Brassica mainly comprises three diploid and three recently derived allotetraploid species, most of which are highly important vegetable, oil or ornamental crops cultivated worldwide. Despite being extensively studied, the origination of B. napus and certain detailed interspecific relationships within Brassica genus remains undetermined and somewhere confused. In the current high-throughput sequencing era, a systemic comparative genomic study based on a large population is necessary and would be crucial to resolve these questions.The genus Brassica materials, which contain 72 accessions and maximally integrated the known Brassica species. The Brassica genomewide cpDNA and mtDNA variations have been identified. Detailed phylogenetic relationships inside and around Brassica genus have been delineated by the cpDNA- and mtDNA- variation derived phylogenies. Different from B. juncea and B. carinata, the natural B. napus contains three major cytoplasmic haplotypes: the cam-type which directly inherited from B. rapa, polima-type which is close to cam-type as a sister, and the mysterious but predominant nap-type. Certain sparse C-genome wild species might have primarily contributed the nap-type cytoplasm and the corresponding C subgenome to B. napus, implied by their con-clustering in both phylogenies. The strictly concurrent inheritance of mtDNA and cpDNA were dramatically disturbed in the B. napus cytoplasmic male sterile lines . The genera Raphanus, Sinapis, Eruca, Moricandia show a strong parallel evolutional relationships with Brassica.The chloroplast DNA and mitochondrial DNA were synchronously resequenced in a selected set of Brassica, which can substantially facilitate the development of novel Brassica germplasms.The overall variation data and elaborated phylogenetic relationships provide further insights into genetic understanding of  Brassica in Brassicaceae family is one of the most agriculturally important plant genera worldwide, which mainly comprises three diploid and three allotetraploid species, as described in the genetic model of U’s Triangle [Brassica napus , B. juncea  and B. carinata  are thought to be generated by interspecific hybridizations between each two of the three basic diploid progenitors: B. rapa , B. oleracea  and B. nigra . The current abundant genomic and phenotypic diversifications have given rise to highly diverse crops of vegetable, oil, ornamental, fodder and fertilizer use types. To date, B. napus (rapeseed) has become to be the second largest vegetable oil crop worldwide [Brassica as an ideal model for studying polyploidy [The genus Triangle . Brassicus AACC, n = 38, Blyploidy .B. napus is supposed to originate from certain kind of hybridization between B. rapa and B. oleracea, which co-existed in European Mediterranean coastwise regions, at approximately 10,000 years ago [B. napus and its potential progenitors revealed that winter type rapeseed might be the original form of B. napus, European turnip ancestor might donate the A subgemone, the origin of C subgenome is mysterious and it was currently supposed to evolve from a common ancestor of cultivated C-genome species  [ears ago . Then itse kale) . The A ase kale) .B. napus remain largely unresolved. The frequent post-formation introgression events occurred during human breeding consequentially confused the recovery of the originating trajectory of B. napus at nuclear genome level. Cytoplasmic DNA in plant cell, especial for chloroplast DNA (cpDNA), are structurally simple with a small genome size (100–300 kb) and stably inherited mostly in a uniparental pattern with nearly none recombination [B. napus [B. napus of this same cpDNA haplotype generally formed an ambiguous clade, which did not group with the investigated B. rapa or B. oleracea accessions [B. napus accessions were grouped with the majority of B. rapa accessions suggested another independent cytoplasmic origin from B. rapa [B. napus has drawn much more attention for the extensive application of its cytoplasmic male sterility (CMS) lines in the heterosis-driving hybrid breeding, mainly containing polima (pol), cam and nap mitotypes in the natural resources [. Nap mitotype is predominant in natural B. napus, However, it remains unsolved and were supposed to be from an unidentified or lost mitotype of B. rapa [nap mitotype was further judged to be derived from B. oleracea, since it was phylogenetically grouped with botrytis-type and capitata-type B. oleracea [To date, the genuine originating mechanisms of ome size 0–300 kb bination –14. GenoB. napus , 16. Whicessions , implyin B. rapa , 18, indesources . Nap mit B. rapa . The napoleracea .nap-type B. napus are controversial and ambiguous. Previous cpDNA and mtDNA-based studies were separated and never been corresponded and integrated to accurately explore the multiply origin of B. napus. Cytoplasmic DNA and its corresponding cytonuclear interactions, are highly valuable for crop breeding not only due to its cause of cytoplasmic male sterility [nap-type rapeseed [B. napus have been synchronously resequenced at the cpDNA and mtDNA level, a systematic genetic investigation and an elaborate phylogenetic pedigree at intraspecific level have been constructed, with the purpose of improving our understanding of the whole Brassica genus.Apparently, the current above conclusions regarding the origin of terility , but alsrapeseed  and planBrassica genus, genotyping analysis through High Resolution Melting (HRM) method were performed in our germplasm collections , certain wild C-genome relatives and three B. napus cytoplasmic male sterility (CMS) lines were treated as each with a distinct haplotype for the subsequent genome sequencing. B. cretica, B. incana, B. insularis and B. villosa represent the wild C-genome relatives. Polima [nsa [mori [Eruca sativa (2n = 22), Raphanus sativus (2n = 18), Sinapis arvensis (2n = 24) and Moricandia arvensis (2n = 28), were also included to enrich this study (Table To distinguish the cytoplasmic DNA (cpDNA and mtDNA) haplotypes within s Figure . Primerss Figure . Three m. Polima , 24, nsaima [nsa  and morisa [mori , 27 are dy Table .Brassicaceae family. The mapped reads were extracted and de novo assembled by SOAPdenovo software package [Cytoplasmic DNA was synchronously isolated from 72 accessions that represent for all major cytoplasmic haplotypes and morphological varieties Table , using a package . GeneralB. napus strain 51,218 [nap mitotype, were respectively used as reference sequences to call the overall cpDNA and mtDNA basic variants. The calling was conducted by standard BWA/Genome Analysis Toolkit (GATK) pipeline with manual inspection [Brassica genus. While, approximately 3400 polymorphic sites (2700 SNP and 700 InDels) were identified for the mitochondrial haplotypes , another branch further split into two little branches, containing cam-type  and polima-type  lines, respectively  and part of the wild B. oleracea were shown with one nearly identical chloroplast genome sequence. However, the C-genome wild relatives  each contains a distinct haplotype. All the C-genome species demonstrated a hierarchically clear pedigree, from B. villosa stepwise to the cultivated B. oleracea , Sarsons-1 (B. rapa), Broccoletto-3 (B. rapa), Black mustard (B. juncea) and Ethiopian mustard (B. carinata) were added from Li et al., [Brassica genus was mainly divided into three clades, from which the maternal origin of the three natural allotetraploid species can be clearly inferred  and polima-type B. napus, respectively. Three B. juncea accessions clustered only in Clade I without any further divergences from their co-clustered B. rapa accessions, thus indicating that the investigated B. juncea has a monophyletic maternal origin from cam-type B. rapa. Clade II comprises all the B. oleracea lines and other wild C-genome species, parallelly branched with Clade I. The branch, which comprises only the B. napus accessions with a same nap cytoplasmic type, is inserted in the middle of Clade II and separated certain C-genome wild relatives (B. insularis and B. villosa) from the remaining part, which contains all B. cretica, B. incana and the cultivated B. oleracea. Clade III comprises mainly B. nigra, B. carinata and B. maurorum accessions, indicating that the investigated B. carinata has a monophyletic maternal origin from B. nigra. The major cytoplasmic haplotype of B. nigra was designated as nigra-type cytoplasm. The wild species B. maurorum had been reported to be close to the B-genome species [B. juncea and B. caritana, B. napus was dispersedly distributed in the B. rapa and B. oleracea clusters, suggesting its multiple maternal origins from A-genome B. rapa or certain C-genome Brassica species (2n = 18).A part of the above intra-specific materials were selected capable of maximumly representing each their intraspecific genetic diversities, and then together with  et al.,  to enric species  and seemRaphanus sativus was inserted between Clade II and Clade III and bidirectionally close to B. villosa and B. maurorum in the Brassica phylogenetic tree  chloroplast genome sequences in Brassicaceae family was constructed  accessions displayed scattered distribution that is fully merged together with the B-genome containing species in Clade III. These findings imply a tight evolutionary association among Brassica and these relatives. Cakile arabica, Orychophragmus diffusus, Alliaria grandifolia, Isatis tinctona and Scherenkiella parvula in Clade IV were shown to be close to Brassica cluster at cytoplasmic DNA level. Successful germplasm development through inter-specific sexual or somatic hybridization between Brassica species with Orychophragmus violaceus or Isatis tinctona [Brassica.Intriguingly, ree Fig. , suggest studies , 34. Anotinctona , 36 coulBrassica phylogeny if being inferred based on mitochondrial genomes, the segmented sequences containing the mitochondrial allelic variants from each corresponding material inside and around Brassica genus were extracted and concatenated as each separate intact sequence. All the assembled sequences were subjected to phylogenetic analysis according to the above same procedure used for chloroplast genomes. The obtained mitochondrial tree  in the mtDNA derived tree were also integrated into Brassica genus, demonstrating that mtDNA evolved parallelly linked with cpDNA in Brassica genus. Nevertheless, differences happened for the B. napus cytoplasmic male sterile lines, i.e., mori [nsa [Mori and nsa lines located in the cam-type and nap-type B. napus clusters, respectively, in the cpDNA deriving tree , most of the genus speciated and formed several large clades. Brassica genus emerged approximately at 4.85 Mya, and began maybe as a kind of B. nigra or B. rapa. Moricandia arvensis, Eruca stivus, Brassica maurorum, Raphanus sativus and Sinapis arvensis speciated at 3.15 Mya, 2.85 Mya, 2.17 Mya, 2.05 Mya and 1.42 Mya, respectively. The Brassica C-genome species  separated from A-genome species (B. rapa) since 1.12 Mya. Three allotetraploid species  speciated during the period 0.17–0.01 Mya or much later, which are consistent with the estimated originating time of ~ 7500 years ago for B. napus [B. juncea [Brassica tetraploid species are much younger than certain other polyploidy crops, e.g., the emerge of cotton (Gossypium hirsutum) at 1–2 Mya [Glycine max) at 0.8 Mya [The phylogenetic tree containing 54 chloroplast genome sequences in  Reltime . It was ectively , 38. Euce Figure  indicate 1–2 Mya , 40 and  0.8 Mya .B. rapa mainly contains a predominant haplotype (cam-type) and a newly identified sarson-type cytoplasm, which presents merely approximately 50 cpDNA and 20 mtDNA basic variations  clustered next to B. nigra. None cytoplasmic DNA types of BB-genome and CC-genome diploid species have ever been detected in the germplasm collections of the natural B. juncea or B. carinata, respectively. These results ascertain that B. juncea and B. carinata each has a monophyletic maternal origin from B. rapa and B. nigra, respectively.As mentioned above, ns Table . FourteeB. napus collection. Of the 24 sequenced B. napus accessions, 7 lines tightly clustered with the majority of B. rapa in Clade I  also clustered in Clade I but adjacent to sarson-type B. rapa (Sarson-1) with minor divergence, suggesting that the polima haplotype may inherit from certain sarson-type like B. rapa, which is different from the previous assumption that polima haplotype likely diverge from Cam haplotype. Nap-type cytoplasm, which is predominant with a population frequency of 87.1% in our B. napus collection, resides in numerous elite rapeseed cultivars worldwide . The cluster of nap-type B. napus is inserted in the middle of C-genome Clade II, appears like a separate haplotype that is parallel to B. cretica, B. incana, B. insularis, B. villosa and B. oleracea. This was also supported by our mtDNA-based phylogeny  demonstrated that most GB associations in Brassica species could be detected in Raphanus sativus [Raphanus and Brassica species potentially shared a common hexaploid ancestor after whole genome triplication (WGT). Common translocation Proto-Calepineae Karyotype (tPCK)-like ancestors were deduced to be the likely common ancestor of all current Brassiceae species that had undergone WGT and repetitive chromosomal rearrangements. Phylogenetic analysis based on 32 mitochondrial protein-coding genes suggested that Eruca sativa is closer to the Brassica species and Raphanus sativus than to Arabidopsis thaliana [Raphanus, but also Eruca, Moricandia and Sinapis species as basic diploid species as suggested herein by our studies  was primarily obtained by protoplast fusion between Moricandia arvensis  and B. juncea [B. napus through several rounds of sexual hybridization. Nsa sterile line (AC500) was developed primarily also from protoplast fusion between B. napus and Sinapis arvensis [Ogura sterile line, which was also developed through somatic hybridization of Raphanus sativus and B. napus [Brassica mitochondrial genome sequences belonging to U’s Triangle. Therefore, both the mori and nsa lines ought to contain plenty of mitochondrial genome regions from their incipient donor parents, thus clustered close to Moricandia arvensis and Sinapis arvensis, respectively, in the mtDNA based phylogenetic tree. It seems that somatic hybridization through protoplast fusion is an effective means to induce the recombination of mitochondrial genomes. Intergenomic recombinations and DNA rearrangements had been frequently identified within mitochondrial genomes [Generally, mitochondrial and chloroplast genomes demonstrate consistent evolutionary relationships in higher plants, because of their coupled inheritance in a uniparental manner. The inconsistent locations of ree Fig.  and S5 r. juncea , 44, andarvensis . SequencB. napus , 46, revB. napus . Nine re genomes , 49, sugnsa and mori lines possess the identical nap- and cam-type chloroplast genomes from each of their recipient B. napus and B. juncea lines  is a diploid edible plant and its medicinal properties have various promoting effects to health [Moricandia arvensis (2n = 28) is reported to be a C3-C4 intermediate species, transferring this feather of strong photosynthetic efficiency into Brassica crops have been tried by means of hybridization [Sinapis arvensisis is a wild weedy plant of the genus Sinapis, both Sinapis arvensisis and Sinapis alba (2n = 24) possess high resistances to drought, leanness, multiple diseases, herbicides and pod shattering [Raphanus species were identified to be immune to clubroot [Brassica present a potential guidance for improving the current Brassica crops and even the development of certain novel allotetraploid gerplasms by intercrossing the corresponding diploid species.The diversified o health . Moricandization . Sinapisattering . Certainclubroot . The intBrassica populations  remain rather low, which may be a key limiting factor for crop improvment. To create extensive germplasms with various novel cytonuclear combinations may be of great significance for both the fundamental studies and the crop breeding in the future.Natural cytoplasmic variations could interact with nuclear genomes to shape a large proportion of phenotypic traits that contributed to adaptation . The cytBrassica cytoplasmic genomes has been systematically identified by the synchronous resequencing of the chloroplast and mitochondrial genomes. The whole Brassica phylogeny has been refined and enriched, providing further insights into the understanding of the origin of the important B. napus nap-type cytoplasm. Human interference has remodeled the cytoplasmic inheritance in B. napus. The obtained genetic resources can substantially support the further research on the Brassica evolution, the development of novel germplasms.Compared with the huge nuclear genomes, cytoplasmic DNA is a primary and easy means to evaluate the evolutionary relationships. Meanwhile, it is also highly effective to dissect the maternal origins and to infer the primary originating events. Herein, the overall genetic diversity of B. napus accessions were collected from the National Mid-term Genebank for Oil Crops of China, it has been repeatedly used as a core rapeseed collection in our previous studies [B. rapa and B. juncea populations contain primarily landraces, which were collected across China. The cultivated B. oleracea inbred lines were obtained commercially from market, the wild B. oleracea and other C-genome wild species which are native to coastal southern and western Europe were collected from rocky Atlantic coasts of Spain (Bay of Biscay) and the Centre for Genetic Resources, The Netherlands (CGN). Detailed information in regard to all the above materials and other materials in Brassica genus and its relative genera are provided in Table A set of 480 worldwide  studies . The B. Leaf total DNA of the corresponding accessions were directly extracted using the cetyltrimethylammonium bromide (CTAB) method described by Lutz et al.,  and thenIsolation of the cytoplasmic DNA was performed according to Hao et al.,  with minBrassica chloroplast genomes through BLAST alignments, and then sutured by manually jointing the overlapping ends between each two contiguous contigs. Basic variants (SNP and short InDels) were called using standard BWA/Genome Analysis Toolkit (GATK) pipeline [B. napus line 51,218 (GenBank: KP161617.1 and KP161618.1) were used as the cpDNA and mtDNA reference genomes, respectively.High-throughput sequencing of the cytoplasmic DNA was performed according to our previous study . The DNApipeline , the chlChloroplast genome sequences were trimmed with aligned beginning sequences, and then subjected to alignment, which was conducted by ClustalW . MaximumAdditional file 1 Figure S1. Representative genotyping results by HRM analysis. (A) The normalized and temperature-shifted difference plot indicated that three site-specific haplotypes were identified in a plate of 96 plant DNA samples using HRM407 primers. (B) The normalized and temperature-shifted difference plot showed that two site-specific haplotypes were identified in a plate of 96 plant DNA samples using HRM727 primers. Figure S2. Representative genotyping results in a plate of 384 plant DNA samples by KASP analysis for primers mP1858 (A) and cP1225 (B). Figure S3. Phylogenetic tree of Brassica rapa. This tree structure was inferred using Maximum Likelihood method based on the entire chloroplast genomes from representative B. rapa materials. The sequence data for materials Zicaitai-1, Turnip-3 and Sarsons-1 from Li et al. (2017) were added. Figure S4. Phylogenetic tree of Brassica juncea. This tree structure was inferred using Maximum Likelihood method based on the entire chloroplast genomes from representative B. juncea materials. Figure S5. Phylogenetic tree of Brassica napus. This tree structure was inferred using Maximum Likelihood method based on the entire chloroplast genomes from representative B. napus materials. Figure S6. Phylogenetic tree of Brassica C-genome species. This tree structure was inferred using Maximum Likelihood method based on the entire chloroplast genomes from representative B. oleracea materials. Figure S7. Timetree analysis using the RelTime method. The timetree was computed based on the phylogenetic tree of Brassicaceae family in Fig. Eucalyptus verrucata labeled with lightgray was set as outgroup.Additional file 2 Table S1 Primers used for HRM and KASP genotyping analysis. Table S2 List of plant materials investigated in this study. Table S3 Total cpDNA and mtDNA variant data.Additional file 3 Appendix A The dataset for chloroplast genome sequences of 72 Brassica accessions. Appendix B Accessions of the public sequence data.


Cryphonectria parasitica colonized Europe. After establishment, the pathogen population gave rise to a highly successful lineage that spread rapidly across the continent. Core to our understanding of what makes a successful pathogen is the genetic repertoire enabling the colonization and exploitation of host species. Here, we have assembled >100 genomes across two related genera to identify key genomic determinants leading to the emergence of chestnut blight. We found subtle yet highly specific changes in the transition from saprotrophy to latent pathogenicity mostly determined by enzymes involved in carbohydrate metabolism. Large-scale genomic analyses of genes underlying key nutrition modes can facilitate the detection of species with the potential to emerge as pathogens.Forest and agroecosystems, as well as animal and human health, are threatened by emerging pathogens. Following decimation of chestnuts in the United States, the fungal pathogen  Cryphonectria, including the prominent invasive pathogen Cryphonectria parasitica, the causal agent of chestnut blight on Castanea species. We assembled and compared the genomes of pathogenic and putatively nonpathogenic Cryphonectria species, as well as sister group pathogens in the family Cryphonectriaceae , to investigate the evolution of genome size and gene content. We found a striking loss of genes associated with carbohydrate metabolism (CAZymes) in C. parasitica compared to other Cryphonectriaceae. Despite substantial CAZyme gene loss, experimental data suggest that C. parasitica has retained wood colonization abilities shared with other Cryphonectria species. Putative effectors substantially varied in number, cysteine content, and protein length among species. In contrast, secondary metabolite gene clusters show a high degree of conservation within the genus. Overall, our results underpin the recent lifestyle transition of C. parasitica toward a more pathogenic lifestyle. Our findings suggest that a CAZyme loss may have promoted pathogenicity of C. parasitica on Castanea species. Analyzing gene complements underlying key nutrition modes can facilitate the detection of species with the potential to emerge as pathogens.Emerging fungal pathogens are a threat to forest and agroecosystems, as well as animal and human health. How pathogens evolve from nonpathogenic ancestors is still poorly understood, making the prediction of future outbreaks challenging. Most pathogens have evolved lifestyle adaptations, which were enabled by specific changes in the gene content of the species. Hence, understanding transitions in the functions encoded by genomes gives valuable insight into the evolution of pathogenicity. Here, we studied lifestyle evolution in the genus IMPORTANCE Forest and agroecosystems, as well as animal and human health, are threatened by emerging pathogens. Following decimation of chestnuts in the United States, the fungal pathogen Cryphonectria parasitica colonized Europe. After establishment, the pathogen population gave rise to a highly successful lineage that spread rapidly across the continent. Core to our understanding of what makes a successful pathogen is the genetic repertoire enabling the colonization and exploitation of host species. Here, we have assembled >100 genomes across two related genera to identify key genomic determinants leading to the emergence of chestnut blight. We found subtle yet highly specific changes in the transition from saprotrophy to latent pathogenicity mostly determined by enzymes involved in carbohydrate metabolism. Large-scale genomic analyses of genes underlying key nutrition modes can facilitate the detection of species with the potential to emerge as pathogens. Pyrenophora tritici-repentis, a former saprophyte or weak pathogen on grass species including wheat, which became highly pathogenic on wheat through acquisition of the virulence gene ToxA from the wheat pathogen Stagonospora nodorum   , 6. HencTo be successful, pathogens must overcome physical and chemical barriers deployed by the host . Plant-p8–14–The emergence of pathogenic lifestyles has often required the ability to secrete effector proteins and secondary metabolites during contact with the host. Effectors are characterized as quickly evolving small, cysteine-rich secreted proteins, which are produced to manipulate plant host immune responses , 26. BioChrysoporthe species affecting hosts in the order Myrtales , as well as Cryphonectria parasitica (Murr.) Barr., the causal agent of chestnut blight on Castanea (Fagaceae) species , where it occurs as a weak pathogen on Chinese (Castanea mollissima Blume) and Japanese (Castanea crenata Siebold & Zucc.) chestnuts. However, C. parasitica was first described after its discovery in 1904 on American chestnut [Castanea dentata (Marsh.) Borkh.] in the United States , maples (Acer spp.), and European hornbeam (Carpinus betulus L.)  includes mainly bark-inhabiting species ranging from weak to severe pathogens , 35. The species , 37. C.  America . In Euro lineage . The inv lineage . BesidesC. parasitica has closely related sister species, which are considered weak pathogens or saprophytes (Cryphonectria japonica Tak. Kobay. & Kaz. Itô (previously named Cryphonectria nitschkei) was isolated from Ca. crenata in Japan . The diversity of lifestyles within the Cryphonectriaceae, including the emergence of new pathogens, raises important questions of whether genetic factors facilitate pathogenic lifestyles.Both in the native and in the invasive range, rophytes . Among tin Japan , 42 and in Japan . The Eurecipiens ) was iso Algeria –47. Inocrasitica , 43. Two species . Interesntinents . C. carpC. parasitica, C. radicalis, C. naterciae, and C. japonica and a recently detected European Cryphonectria species named C. carpinicola . We analyzed orthology among the gene sets of the species and constructed a robust phylogenomic tree. We find that Cryphonectriaceae share similar trophic lifestyle traits. However, the chestnut pathogen C. parasitica has a substantially reduced complement in CAZymes. In contrast, the capacity to produce secondary metabolites is reduced among Cryphonectria species but is broadly conserved within the genus. Effector candidate proteins show genus and species specificity consistent with faster evolvability of the underlying genes.In this study, we assembled and analyzed 104 genomes of the Cryphonectriaceae family including the major representatives Cryphonectria species isolates of Asian, European, and North American origin, in addition to the previously assembled genome of C. parasitica reference genome EP155. As a near outgroup to the genus Cryphonectria, we analyzed previously assembled draft genomes of 3 Chrysoporthe species from South Africa, Colombia, and Indonesia. To assemble Cryphonectria genomes de novo, we used Illumina sequencing data at 9 to 53× coverage (Cryphonectria and Chrysoporthe genome assemblies showed >95% completeness for BUSCO genes (ascomycota_odb9 database) with the C. parasitica isolate M7832 having the lowest score at 95.9% (C. japonica) to 41.9 Mb (C. carpinicola). Pathogenic species had slightly larger genomes ranging from 43.7 Mb (C. parasitica and Chrysoporthe austroafricana) to 45 Mb (Chrysoporthe cubensis) (We assembled draft genomes of 100 coverage . All Cryat 95.9% . Based oubensis) ; Fig. 1Aubensis) . This shCryphonectria species compared to ∼12,800 to 13,170 genes in Chrysoporthe spp.  shows close proximity of analyzed Cryphonectria and Chrysoporthe species, clustering at the verge with other hemibiotrophic and necrotrophic species  . We anal species . Lifestylsa mali .C. parasitica  than in Chrysoporthe spp. (n = 12 to 14) and saprophytic Cryphonectria (n = 15 to 16). Analyzing CAZymes for which at least one species is missing an ortholog, Cryphonectria species share a relatively conserved set of ortholog CAZyme genes as expected from their short phylogenetic distance , which is present only in Asian Cryphonectria species, as well as a single GH orthogroup  present only in European Cryphonectria species (C. parasitica displayed a particularly high degree of intraspecific presence/absence variation for four auxiliary activity (AA) and GH enzymes, which are otherwise well conserved   . The fouCryphonectria species and a member of the genus Chrysoporthe (Chr. cubensis), we conducted an inoculation experiment on dormant and healthy chestnut stems. We performed the experiment with and without prior removal of the bark. None of the species were able to colonize dormant chestnut logs without artificial wound induction. After 2 weeks of incubation, C. japonica showed signs of mycelial growth on the bark at a maximum of 1 cm beyond the inoculation point. No bark penetration was detected. For inoculations with bark removal, C. parasitica expectedly showed the fastest and most extensive lesion growth. Other Cryphonectria species, with the exception of one C. radicalis isolate (M4733), developed only minimal lesions  or variable substrate conditions  (Chr. cubensis showed growth on nonhost chestnut (Ca. sativa) logs; however, lesions developed at a comparatively slow pace (C. parasitica.To assess the wood-colonizing capabilities of different  lesions . We founickness) . The euclow pace . After 410.1128/mSphere.00737-20.1FIG S1Castanea sativa) with bark removal. Measurements were taken once per week during four weeks. Download FIG S1, PDF file, 1.2 MB.Lesion length on dormant chestnut stems  revealed 28 clusters conserved among all analyzed Cryphonectriaceae  and all four (Chr. cubensis) core genes -like, and NRPS-T1PKS clusters on scaffolds 4, 6, and 11  (C. parasitica isolates from China (TA51), Georgia (M7776 and M7832), Japan (WB-3), and the United States (MD-1). Generally, we identified only weak homology with secondary metabolite clusters in other species. A notable exception includes two gene clusters potentially underlying emodin production (Secondary metabolites (SMs) can play important roles in pathogenicity and the interaction with microbes , 52. We  species . Among t species . The cla species , was exc species . The pretriaceae . Additiore genes . Overall0009181) . The cluoduction .10.1128/mSphere.00737-20.3FIG S3C. parasitica reference genome cluster to emodin and orthologs in other Cryphonectriaceae (gray = gene is absent). Download FIG S3, PDF file, 0.4 MB.Two PKS gene clusters (biosynthetic core gene orthologs OG0009468 and OG0002648) potentially underlying emodin production. Synteny plots of emodin and identified homologous gene clusters among species. Heatmaps show similarity (%) of genes in the Copyright © 2020 Stauber et al.2020Stauber et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Cryphonectria species encode slightly more putatively secreted proteins (n = 777 to 796) than pathogenic Chrysoporthe spp. (n = 751 to 772). Surprisingly, C. parasitica encodes markedly fewer secreted proteins (n = 619) than all other species  conserved orthologs in all Cryphonectriaceae, and 91 orthologs showed presence/absence variation among species   . For eigand S35) .Cryphonectria, we detected striking CAZyme gene loss in the invasive pathogen C. parasitica. In spite of the substantial CAZyme gene loss, C. parasitica shares wood colonization strategies with the other Cryphonectria species and has retained the ability for early saprotrophic wood decay. In contrast, secondary metabolite gene clusters diverged at the genus level but were largely conserved among Cryphonectria species. Putative effector content varied substantially among species with differences in cysteine content and protein length.We assembled and analyzed genomes of eight bark-inhabiting Cryphonectriaceae species to retrace the evolution of genome size and gene content. Based on CAZyme content, all analyzed species are predicted to share a similar trophic lifestyle. In the genus C. japonica, C. naterciae, and C. radicalis as predominantly saprotrophic species  Castanea species, which seems to be absent in other Cryphonectria species. Many CAZymes play a role in plant cell wall degradation and can be important virulence factors in necrotrophic fungi. Reductions in CAZyme genes have been observed in biotrophic pathogens and are thought to be an adaptation to reduce the exposure of molecular patterns, which can trigger host defenses  can play key roles in the emergence of new pathogens. We identified a broad pool of putative effector orthologs among Cryphonectriaceae. The size of the effector gene pool did not correlate with genome size or lifestyle as seen in other clades of plant pathogens , the chestnut blight fungus C. parasitica causes only mild symptoms, which has been attributed to host-pathogen coevolution (Ca. dentata and Ca. sativa), the pathogen causes lethal bark cankers . ary host . The weacrobiome . Subsequdophytes –74. Lateophytes – found thus trees . Hence, us trees , 76. C. C. parasitica, 3 C. japonica, 3 C. radicalis, 2 C. naterciae, and 2 C. carpinicola isolates covering the global distribution range  using t v0.11.0  and EMBO v0.11.0 . We set Cryphonectria and Chrysoporthe isolates, we used OrthoFinder v2.3.7 , Phanerochaete carnosa (Phlebia centrifuga (Heterobasidion annosumsensu lato (Neonectria ditissima (Nectria canker on apple and pear trees) (Ophiostoma novo-ulmi (Dutch elm disease)  (https://github.com/ccdmb/catastrophy-pipeline), choosing the options -profile conda and –dbcan_version 8. The CATAStrophy literature-derived nomenclature  was used for defining trophic lifestyles of species included in the CATAStrophy training set. Moreover, we selected principal components PC1 and PC2, which separate most training set species according to lifestyle for visualization (We inferred trophic lifestyles of Cryphonectriaceae according to carbohydrate-active enzyme (CAZyme) gene content using the CAZyme-Assisted Training And Sorting of -trophy (CATAStrophy) prediction tool . CATAStr carnosa , and Phlntrifuga . Moreovensu lato , associar trees) , Ophiostdisease) , 103, ane trees) . For trolization .For the identification of CAZyme genes, we ran dbCAN v2.0.0  on the sCa. sativa; length, 50 cm; diameter, 3.3 to 6.7 cm), which were cut in a healthy state during winter from chestnut stands in Ticino, Switzerland, a week prior to the experiment. The logs were surface sterilized with 70% ethanol and sealed on both ends with paraffin to prevent desiccation. We selected 3 C. parasitica , 2 C. japonica (M9249 and IF-6), 2 C. naterciae (M3664 and M3656), 2 C. radicalis (M4733 and M283), and 2 C. carpinicola (M9290 and CS3) isolates and 1 Chr. cubensis (CBS115724) isolate for inoculation. For all isolates except CBS115724, full genome sequences were available for this study. Prior to inoculation, all isolates were freshly inoculated from glycerol stocks onto potato dextrose agar  and incubated at 25°C in complete darkness for 5 days to induce mycelial growth. For inoculation of the first batch of chestnut logs (n = 13), we removed the bark on 5 equally distanced spots  on each log, placed a mycelial plug  into the wound, and sealed it with tape. For the second batch of chestnut logs (n = 13), we directly placed five mycelial plugs onto the bark of each log with equal distance  and sealed the inoculation spots with tape. For each treatment batch with and without wound, we selected 5 replicates per isolate and 5 negative controls (mycelium-free agar plugs), resulting in a total of 130 completely randomized inoculation spots (n = 65 per treatment). All chestnut logs were randomly placed onto racks in plastic containers, separated by treatment, filled with 2 liters of demineralized water to avoid drying out, and sealed with plastic lids (To analyze the wood colonization capabilities of the different species, we set up an inoculation experiment. We selected 26 dormant chestnut logs (tic lids . IncubatWe performed effector gene prediction with the same isolates as in the secondary metabolite and CAZyme analysis, using a machine-learning approach. First, secreted proteins were predicted with SignalP v5.0b , choosinde novo assemblies of Cryphonectria genomes, are available in Cryphonectria were obtained for Chrysoporthe cubensis, Chr. deuterocubensis, and Chr. austroafricana from NCBI BioProjects PRJNA279968, PRJNA265023, and PRJNA263707, respectively (All sample accession numbers for the NCBI Short Read Archive, as well as NCBI accession numbers for all ectively , 109.


Helicobacter pylori (H. pylori) eradication. There is limited data about H. pylori strains in Cambodia. This study aimed to evaluate H. pylori prevalence and antibiotic resistance in Koh Kong, Cambodia. Gastric cancer, which is the leading cause of cancer mortality in Cambodia, can be prevented by H. pylori culture with E-test and GenoType® HelicoDr . 3-mL blood sample was collected for CYP2C19 genotyping. 118 Cambodian dyspeptic patients were scheduled to enter this study and 58 were enrolled between July and September 2019. All patients underwent upper GI endoscopy. 3 gastric biopsies were obtained for rapid urease test, H. pylori prevalence was 31.0%. Antibiotic resistance rates were 78.6% for metronidazole, 50.0% for fluoroquinolones, and 27.8% for clarithromycin. There was no amoxicillin and tetracycline resistance. More than half of H. pylori strains (57.1%) were multidrug-resistant. Most (35.7%) were resistant to metronidazole and quinolone. Poor, intermediate and rapid metabolizers were 5.5%, 38.9% and 55.6%, respectively. 58 subjects were enrolled . Overall H. pylori infection remains common infection in Cambodia. High prevalence of clarithromycin, metronidazole, levofloxacin and multidrug-resistant H. pylori is still major problems in Cambodia. Treatment regimens without clarithromycin and quinolone such as 14-day bismuth-based quadruple therapy might be an appropriate choice for H. pylori eradication in this particular area.  Helicobacter pylori (H. pylori) is a gram-negative, spiral-shaped bacterium causing persistent gastric mucosal infection. Various H. pylori-associated conditions range from gastritis and gastroduodenal ulcers to more severe diseases including mucosa–associated lymphoid tissue (MALT) lymphoma, and gastric cancer . Considered as carcinogens, H. pylori promote non-cardia gastric carcinogenesis through chronic gastric mucosal inflammation in Correa’s precancerous cascade . H. pylori eradication resulted in reduced gastric cancer incidence, regression of gastric premalignant lesion, and healing of peptic ulcers . However, there are several factors affecting eradication success rate such as host genetics, bacterial virulence, and environmental factors . Recently, antibiotic-resistant strains have been increasing worldwide and subsequently had an effect on treatment failure rate . Southeast Asian countries are currently facing major problems about drug-resistant H. pylori and appropriate treatment regimen for each country should be reviewed depending on antimicrobial susceptibility testing .H. pylori infection can be prevented by bacterial eradication . However, there has been limited research determining the prevalence of H. pylori infection and antibiotic susceptibility testing cannot be performed in Cambodia . Cambodia is the Southeast Asian sovereign state bordered by Thailand to its northwest, Laos to its northeast, and Vietnam to its east. Koh Kong province is located in the southwestern part of Cambodia. Its geography is characterised by mountains, rainforests and coastal areas. Koh Kong’s population has been increasing since the improvement of infrastructure in 2002. According to global cancer statistics 2018, gastric cancer is the eighth leading cause of cancer-related deaths in Cambodia . As diagnostic tools and treatment facilities are not easily accessible for Cambodian people, health care providers need to implement cancer prevention strategies to decrease the incidence of specific cancers . Regarding as one of preventable cancers, most of gastric cancers associated with H. pylori infection in Cambodia is still lacking. This study aimed to evaluate prevalence and antibiotic resistance patterns of H. pylori infection in Koh Kong, Cambodia. The result of this study could be used for developing appropriate regimen for H. pylori treatment in Cambodia. Nowadays, the information about Patients H. pylori culture with Epsilometer test (E-test) and GenoType® HelicoDr . 3 mL of blood sample was collected for CYP2C19 genotyping. The exclusion criteria were as follows; patients receiving H2 receptor antagonists, proton pump inhibitor (PPI), bismuth compound, and antimicrobial agents within 4 weeks prior to the study, using NSAIDs and anticoagulant, having history of stomach surgery, or having significant comorbidities such as renal failure, advanced cirrhosis, advanced or metastatic cancer, or cardiac arrhythmia, immunocompromised patients, pregnant women, and breastfeeding women. Informed consent was obtained from study volunteer before upper GI endoscopy was performed.118 Cambodian patients with dyspepsia were scheduled to enter this study and 58 patients were enrolled between July and September 2019. All 58 patients  underwent upper GI endoscopy and 3 gastric biopsies were obtained for rapid urease test , Rapid urease testH. pylori in gastric specimen could hydrolyze urea to ammonia and raise the pH of the medium. Consequently, the color of pH indicator changed from yellow to pink or red as a positive result. This is a diagnostic test for detection of urease-secreting bacteria. After the gastric biopsy had been placed in the center of the well containing urea and pH indicator, the test was kept at room temperature for 60 minutes. Urease produced by H. pylori cultureH. pylori culture were contained in transport media in the Eppendorf tubes at -40°C in the freezer during 446-kilometer transportation from Cambodia to Thammasat University Hospital, Pathumthani, Thailand. In the laboratory, gastric biopsies were minced in the broth. We used a sterile heated wire loop to streak the fluid mixture on a Mueller Hinton - Agar medium. The media were then placed in the candle jar and incubated at 37°C in microaerophilic condition. H. pylori became visible as translucent colonies on blood agar approximately 3 to 5 days after incubation. Gram staining of these bacteria demonstrated curved gram-negative rods with positive oxidase, catalase, and urease tests.Antral biopsies obtained for Antimicrobial susceptibility testingH. pylori was considered resistant when MIC values were > 0.12 μg/mL for AMX, > 0.5 μg/mL for CLR, > 8 μg/mL for MNZ, > 1 μg/mL for LVX, and > 1 μg/mL for TET. The Epsilometer test (E-test) directly quantifies susceptibility of bacteria to specific antibiotics by determining the minimum inhibitory concentrations (MICs) of antibiotics including amoxicillin (AMX), clarithromycin (CLR), metronidazole (MNZ), tetracycline (TET), and levofloxacin (LVX). An E-test strip with antibiotic was placed on a previously inoculated plate. On the subsequent 3 to 5 days, the MIC value could be defined as the intersection of an ellipse and the scale on the E-test strip. The MIC is the lowest concentration of antibiotics that can inhibit visible bacterial growth. GenoType® HelicoDr testH. pylori culture to determine whether they had CLR or quinolone-resistant strains. First, H. pylori genomic DNAs were extracted from gastric biopsies using QIAamp DNA Mini Kit . The extracted DNAs were then amplified by polymerase chain reaction (PCR). If amplified DNA regions involving clarithromycin (rrl gene) and fluoroquinolone (gyrA gene) resistance were present, specific probes would bind them during hybridization. The positive band was visibly detected by the colorimetric assays .GenoType® HelicoDr test is a molecular test for detection of genetic mutations resulting in CLR and quinolone resistance. In our study, the tests were used in patients with negative Statistical analysisAll data were analysed by using SPSS version 22 . The demographic data were analysed by using the Fisher’s exact test, and Chi-square test where appropriate. Statistical significance was defined as a two-tailed p-value cut point of less than 0.05. H. pylori infection was 31.0%. Baseline characteristics were not different between groups of positive and negative H. pylori infection. Demographic data including gender, age, comorbidities, and endoscopic findings were demonstrated in Total of 58 dyspeptic Cambodian patients were enrolled in the study. There were 18 males (31.0%) and 40 females (69.0%) with the mean age of 43.8 years. Median duration of dyspepsia was 36 months. Patients mostly had gastritis (91.4%) on endoscopic findings, whereas 8.6% had peptic ulcer diseases. The prevalence of H. pylori infection, 14 patients could achieve H. pylori cultures. Another 4 patients subsequently performed HelicoDr tests. Antibiotic resistance rates were 78.6% for metronidazole, 50.0% for fluoroquinolones, and 27.8% for clarithromycin as demonstrated in H. pylori strains (57.1%) were multidrug-resistant and most of them (35.7%) were resistant to MNZ and quinolones. H. pylori strains with triple drug resistance to CLR, MNZ, and quinolones were found in 14.3% of all patients. CYP2C19 genotyping was performed and revealed 5.5%, 38.9% and 55.6% for poor, intermediate and rapid metabolizers, respectively as demonstrated in Of 18 patients with H. pylori strains in Cambodia. The overall prevalence of H. pylori infection in our study was 31.0% which was quite low compared to neighbouring countries’. Since Thai-Koh Kong bridge was built and Chinese-Cambodian deep-water port project was developed, Koh Kong’s economy has been growing and this might be the cause of improved sanitation resulting in reduced prevalence of H. pylori infection. The other study conducted in Phnom Penh, Cambodia’s capital city demonstrated slightly lower H. pylori prevalence (26.7%) but almost similar baseline characteristics to our study including female predominance (58.2%) and mean age of 43.9 years in the positive culture group . Tuan et al. study revealed higher antibiotic resistance rates of metronidazole (96.4%), levofloxacin (67.3%), and multidrug (76.4%) but lower rate of clarithromycin (25.5%) than our study. However, we did not observe any amoxicillin resistance, whereas there were 9.1% of amoxicillin resistance in the previous study. Multidrug-resistant strains in Tuan et al., (2019) study demonstrated the highest prevalence of resistance to MNZ and quinolone (40.0%), followed by triple resistance to MNZ, CLR, and quinolone (18.2%) which were comparable to our study resistance rates of 35.7%, and 14.3%, respectively. In conclusion, H. pylori strains in the capital city exhibited higher antibiotic resistance than strains from rural area. Higher resistance rates in urban area compared to rural areas were also observed in previous studies in Thailand .Cambodia has been reckoned as a mysterious country with vulnerable economic growth despite encouraged international tourism since the late 1990s. The healthcare service and medical research are still under-subsidized requiring international collaboration to refine the country’s healthcare system. Although there was limited access to healthcare service and antibiotics were presumed to be used infrequently, our study demonstrated extremely high prevalence of multidrug-resistant H. pylori prevalence as in eastern Thailand, which was the Thai region sharing main boundaries with Cambodia . Our study disclosed the extremely high prevalence of fluoroquinolone-resistant H. pylori strains and also considerably higher than other countries in the same region . Moreover, MNZ and CLR resistance were among the highest in Southeast Asia . Abundant quinolone resistance in Cambodia was probably because of antibiotic use in treatment for common infectious diseases. Lower respiratory infection was the second leading cause of deaths while diarrhea was also the leading cause of mortality in children under 5 years old in Cambodia . Diarrhea and pneumonia were commonly treated by quinolones and this could consequently induce high quinolone resistance rate. In addition, over-the-counter antibiotic dispensing in pharmacies could contribute to increased antibiotic resistance rates in Cambodia. At present, H. pylori antibiotic resistance is a substantial global issue resulting in treatment failure in many countries . In 2018, CLR-resistant H. pylori ranked first for community-acquired organisms on the WHO priority list of antibiotic-resistant bacteria in search of new antibiotic development to combat them . Standard triple therapy containing CLR is not recommended to be used if CLR resistance is greater than 15% . CLR resistance of 27.8% in our study suggested that standard triple therapy should not be used as first-line treatment for H. pylori infection in this country. In contrast, even though there was MNZ resistance, successful eradication could be achieved by extending duration of therapy and addition of other effective antibiotics . Compared to other countries in Southeast Asia, this study revealed approximately the same H. pylori treatment outcomes. According to US Food and Drug Administration (USFDA), serious drug interactions can occur attributed to concomitant use of a drug interfering CYP2C19 metabolism such as PPI and a drug requiring metabolism into its active form such as clopidogrel especially in CYP2C19 poor metabolizers. However, there was no prior study of CYP2C19 genotyping in Cambodia. CYP2C19 genotyping in our study revealed majority of rapid and intermediate metabolizers which were comparable to the number of metabolizers in the previous Thai study . Larger clinical study with proper H. pylori treatment regimen should be performed in the future to clarify important role of CYP2C19 genotype on H. pylori eradication.CYP2C19 is an important host factor regulating metabolism of all proton pump inhibitors (PPI) causing variability in H. pylori infection remain common infection in Cambodia. Clarithromycin, levofloxacin, metronidazole and multidrug resistance of H. pylori strains are major problems in Koh Kong, Cambodia. The treatment regimen without clarithromycin and quinolone such as 14-day bismuth-based quadruple therapy might be an appropriate choice for H. pylori eradication in this particular area and might be helpful to decrease fatal H. pylori-associated diseases especially gastric cancer in this country.


Toxoplasma gondii (T. gondii) can cause zoonotic toxoplasmosis worldwide. Neutrophil extracellular traps (NETs) have been known as a novel effector mechanism against T. gondii infection in the innate system of humans, cats, and sheep. Dogs are the intermediate host of T. gondii, in which the use of NETs against T. gondii infection remains unclear. Thus, this study aims to examine the effects of T. gondii on NETs release in dogs, and to further investigate the mechanism involved in the process. T. gondii-triggered NETs were analyzed by scanning electron microscopy (SEM) and fluorescence confocal microscopy, and the mechanism of T. gondii-triggered NETs release was determined by using inhibitors and a fluorometric reader. The results showed that T. gondii tachyzoites significantly triggered NETs-like structures, which consisted of DNA decorated with neutrophil elastase (NE) and myeloperoxidase (MPO). Further investigations revealed that reactive oxygen species (ROS)-, NADPH oxidase-, Rac 1- or p38 mitogen-activated protein kinase (MAPK)-signaling pathways were relevant to T. gondii tachyzoites-triggered NETs release. Moreover, zymosan-triggered NETs release was strikingly degraded by T. gondii tachyzoites treatment, indicating that T. gondii may escape from the NETs-based capture strategy. Taken together, promoting NETs release is suggested to limit motility and evade infection of T. gondii in dogs. Toxoplasma gondii is considered as one of the most prevalent zoonotic parasites worldwide  are reported as a novel effector mechanism of polymorphonuclear neutrophils (PMNs) in the host immune system against infection. Since NETs were first discovered to entrap and kill extracellular bacteria -dependent process . Finally, these tachyzoites were centrifuged at 3,000 r/min for 10 min and washed two times.Blood was collected from three healthy adult dogs, and the PMNs were isolated in the available dog PMN isolation kit®  as previously described , they were observed under scanning electron microscope .T. gondii tachyzoite-triggered NETs were detected with fluorescence confocal microscopy analysis as described elsewhere  and T. gondii tachyzoites for 120 min. The NETs specific antibodies included the anti-histone antibody  and the anti-myeloperoxidase (MPO) antibody . DNA in T. gondii tachyzoites-triggered NETs structures were stained with 5 μM Sytox Orange (Invitrogen), and the images were observed and analyzed with a scanning confocal microscope (Olympus FluoView FV1000).The components of T. gondii tachyzoites  for 90 min. To determine if T. gondii-triggered NETs were time-dependent, PMNs were incubated with viable T. gondii tachyzoites (ratio 1:1) for 10, 30, 60, 90, or 120 min. In inhibition tests, cells were pre-treated with 10 μM of the NADPH oxidase inhibitor Diphenyleneiodonium chloride , 10 μM of the p38 MAPK (Sigma-Aldrich) signaling pathway inhibitor SB202190, 100 μM of the Rac1 activation inhibitor NSC23766, or 8.0 nM of the NLRP3 inhibitor MCC950 for 15 min. DNase I (90 U) was also pre-treated with cells for 15 min. Zymosan (1 mg/mL) was used as positive control. Finally, the release of T. gondii-triggered NETs was quantified with Pico Green® (Invitrogen) and the fluorometric reader Infiniti M200® .PMNs were incubated with viable T. gondii-triggered NETs release, PMNs were incubated with viable T. gondii tachyzoites (ratio 1:1 or 1:2) for 90 min. T. gondii tachyzoites-induced ROS levels in the process of NETs release were tested by using 2, 7 dichlorofluorescein diacetate (Sigma-Aldrich) and the fluorometric reader Infiniti M200® .To determine ROS levels in the process of T. gondii tachyzoites-triggered NETosis from necrosis, LDH levels were tested. PMNs were incubated with viable T. gondii tachyzoites  for 90 min. T. gondii tachyzoites-induced LDH levels in the process of NETs release were tested by the LDH Cytotoxicity Assay kit® .To further differentiate P ≤ 0.05 was considered as significant.Data were expressed as the means ± standard deviation (SD). One-way analysis of variance (ANOVA) with Tukey's multiple comparison tests was performed by using the GraphPad 5.0 software to analyze the differences among groups. The level of T. gondii tachyzoites significantly triggered NETs-like structures . Further experiments revealed that T. gondii tachyzoites-triggered NETs release within 60 min which was in a time-independent manner , and it was of great interest that the increasing of NETs release significantly decreased at the time point of 90 min  and 120 min , but lower than that of T. gondii tachyzoites , the p38 MAPK signaling pathway inhibitor SB202190 (P < 0.01), and the Rac1 activation inhibitor NSC23766  significantly decreased the release of T. gondii tachyzoites-triggered NETs in varying degrees , but LDH levels were significantly increased by the lysis .LDH is a typical indicator of cell necrosis. To further investigate if cell necrosis also exists in the process of T. gondii tachyzoites to clarify if T. gondii could escape from NETs. As shown in T. gondii tachyzoites was slightly observed at the time point of 120 min, while zymosan significantly triggered NETs compared to negative controls. However, the release of zymosan-triggered NETs was strikingly degraded by T. gondii tachyzoites treatment (T. gondii tachyzoites consisted of NE (PMNs were co-treated with zymosan and reatment , 7L. Theed of NE  and MPO ed of NE  with DNAed of NE , 7E,H,K.T. gondii tachyzoites has been demonstrated in dogs for the first time, just like that in humans, sheep, or harbor seals (Abi Abdallah et al., T. gondii tachyzoites were captured by NETs-like structures (Neospora caninum (Wei et al., Eimeria bovis (Munoz-Caro et al., Cryptosporidium parvum (Munoz-Caro et al., Eimeria arloingi (Silva et al., T. gondii tachyzoites triggered NETs release was in a dose-dependent manner. These results have been suggested that one part of T. gondii tachyzoites would be captured and then killed by NETs in dogs. A further quantitative experiment also revealed that T. gondii tachyzoites-triggered NETs release was in a time-independent manner, and it was of great interest that the increase in NETs release was significantly decreased at the time point of 120 min. In addition, the release of NETs triggered T. gondii tachyzoites was also degraded by DNase I (T. gondii tachyzoites could also evade capture by these NETs after 90 min.In the present study, NETs release triggered by ructures , and theructures , which c DNase I . All theT. gondii infection (Gorfu et al., T. gondii tachyzoites-triggered NETs formation. It was found that inhibitors of the NADPH oxidase, Rac 1, or p38 MAPK signaling pathways but not the inhibitor of NLRP3 significantly inhibited NETs release (T. gondii tachyzoites-triggered NETs. Further results showed that T. gondii tachyzoites significantly increased ROS levels but not LDH levels in the process of NETs release, proving that ROS was also related to T. gondii tachyzoites-triggered NETs. Zymosan used as the positive stimuli could also significantly induce ROS production in NETs release as shown in previous research (Wei et al., T. gondii tachyzoites. We deduced that there are possibly two main reasons for the results. The first one is that the type of stimuli are different for inducing NETs release. NETs release is a ROS -dependent or -independent process, these two ways may both exist in NETs release. The other reason is that the amount of the same stimuli significantly affects ROS production in the process of NETs release. Taken together, the mechanism of T. gondii tachyzoites-triggered NETs release was a ROS-, NADPH oxidase-, Rac 1-, or p38 MAPK- signaling pathway dependent process. In this aspect, no significant species-difference of T. gondii tachyzoites-triggered NETs were found between harbor seals and dogs.Given that ROS, NADPH oxidase, Rac, or p38 signaling pathways are involved in the mechanism of several pathogen-induced NETs releases (Lim et al.,  release , which sT. gondii tachyzoites was markedly decreased at the time point of 120 min, we speculated that T. gondii tachyzoites could degrade NETs structures and escape from the NETs-based capture strategy. To provide further evidence, PMNs were co-treated with zymosan and T. gondii tachyzoites for fluorescence confocal microscopy analysis. Interestingly, zymosan-triggered NETs release was strikingly degraded by T. gondii tachyzoites treatment that confirmed our suspicions. In other words, there must be one or more DNase-like enzyme existing in T. gondii to degrade NETs structures. However, predicting and identifying these DNase-like enzymes in T. gondii still needs further research, which will help understand the biological functions and mechanisms of T. gondii escape from the NETs-based capture strategy.On the basis of the quantitative results that NETs release triggered by T. gondii infection, and the regulatory mechanism undergoing T. gondii-triggered NETs release was associated with ROS-, NADPH oxidase-, Rac 1-, or p38 MAPK- signaling pathways. Moreover, it was found that T. gondii escaped from the NETs-based capture strategy, but the escape mechanism still needs careful study.In conclusion, the NETs-based effector mechanism worked in dogs during All datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by Animal experiments were approved by the Ethics Committee on the Care and Use of Laboratory Animals of Foshan University. Written informed consent was obtained from the owners for the participation of their animals in this study.ZY and QL conceived and designed the project and experiments. ZWe, ZWa, XL, CW, ZH, DW, and YZ carried out the experiments. ZWe and XZ performed the statistical analysis. ZWa wrote the manuscript. QL revised the manuscript. All authors reviewed 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.


Fusarium spp. are significantly important plant pathogens, and some of them are ethanol-producing strains. During infection and/or ethanol production, Fusarium requires a plenty of energy that is mainly provided by mitochondria. Here we report the first mitogenome from a selected Fusarium oxysporum strain mh2-2 that produces ethanol from glucose and xylose. The size of this mitogenome, 46 kb, is different from the size of any reported Fusarium mitogenome. Our results provide insight into the functions and evolution of mitochondrial genes and genomes. Fusarium species include plant pathogens, opportunistic pathogens of humans and other animals, and non-pathogenic strains  that produces ethanol from glucose and xylose. The fungus was originally isolated from the root of cotton in Urumqi, Xin Jiang, China  and confirmed using ITS sequences. This isolate was deposited into the Guangdong Microbial Culture Collection Center (GDMCC 3.632). We find that the complete mitogenome of FoxM is 46 kb (GenBank accession no. MF155191) and it encodes 18 protein-coding genes, including 15 typical genes  that are commonly found in fungal mitogenomes in Hypocreales . Compared with other three, F. oxysporum mitochondrial genomes have been reported . There were four major clades: clade A includes four F. oxysporum strains, clade B includes F. circinatum (31 proteins) and F. verticillioides (21 proteins) , F. gerlachii (53 proteins), and F. graminearum (50 proteins)  (Al-Reedy et al. Fusarium mitogenomes encode 15 typical protein-coding genes, but they encode different numbers of LAGLIDADG endonucleases, GIY-YIG endonucleases, and hypothetical proteins. The difference between phylogenetic clusters and gene numbers indicates significant differences in gene gains and losses in Fusarium mitogenomes during evolution. For example, compared with F. solani, strains in clade C gain more introns and endonucleases, F. verticillioides in clade B and four F. oxysporum strains in clade A lose many introns and endonucleases, while F. circinatum in clade B has similar numbers of introns and endonucleases. This study reports the complete mitogenome of ethanol production fungus FoxM for the first time and provides valuable information for further exploration of mitochondrial evolution.Moreover, we determined the phylogenetic relationships between


We applied the phylogenomics to clarify the concept of rice species, aid in the identification and use of rice germplasms, and support rice biodiversity.Oryza) is one of the most important crops in the world, supporting half of the world’s population. Breeding of high-yielding and quality cultivars relies on genetic resources from both cultivated and wild species, which are collected and maintained in seed banks. Unfortunately, numerous seeds are mislabeled due to taxonomic issues or misidentifications. Here, we applied the phylogenomics of 58 complete chloroplast genomes and two hypervariable nuclear genes to determine species identity in rice seeds. Twenty-one Oryza species were identified. Conspecific relationships were determined between O. glaberrima and O. barthii, O. glumipatula and O. longistaminata, O. grandiglumis and O. alta, O. meyeriana and O. granulata, O. minuta and O. malampuzhaensis, O. nivara and O. sativa subsp. indica, and O. sativa subsp. japonica and O. rufipogon.D and L genome types were not found and the H genome type was extinct. Importantly, we evaluated the performance of four conventional plant DNA barcodes , six rice-specific chloroplast DNA barcodes , two rice-specific nuclear DNA barcodes (NP78 and R22), and a chloroplast genome super DNA barcode. The latter was the most reliable marker. The six rice-specific chloroplast barcodes revealed that 17% of the 53 seed accessions from rice seed banks or field collections were mislabeled. These results are expected to clarify the concept of rice species, aid in the identification and use of rice germplasms, and support rice biodiversity.Rice (genus The online version of this article (10.1007/s11103-020-01054-3) contains supplementary material, which is available to authorized users. Akin to African rice (O. glaberrima), its accessions are intermingled genetically with those of its wild progenitor  was extracted from dry leaves using the mCTAB method . The sequences were assembled using Sequencher 5.4 and edited if necessary to correct some nucleotide calling mistakes.The PCR reaction mixture contained 1 × Taq buffer with MgThe newly determined chloroplast genomes (Table S2) were combined with 37 chloroplast genomes (together with chloroplast fragments of three species) downloaded from GenBank (Table S3), aligned using mafft-win  or genomes of relatively low quality (longer branch lengths). This dataset was used to delimit the circumscription of species together with dataset 6 and a super barcode of Oryza.Dataset 1 contained 58 chloroplast genomes, representing all rice species (1–3 per species), together with three matK), dataset 3 (rbcL), dataset 4 (psbA-trnH), and dataset 5 (ITS) represented conventional DNA barcodes. The psbA-trnH sequence is interrupted by rps19 in Poaceae. Dataset 6 represented the concatenation of two single-copy nuclear genes (N78 and R22) selected from 142 genes . The eight clades in the complete chloroplast genome phylogeny matched exactly the eight genome types  × O. punctata (B) and O. schweinfurthiana = O. punctata (B) × O. eichingeri (C).Phylogeny based on the nuclear NP78 and R22 genes clarified the origins of allotetraploid species. Haplotypes of the same genome types formed monophyletic clades  were picked based on nucleotide diversity. Further evaluation of these 36 regions was carried out using the tree building method, and six high-resolution regions .The rbcL gene had an aligned length of 1428 sites with 50 parsimony-informative characters when outgroups were considered. This marker also failed to discriminate between species of the A, B, and C genomes .The psbA-trnH region had an aligned length of 515 sites with 10 parsimony-informative characters when outgroups and partial rps19 were included. This marker could successfully identify only O. brachyantha and O. sativa subsp. indica .The H or J genome types originated from the F genome type , a finding not supported by the other two nuclear genes. The ITS failed to discriminate between species of the A and C genome types.The nuclear ITS (including 5.8 s) had an aligned length of 713 sites with 162 parsimony-informative characters when outgroups were considered. The samples used for this marker differed slightly from those subjected to chloroplast markers because the sequences were difficult to amplify. Only one ITS copy was detected in several allotetraploid species. Phylogeny data based on ITS suggested that the A, B, C, H, and J genome types .The nuclear NP78 + R22 gene combination had an aligned length of 2218 sites with 722 parsimony-informative characters when outgroups were included. This marker combination failed to discriminate between species of the O. punctata and O. minuta of the B genome type  and O. glumipatula. Similarly, in the C genome type, there is confusion between diploid and tetraploid O. punctata, and among tetraploid O. alta, O. latifolia, and O. minuta.Considering that the rice-specific barcode resolved almost all rice species, we used it to identify 53 accessions of seeds from seed banks or field collections. Nine (17%) mislabeled samples were found  and F (O. brachyantha) genome types are monospecific and relatively isolated from other species. Species pairs have been found between O. meyeriana and O. neocaledonica of the G genome type, between O. longiglumis and O. ridleyi of the HJ genome type, and between O. coarctata of the KL genome type and O. schlechteri of the HK genome type .There is a general correlation between molecular divergence and species delimitation  and O. nivara subsp. indica (syn. O. sativa subsp. indica). The names of their wild progenitors, O. nivara and O. rufipogon, have to be changed accordingly to O. sativa subsp. rufipogon (syn. O. rufipogon) and O. nivara subsp. nivara (syn. O. nivara). In 1970, a male sterile interspecific hybrid between O. nivara subsp. indica (= O. sativa subsp. indica) and O. sativa subsp. sativa (= O. sativa subsp. japonica) was discovered at a farm in Hainan province, China. The reproductive isolation between these subspecies was broken artificially and partially fertile F1 hybrid rice was used to produce fertile F2 hybrids as a new cultivar, which exhibited considerable hybrid vigor. Subsequent hybridization, however, created taxonomic problems regarding the correct identification of the two kinds of rice and their wild progenitors, resulting in many incorrectly labeled sequences being deposited in GenBank.A second confusing case involves the Asian cultivated rice O. longistaminata under O. glumipatula and including Porteresia coarctata (Roxb.) Tateoka into Oryza (= O. coarctata Roxb.), 21 species are now recognized in the Oryza genus (supporting text S1).After synonymizing matK, psbA-trnH, and rbcL) and one nuclear region (ITS) represent conventional DNA barcodes for higher plants  resolvable. Both barcodes failed to discriminate between species of the A, B, and C genome types. Moreover, a combination of matK + rbcL did not improve the situation, because both barcodes resolved almost the same species without complementation. The psbA-trnH intergenic spacer, one of the most variable regions in chloroplast genomes, performed similarly poorly with only one identifiable species. This is probably due to the insertion of rps19, which replaced the spacer with rps19 sequences.Three chloroplast regions , O. ridleyi (HJ), and O. schlechteri (HK). However, two kinds of sequences were observed in O. longiglumis (HJ), one of them was similar to that of O. ridleyi, and the other was similar to that of O. brachyantha, a phenomenon never reported previously. Similarly, only the C genome type was confirmed in O. alta and O. grandiglumis, whereas the B genome type defined O. malampuzhaensis. Both B and C genome types were detected in O. schweinfurthiana. The sequences deposited in GenBank include only one kind of sequence for species of the BC and CD genome types, which is probably because of concerted evolution of the ITS in relatively old tetraploids. Only newly formed tetraploids such as O. schweinfurthiana maintain both B and C genome types.The nuclear ITS afforded similar resolution as conventional chloroplast regions. Although there are 10 allotetraploid species in Oryza genus have an evolutionary history of only a few million years. Very limited genetic variation has accumulated within such a short time and conventional DNA barcodes do not work well at species level, especially for those belonging to the A, B, and C genome types. The two most variable genes (NP78 and R22) picked out from 142 nuclear genes tested by Zou et al. Figure S1. The maximum parsimony strict consensus tree based on the conventional DNA barcode rbcL sequences of all species in Oryza, demonstrating the resolution of the marker. The figures beside branches are bootstrap values 2 (PDF 145.6 kb)Figure S2. The maximum parsimony strict consensus tree based on the conventional DNA barcode psbA-trnH sequences of all species in Oryza, demonstrating the resolution of the marker. The figures beside branches are bootstrap values (PDF 137.2 kb)Figure S3. The maximum parsimony strict consensus tree based on the conventional DNA barcode Oryza, demonstrating the resolution of the marker. The figures beside branches are bootstrap values (PDF 164.5 kb)Figure S4. The maximum parsimony strict consensus tree based on the conventional DNA barcode ITS sequences of all species in Oryza, demonstrating the resolution of the marker. The figures beside branches are bootstrap values (PDF 41.4 kb)Figure S5. The maximum parsimony strict consensus tree based on the rice-specific nuclear DNA barcode of NP78 + R22 sequences of all species in Supplementary material 6 (DOCX 15.0 kb)Supplementary material 7 (DOCX 16.9 kb)Supplementary material 8 (DOCX 15.8 kb)Supplementary material 9 (DOCX 19.1 kb)Supplementary material 10 (DOCX 17.6 kb)Below is the link to the electronic supplementary material.


Streptomyces as an example, we developed a combinatorial strategy for systematic identification of large and dispensable genomic regions in Streptomyces based on multi-omics approaches.Large-scale genome reduction has been performed to significantly improve the performance of microbial chassis. Identification of the essential or dispensable genes is pivotal for genome reduction to avoid synthetic lethality. Here, taking S. coelicolor A3(2), S. albus J1074 and S. avermitilis MA-4680 were preferred reference for comparative analysis of candidate genomes. Multiple genome alignment suggested that the Streptomyces genomes embodied highly conserved core region and variable sub-telomeric regions, and may present symmetric or asymmetric structure. Pan-genome and functional genome analyses showed that most conserved genes responsible for the fundamental functions of cell viability were concentrated in the core region and the vast majority of abundant genes were dispersed in the sub-telomeric regions. These results suggested that large-scale deletion can be performed in sub-telomeric regions to greatly streamline the Streptomyces genomes for developing versatile chassis.Phylogenetic tree analysis revealed that the model strains including Streptomyces genomes, but also work as a universal method for systematic analysis of removable regions in other microbial hosts in order to generate more miscellaneous and versatile chassis with minimized genome for drug discovery.The integrative approach of comparative genomics, functional genomics and pan-genomics can not only be applied to perform a multi-tiered dissection for  Streptomyces albus J1074 by antiSMASH analysis. By removing the 15 BGCs spanning 503 kb in sequence, a cluster-free mutant S. albus Del14 was constructed and the mutant Del14 has successfully served as chassis for improving the production of microbial drugs and activating cryptic gene clusters [Escherichia coli Data Banks, the IS-free E. coli MS56 was constructed by sequentially deleting all ISs and K-islands, and the mutant MS56 harboring 23% genomic deletion showed enhancing genomic stability and recombinant protein production [E. coli MG1655 and EDL933, 12 K-islands were identified and deleted to construct a genome-reduced host MDS12 [Streptomyces hosts Streptomyces avermitilis SUKAs with large-deletion. Comparative analysis of S. avermitilis, Streptomyces coelicolor A3(2) and Streptomyces griseus revealed two sub-telomeric regions at the left and right chromosomal ends of S. avermitilis. A > 1.4-Mb segment from the left sub-telomeric region was deleted directly by Cre/loxP recombinant system and further endogenous gene clusters were removed one by one to construct a series of genome-streamlined S. avermitilis mutants SUKAs which have been widely used to efficiently express a variety of heterologous gene clusters [E. coli, Bacillus subtilis, Pseudomonas putida, Cyanobacterium, Aspergillus nidulans, Schizosaccharomyce pombe and Streptomyces [Large-scale genome reduction has been performed to streamline microbial genomes in order to decrease metabolic burden as far as possible, and to further develop simplified and versatile chassis for producing valuable amino acids, peptides, fuels or drugs –4. Identclusters . Based ooduction . By compst MDS12 . Anotherptomyces . As expeptomyces . The favStreptomyces spp. are the main antibiotic-producers which can biosynthesize antibiotics, antitumor agents, immunesuppressor, antioxidants, antihypertensive and hypoglycemic drugs widely used in clinical practice [Streptomyces species can synthesize a variety of precursors and elements, and harbor 20–40 gene clusters on average in their genomes which mightily indicates their robust capabilities of primary and secondary metabolism. Therefore, Streptomyces species are the preferred chassis for heterologous activation or overproduction of valuable microbial or plant drugs [Streptomyces by phage infection or horizontal gene transfer. The MGE like insertion sequences (ISs), transposons can randomly move and insert into other loci of genomes, and may result in genome rearrangement, gene inactivation or deletion, which affect genomic stability [Streptomyces hosts for drug discovery and high-yield.All the time, practice , 23. Gennt drugs . Howevertability . The endtability . TherefoStreptomyces as an example, we developed an integrative strategy for systematically analyzing large and dispensable genomic regions in Streptomyces based on multi-omics approaches. The analytical hierarchy process was completed according to the flowchart in Fig. Streptomyces hosts, but also can be widely used in other microbial cells to systematically analyze abundant genes for generating more miscellaneous and versatile chassis with minimized genomes.Here, taking Streptomyces genomes were selected to construct phylogenetic tree using neighbor-joining algorithm in the MEGA software [Streptomyces, S. albus J1074, S. lividan TK24 and S. coelicolor A3(2), S. avermitilis MA-4680 and S. griseus belonged to Group I, II and III, respectively. As is well-known, S. albus J1074 and S. xiamenensis 318 were two naturally genome-minimized strains harboring 6.84 Mb- and 5.96 Mb-size genomes, respectively. Besides, S. coelicolor A3(2), S. albus J1074, and S. avermitilis MA-4680 are three well-studied models and have been genetically engineered to generate genome-streamlined mutants which work as versatile hosts for heterologous expression of value-added natural products. Therefore, the genomes of S. coelicolor A3(2), S. albus J1074 and S. avermitilis MA-4680 are preferred references for comparative analysis of target genomes.In theory, essential genes are more evolutionarily conserved than non-essential genes among different strains. However, non-essential genes may be well conserved between taxonomically closely related species which will make us overestimate the amount of essential genes. Therefore, in order to systematically predict the essential genes by computational approaches, we need to firstly investigate the evolutionary relationship between different strains by phylogenetic tree analysis. Here, 50 16S ribosomal DNA (rDNA) sequences of software . The gensoftware . We can Streptomyces genomes which revealed highly conserved core region and variable sub-telomeric regions  were located at the end of its genome, and there were no apparent and large sub-telomeric regions, and most genes are highly conserved. However, two distinct and less conservative regions were observed in the genomes of S. coelicolor A3(2), S. griseus and S. avermitilis MA-4680. And the size of sub-telomeric regions was approximately same (1 Mb) in the genomes of S. coelicolor A3(2) and S. griseus, but was unequal (1.5 Mb and 0.5 Mb) in the genome of S. avermitilis MA-4680. Similar asymmetric distribution was also noticed in the genome of S. chattanoogensis L10 according to our previous study [S. avermitilis MA-4680 and S. chattanoogensis L10 may attribute to the deviation (0.7 Mb and 0.77 Mb) of oriC to the center of chromosome. In order to make it more convincing, we performed multiple genome alignment with Streptomyces bingchenggensis BCW-1 in which the oriC had a 0.64 Mb deviation to the center , genomic islands (GIs) and insertion sequences (ISs) mainly from horizontal gene transfer or bacteriophage infection, belong to well-known non-essential elements. The antiSMASH , IslandVS. albus genome, 11% for S. coelicolor genome, 19% for S. griseus and 17% for S. avermilitis genome) and lots of BGCs were distributed in sub-telomeric regions . Although the GIs and ISs took up a smaller proportion, 30-70% of them were also concentrated in the sub-telomeric regions of each genome except S. albus genome. The concentrated distribution of non-essential elements suggested the plasticity and editability of Streptomyces genomes which impelled us to engineer and refactor the genomes for improving their performances. As previously reported, in certain Streptomyces species like S. albus BK3-25 [S. ambofaciens [E. coli [S. chattanoogensis may decrease IS-mediated random mutagenesis and increase its genetic stability [Streptomyces genomes was more than that in model ones which maybe the reason why many industrial Streptomyces were more unstable. Although large-scale deletion of sub-telomeric regions had been performed to generate genome-minimized mutant in S. avermilitis, this method may bring about risk of lethality because some essential genes may be dispersed in sub-telomeric regions. Therefore, it is very necessary to further identify essential genes and their distributions.We can see that the BGCs accounted for a great proportion in the whole genomes  genes in given genomes. According to the phylogenetic tree of 50 ith BPGA . We can ith BPGA . The resStreptomyces genomes were submitted to ARTS to analyze well-known essential genes and their duplications. The well-known essential genes were identified using ARTS with its comparative pipeline and TIGRfam Equivologs. Generally, these genes belonged to core genes which were responsible for fundamental cellular functions like DNA replication, transcription, translation, primary metabolic pathways and cell architecture. These genes were generated by comparative genomic analysis and the reference genes in ARTS were from published literatures. The result was shown in Table Analysis of COG distribution suggested that the overall proportion of secondary metabolic process-associated functions among the accessory and unique genes was approximately 80%, compared with the core genes Fig. a. The KES. avermilitis, 96% in S. griseus, 94% in S. coelicolor and 97% in S. albus) and some copies of several essential genes were also located at the sub-telomeric regions. To further analyze the locations of these copies, we found that different essential genes and their copies did not appear in two sub-telomeric regions simultaneously which indicated that large-scale deletion of sub-telomeric regions did not result in synthetic lethality , S. albus J1074 and S. avermitilis MA-4680 are preferred references for comparative analysis of target genomes, for different candidate genomes, a new phylogenetic analysis should be made to choose taxonomically distinct genomes with the target one. Secondly, comparative genomic analysis and location of oriC were performed to determine the genome structure and non-essential elements. Our results suggested that there exist a highly conserved core region (~ 6 Mb) around the oriC as its axis of symmetry and two sub-telomeric regions at the end of chromosomes. Different arms might have gone through rounds of deletion, insertion and/or duplication to achieve the balance of replication [Streptomyces genomes, which may greatly improve the performance of strains. The expansion of the genetic repertoire of an organism by gene duplication can aid adaptation. Expanding gene families can help maintain cell functionality during metabolic perturbation [S. chattanoogensis L10 genome and successfully removed them separately by Cre/loxP system [As mentioned above, the first step to streamline genomes is to design the removable segments systematically. Comparative genomic analysis has been performed to identify non-essential elements in genomes like BGCs, ISs, GIs or less conservative regions. However, previous comparative genomic approaches are mainly based on the multiple alignments of nucleotide sequences among different genomes, which is hard to define the removable segments systematically. lication . For asyurbation . HoweverP system . This deWith the rapid development of metabolomics, the genome-scale metabolic network model can also be integrated into multi-omics analysis . For exaStreptomyces genomes suggests that dispensable genomic regions take up a great proportion in the genomes. The integrative approach of comparative genomics, functional genomics and pan-genomics can not only be applied to perform a multi-tiered dissection for Streptomyces genomes, but also work as a universal method for systematic analysis of removable regions in other microbial hosts in order to avoid synthetic lethality of large-scale deletion and generate more miscellaneous and versatile chassis with minimized genome for drug discovery.In summary, the systematic analysis of Streptomyces, S. albus J1074, S. lividan TK24 and S. coelicolor A3(2), S. avermitilis MA-4680, S. griseus NBRC 13350 and other 45 Streptomyces genomes were selected to construct phylogenetic tree using neighbor-joining algorithm in the MEGA software. Mycobacterium tuberculosis H37Rv was used as outgroup. The phylogenetic tree was further visualized and edited by the EvolView online tool.16S ribosomal DNA (rDNA) sequences of five model representatives of S. albus J1074, S. coelicolor A3(2), S. avermitilis MA-4680, S. griseus NBRC 13350 were obtained from NCBI  and used for genome comparison with the progressive algorithm in Mauve 2.3.1 [oriC) was analyzed by BLAST Program in DoriC [oriC and center of chromosome.The complete genome sequences of ve 2.3.1 . The matin DoriC . The expS. albus J1074, S. coelicolor A3(2), S. avermitilis MA-4680, S. griseus NBRC 13350, S. rapamycinicus and S. cattleya were also obtained from NCBI database and used for proteome comparison with OrthoVenn. The E-value was set to 1e-5 and inflation value was 1.0, and cluster relationship network was selected in OrthoVenn. The whole genome sequences of S. albus J1074, S. coelicolor A3(2), S. avermitilis MA-4680, S. griseus NBRC 13350, S. rapamycinicus, S. cattleya, S. natalensis and S. clavuligerus were used to perform pan-genome analysis with BPGA. ONE CLICK MODE was performed in BPGA program and all the analyses were performed in single step using all default parameters with identity cut off = 50% and No. of combination = 30. The KEGG/COG functional analysis was also selected. Other values in OrthoVenn and BPGA were default.The whole protein sequences of The BGCs were predicted by antiSMASH bacterial version, the GIs were analyzed by IslandViewer 4 and the ISs were determined by ISsaga2 in ISFINDER. In the antiSMASH, the detection strictness was relaxed and extra features were all on including ClusterBlast, Cluster Pfam analysis and Pfam-based GO term annotation. All values in IslandViewer 4 were default. In the ISsaga2, the number of replicon to annotate was set to 1. The essential genes and their duplications were predicted by DEG10 and ARTS. In the DEG10, the expect value of BLAST parameter was set to 1E−05. In the ARTS, the reference set was Actinobacteria and exploration mode was selected, and the HMM search mode was trusted cutoff (TC). Other parameters in antiSMASH, ISsaga2, DEG10 and ARTS were default. The circular genome map was generated by Circos  to visuaThe COG  and KEGGAdditional file 1. Determination of genome structure of Streptomyces bingchenggensis BCW-1. Multiple genome alignment suggested that the genome of Streptomyces bingchenggensis BCW-1 is asymmetric. IS. albus J1074 genome; IIS. coelicolor A3(2) genome; IIIS. griseus NBRC 13350 genome; IVStreptomyces bingchenggensis BCW-1 genome.