Copy number variations (CNVs) are structural variants consisting of large-scale insertions and deletions of genomic fragments. Exploring CNVs and estimating their effects on phenotypes are useful for genome selection but remain challenging in the livestock.P value < 0.05) associated with at least one out of 41 complex traits. Within them, 39 CNVRs were simultaneously associated with at least 2 complex traits. Notably, 24 CNVRs were markedly related to daughter pregnancy rate (DPR). For example, CNVR661 containing CYP4A11 and CNVR213 containing CTR9, respectively, were associated with DPR and other traits related to reproduction, production, and body conformation. CNVR758 was also significantly related to DPR, with a nearby gene CAPZA3, encoding one of F-actin-capping proteins which play a role in determining sperm architecture and male fertility. We corroborated these CNVRs by examining their overlapped quantitative trait loci and comparing with previously published CNV results.We identified 1043 CNV regions (CNVRs) from array comparative genomic hybridization (CGH) data of 47 Holstein bulls. Using a probe-based CNV association approach, we detected 87 CNVRs significantly  contains supplementary material, which is available to authorized users. Enhancement of dairy sector is required to meet the increasing demands of animal protein in the world. Since Holstein is the largest milk-producing dairy breed, improving its performance, e.g. production, reproduction, growth, and disease resistance, is crucial for the global agriculture. As one of the most important phenotypes, cattle fertility is affected by both genetic and environmental factors . For exaGenomic structural variants are comprised mainly of copy number variation (CNV) in the form of large-scale insertions and deletions, as well as inversions and translocations . CNVs haBos taurus and Bos indicus , body depth, rump angle, rump width, fore udder attachment (Fore_udder_att), rear udder height (rear_ud_height), udder depth, udder cleft, front teat placement (Front_teat_pla), teat length, Rear legs(rear view) [Rear_legs(rear)], feet/legs score (Feet_and_legs), rear teat placement (Rear_teat_pla). These PTA were predicted additive genetic effects after removing fixed non-genetic effects, and the reliabilities of the PTA were used to quantify the amount of information available for different individuals.apply.pca. Through clustering the PCA transformed data (first 3 components), a copy number genotype  was then assigned to animal for the association testing based on a standard regression approach [apply.pca function and phenotypes .Each CNV was analyzed for the association with each complex trait using the R package CNVtools separately, which implements a mixture model . Brieflyapproach . The CNVAdditional file 1:Figure S1. Characteristics of CNV distribution on each autosome. A. Distributions of CNV length per individual. B. Distributions of CNV count. Figure S2. Hierarchical clustering of 41 complex traits based on P values from association results between CNVRs and phenotypes. Pearson correlation was used to measure distances. Different colors represent various types of phenotype traits. (PDF 294 kb)Additional file 2:Table S1. CNVRs identified in this study and overlapping with QTL, OMIA, and CNVRs in previous studies. Table S2. Gene Ontology terms generated by PANTHER analysis. Table S3. Results of GWAS between CNVRs and 41 phenotypes of interest in 39 Holstein cattle. Table S4. Characterizes for significant CNVs associated to Holstein phenotypes. (XLSX 395 kb)


Scientific Reports 10.1038/s41598-017-07719-2, published online 02 August 2017Correction to: The Acknowledgements section in this Article is incomplete.“This work was funded by National Science Foundation (NSF) DMREF/GOALI 1436268. The authors are grateful for characterization facilities provided by NSF MRSEC DMR 1119826 (CRISP) and the Yale Institute for Nanoscience and Quantum Engineering (YINQE). The authors would like to thank Glenn Weston-Murphy for his help with the laser equipment. The authors would also like to thank Sungwoo Sohn, Frederick J. Walker, Chinedum O. Osuji, Michael Loewenberg, and Corey S. O’Hern for fruitful discussions.”should read:“This work was funded by National Science Foundation (NSF) DMREF/GOALI 1436268. The development of the technique was funded through NSF DMR 1609391. The authors would like to thank Glenn Weston-Murphy for his help with the laser equipment. The authors would also like to thank Sungwoo Sohn, Frederick J. Walker, Chinedum O. Osuji, Michael Loewenberg, and Corey S. O’Hern for fruitful discussions.”


Pionoconus magus, from Okinawa (Japan) were sequenced, assembled, and annotated. In addition, RNA-seq raw reads available at the SRA database from one additional specimen of P. magus from the Philippines were also assembled and annotated. The total numbers of identified conotoxin precursors and hormones per specimen were 118, 112, and 93. The three individuals shared only five identical sequences whereas the two specimens from Okinawa had 30 sequences in common. The total number of distinct conotoxin precursors and hormones for P. magus was 275, and were assigned to 53 conotoxin precursor and hormone superfamilies, two of which were new based on their divergent signal region. The superfamilies that had the highest number of precursors were M (42), O1 (34), T (27), A (18), O2 (17), and F (13), accounting for 55% of the total diversity. The D superfamily, previously thought to be exclusive of vermivorous cones was found in P. magus and contained a highly divergent mature region. Similarly, the A superfamily alpha 4/3 was found in P. magus despite the fact that it was previously postulated to be almost exclusive of the genus Rhombiconus. Differential expression analyses of P. magus compared to Chelyconus ermineus, the only fish-hunting cone from the Atlantic Ocean revealed that M and A2 superfamilies appeared to be more expressed in the former whereas the O2 superfamily was more expressed in the latter. The transcriptomes of the venom glands of two individuals of the magician’s cone,  Cones are marine venomous snails that live in tropical and subtropical waters around the world hunting on worms, snails, and fish . The venFor many years, mature conotoxins were purified from secreted venoms using different chromatographic methodologies, their amino acids were sequenced, and their physiological roles were characterized . This wChelyconus ermineus [Cylinder gloriamaris [Dendroconus betulinus [Pionoconus magus , one of the earliest described and most well-known cone species [P. magus from the Philippines was recently sequenced, it was used  to test the performance of a new method for identifying highly divergent conotoxin precursors using machine learning algorithms, and not to describe in detail the venom repertoire of this emblematic species [P. magus [P. magus, belonging to the superfamilies A, O1, and O2 as retrieved from ConoServer [http://www.conoserver.org/index.php?page=list&table=protein&Organism_search=Conus%20magus&Type=Precursor). Six of these precursors corresponded to conotoxins isolated from the milked venom of a Northern Australian individual [P. magus from the Philippines using similarity searches reported 49 canonical conotoxin precursors and about 950 additional highly divergent candidates identified using machine learning methodologies [Pionoconus [P. catus [P. consors [P. striatus [To date, the list of sequenced venom gland transcriptomes has grown, steadily improving our knowledge of the vast diversity of conotoxins produced by different cone species such as, e.g., ermineus , Cylinderiamaris  or Dendretulinus , just to species . Althoug species . This isP. magus ,32. The P. magus . The proP. magus  and eggsP. magus , both feP. magus , the aduP. magus . Thus fanoServer  on July,dividual . Also, cdologies . The magonoconus . Within P. catus  and P. c consors , Expressstriatus , and thestriatus .P. magus from the Okinawa archipelago (Japan). We annotated all conotoxin precursors found in these two individuals as well as those found in the one reported from the Philippines [Chelyconus ermineus, the only piscivorous cone species in the Atlantic Ocean [Here, we sequenced and analyzed the venom gland transcriptomes of two individuals of lippines  using siic Ocean .P. magus from Okinawa (Japan) were sequenced. A total of 30–40 million raw reads were produced per sample (P. magus from the Philippines was assembled from raw reads available in the SRA database (SRX5015024) [P. consors, [C. ermineus [The transcriptomes of the venom ducts of two specimens of r sample . After qr sample . In addi5015024)  as that 5015024) . Up to 3ermineus ). D. betulinus [C. ermineus [P. magus [Virroconus ebraeus in Okinawa, Hawaii, and Guam [P. magus individuals from Okinawa were from nearby populations within the same island, thus other factors than geographic distance such as differences in age/size [Annotation of the transcripts expressed in the venom duct using BLASTX searches against a curated reference database rendered 118, 112, and 93 distinct (with at least one amino acid difference) conotoxin precursor and hormone sequences in magus1, magus2, and magus3 individuals, respectively . Only fiermineus , respectermineus , in agreP. magus  and couland Guam . Howeverage/size  or physiage/size  could beP. magus available at ConoServer were recovered corresponding to different members of A and O1 superfamilies , three rfamily; ), and onrfamily; ), expandC. ermineus [C. gloriamaris [D. betulinus [+ channels, κM-conotoxins that block voltage-gated K+ channels, and ψ-conotoxins that are non-competitive antagonists of nicotinic-acetylcholine receptors  channels [Overall, the superfamilies that were represented by the highest number of conotoxin precursors were M (42), O1 34), T 27), A (18), O2 (17), and F (13), accounting for 55% of the total diversity . All the, A (18),riamaris , and theetulinus . Therefo4, T 27),G. geographus [Textilia bullatus [C. ermineus . The diversity within the A superfamily can be subdivided into two groups: (i) kappa, which contains conotoxins targeting selectively K+ channels, producing an excitatory effect [P. magus produce mature peptides with the α3/5 cysteine spacing. Instead, the only piscivorous cone species of the Atlantic Ocean, C. ermineus exhibits high expression of mature peptides with the α4/4 cysteine spacing. This distinct pattern together with the distant relative phylogenetic position of C. ermineus with respect to Indo-Pacific piscivorous cones support a convergent evolution of fish hunting in cones [Rhombiconus [P. magus , and sponoconus  includinP. catus , P. cons consors , and P. y effect  and thaty effect . All conin cones . Furthermbiconus , whose smbiconus . HoweverP. magus .P. magus but neither in the other studied individuals nor in other cone species. Other conotoxin superfamilies have also display experienced bursts of diversification like P superfamily in Turriconus [d in Rhizoconus vexillum [The F superfamily, whose function remains unknown , shows arriconus  and d invexillum . The evoPionoconus delimited at least four paralog groups differing in their cysteine patterns  and in the spacing between cysteines in the alpha group . Phylogparalogs . Notablyparalogs . MoreoveR. vexillum [R. vexillum is produced in the proximal region of the venom duct and it is almost exclusively composed of d conotoxins [Rhizoconus capitaneus, Rhizoconus rattus, R. miles, and Rhizoconus mustelinus [Strategoconus vitulinus, Strategoconus generalis [Strategoconus planorbis [Ductoconus princeps [C. ermineus [P. magus showed no propeptide region and a larger and highly divergent mature region compared to those of R. vexillum. Hence, it was assigned to a different paralog group . The mature peptide in this paralog has a new cysteine framework (C-C-C-CC-C-C-C-C-C). The function of this second paralog and whether it also has defensive role is unknown.Among less diverse superfamilies, the presence of a member of the D superfamily is remarkable . This suvexillum ,57. In tvexillum ), has thvexillum . The defnotoxins . This sustelinus ,57, but eneralis , and Strlanorbis , the Easprinceps  and mostog group . This paperialis  and founPionoconus but others had identical sequences in distantly related species such as the vermivorous E. litteratus, Lithoconus leopardus, Conus marmoreus, and Darioconus episcopatus; R. imperialis (which hunts fireworms), and the molluscivorous Cylinder textile. Moreover, in some cases the 100% sequence identity occurred not only at the amino acid level but also at the nucleotide level  dominated in magus1  but had much lower representation in the other individuals . This ischannels  and is astriatus . Finallystriatus ,71. Witherfamily .P. magus from Japan, collected in Ishigaki Island in 2017  and OK206 (magus2) of  in 2017 . Their s in 2017  and methEach individual, in a resting stage, was extracted from the shell and dissected to remove the complete venom duct, which was stored in 1 mL RNAlater  at −20 °C. For RNA extraction, each venom duct was grinded in 300 µL of TRIzol  and mixed with 60 µL of chloroform. After centrifugation, the aqueous phase was recovered and RNA precipitated in one volume of isopropanol and incubated overnight at −80 °C. The Direct-zol RNA miniprep kit  was used to purify total RNA (5–15 µg) following manufacturer’s instructions. Dual-indexed cDNA libraries (307–345 bp insert average size) for each sample were constructed after isolation of mRNA using the TruSeq RNA Library Prep Kit v2  and following the manufacturer’s instructions. The quality of the libraries was analyzed with the 4200 TapeStation and the High Sensitivity D1000 ScreenTape assay ; libraries were quantified using real-time PCR in a LightCycler 480 . The pool of indexed libraries (including samples from other projects) was loaded into different lanes and sequenced by paired-end sequencing (2 × 100 bp) in an Illumina HiSeq2500 (two flow cells) following standard procedures at Sistemas Genómicos .Following , the rea−5). Selected sequences were translated into amino acids using the universal genetic code. TBLASTX similarity searches against the GenBank NR database and manual inspection were performed in order to discard false positives  or assembly artifacts . Duplicate and highly truncated  peptide sequences were removed to produce the final working list of conotoxin precursors and associated proteins of the three individuals of P. magus (provided in P. magus conotoxin precursor sequences were deposited (as nucleotide sequences) in GenBank under accession numbers .The amino acid sequences of all conotoxin precursors and other proteins produced in the venom gland of cone species available in GenBank release 226 , UniprotWe performed a phylogenetic analysis of the highly diverse O1 conotoxin precursor superfamily using the signal and propeptide domains. Multiple sequence alignment was performed with mafft v7  using thP. magus and three individuals of C. ermineus [Clean reads were mapped onto the assembled precursors and transcript abundance was calculated in transcripts per million (TPM), a metric which normalizes for gene length and sequencing depth. We run RSEM within Trinity v.2.6.6, which internally uses Bowtie2 . TPM estermineus  as bioloermineus  as impleP. magus is dominated by high intraspecific variability, which increases with geographic distance. Yet, several conotoxin precursors of the most diverse superfamilies in P. magus are highly conserved at the amino acid and nucleotide levels not only compared to orthologs from other species of the genus Pionoconus but also to distantly related cone species. As on other cones, the most diverse superfamilies were M, T, O1, O2, and A. Particularly remarkable was the detection of precursors of the D superfamily (formerly associated to vermivorous cones) and of the A alpha 4/3 conotoxins (formerly associated to Rhombiconus). The detection of the ω-conotoxin MVIIA (Ziconotide) only in the Philippines individual may indicate that paradoxically its ecological role may not be essential for the cone despite its relevant clinical value for humans.The composition of the venom repertoire of


Legionella pneumophila serogroup 6, sequence type 185, and used whole-genome sequencing to confirm the environmental and clinical isolates were of common origin.Legionellosis was diagnosed in an immunocompromised 3-year-old girl in Canada. We traced the source of the bacterium through co-culture with an ameba collected from a hot tub in her home. We identified  Legionella pneumophila is a waterborne bacterium responsible for Legionnaires’ disease, a potentially fatal respiratory disease acquired through environmental exposure to aerosolized water. According to the World Health Organization, L. pneumophila is the most common cause of legionellosis worldwide , Edmonton, successfully isolated Legionella sp. from clinical samples by using buffered charcoal yeast extract (BCYE) medium, with and without antimicrobial drugs, including polymyxin B, cycloheximide, and vancomycin. The clinical isolate was identified as L. pneumophila by using Legionella pneumophila Direct DFA Kit  direct fluorescent antibody assay. The National Microbiology Laboratory in Winnipeg, Manitoba, typed the isolate as serogroup 6, sequence type 185 (ST185), confirming Legionnaires’ disease. In December 2016, an immunocompromised 3-year-old girl was admitted to Alberta Children’s Hospital in Calgary, Alberta, Canada, with acute respiratory distress syndrome and septic shock requiring extracorporeal membrane oxygenation. She was transferred to a pediatric intensive care unit in Edmonton, Alberta, Canada, where examination of the lungs confirmed pneumonia in the left lower lobe segment with a lung abscess. Empiric treatment for infections was started immediately with meropenem, vancomycin, tobramycin, azithromycin, and trimethoprim/sulfamethoxazole. Subsequent bronchoalveolar lavage and bronchoscopy were performed, along with microbial culture for identification of fungi, mycobacteria, mycoplasma, viruses, and The patient was treated with levofloxacin and a prophylactic dose of trimethoprim/sulfamethoxazole. She also received vancomycin, meropenem, and tobramycin for 8 days and azithromycin for 5 days. Her condition steadily improved, and she was discharged from the hospital a few days after treatment.L. pneumophila sources, the Infection Prevention Control Research Laboratory of Alberta Health Services collected several first-flush water samples from sinks, a shower head, and a hot tub at the patient’s house and from sinks in the admitting hospital. All samples were negative for L. pneumophila by culture, but quantitative PCR results indicated the home hot tub was the likely source of the bacterium. To investigate possible Acanthamoeba polyphaga (ATCC30461) . We isolated 2 free-living amebae, an Acanthamoeba sp. and a Vermamoeba vermiformis, and identified species by morphology  containing 5 mL serum casein glucose yeast extract medium at 37°C with 10% fetal calf serum. Before experiments, we performed subcultures of amebae every 3–4 days to ensure that trophozoites were in an exponential growth phase.For co-culture experiments, we established amebae in axenic cultures in Nunc 25-cmL. pneumophila by using 16S rRNA gene sequencing . We prepared libraries according to the protocol for the Nextera XT DNA Library Prep Kit  and sequenced on an Illumina MiniSeq by using 2 × 150-nt reads. We deposited sequence information into BioProject (https://www.ncbi.nlm.nih.gov/bioproject) under accession no. PRJNA482644. For confirmation, we performed whole-genome sequencing on clinical and environmental isolates. We extracted genomic DNA by using the NucleoSpin Tissue Kit , then processed reads with Picard  and identified single-nucleotide polymorphism insertions/deletions by using FreeBayes version 1.2.0 (https://www.geneious.com/plugins/freebayes). We filtered resulting polymorphisms with VCF.Filter version 4.2  by using QUAL >10 & DP >20 & QUAL/AO >10 & SAF >0 & SAR >0 & RPR >1 & RPL >1. To rule out assembly errors or other spurious calls, we visually inspected the location of each putative polymorphism in reference assemblies of each isolate and traced back to the clinical contigs. We trimmed sequence reads by using Trimmomatic version 0.36 (https://orthomcl.org), alongside the complete ORFeomes of 6 other L. pneumophila strains: Philadelphia-1 NC_002942.5, Lens NC_006369.1, Thunder Bay CP003730.1, 570-CO-H NC_016811.1, Toronto-2005 NZ_CP012019.1, and Calgary-2012 SAMN03944918. We individually aligned 2,403 identified orthologs  across all strains by using the MUSCLE algorithm (https://www.ebi.ac.uk/Tools/msa/muscle) and concatenated orthologs into a superalignment for tree construction. We adopted RAxML version 8.2.12  with a general time-reversible nucleotide substitution model for 1,000 bootstraps to generate a maximum-likelihood phylogenetic tree.We constructed a phylogenetic tree by submitting assembled scaffolds to the RAST server for genome annotation (Results of whole-genome sequencing analysis strongly suggest that clinical isolate 2017a and environmental isolate 2017b from the patient’s home hot tub were of common origin. With only a few single-nucleotide polymorphism differences , these dL. pneumophila serogroup 6 has not been identified in previous hot tub–associated infections, and this case might have been a result of the patient’s immunocompromised status. A commercial hot tub cleaning product was subsequently used for disinfection and maintenance of the hot tub. In a 1-year follow up analysis, water samples from the hot tub were negative for amebae and any Legionella spp.Of note, we were not able to recover any legionellae from environmental samples with initial BCYE and other direct culture approaches. L. pneumophila from environmental samples. Our findings also suggest that investigations should include free-living ameba to indicate the presence of potentially pathogenic Legionella spp. and as a potential factor to minimize the need for remediation actions associated with contaminated environments.Our report demonstrates the utility of ameba co-culture and emphasizes the use of locally sourced ameba to recover the source of


Brucella (B.) spp. isolated from animal populations located in different districts of Egypt and to determine their antimicrobial resistance. In total, 34-suspected Brucella isolates were recovered from lymph nodes, milk, and fetal abomasal contents of infected cattle, buffaloes, sheep, and goats from nine districts in Egypt. The isolates were identified by microbiological methods and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Differentiation and genotyping were confirmed using multiplex PCR for B. abortus, Brucella melitensis, Brucella ovis, and Brucella suis (AMOS) and Bruce-ladder PCR. Antimicrobial susceptibility testing against clinically used antimicrobial agents  was performed using E-Test. The antimicrobial resistance-associated genes and mutations in Brucella isolates were confirmed using molecular tools. In total, 29 Brucella isolates (eight B. abortus biovar 1 and 21 B. melitensis biovar 3) were identified and typed. The resistance of B. melitensis to ciprofloxacin, erythromycin, imipenem, rifampicin, and streptomycin were 76.2%, 19.0%, 76.2%, 66.7%, and 4.8%, respectively. Whereas, 25.0%, 87.5%, 25.0%, and 37.5% of B. abortus were resistant to ciprofloxacin, erythromycin, imipenem, and rifampicin, respectively. Mutations in the rpoB gene associated with rifampicin resistance were identified in all phenotypically resistant isolates. Mutations in gyrA and gyrB genes associated with ciprofloxacin resistance were identified in four phenotypically resistant isolates of B. melitensis. This is the first study highlighting the antimicrobial resistance in Brucella isolated from different animal species in Egypt. Mutations detected in genes associated with antimicrobial resistance unravel the molecular mechanisms of resistance in Brucella isolates from Egypt. The mutations in the rpoB gene in phenotypically resistant B. abortus isolates in this study were reported for the first time in Egypt.Brucellosis is a highly contagious zoonosis worldwide with economic and public health impacts. The aim of the present study was to identify  Brucella (B.) that are genetically highly related Brucella The PCR products from each PCR were separated by electrophoresis using 1.5% agarose gels . Gels were stained with ethidium bromide and photographed using a gene snap camera .B. melitensis and B. abortus isolates was performed against eight clinically relevant antimicrobial agents  using gradient strip method  as described previously [2 for 48 h before reading. As MIC breakpoints for clinically used antimicrobials are not yet established for brucellae, the guidelines for slow-growing bacteria (Haemophilus influenzae) were used as an alternative [E. coli . The susceptibility profiles of Brucella isolates are presented as resistant and susceptible using minimum inhibitory concentrations (MIC), MIC50 and MIC90. The interpretations were performed using CLSI  [The antimicrobial susceptibility of eviously . Brieflyernative . Qualitystitute)  and EUCAstitute)  using thcatB, gyrA and gyrB, rpoB, Aac genes and tet genes for chloramphenicol, ciprofloxacin, rifampicin, streptomycin, gentamicin and tetracycline, respectively (rpoB gene were designed by using submitted sequences for the rpoB gene of B. abortus (accession number AY562181) [The PCR assays were performed as described previously ,49,52,59ectively . The priY562181) . PCR wasgyrA, gyrB and rpoB genes were purified using Qiagen QIAquick Gel extraction kit  and sent for sequencing . All consensus sequences were aligned and compared to the reference Brucella genes obtained from NCBI for detection and evaluation of nucleotide diversity and mutations using the software Geneious® R11.1.5 (https://www.geneious.com). The sequences of gyrA (CP034103 and AE017223), gyrB (CP007760 and SDWB01000001) and rpoB (AY562181 and AY540346) genes of B. melitensis and B. abortus were geared from Gene bank and used as reference. Amino acid sequences were determined along with nucleotide sequences to identify missense mutations using BLAST.Amplified PCR products for B. melitensis biovar 3, eight strains were B. abortus biovar 1 and five samples were identified as Achromobacter species  and 8 isolates as B. abortus . All isolates were confirmed as field strains ( strains .Brucella isolates were determined by the gradient strip method (E-test). The MIC values along with MIC50 and MIC90 are summarized in The in vitro MIC values against eight antimicrobial agents of all 29 B. melitensis isolates were resistant to ciprofloxacin, erythromycin, imipenem, rifampicin/rifampin and streptomycin, respectively. While, 25%, 87.5%, 25%, and 37.5% of B. abortus isolates were phenotypically resistant to ciprofloxacin, erythromycin, imipenem and rifampicin/rifampin, respectively. All 29 Brucella isolates were sensitive to chloramphenicol, gentamicin, and tetracycline. Four isolates of B. melitensis (19.04%) and one B. abortus isolate showed multidrug resistance against ciprofloxacin (fluoroquinolones), erythromycin (macrolides), imipenem (carbapenems) and rifampicin (ansamycins).In this study, 76.19%, 19.04%, 76.19%, 66.66%, and 4.76% of the catB, Aac and tet  conferring resistance to chloramphenicol, streptomycin/gentamicin and tetracycline, respectively) were not identified either in resistant or sensitive isolates. The gyrA, gyrB and rpoB genes were amplified in all isolates.Genes associated with antimicrobial resistance (rpoB gene associated with a rifampicin-resistant B. melitensis and B. abortus phenotypes were detected at different positions (Mutations in ositions .gyrA gene associated with phenotypic-ciprofloxacin resistance were detected at positions 167 (ATG to AGG/methionine to arginine), 197 (CCC to CGC/proline to arginine), 202 (CGC to AGC/arginine to serine), 235 (GGT to CGT/glycine to arginine), 941 , 944 , 944-945 , 946  and 962 (AAC to ACC/asparagine to threonine) in B. melitensis (Mutations in litensis .gyrB gene at position 1141 (AAG to GAG/Lysine to Glutamine), 1144 (ATC to CTC/Isoleucine to leucine) and 1421 (TCA to TTA/Serine to Leucine) in phenotypically resistant B. melitensis isolates  and 1435 (AAG to CAG/lysine to glutamine) in the rpoB gene of phenotypic resistant B. melitensis isolates while the same was recorded at position 2890 (CGT to GGT/arginine to glycine) in the rpoB gene of B. abortus isolates. No mutation was detected in gyrA and gyrB gene of B. abortus strains.Repeated mutations were detected at positions 676, 677  recommended treatment include high oral doses of rifampicin, doxycycline or tetracycline and trimethoprim-sulfamethoxazole. Although streptomycin and tetracycline are considered as powerful therapeutic agents against brucellosis, their higher toxicity limits their use ,61. QuinB. melitensis isolates were also reported from Norway in imported cases from the Middle East, Asia or Africa [B. melitensis isolates from humans in Egypt between 1999 to 2007 [Only one study from Brazil reported reduced antimicrobial sensitivity in brucellae isolated from cattle . Howeverr Africa . Probabl to 2007 . HoweverB. melitensis strains isolated from animals. In contrast, none of the mentioned studies reported ciprofloxacin resistance in clinical isolates of humans and animals before. However, antimicrobial resistance against quinolones has been reported in in vitro studies of B. melitensis from Greece and France [In this study, a notable phenotypic resistance against ciprofloxacin (76.19%) was detected in d France ,52.B. melitensis isolates was found in this study. Previous reports from Egypt (19%), [B. melitensis strains were also reported from Iran, Malaysia, China, and Kazakhstan [An alarming high number of rifampicin resistant 66.66%) t (19%), , Norway t (19%),  and Kaza.66% t (1t (19%), . Reducedzakhstan ,63,64,66B. melitensis isolates. However, the increased use of these antimicrobials in Egypt in veterinary and human practices may be the cause of the emerging of this resistance [The most striking finding of the present study was the emergence of phenotypic antimicrobial resistance against erythromycin (19.04%), imipenem (76.19%) and streptomycin (4.76%) in sistance .B. abortus isolated in this study was not proved previously. Multidrug resistant strains of B. abortus isolated from cattle in this study were reported previously in Brazil [B. melitensis and one isolate of B. abortus showed multidrug resistance against ciprofloxacin, erythromycin, imipenem and rifampicin. These findings are in agreement with the results of Barbosa Pauletti et al. who find corresponding resistance among B. abortus isolates from cattle in brazil [B. melitensis and B. abortus isolates in this study were sensitive to chloramphenicol, gentamicin and tetracycline. These findings are comparable to previously published reports in Egypt, China, Qatar and Kazakhstan [The phenotypic antimicrobial resistance against ciprofloxacin (25%), erythromycin (87.5%), imipenem (25%) and rifampicin (37.5%) of n Brazil . Four isn brazil . All B. zakhstan ,48,65,66Brucella as well as in other bacteria is the beta-subunit of the DNA dependent RNA polymerase (RNAP) encoded by rpoB gene [rpoB gene associated with phenotypic rifampicin resistant Brucella strains isolated from clinical specimens of animals in Egypt. Mutations were detected in all phenotypically resistant brucellae. Multiple and variable mutations were noted in each isolate along with few commonly shared mutations among many isolates. Frequent mutations at positions 676, 677-TAC to CTC  and 1435-AAG to CAG  in the rpoB gene of phenotypically resistant B. melitensis were detected. These mutations are different from previously reported mutations (in vitro mutations) associated with rifampicin resistance in Brucella [The target for rifampicin action in poB gene ,51. In tBrucella .B. melitensis isolates [B. melitensis.Johansen et al. reported mutations in phenotypic rifampicin resistant or intermediately resistant isolates , which irpoB gene of rifampicin resistant B. abortus strains. Frequent mutations were detected at position 2890-CGT to GGT .To the best of our knowledge, this study is the first report that proved mutations in the Brucella is multifactorial by nature in addition to obvious mutations of the gyrA, gyrB, parC and parE genes [gyrA and gyrB genes in phenotypically resistant B. melitensis and B. abortus to ciprofloxacin were investigated. The mutations in gyrA did not correspond with fluoroquinolone resistance mutations described by Turkmani et al. [Brucella mutants. The mutations in the gyrB gene detected at positions 1141-AAG to GAG (lysine to glutamine), 1144-ATC to CTC (isoleucine to leucine) and 1421-TCA to TTA (serine to leucine) of B. melitensis considered as novel findings of this study. None of these mutations was detected in B. abortus strains in gyrA or gyrB genes. However, the role of parC, parE and efflux systems cannot be ruled out for fluoroquinolone resistance [parC and parE genes.Fluoroquinolone/quinolone resistance in rE genes ,52. In ti et al. , althougsistance  as we dicatB), gentamicin (Aac) and tetracycline  were not detected in all investigated Brucella isolated in this study, which in accordance with the phenotypic antimicrobial susceptibility results of isolated Brucella isolates. It is also worth mentioning that all resistant Brucella strains were isolated from animals and they showed resistance to antimicrobials clinically used in humans practice, suggesting that the source of these Brucella strains may be of human origin. These findings point to the fact that inter-species and intra-host species Brucella transmission is common, but spillback may occur also when chronic human brucellosis is mistreated and resistant strains are shedded [Genes responsible for resistance against chloramphenicol  in bacteria is a public health issue globally and already compromises the treatment options regarding effectiveness of antimicrobials and control of several bacterial infections especially caused by gram-negative bacteria . Wide spBrucella . Higher od chain . Being aB. melitensis and B. abortus have been reported as the main causative agents of brucellosis in humans and animals. High phenotypic resistance against ciprofloxacin, erythromycin, and imipenem were detected in Brucella spp. isolated from different districts and animals species reflecting a broad geographical distribution. The molecular identification of mutations in antimicrobial resistance associated genes highlight the mechanism of resistance in Brucella spp. There is a need for further insights into the epidemiology and spread of antimicrobial resistant Brucella in Egypt. The WHO regimes have to be reevaluated and awareness among physicians about AMR needs to be raised.Brucellosis is a contagious and often communicable worldwide zoonosis with high morbidity and low mortality. There has been a tremendous increase in inter host-species infection in the recent decades, especially in developing countries when farm animal species are kept on the same premises without biosecurity precautions. The disease is endemic in Egypt and


Pyrus betulifolia is limited compared with studies of functional gene expression. Using high-throughput sequencing technology, the transcriptome of P. betulifolia under drought stress was analyzed to identify lncRNAs. A total of 14,478 lncRNAs were identified, of which 251 were found to be drought-responsive. The putative target genes of these differentially expressed lncRNAs were significantly enriched in metabolic processes, organic substance metabolic processes, macromolecule metabolic processes, and heterocyclic compound binding. Real-time quantitative polymerase chain reaction validation suggested that the results of the RNA sequencing data analysis were reliable. This study will provide genetic resources for pear breeding and provide reference to other pomological studies.Increasing evidence shows that long noncoding RNAs (lncRNAs) play important roles in developmental regulation and many other biological processes in plants. However, identification of lncRNAs in  Pyrus spp. L) is one of the most important and popular fruit crops cultivated commercially in most temperate regions [Pear  are a class of RNA transcripts that do not encode proteins . They arthaliana . In maizthaliana .A. thaliana [Medicago truncatula [Gossypium spp. [Triticum aestivum [Oryza sativa [Zea mays [A. thaliana, 6480 lincRNAs have been identified using bioinformatics [In order to identify novel lncRNAs, several strategies have been employed, including both computational and experimental screening methods ,22. Withthaliana , Medicaguncatula , Gossypiium spp. , Triticuaestivum , Oryza sZea mays ,28. For ormatics , and in ormatics .Pyrus betulifolia Bunge exhibits both drought resistance and salt tolerance [P. betulifolia will be important for improving the drought resistance abilities of pears. The genome of the pear (Pyrus bretschneideri Rehd.) has been sequenced recently [P. betulifolia. In this study, RNA-seq was used to analyze differentially expressed lncRNAs during drought stress. Identification of drought-resistance lncRNA genes will be beneficial for pear breeding programs.Pears are reproduced primarily through grafting. As one of the main pear rootstocks, olerance ,32 and irecently , but no P. betulifolia Bunge) seedlings were grown in the seedling beds of the Institute of Pomology at Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu, China. Seedlings were placed in a growth chamber under a 24-h cycle: 14 h at 25 °C in the light and 10 h at 20 °C in the dark, as per our previous report [Birch-leaf pear  according to the manufacturer’s recommendations. Nine strand-specific RNA libraries were submitted to the Novogene  for 150 base pair (bp) paired-end sequencing on the Illumina HiSeq 4000 platform , at a depth of 120 million reads per library.To construct RNA libraries, leaves were collected after 0, 24, and 48 h of PEG- treatment of six-leaf stage seedlings, using three biological replicates. Total RNA was extracted from these samples using the Total RNA Kit  according to the manufacturer’s instructions. RNA degradation and contamination was monitored using 1% agarose gels. RNA integrity was assessed using the RNA Nano 6000 Assay Kit with the Bioanalyzer 2100 system . Because some lncRNAs lack a poly(A) tail, total RNA was treated to remove ribosomal RNA (rRNA) using the Epicentre Ribo-zero rRNA Removal Kit . Finally, sequencing libraries were generated using rRNA-depleted RNA with the NEBNext UltraN and low-quality sequences (Q20 < 20) from the raw data, clean data were obtained for further analysis. A reference genome and gene annotation files were downloaded from the pear genome website (http://peargenome.njau.edu.cn/). The index of the reference genome was built using Bowtie v2.0.6 [After removing reads that included adapter sequences or poly-e v2.0.6  and cleae v2.0.6 . Based oe v2.0.6  with a rTo identify the noncoding transcripts, we used CNCI (Coding-Non-Coding-Index) , CPC (Cohttp://www.mirbase.org/) using BLAST with default parameters [To identify lncRNAs acting as precursors of miRNA in birch-leaf pear, the lncRNAs were aligned with the precursors of known microRNA (miRNAs) in the miRNA Base 21.0  software  to calcuery rate . lncRNAspression .p-values less than 0.05 were considered significantly enriched. KEGG orthology based annotation system (KOBAS) software was used to test for statistical significance of the differentially expressed lncRNA putative target genes in KEGG pathways (http://www.genome.jp/kegg) [To predict the putative target genes of significantly differentially expressed lncRNAs two approaches were used, coexpression and colocation method. Coexpression were predicted based on the expression correlation coefficient (Pearson’s correlation > 0.95 or < −0.95). In colocation, we searched for all the protein-coding genes 10 kb upstream or downstream of the differently expressed lncRNAs . Gene onjp/kegg) .2O. The primers sequence used in our PCR experiments are described in Ct−ΔΔ method was used to calculate relative changes in gene expression between control and treatment plants [UBQ) gene was used as housekeeping gene for normalization [The expression profiles of drought-responsive lncRNAs were validated through quantitative PCR. Total RNA for use as a template was extracted from leaves using the Total RNA Kit (TIANGEN) according to the manufacturer’s instructions, the first cDNA strand was synthesized from 1000 ng total RNA in a volume of 20 μL using the PrimeScript RT Reagent Kit with genomic DNA (gDNA) eraser , , according to the manufacturer’s protocol. Twelve lncRNAs were randomly selected from the differentially expressed lncRNA and analyzed using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). Primers were designed using Primer Premier 5.0 software , and RT-t plants . Ubiquitlization .P. betulifolia, we performed Illumina sequencing of RNA from the PEG-stressed pear using Hiseq2500. In total, we obtained 877.97 million raw reads from nine samples. After filtering out adapter sequences and low-quality reads, we obtained 853.77 million clean reads for further analysis  showed the reproducibility of the lncRNA expression, the h24–3 sample do not cluster with its h24–1 and h24–2 replicates  by examining the paired lncRNA and mRNA expression profiles. For differently expressed lncRNAs and their putative target genes between 24 h and 0 h. a total of 3139 positive and 1939 negative interaction relationships putative target genes were obtained, for 48 h and 0 h a total of 3967 positive and 1486 negative interaction relationship putative target genes were detected. GO enrichment A,B analyPrevious studies showed that lncRNAs could play a cis-regulatory role by mediating the expression of neighboring genes. We sought the 10 kb upstream or downstream of the differentially expressed lncRNAs to find protein-coding genes. We found 3010 protein-coding genes close to 185 differentially expressed lncRNAs between 24 h and 0 h, and 2158 protein-coding genes were located close to 126 differentially expressed lncRNAs between 48 h and 0 h. GO term analysis of the putative target genes were annotated as regulating biological process, response to stimulus, and catalytic activity . KEGG paThe response to drought stress in plants is a complicated process, involving several genes and metabolic network, hormones synthesis is one of important factor . In totaTo validate the reliability of the transcriptome gene expression profiles, 10 differentially expressed lncRNAs were randomly selected for expression analysis through RT-qPCR. The expression patterns shown in the RT-qPCR results were consistent with the RNA-seq results . For exaGossypium spp. [Arabidopsis [Zea mays [Arabidopsis, rice, but fewer than in Gossypium spp. and Z. mays. In Gossypium spp. and Z. mays, cDNA libraries were generated from multiple tissue types, whereas the cDNA libraries in the present study were generated only from leaves. Therefore, we likely identified only a subset of the total lncRNAs in birch-leaf pear, and we believe that more lncRNAs may be identified using different organs and under different stresses.We obtained a large number of lncRNAs from the birch-leaf pear: 14,478 lncRNAs were reliably identified. Among other plants, 50,566, 6480, 2224, and 20,163 lncRNAs were previously identified in ium spp. , Arabidobidopsis , rice [2Zea mays , respectPopulus and wheat [A. thaliana [In contrast to mRNAs, lncRNAs are expressed at a low level in a tissue-specific manner, and generally exhibit poor conservation across different species . lncRNAsnd wheat ,56. lncRthaliana . In wheathaliana . In our Cunninghamia lanceolata), the largest proportion of differentially expressed genes were related to metabolic pathways [Plants are frequently affected by a wide range of environmental stressors . To copepathways . Thus, iIn summary, we identified 14,478 lncRNAs in birch-leaf pear and showed that 251 were dehydration-responsive. These results indicate that the drought stress affected the expression of some lncRNAs. The putative target genes of different expressed lncRNA were identified, some of these putative target genes are involved in desiccation-response processes. The results of RT-qPCR validated the reliability of the RNA-seq. Our results provide a rich genetic resource for discovery of genes related to drought stress and can be readily applied to other fruit tree species.


Dendrobium are tropical orchid plants that host diverse endophytic fungi. The role of these fungi is not currently well understood in Dendrobium plants. We morphologically and molecularly identified these fungal endophytes, and created an efficient system for evaluating the pathogenicity and symptoms of endophytic fungi on Dendrobium nobile and Dendrobium officinale though in vitro co-culturing. ReThe colony morphological traits of Dendrobium myco-endophytes (DMEs) were recorded for their identification. Molecular identification revealed the presence of Colletotrichum tropicicola, Fusarium keratoplasticum, Fusarium oxysporum, Fusarium solani, and Trichoderma longibrachiatum. The pathogenicity results revealed that T. longibrachiatum produced the least pathogenic effects against D. nobile protocorms. In seedlings, T. longibrachiatum showed the least pathogenic effects against D. officinale seedlings after seven days. C. tropicicola produced highly pathogenic effects against both Dendrobium seedlings. The results of histological examination of infected tissues revealed that F. keratoplasticum and T. longibrachiatum fulfill Koch’s postulates for the existence of endophytes inside the living tissues. The DMEs are cross-transmitted inside the host plant cells, playing an important role in plant host development, resistance, and alkaloids stimulation. Dendrobium orchids are horticultural plants and have been used for profitable trade due to their flowering profusion, extensive variety of flower colors, shapes, sizes, and year-round producibility, along with long lifespan [Dendrobium officinale has been extensively used in traditional medicines for over 2000 years to decrease fever, inhibit tumors, increase antioxidant activity, treat hypoglycemia, recover from loss of eyesight, and control the immune system, according to the best of China Pharmacopeia [The Orchidaceae is the chief family of plants, with over 25,000 plant species worldwide. They are also some of the most vulnerable flowering plants, as numerous genera are endangered and nearly all genera are at risk of habitat harm and over-assortment . China ilifespan . Most splifespan ,5. Dendrmacopeia ,7.Dendrobium plants parts, for example, roots, stems, buds, and leaves of tropical orchid, harbor diverse fungal taxa, including mutualistic mycorrhiza, and endophytic fungi and considerably diverse nonmycorrhizal fungal associates. The role of the root-allied fungi is not well understood. They typically originate in the velamen, without causing any disease symptoms [Dendrobium by activating soil chemicals in the rhizosphere. The impact of quantities or variations of secondary metabolite have been investigated. At large, they act as a supply for bioactive molecules that defend the host from rhizospheric pathogens [symptoms . They maathogens . Endophyathogens . The relathogens .Dendrobium nobile and D. officinale) protocorms initiating from the orchid seeds are typically very small, similar to dust, and deficient an endosperm. Subsequently, orchid seed incubation and seedling expansion require well-matched endophytic fungi to deliver the carbon, nutrients, and water to the seeds under usual plant conditions [Protocorm and seedling expansion is an important step in commercial orchid production, and its conservation is crucial tool for maintaining the genetic diversity of the orchid plant . Orchid nditions . Consequnditions .Dendrobium myco-endophytes (DMEs) focused on symbiotic in vitro practices using fungal endophyte strains obtained from fully-grown dendrobium roots, buds, stems, and leaves [Most examinations of d leaves . Understd leaves . Dendrobium–fungus connections at the protocorm and seedling phase are available at present, which places major constraints on understanding the host–endophyte relationships [A steady state between the fungal pathogen and its host plant is achieved when the pathogen resides in equilibrium with the surrounding host tissues and causes little damage, which is also called least pathogenic. The virulence expression of the pathogen is dependent on the particular host environment. When the pathogen is isolated from an asymptomatic host and introduced into a new host, strong pathogenic reactions may be observed . Commonlionships . D. nobile plant parts, which were inoculated onto well-developed protocorms and seedlings of D. nobile and D. officinale for the evaluation of pathogenicity using in vitro inoculation and histopathological examination of infected tissues. The test endophytic fungus was re-isolated from the leaves tissues for examining Koch’s postulates. In this study, we focused on species-specific endophytic fungi pre-isolated from D. nobile. The colony morphological traits of the DME isolates, such as hyphal structures and spore arrangements, were used for their identification. The isolated endophytic fungal colony characteristics and microscopic characteristics findings were found as follows: (1) ZF01: fast grower (35 ± 0.2 mm), flat and buff growth surface, whitish color, reverse whitish to grey/orange, margin irregular mycelium with conidiogenous cells hyaline to pale brown, conidia hyaline with round ends, smooth-walled; (2) ZF02: moderate grower (16 ± 0.2 mm), colony flat velvety, white color with no diffusing pigment coloration, margin irregular, white rims, mycelium with microconidia oval and elongated and clavate, sickle-shaped conidia containing numerous septations, septate hyaline hyphae; (3) ZF03: fast grower (25 ± 0.2 mm), colony cottony white to light violet color, aerial white margins septate and hyaline, macroconidia slightly curved and thick with an attenuated apical cell, three to five septa, short aerial conidiophore, unbranched, microconidia non-septate, aerial conidia, ellipsoidal to cylindrical, slightly curved; (4) ZF04: fast grower (28 ± 0.2 mm), colony cottony white to light white with creamy to white-greyish and reverse light yellow color, irregular white margins with cream yellow edges with long and narrow macroconidia, cylindrical, dorsal and ventral surface parallel, three to five septa, phyalide long and thin; and (5) ZF05: fast grower (34 ± 0.2 mm), colony cottony off white to greenish, later green tufts sporulation, irregular white margins with very hyaline hyphae, long conidiophores, flask-shaped single phialides, small-walled ellipsoidal conidia, formed chlamydospores  in terminal or intercalary (Colletotrichum tropicicola (ZF01), Fusarium keratoplasticum (ZF02), Fusarium oxysporum (ZF03), Fusarium solani (ZF04), and Trichoderma longibrachiatum (ZF05)  from leaf segments of the wild ercalary . Moleculm (ZF05) . ResultsD. nobile and D. officinale protocorm revealed that T. longibrachiatum (ZF05) showed the least pathogenicity (11%) against the D. nobile protocorm after 7, 14, and 21 days, whereas it showed moderate pathogenic (33%) effects at 21 days of incubation against D. officinale. We found that most of the plant pathogens are latent pathogens or conditional pathogens, causing symptoms some weeks later when the plant defense mechanism has weakened. Many environmental factors function together to trigger the virulence factors or genes of the endophytic fungi. T. longibrachiatum (ZF05) started to change the color of the D. officinale protocorm from green to light yellowish at 14 days, which changed to brown to blackish at 21 days; whereas no color change symptoms were observed in the D. nobile protocorm. The second least pathogenic strain was F. keratoplasticum (ZF02), which was found to be non-pathogenic against D. nobile after 7 and 14 days of incubation, and least pathogenic (11%) against D. officinale after seven days of incubation, but showed moderate pathogenic effect after 21 days incubation on both D. nobile and D. officinale. The symptoms on the protocorms of D. nobile and D. officinale by F. keratoplasticum (ZF02) included the color of D. officinale protocorm starting to change from green to light yellowish at 14 days, then turning yellow at 21 days, whereas no color change symptoms were observed in the D. nobile protocorm due to F. keratoplasticum (ZF02). F. oxysporum (ZF03) and F. solani (ZF04) were moderate pathogens at seven days of incubation and strong pathogens (100%) at 21 days of incubation. C. tropicicola (ZF01) showed high pathogenicity (77–100%) effects against both D. nobile and D. officinale seedlings. F. oxysporum (ZF03) produced no symptoms on the protocorms of D. nobile and D. officinale at 7 and 14 days, indicating these are latent pathogens because they demonstrated high pathogenicity at 21 days and their symptoms included color change of the green protocorm to brownish for both D. nobile and D. officinale. F. solani (ZF04) produced moderate symptoms on the protocorms of both D. nobile and D. officinale by changing their green color to light reddish to brown in the initial days and then turned dark reddish-brown at 21 days. C. tropicicola (ZF01) significantly affected the protocorms of D. nobile and D. officinale by changing their green color to dark brown at 7, 14, and 21 days  produced the least pathogenic effects on D. officinale protocorm after seven days and increased gradually with increasing incubation period , which was stable at 21 days (33% disease index). For D. nobile, the fungus showed highly pathogenic (77% disease index) effects from the start to the end of the incubation period. The second least pathogenic strains were F. keratoplasticum (ZF02) and F. solani (ZF04), which showed least pathogenic effects against D. officinale, whereas F. keratoplasticum (ZF02) and F. oxysporum (ZF03) showed stable moderate pathogenic (33%) effects against D. nobile and D. officinale, respectively, throughout the incubation period. C. tropicicola (ZF01) showed high pathogenicity (77–100%) against both D. nobile and D. officinale seedlings. The seedlings symptoms included a color change of D. nobile seedlings from green to black spots and brownish to blackish spots on the D. officinale seedlings with T. longibrachiatum (ZF05) at 7, 14, and 21 days. F. keratoplasticum (ZF02) produced symptoms on the D. nobile and D. officinale seedlings including color change of the D. officinale seedlings from green to yellowish at 7 days, then to light brown at 14 days, then to black at 21 days; similar symptoms were observed in the D. nobile seedlings with F. keratoplasticum (ZF02). F. oxysporum (ZF03) produced light brown color symptoms on the seedlings of D. nobile and D. officinale at 7 and 14 days, which later changed to brownish to yellowish. F. solani (ZF04) produced moderate symptoms on the seedlings by changing its green color to light yellowish to brown at 7 days, changing to dark brown at 14 days, then to blackish brown at 21 days on the seedlings of D. nobile and D. officinale. C. tropicicola (ZF01) produced significant symptoms on the seedlings of D. nobile and D. officinale by changing the seedling color from green to brown at 7, 14, and 21 days (The DME pathogenicity results for  21 days .C. tropicicola (ZF01), F. keratoplasticum (ZF02), F. oxysporum (ZF03), F. solani (ZF04), and T. longibrachiatum (ZF05). All isolated endophytic fungi were confirmed as the same fungi via microscopic examination as described in After the coculturing incubation periods (21 days), the endophytic fungi were re-isolated from the plant tissues to confirm the presence of fungi inside the plant tissues by re-inoculating the samples into the growth media (PDA: potato dextrose agar media). The results showed that the same endophytic fungi were re-isolated from the uninoculated plant tissues, which were identified based on characteristics: D. nobile and D. officinale seedling tissues  produced the least pathogenic effects on D. nobile and D. officinale, which provided the plants with nutrition and helped them build an active defense mechanism to survive without the presence of any nutrients in the media. This phenomenon was observed as asymptomatic colonization due to the balanced antagonism between the host and the endophyte [Dendrobium defense are well balanced, the relationship is avirulent and asymptomatic. This stage is only a transitory time where environmental influences play a key role in destabilizing the delicate equilibrium of antagonisms. [Beauvaria and Fusarium species are endophytic fungi, which we also recovered, that may play a role in the growth and survival of the plants like D. friedericksianum. Though grown in orchid stems and roots, the fungi may be non-casual and non-mycorrhizal endophytes. Many of these endophytes were reported in many white rot fungi and are incapable of phytopathogenicity [Dendrobium protocorms and seedlings have the ability to cause disease symptoms. Zi et al. [Epulohiza, an anamorphic phase of Tulasnella, in the Dendrobium protocorms. Athipunyakom et al. [Trichosporiella multisporum is present in Paphiopedilum roots. In an ecosystem, orchids depend on orchid fungal endophytes to provide needed nutrients for growth, a process termed an asymptomatic relationship. The symbiotic plant relationship, specifically in vitro approaches, is adopted because it enables higher growth rates and/or symbiotic seedlings and protocorms progress faster than without this relationship [This study provides valuable information about the orchid bionetwork with endophytic fungi associated with ndophyte . Endophygonisms. . Endophygonisms. . Khamchagonisms.  also stagenicity . In anotgenicity  found thi et al.  describem et al.  reportedtionship . Althougtionship .C. tropicicola (ZF01) is a highly pathogenic strain for the protocorms and seedlings of both D. nobile and D. officinale. Dendrobium endophyte contact might, in addition to balancing between defense and virulence, might more precisely control this complex contact [T. longibrachiatum (ZF05) mycelium may migrate from inoculated to uninoculated tissues of host plants. The histological image of D. nobile and D. officinale seedlings clarified that the fungus infects tissues. This paper is the first report on the cross-transmission of T. longibrachiatum (ZF05) from inoculated tissues to uninoculated tissues confirmed by re-isolated from host plant segments that fulfilled the Koch postulates. Colletotrichum, Fusarium, and Trichoderma include morphologically similar taxa that are commonly found as endophytes, saprobes, and plant pathogens [From this study, we found that  contact . Plant–p contact . Many fu contact . We hypoathogens ,14,15. D. nobile and D. officinale seedlings. This means that these endophytic fungi are cross-transmitted intracellularly from one cell to another healthy cell, creating comprehensive endophytic molecular connections, cross-species appearance, and on/off switching of the compulsory gene cascades that constantly yield a chosen molecule [As endophytes exist in within plant tissues and endlessly network with their host tissues, fungal endophytes may be linked intracellularly, which is responsible for the cross-transmission of the fungus into the new cells. Along this line, we strongly suggest that endophytic fungus is re-isolated from the uninoculated infected segments of the molecule . D. nobile plants were collected  from Jinshishi, Chishui, Guizhou, China and processed in the Bioresource Institute for Health Utilization, Zunyi Medical University, Zunyi, China, and then inoculated aseptically into PDA media. The samples were processed according to a method modified from Novotná et al. [D. nobile were used for surface sterilization processing. All the leaves samples were washed with tap water to remove the dirt spots and connective tissue of stems. Thereafter, the leaves were washed with tap water to further clean the surface. After cleaning, all the plant samples were placed into laminar air flow and exposed to UV rays for 20 min. Under sterilized conditions in the laminar air flow, the samples (1.5 cm pieces) were inoculated onto PDA media plates. The sample plates were placed in a fungal incubator at 25 °C for 5 days. After 5 day of incubation, the fungal hyphal tips emerging from the samples were recovered and purified by sub-culturing from the grown hyphal tips.Endophytic fungi were isolated from leaves parts. Five healthy wild á et al. . All theThe endophytic fungal genus was identified using lactophenol cotton blue staining according to the protocol followed by Barnett and Hunter  and ElliTM Basic, Henderson, Singapore) for 30 min at 120 V and 250 mA using 1× TAE (Tris-acetate-EDTA) buffer. The gel was marked with ethidium bromide (0.1 μg/mL). A Molecular Imager Gel DocTM XR+ with Image Lab Software system  was used to capture images using Image Lab software (version 4.1).The PCR samples were analyzed using electrophoresis on a 1% (w/v) agarose gel  . A DNA mColletotrichum tropicicola ZF01: MN826680; Fusarium keratoplasticum ZF02: MN832906; Fusarium oxysporum ZF03: MN826681; Fusarium solani ZF04: MN826682; and Trichoderma longibrachiatum ZF05: MN826683. MEGA 7.0 software was used for phylogeny tree study.The amplified ITS regions of PCR samples were sequenced by Tsingke, Beijing, China. Sequence data were subjected to BLAST analysis on the NCBI  web tool to validate the characteristics of the amplified sequences along with isolates. All the sequences have been deposited in the GenBank under accession numbers i.e., D. nobile and D. officinale (both protocorms aged 4 months) and seedlings (aged 8 months) were obtained from the Bioresource Institute for Healthy Utilization (BIHU), Zunyi Medical University  for experiments and authenticated per previous work [D. nobile and D. officinale were inoculated into separate bottles under aseptic conditions. For seedlings, we cut marks into the top leaf of the seedlings where it joined then stem using a sterilized scalpel blade over sterilized paper, as shown in Water agar (0.65% w/v) was used as media and relocated into the glass bottles for the coculturing experiments. No additional nutriments were added into the media; only twofold distilled water and agar were used. ous work ,30,31,32D. nobile and D. officinale. The control was set in triplicates under the same conditions without added any endophytic fungal cultures. After the inoculation, the test co-culturing bottles were transferred into the plant growth chamber instrument and maintained at 25 °C along with a 14/10 h light/dark cycle with 2000 lux light intensity. All the test bottles were examined in 7-day intervals up to 21 days. For D. nobile and D. officinale seedlings, the contents of endophytic fungal flask cultures were filtered to separate the mycelium suspension. The mycelium was concentrated and injected onto cut marks at terminal leaves at the link to the stem. The sterilized cotton plug served as a control for seedlings co-culturing pathogenicity and symptoms evaluation experiments. The disease index was measured using the disease scale and disease index formula. The scale of the disease index is described as follows: 0 indicates no disease (non-pathogens), 1 indicates small spots < 1% (pin brown spots: fewest pathogens), 3 indicates spots (1–11%: least pathogens), 5 indicates spots with 12–25% coverage (moderate pathogens), 7 indicates circular spots (26–55% coverage: moderate pathogens), and 9 indicates circular to irregular spots (>75% coverage: highly pathogenic) [For pathogenicity experiments, the identified endophytic fungal mycelium was used for the pathogenicity evaluation. For protocorms, 0.5-cm fungal discs were transferred into the protocorm bottles of hogenic) .Disease2 was immersed into the glass bottle for 3 min with continuous shaking. The HgCl2 water was removed after 3 min and then the samples were washed 3 times using sterilized distilled water. Brown paper sterilized in a hot air oven at 160 °C for 1.5 h was used for cutting the samples into smaller pieces. The samples (seedling) were moved onto the potato dextrose agar media plates. Two pieces of samples were transferred onto each Petri plate with the surface touching the media. The plates were incubated for five days at 25 °C in the incubator. After the fifth day of incubation, fungal hyphal tips emerging from the samples were recovered and purified using sub-culturing from growing hyphal tips. The growth, colony properties, and microscopic features of DMEs were recorded from the strains grown on the PDA media [All the samples (seedlings) were washed with sterilized distilled water to eliminate surface fungal growth. Thereafter, the seedlings were placed into laminar air flow and exposed UV rays for 20 min to eliminate surface fungi. After UV exposure, 75% alcohol was added into the sample bottles for 30 s and mixed using gentle shaking, which was then washed twice with sterilized distilled water under laminar airflow. Thereafter, HgClDA media ,35. MicrD. nobile and D. officinale samples were used for visual histopathological assessment of endophytic fungi by staining the infected tissues using a method modified from Ding et al. [D. nobile and D. officinale leaves and stems. Photographs were captured under different magnifications (10× and 40×).Infected g et al. . InfecteC. tropicicola (ZF01), F. keratoplasticum (ZF02), F. oxysporum (ZF03), F. solani (ZF04), and T. longibrachiatum (ZF05) were calculated using SAS CORR procedure.Statistical calculations were completed using SPSS11 software (USA) and the Fisher’s least significant difference procedure. Correlations of disease index caused by T. longibrachiatum (ZF05) was found to be the least pathogenic or a conditional pathogen that supports the development of the D. nobile protocorms and D. officinale seedlings without the presence of any nutrients in the media. C. tropicicola (ZF01) is a highly pathogenic strain, responsible for the host death. We concluded that endophytic fungi were cross-transmitted from host plant inoculated to uninoculated cells, which was confirmed by histopathological examination and re-isolation of the same endophytic fungi from uninoculated plant tissues. Future investigations should determine what role, if any, the plant host specificity plays in the interior plant passage and differential tissues establishment by test fungal endophytes. How fungi are able to precisely move within tissue (host plant) should also be examined. The symbiotic seedling and protocorm growth are advantageous and expedient method to improve orchid growth under the experimental conditions and could help with the reintroduction of Dendrobium orchids to the natural environment.In this study, we evaluated fungal pathogenicity and colonization inside plant tissues under in vitro conditions. Firstly,


Rickettsiajaponica strain YH. R. japonica infection should be considered for febrile patients in China.We investigated 16 Japanese spotted fever cases that occurred in southeastern China during September–October 2015. Patients had fever, rash, eschar, and lymphadenopathy. We confirmed 9 diagnoses and obtained 2 isolates with high identity to  Rickettsia japonica is a member of the spotted fever group rickettsiae (SFGR) that causes tickborne Japanese spotted fever (JSF). First reported in Japan’s Tokushima Prefecture in 1984 . All these patients lived in the Xitianmu Mountain area of Linan in Zhejiang Province. Besides fever (38.8°C–40.3°C), clinical signs of disease in these patients included rashes on the trunk and limbs (15/16) and an eschar (11/16) . In thosRickettsia infection. We extracted DNA from acute-phase blood specimens by using the DNeasy Blood & Tissue Kit . We used nested PCR to amplify the partial groEL genes of SFGR, typhus group rickettsiae, and Orientia tsutsugamushi bacteria (http://www.ncbi.nlm.nih.gov/BLAST), and all had 100% identity to R. japonica YH prototype strain (GenBank accession no. NC016050)  and convalescent-phase (n = 14) whole blood specimens and sent them to Zhejiang Province Center for Disease Control and Prevention for laboratory confirmation of bacteria . The tarbacteria . We sequgroES and groEL (GenBank accession nos. KY073364–5) and a 609-bp fragment containing the partial rickettsial ompA gene sequence  every 2 days to access SFGR growth . Two of R. japonica (HL-60 cells infected with LA4/2015) and R. rickettsii  to test patients for specific antibodies, and in all 16 patient serum samples, we detected SFGR IgG. All paired serum samples (n = 14) showed a >4-fold increase in titer against SFGR (R. japonica by PCR.We used IFAs with bacterial substrate antigens nst SFGR . The 2 pO. tsutsugamushi IgG. Some convalescent-phase serum specimens had low-titer reactions to R. typhi bacterial antigen.All serum specimens were negative for R. japonica, R. heilongjiangensis, R. rhipicephali, and R. massiliae have been identified in Haemaphysalis longicornis and Rhipicephalus haemaphysaloides ticks in Zhejiang Province (–R. japonica rickettsiae. The prototype strain R. japonica YH was isolated in Japan in 1985 (R. japonica isolates have been isolated from patients in China: 2 from our research and 1 from Li et al. (groES and groEL genes and the partial ompA gene sequences were 100% identical to R. japonica YH, suggesting that the R. japonica genome has been relatively conserved. Nine patients had clinically confirmed JSF, displaying fever, rash, eschar, and lymphadenopathy; these signs and symptoms were similar to those seen in JSF patients in Japan (The 4 SFGR species R. japonica.Of the vectorborne rickettsial diseases in China, human scrub typhus and murine typhus frequently occur in Zhejiang Province, and spotted fever group rickettsiosis probably occurs but has gone relatively unnoticed. Because the clinical symptoms of spotted fever and scrub typhus are similar, some SFGR infections have likely been diagnosed as scrub typhus. We found that the blood specimens of 7 febrile patients were negative for the targeted PCR fragments but showed a >4-fold increase in antibody titer to SFGR. Although these results suggest SFGR infection, we cannot conclude these 7 patients were infected with R. japonica infections occur in Zhejiang Province, China. These infections are likely more broadly distributed throughout the mainland areas than had been previously realized. Improvements in JSF clinical diagnosis and human epidemiologic surveillance are urgently needed in China.In summary, Rickettsia japonica infections, China, 2015.Primer information and description of methods used for febrile patients with


Aegilops is considered to be an origin of B-genome in cultivated tetraploid and hexaploid wheat species, although its actual donor is still unclear. Here, we attempted to elucidate phylogenetic relationship among Sitopsis species by performing RNA sequencing of the coding regions of each chromosome. Thus, genome-wide polymorphisms were extensively analyzed in 19 accessions of the Sitopsis species in reference to the tetraploid and hexaploid wheat B genome sequences and consequently were efficiently anchored to the B-genome chromosomes. The results of our genome-wide exon sequencing and resultant phylogenetic analysis indicate that Ae. speltoides is likely to be the direct donor of all chromosomes of the wheat B genome. Our results also indicate that the genome differentiation during wheat allopolyploidization from S to B proceeds at different speeds over the chromosomes rather than at constant rate and recombination could be a factor determining the speed. This observation is potentially generalized to genome differentiation during plant allopolyploid evolution.Dramatic changes occasionally occur in intergenic regions leading to genomic alterations during speciation and will consequently obscure the ancestral species that have contributed to the formation of allopolyploid organisms. The S genome of five species of section Sitopsis of genus  Triticum aestivum L., genome constitution AABBDD), a major food crop, is an allohexaploid species derived via allopolyploid speciation through interspecific crossing between cultivated tetraploid wheat Triticum turgidum L. (AABB) and its diploid relative, Aegilops tauschii Coss. (DD).T. turgidum subspecies dicoccoides (AABB), which was thought to be derived through interspecific hybridization between wild diploid progenitors of the A and B genomes. The A genome donor was the wild diploid wheat T. urartu,,Ae. speltoides Tausch (SS).Ae. speltoides,,Ae. speltoides contributed to establishment of the nuclear genome of allopolyploid wheat.Common wheat , Ae. searsii Feldman et Kislev ex Hammer (SsSs), Ae. sharonensis Eig (SlSl), and Ae. longissima Schweinf. & Muschl. (SlSl), except Ae. speltoides. Of the five Sitopsis species that share the S genome,Ae. speltoides of the subsection Truncata is cross-pollinating, whereas the other four subsection Emarginata species are self-pollinating. Two subspecies of Ae. speltoides  have been defined to date,,Lig, on chromosome 3S, which controls spike morphology.Ae. longissima and Ae. sharonensis, are quite closely related and recognized as forming one complex.1 hybrid plants among Sitopsis species show incomplete homoeologous pairing during meiosis,,,Ae. speltoides. Constitutive heterochromatic regions detected by chromosome staining are much more abundant in B-genome chromosomes than those of the A and D genomes,,Ae. speltoides.,The indefinite origin of the wheat B genome is due to failure of homoeologous chromosome pairing between the B genome of allopolyploid wheat and the S genome of ,,Ae. speltoides than to the other modified S genomes of subsection Emarginata species.,Ae. speltoides is the donor of the B genome in allopolyploid wheat.,Ae. searsii among all Sitopsis species.Ae. speltoides, and this divergence is thought to be a result of polyploidization events affecting B-genome evolution.Molecular phylogenetic studies based on nuclear DNA polymorphisms (e.g. restriction fragment length polymorphisms [RFLPs] and amplified fragment length polymorphisms [AFLPs]) have revealed that the two subsections of Sitopsis are extensively differentiatedAegilops species.Aegilops species, polymorphisms identified without any reference genome information can be efficiently anchored to the homoeologous chromosomes of related species, such as common wheat and barley, based on conserved chromosomal synteny.RNA sequencing is an effective approach for surveying large number of genome-wide polymorphisms derived only from the exon sequences in 2.1.Ae. speltoides ssp. ligustica (SS genome), four accessions of Ae. speltoides ssp. speltoides (SS genome), two accessions of Ae. bicornis (SbSb genome), three accessions of Ae. longissima (SlSl genome), three accessions of Ae. sharonensis (SlSl genome), and four accessions of Ae. searsii (SsSs genome) were chosen as representatives of each species from the collection of the section Sitopsis at the National Bio Resource Project–Wheat, Japan (T. turgidum) cultivar Langdon (AABB genome) was also used in this study. Triticum urartu KU-199-5 (AA genome), Ae. umbellulata KU-4017 (UU genome), and Ae. tauschii KU-2075 (DD genome) were used as outgroup species.Three accessions of t, Japan . These at, Japan . A tetra2.2.Ae. tauschii KU-2075 and DRA006404 for Ae. umbellulata KU-4017.Total RNA was extracted using Sepasol-RNA I Super G  from leaves of 2- to 3-month-old plants grown in a glass house. The extracted RNA was treated with DNase I at 37 °C for 20 min, after which paired-end libraries for RNA sequencing were constructed from 6 to 10 µg of total RNA using a TruSeq RNA Library Preparation kit v2  according to a previously reported protocol2.3.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) was used to evaluate sequencing quality of the reads from each of the sequenced samples. Adapter sequences, low-quality bases with an average quality score per 4 bp of <30, and reads of less than 100 bp were removed using Trimmomatic software, version 0.33,T. aestivum cv. Chinese SpringDe novo transcriptome assembly of Ae. speltoides ssp. ligustica KU-7716 was performed using Bridger.Ae. speltoides ssp. ligustica KU-7716 were aligned to its assembled transcripts using Bowtie.T. aestivum cv. Chinese Spring (iwgsc_refseqv1.0)FASTQC software (2.4.Neighbour-joining (NJ) and maximum-likelihood (ML) phylogenetic trees were constructed using Molecular Evolutionary Genetics Analysis (MEGA) software, version 5.05.2.5.T. aestivum, the chromosomal regions of the B genome were divided into ranges of 20 Mbp each. A total of 262 segments of subsets of non-redundant SNPs in Sitopsis species and the B genome of T. aestivum cv. Chinese Spring were obtained. The fixed nucleotide differences and average number of nucleotide differences between Sitopsis species and the B genome were estimated for each subset of non-redundant SNPs. Non-synonymous fixed nucleotide differences were estimated using the variant annotation and effect prediction tool SnpEff.To estimate nucleotide divergence between Sitopsis species/subspecies, two distance parameters were calculated: fixed substitutions between species3.1.Ae. bicornis, whereas the other four species had similar numbers (To identify genome-wide SNPs and indels in Sitopsis species and the wheat B genome, RNA sequencing of 19 representative accessions of the five Sitopsis species was performed, generating 2–3 million filtered paired reads for each species . Of thes numbers . High wi numbers . To exam numbers .To confirm that RNA sequencing could identify genome-wide SNPs and indels, the chromosomal distribution of SNPs and indels was examined . SNPs an3.2.T. urartu, Ae. umbellulata, and Ae. tauschii. Without the outgroup species, 30,589 non-redundant SNPs were obtained. When the outgroup species were included, 39,148 non-redundant SNPs were identified. These sets of non-redundant SNPs covered all of the chromosome of the B genome (To clarify the phylogenetic relationship and nucleotide divergence between species, we estimated sets of non-redundant SNPs anchored to each chromosome of the B genome in the 19 tested accessions of Sitopsis species and the B genome of Chinese Spring, with/without three outgroup species: B genome , allowinAe. speltoides ssp. speltoides and Ae. speltoides ssp. ligustica, and the other clade included Ae. longissima, Ae. sharonensis, Ae. bicornis, and Ae. searsii; the two clades corresponded to subsections Truncata and Emarginata, respectively. This result was consistent with the results of previous studies based on RFLPs and AFLPs.,,Ae. tauschii and Ae. umbellulata than the Truncata clade. The B genomes of T. aestivum and T. turgidum were closely related to Ae. speltoides ssp. speltoides and Ae. speltoides ssp. ligustica in the Truncata clade. The average number of nucleotide differences and fixed nucleotide differences between Ae. speltoides ssp. and the B genome were the lowest in pairwise comparisons between species of section Sitopsis and the B genome (Ae. speltoides. Considering that the wheat B genome was not nested within the Truncata clade, the most recent common ancestor of Ae. speltoides ssp. speltoides and Ae. speltoides ssp. ligustica is likely the direct donor of the wheat B genome.NJ and ML phylogenetic trees and a phylogenetic network tree were constructed based on the set of non-redundant SNPs with the outgroup species . The SitB genome . These rAe. speltoides were not clearly divided in the Truncata clade (Ae. speltoides is highly outcrossing.,Ae. speltoides contained a higher proportion of heterozygous loci compared with other self-pollinating species of Sitopsis.Ae. speltoides would harbour nucleotide variations as heterozygous states. The tested accessions of Ae. speltoides had been maintained by self-pollinating for several decades in the Japanese gene bank, which could have led to fixation of one of the alleles in heterozygous sites, increasing the number of singletons within the Ae. speltoides accessions. This resulted in detection of a relatively large number of SNPs in the Ae. speltoides accessions (Ae. searsii was monophyletic and separated from the other three species, whereas Ae. bicornis nested within Ae. sharonensis and Ae. longissima. Aegilops longissima diverged from Ae. sharonensis. The close relationship between Ae. sharonensis and Ae. longissima was consistent with previous reports,Ae. sharonensis is a subspecies of Ae. longissima.The two subspecies of ta clade . The Trucessions . In the 3.3.Ae. speltoides ssp. speltoides and Ae. speltoides ssp. ligustica, whereas the subspecies of Ae. longissima and Ae. sharonensis were distinguished. These subspecies are morphologically classified. Notably, differences in spike morphology between Ae. speltoides ssp. speltoides and Ae. speltoides ssp. ligustica can be explained by a single locus, Lig, located on chromosome 3S.Ae. speltoides ssp. speltoides and Ae. speltoides ssp. ligustica, 99 fixed nucleotide differences were detected (The phylogenetic tree did not discriminate well between subspecies of detected . The posdetected . Ten of detected .Ae. longissima and Ae. sharonensis, 121 fixed nucleotide differences were found (Ae. sharonensis from Ae. longissima, as Ae. longissima lacks a lateral awn.l, in which many fixed nucleotide differences accumulated, might control the morphologic and physiologic differences between Ae. sharonensis and Ae. longissima. Identification of the causal genes is a focus for future research.Between re found . All of re found , and there found . Lateral3.4.Ae. speltoides (Some previous studies suggested the possibility of introgression from several parental Sitopsis species, supporting a polyphyletic origin of the wheat B genome.eltoides . Two treeltoides . These iE-B − DT-B]/DT-B × 100 > 0 in We estimated the average number of nucleotide differences between the B genome of Chinese Spring and each of the Truncata and Emarginata clades as a parameter of genetic divergence between the B genome and each clade . In the ,,,In common wheat, the recombination rate and chromosomal gene density increase as the centromeric region recedes.Ae. tauschii and Ae. umbellulata.,In the present study, a large number of SNPs and indels were discovered in the wheat B genome and five Sitopsis species based on RNA sequencing of leaf-derived transcripts. The polymorphic data would be useful for developing genome-wide markers on the S-genome chromosomes as performed for the wild diploid relatives, Ae. speltoides. A few chromosomal regions demonstrated the clearly exceptional relationship between the B genome and Sitopsis species, whereas the irregular topology observed could be explained by higher recombination rates in the distal regions of wheat chromosomes. Therefore, based on genome-wide polymorphisms identified from the RNA sequencing data, all of the chromosomal regions of the wheat B genome could have originated from the S genome of Ae. speltoides. Moreover, the failure of pairing between homoeologous chromosomes between the B and S genomes during meiosis could be due to factors associated with highly evolved regions or intergenic regions. The alignment rate to B genome of Ae. speltoides was not as high as those of Ae. bicornis and Ae. searsii (In conclusion, the present phylogenetic analyses based on genome-wide polymorphisms suggest that the B genome of common wheat was derived from the S genome of  searsii . Of the  searsii  S1. PosiDRA007097. RNA sequencing data for the outgroup species were used in the DDBJ/GenBank/MMBL database: BioProject PRJDB4683 for Ae. tauschii KU-2075 and DRA006404 for Ae. umbellulata KU-4017.All read sequences were deposited into the DDBJ/GenBank/MMBL database with accession number Supplementary DataClick here for additional data file.


Phyllosticta citricarpa, responsible for citrus black spot, and Phyllosticta capitalensis, an endophytic species, are examples of closely related species with different behavior in citrus. Both species are always biologically associated and are morphologically very similar, and comparing their genomes could help understanding the different lifestyles. In this study, a comparison was carried to identify genetic differences that could help us to understand the biology of P. citricarpa and P. capitalensis.Citrus are among the most important crops in the world. However, there are many diseases that affect Citrus caused by different pathogens. Citrus also hosts many symbiotic microorganisms in a relationship that may be advantageous for both organisms. The fungi P. citricarpa and P. capitalensis, respectively. Even though the functional categories of these coding sequences is similar, enrichment analysis showed that the pathogenic species presents growth and development genes that may be necessary for the pathogenicity of P. citricarpa. On the other hand, family expansion analyses showed the plasticity of the genome of these species. Particular families are expanded in the genome of an ancestor of P. capitalensis and a recent expansion can also be detected among this species. Additionally, evolution could be driven by environmental cues in P. citricarpa.Drafts genomes were assembled with sizes close to 33 Mb for both fungi, carrying 15,206 and 14,797 coding sequences for P. citricarpa and P. capitalensis. Although the idea that these differences could explain the different lifestyles of these fungi, we were not able to confirm this hypothesis. Genome evolution seems to be of real importance among the Phyllosticta isolates and it is leading to different biological characteristics of these species.This work demonstrated genomic differences between The online version of this article (10.1186/s12864-019-5911-y) contains supplementary material, which is available to authorized users. Phyllosticta citricarpa McAlpine (Synonym: Guignardia citricarpa Kiely) is the fungus responsible for causing citrus black spot (CBS), which is one of the most important diseases affecting the citrus industry worldwide [orldwide , 2. Thisorldwide . Since torldwide , 2, 4–6.CBS affects almost all commercial varieties of citrus with the main symptom associated with this disease being the development of hard spot lesions in the fruit peel . AdvancePhyllosticta are plant pathogens of a wide range of hosts [P. citricarpa is pathogenic, other endophytic and saprophytic species have also been reported for citrus [P. capitalensis is among the endophytic species that lives within citrus and other hosts with a wide geographic distribution [P. capitalensis is commonly found to be associated with lesions in plants; from an economic point of view, this association can be very negative since P. capitalensis is normally confused with the pathogen that is actually responsible for causing the disease. This is an even worse problem if the actual pathogen is a quarantine organism like P. citricarpa that has phytosanitary restrictions, being classified as quarantine A1 in the European Union and A2 in the United States [P. citricarpa and P. capitalensis is so close that CBS was erroneously reported in New Zealand [Most of the species in the genus of hosts , 11 and r citrus –15. P. cribution , 16, 17.d States , 18. The Zealand , with th Zealand . Due to  Zealand , 22.P. citricarpa was compared to the genome of P. capitalensis, the endophytic species, in order to identify genetic differences that could help understand their different lifestyles.In recent years, the number of works using comparative genomic analysis to understand the genetic basis of the lifestyle of pathogenic and endophytic or symbiont organisms has grown considerably –26. In tP. citricarpa and P. capitalensis genomes generated a total of 179,880,616 and 148,831,020 paired-end reads, respectively, with more than 90% showing Phred quality > 20 (Q20) for both species. The CG content was slightly lower for P. citricarpa (48.72) compared to P. capitalensis (51.43).The Illumina sequencing of P. citricarpa and N50 of 3049. The assembly of P. capitalensis reads resulted in 11,080 contigs and 33.2 Mb with a N50 of 4925. A Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the BioProject number PRJNA486917.A de novo assembly was done for both genomes, generating drafts of 19,143 contigs in 32.6 Mb for Botrytis cinerea, an ascomycete necrotrophic plant pathogen with a broad host range [P. citricarpa, 15,206 proteins were identified, while for P. capitalensis, the total was 14,797. These proteins were annotated using blastp [https://www.ncbi.nlm.nih.gov/protein/) .The contigs of both fungi were used for the prediction of coding sequences using st range  as a refst range . For P. g blastp  to find P. citricarpa and P. capitalensis showed the same pattern for most of the subcategories, with a few additional genes in the latter compared to the former, there were some differences that could be representative for understanding the behavior of both organisms . The phylogenetic tree obtained using the 3185 single copy-ortholog genes . LysM domain proteins bind chitin and are important for fungus defense against the host response [P. citricarpa, as known for other filamentous fungi [In the other clade, fewer families were expanded and besides, most of them encodes uncharacterized proteins Fig. . Howeverresponse . Becauseus fungi .P. citricarpa and P. capitalensis are distant species as previously reported. Therefore, the changes observed in their genomes may result from the evolution of the species, including gain and loss of genes. Even distant, a comparative genomics analysis is appealing because of the different lifestyle and it was carried out to identify genes exclusively found in the genomes of P. citricarpa LGMF06 and P. capitalensis LGMF01. A reciprocal blastp analysis was carried out using the protein sequences encoded in both genomes. The exclusive proteins were considered those that either had a positive hit with an E-value above 1 e− 30 or resulted in a “no hit”. These proteins were reciprocally re-evaluated in blastp using the proteomes from P. citricarpa CBS141350 and P. capitalensis CBS128856 to make sure that the differences were not a problem resulting from the incompleteness of the genomes .To identify putative genes involved in pathogenicity, we analyzed the P. citricarpa , signaling, detoxification systems, virulence factors and transcription factors were identified , CEs (carbohydrate esterases), GHs (glycoside hydrolases), GTs (glycosyl transferases) and AAs (auxiliary activities) families , and 3 of the GH family  . These findings seem to be related to the evolution of the species detected through the genome analyses but drivers are acting in the genome of the different species that could leads to the formation of new species in the future.There are many different genomes sequenced for P. citricarpa (LGMF06) was isolated from CBS lesions in fruits from a plant grown in the State of São Paulo, Brazil, and tested for pathogenicity according to [P. capitalensis (LGMF01) was isolated from asymptomatic fruits from a plant also grown in the State of São Paulo, Brazil. These fungi were grown in liquid Citrus Medium Fabris-Nishimura (CFN) [The fungi isolates used for genome sequencing are from the biological bank of the Laboratory of Microorganisms (LabGeM) at the Federal University of Paraná, Curitiba, Paraná, Brazil. The pathogenic species rding to . The endra (CFN)  and incura (CFN) .P. citricarpa and P. capitalensis) was sent to Macrogen Inc. (South Korea) for sequencing using the HiSeq 2000 platform (Illumina Inc.). All procedures were performed according to Illumina’s protocols. Paired-end fragments were generated with 101 base pairs (bp). One lane was used per library. The results were sent in fastq format. De novo assembly was carried out using CLC Genomics Workbench software (https://www.qiagenbioinformatics.com/products/clc-genomics-workbench/) with default parameters.A total of 10 μg of DNA from each fungus (Botrytis cinerea (Botryotinia fuckeliana) as the model species. The annotation and categorization of ORFs were automatically carried out using Blast2GO with an E-value cut-off ≤10− 6 [The prediction of open read frames (ORFs) was conducted using Augustus software v3.2  with Botf ≤10− 6 . The toof ≤10− 6 . The comf ≤10− 6  softwareP. capitalensis strains: CBS128856, Gm33, LGMF01; P. citricarpa strains: CBS141350, Gc12, LGMF06, P. citribraziliensis strain CBS100098, P. citrichinaensis strain CBS130529), in total, we used 3.185 single copy-ortholog genes identified with OrthoFinder [To reconstruct a phylogenetic hypothesis for nine Phyllosticta species  using blastp. Once again an E value filter (1e-50) was used and only the sequences showing results below the cutoff were kept for further analyses, being considered as the exclusive sets for each species.Comparative genomics analysis between of BLAST . InitialP. citricarpa and P. capitalensis, exclusive gene sequences for each, were used to investigate whether they had been verified to be pathogenic genes using annotations from the PHI-base using blastp  [The < 0.001) .The CAZymes are involved in plant cell wall degradation. For this reason, the genes of both fungi, pathogenic and endophytic, were subjected to CAZy annotation using dbCAN , which iTo verify which proteins are secreted the following pipeline was used: the secreted proteins were identified using SignalP 3.0 , which dAdditional file 1:P. citricarpa proteins. The proteins obtained after de novo assembly of P. citricarpa genome were used for coding sequences prediction using Botrytis cinerea as reference in Augustus, when 15,206 proteins were obtained. (TXT 5151 kb)Sequences of Additional file 2:P. capitalensis transcripts. The proteins obtained after de novo assembly of P. capitalensis were used for proteins prediction using Botrytis cinerea as reference in Augustus, when 14,797coding sequences were obtained. (TXT 5895 kb)Sequences of Additional file 3:P. capitalensis LGMF01 was C: 65.2%  and P. citricarpa LGMF06 was C:46.9% . The corresponding protein-level benchmarking value was C: 57.5%  and C: 40.0%  and single-copy (S) genes; dark-blue: complete and duplicated genes (D); yellow: fragmented genes (F); red: missing genes (M) and n: total BUSCO groups for searching. P. citriasiana was used with genome control. (PDF 592 kb)BUSCO assessment of genomes from Phyllosticta. The genome-level benchmarking value of Additional file 4:Comparative analysis of gene gain and loss rates. Comparative analysis of gene gain and loss rates is represented in relation to different branches from the pathogenic and endophytic Phyllosticta species. (XLSX 41 kb)Additional file 5:P. citricarpa. Unique proteins identified after comparison of the P. citricarpa and P. capitalensis sequences by Blastp. (FA 654 kb)Unique sequences of proteins of Additional file 6:P. capitalensis. Unique proteins identified after comparison of the P. capitalensis and P. citricarpa sequences by Blastp. (FA 992 kb)Unique proteins of genes of Additional file 7:P. citricarpa. List of P. citricarpa exclusive proteins displaying similarity with subjects in the pathogen-host interaction gene database (PHI-base). (XLSX 18 kb)Pathogenicity proteins of Additional file 8:P. capitalensis. List of P. capitalensis exclusive proteins displaying similarity with subjects in the pathogen-host interaction gene database (PHI-base). (XLSX 22 kb)Pathogenicity proteins of Additional file 9:P. citricarpa and P. capitalensis. List of exclusive and common pathogenicity proteins identified after comparison of P. citricarpa and P. capitalensis. (XLSX 18 kb)Comparison of the pathogenicity proteins of Additional file 10:P. citricarpa and P. capitalensis. List of secreted CAZymes identified among the exclusive proteins of each species. (XLSX 9 kb)Secreted CAZymes by