Streptococcus, Prevotella, Rothia, Veillonella and Actinomyces. CF-associated pathogens such as Pseudomonas, Burkholderia, Stenotrophomonas and Achromobacter are less prevalent than core genera, but have a strong tendency to dominate the bacterial community when present. Community diversity and lung function are greatest in patients less than 10 years of age and lower in older age groups, plateauing at approximately age 25. Lower community diversity correlates with worse lung function in a multivariate regression model. Infection by Pseudomonas correlates with age-associated trends in community diversity and lung function.Understanding the significance of bacterial species that colonize and persist in cystic fibrosis (CF) airways requires a detailed examination of bacterial community structure across a broad range of age and disease stage. We used 16S ribosomal RNA sequencing to characterize the lung microbiota in 269 CF patients spanning a 60 year age range, including 76 pediatric samples from patients of age 4–17, and a broad cross-section of disease status to identify features of bacterial community structure and their relationship to disease stage and age. The CF lung microbiota shows significant inter-individual variability in community structure, composition and diversity. The core microbiota consists of five genera -  Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) has been associated with the frequent exacerbations of airway dysfunction and progressive functional decline that are the hallmarks of the disease12Cystic fibrosis (CF) is characterized by recurrent airway infection, inflammation and progressive decline in lung function. Infection with key organisms such as Gemella spp. and the Streptococcus anginosus group), ecological diversity, and community stability are potentially associated with disease status810Over the last decade, cross-sectional and longitudinal studies of bacterial taxa using culture-independent microbial detection methods such as terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene sequencing, have identified polymicrobial communities in the airways of CF patients that exceed the complexity captured by traditional culture45678910111256P. aeruginosa. Molecular methods have demonstrated that not only their presence or absence, but also their relative abundance, dominance over other taxa, and temporal stability are potentially important determinants of ecological and clinical change in lung disease1016Culture-based methods have demonstrated the clinical significance of infection with pathogens such as P < 0.001). A larger proportion of adult patients had intermediate (FEV1 < 70% predicted) or advanced (FEV1 < 40% predicted) disease  and the median FEV1 was lower in the adult cohort, reflecting the progression of disease with increasing age (P < 0.001). Adult patients were more likely than pediatric patients to be infected by Bcc or Aspergillus species by sputum culture , with a trend towards increased infection with P. aeruginosa (P = 0.085).During the study period, 76 pediatric (<18 years old) and 193 adult CF patients provided at least one expectorated sputum sample for analysis. Complete clinical data was available for 73 pediatric and 191 adult patients. The characteristics of these patients are reported in Streptococcus, Prevotella, Rothia, Veillonella and Actinomyces were core genera in both groups, while Neisseria, Haemophilus and Gemella were unique core genera in pediatric samples and Pseudomonas was unique to the adult core microbiota. In both groups, the genera with the greatest prevalence also had the greatest mean relative abundance  was present in 45% of pediatric samples and 57% of adult samples. n adults . The medRothia, Prevotella, Gemella, Actinomyces and Veillonella were prevalent in both pediatric and adult samples, but were seldom or never the dominant genus. Pseudomonas was more prevalent in adults but tended to be dominant in both pediatric and adult samples in which it was present , Pseudomonas and Burkholderia, when dominant, accounted for a larger proportion of the community than Streptococcus in Streptococcus-dominant samples . Only five genera  comprised more than 90% of any sample, and only in samples from adult patients. present . StreptoP = 0.004, P = 0.002, Streptococcus dominance was associated with higher community diversity in both datasets  using unsupervised principal coordinates analysis (PCoA) of Bray-Curtis dissimilarity . The reldination . Samplesdination . This an1 was lower in older age groups, with the lowest median FEV1 in patients aged 30-34 years (10 ng/μL of 16S rRNA gene by quantitative PCR) was greatest at ages 20-24 .Patients were stratified into age groups and compared based on lung function, community diversity and dominant genera . As expe34 years . Bacteries 20-24 . Diversies 20-24 . In addint genus . If dich10 16s rRNA gene by quantitative PCR) and dominant taxa based on the strategy outlined in the methods section. The final model included age, BMI z-score, pancreatic insufficiency, SDI and Pseudomonas dominance , bacterial density . Given the highly variable bacterial community structure in CF which we and others56Furthermore, our analysis was not designed to assess the impact that rare or very low abundance taxa (<1% relative abundance) may have on the biology of the CF airway, but rather to assess trends in community structure over a broad range of ages and disease stage. Very low abundance and rare taxa may be critical determinants of pathological processes in the CF lung. Our observations highlight the heterogeneity between patients and confirm the need for longitudinal analysis in individual patients and groups of patients to identify factors associated with disease exacerbations, disease progression and response to treatment. Our method resolved community composition to the genus level only. Significant within-genus or intra-species variability in community composition may have important effects on community behavior and disease pathogenesis and has the potential to explain cross-sectional variability in disease status that is not apparent at the genus level.Despite these caveats, our data agree with prior reports of CF microbiota, and extend these observations to a broader age range of patients, particularly those less than 18 years of age, a group that is not well represented in previously published reports. Our principal findings agree with and extend those of smaller studies of mostly adult patients showing high inter-individual variability in community structure5616811121316171819Pseudomonas and Burkholderia, and mechanistic studies elucidating the interactions of these taxa with other colonizing organisms, as well as exploration of possible causal relationships between community diversity and disease manifestations would be illuminating.Longitudinal studies identifying microbial and host risk factors for infection with patho-adapted taxa such as 1), forced vital capacity (FVC), age, sex, disease stage, antimicrobial therapy, clinical status, cystic fibrosis transmembrane conductance regulator (CFTR) mutation, pancreatic status, and visit outcome were recorded for the day of sample collection. Expectorated sputa were collected for all patients - no oropharyngeal samples were used. Pediatric samples were split into aliquots, one for culture and one for nucleic acid extraction, while adults provided two samples in parallel at each clinic visit. Samples for sequencing were treated with sputolysin and frozen for subsequent processing. Quantitation of 16S rRNA gene copy number was performed by quantitative PCR based on published protocolsThis study received approval from the research ethics boards of both St. Michael’s Hospital and the Hospital for Sick Children in Toronto, Canada. Patients provided informed consent and were enrolled prospectively at The Hospital for Sick Children and St. Michael’s Hospital between 2010 and 2013. Height, weight, forced expiratory volume in one second (FEVPseudomonas aeruginosa controls)Sequencing was performed at the Centre for the Analysis of Genome Evolution and Function (CAGEF) at the University of Toronto as previously described using an Illumina GAIIx1 (% predicted) was built by entering all variables, then using backwards removal to identify variables in the final model.Statistical analyses were performed using SPSS version 22 (IBM). Means were compared using T-tests or ANOVA with Bonferroni correction as needed. Chi-squared tests were used to test proportions. A multivariate linear regression model predicting FEV1, and FVC were defined as previously reported32et al.www.cdc.gov/growthcharts/percentile_data_files.htm). For adults over 20 years old, the age values for BMI z score were calculated based on an age of 20et al.Disease stage, pancreatic insufficiency, FEVAll methods were performed in accordance with the approved guidelines.B.C., D.M.H. and D.S.G. drafted the manuscript. B.C. created all figures and tables. D.E.T., S.D., S.T.C., V.B., Y.C.W.Y., V.J.W. and Y.Z. enrolled patients, collected clinical data and samples, and processed samples for sequencing. P.W.W., A.S., Y.G., J.D.C., S.T.C. and B.C. sequenced samples and processed sequence data. B.C., J.D.C., A.S., Y.G., V.J.W., D.M.H. and D.S.G. analyzed and interpreted the data. B.C., Y.C.W.Y., V.J.W., D.M.H., D.S.G. conceived the study design. All authors reviewed the manuscript.How to cite this article: Coburn, B. et al. Lung microbiota across age and disease stage in cystic fibrosis. Sci. Rep.5, 10241; doi: 10.1038/srep10241 (2015).


Helicobacter pylori (H. pylori) infection and colorectal polyps are more prevalent in African Americans than in the general population. We aimed to investigate whether gastric H. pylori infection is associated with colorectal polyps in African Americans.Gastric H. pylori status was assessed by immunohistochemistry on gastric specimens. Colorectal polyps were confirmed by histological examination of colorectal biopsies. A subset of serum samples from healthy and polyp-bearing patients (n = 163) were analyzed by ELISA for anti-H. pylori and anti-CagA antibodies. The crude and adjusted effect of H. pylori on the risk of colorectal adenoma and polyp were computed by logistic regression models.Medical records of African Americans, 40 years and older (n = 1256) who underwent bidirectional gastrointestinal endoscopy on the same day were reviewed. H. pylori infected than non-infected subjects . Patients with H. pylori-associated chronic active gastritis were at high risk to have adenomas . There was no difference in histopathology, size, or location of polyps with respect to H. pylori status. Gastric H. pylori infection, age, male gender and high risk clinical presentations were independent risk factors for colorectal polyps. Serological testing also revealed a higher prevalence of H. pylori and its toxin Cag-A in polyp patients vs. non polyp patients’ sera, although in a non-statistically significant manner.The prevalence of colorectal polyps and adenomas were 456 (36%) and 300 (24%) respectively. Colorectal polyps were more prevalent in gastric H. pylori infection is associated with an increased risk of colorectal polyps in African Americans. Patients with H. pylori induced gastritis may benefit from early screening colonoscopy as a preventative measure for colorectal cancer.This study showed that current gastric  H. pylori’s infection to colorectal neoplasm either through high prevalence of H. pylori seropositivity among CRC or colorectal polyp patients [H. pylori in the stomach [Colorectal cancer (CRC) is the third most common cancer and the third most common cause of cancer deaths in both men and women in US . In its patients -14, or tpatients -18, whilpatients -22. More stomach  or colon stomach -29 with H. pylori predisposes to the development of gastric cancer precursor lesions, thus it has been classified as class 1 carcinogen [H. pylori on the gastrointestinal tract with diseases that are inversely associated with H. pylori, such as reflux disease, erosive oesophagitis, Barrett’s oesophagus, and oesophageal adenocarcinoma, showing a striking rise during the recent decline of H. pylori infection in the general population [H. pylori’s effect on gastric mucosa predicts its effect on colon mucosa is still controversial. Indeed, a recent meta-analysis of the correlation between H. pylori and extra-gastric malignancies revealed a modest statistically significant relationship of H. pylori infection with both colon cancer and polyps [H. pylori’s infection and colorectal lesions appear to be more common in African Americans compared to the Caucasian population in the US [H. pylori infection was associated with the presence of colorectal polyps in a population at high risk for colorectal lesions.It is well known that rcinogen . A recenpulation . Whetherd polyps . H. pylon the US . We sougH. pylori immunohistochemistry (IHC) status of gastric biopsies, histo-pathological diagnosis of gastric specimens, and colorectal polyps’ type, size, grade of dysplasia and location. We divided our patients into high and average risk for colorectal polyps based on their presentations [H. pylori immunohistochemistry examination of gastric biopsies.We retrospectively reviewed the medical records of 1920 patients of which 1256 were included in the present study. The 1256 retained records correspond to African American patients, 40 years and older who underwent bidirectional endoscopy (complete colonoscopy and gastroscopy) at the same day from January 2005 to August 2009. The study was conducted at Howard University Hospital, a tertiary hospital serving predominantly African Americans in the District of Columbia, USA. The study was approved by the Howard University Hospital Institutional Review Board and we obtained consent from patients who provided blood samples for the serological analysis. Demographic variables included gender, race and age. Clinical and pathological data were collected with respect to reasons for undergoing bidirectional endoscopies, ntations ,35. Highntations ,35, and/ntations . AverageH. pylori status was identified using immunohistochemistry staining on gastric biopsies. A Novocastra Liquid mouse monoclonal Anti-H. pylori antibody was used . An experienced gastrointestinal pathologist examined the specimens and made the histo-pathological classification of gastric biopsies and colorectal polyps. We classified H. pylori associated gastric lesions (independently of their distribution or severity) into chronic active (non-atrophic) gastritis, chronic atrophic gastritis with intestinal metaplasia, reactive gastropathy with foveolar hyperplasia, hyperplastic gastropathy and normal gastric mucosa [Gastric biopsies were taken during gastroscopy and were labeled as antrum, body, and fundus. Both gastric biopsies and colorectal polyps (when encountered) were harvested by biopsy, snare, piecemeal excision, or saline assisted endoscopic mucosal resection. Colorectal polyps were divided by location. Polyps located in cecum, ascending, and transverse colon were classified as “right sided”. Those located in descending colon, sigmoid, and rectum were classified as “left sided”. Patients with multiple polyps all over the colon were classified as having “both” right and left colon polyps. All specimens were sent to the pathology department after immersion in formalin. The colorectal polyps size was measured after tissue fixation. c mucosa . We exclc mucosa .H. pylori antibodies in serum using 96-well plates coated with H. pylori whole cell antigens, according to previously described methods [We determined the presence of anti- methods ,39. All  methods ,41. The  methods ,42.For the detection of antibodies against CagA, 96-well plates coated with recombinant CagA protein prepared according to specifications previously reported ,44. All t-test to compare the distribution of continuous variables between H. pylori positive and negative subjects. For categorical variables, we used Chi-square test. Then we computed unadjusted Odds Ratio (OR)  for potential predictors of colorectal polyps and adenomas. Multivariate logistic regression analysis was applied to compute the adjusted OR (95% CI) for predictors of colorectal polyps and adenomas. All variables with P < 0.2 from bivariate analysis were selected for multivariate logistic regression. The final model was developed with a stepwise backward approach. All variables with P value < 0.05 were considered statistically significant and remained in final model. All analyses were done using SPSS 17.0 .We used Student’s H. pylori IHC stain (n = 33) were excluded. The prevalence of H. pylori infection was 366/1256 (29.1%) while the prevalence of colorectal polyps and adenomas were 456 (36%) and 300 (24%) respectively. The frequency of males was 433 (34%). The frequency of colorectal adenomas increased with age . The same trend was observed for polyps . The mean age (SD) 57 years (9.6) was the same for H. pylori infected and non-infected subjects (p = 0.9). The frequency of male gender in H. pylori negative and positive patients were 32% and 41%, respectively (p = 0.004). The frequency of chronic active gastritis (p <0.001) and colon polyps (p = 0.001) were higher in H. pylori positive patients. Polyp histology, size and location were not correlated to H. pylori status  had colorectal polyps.Among 1920 potential participants, 1256 African Americans, aged forty or above, were eligible for this study. Non-African American patients (n = 100), as well as patients without bidirectional endoscopy (n = 385), patients with inflammatory bowel disease (n = 146) or lacking the H. pylori positivity (p = 0.003) and chronic active gastritis (p = 0.04) were significantly associated with higher frequency of adenoma , male gender (p < 0.001), ma Table .a. In a ps Table .b.H. pylori positivity (adjusted OR = 1.5) were independent risk factors for colorectal adenoma , male gender (adjusted OR = 1.7), and ma Table .a, with ma Table .b.Using combination criteria of baseline high risk clinical presentations- such as lower GI blood loss, abdominal mass and/or family/personal history of colorectal polyps or cancer, was more sensitive and specific than using each presentation alone in the prediction of colorectal polyps, but not adenoma Table . In multH. pylori and anti-Cag-A antibodies. In these two groups, 85 subjects (45%) were positive for anti-H. pylori. Of these 85 sera, 60 (71%) were anti-Cag-A positive. The anti-H. pylori positive number in non polyp patients were 40 (49%) while they were 45 (56%) in the polyp patients’ sera (p = 0.3) while anti-Cag-A positivity was 73% in polyp patients vs. 68% in non polyp controls (p = 0.5). Thirty three (55%) patients who were positive for both H. pylori and Cag-A had polyps while 33 (47%) patients of those negative for both H. pylori and Cag-A had polyps . While the p values were not statistically significant, the observed pattern of higher polyp prevalence in H. pylori/Cag-A patients is consistent with the overall epidemiological results.One hundred sixty three serum samples (including 81 polyp and 82 without polyps gender and age matched subjects) were analyzed for anti-subjects 5% were pH. pylori infection, confirmed by immunohistochemistry staining of gastric biopsies, associates with an increased risk of colorectal lesions in African Americans. The same association was true in 3 meta-analyses which included studies from different ethnic groups. In particular, Zumkeller et al. [H. pylori, and Zhao et al. [H. pylori to demonstrate previous H. pylori infection in patients with colonic lesions. Jones et al. [H. pylori do reside in the subjects’ colon biopsies and associates with colorectal neoplasm.Our study indicates that gastric r et al.  includedo et al.  includeds et al.  used immH. pylori positivity which is consistent with findings in other ethnic groups [H. pylori infection.Our previous report showed colorectal polyps’ incidence begins to increase significantly at age forty in African Americans  with or c groups . In thisH. pylori and colorectal adenoma which was not altered even after adjustment for confounding factors [Other confounders such as BMI, smoking, alcohol consumption, diabetes, socioeconomic status, diet and lack of physical activity are known risk factors for colorectal polyp/cancer. Our retrospective epidemiological study did not adjust for these factors because of lack of corresponding information. Previous studies reported an association between  factors ,50.H. pylori infection with colorectal polyps or cancer in African Americans. The higher prevalence of colorectal polyps in H. pylori infected patients and the higher seropositivity could be explained by their environmental and genetic differences as well. Potentially, such differences may alter host gastric and/or colorectal mucosa in response to H. pylori carcinogenic effect among some ethnic groups. For instance, Indians have a low rate of gastric cancer and high rate of H. pylori infection which was not significantly associated with intestinal metaplasia, gastric tumor site, and patient’s age [H. pylori infection with colorectal cancer among Finnish male smokers [To our knowledge, no previous study has linked nt’s age . Moreove smokers ,52.H. pylori positive subjects was found among those who had chronic active gastritis. Such polyps were more likely to be neoplastic (adenomatous). Due to the retrospective nature of the study, the time lapse between the H. pylori infection and the colonic lesions occurrence is unknown. Meira et al. [H. pylori, the chronic inflammation induced DNA damage in alkyladenine DNA glycosylase deficit mice and enhances inflammation-associated colon tumorigenesis. It also predisposed to the development of gastric cancer precursor lesions.High risk features when combined at presentation were more predictive for colorectal polyps, As such, they can be useful clinical criteria to prioritize access to colonoscopy. The risk prediction of colorectal polyps based on gastric lesions recovered from gastroscopy depend on the nature of such lesions. The highest prevalence of colorectal polyps among a et al.  reportedH. pylori positivity decreased and the risk of colorectal polyps increased, being highest in patients with chronic atrophic gastritis with intestinal metaplasia, but the number of associated colorectal polyps was too small to make meaningful conclusions. The annual progression from chronic non-atrophic gastritis to atrophic gastritis is 1 to 3% [H. pylori in advanced gastric lesions has probably decreased because of migration through the gastrointestinal tract [H. pylori status would be appropriate in the prediction of colorectal polyps [H. pylori in contrast to 6% positive normal tissues from cancer patients. This could be explained by migration phenomena too [H. pylori gastric effect could be similar to its colonic effect. Once H. pylori’s infection is established, it likely elicits robust and lasting inflammatory and immune responses that may potentially influence the development of diseases that occur later in life such as cancer.As gastric lesions progressed,  1 to 3% ,54. H. pal tract . This sul polyps ,56,57. Bl polyps  found onmena too  and woulH. pylori, the symptoms it may produce, and the associated gastric lesions with its extra-gastric colonic effects [H. pylori infection (gastric biopsy). We used immunohistochemistry to diagnose H. pylori in gastric biopsies as it is highly sensitive and specific particularly in patients who have been partially treated. Besides, we had a fairly large sample size (n = 1256). Moreover, the findings with this large sample were further confirmed serologically.The strengths of our study; are in its comprehensive nature since it linked the real time presence of gastric  effects . Gold stH. pylori’s prevalence and its associated gastric lesions in the prediction of colorectal polyps could be explained by gastric sampling errors and unknown received treatments. Also, our serology testing sampling was smaller (n = 163) than the sampling for the epidemiological study (n = 1256). Despite that, we were able to prove positive association between H. pylori and colorectal polyps consistent with the colon cancer mouse models studies [H. pylori infections.Because our study was retrospective and hospital-based rather than population-based, it has its own limitations. The underestimation of  studies . Also, wH. pylori infection were at high risk of neoplastic and non-neoplastic colonic lesions. H. pylori associated chronic active gastritis and alarming clinical features at presentation may necessitate early screening colonoscopy and/or H. pylori eradication. Prospectively designed studies are needed to establish the conditions in which the current H. pylori infection in gastric mucosal lesions might participate in the colon carcinogenic transformation, either through its colonization of the colon and/or through its metabolites and whether the eradication of H. pylori would reduce colon polyp incidence in African Americans in the future.In conclusion, forty years and older African Americans with gastric AA: African Americans; CRC: Colorectal cancer; IHC: Immunohistochemistry; GI: Gastrointestinal.The authors declare they have no competing interests.MZ and HB contributed equally in acquisition and interpretation of the data, EL contributed in pathology interpretation of results, DS and AOL contributed in sample recruitment, MN and HR performed the statistical analysis, GPP performed H. pylori Cag-A analysis, and HA designed and wrote the paper and submitted the manuscript for publication. All authors read and approved the final manuscript.HA is the director of microarray lab at Howard University Cancer Center.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/296/prepub


Fusarium graminearum to elucidate the significance of non-conserved regions.Genome comparisons between closely related species often show non-conserved regions across chromosomes. Some of them are located in specific regions of chromosomes and some are even confined to one or more entire chromosomes. The origin and biological relevance of these non-conserved regions are still largely unknown. Here we used the genome of F. graminearum harbours thirteen non-conserved regions dispersed over all of the four chromosomes. Using RNA-Seq data from the mycelium of F. graminearum, we found weakly expressed regions on all of the four chromosomes that exactly matched with non-conserved regions. Comparison of gene expression between two different developmental stages (conidia and mycelium) showed that the expression of genes in conserved regions is stable, while gene expression in non-conserved regions is much more influenced by developmental stage. In addition, genes involved in the production of secondary metabolites and secreted proteins are enriched in non-conserved regions, suggesting that these regions could also be important for adaptations to new environments, including adaptation to new hosts. Finally, we found evidence that non-conserved regions are generated by sequestration of genes from multiple locations. Gene relocations may lead to clustering of genes with similar expression patterns or similar biological functions, which was clearly exemplified by the PKS2 gene cluster.The genome of Our results showed that chromosomes can be functionally divided into conserved and non-conserved regions, and both could have specific and distinct roles in genome evolution and regulation of gene expression.The online version of this article (doi:10.1186/1471-2164-15-191) contains supplementary material, which is available to authorized users. Fusarium includes a large group of phytopathogenic fungi, each having a different or partially overlapping host range. F. graminearum is an important pathogen on wheat, barley and maize, causing Fusarium head blight[F. verticillioides mainly infects maize, causing rot and wilting[F. oxysporum causes disease on more than 100 different plant species, but specific strains usually infect only a single host species[forma specialis concept in F. oxysporum, where strains are categorized according to the host plant they infect, such as tomato or banana[F. oxysporum may also cause infections in humans[Fusarium species have been generated, including F. graminearum (strain PH-1), F. verticillioides (strain FGSC 7600), F. pseudograminearum (strain CS3096), F. solani (strain FGSC 9596), F. fujikuroi (strain IMI58289) and F. oxysporum f. sp. lycopersici strain 4287. In addition, 11 different F. oxysporum strains, including pathogens from different hosts as well as a biocontrol strain have been sequenced[F. fujikuroi is absent in the genome sequence of related species F. verticillioides, while 285 and 820 kb genome sequences at both ends of chromosome IV of F. verticillioides are absent in F. fujikuroi[F. pseudograminearum with F. graminearum showed that 89.8% of genomic sequence could be aligned at >70% nucleotide identity[F. oxysporum 4287 includes 11 core chromosomes and four lineage-specific (LS) chromosomes. LS chromosomes are specific to F. oxysporum strain 4287 and have no collinear chromosomes in either F. graminearum, F. verticillioides or other F. oxysporum strains. The LS chromosomes in F. oxysporum 4287 are suggested to have resulted from horizontal transfer from unknown sources[The genus d blight, 2, whil wilting. In cont species. This har banana. In addin humans. Genomic sources–14.F. verticillioides (11), F. oxysporum (15) and F. fujikuroi (12), F. graminearum has only four chromosomes, which probably resulted from fusions of ancestral chromosomes[F. graminearum isolate PH-1 with F. graminearum isolate GZ3639 revealed several regions with high SNP density[F. graminearum with the closely related species F. verticillioides and F. oxysporum revealed several non-conserved regions[F. graminearum to investigate the putative effect of gene locations (in conserved or non-conserved regions) on their expression patterns. In addition, by comparing the genome of F. graminearum with those of F. verticillioides and F. oxysporum, we show a possible mechanism for the generation of non-conserved regions.In contrast to the high number of chromosomes present in omosomes. Compari density. In addi regions, the oriFusarium graminearum, F. verticillioides and F. oxysporum.Synteny block analysis between the genomes of F. graminearum (Fg) and F. verticillioides (Fv), we performed synteny block analysis presented in Figure F. graminearum chromosome 1 (Fgchr 1) is largely collinear with F. verticillioides chromosomes (Fvchrs) 1, 5 and 8; Fgchr 2 is collinear with Fvchrs 6, 9, 10 and 11; Fgchr 3 is collinear with Fvchrs 2, 4 and 7; while Fgchr 4 is largely collinear with Fvchrs 2 and 3  values. The averaged expression levels of genes on chromosome 1 are significantly higher than other chromosomes, while the expression levels of genes on chromosome 2 are significantly lower   when compared with other parts of the same chromosome or with other chromosomes. In addition, four regions on Fgchr 1, three regions on Fgchr 3 and three regions on Fgchr 4 showed significantly lower gene expression levels. These gene expression patterns were observed for each of three biologically independent replicates  and Nc6 (5.44 Mb - 8.89 Mb) exhibited significantly lower expression levels , while for 4024 genes in F. graminearum, no conserved ortholog in F. verticillioides was identified . When calculating the number of non-conserved genes in each 20 kb window, we found that non-conserved genes are more abundant in chromosomal regions, where genes are weakly expressed . Compared to the other three chromosomes, Fgchr 2 contains a higher proportion of non-conserved genes   are located at one of the terminal regions of Fgchr 3.To investigate whether non-conserved genes show lower levels of expression, the expression levels of all conserved and non-conserved genes were categorized according to their RPKM values. On each chromosome, the conserved genes showed much higher gene expression levels than non-conserved genes Figure A. Furthe Figure B. In add Figure C, as gene et al. were ana Figure D. FurtheF. graminearum isolate PH-1 produced in mung bean medium. Three biologically independent replicates were analyzed and in total 40,615,406 reads were obtained. Gene expression patterns on each chromosome of F. graminearum were analyzed with the obtained RNA-Seq data. Firstly, similar gene expression patterns on each chromosome of F. graminearum were observed for the three biological replicates . However, some parts of non-conserved regions, especially in regions Nc4 and Nc7 showed expression levels similar to conserved regions  . Moreover, a higher proportion of genes encoding secreted proteins were observed on chromosome 2  . In contrast, ribosomal genes that are essential for growth are enriched in conserved regions  and fewer ribosomal genes are identified on chromosome 2 of F. graminearum .To further understand the biological relevance of non-conserved regions, we examined which categories of genes are enriched in the non-conserved regions. Firstly, we studied the distribution of all genes encoding secreted proteins in n et al. identifim genome. Distrib Figure A, suggesF. graminearum have homologs in F. verticillioides. To further demonstrate that genes with their putative homologs in F. verticillioides are likely to be true orthologs, the homologs in F. verticillioides were aligned against the genes in F. graminearum using BLASTn and we found that 9208 gene pairs are the reciprocal best blast hits, suggesting that they are orthologs  chromosomes[F. oxysporum with those of F. verticillioides through synteny analysis revealed several non-conserved regions  there is a strong correlation between the degree of gene conservation and gene expression level and (ii) genes in the non-conserved regions showed lower expression levels than genes in the conserved regions. In addition, comparing gene expression levels between conidia and mycelium, we found that the expression of genes in non-conserved regions is highly variable, while the expressions of genes in conserved regions is surprisingly stable. This indicates the expression of genes in conserved regions is less dependent on fungal development (e.g. in conidia vs. mycelium), while the expression of genes in non-conserved regions seems more developmentally regulated. Furthermore, house-keeping genes are more abundant in conserved regions, while genes required in specific developmental stages or environmental conditions are found more often in non-conserved regions. This conclusion was supported by the fact that genes encoding secreted proteins or involved in the production of secondary metabolites, which are induced under specific conditions[The development of RNA-Seq technology allowed nditions, 30–32, Aspergillus species. The rapid rearrangement of subtelomeric regions may promote the rapid evolution of these genes to become species-specific attributes[Verticillium dahliae, in planta-expressed genes are enriched in lineage-specific genomic regions that have developed by extensive chromosomal reshuffling, which was suggested to drive evolution of virulence[F. oxysporum, genes encoding secreted effectors and virulence factors are more abundant in LS chromosomes, while few house-keeping genes are identified on LS chromosomes[Gene enrichment in specific chromosomal regions has also been studied in other species. For instance, secondary metabolite genes are enriched in subtelomeric regions of tributes, 33. In irulence. In F. oomosomes. The driomosomes–38. Secoomosomes, 40, so F. graminearum and non-conserved regions could form specific sub-compartments of the nucleus with repressed gene expression.Our data indicate that genes in non-conserved regions are weakly expressed, but how the expression of these genes is suppressed is still unknown. Previous studies have shown that gene position in the nucleus is associated with their transcriptional regulation, for instance, the nuclear periphery was considered as a zone for transcriptionally repressed genes. This tyF. graminearum, in contrast to conserved regions. Gene relocations have been described previously in S. cerevisiae where the DAL gene cluster, including six genes, was formed by gene relocations from six different loci[F. graminearum and F. sporotrichioides, trichothecene biosynthesis requires genes at three loci: the 12-gene TRI cluster, a second locus with two genes (TR1 and TRI16), and a third locus with one gene (TRI101). However, in the more distantly related species F. equiseti, both TRI1 and TRI101 are located within the TRI core cluster suggesting that the latter two genes have been relocated during evolution of F. equiseti[F. graminearum. However, how these genes are marked for relocation is still obscure. Based on our findings that genes with low similarity to their homologs have low expression levels  are significantly abundant in non-conserved regions. This type of genome arrangement may not only facilitate the co-regulation of specific sets of genes, but could also enable fungi to maintain on the one hand the required conservation of house-keeping genes and on the other to accelerate the evolution of species-specific genes to rapidly adapt to new environments or new hosts. Moreover, due to the selective transcription of genes in non-conserved regions, this could prevent organisms spending too much energy in transcription and translation of evolving genes that do not have acquired full functionality yet.Taken together, our data demonstrate that chromosomes of Fusarium graminearum wild-type isolate PH-1 was used in this study. To prepare conidia and mycelium for RNA isolation, PH-1 was grown in 400 ml liquid mung bean medium for 3 days to produce conidia . The conidia were collected by centrifugation. 10e5 conidia of PH-1 were transferred to 400 ml liquid complete medium and subsequently incubated for 30 h to produce mycelium . Mycelium was harvested from liquid CM medium by filtration and grounded in liquid nitrogen using a mortar and pestle. The conidia and mycelium were used for RNA extraction using TRIzol reagent  according to manufacturer’s instructions. The quality of RNA was analyzed by Agilent 2100. RNA-Seq was performed according to protocols described previously[eviously.F. graminearum, F. verticillioides and F. oxysporum were downloaded from Broad Fusarium Comparative Database. The gene database (fasta file format) of each Fusarium species were imported into CLC, and “BLASTn” option was used to align the genes of F. graminearum against the genes of F. verticillioides or F. oxysporum using default settings. Genes of F. graminearum were grouped according to their p value after aligning with the genes of F. verticillioides by using BLASTn.Gene homology was evaluated in CLC genomic workbench. The gene database of Fusarium species was based on two criteria: (i) a cutoff of p value of 1E-5, and (ii) reciprocal best blast hits. For the genes in conserved chromosomal regions, synteny block analysis was also explored for the definition of orthologous relationship.The identification of orthologs between MCScanX with a small modification[F. graminearum, F. verticillioides and F. oxysporum were downloaded from the Broad Fusarium database. The local blast database pools of F. verticillioides and F. oxysporum were created by using program formatdb. All genes in F. graminearum were analyzed against the gene database pool of F. verticillioides and F. oxysporum, respectively, by using BLAST tool. The BLAST results were exported in m8 format. Besides, gff file containing the information of the chromosome number (e.g. Fg1), gene name (e.g. FGSG_00001), start position and stop position of each gene on each chromosome of Fusarium species was prepared. The BLAST file and gff file were imported for synteny block analysis according to the procedure described in the manual of MCScanX. The criteria for the synteny analysis are as follow: match score 50, match size > 5, gap_penalty −1, overlap_window 5, max gaps 25. Finally, two types of output  were obtained by using two downstream programs.Synteny block analysis was performed according to the program fication. The genF. graminearum genes with high similarity  in F. verticillioides or F. oxysporum were collected, from which we selected genes that have their putative orthologs on non-collinear chromosomes of F. verticillioides or F. oxysporum. The map of these genes to their orthologs was performed by using MCScanX software[F. verticillioides or F. oxysporum were collected and matched with all predicated genes in F. graminearum using BLASTn tool to identify the best hits to show that they are the bidirectional best hits.According to the BLASTn result, all software. FurtherF. graminearum using software available in the CLC Genomics Workbench. The RNA-Seq reads were mapped to each chromosome by using “RNA-Seq analysis” option with default settings. The number of reads matched to each chromosome was calculated and subsequently the expression level of each chromosome was evaluated by using RPKM (reads per kilobase per million mapped reads) values. Similarly, to evaluate the expression of each gene, the transcript database of F. graminearum were imported in CLC and the expression level of each gene was evaluated by RPKM value.To evaluate gene expression along chromosomes, RNA-Seq reads were mapped to chromosome sequences of To draw the gene expression level along each chromosome, we divided the chromosomes into portions of 20 kb. The read coverage of each 20 kb window was calculated and log2-transformed reads coverage in each window was used to compare gene expression levels. To compare the gene expression level between conidia and mycelium, reads coverage of each window was compared and log2-transformed reads coverage fold change was used to evaluate gene expression differences. The total gene number and the number of non-conserved genes in each 20 kb window were calculated manually based on the criteria described above.To analyze the expression levels of conserved and non-conserved genes, the transcript sequences of conserved and non-conserved genes were collected and assembled, respectively. Also in this case the PRKM value was used to evaluate the expression levels of conserved and non-conserved genes. Box plot analysis of gene expression was performed by using SPSS software.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55477).The data sets supporting the results of this article are included within the article and its additional files. Six Illumina sequence data used in this study are available in the NCBI GEO repository Additional file 1: Genome-wide gene expression analysis. Excel file showing the expression of all predicted genes in both conidia and mycelium of Fusarium graminearum . (XLS 2 MB)Additional file 2: Gene expression pattern analysis using RNA-Seq data obtained from mycelium ofF. graminearum. Each chromosome of F. graminearum was divided into 20 kb windows. For each window, the log2-transformed reads coverage was drawn to show gene expression patterns along each chromosome of F. graminearum. The gene expression patterns analyzed by three biologically independent RNA-Seq data obtained from mycelium of F. graminearum are shown. Green boxes represent non-conserved regions identified in F. graminearum. (PDF 146 KB)Additional file 3: Putative orthologs ofFusarium graminearumgenes inF. verticillioidesandF. oxysporum. All predicted genes of F. graminearum were matched to their orthologs in both F. verticillioides and F. oxysporum by using BLASTn. (XLS 2 MB)Additional file 4: Distribution of genes on each chromosome ofF. graminearumaccording to their similarity to genes inF. verticillioides. Genes on each chromosome were divided into five groups according to their degree of similarity. Genes with a low degree of similarity to their orthologs are enriched in weakly expressed regions, while genes with a high degree of similarity to their orthologs are enriched in highly expressed regions. Green boxes represent non-conserved regions identified in F. graminearum. Note the absence of conserved genes  at the telomeric region of Chr3, corresponding to Nc10. (PDF 182 KB)Additional file 5: Gene expression pattern analysis using RNA-Seq data obtained from conidia ofF. graminearum. Each chromosome of F. graminearum was divided into 20 kb windows. For each window, the log2-transformed reads coverage was drawn to show gene expression patterns along each chromosome of F. graminearum. The gene expression patterns analyzed by three biologically independent RNA-Seq data obtained from the conidia of F. graminearum are shown. Green boxes represent non-conserved regions identified in F. graminearum. (PDF 144 KB)Additional file 6: Comparison of the expression of relocated and non-relocated genes. All genes that have orthologs in F. verticillioides were divided into two groups: genes that are relocated to non-conserved regions and genes that are not relocated. Box plot analysis shows that the expression levels of relocated genes in non-conserved regions (B) are lower than non-relocated genes (A). Asterisk indicates significant difference . (PDF 41 KB)Additional file 7: LS chromosomes inF. oxysporumare enriched for gene relocations. Genes that match their orthologs on non-collinear chromosomes of F. verticillioides are distributed on the chromosomes of F. oxysporum. Four LS chromosomes, Fo3, Fo6, Fo14 and Fo15, show multiple gene relocations. In addition, the telomere proximal regions of the core chromosomes, especially chromosome 1 and 2, show multiple gene relocations. (PDF 420 KB)Additional file 8: Group of neighboring genes on LS chromosomes ofFusarium oxysporumthat are collinear with their homologs inF. verticillioides.(DOC 98 KB)Additional file 9:Synteny block analysis of each chromosome against other chromosomes inF. oxysporum. The genomic sequence of each chromosome was used to compare other chromosomes by using program MCScanX. Genomic sequence duplications were identified between four LS chromosomes. Remarkably, LS chromosome 15 is duplicated from the telomere proximal region of core chromosome 1. (PDF 338 KB)Additional file 10:


Spinacia oleracea L.), which originated in central and western Asia, belongs to the family Amaranthaceae. Spinach is one of most important leafy vegetables with a high nutritional value as well as being a perfect research material for plant sex chromosome models. As the completion of genome assembly and gene prediction of spinach, we developed SpinachDB (http://222.73.98.124/spinachdb) to store, annotate, mine and analyze genomics and genetics datasets efficiently. In this study, all of 21702 spinach genes were annotated. A total of 15741 spinach genes were catalogued into 4351 families, including identification of a substantial number of transcription factors. To construct a high-density genetic map, a total of 131592 SSRs and 1125743 potential SNPs located in 548801 loci of spinach genome were identified in 11 cultivated and wild spinach cultivars. The expression profiles were also performed with RNA-seq data using the FPKM method, which could be used to compare the genes. Paralogs in spinach and the orthologous genes in Arabidopsis, grape, sugar beet and rice were identified for comparative genome analysis. Finally, the SpinachDB website contains seven main sections, including the homepage; the GBrowse map that integrates genome, genes, SSR and SNP marker information; the Blast alignment service; the gene family classification search tool; the orthologous and paralogous gene pairs search tool; and the download and useful contact information. SpinachDB will be continually expanded to include newly generated robust genomics and genetics data sets along with the associated data mining and analysis tools.Spinach ( Spinacia oleracea L.), which originated in central and western Asia, belongs to the family Amaranthaceae. Spinach is particularly resistant to cold, has wide adaptability, and is cultivated worldwide, mostly in temperate regions. As one of most important leafy vegetables, spinach has a high nutritional value, and is rich in carotene, vitamin C, amino acids and iron, among others [http://faostat3.fao.org).Spinach  [Spinach possesses a small genome, thus is suitable for basic genomic studies, and many physiologically important genes have been cloned from the species . Recentlssembled ,18. The The workflow was, essentially, gene annotation, SSR and SNP detection, and expression profiling of genes using spinach genomic and transcriptomic data. This was followed by pooling the data into a database and providing the researcher with a user-friendly website interface .Beta vulgaris Resource website (http://bvseq.molgen.mpg.de/Genome/Download/Spinach/), we downloaded the Spinach genome assembly version 1.0.1 and annotation version SpiSet-1 (August 2014) for further analysis. RNA-seq raw data were also downloaded in the NCBI SRA (Sequence Read Archive) database (http://www.ncbi.nlm.nih.gov/sra) [www.uniprot.org) [http://www.genome.jp/kegg) [http://www.ncbi.nlm.nih.gov/COG) [−5. SSRs were identified using the MIcroSAtellite (MISA) identification tool (http://pgrc.ipk-gatersleben.de/misa/) based all the spinach genome sequences. For mono-, di-, tri-, tetra-, penta- or hexanucleotide SSRs, the minimum number of units were set to be 10, 6, 5, 5, 5 and 5 repeats, respectively. The software Primer3 (http://bioinfo.ut.ee/primer3/) was used to design potential primers for each SSR, with default parameters following the previous rules [Since the spinach genomic data was available in The gov/sra) . All of 1 August 14 for fujp/kegg) , and KOGgov/COG)  databaseus rules .http://pfam.xfam.org) on September 26, 2015 [Arabidopsis, rice, grape, poplar, tomato, apple, soybean and sugar beet were also downloaded. The proteins of Arabidopsis and sugar beet were downloaded from TAIR 10 (http://www.arabidopsis.org) and The Beta vulgaris Resource website respectively; the others were downloaded from the Phytozome portal  [http://www.hmmer.org, v3.1b1) [-5. The output of all identified gene families was classified according to the species. The software orthoMCL  [Arabidopsis, grape, rice, sugar beet and spinach.The HMM (Hidden Markov Models) models of gene families were downloaded from the Pfam database   was usedS. oleracea , 3 from S. tetrandra  and 3 from S. turkestania . The fastq-dump program in the SRA Toolkit was used to release fastq format files from SRA files; then NGS QC Toolkit  was used to obtain high quality reads which contained at least 90% bases beyond Phred-Qual 20 [https://ccb.jhu.edu/software/tophat/index.shtml, v2.1.0) [http://cole-trapnell-lab.github.io/cufflinks, v2.2.1) that under the guidance of annotated gene models with a GFF file [http://www.broadinstitute.org/gatk/, v3.5) [http://sourceforge.net/projects/seqgene/, v2.5) [Thirteen SRA files were used to characterize the gene expression profiles and for SNP mining of spinach, including 7 from cultivated -Qual 20 . The pro v2.1.0)  was usedGFF file . Meanwhihttp://www.centos.org) was installed on an HP blade server machine with 80 threads and 700 GB memory. The MySQL database server  was installed for storing all genomic and website data. The Apache web server with PHP (v5.3.3) was used for powerful website service, and the software Joomla  was used for user-friendly website interface construction. Some frequently used bioinformatics programs like GBrowse  and wwwblast  were installed to provide user-friendly search capability for genes of interest. Some Perl and CGI (Common Gateway Interface) scripts were built within to provide additional search services.Linux system CentOS6.6 (http://222.73.98.124/spinachdb). The The genomic and transcriptomic data of spinach are publicly available in SpinachDB  predicted 40309 genes, among which only 21702 were high-evident ones that were collected for the dataset used for final gene assembly. To functionally categorize these genes, there were 15550, 18864, 18659, 7956, 18634 genes assigned annotation after aligning with Swiss-Prot, TrEMBL, KEGG, KOG, NR and NT databases, respectively  while the genome sizes of these two species were similar. A collection of 13229 SSRs that fit the definition of a compound SSR were found in this study. Among all of the SSRs, the Ahttp://www.nipgr.res.in/ngsqctoolkit.html) [Recently, biology has become a data intensive science because of huge data sets produced by high throughput molecular biological experiments in the field of genomics, transcriptomics, proteomics, and metabolomics. These data is useful for constructing molecular biological networks . To charit.html) . After thttps://www.broadinstitute.org/gatk/) and SOAPsnp (http://soap.genomics.org.cn/). Although DNA-oriented sequencing reads were regularly used for SNP prediction for better results, RNA-oriented sequencing reads such as RNA-seq have also been performed occasionally, but increasingly, in many studies. In some studies, the RNA-seq data and EST sequences were also used to predict potential SNPs. The subsequent SNP validation experiment supported the use of RNA-seq and also EST data for contribution to SNP identification. The validation of selected high-confident SNPs in tea plant [SNP detection is usually performed by mapping the DNA sequencing reads to the referent genome sequences with pipelines and software, such as GATK  and 83.9% (Arabidopsis). All five of these species were flowering plants and the orthology they shared consisted of 64184 orthologous gene pairs in 8441 groups. The four eudicots shared more orthologous genes, while rice, the monocotyledonous species, shared the fewest orthologs with the others. The distribution of orthologous gene pairs was totally consistent with the genetic relationship. The species in the same family shared the largest number of orthologous gene pairs, and then with other eudicots, and the least with the monocot  [In order to explain a possible usage for these classified gene families, the genome-wide identification and analysis of BES1/BZR1 in spinach were performed. The BES1 gene family is a class of plant-specific transcription factors that play a key role in the Brassinosteroid signaling pathway. The BES1 gene interacts with the basic helix-loop-helix protein BIM1 to synergistically bind to E box (CANNTG) sequences present in many BR-induced promoters . In thisre.net/)  was usedGBrowse, developed by the Generic Model Organism Database Project, is the most widely used genomic browser among organism databases, because the browser is both universal and customizable. It is possible to quickly and accurately locate regions of interest in spinach genes, and to display sequences and annotations visually. In SpinachDB, the GBrowse tool is highly configurable and portable; users can view eight data tracks including Gene, mRNA, CDS (Coding Sequence), SSR, SNP, 6-frame translation, the DNA/GC Content, and restriction sites of the spinach genome. The annotation and expression of genes that were measured using the FPKM method were integrated inside GBrowse, to provide users a way to comparably study gene function and expression in different samples. Furthermore, all of the useful SNP data are also available in GBrowse, allowing users to view the SNP information from cultivars of interest and referent species. This is robust tool for breeders to identify markers that may relate to traits of interest.All sequences and annotations of spinach genome, genes, proteins and ESTs were deposited in SpinachDB. SpinachDB provides a BLAST search interface which allows users to perform sequence similarity searches against the spinach assembled genome, genes, cDNA, CDS, EST and proteins. Since the annotations using NR, KEGG, KOG, Swiss-Prot and TrEMBL databases were deposited in SpinachDB, users can also perform keyword searches in SpinachDB, which will obtain a list of annotated genes with the specific keyword. Similar searches could also be performed to search orthologs, paralogs and gene families.S1 File(XLSX)Click here for additional data file.


Lactobacillus delbrueckii subsp. bulgaricus CRL871 is the first strain of L. delbrueckii subsp. bulgaricus reported as a folate-producing strain. We report the draft genome sequence of L. delbrueckii subsp. bulgaricus CRL871 . This strain is of great biotechnological importance to the dairy industry because it constitutes an alternative to folic acid fortification. Lactobacillus delbrueckii subsp. bulgaricus is a lactic acid bacterium (LAB) that is widely used as a starter culture in the dairy industry for the manufacture of fermented milk products, such as yogurt, when combined with Streptococcus thermophilus. Historically, L. delbrueckii subsp. bulgaricus was described as a folate consumer that normally took up folates produced by S. thermophilus. L. delbrueckii subsp. bulgaricus CRL871, isolated from an artisanal yogurt in the northwestern region of Argentina, showed the ability to produce folate when grown in folate-free culture medium  strategy  with an Ion Torrent personal genome machine (Life Technologies). Quality filtered reads were assembled using the Ngen (DNASTAR) assembler, giving 63 contigs that are more than 2,000 bp in length. Genome annotation was done using the NCBI Prokaryotic Genome Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/) and from the Rapid Anotations using Subsystems Technology (RAST) server , 1,932 genes with predicted function, 122 structural tRNAs, and 39 rRNA were predicted. Additionally, there are 264 RAST subsystems represented in the chromosome, which represent only 47% of the assigned sequences.L. delbrueckii subsp. bulgaricus CRL871 showed the ability to synthetize folates when grow in a folate-free culture medium (L. bulgaricus showed sequences coding enzymes responsible for folates synthesis. However, none of these strains have been described as folate producers. In silico genomic analysis of L. delbrueckii subsp. bulgaricus CRL871 revealed the presence of folA, folC, folP, and phoA genes, which are responsible for de novo synthesis of folates. The presence of these genes together with the ability to synthetize folate gives this strain a very important biotechnological potential in the dairy industry for the development of yogurt naturally bio-enriched in folate, as was recently reported (As was previously mentioned, e medium . Genomesreported , 6.L. delbrueckii subsp. bulgaricus CRL871 whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. JXRV00000000. The version described in this paper is version JXRV01000000.The


Lolium perenne L.) is one of the most important species for temperate pastoral agriculture, forming associations with genetically diverse groups of mutualistic fungal endophytes. However, only two taxonomic groups (E. festucae var. lolii and LpTG-2) have so far been described. In addition to these two well-characterised taxa, a third distinct group of previously unclassified perennial ryegrass-associated endophytes was identified as belonging to a putative novel taxon (or taxa) (PNT) in a previous analysis based on simple sequence repeat (SSR) marker diversity. As well as genotypic differences, distinctive alkaloid production profiles were observed for members of the PNT group.Perennial ryegrass . In addition, an endophyte-specific gene (perA for peramine biosynthesis) and the fungal-specific MT genes for mating-type control were included. The results supported previous phylogenomic inferences for the known species, but revealed distinctive patterns of diversity for the previously unclassified endophyte strains, which were further proposed to belong to not one but two distinct novel taxa. Potential progenitor genomes for the asexual endophytes among contemporary teleomorphic  species were also identified from the phylogenetic analysis.A detailed phylogenetic analysis of perennial ryegrass-associated endophytes using components of whole genome sequence data was performed using complete sequences of 7 nuclear protein-encoding genes. Three independently selected genes , none of which have been previously used for taxonomic studies of endophytes, were selected together with the frequently used ‘house-keeping’ genes MT gene idiomorphs further supported a predicted independent origin of two distinct perennial ryegrass-associated novel taxa, designated LpTG-3 and LpTG-4, from different members of a similar founder population related to contemporary E. festucae. The analysis also provided higher resolution to the known progenitor contributions of previously characterised perennial ryegrass-associated endophyte taxa.Unique taxonomic status for the PNT was confirmed through comparison of multiple nuclear gene sequences, and also supported by evidence from chemotypic diversity. Analysis of The online version of this article (doi:10.1186/s12862-015-0349-6) contains supplementary material, which is available to authorized users. Lolium, which belongs to the sub-family Pooideae (cool-season grasses) of the grass and cereal family Poaceae, includes several important forage and turf species [Lolium perenne L.) is extensively cultivated for pasture production on a global basis [Neotyphodium), but in accord with general recommendations for fungal taxonomy, have been recently been combined with the sexual (teleomorphic) taxa within a single genus, designated Epichloë [Epichloë endophytes colonise the intercellular spaces of leaf sheaths, culms, and rhizomes, and infrequently the surface of leaf blades, without inducing obvious pathological symptoms [Epichloë species [The genus  species ,2. Perenal basis ,4. Like al basis -7. The aEpichloë . Asexualsymptoms . These asymptoms ,11. The  species , but in  species .Epichloë species form mutualistic associations with their hosts [Asexual ir hosts ,14. Beneir hosts ,16. The ir hosts -19. Convir hosts ,21.Epichloë festucae var. lolii  Bacon and Schardl syn. N. lolii  Glenn, Bacon and Hanlin [Lolium perenne taxonomic group 2 (LpTG-2) [Epichloë endophytes are proposed to have evolved either by direct evolution from a single teleomorphic species, probably due to loss of the sexual state, or through interspecific hybridisation events between either sexual species or distinct sexual and asexual lineages, the latter generating heteroploid genetic constitutions [E. festucae var. lolii has been identified as a direct derivative of Epichloë festucae Leuchtm., Schardl & M. R. Siegel while the heteroploid LpTG-2 arose as an interspecific hybrid between E. festucae var. lolii and E. typhina (Pers.) Tul. & C. Tul. [Perennial ryegrass has been found to be host to two distinct fungal endophyte taxa: d Hanlin  and Loli(LpTG-2) . Distincitutions -27. The  C. Tul. ,26.Epichloë species have largely been based on the use of partial sequences of genes encoding highly conserved proteins such as tefA, tubB and act1 (actin), including both relatively invariant coding sequences and more diverse intronic regions [perA gene, which is responsible for peramine production, has been identified as potentially exhibiting a higher rate of molecular evolution than ‘house-keeping’ genes, permitting resolution of close taxonomic relationships [perA, along with several of the more conserved genes, have previously been used to study relationships between endophytes of perennial ryegrass and members of the closely related grass genus, Festuca (fescues) [Previous phylogenetic characterisation studies of both sexual and asexual  regions ,28,29. Iionships . Gene sefescues) ,31, inclfescues) . In addifescues) . Due to fescues) . HoweverEpichloë endophytes [E. festucae var. lolii and LpTG-2, a third distinct group of previously unclassified endophytes was identified as belonging to one or more PNT. However, SSR variation, although indicative, is of only limited value for definition of taxonomic groupings, due to the generation of data suitable for phenetic, rather than phylogenetic, analysis. Nonetheless, putative novel taxa of fescue-derived heteroploid endophytes that were predicted on the basis of SSR-based phenetic dendrograms [Molecular genetic markers based on sequence polymorphism may also be used to address issues of endophyte diversity, taxonomy and phylogeny . A largedophytes . In prevdophytes . In addidrograms  were demdrograms .E. festucae var. lolii and LpTG-2, and the uncharacterised PNT, as well as several putative ancestral sexual Epichloë species. The phylogenetic analysis was based on 7 full-length gene sequences derived from whole genome sequence datasets, including tefA, tubB and perA which were previously used for a corresponding study of fescue-derived endophytes [MT) genes, which play an important role in evolution of fungal species through control of sexual compatibility, were also included.The present study describes the evolutionary relationships between 19 representative perennial ryegrass-associated endophyte strains belonging to the known taxa dophytes . In addidophytes . Glycosidophytes . MEAB isdophytes . FinallyF. rubra ssp. commutata Gaudin). However, E1 was shown to be capable of stable establishment after introduction into multiple genotypes of perennial ryegrass through artificial inoculation into regenerating meristem-derived callus [E. festucae var. lolii and LpTG-2, and so was included in this study, but is nonetheless distinct in genetic terms [A total of 19 endophyte strains that form associations with perennial ryegrass were used for the phylogenetic analysis  DNA sample preparation kit  were used to generate assemblies of ryegrass-associated endophyte genomes. Low-quality sequence reads were filtered out using a custom python script, which calculates quality statistics and stores trimmed reads in several fastq files. Genome assemblies of endophytes C09, E1, NEA4 and NEA11 were obtained through VelvetOptimiser (version 2.2.0) using default parameters except for hash length (K-mer sizes) which was in the range of 31 to 71. Previously generated genome assemblies from other endophyte strains ,41 were tefA, tubB, perA, DEAD/DEAH helicase, glycosyl hydrolase, MEAB protein and MT genes in each endophyte genome were initially identified through nucleotide BLAST  (version 2.2.25) analysis [tubB: AY722412 from E. festucae Fl1; tefA: FJ660614 from E. festucae E2368; perA: AB205145 from E. festucae Fl1; glycosyl hydrolase, family 92 protein: XM_006669157.1 from Cordyceps militaris CM01; DEAD/DEAH box helicase (Sbp4): XM_007820576.1 from Metarhizium anisopliae ARSEF 23; MEAB protein: XM_007825059.1 from M. anisopliae ARSEF 23; MT genes: FJ717711.1 (mtAA), HQ680588.1 (mtAB), HQ680587.1 (mtAC) from E. festucae E2368 and HQ680590.1 (mtBA) from E. festucae Fl1. Following identification of contigs and gene coordinates for selected genes within the genomes of the haploid strains (E. festucae var. lolii and PNT) using corresponding reference genes, the contig and subsequently, the relevant gene sequence was extracted using fastacmd (version 2.2.24) (www.ncbi.nlm.nih.gov) and EMBOSS extractseq (version 6.3.1) (gwilliam@hgmp.mrc.ac.uk), respectively. A similar approach was used to extract corresponding gene sequences from the genomes of E. typhina 9340, 9636 and E. baconii 9707 based on the relevant databases. Data for representative Epichloë species isolates was extracted from genome sequence data available at http://www.endophyte.uky.edu/ [Presence and copy number of analysis  using couky.edu/ .LpTG-2), due to the presence of two copies for each gene. Corresponding gene sequences of the predicted progenitors, E. festucae var. lolii (SE) and E. typhina (9636) were used as references for mapping of all sequencing reads from LpTG-2 strains using Bowtie (version 1.0.0) (Langmead). Mapped reads were assembled using velvet (version 1.1.06) [LpTG-2 strain.A different approach was used to extract genes from heteroploid strains ( 1.1.06)  using thSequences of all selected endophyte strains were aligned using MUSCLE, built-in version of MEGA (version 5.2.2) . AlignmeLpTG-2 strains were appended with the suffixes GC1 and GC2, reflecting phylogenetic affinity with E. festucae var. lolii and E. typhina, respectively. Megablast searches [tubB, tefA and perA sequences were performed in GenBank nucleotide sequence database of the National Centre for Biotechnology Information (NCBI) to identify the closest matching sequences of Epichloë species, which were added to the sequence alignment. Accession numbers for GenBank-derived sequences are provided in each phylogram next to the isolate name.For the sake of clarity, gene copies of searches  using tuMT, genes were concatenated in the order of tefA, tubB, perA, DEAD/DEAH helicase, glycosyl hydrolase and MEAB protein into a multi-gene alignment which was then aligned using MUSCLE. Phylogenetic topology was constructed using the same methods as described above.Phylogenetic analysis was also performed with the concatenated sequences. The sequences of all, except Statistics for genome assemblies of perennial ryegrass-associated endophyte strains are provided in Additional file Epichloë species. Single copies of each gene were identified for all isolates of E. festucae var. lolii and PNT, reflecting haploid genome structures. In contrast, two copies of each gene were observed for both representatives of taxon LpTG-2. Complete copies of the perA gene were identified in E. festucae var. lolii and LpTG-2 strains, but all PNT representatives showed the presence of a 1223 bp deletion at the 3’-end of the gene. Furthermore, apart from strain E1, all PNT strains contained a premature stop codon at coordinate 2923 bp within the perA gene sequence.BLASTN analysis, as described, was used to identify the presence of the 7 selected gene sequences in the genomes of all candidate strains and reference sexual MT genes, the phylogram structure revealed three major distinct sequence clades. The single gene copies from E. festucae var. lolii and GC1 from LpTG-2 were located in the first clade, while LpTG-2 GC2 was located in the second clade. For all phylograms, the third clade contained single gene copies from PNT representatives, except for that generated from tefA sequences, in which strain E1 was grouped with the E. festucae var. lolii strains in clade 1. The separation of the three clades was supported by >80% bootstrap values in all instances (results not shown). For LpTG2-derived sequences, lower variability was observed between GC1 representatives from the alternate strains than for those from GC2.Phylograms were constructed on an individual gene basis using the ML and NJ methods. In all instances, except for the Epichloë species, and phylograms were constructed using both ML and NJ methods. All trees resulting from phylogenetic analysis using each method were congruent in topology, which indicated similar evolutionary relationships in respect of both E. festucae var. lolii and LpTG-2 .The significant levels of divergence between clades suggest different evolutionary origins. In order to identify potential progenitor genomes among contemporary teleomorphic taxa, phylogenetic analysis was repeated to include representative isolates of  Figures , 5 and 6E. festucae var. lolii and PNT-derived strains were most closely related to those of strains of E. festucae. However, contrasting patterns of affinity with specific E. festucae isolates were observed between the different selected genes. In the tubB-specific phylogram, E. festucae strain E2368- and E. festucae Fl1-derived sequences exhibited a similar degree of affinity to those of each of the haploid taxa. In contrast, in the perA-specific phylogram, E. festucae var. lolii-derived sequences were grouped more closely with E. festucae Fl1, while PNT-derived sequences were located closer to E. festucae strain E2368. Furthermore, strain E1 formed a sister group with E. festucae strain E2368. In the phylograms derived from the glycosyl hydrolase, MEAB protein and DEAD-box helicase genes, both E. festucae Fl1 and E. festucae strain E2368 were closer to the PNT genotypes. Finally, a higher similarity between E. festucae var. lolii and both E. festucae strains (E2368 and Fl1) was observed in the tefA-based phylogenetic tree.In general, the single gene copies characteristic of LpTG-2 endophyte contained two distinct copies for each gene, reflecting the hybrid origin of this taxon. While GC1 was closely related to those of E. festucae var. lolii, GC2 showed affinity to the corresponding sequences from multiple isolates of E. typhina related sequences. In all individual gene-based phylograms (with the exception of that derived from the glycosyl hydrolase), as well as the concatenated gene-based tree, LpTG-2 GC2 was more closely related to E. typhina E8 (strain ATCC_200736) and E. typhina 9636 than the two other E. typhina strains, 9340 and E5819. Furthermore, in the tubB- and MEAB protein-specific phylograms, GC2 of LpTG-2 strain NEA4 was closer to E. typhina E8, while that of NEA11 was closer to E. typhina 9636. In contrast, in the DEAD box-based phylogram, NEA11 GC2 was closer to E. typhina E8, while NEA4 GC2 was closer to E. typhina 9636.The genomes of the heteroploid MT idiomorphs, MTA  and MTB (mtbA), revealed the presence of a single idiomorph in all strains. MTB was detected for all E. festucae var. lolii and PNT strains, with the single exception of E1, which possessed all three genes corresponding to mating type idiomorph MTA. Two copies of mtbA were identified for each of the two LpTG-2 strains  were concatenated and analysed by ML and NJ methods, providing strong bootstrap support for all major clades that were defined by single gene-based phylogenetic analysis  have been previously described. Limited sequence diversity for all selected genes was detected within each taxon, as would be expected for asexual species [E. typhina displayed higher levels of variation, as is further supported by possession of different MT genes and substantial differences between mitochondrial genomes [In the present study, sequence diversity of multiple independently selected genes was used to identify the phylogenetic affinities between perennial ryegrass-associated endophytes, and to interpret potential progenitor relationships with contemporary sexual  species . In cont genomes ,30.E. festucae var. lolii and LpTG-2 have been identified in several previous phylogenetic studies. The most common endophyte of perennial ryegrass, E. festucae var. lolii, has been proposed as a direct haploid descendant of E. festucae, while the heteroploid LpTG-2 was suggested to have evolved through hybridisation of E. festucae var. lolii and E. typhina [E. festucae strains that were used, Fl1 appears to be more closely related to E. festucae var. lolii strains based on the tubB- and perA-specific phylograms, while both strains exhibited similar levels of affinity to E. festucae var. lolii in the tefA-derived tree.The identities of progenitors for  typhina ,47. EquiE. typhina E8, which was isolated from perennial ryegrass, provided the closest match to LpTG-2 GC2 in the phylograms based on both individual and concatenated gene sequence, consistent with a previous study [E. typhina lineages to the origin of LpTG-2 strains, rather than a monophyletic event. Further evidence for complexity in the evolution of LpTG-2 is apparent from the MT gene-based analysis. Of the E. typhina strains that were subjected to analysis, E. typhina 9340 and E. typhina MAFF (AB161000.1) display the MTB idiomorph, while the opposing (MTA) mating type was observed in E. typhina E8. Hence, despite the close relationships between E. typhina E8 and LpTG-2 GC2 revealed by analysis based on other nuclear genes, a lineage similar to, but distinct from E. typhina E8 must have been the direct ancestor of the LpTG-2 strains included in the present study.The origin of heteroploid endophytes may be verified by comparison with multiple candidate progenitor species . E. typhus study . The higMT genes. The PNT individuals were separated from the other two taxa with strong bootstrap support in all phylograms that included sexual Epichloë reference isolates, except for the moderate value obtained for the tubB-based tree. A similar difference between confidence of structure obtained with individual genes was also observed during phylogenetic analysis of annual ryegrass-derived endophytes based on tubB and nuclear ribosomal DNA internal transcribed spacer (rDNA-ITS) sequences. In this analysis, many branches of the rDNA-ITS-based phylogram displayed a lower bootstrap support than the tubB-based phylogram. The discrepancy was interpreted in terms of homoplasy effects and/or the lower number of informative characters that are available from rDNA-ITS gene sequence [Similar patterns of relationships between the three distinct clades were apparent for all independently selected gene sequences, apart from the sequence . SimilartefA gene, and account for the anomalous location of strain E1 in the respective phylogram. Equivalent patterns of relationships have been reported in other phylogenetic analysis studies. For instance, the tefA gene of the meadow fescue-derived endophyte N. uncinatum was most closely related to that of E. bromicola, but the single tubB gene showed a closer relationship to that of E. typhina [Convergence of gene structure between otherwise distantly related genotypes may also apply to the highly conserved  typhina . To addr typhina . InteresE. festucae var. lolii, PNT strains exhibit the closest relationships to E. festucae among the available contemporary sexual Epichloë species. However, in respect to strain diversity within E. festucae, different relationships were observed across different genes. On the basis of the primary concatenated phylogram, the PNT group showed a closer relationship to E. festucae 2368, while E. festucae var. lolii was grouped with E. festucae Fl1. However, this seems likely to be due to the influence of the perA gene, which contributes a length of c. 7.3 kb that is common to all strains in the study, close to the combined length of the other 5 genes (c. 10.3 kb). In the absence of the perA gene, members of the PNT group seem to exhibit a slightly higher affinity to both E. festucae isolates  of an E. festucae-like progenitor which had already undergone the perA deletion, leading to establishment of two parallel lineages of asexual endophyte, further modification of perA being confined to the LpTG-3 genomes. Consequently, it is further proposed that although E1 did not originate from a perennial ryegrass host, it is provisionally assigned (pending further investigation) to a fourth taxonomic grouping of perennial ryegrass-associated endophytes as a sister group to LpTG-3, to be designated LpTG-4. As both of the proposed new taxa are capable of producing the novel alkaloid class of epoxy-janthitrems, which display valuable feeding deterrence effects toward invertebrate herbivores, the definition of genomic properties of such taxa is capable of assisting future targeted endophyte discovery programs.Although the endophyte strains located within the PNT group are phylogenetically distinct from both http://purl.org/phylo/treebase/phylows/study/TB2:S17154) [The data sets supporting the results of this article are included within the article and its additional files. All gene sequences have been deposited in GenBank and sequence alignments are availabe in the TreeBASE repository :S17154) . Details . Detail