Pyricularia oryzae, a well-characterized plant pathogenic fungus, is the causal pathogen of rice blast disease. P. oryzae is also rich in secondary metabolism genes. However, biosynthetic genes for only four groups of secondary metabolites have been well characterized in this fungus. Biosynthetic genes for two of the four groups of secondary metabolites have been identified by activating secondary metabolism. This review focuses on the biosynthesis and roles of the four groups of secondary metabolites produced by P. oryzae. These secondary metabolites include melanin, a polyketide compound required for rice infection; pyriculols, phytotoxic polyketide compounds; nectriapyrones, antibacterial polyketide compounds produced mainly by symbiotic fungi including endophytes and plant pathogens; and tenuazonic acid, a well-known mycotoxin produced by various plant pathogenic fungi and biosynthesized by a unique NRPS-PKS enzyme.Plant pathogenic fungi produce a wide variety of secondary metabolites with unique and complex structures. However, most fungal secondary metabolism genes are poorly expressed under laboratory conditions. Moreover, the relationship between pathogenicity and secondary metabolites remains unclear. To activate silent gene clusters in fungi, successful approaches such as epigenetic control, promoter exchange, and heterologous expression have been reported.  Filamentous fungi, including plant pathogenic fungi, produce a wide variety of secondary metabolites with unique and complex structures. However, the relationship between pathogenicity and secondary metabolites remains unclear in most cases. Filamentous fungi are a rich source of secondary metabolites for drug development. Whole-genome sequencing analyses have revealed that filamentous fungi possess many more secondary metabolism genes than expected, suggesting that most secondary metabolite biosynthetic genes are silent under laboratory conditions. To utilize fungal secondary metabolite production ability, secondary metabolism genes have been activated through many approaches, including epigenetic control, manipulation of global regulators, ribosome engineering, overexpression of pathway-specific transcription factors, co-culture, and heterologous expression of secondary metabolite gene clusters ,2,3,4.Pyricularia oryzae (syn. Magnaporthe oryzae) is the causal pathogen of rice blast disease and is a well-characterized plant pathogen. P. oryzae infects rice plants through an infection-specific organ, the appressorium, and proliferates inside the rice plant via filamentous growth and causes rice blast disease [P. oryzae is also rich in secondary metabolism genes and shown to have 22 polyketide synthase (PKS) genes and eight non-ribosomal peptide synthetase (NRPS) genes [P. oryzae ( disease . P. oryzS) genes ,7. Biosy. oryzae . BiosyntP. oryzae. This review mainly focuses on the four groups of secondary metabolites shown in Here, I review the biosynthesis and biological roles of secondary metabolites in the rice blast fungus P. oryzae produces the black pigment melanin  is biosynthesized using an acetyl-CoA and five malonyl-CoA [Melanin is a well-known black pigment of biological origin. One type of fungal melanin is dihydroxynaphthalene (DHN)-melanin, which is biosynthesized by polymerizing the polyketide compound 1,8-dihydroxynaphthalene  ,13. P. oucidated  [14,15,1onyl-CoA . Then, 1onyl-CoA .Melanin biosynthetic enzymes are targets of agrochemical development, and three types of commercial melanin biosynthesis inhibitors (MBIs) have been developed . These iP. oryzae produces both alcohol and aldehyde analogs. Identification of the genes responsible for this oxidoreductive conversion will help to answer this question.Pyriculol  is a welMGG_10912/MoPKS19) and other genes predicted to be responsible for the biosynthesis of pyriculols have been identified [MGG_10961/MoC19OXR1) responsible for the oxidation of alcohol-type analogs to aldehyde-type analogs has been reported [Pyriculols are polyketide compounds, and the biosynthetic gene cluster has been recently identified . The PKSentified . It has entified . The genNeurospora crassa produces the structurally related salicylaldehyde sordarial  protein, respectively. This genetic modification induced the production of two polyketide compounds, nectriapyrone and its hydroxylated analog.Secondary metabolite production may be strictly regulated to produce under specific environmental conditions. Thus, we predicted that secondary metabolite production may be activated by disturbing signal transduction pathways involved in environmental responses. A two-component system (TCS) is a signal transduction system that regulates various cellular functions in response to environmental signals and is found in bacteria, archaea, plants, slime molds, and fungi ,46. The NEC1/MGG_00806) and an O-methyltransferase gene (NEC2/MGG_14657)  . OverexpG_14657)  [47,48,4G_14657)  from Fus cluster . Nectria cluster ,53,54 frces spp. . A type Streptomyces strains, act as autoregulators of spore germination [Nectriapyrones belong to the class α-pyrone. α-Pyrones have a wide range of biological activities ,57. For mination ,53. Somemination ,36,42,59mination . Nectriamination .Streptomyces spp. Our data have indicated that nectriapyrone can control growth and pigment formation in S. griseus and has a growth-promoting effect on P. oryzae in interactions with S. griseus. Therefore, nectriapyrones may be involved in microbe-microbe interactions with other environmental organisms, including bacteria such as endophytic Streptomyces strains. To identify the active nectriapyrone analogs in this interaction, we analyzed the bioactivity of each analog  are known as TeA producers [P. oryzae also produces a related tetramic acid derivative from valine as a very minor product [Alternaria toxins, TeA is the most toxic and shows acute toxicity to mammals. The oral median lethal doses for male and female mice are 182 or 225 mg kg−1 and 81 mg kg−1 body weight, respectively [−1 body weight day [+-ATPase at micromolar concentrations. Inhibition of the PM H+-ATPase results in depolarization of the membrane potential and eventually necrosis. However, it is still unclear whether TeA is required for plant infection or not. We previously induced TeA production in P. oryzae and identified the biosynthetic gene TAS1, encoding the first reported fungal NRPS-PKS hybrid enzyme [Tenuazonic acid TeA, , a tetra in 1957 . Alternast decay . TeA hasnd crops ,64,65. Troducers ,67. P. o product . Among tectively ,70. The ight day . TeA inhight day . TeA alsight day ,73,74,75ight day ,77,78, aight day . A recenight day  has showd enzyme ,84. In tThe major fungal secondary metabolites, polyketides and nonribosomal peptides, are biosynthesized by PKSs and NRPSs, respectively. Fungal PKSs can be classified into three types. The first type is iterative type I PKSs, which consist of multiple catalytic domains, including ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) main domains, along with several optional β-keto modifying domains, such as β-ketoacyl reductase (KR), dehydratase (DH), and trans-acting enoyl reductase (ER) domains . The secOSM1 gene and 1% dimethyl sulfoxide (DMSO) treatment. OSM1 is the osmosensory mitogen-activated protein kinase (MAPK), which works downstream of the two-component signal transduction system involved in environmental responses. We identified the TeA biosynthetic gene TAS1/MGG_07803 from the induced genes under inducing conditions [TeA was shown to be a hybrid of an isoleucine and two acetates . Becausenditions ,82. TAS1nditions . In othenditions , whereasnditions . In addinditions . These dP. oryzae conducts substrate cyclization [The KS domains of PKS normally catalyze the decarboxylative Claisen condensation of acyl and malonyl blocks to extend the polyketide chain . In contlization . Nonconvlization . KS3 of lization . Furtherlization . Howeverlization . We founlization  althoughlization ,110,111.P. oryzae by TAS1, and its production is induced by osmo-sensory MAPK gene (OSM1) deletion or 1% DMSO treatment. However, the detailed regulatory mechanisms of TeA production have been unknown. We found two positive regulators of TeA production , which regulates TeA production. TAS2 is located in the upstream region of TAS1 (aflR expression) is a well-known global regulator of secondary metabolism identified and characterized in Aspergillus spp. [Monascus pilosus, Cochliobolus heterostrophus, and Fusarium spp. [P. oryzae. Analysis of PoLAE1 deletion and overexpression strains showed that PoLAE1 positively regulated TeA production. We also revealed that two TeA-inducing signals, OSM1 deletion and 1% DMSO treatment, were transmitted via PoLAE1. These results indicated that TeA production was regulated by two specific regulators, TAS2 and PoLAE1, in P. oryzae  is an avirulence gene, and isolates of P. oryzae carrying the ACE1 gene are specifically recognized by rice cultivars carrying the resistance gene Pi33 [ACE1 is a secondary metabolism gene and encodes a PKS-NRPS hybrid enzyme. The secondary metabolite whose synthesis is dependent on ACE1 is predicted to be recognized by resistant rice plants. ACE1 is located in an infection-specific gene cluster consisting of 15 genes [2Cys6-type transcription factor (BC2). The ACE1 gene cluster shows a rice-infection-specific expression pattern. Heterologous co-expression of ACE1 and RAP1 in Aspergillus oryzae causes production of an amide compound similar to the PKS-NRPS-derived backbone of cytochalasans [ACE1-mediated avirulence. These data suggest that the active final product may be a cytochalasin-like compound.ene Pi33 . This re MGG_1244 is an avhalasans . BioactilaeA homolog (MoLAEA/MGG_07964) increases the production of penicillin G compared to the wild type strain. In contrast, the silenced strain does not produce penicillin G. The putative NRPS gene (MGG_14767) for penicillin G biosynthesis is 3-fold upregulated in the overexpression strain, whereas it is 3.8-fold downregulated in the knockdown strain. Transcriptomic data show that MoLAEA regulates genes involved in secondary metabolism. This laeA homolog (MoLAEA/MGG_07964) is different from the laeA homolog (PoLAE1/MGG_01233) involved in TeA production. Multiple laeA homologs may be involved in the control of secondary metabolite production.Penicillin production in rice blast fungus has been recently shown . OverexpABM (MGG_04777) is a monooxidase gene located in a putative secondary metabolite biosynthetic gene cluster containing a polyketide synthase gene (MGG_04775) [P. oryzae. Such fungal 12OH-JA is secreted during infection and helps evade the defense response by inhibiting the induction of JA signaling. In contrast, loss of Abm in P. oryzae causes accumulation of methyl JA (MeJA), which induces host defense and blocks fungal infection. Furthermore, Abm itself is secreted after infection and is predicted to convert plant JA into 12OH-JA to help host colonization. P. oryzae also produces other plant hormones, abscisic acid (ABA) [MoABA4/MGG_07514, homologous to the Botrytis cinerea ABA4 gene responsible for ABA biosynthesis, reduces ABA levels by two-fold [Moaba4 mutant is strongly compromised, suggesting that ABA contributes to the virulence of this fungus. CKS1/MGG_04857 encodes a putative tRNA-Isopentenyl transferase (tRNA-IPT) protein essential for CK biosynthesis [csk1 strain has been characterized. This analysis has shown that P. oryzae-derived CKs are required for full virulence by affecting rice defenses, nutrient distribution, and fungal oxidative stress tolerance.G_04775) . While tid (ABA) , cytokinid (ABA) , and auxid (ABA) , and theid (ABA) ,130. Gentwo-fold . The virP. oryzae is rich in secondary metabolism genes, and some of the secondary metabolites are expected to be involved in rice infection. Here, this review focused on the biosynthesis and biological roles of secondary metabolites in P. oryzae. Five groups of secondary metabolites  have been shown to be produced by P. oryzae. Biosynthetic genes for three  of the five groups of secondary metabolites have been identified by activating secondary metabolism. Nectryapyrones and TeA were induced by manipulating the factors involved in the TCS. Penicillin G was induced by overexpression of a laeA homolog. Activation of secondary metabolism is a useful method for the identification of secondary metabolites. Melanin is a well-characterized secondary metabolite that is essential for rice infection. Identification and gene manipulation of the biosynthetic genes revealed that three groups of secondary metabolites  are not required for rice infection. Nectriapyrones and penicillin G show antibacterial activity and are predicted to be involved in interactions between bacteria. Controlling plant pathogenic fungi is important in agriculture. Unveiling the roles of secondary metabolites of plant pathogens will help in developing agrochemicals.


Amaranthus (A.) hypochondriacus (A.hyp_K_white) and had reported a draft genome in 2014. We selected this landrace because it is well adapted for cultivation in India during the last century and is currently a candidate for TILLING-based crop improvement. More recently, a high-quality chromosome-level assembly of A. hypochondriacus  was reported. Here, we report a chromosome-level assembly of A.hyp_K_white (AhKP) using low-coverage PacBio reads, contigs from the reported draft genome of A.hyp_K_white, raw HiC data and reference genome of Plainsman (A.hyp.V.2.1). The placement of A.hyp_K_white on the phylogenetic tree of grain amaranths of known accessions clearly suggests that A.hyp_K_white is genetically distal from Plainsman and is most closely related to the accession PI619259 from Nepal (Ramdana). Furthermore, the classification of another accession, Suvarna, adapted to the local environment and selected for yield and other desirable traits, is clearly Amaranthus cruentus. A classification based on hundreds of thousands of SNPs validated taxonomy-based classification for a majority of the accessions providing the opportunity for reclassification of a few.In the age of genomics-based crop improvement, a high-quality genome of a local landrace adapted to the local environmental conditions is critically important. Grain amaranths produce highly nutritional grains with a multitude of desirable properties including C4 photosynthesis highly sought-after in other crops. For improving the agronomic traits of grain amaranth and for the transfer of desirable traits to dicot crops, a reference genome of a local landrace is necessary. Toward this end, our lab had initiated sequencing the genome of  Grain amaranth, also known as Ramdana (The God’s grain) or Rajgira or Rajeera, has been in continuous cultivation at least since last century in India. This crop was declared “The Future Crop” by the United States in the 1980s based on a decade of intense research in the 1980s . At a time when gluten-free, protein-rich, high-fiber, and high nutritional values are becoming attractive labels in supermarkets around the globe, grain amaranths deserving all these labels cannot be ignored as a future crop. Furthermore, desirable agronomic traits including drought resistance, C4 photosynthesis, herbicide resistance and high dry-biomass renders grain amaranths as a potential model organism by researchers working on the improvement of other edible dicots. In the context of increasing demand on water and other natural resources from an increasing world population, grain amaranths offer an alternative to other staple cereals such as rice or wheat. With one-sixth of the world population under the poverty line, the value of the seed protein content in amaranth to India cannot be overestimated. Unfortunately, despite India being one of the few countries where multiple landraces of grain amaranths are under continuous cultivation for more than a century, they have received little attention and failed to reach the status of a staple crop.Interestingly, grain amaranths, domesticated around 8000 years ago, enjoyed equal status as corn during the Aztec and Inca civilizations . This prPI558499 (Plainsman), has been deciphered using state-of-the-art technologies including Bionano, HiC, and long PacBio reads  . Such anividuals . Also, aividuals . The draividuals . More reomosomes .Lates calcarifer was assembled from a draft genome using long-read sequencing, transcriptome data, optical/genetic mapping and synteny to two closely related seabasses , 94 accessions for grain amaranths have been classified . While Gde novo assembly of a landrace (A.hyp_K_white) and demonstrate that, in the presence of a reference genome for a distal variety, a chromosome-level assembly can be generated at a reasonable cost. Also, we normalized the variants from GBS and WGS data for various accessions enabling decoration of the phylogenetic tree including many accessions with the landraces of interest from India.In this article, we report a This section is divided into three parts. The first section describes the results from our efforts to assemble a near-chromosome level assembly for a landrace A.hyp_K_white using contigs from previously reported draft genome of A.hyp_K_white , low-covPacBio reads using RSII technologies sequenced in 2013 with an average length of 7.5 kb with a coverage of 25X for A.hyp_K_white were assembled using state-of-the-art tools CANU and FLYE Amaranthus hypochondriacus sequenced here are different from the A.hyp_Plainsman variety .We have attempted to classify the landraces reported here using multiple approaches. In the first approach we have attempted to create a classification tree with WGS data for landraces sequenced here and only those accessions for which WGS data was available , A.hyp_Mexico_PI511731(GBS). Interestingly, A.hyp_Mexico_ PI511721 clusters with A.hyp_Plainsman_PI558499, which does not show any pink components.ADMIXTURE analysis shown in ArabidopsisArabidopsis to visualize the expression profile of the corresponding orthologs on AhKP.In a previous report, our lab had sequenced and reported developmental transcriptome of A.hyp_K_white from several tissues , 2017. HArabidopsis gene model as shown in Here, a chromosome level assembly (AhKP) of a landrace, A.hyp_K_white, under contiguous cultivation in India for over several centuries is reported. A draft genome for the same landrace was reported by our group in 2014 . The assA. hypochondriacus from Mexico and A. hybridus from Pakistan (A. hybridus accession from Greece (A.hyb_Greece_PI605351) included in the classification is not in the same clade as Plainsman but, instead, clusters along with all accessions from A. cruentus  from A. cruentus. All the accessions from A. quitensis and A. caudatus clusters together in a single clade with two branches of A. quitensis enclosing A. caudatus, which is also reported using only GBS data using A.hyp.V.2.1 as reference , a pureline released by University of Agricultural Sciences (UAS), Bangalore-1992 from the material “Rodale Plus” received from Rodale Institute  has prevWGS data . Also, mWGS data  supportsK = 5 and 6 , Bengaluru, Karnataka, India.1A. hypochondriacus genome V.2.1(A.hyp.V.2.1) (2.Plainsman reference: Phytozomep.V.2.1)  GBS: Blap.V.2.1)  WGS (Ligp.V.2.1) 2.Amaranth A.hyp_K_white, A.hyp_K_red, A.cru_ornamental, A.cau_ornamental, and A.cru_Suvarna variety were grown at IBAB . GenomicLibrary preparation was done for Illumina whole genome sequencing in-house and outsourced for PacBio RSII sequencing.Whole Genome libraries for A.hyp_K_white, A.hyp_K_red, A.cru_ornamental, A.cau_ornamental and A.cru_Suvarna were prepared using the TruSeq DNA Sample Preparation Kit (Illumina) by following the manufacturer’s low throughput protocol. One microgram and 10 μg of the DNA were used for the preparation of Paired-End (PE) and Mate-Pair (MP) libraries, respectively. DNA was sheared using Adaptive Focused Acoustic technology  to generate fragments of desired insert size. The average insert size was around 200 bp for PE libraries and 1.75, 3, 5, and 10 kb for four MP libraries.TM M-280 Streptavidin, Invitrogen), the fragments were end-repaired, 3′-adenylated and ligated with Illumina adapters. Further, the biotinylated, adapter-ligated immobilized DNA were enriched by PCR. The size selection for all the libraries were done using solid-phase reversible immobilization (SPRI) beads (Agencourt AMPure XP Beads) from Beckman Coulter. The quality, quantity, and size distribution of the libraries were evaluated using Qubit (Invitrogen) and TapeStation (Agilent)  . The cluWhole-genome PacBio sequencing was done by Molecular Biology and Genomic Core, Washington University using P5/C3 chemistry on the Pacific Biosciences RSII platform. This platform is a single-molecule, real-time (SMRT) sequencing machine that uses a sequencing-by-synthesis method to generate good quality very long reads.The raw PacBio data was assembled using Canu  and FlyeThe Illumina data of all the plants with accessions listed in For the 27,658 SNP positions 150 base pair sequences were downloaded from public sources and coordinates for all 27,658 positions on A.hyp.V.2.1 were extracted by BLAST alignment. A separate VCF file was made for all the 13 datasets as listed in A. palmeri was excluded from the analysis because of the reported high level of missing data. The reads were demultiplexed using GBSX . Only 97 out of 107 samples from both GBS and WGS data were filtered based on their good clustering and bigger sample size. The merged SNP file was processed using PLINK  error value plot as reported by ADMIXTURE(v1.3) where we see a dip in the c.v value at K = 4 and 6  MAKER  (Annotation of AhKP was done using multiple approaches (i) Augustus  (v3.2.3)i) MAKER  genome a(TAIR10) . SNAP (K(TAIR10)  and Augu(TAIR10) .Arabidopsis proteins.Genes involved in lysine biosynthesis pathway were identified by BLASTP  analysisRaw transcriptome reads generated in our earlier work and reported in The Amaranth database is running on EC2 instances of Amazon cloud service (AWS). The database is built using HTML5, bootstrap and JavaScript. The database consists of a landing page, genome browser and NCBI BLAST  tool. ThJPXE00000000. The version described in this paper is version JPXE02000000.This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession SPD: classification, characterization and writing of the manuscript. SJ: assembly of AhKP. SR: library preparation and aiding writing of manuscript. KRR: assembly and analysis of other landraces. SW: development of genome browser. NH: transcriptome analysis. SD: DNA isolation and repeat analysis. MS: PacBio data, developmental transcriptome and taxonomic classification of landraces. ER: forinitiating translational work and validating transcripts. BC: for overseeing the experimental component of the project. PGS: for guidance throughout the project. SS: for overseeing the project and writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Helicobacter pylori and mozuku consumption with fucoidan absorption. Overall, 259 Japanese volunteers consumed 3 g fucoidan, and their urine samples were collected to measure fucoidan values and H. pylori titers before and 3, 6, and 9 h after fucoidan ingestion. Compared to the basal levels (3.7 ± 3.4 ng/mL), the urinary fucoidan values significantly increased 3, 6, and 9 h  after fucoidan ingestion. The basal fucoidan levels were significantly lower in H. pylori-negative subjects who rarely ate mozuku than in those who regularly consumed it. Regarding the ΔMax fucoidan value  in H. pylori-positive subjects who ate mozuku at least once a month, those aged ≥40 years exhibited significantly lower values than <40 years old. Among subjects ≥40 years old who regularly consumed mozuku, the ΔMax fucoidan value was significantly lower in H. pylori-positive subjects than in H. pylori-negative ones. In H. pylori-positive subjects who ate mozuku at least once monthly, basal fucoidan values displayed positive correlations with H. pylori titers and ΔMax fucoidan values in subjects <40 years old. No correlations were found in H. pylori-positive subjects who ate mozuku once every 2–3 months or less. Thus, fucoidan absorption is associated with H. pylori infection and frequency of mozuku consumption.We examined the associations of  Helicobacter pylori (H. pylori) agent based on its ability to disrupt the adhesion of the microbe to the gastric epithelium in vivo and in vitro [Cladosiphon okamuranus (Okinawa mozuku) on H. pylori was demonstrated in vitro by Shibata et al. [H. pylori binding to human gastric cell lines was inhibited more by Cladosiphon fucoidan than by fucoidan procured from Fucus. In addition, fucoidan blocked both Leb- and sulfatide-mediated attachment of H. pylori to gastric cells. They concluded that the inhibitory effect of Cladosiphon fucoidan on the binding of H. pylori and gastric cells might be caused by the coating with this component of the bacterial surface. However, no bacteriostatic or bactericidal activity was observed against H. pylori for any fucoidan preparation [Fucoidan is a complex sulfated polysaccharide that is mostly found in brown marine algae. Fucoidan exhibits a broad spectrum of biological activities, including anti-inflammatory, immunomodulatory, anti-oxidant, anti-tumor, and anti-infection effects ,2,3,4,5.in vitro ,7,8,9. Ta et al. . Their sCladosiphon okamuranus Tokida), a brown seaweed containing fucoidan, is an important factor associated with fucoidan absorption. Based on a previous report by Hehemann et al. [Fucoidan is reported to be absorbed across the intestinal tract via energy-dependent processes and pinocytosis ,11,12. In et al. , we specH. pylori is a Gram-negative, spiral-shaped, microaerophilic bacterium. It colonizes the entire gastric mucosa in approximately half of the world’s human population, and a poor socioeconomic condition is an important risk factor for infection [H. pylori causes peptic ulcer disease and atrophic gastritis, and it is associated with primary gastric B-cell lymphoma and gastric adenocarcinoma. The host immune system cannot clear the infection, and it persists without treatment.nfection ,16,17,18H. pylori infection. For example, H. pylori infection can lead to the deficiency of vitamins, such as vitamin C, vitamin A, α-tocopherol, vitamin B12, and folic acid, as well as essential minerals [H. pylori infection affects local and distant microbial populations and host responses.Many studies have focused on the modification of the gastric environment induced by minerals ,20,21. MH. pylori and disrupt its attachment to the gastric epithelium [H. pylori infection is assumed to affect fucoidan absorption. In this study, we examined the effects of H. pylori infection on the absorption of fucoidan extracted from Okinawa mozuku in Japanese volunteers. Although fucoidan absorption is extremely low in humans, the fucoidan concentration after oral administration is approximately 10-fold higher in urine than in serum [Because fucoidan can bind to ithelium ,7,8, H. in serum . TherefoH. pylori infection is shown in H. pylori infection was detected in 60.0%, 58.7%, 61.9%, 77.8%, and 88.5% of participants aged 20–29, 30–39, 40–49, 50–59, and ≥60 years old, respectively.The relevance of mozuku consumption and age to H. pylori infection, as the risk of infection was significantly higher in patients ≥40 years old than in those <40 years old. Mozuku consumption was not a significant risk factor for H. pylori infection . To determine relevance of age to fucoidan absorption, the subjects were divided into two age groups (<40 and ≥40 years).H. pylori-positive participants. No effect of age on ΔMax fucoidan values was observed among H. pylori-negative subjects. No significant difference of ΔMax fucoidan values was found according to the presence of H. pylori infection in either age group.ΔMax fucoidan values were significantly lower in subjects aged ≥40 years than in younger subjects among H. pylori-positive subjects but not in H. pylori-negative subjects  and H. pylori-negative subjects (32.1 ± 17.6) among those who ate mozuku at least once a month. However, no difference in ΔMax fucoidan values was noted according to age or frequency of mozuku consumption among H. pylori-negative participants.Among H. pylori-positive subjects who regularly consumed mozuku, a significant positive correlation between the basal and ΔMax fucoidan values was found for those aged <40 years but not those aged ≥40 years. No significant correlation was found between the basal and ΔMax fucoidan levels among participants who rarely ate mozuku (Among e mozuku .H. pylori-negative subjects regardless of the frequency of mozuku consumption (data not shown).In addition, no significant correlations were found between the basal and ΔMax fucoidan levels in H. pylori infection and the absorption of fucoidan. Specifically, fucoidan absorption was significantly diminished among H. pylori-positive subjects aged ≥40 years who ate mozuku at least once monthly, whereas no association was found among H. pylori-negative subjects irrespective of the frequency of mozuku consumption and age. In addition, fucoidan absorption was not diminished among H. pylori-positive subjects who ate mozuku once every 2–3 months or less; therefore, mozuku consumption affects the absorption of fucoidan. Although the precise mechanisms by which H. pylori infection and mozuku consumption reduce fucoidan absorption have not been determined, a few possibilities have been postulated.The present study revealed an association between H. pylori-negative and H. pylori-positive subjects among participants aged ≥40 years, H. pylori was less likely to directly diminish the absorption of fucoidan in this age group. Excluding H. pylori-positive participants aged ≥40 years who ate mozuku regularly, ΔMax fucoidan values were similar between H. pylori-positive and H. pylori-negative subjects. In addition, the frequency of mozuku consumption among H. pylori-positive participants was similar between subjects aged <40 years and those aged ≥40 years; therefore, the frequency of mozuku consumption is not directly associated with the absorption of fucoidan. Given that H. pylori positivity and regular mozuku consumption were associated with diminished fucoidan absorption among subjects aged ≥40 years but not among younger subjects, the duration of H. pylori infection and the frequency of mozuku appear important for fucoidan absorption.As ΔMax fucoidan values were similar between H. pylori infection disturb fucoidan absorption in subjects aged ≥40 years? H. pylori can change the secretion and acidification functions of the stomach because it penetrates into this organ. Although nutrient absorption does not occur in the stomach, H. pylori infection can affect the digestion and absorption of nutrients such as vitamin B12, vitamin C, vitamin A, vitamin E, and folate [H. pylori and inhibit its attachment to the gastric mucosa at pH 2.0 and 4.0, but not at pH 7.4. It is well known that H. pylori rarely causes atrophic gastritis in young people (<40 years old), whereas H. pylori-induced atrophic gastritis tends to be rather common in the elderly [H. pylori-induced atrophic gastritis, intragastric pH increases, consequently inhibiting the ability of H. pylori to bind to fucoidan. However, fucoidan absorption was not diminished in H. pylori-positive subjects aged ≥40 years who rarely ate mozuku, suggesting the influence of a long duration of mozuku ingestion on fucoidan absorption. Amornlerdpison et al. reported that fucoidan present in mozuku acts as an antagonist of the H2 receptor (similar to cimetidine), decreasing the acidity of gastric acid and raising the pH in the stomach [H. pylori-induced atrophic gastritis and a long duration of mozuku ingestion may markedly decrease acid secretion, consequently leading to the failure of H. pylori to bind fucoidan and reductions of its absorption in the small intestine in people aged ≥40 years. Thus, the possible mechanism by which H. pylori infection leads to reduced gastric acid secretion and fucoidan absorption has not been sufficiently investigated to draw definite conclusions, and other mechanisms other than hypochlorhydria following H. pylori infection are possible.How do frequent mozuku consumption and d folate ,20,21. Sd folate  reported elderly ,16,17,18 stomach . Taken tH. pylori titers and ΔMax fucoidan values were revealed in H. pylori-positive subjects aged <40 years who frequently consumed fucoidan. Such correlations were not found in the corresponding group of participants aged ≥40 years, nor were they observed in H. pylori-positive subjects who rarely ate mozuku or in H. pylori-negative subjects. Because the significance of the positive correlation observed in H. pylori-positive subjects aged <40 years who frequently consumed fucoidan is unclear, further research is necessary to clarify the relevance of H. pylori infection and mozuku intake to fucoidan absorption using a large number of subjects.Of note, significant positive correlations of the basal fucoidan levels with both H. pylori infection and mozuku consumption. As H. pylori is known to affect the absorption of various nutrients, this stomach bacterium may participate in basal fucoidan absorption. In recent years, “the nutrition-gut microbiome-physiology axis” has attracted substantial attention [H. pylori can induce drastic alterations in the variety of the gastrointestinal microbiota [H. pylori-negative subjects who regularly consumed mozuku than in their counterparts who rarely ate mozuku, which confirmed our previous findings [H. pylori infection and mozuku consumption to basal fucoidan levels is unclear.Interestingly, basal fucoidan levels were significantly increased by ttention ,26,27,28crobiota ,30,31, tfindings . We specH. pylori and underwent gastrectomy prior to the study were not excluded. The study also did not exclude subjects who used complementary and alternative medicines, which can affect the absorption of fucoidan.This study had several limitations. First, subjects who received eradication therapy for H. pylori infection, and the high accuracy of this test was certified by several investigators [H. pylori infection with fucoidan absorption using serum-based ELISA kits or the 13C urea breath test (13C-UBT) in future research.Second, a urine-based ELISA kit (URINELISA) was used to assay tigators ,33. A diFucus vesiculosus fucoidan [Laminaria japonica) and wakame (Undaria pinnatifida) are traditional foodstuffs in Japan, fucoidan contained in these seaweeds may cross-react with our antibody. Further studies are necessary to elucidate the effects of mozuku consumption on the intestinal absorption of fucoidan using ELISA with a monoclonal antibody.In addition, the specificity of our fucoidan ELISA was limited. We assayed urinary fucoidan levels using a polyclonal antibody for Okinawa mozuku fucoidan, which weakly cross-reacted with fucoidan . BecauseH. pylori infection to absorption of mozuku fucoidan” on the Internet and recruited volunteer participants. Two hundred sixty-two Japanese people submitted applications from April 2014 to June 2016. They completed a questionnaire assessing gender, age, and mozuku consumption. We enrolled 259 volunteers who completed questionnaires and collected urine samples as planned. Subjects were divided into five age groups: 20–29, 30–39, 40–49, 50–50, and ≥60 years old.We published pamphlets describing the purpose, methods, and exclusion items of our research titled “The reference of The frequency of mozuku consumption was divided into five groups as follows: approximately 1–3 times weekly, approximately once every 2 weeks, approximately once monthly, approximately once every 2–3 months, and rarely .This study was conducted according to the Declaration of Helsinki. The protocol of the study was approved by the Ethics Committee of South Product Co., Ltd. (UMIN000039117). Following an explanation of the study and its aim, all subjects provided informed consent.Subjects refrained from marine algae and fucoidan supplementation on the day before the test and on the day of the test to avoid the effects of diet. Subjects orally consumed two fucoidan drinks (1500 mg/bottle) at 9:00 in the morning. Urine samples were extracted four times, namely, before (0) and 3, 6, and 9 h after fucoidan ingestion. Urine samples were collected by a parcel delivery service.In this study, the subjects orally consumed 3 g of mozuku fucoidan. The drink was prepared by South Product Co., Ltd.Urine fucoidan levels were assayed using a sandwich ELISA method developed by our laboratory . The repH. pylori were measured using a urine-based ELISA kit  that utilizes a VacA- and CagA-positive H. pylori strain isolated from a Japanese patient with gastritis as the antigen source. This ELISA-based test result was considered positive when a cutoff index of 1.0  or greater was obtained after measurement of the optical density according to the manufacturer’s instructions [Single-void urine samples were obtained and stored at 2–8 °C until use. Urinary IgG antibodies to ructions ,33,34.The ΔMax fucoidan value was calculated by subtracting the basal value (before ingestion) from the highest level of urinary fucoidan following fucoidan ingestion. If the basal fucoidan level was higher than that after fucoidan ingestion, then the ΔMax fucoidan value was recorded as 0.The urinary fucoidan values after fucoidan ingestion were analyzed using two-way analysis of variance (ANOVA) or one-way ANOVA followed by Tukey’s test for multiple comparisons. SAS version 9.4  was used to perform statistical analyses.H. pylori infection as the dependent variable and age and mozuku consumption as the independent variables. The results were expressed as the hazard ratio and 95% confidence interval. Data are expressed as the mean ± SD. P < 0.05 indicated a statistically significant difference.The Mann–Whitney U-test was used to analyze between-group differences. Statistical correlations were analyzed using Spearman’s rank correlation coefficient. In addition, multiple regression analysis was performed with H. pylori infection and mozuku consumption. Fucoidan absorption in H. pylori-positive subjects who regularly consumed mozuku differed by age, being significantly lower in subjects aged ≥40 years than in their younger counterparts. A significant positive correlation between the basal fucoidan level and ΔMax fucoidan value was found among subjects aged <40 who regularly consumed mozuku but not among their older counterparts. Further studies are needed to elucidate the precise mechanisms influencing fucoidan absorption.The present data illustrated that fucoidan absorption is associated with


Lycium ruthenicum is a precious desert plant with high medicinal and ecological values. It contains rich anthocyanins, which are safe natural pigments and could be used for natural food colorants. In this study, the complete chloroplast (cp) genome of L. ruthenicum was assembled on the basis of next generation sequencing. The whole genome is 154,996 bp in length, including two single copy regions  and two inverted repeat regions . The overall G + C content of the genome is 37.9%. The cp genome consists of 111 unique genes, including 30 tRNA genes, 4 rRNA genes and 77 protein-coding genes. Phylogenetic analysis indicates that L. ruthenicum is closely clustered with Atropa belladonna, Przewalskia tangutica, and Scopolia parviflora. Lycium contains several most well-known medicinal and food plants in Solanaceae, such as L. barbarum, L. chinense, and Lycium ruthenicum. There are about 80 Lycium species around the world  in Qinghai Province (http://www.qhys.gov.cn/html/42/202883.html). To scientifically and effectively conserve this plant, collecting more genetic information is important. Complete chloroplast (cp) genome could be applied to studies on population genetics and phylogenetics. It is useful to discuss the occurrence, evolution, and conservation of this precious endangered species.The genus L. ruthenicum were collected from the Yuepuhu County in Xinjiang Autonomous Region of China . Voucher herbarium specimens (GY17001) were deposited at the Herbarium of Xinjiang Normal University (XJNU). The total DNA was extracted using DNA Plantzol Reagent  according to the manufacturer’s protocol, and then sequenced using Illumina HiSeqTM 2000 . The chloroplast sequences were assembled by NOVOPlasty 2.6.3  as the reference. Finally, the whole sequence was annotated with Geneious 11.0.2 . Then the complete circle was accomplished by OGDRAW (http://ogdraw.mpimp-golm.mpg.de/). The complete cpDNA sequence has been submitted to GenBank (MG976805). Lycium ruthenicum sequence was also aligned with the other Solanaceae cp genomes. The phylogenetic tree was constructed by MEGA6 , a small single copy region  and two inverted repeat regions . The overall G + C content of the genome is 37.9%. The cp genome consists of 111 unique genes, including 30 tRNA genes, 4 rRNA genes and 77 protein-coding genes. Seventeen genes are duplicated in the IR regions, including all four rRNA genes, seven tRNA genes and six protein-coding genes. Sixteen genes have one intron, while two genes have two introns. The ycf1, rps19 genes are crossing the SSC/IR and LSC/IR borders. The rps12 gene is trans-spliced, with the 5′ end located in the LSC and the 3′ end duplicated in the IR regions.The whole genome of L. ruthenicum within Solanaceae, we constructed neighbour joining (NJ) tree (with 1000 bootstrap replicates) using all Solanaceae species which have publicly available cp genome, with one species of Convolvulaceae (Ipomoea purpurea: NC009808) as the outgroup. It is found that L. ruthenicum is sister to a clade of Atropa belladonna, Przewalskia tangutica, and Scopolia parviflora (L. ruthenicum, and this information which establish a solid foundation for future conservation genetic and phylogenetic studies.To figure out the phylogenetic position of rviflora . In conc


Haemonchus contortus and Teladorsagia circumcincta are among the two most pathogenic internal parasitic nematodes infecting small ruminants, such as sheep and goats, and are a global animal health issue. Accurate identification and delineation of Haemonchidae species is essential for development of diagnostic and control strategies with high resolution for Trichostrongyloidea infection in ruminants. Here, we describe in detail and compare the complete mitochondrial (mt) genomes of the New Zealand H. contortus and T. circumcincta field strains to improve our understanding of species- and strain-level evolution in these closely related roundworms. In the present study, we performed extensive comparative bioinformatics analyses on the recently sequenced complete mt genomes of the New Zealand H. contortus NZ_Hco_NP and T. circumcincta NZ_Teci_NP field strains. Amino acid sequences inferred from individual genes of each of the two mt genomes were compared, concatenated and subjected to phylogenetic analysis using Bayesian inference (BI), Maximum Likelihood (ML), and Maximum Parsimony (MP). The AT-rich mt genomes of H. contortus NZ_Hco_NP and T. circumcincta NZ_Teci_NP are 14,001 bp (A+T content of 77.4%) and 14,081 bp (A+T content of 77.3%) in size, respectively. All 36 of the typical nematode mt genes are transcribed in the forward direction in both species and comprise of 12 protein-encoding genes (PCGs), 2 ribosomal RNA (rrn) genes, and 22 transfer RNA (trn) genes. The secondary structures for the 22 trn genes and two rrn genes differ between H. contortus NZ_Hco_NP and T. circumcincta NZ_Teci_NP, however the gene arrangements of both are consistent with other Trichostrongylidea sequenced to date. Comparative analyses of the complete mitochondrial nucleotide sequences, PCGs, A+T rich and non-coding repeat regions of H. contortus NZ_Hco_NP and T. circumcincta NZ_Teci_NP further reinforces the high levels of diversity and gene flow observed among Trichostrongylidea, and supports their potential as ideal markers for strain-level identification from different hosts and geographical regions with high resolution for future studies. The complete mt genomes of H. contortus NZ_Hco_NP and T. circumcincta NZ_Teci_NP presented here provide useful novel markers for further studies of the meta-population connectivity and the genetic mechanisms driving evolution in nematode species. H. contortus NZ_Hco_NP and T. circumcincta NZ_Teci_NP field strains to improve our understanding of species- and strain-level evolution in roundworms.- In-depth comparative and phylogenomic analyses of the recently published complete mitochondrial (mt) genomes of the New Zealand Haemonchus and T. circumcincta mt genomes supports their potential as ideal markers for easy species- and possibly strain-level identification with high resolution for future studies.- Analysis of the A+T rich and non-coding repeat regions of the complete - Phylogenetic relationships of complete mt genomes for all Trichostrongyloidea species currently available inferred from Bayesian, Maximum Likelihood and Maximum Parsimony analyses of mitochondrial genes, revealed high levels of intra-species diversity and a panmictic structure observed among Trichostrongylidea.- Our findings indicate that a three-pronged approach incorporating phylogenetic inertia, pangenome structure/features and environmental data are needed in order to understand the mitochondrial genome evolution.H. contortus (barber's pole worm) and T. circumcincta (brown stomach worm), are the most economically important pathogenic nematodes infecting small ruminants (sheep and goats) worldwide. These blood-feeding strongylid nematodes are orally transmitted via contaminated pasture to the host where they infect the fourth stomach (abomasum) reducing animal production and causing anemia, edema, and associated complications often leading to death  genes  genes  of complete nuclear genomes of parasitic roundworms (Nematodes)  whole genome shotgun paired-end (PE) and Single-Molecule, Real-Time (SMRT) long-read sequencing (SRA Runs: SRR11022845 and SRR11022846) were generated using the Illumina HiSeq2500 and Pacific Biosciences (PacBio) platforms, as previously described  whole genome shotgun library (SRA Run: SRR3145390) was generated as previously described . Repeat-rich subsets of the mt genome assemblies were identified with varying degrees of complexity using the mDUST v.2006.10.17 . The coordinates of the found repeats were matched with coding sequences and the proportion of repeats overlapping coding regions was calculated. tRNA genes were further corroborated using the software SPOT-RNA  . Caenorhabditis elegans N2 (JF896456) was used as the outgroup species in this study. All coding sequences (CDS) used in this analysis had correct start and stop codons based on translation (transl_table = 5)The phylogenetic positions of * v.4.0b10 (http://paup.csit.fsu.edu/)  are extremely abundant in eukaryotic proteins that are typically represented by amino acid sequences containing repeats of single amino acids or short amino acid motifs, and may facilitate the formation of novel coding sequences. H. contortus NZ_Hco_NP had 23 LCRs with 15 (65%) in coding regions, while T. circumcincta NZ_Teci_NP had 26 repeats with 21 (81%) found in coding regions  regions, respectively , 22 transfer RNA (trn) genes, and 2 ribosomal RNA genes . The 12 mt PCGs of both H. contortus NZ_Hco_NP and T. circumcincta NZ_Teci_NP were encoded on the heavy strand and transcribed in the same direction. The order of genes observed in H. contortus NZ_Hco_NP and T. circumcincta NZ_Teci_NP are identical to the previously reported H. contortus , TTG (nad2-3), and ATA (nad1 and cox1-3) in H. contortus NZ_Hco_NP, with T. circumcincta NZ_Teci_NP predominantly using ATT and ATA (cytb and cox1)  . Both mttrn genes, size ranged from 54 to 63 bp in length  and rrnL (16S) genes identified in the mt genomes of H. contortus NZ_Hco_NP (703 and 951 bp) and T. circumcincta NZ_Teci_NP (700 and 962 bp) were A+T biased , and mt genome structure as previously reported for trichostrongyloids  gene reported only in Trichinella spiralis , was absent in our data.Comparisons of the gene synteny as well as the nucleotide, codon usage and amino acid compositions revealed a highly conserved GA2 gene arrangement (H. contortus NZ_Hco_NP are very similar to those of T. circumcincta NZ_Teci_NP (≤1 amino acid difference). The sequence variability between Haemonchus species/strains and T. circumcincta strains of the Trichostrongyloidea superfamily was assessed by performing pairwise comparisons for each PCG of the mt genomes  and T. circumcincta strains . In comparison, the nad4 gene appears to be conserved among different Trichostrongyloidea species, for example Trichostrongylus vitrinus and T. axei , while M. digitatus, C. oncophora, and C. elegans N2 all share 1,230 bp nad4 genes  were highly utilized across all PCGs. Among each codon family, the T-ending (49.9%) codons are used most frequently than codons ending in any of the three other nucleotides, with C-ending (2.7%) codons least used. Similarly, for both Haemonchidae and Trichostrongylidae the average nucleotide content of their mt PCGs are 26.0% (A) and 43.7% (T), compared to 25.4% (A), and 44.2% (T), respectively. In particular the nad4L was the most T-rich at 53.5 and 54.9%, in each family, respectively. Also the most commonly codon observed was TAA as the termination codon with the majority of initiation codons also being AT-rich.Analysis of codon usage patterns in Trichostrongyloidea and ial PCGs . As expeH. contortus NZ_Hco_NP and T. circumcincta NZ_Teci_NP mt genomes , TTT , ATT , TTA , TTT , GTT , and TAT , respectively. In addition, T-ending codons are far more frequent than codons ending in any of the other three nucleotides among each codon family. For example, the least used codons included CCA , GCG , along with the C-ending codons GAC  and CTC .The observed nucleotide biases also have substantial effects on codon usage patterns for both  genomes . The mosHaemonchus and Teladorsagia species and strains revealed a common and highly variable A+T-rich non-coding region between 5,500 and 6,100 bp in each mt genome (Haemonchus (89.0%) and Teladorsagia (88.2%) mt genomes, and is located between trnA and trnP (or between genes nad5 and nad6). Among Haemonchus, the A+T-rich region is ~600 bp in H. contortus (89.7%) and 155 bp in H. placei (87.1%), whereas the region is 340 bp in Teladorsagia (88.2%). A pairwise alignment of the nucleotide sequences of the A+T-rich regions of H. contortus NZ_Hco_NP (611 bp) and closely related H. contortus McMaster  resulted in 91.5% identity. Interestingly, the region in H. contortus is among the largest among other nematodes studied to date with the exception of As. suum (886 bp)  units  compared to only one (126 bp) found in T. circumcincta strains mt genomes (T. circumcincta NZ_Teci_NP corresponding to TTATAATTATTATATAATAATTA (23 bp), TATATATATATAAATTAATATAATTATTATTAATAAT (37 bp), and ATATTATATATTATATATTA (20 bp) consensus units were 4, 2, and 4. In general, TRs of Haemonchus mt genomes were much more abundant and distributed randomly throughout the mtDNAs, whereas TRs were mostly in the intergenic regions of T. circumcincta mtDNA. Some TRs were found within the mitochondrial genes, such as those in cox1, nad5, nad6, and nad4, with also nad4L in T. circumcincta mtDNA. Overall, our analyses show that the number of tandem repeats was highly variable among different species and strains of Haemonchus and Teladorsagia and largely account for the variations among the mtDNA sequences lengths.Comparative analysis of the control (A+T-rich) region of  genomes . The numHaemonchus species/strains and T. circumcincta strains mt genomes, we were able to differentiate individual strains with high confidence due to the high mutation rate and sequencing coverage for these regions. We propose that such regions can serve as ideal markers for future studies investigating nematode population structure  strains grouped together and originate from nearby geographic locations, but separate from H. contortus ISE (East Africa). This observation corroborates our hypothesis on the impact of environmental constraints and preferences acting as secondary drivers of within-species diversity. Overall, we are in agreement with previous phylogenetic studies suggesting that ITS rDNA nuclear genetic markers alone may not provide sufficient information to reveal higher taxonomic level relationships within the Trichostrongyloidea. Our findings indicate that a three-pronged approach that incorporates phylogenetic inertia, pangenome structure/features and environmental data in order to understand the mitochondrial genome evolution.The phylogenomic tree based on the concatenated PCGs of the complete mt genomes , placed H. contortus and T. circumcincta, two of the most economically important and common pathogenic nematodes infecting small ruminants worldwide. We explored the mitochondrial pangenome features and phylogenomic relationships to assess species- and strain-level diversity among Trichostrongyloidea nematodes and with available mt genome in public databases. Our analyses corroborate previous studies showing that our H. contortus NZ_Hco_NP and T. circumcincta NZ_Teci_NP strains position in their respective clades. Future work should focus on utilizing our insights on the highly variable regions of Haemoncus and Teladorsagia conspecific strains to develop cost-effective DNA-based approaches for novel parasite management and control strategies. The complete mt genomes of the New Zealand H. contortus and T. circumcincta field strains are important contributions to our understanding of meta-population connectivity as well as species- and strain-level evolution in nematodes. With the continuing improvements in sequencing technology combined with a community effort we may be able to reconstruct the true origins of Haemoncus and Teladorsagia, as well as other parasitic nematodes that are of a global interest.This study compares the recently sequenced complete mitochondrial genomes of sibling species of parasitic roundworms, The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/NP conceived and designed the project, analyzed and interpreted the data, and wrote the manuscript. PM provided bioinformatics support for the project. MM and Y-JC provided resources and critically revised the manuscript. All authors read and approved the final manuscript.NP was employed by the company AgResearch Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Throscidae is a relatively small lineage in the beetle superfamily Elateroidea. The Mesozoic fossil records of this family are sparse. Here we describe three new throscid species found in mid-Cretaceous Burmese amber, all represented by well-preserved specimens. These newly discovered species suggest that Throscidae had a high diversity in the Cretaceous.Captopus depressicepsgen. et sp. nov., Electrothroscus yanpingaegen. et sp. nov. and Pseudopactopus robustusgen. et sp. nov. are reported from the mid-Cretaceous Burmese amber. These new findings greatly extend the Mesozoic diversity of Throscidae, which implies a high degree of morphological disparity for this family in the Cretaceous. Drapetes Dejean and Lissomus Dalman , wh, whDrapen , Hukawng Valley, Kachin State, northern Myanmar. The specimens are deposited in the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences (NIGP), Nanjing, China. The amber piece was trimmed with a small table saw, ground with emery papers of different grit sizes, and finally polished with polishing powder. Photographs under incident light were taken with a Discovery V20 stereo microscope . Widefield fluorescence images were captured with the Zeiss Axio Imager 2 light microscope combined with a fluorescence imaging system. Confocal images were obtained with a Zeiss LSM710 confocal laser scanning microscope. Images under incident light or widefield fluorescence were automatically stacked in Helicon Focus 7.0.2 or Zerene Stacker 1.04. Confocal images were automatically stacked and colour-coded in ZEN 2011 or ZEN 2.3 (Blue Edition), or manually stacked in Adobe Photoshop CC. In the multicoloured images, the colours were coded based on z-depth. Images were further processed in Adobe Photoshop CC to enhance contrast. This published work and the nomenclatural acts have been registered in ZooBank, the official registry of Zoological Nomenclature. The LSID for this publication is urn:lsid:zoobank.org:pub:4797AF94-8912-4DCA-B375-A7D25F16B2A6.Order Coleoptera Linnaeus, 1758Suborder Polyphaga Emery, 1886Superfamily Elateroidea Leach, 1815Family Throscidae Laporte, 1840 Diagnosis. Antennae 11-segmented, with a distinct club; antennomere 1 attached to antennomere 2 apically. Prothoracic antennal grooves running along the notosternal suture and extending posterolaterally along the hind margins of the hypomera. Prosternal process apically acute, fitting into mesoventral cavity. Metacoxal plates medially extending posteriorly. Abdomen with five connate ventrites. GenusCaptopus Li & Cai gen. nov. LSID. urn:lsid:zoobank.org:act:CB5975DC-1566-406B-88C1-B70512D32B40Type species.Captopus depressicepssp. nov.Etymology. The generic name is an anagram of “Pactopus”, another genus in Throscidae. The name is masculine in gender.Diagnosis. Frons without carina, but with a deep cavity on both sides. Eyes large. Lateral pronotal ridge incomplete. Prosternum with subparallel prosternal carinae. Metaventrite with well-developed mesotarsal grooves. Abdomen with metatarsal grooves extending beyond the posterior margin of the third ventrite.Remarks. The new genus Captopus is similar to the extant genus Pactopus Horn and the extinct genus Tyrannothroscus Muona in having both well-developed mesotarsal and metatarsal grooves. Captopus shows some crucial differences from other known extant or extinct species in Pactopus. For example, the metatarsal grooves of Pactopus do not extend beyond the posterior edge of ventrite 3, while in Captopus, the metatarsal grooves extend well beyond ventrite 3 and reach the middle of ventrite 4. More importantly, there is a deep cavity on both sides of frons in Captopus, formed by the expansion of the groove around the ridge bordering antennal insertion. This feature is unique among all known extant and extinct throscids. Captopus differs from Tyrannothroscus additionally in having no supraocular ridges, and lateral pronotal ridge incomplete. Captopus depressiceps Li & Cai sp. nov. , an evolutionary botanist.Material. Holotype, NIGP173916, a well-preserved specimen with elytra partially polished away.Locality and horizon. Amber mine located near Noije Bum Village, Tanai Township, Myitkyina District, Kachin State, Myanmar; unnamed horizon, mid-Cretaceous, Upper Albian to Lower Cenomanian [nomanian ,19.Diagnosis. As for the genus.Description. Body elongate, length 2.3 mm, width 0.8 mm.Head transverse A; frons Pronotal disc about 1.6 times as wide as long along the middle; sides not sinuate, converging anteriorly; posterior angles strongly acute and produced posteriorly; posterior edge bisinuate. Elytra widest behind the base, about 2.2 times as long as wide combined.Prosternum subtrapezoidal; prosternal carinae almost parallel; prosternal process slightly narrowed in middle, apically acute, fitting into mesoventral cavity. Metaventrite without discrimen; mesotarsal grooves absent B. MesocoAbdomen with five connate ventrites; ventrite 5 twice as long as ventrite 4; metatarsal grooves or impressions absent C. GenusPseudopactopus Li & Cai gen. nov. LSID. urn:lsid:zoobank.org:act:8CA0EA80-040B-4924-AF10-F9D0F6453D1CType species.Pseudopactopus robustussp. nov.Etymology. The generic name is derived from the Greek “pseudes”, false, and the generic name Pactopus LeConte. The name is masculine in gender.Diagnosis. Body wide (length/width ratio ~2.1). Frons without modifications. Eyes large. Lateral pronotal ridge incomplete. Prosternum with subparallel prosternal carinae. Protibiae simple. Metaventrite with well-developed mesotarsal grooves. Abdomen with metatarsal grooves reaching or almost reaching the posterior margin of the third ventrite.Remarks. The new genus Pseudopactopus possesses well-developed mesotarsal and metatarsal grooves, which, together with its unmodified head, link the new genus with Pactopus. However, the protibiae of Pseudopactopus are simple , while protibiae are enlarged apically and with tarsal grooves in Pactopus . Such si et al. .Pseudopactopus from Pactopus. The body shape of Pseudopactopus robustus is broader than all other known extant and extinct Pactopus species. The type specimens of Pactopus burmensis Muona and Pactopus americanus Wickham have a length/width ratio of about 2.4–2.5, and Pactopus horni LeConte, Pactopus fasolti Muona and Pactopus fafneri Muona are even much narrower [Pseudopactopus robustus almost reaches half of its length. The sides of pronotal disc are more or less sinuate in P. horni [P. americanus , wherea Pseudopactopus robustus Li & Cai sp. nov. . The divergence between Throscidae and the group of Eucnemidae + Cerophytidae has been dated to Middle Jurassic, approximately 165 Ma [The three throscid species presented in this paper, together with two previous reported species, show a high degree of morphological variation of Throscidae in Burmese amber. Specialized throscids have also been reported in Lower Cretaceous Lebanese amber . Comparey 165 Ma . TherefoAulonothroscus as non-monophyletic, he concluded it was reasonable to opt for the tree supporting a monophyletic Aulonothroscus. However, later Li et al. [Aulonothroscus appeared to be paraphyletic in the majority-rule consensus tree.Despite the wide distribution of the family, throscids are poorly studied and their taxonomy is not well resolved. Though the monophyly of Throscidae has been supported by recent molecular phylogenetic analyses ,23, no mi et al.  performeTrixagus parvulus Muona, was assigned to genus Trixagus. However, the most important diagnostic feature for Trixagus, the vestigial tarsal grooves, is not visible on the specimen: the position for possible tarsal groove is concealed by its femur. The club-forming antennomeres in T. parvulus seems to be rather symmetrical and moniliform , whereas absent . VariousCaptopus depressicepsgen. et sp. nov., Electrothroscus yanpingaegen. et sp. nov. and Pseudopactopusrobustusgen. et sp. nov. in the mid-Cretaceous Burmese amber greatly extends the Mesozoic diversity of Throscidae. These newly discovered species imply a high degree of morphological disparity for this family in the Cretaceous. Further phylogenetic analysis with incorporation of both molecular data and fossil taxa would be helpful for elucidating the early evolutionary history of Throscidae.Our discovery of


Zymoseptoria tritici with its highly variable genome, has emerged as a model organism to study genome evolution of plant pathogens. Here, we compared genomes of Z. tritici isolates and of sister species infecting wild grasses to address the evolution of genome composition and structure.Antagonistic co-evolution can drive rapid adaptation in pathogens and shape genome architecture. Comparative genome analyses of several fungal pathogens revealed highly variable genomes, for many species characterized by specific repeat-rich genome compartments with exceptionally high sequence variability. Dynamic genome structure may enable fast adaptation to host genetics. The wheat pathogen Z. ardabiliae, Z. brevis, Z. pseudotritici and Z. passerinii, together with two isolates of Z. tritici. We report a high extent of genome collinearity among Zymoseptoria species and high conservation of genomic, transcriptomic and epigenomic signatures of compartmentalization. We identify high gene content variability both within and between species. In addition, such variability is mainly limited to the accessory chromosomes and accessory compartments. Despite strong host specificity and non-overlapping host-range between species, predicted effectors are mainly shared among Zymoseptoria species, yet exhibiting a high level of presence-absence polymorphism within Z. tritici. Using in planta transcriptomic data from Z. tritici, we suggest different roles for the shared orthologs and for the accessory genes during infection of their hosts.Using long-read technology, we sequenced and assembled genomes of Z. tritici, we describe here a high level of conservation in genomic, epigenomic and transcriptomic composition and structure across the genus Zymoseptoria. The compartmentalized genome allows the maintenance of a functional core genome co-occurring with a highly variable accessory genome.Despite previous reports of high genomic plasticity in  Co-evolution between plants and pathogens can drive rapid evolution of genes involved in antagonistic interactions . In filaFusarium solani, Fusarium oxysporum and Leptosphaeria maculans [F. oxysporum shows that accessory chromosomes may be acquired horizontally as chromosomes can be transferred between distinct isolates by hyphal fusion [Accessory chromosomes are characterized by intra-species presence-absence polymorphism, low gene density, an enrichment of repetitive sequences and, in some species, a different histone methylation pattern , 7. It hmaculans –11. Littl fusion . Throughl fusion .Genes involved in plant-pathogen interactions may diversify at a higher rate in repeat-rich genome compartments and thereby evolve new virulence specificity faster . These gZymoseptoria tritici has emerged as a model organism in evolutionary genomics of pathogens. This species originated in the Fertile Crescent during the domestication of its host, wheat [Z. tritici have been collected from wild grasses in the Middle East providing an excellent resource for comparative genome analyses of closely related and recently diverged pathogen species. Comparative analyses of genome organization and gene content within and among Zymoseptoria species have previously revealed a wide distribution of accessory chromosomes and dynamic gene content [Z. tritici show a low transcriptional activity in vitro as well as in planta [The ascomycete pathogen t, wheat . Closely content , 22. The content . Some of content –29. Inten planta , 31. Thin planta , 32.Zymoseptoria genus has so far been unknown due to the lack of high-quality genome assemblies and large-scale population sequencing. Assemblies based on short-read data failed to recover complete sequence of accessory chromosomes and “orphan regions” due to their high repeat content [Z. tritici isolates sequenced with PacBio long-read sequencing [Z. tritici high-quality chromosome assemblies revealed the occurrence of “orphan regions” enriched with transposable elements and encoding putative virulence-related genes [In the reference isolate IPO323, the accessory chromosomes comprise more than 11% of the entire genome assembly. To which extent such a high amount of accessory DNA is also found in genomes of other members of the  content . The assquencing , 33. Comed genes , 34.Zymoseptoria species. Beside presenting a new and significantly improved resource for future genomic studies of these fungal pathogens, we specifically ask: 1) how conserved is the genome architecture among Zymoseptoria species? 2) can we identify accessory compartments in other Zymoseptoria isolates? 3) to which extent does variation in genome architecture reflect variation in gene content?In this study, we investigate the genomic architecture and variability among five Z. tritici (Zt05 and Zt10) and one isolate of each of the sister species, Z. ardabiliae, Z. brevis, Z. passerinii, and Z. pseudotritici. We explore the core and non-core genome architecture of Zymoseptoria spp. combining genomic data with transcriptome and histone methylation data and relate this to core and accessory genome compartments. Furthermore, we compare the distribution of orthologous and non-orthologous genes in the Zymoseptoria genomes and one additional Dothideomycete species. Our analyses reveal an overall conserved genome architecture characterized by gene-rich core compartments and accessory compartments enriched in species-specific genes. Finally, we report a remarkably high extent of variation in presence-absence of protein coding genes in a eukaryote genome.To answer these questions, we used high-quality assemblies based on long-read sequence data and new gene predictions in two isolates of Z. ardabiliae, Z. brevis, Z. pseudotritici and Z. passerinii and the genomes of two Z. tritici isolates sampled in Denmark and Iran [Z. tritici isolates comprising 19 and 30 contigs and the most fragmented was of Z. passerinii comprising 103 contigs  but larger than previous short-read based assemblies , Z. brevis (Zb87) and Z. pseudotritici (Zp13) contained, respectively, twelve, nine and five fully assembled chromosomes including both core and accessory chromosomes  and Z. passerinii (Zpa63) genomes included no fully assembled chromosomes, but twelve and ten contigs respectively with telomeres at one of the ends  and up to 71.8% of genes in Z. pseudotritici (N = 8373) lack a predicted function  to 11% of genes in Z. ardabiliae  is three times higher compared to Z. brevis (N = 206). Interestingly, the three Z. tritici isolates also vary considerably in their predicted effector repertoires. The reference isolate IPO323 has a reduced set of predicted effector genes (N = 274) compared to Zt05 and Zt10 that encode approximately 30% more predicted effector genes  from the gene predictions. We categorized 22,341 gene orthogroups identified in the seven Zymoseptoria genomes and in C. beticola according to their distribution among fungal genomes . The genus-specific orthogroups, shared between several Zymoseptoria spp. but not found in the C. beticola genome, represent 45% of the orthogroups . Among the genus-specific orthogroups, 1100 are found in all Zymoseptoria genomes  are found only in individual species. Among the species-specific genes, 205 orthogroups (Ngenes = 414 to 562) are found in all three Z. tritici genomes while the isolate-specific genes in Z. tritici represent 391 (Zt10) to 792 (IPO323) genes.To further characterize variation in gene content among the five Z. tritici isolates independently from the other species, we observe extensive gene presence-absence polymorphisms between the three isolates: 1540 orthogroups are identified in only two strains and 2522 are found in only one . Fifty-six percent of predicted effectors in Z. ardabiliae and up to 78% of predicted effectors in Z. pseudotritici are shared with at least one of the other five Zymoseptoria species  are found in all seven Zymoseptoria genomes. Among the predicted effectors shared by Z. tritici, and at least one other Zymoseptoria species, 32% (N = 112 of 352) are present in all three Z. tritici isolates while 68% (N = 240 of 352) show presence-absence polymorphisms in at least one of the three isolates. These results indicate that the majority of these shared predicted effectors are actually accessory (i.e. presence-absence polymorphism) in Z. tritici.Interestingly, we show that predicted effectors are enriched among the ies Fig. c. IndeedZ. tritici by analyzing gene expression patterns. We used previously published in planta expression data of three Z. tritici isolates [in planta expression data into two different infection phases: the biotrophic phase and the necrotrophic phase, a separation supported by principal component analysis of normalized DESeq2 counts . We compared expression levels by mapping RNA-seq reads to the genomes of IPO323, Zt05 and Zt10, using normalized read mappings to transcript per million. We tested differences among gene categories using pairwise comparisons with a Kruskal-Wallis test . Species-specific and isolate-specific gene median expression ranges from 3.2 to 5.6 TPM in IPO323 and Zt10 while median expression of core genes is 12.1 and 10.9, respectively. The Zt05 expression profile does not follow the same trend: the core genes are the lowest expressed gene category (8.9 median TPM), while genus-; species- and isolate-specific genes showed higher transcription levels .Finally, we addressed the functional relevance of accessory and orphan genes in isolates . The expisolates . We sort Figure S. Furtherspecies-specific and isolate-specific genes for all three isolates  when comparing the expression of genes that are differentially expressed  between the biotrophic and necrotrophic phases. Species-specific and isolate-specific DEGs are higher expressed in planta than the core and genus-specific genes . The spZ. tritici is highly variable [Zymoseptoria genus. Thereby, we show that more than 25% of the genes identified as isolate-specific in a comparison including only Z. tritici isolates are actually present in the wild-grass infecting sister species. This observation suggests that a large proportion of the accessory genome of Z. tritici is not specific to this species. Instead, the accessory genome content is shared among Zymoseptoria species. The proportion of accessory Z. tritici genes shared with other Zymoseptoria species was found to be the highest in the Iranian isolate, which is the only isolate sympatric with the four sister-species. A likely explanation for this observation would be inter-specific gene flow, which would allow the different wild species to exchange genes with sympatric Z. tritici isolates. This new finding is consistent with recent findings from population genomic data studies revealing extensive introgression between Zymoseptoria species [Z. tritici.In this study, we confirm previously reported genome comparisons showing that gene content in variable . We furt species , 42. OurZymoseptoria genus. Although the lifestyles of the wild-grass infecting Zymoseptoria are poorly understood, the species share major features of their lifestyles. Thus, as expected, we find similar CAZymes and BGC contents across the genomes studied here. In Zymoseptoria, most of the predicted effectors are shared among all species, although they show presence-absence variation. In the Botrytis genus (Dothideomycetes), sister species infecting different hosts share effectors with confirmed functions [Microbotrym, a fungal plant pathogen including several species specialized on different hosts a large set of genes encoding conserved and shared secreted proteins was identified. This repertoire of effectors is hypothesized to include traits relevant for pathogenicity among Microbotrym lineages [Zymoseptoria. Predicted effector genes conserved across the Zymoseptoria genus are likely core pathogenicity factors potentially targeting key plant defense mechanisms common to all of the grass hosts [species-specific and isolate-specific may reflect different host specificities and rapid evolution of these genes [genus-specific predicted effector genes is shared among all Zymoseptoria species; the majority shows presence-absence polymorphisms suggesting that a variable effector repertoire is an ancestral trait in these plant pathogens. Z. ardabiliae has been isolated from leaves of distantly related grass species in Iran, including Lolium spp., Elymus repens, and Dactylis glomerata and potentially resulting in a broader species-specific effector repertoire of Z. ardabiliae compared to the other species [The genes with predicted functions and, in particular, functions related to pathogenicity are largely shared among species in the unctions . Likewislineages . We hyposs hosts . Variatise genes . Here we species .Z. tritici core genes are generally more expressed compared to accessory genes in planta. Core genes are more likely to encode essential functions, which could explain higher expression pattern during infection. Differentially expressed genes that are specifically induced during the course of the infection are very likely to have functions essential to pathogenicity of the fungus. Interestingly, here we show that within the genes differentially expressed between the biotrophic and the necrotrophic phases of infection, isolate- and species-specific genes have higher expression levels than core genes. These isolate- and species-specific genes could be functionally important and regulate functions linked to infection success in the biotrophic phase or to leaf colonization in the necrotrophic phase. Since these genes show presence-absence polymorphisms in the genus and in the Z. tritici species, they could represent a reservoir for possible adaptations to host species, host cultivars or local environments.Consistent with a previous study , we founZ. tritici. Comparing genome content and genome structure, we identified a large shared predicted effector repertoire characterized by inter- and intraspecies presence-absence polymorphisms. Major features of genomic, transcriptomic and epigenetic compartmentalization, distinguishing accessory and core compartments, were shared among wheat and wild-grass infecting Zymoseptoria species. We conclude that compartmentalization of genomes is an ancestral trait in the Zymoseptoria genus.We investigated the genomic architecture in a genus of plant pathogens, including the economically relevant wheat pathogen Zymoseptoria isolates can be found in Table Z. tritici isolates IPO323 (reference), Zt05 and Zt10, one Z. ardabiliae isolate (Za17), one Z. brevis isolate (Zb87), one Z. passerinii isolate (Zpa63), and the Z. pseudotritici isolate (Zp13). For transcriptomic and epigenomic data we used Z. tritici Zt09 (IPO323 ΔChr18) a derivate of the reference isolate IPO323 deleted with the chromosome 18 [Details regarding the individual osome 18 .Z. tritici isolates Zt05 and Zt10 were described and published previously [Z. pseudotritici, Z. ardabiliae, Z. brevis and Z. passerinii were maintained in liquid YMS medium  at 200 rpm and 18 °C. DNA extraction was conducted as previously described [Long read assemblies of the eviously . For DNAescribed . PacBio escribed , 46. TheAfter selection, the average fragment length was 15 kb. Sequencing of the isolates Za17, Zb87, and Zp13 was run on a PacBio RS II instrument at the Functional Genomics Center, Zurich, Switzerland. Sequencing of the Zpa63 isolate was performed at the Max Planck-Genome-Centre, Cologne, Germany.For each isolate, we assembled the genome de novo using SMRT Analysis software v.5 (Pacific Bioscience) with two sets of parameters: default parameters and “fungal” parameters. We chose the best assemblies generated by comparison of all assembly statistics produced by the software Quast such as the number of finished contigs, the size of the assembly and the N50 . SummaryZ. ardabiliae Za17, Z. brevis Zb87, Z. pseudotritici Zp13, Z. passerinii Zpa63, and the three Z. tritici isolates IPO323, Zt05 and Zt10 . Fohttps://gitlab.gwdg.de/alice.feurtey/genome_architecture_zymoseptoria).We used previously published RNA sequencing data to increase the quality of the gene prediction and combined three distinct methodologies , 30, 32.Pezizomycotina. The predicted genes were also used to create a phylogeny with the online implementation of CVtree3, using kmer sizes of 6 and 7 as recommend for fungi [The predicted gene sequences were the basis for an evaluation of the completeness of the assembly and gene prediction by the program BUSCO v.3 . We usedor fungi . We geneor fungi .Z. tritici genome and the reference Cercospora beticola genome, a related Dothideomycete, which we used as outgroup to identify genes with orthologs restricted to the Zymoseptoria genus [We predicted orthologs between the newly assembled genomes, the reference ia genus . For thiia genus , 61 whicia genus . These oia genus .The whole-genome assemblies were used to create a matrix distance with the software andi, from which we generated a tree . A seconWe used several tools to predict the putative functions for the gene models. First, we used the eggnog-mapper which provide COG, GO and KEGG annotations . The onlhttps://gitlab.gwdg.de/alice.feurtey/genome_architecture_zymoseptoria. Briefly, we gathered outputs of several software to predict the cellular location, transmembrane domain and secretion and created a consensus based on the different output to prevent the pitfalls of any one of these methods. From this consensus, we extracted the gene products predicted to be secreted and without a transmembrane domain. The comparisons of genes functions repartition were done by combining predictions of COG categories, secondary metabolite genes with pathogenicity-related gene functional categories such as CAZymes and effector predictions.Additionally, we designed a pipeline to predict protein cell localization and to identify effector candidates. The pipeline for effector prediction is outlined in Figure SZ. tritici isolates IPO323, Zt05 and Zt10, we used previously generated RNA-seq data from in planta and in vitro growth [in planta RNA-seq data was obtained from infected leaves at four different stages corresponding to early and late biotrophic and necrotrophic stages of the three Z. tritici isolates [in planta infection, considering only two infection stages; biotrophic and necrotrophic [To update expression profiles on the new genome assemblies and new gene predictions of the three o growth , 32. Theisolates . Strand-isolates , using gisolates . The reaotrophic . Gene exotrophic . For genZ. ardabiliae (Za17) and Z. pseudotritici (Zp13) cells were grown in liquid YMS medium for 2 days at 18 °C until an OD600 of ~ 1 was reached. Chromatin immunoprecipitation and library preparation were performed as previously described [http://hannonlab.cshl.edu/fastx_toolkit/), mapping was performed using bowtie2 [escribed . We sequ bowtie2  and peak bowtie2 . Peaks w bowtie2 .Additional file 1: Table S1. Summary metrics of assemblies filtering process in function of contig coverage quality, presence of telomeric repeats and mitochondrial DNA.Additional file 2: Table S2. Summary of gene expression in Z. tritici isolates during infection stages.Additional file 3: Table S3. Enrichment of species-specific and isolate-specific genes per chromosome/contig.Additional file 4: Table S4. Summary metrics of Z. tritici IPO323, Zt10 and Zt05 expressed genes during in planta infection stages: biotrophic versus necrotrophic.Additional file 5: Table S5. Summary metrics of Zymoseptoria genome sequencing coverage, assembly size and median coverage per contig.Additional file 6: Figure S1. Plant-associated genes compartmentalization along the chromosomes. The first track represents core (dark grey) and accessory (light grey) chromosomes/contigs. Telomeric repeats are indicated in orange. Circles from outside to inside represent the position of: predicted effector genes (blue), biosynthetic gene clusters  and CAZymes (yellow) per 100 kb windows, respectively.Additional file 7: Figure S2. A) Intra-species synteny between the reference genome of Z. tritici and the genome of the Z. tritici isolate Zt05. Each color represents a different chromosome as based on the reference Z. tritici genome, except for accessory chromosomes, which are in grey. The connecting lines represent orthologs between each genome. The track between the chromosomes and connecting lines are predicted effector genes. Telomeric repeats are indicated in orange. B) Inter-species synteny between reference genome of Z. tritici and Z. pseudotritici. The arrows represent the large-scale inversions identified between the genomes of these two species.Additional file 8: Figure S3. Inter-species synteny between genomes of Z. pseudotritici and Z. brevis. A) High level of nter-species synteny at the whole genome level between Z. pseudotritici (dark grey) and Z. brevis (blue). The arrows indicate the contigs identified as carrying rearrangement with Z. tritici but not between Z. pseudotritici and Z.tritici. B – D) Zoom-in on the contigs highlighted with the arrow in part A, showing the details of the pair-wise synteny of both Z.pseudotritici (Zp13) and Z. brevis (Zb87) with the Z.tritici reference (IPO323) chromosomes.Additional file 9: Figure S4. Genome architecture in A) Z. tritici Zt05, B) Z. tritici Zt10. C) Z. brevis Zb87 and D) Z. ardabiliae Za17. Circles from the outside to the inside represent respectively: gene density along chromosomes/contigs; gene expression in vitro (TPM); H3K4me2 distribution in vitro (only for Za17) and species-specific gene density per 100 kb windows. Contigs are ordered based on synteny with chromosomes of the reference strain IPO323. Telomeric repeats are indicated in orange.Additional file 10: Figure S5. Simplified diagram of the pipeline used to predict the functions and subcellular localization of gene model products.Additional file 11: Figure S6. Functional gene categories in Zymoseptoria spp. genomes. A) The number of genes in Eggmapper COG categories in addition to Effectors and CAZymes. B) Pathogenicity-related genes of interest: secreted proteins, predicted effectors, secreted CAZymes and non-secreted CAZymes. C) Subcellular localization of predicted gene products.Additional file 12: Figure S7. Principal component analysis of DESeq2 rlog transformed expression data. Principal component analysis (PCA) of Zymoseptoria tritici IPO323, Zt05 and Zt10 transcripts levels measured in biotrophic (red) and necrotrophic (blue) stages using RNA-sequencing. Reads detected per transcript (counts) were normalized using the size factor method used by DESeq2 package [ package . The PCA


Zostera marina is limited. In this study, a systematic analysis was conducted on the characteristics of the SOD genes in Z. marina at the whole-genome level. Five SOD genes were identified, consisting of two Cu/ZnSODs, two FeSODs, and one MnSOD. Phylogenetic analysis showed that ZmSOD proteins could be divided into two major categories (Cu/ZnSODs and Fe-MnSODs). Sequence motifs, gene structure, and the 3D-modeled protein structures further supported the phylogenetic analysis, with each subgroup having similar motifs, exon-intron structures, and protein structures. Additionally, several cis-elements were identified that may respond to biotic and abiotic stresses. Transcriptome analysis revealed expression diversity of ZmSODs in various tissues. Moreover, qRT-PCR analysis showed that the expression level of most ZmSOD genes trended to decreased expression with the increase of temperature, indicating that heat stress inhibits expression of ZmSODs and may result in reduced ability of ZmSODs to scavenge ROS. Our results provide a basis for further functional research on the SOD gene family in Z. marina, which will help to determine the molecular mechanism of ZmSOD genes in response to environmental stress.Superoxide dismutases (SODs) serve as the first line of defense in the plant antioxidant enzyme system, and play a primary role in the removal of reactive oxygen species (ROS). However, our understanding of the functions of the SOD family in  Anothern plants . For thear space . FeSOD ilization . With thlization . HoweverZmMnSOD enzyme has been shown to play an important role in minimizing oxidative damage in response to temperature stress . Initial up-regulation of superoxidase dismutase was found, indicating that Z. marina can up-regulate anti-oxidative machinery , and that of Z. muelleri was obtained from Applied Bioinformatics Group (http://www.appliedbioinformatics.com.au).The SOD genes were identified as follows. Firstly, the sequences of published SOD genes of Arabidopsis thaliana and rice were used as query files against the local database of Z. marina, Z. muelleri, and S. polyrhiza, using the BlastP method (e < 1e−10). Next, Hidden Markov Model (HMM) profiles of Fe/Mn-SOD (PF02777 and PF00081) and Cu/ZnSOD (PF00080) were downloaded from the Pfam database (https://pfam.xfam.org/). These files were converted to HMM format by using the Hmm build command, and then applied to local protein retrieval by using the Hmm search command. Sequences of candidate ZmSOD genes were identified using the above two methods. Finally, the presence of SOD domains in identified candidate genes was assessed through SMART (http://smart.embl-heidelberg.de/), NCBI Conserved Domain Search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi), and Pfam, and those lacking apparent SOD domains were excluded from further study (The whole genome sequence of er study .ZmSOD protein using the ProtParam tool (https://web.expasy.org/protparam/) (http://cello.life.nctu.edu.tw/)   and condedu.tw/) .The identified SOD genes were aligned using the MUSCLE software , and thehttp://meme-suite.org/tools/meme) tools were used to detect conserved motifs among SOD proteins of Z. marina. This was done with the default settings except that the number of motifs was set to 9, and the minimum and maximum motif widths were changed to 20 and 150 amino acids, respectively. Then, the identified motifs were detected by SMART software (ZmSODs (Multiple Expectation Maximization for Motif Elicitation (MEME) (software . TBtools (ZmSODs .ZmSOD genes were downloaded from the Z. marina genome database. The cis-regulatory elements in these sequences were then predicted using the PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)  protein database using Blast2GO software  .The predsoftware  with theZ. marina genome were obtained (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) (http://www.pymol.org).Illumina RNA-seq data from a previous study of the obtained . The RNAobtained  includedTASSER/) . FinallyZ. marina were collected in May 2019 from the subtidal zone in Huiquan Bay. Healthy shoots were planted at aquaria bottoms, provided aerated and filtered natural seawater at a constant temperature of 15 °C, and allowed to acclimatize for 7 days. Plants were evenly spaced and positioned upright in each of 4 glass aquaria (200 L) at different temperatures  for 96 h. The aquaria were aerated and illuminated with light intensity conditions of 150 μmol m−2 s−1, and a light:dark ratio of 14:10. Three biological replicates were performed for each process. Leaf samples were immediately harvested after treatments, cooled in liquid nitrogen, and then stored at −80 °C until further use.Entire shoots (with attached roots) of ZmSOD genes at different temperatures were detected using a StepOnePlus™ Real-Time PCR System . The qPCR analysis was conducted using a SYBR Green qPCR kit . The primers of all ZmSOD genes are listed in −ΔΔCt method (n = 3).Total RNA was extracted from leaf samples with the Omega Plant RNAkit  according to the manufacturer’s instructions. The quality and integrity of total RNA samples were assessed by NanoDrop  and 1% agarose gel electrophoresis. The raw RNA (0.5 µg) was used as templates to synthesize cDNA using the FastQuantity RT Kit . The reactions were performed at 55 °C for 1 h, and terminated by heating at 70 °C for 5 min. The mRNA expression levels of t method . Three bZmSOD genes were identified in the Z. marina genome, including two Fe-SOD, three Cu/Zn-SOD, and one Mn-SOD , isoelectric points (pI), genomic location, instability index values, and subcellular localizations  tree was constructed based on 38 SOD proteins. The ML results showed that the SOD proteins from different plants could be divided into two major clades, Cu/ZnSODs and Fe-MnSODs . Motifs 1, 3, 4, and 9 show relation to the Fe- and MnSOD domain .To study the motif composition and phylogenetic relationship of  by MEME . Among tZmSOD genes were examined using TBtools. Generally, the ZmSOD family can be divided into three main groups: ZmCZSOD, ZmMnSOD, and ZmFeSOD. The numbers of introns in the ZmSOD genes ranged from five to eight introns, and genes gathered in the same group tended to exhibit similar structural features  and the least abundant one is the defense stress-related response element (TC rich repeats), with one in ZmCZSOD-2 and one in ZmFeSOD-3. Low-temperature stress-related response elements (LTR) and drought-related elements (MBS) were also identified, but no high-temperature stress-related response elements (HSE) were found.The regulatory controls of ARE tool . The resOD genes . A totalZmSOD proteins were predicted by GO annotation analysis. Based on amino acid similarity, five ZmSOD proteins were categorized into eight functional categories , and the expression of the ZmFeSOD-3 gene was the lowest in most tissues . The other genes exhibited different expression patterns.To gain insight into the functions of  tissues . All fivZmSOD genes under temperature stress, qRT-PCR analysis was used to determine the transcription level of each ZmSOD gene. After 96 h treatment, the relative expression levels of the ZmSOD genes fluctuated under temperature stress , potentially indicating a key role in Z. marina development or environment adaptation. Similar observations have also been reported in durum wheat, the TdMnSOD was induced by different abiotic stresses, and recombinant yeast cells and TdMnSOD-transgenic Arabidopsis plants exhibited enhanced tolerance to multiple abiotic stresses  has long been viewed as the most sensitive component to high temperature in plants. Heat stress can cause photoinhibition of PSII and promotes the accumulation of ROS via the Mehler reaction in chloroplasts, which accelerates oxidative stress , indicating that ZmMnSOD-5 might help clear mitochondrial ROS and protect cells against oxidative stress at high temperature. These results support a role of ZmSOD genes in temperature stress response, however, the actual role of each ZmSOD gene needs to be validated in future studies.Temperature stress affects the ability of plant defense systems to counterbalance ROS accumulation and reduce oxidative stress . In our na cells . The optbe fatal . In our  at 5 °C . Thus, Ze stress . Low exp studies , among tand lead . In Musat stress . In our Z. marina genome, which identified two Cu/ZnSODs, two FeSODs, and one MnSOD. Subsequently, we investigated the genomic organization, gene structure, phylogenetic relationships, conserved domains, gene structures, cis-regulatory elements, GO annotations, and tissue-specific expression profiles for the identified genes. The qRT-PCR analysis showed that ZmSOD genes could respond to temperature stress. These results provide several new and useful findings to increase understanding of the functions of individual ZmSOD genes in response to environmental stress.In the current study, a comprehensive analysis of the SOD gene family was conducted with the 10.7717/peerj.9063/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.9063/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.9063/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.9063/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.9063/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.9063/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.9063/supp-7Supplemental Information 7Click here for additional data file.