decreases in photosynthesis and photosynthetic carbon fixation as the duration of LI increased are the key reasons for retarded blade growth under LI conditions. A potential self-amplifying loop involving calcium signaling, phosphatidylinositol signaling, reactive oxygen species signaling, and MAPK signaling may be triggered in blades in response to LI stress. These signaling pathways might activate various downstream responses, including improving light energy use, maintaining cell membrane stability, mitigating oxidative damage, to resist LI stress. Additionally, the LIT strain maintained transcriptional homeostasis better than the LIS strain under LI stress. Specifically, photosynthesis and energy production were relatively stable in the LIT strain, which may help to explain why the LIT strain was more tolerant to LI stress than the LIS strain. The findings of this study provide the basis for future investigations on the precise mechanisms underlying the LI stress tolerance of P. haitanensis

Minimum bias measurement at 13 TeV

The modelling of Minimum Bias (MB) is a crucial ingredient to learn about the description of soft QCD processes and to simulate the environment at the LHC with many concurrent pp interactions (pile-up). We summarise the ATLAS minimum bias measurements with proton-proton collision at 13 TeV center-of-mass-energy at the Large Hadron Collider

Additional file 4: Table S4. of Construction of a dense genetic linkage map and mapping quantitative trait loci for economic traits of a doubled haploid population of Pyropia haitanensis (Bangiales, Rhodophyta)

The bin markers located on the five linkage groups and their genetic distances. (XLSX 25 kb

Floridean Starch and Floridoside Metabolic Pathways of <i>Neoporphyra haitanensis</i> and Their Regulatory Mechanism under Continuous Darkness

Floridean starch and floridoside are the main storage carbohydrates of red algae. However, their complete metabolic pathways and the origin, function, and regulatory mechanism of their pathway genes have not been fully elucidated. In this study, we identified their metabolic pathway genes and analyzed the changes in related gene expression and metabolite content in Neoporphyra haitanensis under continuous dark conditions. Our results showed that genes from different sources, including eukaryotic hosts, cyanobacteria, and bacteria, were combined to construct floridean starch and floridoside metabolic pathways in N. haitanensis. Moreover, compared with those in the control, under continuous dark conditions, floridean starch biosynthesis genes and some degradation genes were significantly upregulated with no significant change in floridean starch content, whereas floridoside degradation genes were significantly upregulated with a significant decrease in floridoside content. This implies that floridean starch content is maintained but floridoside is consumed in N. haitanensis under dark conditions. This study elucidates the “floridean starch–floridoside” metabolic network and its gene origins in N. haitanensis for the first time