On microbial community of Pyropia haitanensis by metagenomic analysis

Microorganisms plays an important role in the growth of Pyropia haitanensis.To understand the structural and functional diversity of the microorganism community of P.haitanensis(PH40),the associated metabolic pathway network in cluster of orthologous groups(COG)and Kyoto Encyclopedia of Genes and Genomes(KEGG),and carbohydrate-active enzymes(CAZymes)were explored in metagenomic analysis.DNA extraction from gametophytes of P.haitanensis was performed first,followed by library construction,sequencing,preprocessing of sequencing data,taxonomy assignment,gene prediction,and functional annotation.The results show that the predominant microorganisms of P.haitanensis were bacteria(98.98%),and the phylum with the highest abundance was Proteobacteria(54.64%),followed by Bacteroidetes(37.92%).Erythrobacter(3.98%)and Hyunsoonleella jejuensis(1.56%)were the genera and species with the highest abundance of bacteria,respectively.The COG annotation demonstrated that genes associated with microbial metabolism was the predominant category.The results of metabolic pathway annotation show that the ABC transport system and two-component system were the main pathways in the microbial community.Plant growth hormone biosynthesis pathway and multi-vitamin biosynthesis functional units(modules)were the other important pathways.The CAZyme annotation revealed that the starch might be an important carbon source for microorganisms.Glycosyl transferase family 2(GT2)and glycosyl transferase family 3(GT3)were the highly abundant families in glucoside transferase superfamily.Six metagenome-assembled genomes containing enzymes involved in the biosynthesis of cobalamin(vitamin B 12)and indole-3-acetic acid were obtained by binning method.They were confirmed to belong to Rhodobacterales and Rhizobiales,respectively.Our findings provide comprehensive insights into the microorganism community of Pyropia.

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

The genus Pyropia contains several important cultivated species.Genetic research in nori species has mainly focused on the cell nucleus,with few studies on organelles(chloroplast and 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.

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

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

Cytological and transcriptional analysis reveal phosphatidylinositol signaling pathway plays key role in mitotic division of Pyropia yezoensis

The phosphatidylinositol(PI)signaling system,a central regulator of eukaryotic metabolism,is widely found in eukaryotes for regulating a variety of cell activities.Most of the genes in the PI signaling system were found conserved in Pyropia yezoensis.In this experiment,wortmannin was used as an inhibitor to inhibit the activity of phosphatidylinositol-3 kinase(PI3K),an important regulator of the PI signaling system.After wortmannin treatment,the mitotic division of P.yezoensis was signifi cantly inhibited in a dose-dependent manner,and the mitotic division percentage was reduced by 68.1%and 91.9%in the 5-and 10-μmol/L groups,respectively.When thalli were treated with wortmannin,the levels of reactive oxygen species(ROS)were signifi cantly decreased.Furthermore,the expression level of PI3K was inhibited and the expression levels of downstream genes regulated by PI3K was signifi cantly changed.In the PI3K-AGC signaling pathway,the expression levels of Serine/threonine protein kinase(AGC)and cyclindependent kinases A(CDKA)were downregulated,while WEE1 kinase gene(WEE1)was upregulated.Three nicotinamide adenine dinucleotide phosphate(NADPH)oxidase genes were downregulated after wortmannin treatment.These results indicate that the PI signaling system plays an important role in the regulation of cell activity in P.yezoensis.It was speculated that the growth and development of P.yezoensis might be regulated by P.yezoensis PI3K,which promoted the expression of the AGC gene and further regulates the expression of downstream WEE1 and CDKA genes to advance mitotic division,and also promoted the expression level of NADPH oxidase that regulates ROS homeostasis.

Quantification of Photosynthetic Pigments in Neopyropia yezoensis Using Hyperspectral Imagery

Phycobilisomes and chlorophyll-a(Chla)play important roles in the photosynthetic physiology of red macroalgae and serve as the primary light-harvesting antennae and reaction center for photosystem Ⅱ.Neopyropia is an economically important red macroalga widely cultivated in East Asian countries.The contents and ratios of 3 main phycobiliproteins and Chla are visible traits to evaluate its commercial quality.The traditional analytical methods used for measuring these components have several limitations.Therefore,a high-throughput,nondestructive,optical method based on hyperspectral imaging technology was developed for phenotyping the pigments phycoerythrin(PE),phycocyanin(PC),allophycocyanin(APC),and Chla in Neopyropia thalli in this study.The average spectra from the region of interest were collected at wavelengths ranging from 400 to 1000 nm using a hyperspectral camera.Following different preprocessing methods,2 machine learning methods,partial least squares regression(PLSR)and support vector machine regression(SVR),were performed to establish the best prediction models for PE,PC,APC,and Chla contents.The prediction results showed that the PLSR model performed the best for PE(R_(Test^(2))=0.96,MAPE=8.31%,RPD=5.21)and the SVR model performed the best for PC(R_(Test^(2))=0.94,MAPE=7.18%,RPD=4.16)and APC(R_(Test^(2))=0.84,MAPE=18.25%,RPD=2.53).Two models(PLSR and SVR)performed almost the same for Chla(PLSR:R_(Test^(2))=0.92,MAPE=12.77%,RPD=3.61;SVR:R_(Test^(2))=0.93,MAPE=13.51%,RPD=3.60).Further validation of the optimal models was performed using field-collected samples,and the result demonstrated satisfactory robustness and accuracy.The distribution of PE,PC,APC,and Chla contents within a thallus was visualized according to the optimal prediction models.The results showed that hyperspectral imaging technology was effective for fast,accurate,and noninvasive phenotyping of the PE,PC,APC,and Chla contents of Neopyropia in situ.This could benefit the efficiency of macroalgae breeding,phenomics research,and other related applications.

Discrepancy in photosynthetic responses of the red alga Pyropia yezoensis to dehydration stresses under exposure to desiccation,high salinity,and high mannitol concentration

Macroalgae that inhabit intertidal zones are exposed to the air for several hours during low tide and must endure desiccation and high variations in temperature, light intensity, and salinity. Pyropia yezoensis (Rhodophyta, Bangiales), a typical intertidal red macroalga that is commercially cultivated in the northwestern Pacific Ocean, was investigated under different dehydration stresses of desiccation, high salinity, and high mannitol concentration. Using chlorophyll fluorescence imaging, photosynthetic activities of P. yezoensis thalli were analyzed using six parameters derived from quenching curves and rapid light curves. A distinct discrepancy was revealed in photosynthetic responses to different dehydration stresses. Dehydration caused by exposure to air resulted in rapid decreases in photosynthetic activities, which were always lower than two other stresses at the same water loss (WL) level. High salinity only reduced photosynthesis significantly at its maximum WL of 40% but maintained a relatively stable maximum quantum yield of photosystem II (PSII) (Fv/Fm). High mannitol concentration induced maximum WL of 20% for a longer time (60 min) than the other two treatments and caused no adverse influences on the six parameters at different WL except for a significant decrease in non-photochemical quenching (NPQ) at 20% WL. Illustrated by chlorophyll fluorescence images, severe spatial heterogeneities were induced by desiccation with lower values in the upper parts than the middle or basal parts of the thalli. The NPQ and rETRmax (maximum relative electron transport rate) demonstrated clear distinctions for evaluating photosynthetic responses, indicating their sensitivity and applicability. The findings of this study indicated that the natural dehydration of exposure to air results in stronger and more heterogeneous effects than those of high salinity or high mannitol concentration.