Venturia inaequalis, is one of the world’s most commercially significant apple diseases. The fungi have a catastrophic impact on apples, causing considerable losses in fruit quality and productivity in many apple-growing locations despite numerous control agents. Fungi secrete various effectors and other virulence-associated proteins that suppress or alter the host’s immune system, and several such proteins were discovered in this work. Using state-of-the-art bioinformatics techniques, we examined the V. inaequalis reference genome (EU-B04), resulting in the identification of 647 secreted proteins, of which 328 were classified as small secreted proteins (SSPs), with 76.52% of SSPs identified as anticipated effector proteins. The more prevalent CAZyme proteins were the enzymes engaged in plant cell wall disintegration (targeting pectin and xylanase), adhesion and penetration (Cutinases/acetyl xylan esterase), and reactive oxygen species formation (multicopper oxidases). Furthermore, members of the S9 prolyl oligopeptidase family were identified as the most abundant host defense peptidases. Several known effector proteins were discovered to be expressed during the V. inaequalis infection process on apple leaves. The present study provides valuable data that can be used to develop new strategies for controlling apple scab.Apple scab, caused by  Venturia inaequalis Cooke (Wint.) is one of the most devastating diseases of apples. The fungus infects the leaves, fruits, branches, petioles, and stems, but leaves and fruits are the most affected parts of the plant [Apple scab by he plant . In sevehe plant . Differehe plant . This rahe plant ; howeverhe plant ,6. ThereV. inaequalis is a semi-biotropic fungus that survives the winter as pseudothecia on rotting leaves [V. inaequalis [g leaves . Crossovg leaves . The fung leaves . Severalaequalis ,10. High2O2 homostat, protein stability, ubiquitin proteasome, sRNA silencing machinery, and plant hormones [Venturia apple pathosystem [Plants have a sophisticated innate immune system to keep pathogens away . Howeverhormones . Howeverhosystem .V. inaequalis. Changes in phytohormone levels have been noted during the infection process [V. inaequalis [V. inaequalis, leading to the identification of many secreted proteins associated with virulence [Venturia genome has been sequenced and annotated from different isolates in varied geographic regions of the world [V. inaequalis and V. pirina genomes and predicted several effectors associated with pathogenesis [Venturia and also reported the genome sequence of V. inaequalis reference genome (EU-B04) using PacBio technology [V. inaequalis during the apple leaf infection process. Therefore, the present study will help us to better understand the mechanisms implemented by V. inaequalis during apple colonization.A number of virulence-associated proteins have been identified and characterized in  process . Interesaequalis ,19. Advairulence ,21. The he world ,23,24,25We used RepeatModeler2  with defhttps://v100.orthodb.org/download/odb10_fungi_fasta.tar.gz, accessed on 16 February 2022) as a resource of benchmark protein sequences to train GeneMark-EP+ [Venturia inaequalis as a binomial name, were utilized to train Augustus to produce the final gene models. BUSCO was then used to check the completeness of predicted gene models using the dataset ascomycota_odb10 (10 September 2020).Gene models were inferred using BRAKER2 . The iniMark-EP+ . FinallyA well-known protein sorting signal, the secretory signal peptide, was discovered using a combination of SignalP-6.0  and PhobV. inaequalis secretome.To predict subcellular protein localization, the BUSCA program was used . The proEffectorP version 3.0 , a machiGene annotation was performed with default settings using Funannotate v1.8.10. Annotation of functional domains was conducted using EggNOG-mapper v5, InterProScan v5, and HMMer scans against the Pfam-A database. Carbohydrate-activating enzymes (CAZymes) and peptidases were identified using the dbCAN CAZYmes database and the MEROPS protease database. BlastP searches were also performed against the Uniport/Swissport and NCBI non-redundant (NR) and Refseq databases.V. inaequalis were obtained from the Kashmir Valley cultivar Red Delicious. Single spore isolation was carried out, and cultures were grown on a potato dextrose agar (PDA) medium. Mycelium harvested from PDA plates was immediately stored in liquid nitrogen and stored at −80 °C until further use. Mature leaves were cut from the apple plant, surface sterilized and placed in the correct orientation on moist paper in a Petri dish. V. inaequalis mycelium was washed with distilled water and placed onto cut apple leaves [Isolates of e leaves . After 5® Fluorometer. Next-generation transcriptome sequencing was performed on the Illumina HiSeq 2500 platform.Total RNA isolation was performed using the modified Trizol method . QualityV. inaequalis genome, we used the genomic sequence of the EU-B04 genome and the required GTF/GFF from the BRAKER2 study. Proper indices and FASTQ read alignments were generated using STAR (v2.6.1b), and the resulting SAM files were sorted by coordinate using samtools (v1.14) and exported to BAM format for further processing. A count matrix was generated with HTSeq software (v1.99.2) and the resulting counting data was used for differential expression analysis. The trimmed mean method was used for statistical modelling of the count data using the R computing environment (4.1.2) and the Bioconductor package DESeq2 (v1.20).We used the Fastp tool  to removV. inaequalis genome is expected to be around 100 MB in size [V. inaequalis assembly sizes ranging from 40 Mb to 72.3 Mb [The  in size . Various 72.3 Mb ,23,24. L 72.3 Mb .V. inaequalis has received substantial research as a model for infections on woody hosts. There are several genome sequences available for V. inaequalis, and researchers determined that pathogenic strains of this model have a higher repeat content than other species, with repeat regions occupying up to 47% of their genome sequence [sequence ,24.V. inaequalis genome belongs to a non-repetitive region containing all biological genes and other regulatory sequences.We employed RepeatModeler2 to annotate transposable elements (TE) in the EU-B04 genome assembly. The approach overcomes several drawbacks of the previous RepeatModeler method, notably the lack of a comprehensive, nonredundant library of full-length consensus sequences . We alsoV. inaequalis, the number of gene models in V. inaequalis isolates ranges from 11,600 to 13,761 [V. inaequalis, we were able to predict 1363 additional genes. In total, the BRAKER2 pipeline identified 12,970 gene models in the EU-B04 genome. The quality of the gene prediction was evaluated by comparing universal single-copy orthologs (BUSCO) with the available Ascomycota dataset. According to the BUSCO results, the predicted genetic model represents most of the genome, with a BUSCO completeness score of 99.1% (86.1% single-copy complete genes and 13.0% duplicated complete genes), with only 0.2% fragmented and 0.7% missing genes, while Le Cam et al. reported 98.47% completeness in their study [According to the published reports on o 13,761 ,24,25. UV. inaequalis proteome  was examined for secretory proteins, SignalP-6.0 results showed that a total of 1334 proteins contain signal peptides. Among them, TMHMM2.0 identified 245 proteins as having transmembrane signaling, while the remaining 1089 proteins were chosen for further investigation. The Phobius program which predicts both signal peptides and transmembrane topology discovered 1503 non-transmembrane classical secreted proteins in V. inaequalis. Finally, the 1046 non-transmembrane signaling proteins identified as common predictors by SignalP/TMHMM and Phobius were selected and considered to be released via the classical secretory pathway. To predict unconventionally secreted proteins, we employed the SecretomeP and OutCyte prediction algorithms. Unlike SecretomeP, which utilizes conventional proteins by eliminating signal peptides, the OutCyte module (OutCyte-UPS) uses an experimental secretome data source to detect potential unconventional protein secretions (UPS) using the characteristics obtained directly from protein sequences. Outcyte detected 1233 proteins secreted via non-canonical pathways, while SecretomeP identified 5723 proteins. Both SecretomeP and Outcyte predicted 549 proteins as common non-classical secreted proteins. After combining classical (1046) and non-classical (549) proteins, a total of 1595 proteins were predicted as secreted proteins. Deng and colleagues [V. inaequalis isolates. Despite the fact that the bioinformatics tools used in this work are more in number and newer   was obtained by filtering GPI-anchored proteins (17) that facilitate protein binding to the outer face of the lipid bilayer [When the entire alP 6.0) , they yiIn the secretome, several small secreted proteins (SSPs) are known to act as virulence factors involved in disease development or as avirulent factors that induce resistance (R) gene-mediated protection. SSPs were defined as secreted proteins with a maximum length of 300 amino acids. A total of 328 proteins representing 50.69% of the secretome were identified as SSPs. Researchers have identified several potential small secreted proteins known as effectors that contribute to virulence. These effectors develop a wide range of plant microbial associations, ranging from beneficial to harmful. A total of 225 proteins (68.59%) from SSP were recognized as effectors by EffectorP 3.0. Of these, 39 showed homology to known effectors available in the PHI-base. In addition, 26 SSPs identified as non-effectors by EffectorP showed homology to known effectors in PHI-base. These 26 proteins were added to the final effector list recognized by EffectorP, bringing the number of effectors to 251. Localization analysis showed that 176 effectors  are apoplastic in nature, although 18.18% of them could also target the host cytoplasm . The longest secretome protein was approximately 918 amino acids (aa) and the smallest was 67 aa. The shortest protein lengths of SSP and effector were also the same (67 aa), while the longest protein lengths of SSP and effector were 300 and 299 aa. The average length was 294, 174, and 165 aa in the secretome, SSP, and effectome, respectively .V. inaequalis genome, GH predominated (222), followed by AA (95) and GT (94) in approximately equal numbers. CE was the third-abundant class (88) in CAZymes. PL and CBM also showed minimal presence . GH predominated in the secretome (79) as well, followed by AA (46) and CE (33). The GH family plays an important role in pathogen infection and host colonization. Secretome analysis of V. inaequalis revealed the presence of several GH families , GMC oxidoreductase (AA3), and AA9 (endoglucanase activity) showed similar abundance (15.21%), although three GMC oxidoreductases with combined AA3 and AA8 domains were also present in the secretome. Secretome analysis also revealed the presence of PL genes such as PL1(2), PL3(1), and PL4(4).The GH43 family included known enzymes with β-xylosidase, α-L-arabinofuranosidase, arabinanase, and xylanase activities, whereas the GH28 family included polygalacturonase and rhamnogalacturonase. The enzymes of the GH28 family promote host colonization . GH28 porown rot . A GH3 frown rot . AA1 domV. inaequalis secretome. Furthermore, among the identified peptidases, 9.09% were asparagine peptidases, 6.81% were metallopeptidases, and 2.27% were threonine peptidases. In addition, a single copy of a serine carboxypeptidase Y inhibitor (phosphatidylethanolamine-binding protein) was also identified. Deng and coworkers [V. inaequalis isolates. Carboxylesterase (S09X) was the most prominent protease in the genome and secretome of V. inaequalis. S33 and C19, the second and third most abundant peptidases in the V. inaequalis genome showed no dominance in the secretome. C19 was completely absent from the secretome, but there was one copy of the alpha/beta hydrolase enzyme from the S33 family. A subtilase family (S08A), a serine carboxypeptidase (S10), and an endopeptidase with a prokumamolisin activation domain (S53) were other predominant peptidases identified in the V. inaequalis secretome. In addition, small numbers of gamma-glutamyl transpeptidase and aspartyl protease were also detected.Peptidases are known to play a role as virulence factors in fungal infections, and their participation in pathogenicity has been described in various fungal pathogens . In the oworkers  also repV. inaequalis effectome revealed the presence of 21 CAZymes. Of these, 42.85% were CE, 23.80% AA, and 19.04% GH. We also observed the presence of one PL (PL3) and two CBMs: CBM50 (LysM domain) and CBM63 (Lytic transglycolase). The LysM protein has been found to scavenge chitin oligosaccharides from fungal pathogens and evade host defense responses [S. scitamineum, a fungal pathogen that causes smut in sugarcane [V. inaequalis may be releasing these virulence factors to counteract it. Members of the GH11 family have also been identified as virulence factors in Botrytis cinerea, and its mutants have shown reduced virulence [Of the predicted effectors, functional annotation was available for only 23.90% of effector proteins. Functional characterization of the esponses . Of the ugarcane ,54. It birulence . The GH1irulence . FurtherV. inaequalis secretome. The CFEM domain, a conserved eight-cysteine sequence, is found only in fungi, but not in all fungi, and has been implicated in multiple aspects of virulence [Magnaporthe oryzae, has been reported to be required for pathogenesis, and the proper formation of appressorium and appressorium-like structures [Alternaria alternata AltA1-like effector was observed in the V. inaequalis secretome. AltA1 is implicated in fungal pathogenesis and has been reported to act as a virulence factor which blocks multiple plant defense mechanisms and promotes fungal infection [V. inaequalis secretome. This domain has been reported to play a role in stress response or resistance to stress, especially to toxicants [Verticillium longisporum targets the host nucleus and suppresses host PTI defense responses. Moreover, a necrosis-inducing effector protein (NPP1) was also identified in the V. inaequalis secretome. These proteins are widely distributed in different taxa of plant pathogens and act as virulence factors for necrotic or semi-biotrophic plant pathogens [Five genes encoding CFEM  effector proteins were identified in the irulence . The CFEructures . An Altenfection . Egh16-lnfection . Effectooxicants ,62. Effeoxicants  reportedathogens . V. inaequalis infection of apple leaves. Subtilase, the best-known serine protease important for critical processes such as immunity, symbiosis and plant-programmed cell death, showed a 10.22-fold increase in expression levels. Studies have shown that pathogens secreting subtilisin inhibitors can indirectly prevent immune protease activation [V. inaequalis might be increasing the tryptophan synthase expression to reduce the host immune response. Gene expression of the RsRlpA effector was also found to be upregulated 6.59-fold. RlpA-like proteins are protease inhibitors that promote virulence by suppressing hypersensitivity reactions [V. inaequalis. Annotated effectors such as cutinase/acetyl xylan esterase, laccase 3, phospholipase A2, TAP-like protein, amidohydrolase, hydrophobic surface binding protein A (HsbA), CHRD domain, domain of unknown function (DUF4360), and CFEM domain-containing effectors were all differentially expressed and down-regulated. In addition, several other peptidases and CAZymes were also downregulated  required for the tivation . Cellulativation . We hypoeactions . Furtheregulated . V. inaequalis, a fungal pathogen responsible for apple scab disease. A comprehensive study of the V. inaequalis genome, transcriptome, and secretome has identified several extracellular enzyme systems present in this pathogen which should aid in the search for antifungal or plant disease-resistant ingredients active against this pathogen. This study sheds light on the diversity of plant cell-wall-degrading enzymes in this fungus that may be crucial for its interaction with host. The vast majority of proteins in the V. inaequalis secretome repertoire were hypothetical proteins. More research is needed to functionally characterize them, which will aid in the discovery of additional virulence determinants of this pathogen, and understanding their role in the infection process will undoubtedly provide new insight into the underappreciated intricacy of host colonization by this plant pathogen.The present study analyzed the secretome architecture of


Helicobacter pylori is intensively associated with diverse gastric diseases, including gastric ulcers, chronic progressive gastritis, and gastric cancer. Aside from its pathogenic effect on gastric diseases, growing evidences reveal that H. pylori may be related to numerous extragastric diseases. In this article, we reviewed recent studies and systematically elucidated that H. pylori may interfere with many biological processes outside the stomach and influence the occurrence of various extragastric diseases. Many epidemiological studies have indicated that H. pylori plays a pathogenic role in COVID-19, atherosclerosis, hyperemesis gravidarum and several other extragastric diseases, while the effect of H. pylori is currently under investigation in gastroesophageal reflux disease, asthma, and inflammatory bowel disease. Moreover, we also summarized the possible pathogenic mechanisms of H. pylori that may be related to chronic systemic inflammation and molecular mimicker. Taken together, this review provides a new perspective on the role of H. pylori in extragastric diseases and explores the possible mechanisms, which may help guide clinical treatment.Considered as the most popular pathogen worldwide,  Helicobacter pylori is recognized as the most popular human pathogen, which infects nearly half of the population worldwide (approximately 4.4 billion people) , and mucosa-associated lymphoid tissue (MALT) lymphoma  . Exposurlymphoma . The Worcinogens . Previouce of GC  and atroce of GC . Managemce of GC  recommenH. pylori infection may be related to numerous extragastric diseases of various systems throughout the human body in addition to the pathogenetic effects on gastric diseases. For example, H. pylori has been described to be related to some blood system diseases. A separate meta-analysis of 15 observational studies proved that iron deficiency anemia (IDA) was more common among H. pylori-positive individuals than H. pylori-negative controls   . H. pylofrom IDA . In the analysis . Some enpatients . Apart f systems . Moreovece, etc. . Similarclerosis . The latpractice .H. pylori and many extragastric diseases, which is necessary to refine the understanding of the pathogenic processes of H. pylori and help improve clinical prognosis and guide management. We reviewed latest studies and found that H. pylori may be associated with several extragastric diseases of various systems throughout the human body. In addition, we also explored the promising pathogenic mechanisms of H. pylori infection. Ultimately, we sought to improve and refine clinical guidelines and benefit patients suffering from the mentioned extragastric diseases and H. pylori infection.In this article, we aim to elucidate the correlation of H. pylori infection with asthma has attracted extensive attention. For example, H. pylori had a protective effect on allergic asthma by regulating Thl7/Tregs and the Th1/Th2 balance, reducing HSP70 and DCs, stimulating TLRs, and inhibiting gastroesophageal reflux. There are three well-known hypotheses related to the pathogenesis, including the gut-lung axis theory, the “disappearing microbiota” hypothesis and the hygiene hypothesis, all of them supporting the protective effect of H. pylori. In addition, therapeutic products made by H. pylori (such as H. pylori extract) have also been utilized to treat and prevent asthma. Perinatal H. pylori exposure reduced inflammation of the allergic airway in the offspring as well, providing a promising target for interventional therapy of asthma (H. pylori can modulate anti-Th2 inflammation activity through neutrophil-activating protein (NAP) and contribute to allergic asthma, and purifying rNAP before sensitization can significantly reduce the accumulation of eosinophils in the lung tissue of asthmatic mice. It is worth noting that H. pylori treatment decreases the levels of IL-4, IL-13, and serological IgE, and increases the levels of IL-10 and IFN-γ . H. pylori-positive subjects showed a lower FEV1 (L) at baseline than H. pylori-negative patients, although no significant discrepancy in the decline rate between the two groups  was shown  and GOLD 2 severity , which is a serious complication threatening numerous COVID-19 patients  are found in lung biopsy specimens and bronchoalveolar lavage fluid of lung cancer. These proteins can aggravate the progress of airway diseases, promote the H. pylori infection inflammatory status (anti-H. pylori IgG and IgM) and recruit B cells, and finally accelerate the occurrence of lung cancer. Besides, eradication of H. pylori was significantly correlated with the decrease of lung cancer marker CEA. This explained that H. pylori may be of benefit for the treatment of lung cancer (H. pylori strain specific blood biomarkers and lung cancer risk (PLCO2019-1026), which may help understand of H. pylori infection and lung cancer risk, identify markers for lung cancer risk, and provide new information for a feasible cancer prevention strategy.Studies have found that g cancer . Of concH. pylori infection and respiratory diseases, further studies are necessary to confirm a causal relationship. Moreover, the roles of other risk factors, such as air pollution or smoking habits, as well as the latent molecular mechanisms should also be considered  and prostaglandin to induce platelet aggregation. In addition, H. pylori releases many cytokines, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-1 and free radicals, causing atherosclerosis and oxidative stress. Furthermore, an aberrant immune reaction is considered to play a role in atherosclerotic plaque rupture and destabilization by the cross-reactivity between antibodies and CagA vascular wall antigens  is the most dire and serious outcome for patients with CAD due to its fatal influence on survival quality. A meta-analysis including more than 20,000 subjects and 26 studies found that icipants .H. pylori may also significantly increase the risk of carotid atherosclerosis in Chinese men under 50 years old . Compared with individuals without H. pylori infection, infected subjects showed a 0.735 mmHg increase in diastolic blood pressure (95% CI = 0.101–1.369) and a 0.723 mmHg increase in mean arterial pressure (95% CI = 0.034–1.413)  .H. pylori infection is one of the protective factors against EoE. However, in 2018, a prospective case–control study conducted in 23 centers reported that H. pylori was not negatively associated with EoE, neither in adults nor in children (H. pylori infection on EoE still needs further study.Eosinophilic esophagitis (EoE) is a kind of disease mediated by the immune response. A meta-analysis by children . Thus, tH. pylori infection contributes to the incidence of esophageal squamous cell carcinoma. A meta-analysis of 35 studies with 345,886 participants indicated that there was no crucial association between esophageal squamous cell carcinoma and H. pylori infection . An analysis of GERD patients found a higher prevalence of H. pylori infection among patients with peptic ulcers  and tionship .H. pylori infected gallbladder than the control group in 17 studies .Non-alcoholic fatty liver disease is a kind of liver injury that is induced by metabolic stress. A meta-analysis of 21 studies indicated that pulation , but H. in China . One hypipidemia . NotablyHelicobacter pylori infection might play a protective role in inflammatory bowel disease (IBD) reported by a meta-analysis -related chronic hepatitis. A multivariate analysis further indicated that positive anti-H. pylori antibody was independently and significantly related to cirrhosis in individuals with HCV-related chronic hepatitis  may lead to the proliferation of some harmful bacteria and damage gut barrier function. This also causes the immune imbalance and mediates several chronic inflammatory diseases mentioned above  , t  , and t  , activating NF-κB signaling and regulating apoptosis, inflammation, and immunity .It has been reported that the lack of vitamin B12 absorption contributes to pernicious anemia and sorption . In addisorption . BesidesH. pylori in Idiopathic or Immune Thrombocytopenic Purpura (ITP) has also been investigated. A meta-analysis of six studies involving 241 patients proved that H. pylori eradication is an effective treatment for ITP patients  were 1.19 (95% CI = 0.98–1.45), 1.43 (95% CI = 1.11–1.85) and 1.17 (95% CI = 0.94–1.45), respectively, indicating that H. pylori-infected individuals would have a higher risk of diabetes. According to an analysis of geographical subpopulation regions, the infection risk of H. pylori in the Asian population was slightly higher than that in other populations .Diabetes is the most prevalent metabolic disorder worldwide, killing approximately four million people each year. A meta-analysis of 9,559 individuals found that the effects of ulations . In cont females . MoreoveH. pylori on obesity or overweight is still unclear. A meta-analysis including 22 articles and 178,033 samples showed that obesity was associated with H. pylori, which may increase the risk of obesity (OR = 1.2) (H. pylori was not related to obesity or overweight observed in a Chinese population (P = 0.321)  . However= 0.321) . More inH. pylori with autoimmune thyroid diseases (AITDs) also needs more research to clarify. A meta-analysis of 15 articles that included 3,046 cases showed that H. pylori was positively correlated with HT and GD , and CagA+ H. pylori was positively related to AITD  (The relation of  = 1.99) . Neverth = 1.99) .H. pylori infection. H. pylori seropositivity in their retrospective cohort study that included 16,970 participants. In addition, a systematic study also revealed that AD may be associated with gastrointestinal microbiota dominated by H. pylori (Alzheimer’s disease (AD), as a kind of nerve disease characterized by neurodegeneration, has also been studied for a possible association with . pylori .H. pylori eradication was found to intensively improve the clinical symptoms of PD in a prospective cohort study. H. pylori eradication not only increased the normal motor function time  of the day, but also improved gastrointestinal symptoms and reduced fatigue symptoms  and severer motor symptoms in PD patients (Parkinson’s disease (PD) is the second most common neurodegenerative disorder in the world. Although the pathogenesis of PD remains unclear, symptoms . Anotherpatients . A meta-patients .H. pylori was related to the etiology of restless legs syndrome (RLS). Proinflammatory cytokines released by H. pylori infection, such as IL-6, have been shown to increase production of hepcidin, which affects iron transport in healthy human, resulting in an iron deficiency in the CNS and causing RLS  but negative in patients with MS (H. pylori may be a protective factor by manipulating pattern-recognition receptors (PRRs)  is the complex interaction of environmental and genetic factors. Bacterial exposure has been identified as one of the many pathogenic factors of MS . As show with MS . The abos (PRRs)  and inhis (PRRs) . A recens (PRRs) . Aside fnal axis .H. pylori antibodies, especially in cerebrospinal fluid, suggesting that H. pylori is significant in GBS pathophysiology (Guillain–Barré syndrome (GBS) is a serious peripheral nerve autoimmune demyelinating disease that often occurs after bacterial infection. A meta-analysis revealed that there was an intensive relationship between GBS and ysiology .H. pylori infection was associated with non-heterogeneous glaucoma  reduction in intraocular pressure (IOP) was found after 2 months of follow-up, showing that H. pylori eradication may be positive in glaucoma therapy  patients   . Howeversoriasis . Thus, mH. pylori was related to the rosacea process  nephropathy, membranous nephropathy, Henoch–Schonlein purpura nephritis, diabetic nephropathy and other urinary diseases  reacted with the equatorial segment and the flagella  of sperm  is characterized by excessive vomiting and severe nausea that begins before the end of 22 weeks of pregnancy . A studysymptoms .H. pylori ureA gene-positive laryngeal cancer, 46.7–49.3% of 75 were also CagA positive. The CagA gene in laryngeal cancer greatly reduced the survival rate and increased the possibility of recurrence  is regarded as a recurrent painful ulcerative disease that regularly impacts mucosa in the oral cavity. H. pylori, indicating that eradication of H. pylori might relieve RAS symptoms and is a promising RAS therapy.Some studies revealed an association between oral diseases and tal pulp . The preH. pylori eradication is not quite explicit. A meta-analysis that included three RCTs, two review articles, one cohort study, four prospective studies, and eight cross-sectional studies found that individuals with functional dyspepsia who did not improve after H. pylori eradication   . A cohor not men .H. pylori infection was mostly considered as a risk factor for gastric disorders. However, growing evidences show that H. pylori infection presents more complexity and tends to be associated with almost every system in the human body. From our perspective, H. pylori can produce many kinds of bacterial toxins and induce numerous extragastric diseases in the human body, such as asthma, COPD, ITP and psoriasis. We summarized these diseases in terms of the human system, listed them methodically in this article and showed a schematic diagram (H. pylori infection. We synthesized the results of these studies and proposed two promising hypotheses. (i) Since H. pylori can induce several inflammatory factors, such as IL-1/2/6/8/10, TNF-α and IFN-γ, these factors may lead to chronic low-level systemic inflammation in the human body and ultimately represent diseases. Typical disorders due to H. pylori-induced inflammatory factor turbulence include atherosclerosis, insulin resistance, blood-brain barrier damage, brain neurodegenerative disease and decreased sperm motility ; and cross-antigen reactivity between spermatogenesis related proteins, sperm motility related proteins and H. pylori contributes to hypomotility of sperm  H.of sperm . In a woman body .H. pylori-related extragastric diseases in most studies is generally insufficient. Larger sample sizes and broader clinical trials are beneficial to decipher the correlation between various clinical diseases and H. pylori, and the control of confounding factors is necessary. In addition, the pathogenic effect of H. pylori in some extragastric diseases, such as gastroesophageal reflux disease, asthma, and IBD, are still controversial (H. pylori may have a certain protective effect on some diseases (such as GERD) (H. pylori infection model based on specific inflammatory markers (such as CRP and PLR) and the study of inflammatory-activated pathways are of great significance to reveal the systemic effects of H. pylori. Furthermore, there is high heterogeneity in the research on the relationship between H. pylori and the development of autoimmune diseases, and the differences of their distribution patterns make the research results controversial. At present, it is found that H. pylori infection may increase susceptibility to autoimmune diseases by stimulating cell damage, chronic inflammatory, and polyclonal lymphocyte activation  . And mosas GERD) . Therefotivation . Aside foutcomes . Thus, ion means .Helicobacter pylori infection can induce several extragastric diseases through many pathways, and different types of H. pylori may contribute to different kinds of diseases because of their specific bacterial toxins and pathogenies. It is generally accepted that the systemic effects of H. pylori infection should not be neglected. Although H. pylori has been discovered over more than 100 years ago, many aspects of H. pylori still need further studies. For clinical practitioners, the impact of H. pylori infection on extragastric diseases should be taken into more consideration.


Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common opportunistic intestinal pathogens in humans. In China, despite the number of HIV/AIDS patients being extremely large, only a few studies have investigated opportunistic infections caused by intestinal pathogens in this patient population. The aims of this study were to elucidate the occurrence and genetic characteristics of Cryptosporidium spp., G. duodenalis, and E. bieneusi in HIV/AIDS patients.Opportunistic infections are a ubiquitous complication in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients. Cryptosporidium spp.; the triosephosphate isomerase, β-giardin and glutamate dehydrogenase genes for G. duodenalis; and the internal transcribed spacer region of the rRNA gene for E. bieneusi. The Cryptosporidium-positive specimens were further subtyped by polymerase chain reacion and sequencing of the 60-kDa glycoprotein gene.We collected fecal specimens from 155 HIV/AIDS patients (one from each patient). All of the specimens were examined for the presence of the pathogens by genotyping using polymerase chain reaction and sequencing of the small subunit ribosomal RNA gene for Cryptosporidium spp., G. duodenalis, and E. bieneusi, respectively. No statistical differences were observed in occurrence rate between the groups by gender, clinical symptom (diarrhea), and CD4+ cell count. Four Cryptosporidium species were identified: Cryptosporidium hominis (n = 2), Cryptosporidium parvum (n = 1), Cryptosporidium meleagridis (n = 1), and Cryptosporidium andersoni (n = 2). Furthermore, two C. hominis subtypes (IeA12G3T3 and IaA28R4) were detected. Three G. duodenalis-positive specimens were successfully amplified and sequenced at the triosephosphate isomerase and β-giardin loci, which led to the identification of assemblages C and B, respectively. Seven genotypes  were identified in E. bieneusi-positive specimens.Six (3.9%), three (1.9%), and eight (5.2%) HIV/AIDS patients were positive for Cryptosporidium spp., G. duodenalis, and E. bieneusi. Homology analyses of the three intestinal pathogens at the nucleotide and/or amino acid levels indicated their zoonotic potential.Our findings should increase awareness of AIDS-related opportunistic intestinal pathogens, and indicate the need for routine examination in clinical practice for the detection of  Cryptosporidium spp., Giardia duodenalis , and Enterocytozoon bieneusi are common opportunistic intestinal pathogens in humans    and 60-k–850 bp) , respect 530 bp) , the β-g 510 bp) , and the 510 bp) . Enteroc(243 bp) . PCR washttp://www.ncbi.nlm.nih.gov/blast/) and then aligned and analyzed with reference sequences downloaded from GenBank, using Clustal X 1.83  to determine the genotypes and subtypes of Cryptosporidium-, G. duodenalis-, and E. bieneusi-positive specimens. If the obtained nucleotide sequences were different from published sequences, two separate PCR amplicons of the same DNA preparation were sequenced to ensure accuracy.All of the PCR amplicons of expected size were purified and then directly sequenced using the corresponding primers on an ABI PRISMTM 3730 DNA Analyzer  with a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). Sequencing data accuracy was validated by sequencing in both directions. Nucleotide sequences were subjected to Basic Local Alignment Search Tool (BLAST) searches  was more prevalent than Cryptosporidium spp. (3.9%) and G. duodenalis (1.9%). One patient had a mixed infection with G. duodenalis and E. bieneusi.PCR and sequence analyses revealed that, of the 155 HIV/AIDS patients, six, three, and eight patients were positive for Cryptosporidium than males (7.1% vs. 3.6%). Giardia duodenalis and E. bieneusi were only found in males. All Cryptosporidium- and E. bieneusi-positive cases were of non-diarrheal patients. G. duodenalis was identified in both diarrheal and non-diarrheal patients. However, no statistical difference in occurrence rate of diarrhea was found between them , Cryptosporidium parvum (n = 1), Cryptosporidium meleagridis (n = 1), and Cryptosporidium andersoni (n = 2). None of the SSU rRNA gene sequences had been previously described (GenBank accession numbers MT757967–MT757972). Table C. hominis, C. parvum, or C. meleagridis were further subjected to subtyping by sequence analysis of the gp60 gene. However, only two of these specimens, from C. hominis, were successfully subtyped and identified, namely IaA28R4 (GenBank accession no. OM212052) and IeA12G3T3 (GenBank accession no. OM212051).Sequence analysis of the SSU rRNA gene revealed the presence of four inis n = , CryptosG. duodenalis-positive specimens were successfully amplified and sequenced at the tpi and bg loci, and all of them showed mixed infections of assemblages C (at the tpi locus) and B (at the bg locus). Three of the tpi gene sequences of assemblage C had not been previously described. However, at the amino acid level, the tpi sequences showed 100% similarity with the sequences of dog-derived G. duodenalis: OM212053 and OM212054 were identical to KY979493, and OM212055 was identical to MW561663. In contrast, three bg gene sequences of assemblage B were identical and shared 100% similarity with the sequence of a squirrel monkey-derived G. duodenalis (GenBank accession no. KJ888974). Table Three E. bieneusi were identified: D (n = 2), EbpC, TypeIV, Peru11, A, and EbpD (one each) of group 1, and I (n = 1) of group 2.Based on sequence analysis of the ITS region, seven genotypes of Cryptosporidium spp., G. duodenalis, and E. bieneusi in HIV/AIDS patients in Shanghai, China.HIV/AIDS patients are at high risk of OIs , which aCryptosporidium spp., G. duodenalis, and E. bieneusi were 3.9%, 1.9%, and 5.2% in the HIV/AIDS patients, respectively, which are lower than those reported by most previous studies, particularly those from African countries. For example, the rates of Cryptosporidium spp., G. duodenalis and E. bieneusi in HIV-infected patients were reported to be as high as 79.0% in Nigeria  [Sequence analysis of the ectively . Meanwhiin Qatar  and assen China] .bg gene sequences of assemblage B were identical. The same bg gene sequence has been previously described in squirrel monkeys from China [tpi gene sequences of assemblage C obtained in the present study have not been previously described. However, at the amino acid level, the tpi sequences showed 100% similarity with those derived from dogs: OM212053 and OM212054 were identical to KY979493 (China), and OM212055 was identical to MW561663 (Thailand). The results of the homology analysis indicated the zoonotic potential of assemblages B and C detected in the present study in patients from Shanghai. Although we identified assemblage C in the present study, it is usually found in canines, and only occasionally in humans. To date, assemblage C has been found in humans in Brazil [The homology analysis indicated that three om China . Three dn Brazil , Egypt [n Brazil , and Chin Brazil . In Chinn Brazil .E. bieneusi-positive specimens. Among these, genotypes D, EbpC, and Type IV of group 1 have been most frequently identified in humans and numerous animal species worldwide, and display a low level of host specificity and zoonotic or cross-species transmission potential [E. bieneusi infection) [E. bieneusi [E. bieneusi has been found in animal species such as pigs, deer, takins, cats, meerkats, rabbits, and bats, as well as in diarrheal children in China [Sequence analysis of the ITS region revealed seven genotypes  in the fection) , and in fection) . Genotypfection) . Genotypfection) . Genotypbieneusi , but hasbieneusi  and in dbieneusi . In Chinbieneusi ; howeverbieneusi . E. bienin China , 74. TheCryptosporidium spp., G. duodenalis, and E. bieneusi need to be conducted using a sufficient number of human specimens, a greater variety of animal hosts and environmental specimens, to assess and validate the zoonotic potential of these pathogens. The types of data thus collected should facilitate the development of efficient control strategies for the management and prevention of OIs in HIV/AIDS patients.This study has some limitations. The number of analyzed specimens was small, and the number of positive specimens was low. In accordance with the One World—One Health Manhattan Principles, we believe that future molecular studies on the epidemiology of Cryptosporidium spp., G. duodenalis, and E. bieneusi in HIV/AIDS patients in Shanghai, China. Our findings should increase awareness of opportunistic intestinal pathogens in AIDS patients, and indicate the importance in clinical practice of routine examination of patients for the detection of the common pathogens discussed here. The high diversity of Cryptosporidium spp. and the rare gp60 subtypes of C. hominis identified in this study possibly reflect the genetic characteristics of Cryptosporidium endemic to China. Mixed infections with different assemblages were indicated by identification of assemblages B and C in three identical G. duodenalis-positive specimens. Seven zoonotic genotypes of E. bieneusi, including genotypes D, EbpC, Type IV (high frequency), Peru 11, EbpD, and A (commonly found), and I (rarely found), were also identified. Homology analysis of nucleotide and/or amino acid sequences of these pathogens revealed their zoonotic potential.Herein we report the occurrence and genetic characteristics of


Viola philippica Cav. is the only source plant of “Zi Hua Di Ding”, which is a Traditional Chinese Medicine (TCM) that is utilized as an antifebrile and detoxicant agent for the treatment of acute pyogenic infections. Historically, many Viola species with violet flowers have been misused in “Zi Hua Di Ding”. Viola have been recognized as a taxonomically difficult genera due to their highly similar morphological characteristics. Here, all common V. philippica adulterants were sampled. A total of 24 complete chloroplast (cp) genomes were analyzed, among these 5 cp genome sequences were downloaded from GenBank and 19 cp genomes, including 2 “Zi Hua Di Ding” purchased from a local TCM pharmacy, were newly sequenced.Viola cp genomes ranged from 156,483 bp to 158,940 bp in length. A total of 110 unique genes were annotated, including 76 protein-coding genes, 30 tRNAs, and four rRNAs. Sequence divergence analysis screening identified 16 highly diverged sequences; these could be used as markers for the identification of Viola species. The morphological, maximum likelihood and Bayesian inference trees of whole cp genome sequences and highly diverged sequences were divided into five monophyletic clades. The species in each of the five clades were identical in their positions within the morphological and cp genome tree. The shared morphological characters belonging to each clade was summarized. Interestingly, unique variable sites were found in ndhF, rpl22, and ycf1 of V. philippica, and these sites can be selected to distinguish V. philippica from samples all other Viola species, including its most closely related species. In addition, important morphological characteristics were proposed to assist the identification of V. philippica. We applied these methods to examine 2 “Zi Hua Di Ding” randomly purchased from the local TCM pharmacy, and this analysis revealed that the morphological and molecular characteristics were valid for the identification of V. philippica.The V. philippica that may facilitate the application of a super-barcode in TCM identification and enable future studies on phylogenetic evolution and safe medical applications.This study provides invaluable data for the improvement of species identification and germplasm of The online version contains supplementary material available at 10.1186/s12864-022-08727-x. Viola is mainly distributed in temperate and tropical regions, and China is one of the distribution centers [Viola species have been recorded as medicinal plants with definite efficacy and indications [V. philippica (Herba Violae) possesses significant and unique efficacy in clinical antiviral therapy. Research has shown that V. philippica extract possess a wide range of pharmacological and biological activities, including antiviral, antifungal, anticoagulant and anticancer functions [V. philippica extracts are remarkably stable and tolerate harsh thermal, chemical, and enzymatic conditions with high biological activity, including insecticidal, cytotoxic, and neurotensin antagonistic activities. These characteristics make V. philippica ideal for potential agrochemical or pharmaceutical applications [V. philippica is a small perennial herb with violet flowers. The dried whole plant (including the roots) is an important TCM named “Zi Hua Di Ding” in Chinese [Viola species described in the Chinese Pharmacopoeia in 1977, in which V. philippica is the only source of “Zi Hua Di Ding”; it has been widely used ever since [V. philippica as traditional medicine was described in the 1989 World Health Organization (WHO) bulletin [As the largest genus of Violaceae,  centers –3. More ications –7. V. phunctions , 9. Notaications –12. V. p Chinese , 14. “Zier since . Furtherbulletin , 17. Impbulletin –21.Viola is traditionally considered to be a morphologically difficult genus to classify [Viola species exhibit high variability [Viola species are difficult to identify. Unclear phylogenetic relationships and highly similar morphological characteristics among Viola species restrict the breeding and development of V. philippica germplasm resources. One or a more chloroplast DNA molecular markers, the nuclear intergenic transcribed spacer (ITS), or the inter-simple sequence repeat (ISSR) have previously been used to infer the phylogenetic relationships within Viola [Viola are still controversial due to insufficient informative sites and incomplete taxon sampling. Most prior studies have focused on the Viola species of North America, Korea, and Japan, while there are limited studies regarding Viola species from China. Flora of China proposed that the Viola species in China are divided into 14 sections based on morphological characteristics, including sections Adnatae, Bilobatae, Brevicalcaratae, Caudicaules, Diffusae, Erectae, Longicalcaratae, Noverculae, Pinnatae, Plagiostigma, Serpentes, Trigonocarpae, Vaginatae, and Viola [Viola based on molecular inference are frequently inconsistent with the conclusions of traditional views. The delimitation of sect. Adnatae and the phylogenetic position of the species within it are widely disputed. V. philippica belongs to the sect. Adnatae, which is a group of about 35 species in China. It is very difficult to accurately identify V. philippica for use in TCM by morphological characteristics or organoleptic methods. Ensuring the authenticity of source plants is a key issue in the use of herbs. Some species have been misidentified due to their violet-colored flowers being the same color as those of V. philippica, e.g., Corydalis bungeana or Gueldenstaedtia verna. Common adulterants in “Zi Hua Di Ding” are often Viola species, i.e., V. patrinii, V. inconspicua, V. yezoensis, V. phalacrocarpa, and V. prionantha. In practice, some micromorphological features are difficult to identify, especially those concentrated in the floral organs. The result is that many Viola species with violet flowers were often treated as V. philippica. Folk lacks botanical knowledge, confusing the safety and effectiveness of drug use. Consumers are often unable to verify original voucher specimens of herbs when they purchase them in pharmacies. Therefore, accurate and efficient identification of the source plants of “Zi Hua Di Ding” is necessary.classify , 2, 22. iability –26. The in Viola –29. Howend Viola , 30. TheViola species, in particular V. philippica. Furthermore, molecular markers in highly divergent cp genome regions were screened for the identification of Viola and phylogenetic studies.DNA barcoding is an emerging technology of molecular identification and classification; it significantly enhances the safety and efficacy of medicinal herbs –33. DNA Viola samples were obtained, for which cp genome sequences of three species were downloaded from GenBank and the cp genomes of 19 Viola samples were newly sequenced. The common adulterants in “Zi Hua Di Ding” belonging to the Viola were covered. The purpose of this study was to 1) compare the chloroplast genome structures of the sampled Viola species; 2) find an effective means to by which to distinguish V. philippica from other Viola species; and 3) resolve the infrageneric relationships within Viola species, especially section Adnetae, Pinnatae, and Bilobatae, using complete plastomic sequences and highly diverged sequences. This study provides invaluable data for species identification and improvement in germplasm generation of V. philippica; it will facilitate the application of super-barcode cp genomics in TCM identification, allowing for future studies on phylogenetic evolution and safe medical applications of Viola species.Herein, a total of 22 Viola species analyzed in the current study that have been accurately identified possessed a similar genome structure, gene order, and orientation. Plastome size ranged from 156,483 bp  to 158,940 bp (V. acuminata) , SSC region , and a pair of IRs . The overall guanine-cytosine (GC) content was approximately 36%. The GC contents in the LSC and SSC regions of all 17 species were lower than in the IRs. Overall, 128 genes were annotated in the 17 Viola species, of which 110 were unique, consisting of 76 protein-coding, 30 tRNA, and four rRNA genes  were completely degraded. There were 17 intron-containing genes in each of the Viola species, of which two PCGs (ycf3 and clpP) had two introns, 9 PCGs  and 6 tRNAs  had a single intron each.All rps19 gene extended 2-73 bp into the IRs at the junction of the LSC/IRb (JLB), resulting in the duplication of 3’-ends of this gene in the IRa region. The ndhF gene was located in the SSC at a 3-33 bp distance from the SSC/IRb (JSB) border. Meanwhile, in V. mongolica and V. yunnanfuensis the ndhF gene traverses the JSB by extending 55 and 51 bp, respectively, into the IRb region. In V. philippica, the ndhF gene was located in the SSC at a 33 bp distance from the SSC/IRb (JSB) border. In all cp genomes, the SSC/IRa (JSA) junction was located within the ycf1 gene, with 1091 to 1866 bp of ycf1 duplicated in the IRb region. In addition, the trnH genes were all located in the LSC region, with the distance between trnH-GUG and the LSC/IRa (JLA) border varying from 26 to 85 bp.The locations of IR/SC junctions were conserved among all cp genomes Fig. S. In geneViola cp genomes , while V. mongolica had the least (33). Conversely, such complementary repeats were not detected in the cp genomes of V. mongolica and V. yezoensis. Additionally, tandem repeats that ranged from 30 to 33 bp were the most abundant, followed by those that ranged from 38 to 41 bp. The number of repeats in V. philippica was 46, comprising 19 forward repeats, 8 reverse repeats, 1 complementary repeat, and 18 palindromic repeats  and 6 dinucleotide repeats (2 AT repeats and 4 TA repeats).The number of repeat sequences was calculated and the threshold of repeat length was set to ≥30 bp. A total of 729 repeats were detected in the 17 Viola species. However, it was observed that the LSC and SSC regions were more divergent than IRs. In addition, intergenic regions exhibit greater divergence than coding regions  generated phylogenetic trees with strong support , high-performance liquid chromatography (HPLC), near-infrared spectroscopy (NIRS), and metabolomic approaches [V. philippica from closely related species previously, however, fewer adulterants of V. philippica were sampled and strong support for some nodes in the phylogenetic tree was not acquired [Viola exhibit a low level of genetic differentiation as revealed by the ITS analysis [V. philippica were analyzed by Blast online comparison in the NCBI database. The results of gene sequence similarity comparison showed that the V. philippica sequences in this study had highest homology with V. philippica sequences that had been registered on GenBank. The similarity results reach more than 98%. This suggests that validation of the chloroplast genome is effective. The use of genetic distances using standardized gene regions (DNA barcodes) has provided complementary or alternative support for species identification, which is especially useful when distinct morphological characters are scarce or subtle [Viola species. Among them, V. philippica and V. monbeigii had the smallest genetic distance, indicating that they are both closely related, which is consistent with the phylogenetic tree results. The identity of genes between populations within species is quite high, and consequently, genetic distance is small  and nuclear ITS were used to infer the phylogeny of Viola, however, the majority of interspecies relationships are not currently well resolved [Viola species are traditionally morphologically difficult taxon to classify due to their very similar morphological characteristics, and there is some synapomorphy in Viola species. This may be why phylogenetic analyses of Viola based on molecular inference are often inconsistent with the results of the traditional morphological study. Complete cp genomes contain a wealth of genetic variation and is an ideal source of data to study phylogeny among species [Viola were resolved in this study, and both ML and BI trees were strongly supported. Therefore, we suggested that the complete cp genome can be used as a super-barcode to distinguish closely related Viola species. Furthermore, relationships among sampled Viola species were also resolved based on only 16 molecular markers with high Pi values, their topologies are highly consistent with the complete cp genome. Therefore, these identified regions  could be used as markers for elucidating phylogenetic relationship within Viola species. These findings provide additional information for the selection of effective molecular markers to detect intra- and interspecific genetic polymorphisms , for a total of 24 individuals. Plant materials used in this study were collected and deposited at the herbarium of the College of Life Sciences, Shandong Normal University. The sampling newly sequenced species were collected from Shandong Province, China. FSJ and ZXJ undertook the formal identification of the samples  [https://github.com/quxiaojian/PGA) [Viola species were downloaded from GenBank, including V. mirabilis L. (NC_041582), V. raddeana Regel (NC_041584), and V. websteri Hemsl. (NC_041585)  . Annotatian/PGA) . Geneiouian/PGA) . The cirian/PGA) . All chl NC_04158, V. raddhttps://irscope.shinyapps.io/irapp/) [Viola species.The expansion and contraction of IRs were analyzed by IRscope (/irapp/) , coupledhttps://bibiserv.cebitec.uni-bielefeld.de/reputer/) [SSR markers are valuable in study of genetic diversity and molecular marker selection. The size and position of the repeat sequences were detected using REPuter (eputer/) , includieputer/) , with rehttp://genome.lbl.gov/vista/index.shtml) [https://github.com/quxiaojian/Bioinformatic_Scripts/get_mVISTA_format_from_GenBank_annotation.pl) was used to convert GenBank annotation files to mVISTA format files. Then we aligned the complete cp genomes using the Shuffle-LAGAN mode of mVISTA, with V. acuminata as reference.mVISTA (x.shtml)  is a comSingle nucleotide polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by single nucleotide variation at the genome level. The percentage of parsimony information sites (Pi) of the coding and intergenic regions were calculated by using DnaSP v6.0 . The scrV. philippica with the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) by Blast online. The Viola genetic distances were calculated in MEGA, using Kimura’s 2-parameter model [V. philippica, e.g., Corydalis bungeana or Gueldenstaedtia verna. To improve the reliability and accuracy of the identification results, we added closely related species of both, Corydalis tomentella (NC_060366) and Oxytropis arctobia (NC_050861). Paspalum paniculatum (MF563367) was set as the outgroup. The ML trees were reconstructed by RAxML v8.0.26 with the GTRGAMMA substitution model and 1000 bootstrap replicates [matK, ndhF, ycf1, rpl22, rps15, ndhA, petN-psbM, petA-psbJ, ccsA-ndhD, trnG-UCC-2-trnR-UCU, rps8-rpl14, trnD-GUC-trnY-GUA, trnG-GCC-trnfM-CAU, trnH-GUG-psbA, psbZ-trnG-GCC, and rbcL-accD).We blast er model . The coder model  with defplicates . Bayesiaplicates . PhylogeV. philippica from other Viola species were screened by examining the alignment matrix of 76 genes for 17 Viola species. To ensure the accuracy of the four selected specific variation sites in V. philippica, Sanger sequencing of PCR amplicons was performed on 14 newly sequenced Viola species. Primers were designed using Primer3 v0.4.0 [V. philippica cp genome were used as the templates for primer design. The forward primers for these four fragments were ATTCAATATCTGTATGGGGTAAAG, AATAATTGATCAGATTCGTGGACG, CGTCTAAAACCTTGGCACAAATCG and AACTCATCCATTTATCGATTACCA and the reverse primers were TGTTACAAATTCATACCAATCCAC, TTTGTGGACTTCTTTATGCACCTC, GGTTCGTTTGAGTAACGGTTGTCA and GTATTTCGTCATCGTCATTCATTC, respectively . We coded five-character, including lobe, length of stipules adnate to petioles, leaf blade baes, stigma type, and fruit shape. lobe: 0 = entire leaf, 1 = lobed leaf; length of stipules adnate to petioles: 0 = stipule adnate with petioles shorter than 1/2, 1 = stipule adnate with petioles longer than 1/2; leaf blade baes: 0 = explanate, 1 = reflexed; stigma type: 0 = immarginate, 1 = margined; fruit shape: 0 = capsule ellipsoid, 1 = capsule globose. We mapped these traits onto a cluster analysis tree and represented each trait with a different shape  and Bayesian Inference (BI) phylogenetic trees are based on 16 highly diverged regions. Fig. S4. The variable sites in ndhF, rpl22, and ycf1 of Viola philippica. Fig. S5. Maximum Likelihood (ML) and Bayesian Inference (BI) phylogenetic trees are based on complete chloroplast genome. Fig. S6. Original electrophoretogram for four sequence fragments with unique variable sites in 14 newly sequenced Viola species.Additional file 2: Table S1. A list of genes found in the cp genomes of 17 Viola species. Table S2. SSR distributed situation in the 17 Viola cp genomes. Table S3. Results of genetic distance analysis. Table S4. Summary of amplified nucleotide sequences and GenBank accession numbers. Table S5. Morphological characteristics for analysis.


Escherichia coli, Klebsiella, and Enterococcus species. Streptococcus gallolyticus subsp. pasteurianus, previously known as Streptococcus bovis biotype II/2, rarely causes endocarditis, meningitis, and septicemia, mainly in children. Biliary tract infections by Streptococcus gallolyticus subsp. pasteurianus are extremely rare. There have been no reports of cases in Japan. Here, we describe the first case in Japan of acute calculous cholecystitis caused by Streptococcus gallolyticus subsp. pasteurianus infection. A 63-year-old man was admitted to our hospital with epigastric pain and vomiting. He had moderate tenderness and a full sensation in the epigastrium. Abdominal imaging revealed multiple stones in the gallbladder. After admission, he had a high fever that did not improve with antibiotics. Percutaneous transhepatic gallbladder drainage was performed. The patient underwent open cholecystectomy. During surgery, several small stones in the gallbladder and an abscess were observed at the gallbladder base. Streptococcus gallolyticus subsp. pasteurianus was detected by bacterial culture of the bile juice. The gallstones were bilirubin calcium stones. The endoscopic study showed three adenomas in the colon, but the histopathological examination demonstrated no malignant cells. Although infection by this bacterium may not be rare, this is the first reported case in Japan of acute calculous cholecystitis caused by Streptococcus gallolyticus subsp. pasteurianus infection.Acute cholecystitis is an infectious disease of the gallbladder caused mainly by  Streptococcus gallolyticus subsp. pasteurianus (SGSP) belongs to the group D Streptococcus, formerly known as Streptococcus bovis type II/2 [S. bovis was originally classified into type I (mannitol-fermentation-positive), type II/1 (mannitol-fermentation-negative and β-glucuronidase-negative), and type II/2 (mannitol-fermentation-negative and β-glucuronidase-positive) [S. bovis-group bacteria based on genetic differences [S. bovis. Organisms from the S. bovis group have been associated with endocarditis, meningitis, sepsis, and colorectal cancers [ype II/2 . Based o cancers ,4,5,6. H cancers . ReportsA 63-year-old male with a history of herpes zoster at the age of 24 and acute gastroenteritis after the ingestion of raw egg at the age of 61 was admitted to our hospital for upper abdominal pain and vomiting. The day before admission, two hours after eating a pork cutlet for dinner, he felt a burning pain in the epigastrium. Less than 24 h after the start of his symptoms, the patient consulted our institution. He continued having epigastric pain and started vomiting after admission. Physical examination revealed marked tenderness in the epigastrium and right upper quadrant but no rebound tenderness. His vital signs were within the normal range except for a body temperature of 37.3 °C. The blood analysis showed a slight elevation of leukocytes, neutrophils, C-reactive protein, triglycerides, low-density lipoprotein cholesterol, and liver enzymes . No anemAbdominal ultrasonography and computed tomography (CT) findings showed a moderately fatty liver and multiple stones in an enlarged gallbladder with suspicion of stone impaction in the gallbladder neck A,B. HoweAfter admission, the patient received antibiotic therapy, including 1 g of ceftriaxone sodium hydrate (CTRX) twice daily. Percutaneous transhepatic gallbladder drainage (PTGBD) was performed on the same night. Sixty milliliters of turbid concentrated bile was aspirated from the PTGBD tube. The PTGBD is recommended by the Tokyo Guidelines 2018 in cases with complications or comorbidities . CholecyOn the 11th day of hospitalization, the patient underwent an open cholecystectomy through a right subcostal incision. More than 30 dark brown stones 2–3 mm in diameter were found in the gallbladder, and the gallbladder base had partially melted due to necrotizing cholecystitis, forming an abscess, which was coated with a large mesh A. In addS. bovis group are commonly associated with colorectal cancer [The histological specimens from the cholecystectomy showed erosions, fibrosis, and a moderate inflammatory cell infiltration with neutrophils, but no malignant findings B. Infrarl cancer . A colonAfter removing the drain on the 12th postoperative day, the patient was discharged on the 17th postoperative day.S.bovis II/2) infection in a 63-year-old male. Microorganisms of the S. bovis group are commensal intestinal bacteria that can infect humans and animals, including birds [S. bovis from cows to people [S. bovis group members are increasingly isolated as predominant species from fermented products in Europe, Asia, and Africa [In this report, we described a case of acute calculous cholecystitis caused by an SGSP  [S. bovis group, including SGSP, can live in bile. Lee et al. evaluated 37 cases of S. bovis bacteremia and found that 14 (38%) had biliary tract disease, indicating that biliary tract infection was the main source of bacteremia by S. bovis [S. bovis, 9 cases (23.7%) of biotype II  had a hepatobiliary source of infection [S. bovis and S. salivarius and found that S. bovis-biotype-II-associated bacteremia had a hepatobiliary origin in 50% of the patients [S. infantarius (biotype II/1), 20 (39%) cases of SGSP (biotype II/2), and 2 (4%) cases of S. gallolyticus subsp. gallolyticus (biotype I). They concluded that biotype II bacteria of the S. bovis group are predominant in biliary tract infection.Microorganisms that degrade gallic acid have been isolated from koalas’ intestines and named olyticus . This bas (SGSP) . The lats (SGSP) . TherefoS. bovis . Ruoff enfection . Similarpatients . In 2013Escherichia coli (25.0%), Enterococcus spp. (13.4%). Klebsiella spp. (11.1%), Pseudomonas spp. (11.1%), and coagulase-negative Staphylococcus (9.7%) [Acute cholecystitis is a complication of cholelithiasis in more than 90% of patients . Gallstos (9.7%) . Most ofs (9.7%) ,22. In tS. bovis group of bacteria has long been associated with colorectal cancer, although not all genospecies are closely related to this malignant complication. The meta-analysis reported by Boleij et al. reported four subspecies of S. gallolyticus and demonstrated a strong association of S. bovis biotype I  with the development of colorectal adenoma/cancer [The a/cancer . The assa/cancer . We perfAlthough infection by this bacterium may not be rare, this is the first reported case of acute calculous cholecystitis caused by SGSP infection in Japan. In addition, careful clinical follow-up is recommended due to a potential association between SGSP and the development of colorectal cancer.


Population density can be an environmental cue to induce modification of insect morphology, physiology, and behavior. This phenomenon is called density-dependent plasticity. Aphids and locusts exhibit textbook examples of density-dependent plasticity but there is a lack of integrative understanding for insect density-dependent plasticity. To address this problem, we combined public gene expression data obtained from multiple studies and re-analyzed them . The present study provides additional insight into the regulatory mechanisms of density-dependent plasticity, demonstrating the effectiveness of meta-analyses of public transcriptomes.With increasing public data, a statistical analysis approach called meta-analysis, which combines transcriptome results obtained from multiple studies, has succeeded in providing novel insights into targeted biological processes. Locusts and aphids are representative of insect groups that exhibit density-dependent plasticity. Although the physiological mechanisms underlying density-dependent polyphenism have been identified in aphids and locusts, the underlying molecular mechanisms remain largely unknown. In this study, we performed a meta-analysis of public transcriptomes to gain additional insights into the molecular underpinning of density-dependent plasticity. We collected RNA sequencing data of aphids and locusts from public databases and detected differentially expressed genes (DEGs) between crowded and isolated conditions. Gene set enrichment analysis was performed to reveal the characteristics of the DEGs. DNA replication (GO:0006260), DNA metabolic processes (GO:0006259), and mitotic cell cycle (GO:0000278) were enriched in response to crowded conditions. To date, these processes have scarcely been the focus of research. The importance of the oxidative stress response and neurological system modifications under isolated conditions has been highlighted. These biological processes, clarified by meta-analysis, are thought to play key roles in the regulation of density-dependent plasticity. Phenotypic plasticity is the ability to respond adaptively to environmental variations by producing more than one phenotype for a single genotype ,2. If twPhysiological mechanisms underlying density-dependent polyphenism have been revealed in aphids  and locuSchistocerca species, the pea aphid Acyrthosiphon pisum, and the migratory locust Locusta migratoria. We identified DEGs between crowded and isolated conditions and performed gene set enrichment analysis to understand the characteristics of the DEGs.Meta-analysis that combines the transcriptome results from multiple studies is thought to be effective in providing additional insights into density-dependent polyphenisms because a meta-analysis can uncover new information that cannot be achieved with conventional hypothesis-driven research methods. Meta-analysis of transcriptomes is effective in revealing novel gene expression profiles ,18,19,20https://sra.dbcls.jp/ (accessed on 16 April 2021)), and RNA-seq data archived in the Sequence Read Archive (SRA) [Expression data were searched for in the public databases Gene Expression Omnibus (GEO)  and DBCLve (SRA)  using thSRA retrieval and conversion to FASTQ files was performed using the prefetch and fasterq-dump programs in the SRA Toolkit (v2.9.6) , respectAcyrthosiphon pisum and the migratory locust Locusta migratoria), RNA-seq reads were mapped to reference genome sequences using HISAT2 (v.2.2.1) [Acyrthosiphon pisum: GCF_005508785.1 or L. migratoria: GCA_000516895.1). Quantification of the mapped data was performed using StringTie (2.1.5) [In species whose genomic information is available (the pea aphid v.2.2.1)  with par (2.1.5) , and theSchistocerca), TPM values were calculated using Salmon (v.1.5.0) [In species with genomic information not available   with parv.1.5.0) . TrinityTo evaluate the changes in expression of each gene, the expression ratio was calculated using TPM paired with crowded and isolated transcriptomes (CI ratio). The CI ratio for each gene was calculated using Equation (1):This calculation was performed for all pairs of crowded and isolated transcriptomes. Pairs of transcriptome data used for comparison are shown in https://github.com/no85j/hypoxia_code/blob/master/CodingGene/HN-score.ipynb (accessed on 17 March 2022)). Visualization of CI ratio was performed with heatmap2 (R ver 4.2.0).The calculation method for CI ratios and CI scores was based on a previous study  (https:/Acyrthosiphon pisum: GCF_005508785.1 and L. migratoria: GCA_000516895.1) to extract the transcript sequence from the genome data. In species whose genomic information is not available , transcript sequences were collected from the TSA database . TransDecoder (v5.5.0) (https://transdecoder.github.io/ accessed on 22 June 2021) was used to identify coding regions and translate them into amino acid sequences of all species. Open reading frames were extracted by TransDecoder.Longorfs with parameters -m 100, and coding regions were predicted by TransDecoder.Predict with default parameters. Then, the protein BLAST (BLASTP) program with parameters -evalue 0.1 -outfmt 6 -max_target_seqs 1 in the NCBI BLAST software package (v2.6.0) was used to determine the orthologous gene relationship among species. Ensembl Biomart was used to obtain stable protein IDs and gene names from D. melanogaster gene set (BDGP6.32).In species whose genomic information is available, GFFread (v0.12.1)  was useddatabase . HoweverGene set enrichment analysis was performed by Metascape  using thTo compare gene expression under crowded and isolated conditions, we retrieved transcriptome data acquired under different density conditions. Consequently, 66 pairs of transcriptome data were collected from the public databases . Full inS. gregaria as queries against those in each species examined in this study. Since density-dependent polyphenisms have been extensively studied using this species, this species was selected as a reference for the determination of orthologous relationships among species. Next, we performed searches for all genes vs. all BLASTP between D. melanogaster and S. gregaria to annotate the gene names of each species into those of D. melanogaster.After the expression ratio (CI ratio) was calculated for each species, as described in the Materials and Methods ebony, Mcm2, and Mcm7 were upregulated in response to crowded condition, and Duox, Mdr49 and Cyp6a14 were downregulated in response to crowded condition  [obst-E, GO:0006979; spz, Glaz, srl) showed high or low values (ebony (e) showed a high CI score for the upregulated DEGs .CI scores were calculated based on the CI ratio table . The prendition) ,34. In tndition) ,34. CI sw values . These ron ratio , the genCG14803, CG2990, Psf3, CG8142, RnrL, Psf1, Pol31, Mcm7, Mcm2, E2f1, DNApol-alpha60, dup), “DNA metabolic process” , and “mitotic cell cycle”  were enriched significantly in upregulated DEGs (We selected 97 upregulated genes and 199 downregulated genes as DEGs (CI score thresholds:8 and −8). Using these DEGs, we performed gene set enrichment analysis using Metascape to identify their characteristics. Metascape showed that “DNA replication”  was significantly enriched in the downregulated DEGs  were included in GO: 1,901,700.The response to oxygen-containing compounds  gene, encoding tyrosine hydroxylase, which is involved in pigmentation of the cuticle and catecholamine biosynthesis [ple was higher in many isolated transcriptomes than in crowded transcriptomes. ple genes are known to be highly expressed under crowded conditions, inducing gregarious color and behavior [The CI score of the ynthesis , was thebehavior .CG9657: [Lrp4: [GluClalpha: [Syt4: [Among the downregulated DEGs, the genes that are reported to function in the nervous system were conspicuous .A. pisum. This result may reflect the use of different developmental stages. The aphids used in this study are derived from adults, whereas the other samples are derived from larval stages. However, we believe that our meta-analysis of public RNA sequencing using this vast amount of data has led to important insights into the gene expression profile underlying density-dependent polyphenism, as discussed below.As various data acquired under different conditions are registered, manual curations are needed for meta-analysis. In this study, we collected RNA sequencing data (66 pairs) from public databases and compared their expression levels in various insect species. We aimed to identify the common genes that can explain the regulation of the density-dependent polyphenism of all or multiple species. However, in the gene list with highest or lowest CI score , almost Nilaparvata lugenes. Together with this study, our results emphasize the importance of DNA replication and the cell cycle as the regulatory mechanisms of density-dependent polyphenism. Although wing polyphenism was also observed in A. pisum, it was not observed in locusts. Therefore, the regulation of DNA replication and cell cycle is expected to be involved in other developmental processes in locusts. Although the role of JH in aphid wing polyphenism is controversial, JH content is lower under crowded conditions than under isolated conditions in locusts [Gene set enrichment analysis showed that DNA replication, DNA metabolic processes, and the mitotic cell cycle were enriched in response to crowded conditions . DNA rep locusts ,43. Therple gene under isolated conditions was not consistent with a previous study. Although ple genes are known to be responsible for gregarious pigmentation in L. migratoria, the relationship between ple genes and body-color polyphenism is controversial [Duox, GLaz, ple, srl, and spz) classified under response to oxygen-containing compounds were related to the response to oxidative stress, and ple was included in that category. A previous microarray study showed that the expression of transcripts encoding proteins against oxidative stress damage  was higher in isolated locusts than in crowded locusts [L. migratoria, leading to variations in flight traits between solitary and gregarious locusts [ple expression under the isolated conditions in this study may result from the response to oxidative stress.The high expression of oversial . Several locusts . Rapid a locusts . TherefoS. gregaria [L. migratoria [D. melanogaster [Lrp4 functions to ensure normal synapse numbers in D. melanogaster [GluClalpha plays an important role in the ON/OFF selectivity of the visual systems in D. melanogaster [2+-binding proteins involved in the presynaptic transmitter release [Neurological system modifications may play an important role in inducing density-dependent phenotypic changes in gregaria  and L. mgratoria . We foungratoria , Lrp4 [3gratoria , GluClalgratoria , and Sytgratoria ). CG9657nogaster . Presynanogaster . GluClalnogaster . Syts ar release . These gWe identified novel genes related to density-dependent polyphenism in insects using a meta-analysis of public transcriptomes. Reliable and general principles should be derived from meta-analysis because this method integrates the results of a number of studies. Therefore, our results can be applied to other species that exhibit density-dependent polyphenisms.


Both the protein domains and transcript structures influence protein functional variation. The genomic location of both protein domains and transcript structural features can be described using the genomic coordinates of their encoded sequences. However, the coordinates of protein domains and transcriptional features often differ greatly, and it is difficult to view them in combination at the genome-wide level. In this paper, we describe the development of a new tool that allows users to visualize domains and transcript features together, using either built-in or uploaded genome datasets, and export publication-ready figures. We developed a user-friendly, independent R package and Shiny web application named “VisProDom”. VisProDom consists of a genome-wide database containing entire annotated transcripts merged with annotated protein domains from the Pfam database. The built-in dataset includes 82 files, which merge genome general feature format (GFF) annotations with rpsblast tabular outputs from protein sequence searches in the Pfam database. Multiple genomes can be simultaneously screened for protein domains or transcript names. VisProDom includes step-by-step introductions and clickable elements for ease of use.VisProDom can display hundreds of transcripts alongside protein domains and export figures in a publication-ready format. This makes it a valuable tool for molecular evolution and comparative genomics.The online version contains supplementary material available at 10.1186/s12864-022-08713-3. Genetic information can be characterized by both the protein domains and transcript structure. Protein domains are distinct functional and/or structural units that are usually responsible for a particular function or interaction that contributes to the overall role of the protein. Transcriptional features refer to features such as untranslated regions (UTRs), exons, coding sequences (CDSs), and introns. Genomic locations (i.e. coordinates) of protein domains are typically identified from protein sequence queries in the Pfam databases , the Simhttp://CRAN.R-project.org/package=shiny), an R (http://CRAN.R-project.org) package used to easily build interactive web applications.We designed VisProDom, a user-friendly, independent package for R, which is a widely used programming language for statistical computing and graphics. Additionally, we created a web application using Shiny (https://CRAN.R-project.org/package=ggplot2), Shiny (https://CRAN.R-project.org/package=shiny), rintrojs (https://CRAN.R-project.org/package=rintrojs), dplyr (https://CRAN.R-project.org/package=dplyr), data.table (https://CRAN.R-project.org/package=data.table), and plotly (https://cran.r-project.org/web/packages/plotly/index.html). The source code for VisProDom is freely available (https://github.com/whweve/VisProDom). We also deployed VisProDom online (https://whweve.shinyapps.io/VisProDom). Users can download and install VisProDom onto a local computer from GitHub by copying and pasting commands onto their R console. Owing to the characteristics of R and Shiny, VisProDom is a package that can be used in any operating system, requiring very limited programming skills.Imported R packages used for the creation of VisProDom include ggplot2 (VisProDom contains 82 built-in genomes (Supplementary Table https://github.com/whweve/VisProDom). The R package, “devtools”, is required to download and install VisProDom from GitHub. VisProDom requires other R packages. When launching RStudio, the required packages and VisProDom can be installed and loaded using the following commands in the R environment: > install.packages). > devtools:install_github(“whweve/VisProDom”). > library(VisProDom).>  runVPDappTo use the VisProDom R package, R (Comprehensive R Archive Network) and RStudio (an integrated development environment for R) have to be downloaded. Currently, the source code of VisProDom is hosted on GitHub (https://whweve.shinyapps.io/VisProDom/). The web application can be accessed from most internet browsers, including Firefox (https://www.mozilla.org), Microsoft Edge (https://www.microsoft.com/en-us/edge), and Chrome (https://chrome.en.softonic.com).To use VisProDom without installing it into the local computer environment, the Shiny-based web application can be used  one or more entered domains regardless of the background domain, 2) all entered domains regardless of the background domain, or 3) all entered domains with no background domain. When complete, the user clicks on the “Update VisProDom” button.In steps three to seven, users can manually adjust visual elements of their generated figure, such as the color and size of text and transcript features. However, this is not necessary since these features are auto-generated to be in proportion to the number of transcripts visualized.Finally, in step eight, users can adjust the file format, size, and resolution as required, and export their generated figures. VisProDom provides step-by-step instructions and clickable elements created using the R package “rintrojs”, making it easy to use.https://github.com/whweve/VisProDom), following the instructions on the web page. The Shiny web application can be accessed online (https://whweve.shinyapps.io/VisProDom) with no installation needed.VisProDom is an easy-to-use stand-alone R package and Shiny-based web application that displays domains and transcripts using built-in genomes. The VisProDom R package can be downloaded and installed from GitHub , irrespective of the background domain  and ain Fig. .Fig. 2ThIn comparison to existing tools –6, VisPrCurrently, VisProDom does not include phylogenetic tree data, and transcript sequences are not ordered according to phylogenetic relationship. However, in the future we aim to expand the capabilities of VisProDom to include phylogenetic data, and extend its applicability in the field of evolutionary biology.VisProDom is capable of visualizing hundreds of transcripts and domains across multiple selected species in a publication-ready format, making it a valuable tool for molecular and evolutionary biology. The built-in genome data improves usability; since many entire genomes are readily available, users are not required to enter protein sequences, and the running speed is high. Its ease of use is further improved by the provision of detailed instructions within the application. While VisProDom is available as an R package, its availability as a Shiny-based web application ensures that it is highly accessible as a platform-free tool.Additional file 1:Supplementary Table 1. The 82 genomes built in to VisProDom.


Picea rubens) for the first time using the single-end, short-reads (44 bp) Illumina sequences, assembled and functionally annotated it, and identified simple sequence repeats (SSRs). The contigs were assembled using SOAPdenovo2 following the retrieval of chloroplast genome sequences using the black spruce (Picea mariana) chloroplast genome as the reference. The assembled genome length was 122,115 bp (gaps included). Comparatively, the P. rubens chloroplast genome reported here may be considered a near-complete draft. Global genome alignment and phylogenetic analysis based on the whole chloroplast genome sequences of Picea rubens and 10 other Picea species revealed high sequence synteny and conservation among 11 Picea species and phylogenetic relationships consistent with their known classical interrelationships and published molecular phylogeny. The P. rubens chloroplast genome sequence showed the highest similarity with that of P. mariana and the lowest with that of P. sitchensis. We have annotated 107 genes including 69 protein-coding genes, 28 tRNAs, 4 rRNAs, few pseudogenes, identified 42 SSRs, and successfully designed primers for 26 SSRs. Mononucleotide A/T repeats were the most common followed by dinucleotide AT repeats. A similar pattern of microsatellite repeats occurrence was found in the chloroplast genomes of 11 Picea species.We have sequenced the chloroplast genome of red spruce ( Populus  chloroplast genome assembly of P. rubens. This length of the P. rubens chloroplast genome is shorter than that reported for sympatric P. mariana  and P. glauca  as well as allopatric P. sitchensis  and P. abies . From the published information, it is apparent that the chloroplast genomes of the Picea species are about 123 to 124 kb in size. Assuming that the size of the chloroplast genome of P. rubens is in this range, we could state that the P. rubens chloroplast genome reported in our study is likely not complete and could be considered as a draft or near-complete genome. The shorter assembled chloroplast genome of P. rubens is likely due to the presence of gaps in the final assembly resulting from the use of single-end short sequence reads (1 × 44 bp) for scaffolding. From mapping the assembled contigs of P. rubens to the chloroplast genome of P. mariana, the missing regions in the chloroplast genome assembly of P. rubens could be identified  and rps16  were identified. The presence of the non-functional rps16 gene fragments, which is similar to the previous findings in Pinus thunbergii, Picea crassifolia, and Picea asperata, further supports the fact of common loss of this gene in Pinaceae  and chloroplast genome sequences of 11 Picea species  as the reference from GenBank   .Picea species and Pinus thunbergii (NC_001631.1) were downloaded from NCBI viz., P. sitchensis (KU215903.2), P. engelmannii (NC_041067.1), P. glauca (MK174379.1), P. chihuahuana (NC_039584.1), P. neoveitchii (NC_043913.1), P. abies (NC_021456.1), P. asperata (NC_032367.1), P. crassifolia (NC_032366.1), P. jezoensis (NC_029374.1), P. mariana (MT261462.1). Then, global alignment of the entire chloroplast genomes of these 10 Picea species and that of P. rubens was performed, and comparative genomic divergence was estimated using mVISTA [https://genome.lbl.gov/vista/mvista/submit.shtml (accessed on 17 October 2022)] (Shuffle LAGAN mode) and P. sitchensis genome as the reference  with search criteria as 10 repeats for mononucleotide, 5 for di, 4 for tri, 3 for tetra, penta and hexanucleotide repeats [Picea species  for comparative analysis and to understand the SSR repeat pattern in the chloroplast genome sequences of these 10 Picea species and P. rubens. Further, Primer3 (https://primer3.ut.ee/ (accessed on 5 October 2022)) [P. rubens chloroplast genome.The Simple Sequence Repeats (SSRs) were identified in the chloroplast genome of  repeats . A similr 2022))  was usedP. rubens and the first phylogenetic analysis among Picea species using the whole chloroplast genome sequences. The short single-end Illumina sequences could be used to assemble near complete draft chloroplast genome in P. rubens but longer and/or pair-end sequences are needed to complete and polish the chloroplast genome. The P. rubens chloroplast genome has the highest sequence similarities with that of P. mariana and the lowest with that of P. sitchensis. The mononucleotide (A/T) repeat is most abundant followed by the dinucleotide (AT) repeat in the chloroplast genome of P. rubens. The chloroplast genomes of 11 Picea species  have similar patterns of microsatellite repeats occurrence. The global alignment between the chloroplast genomes of these Picea species revealed high genome sequence synteny and conservation of coding regions. Our results support a common monophyletic origin of the studied Picea species. Our study substantially adds to understanding the phylogeny of Picea species. The whole chloroplast genome-based phylogenetic analysis we have reported here may assist in understanding the biogeographical patterns and molecular evolution in Picea. Our study provides an important organellar genomic resource for the conifer genomics community. The microsatellites identified in this study may be used for various population and conservation genetics, phylogenetics, phylogeography, and other studies in the genus Picea and Pinaceae family.We report the first assembly and annotation of the chloroplast genome of