The significance of different ages of perianal disease (PD) onset in patients with perianal Crohn’s disease (PCD) remains unknown. We aimed to investigate the impact of paediatric-onset PD (POP) and adult-onset PD (AOP) on the Crohn’s disease (CD) course in a Chinese cohort.The medical records of diagnosed PCD patients from 2008 to 2018 were reviewed retrospectively. The cumulative incidence and predictors of intestinal resection were calculated using the Kaplan–Meier and logistic regression analysis.p = 0.011) and infliximab (IFX) treatment  were more common among the POP patients . A younger PD onset age  and shorter PCD diagnostic delay  was found in the POP cohort. AOP patients  had a higher rate of current smoking , stricturing behaviour  and intestinal resection . The cumulative probability of intestinal resection in AOP patients was higher than that in POP patients (p = 0.007). In multivariable analysis, AOP , stricturing behaviour  and rectal inflammation  were predictive factors for CD-related intestinal resection in all PCD patients. AOP patients with complex perianal fistula  and POP patients with rectal inflammation  were more likely to suffer intestinal resection. The IFX administration significantly decreased the rate of intestinal resection in AOP patients .Complex perianal fistulas (71.7% vs 50.0%, The AOP patients have more complicated luminal disease and higher rate of intestinal resection than COP patients. The perianal diseases onset-age can provide clinical treatment guidance for individual management of CD patients. Crohn’s disease (CD) is a multifactorial systemic inflammatory bowel disease (IBD) with a heterogeneous clinical course. Perianal disease (PD) is a common complication in CD patients and can lead to a low quality of life, a high rate of surgical interventions, and challenging therapeutic situations –3. PatiePatients with PCD usually have a higher risk of abdominal surgery. However, this increased risk only reached statistical significance in patients who developed PD during follow-up, but not in patients who presented with PD before or at luminal CD diagnosis . This fiWe retrospectively evaluated 747 consecutive CD patients in the Tenth People’s Hospital affiliated with Tongji University  from January 2008 to December 2017. Eligible patients were confirmed CD patients having PD involvement during the disease course. Both the PD presented before and after diagnosis of CD were included in the study. The diagnosis of CD in paediatric patients was based on the Porto criteria . PD was The medical records were reviewed for sex, PD onset age, PD type, age at CD diagnosis, age at PCD diagnosis, PCD diagnostic delay, clinical manifestations, Montreal classification, smoking status, family history of IBD, CD-related medications and surgical interventions . CD-related medical treatment included antibiotics (imidazole and quinolones), immunosuppressants (azathioprine and methotrexate), corticosteroids and IFX. IFX was regularly administered intravenously at a dose of 5 mg/kg body weight at 0, 2, and 6 weeks and then every 8 weeks. The duration of IFX treatment was calculated from the first infusion to the last infusion within the follow-up period. The PSIs included perianal abscess incision and drainage, seton insertion, fistulectomy, de-functioning surgery, and proctectomy. CD-related abdominal surgery in this study mainly referred to CD-related intestinal resection, including the partial ileal resection, partial colon resection, ileocolonic resection.POP was defined as PD with an onset age of less than 18 (< 18) years, and AOP was defined as PD with an onset age of greater than or equal to 18 (≥ 18) years . PerianaThe clinical outcomes of PD were categorized as clinical remission, non-remission and recurrence. Clinical remission was defined as follows: (1) no perianal symptoms ; (2) closure of the external opening of perianal fistulas; (3) no visible internal openings, perianal abscesses or anal ulcers; and (4) absence of drainage despite gentle finger compression. Non-remission referred failure to achieve clinical remission after a certain therapeutic strategy or maintain clinical remission for less than 3 months. Recurrence was defined as the presence of new PD after 3 months of clinical remission.p value < 0.2 in the univariate analysis by the enter method . Kaplan–Meier analysis was applied to calculate the cumulative probabilities of IFX treatment and CD-related intestinal resection during the 10-year follow-up period. A log-rank comparison was used to compare the cumulative probabilities between the two groups of patients with POP and AOP. Spearman correlation coefficients were calculated between the rates of CD-related intestinal resection and the percentages of patients treated with IFX. Two-sided p values < 0.05 were considered statistically significant. SPSS 22.0 software  was used for statistical analysis.Categorical variables are described as counts with percentages and were assessed by the chi-square test or Fisher’s exact test. Continuous variables are described as the mean ± standard deviation (SD) or median plus interquartile range (IQR) and were analysed by the independent sample t-test or the Mann–Whitney test. The factors associated with CD-related surgery were analysed by univariate and multivariate analyses. Multivariate logistic regression analysis was performed for factors with a Among 747 well-characterized CD patients, 293 (39.2%) patients with perianal involvement were enrolled. The median follow-up duration among the 293 PCD patients was 72 IQR: 36–108) months. Two hundred and four (69.6%) patients had the one type of perianal diseases, 68 (23.2%) having two types of PD, and 17 (5.8%) patients having three and over three types of PD during the disease course. Perianal fistula was the most common PCD , with complex phenotypes in 57.1% (93/163) of cases. One hundred forty-one (48.1%) PCD patients had perianal abscesses, and 51 (17.4%) had haemorrhoids. Anal ulcers, fissures and rectovaginal fistula and were seen in only 5.1% (15/293), 4.4% (13/293) and 3.4% (10/293) of PCD patients, respectively. No cases of cancer were identified during follow-up. The mean onset age of PD was 25.9 ± 11.3 years old. The AOP cohort comprised 209 (71.3%) patients, and the POP cohort included 84 cases (28.7%). There was a gradual increase in the diagnosis of POP in PCD patients from 2008.1 to 2017.12 but a reduction in the diagnosis of patients with AOP during the same period  and at onset of PD  was significantly younger than that among AOP patients (Table p = 0.033) was shorter in the POP cohort. Compared to AOP patients, POP patients presented with a higher proportion of complex perianal fistulas  and non-stricturing and non-penetrating (B1) behaviour . Stricturing type (B2) behaviour  and current smoking  were more common among AOP patients than POP patients (Table The baseline characteristics of these two groups of patients were summarized in Table p = 0.609). No significant difference in the disease location, PSI frequency, PSI type or family history of IBD was observed between the two groups of patients , then followed by IFX treatment after PSIs , IFX monotherapy , antibiotic monotherapy  and IFX combined with immunosuppressants . Two hundred forty-six PCD patients achieved clinical remission of PD after initial treatment, including 71 POP patients (84.5%) and 175 AOP patients (83.7%). Of the 246 patients who achieved clinical remission, 60 (24.4%) patients experienced PD recurrence during the follow-up period, including 21  POP patients and 39  AOP patients. The types of PD experiencing recurrence included 11 (52.4%) perianal abscess, 9 (42.9%) perianal fistulas, one (4.8) anal fissure, but no rectovaginal fistula in 21 POP patients. These frequencies in 39 AOP patients were 19 (48.7%), 19 (48.7%), 0 (0%) and 1 (2.6%), respectively  receiving IFX treatment for active PD  after the first diagnosis of PCD. Those PCD patients requiring IFX only for luminal disease were excluded. Among them, 30 patients received IFX monotherapy, 36 patients received IFX combined with PSI, and 8 patients were treated with both IFX and immunosuppressants. The average number of IFX infusions was 7.3 ± 0.5.p = 0.044, Table p < 0.01, Table p < 0.001, Fig. p > 0.05, Table The proportion of patients treated with IFX was significantly higher in the POP group  suffered CD-related intestinal resection after the first diagnosis of PD. The percentage of patients who underwent surgery was significantly higher in the AOP cohort , stricturing behaviour  and presence of rectal inflammation  were associated with a higher risk of CD-related intestinal resection among the 293 PCD patients. Other factors, including sex, age at CD diagnosis, PCD diagnostic delay, current smoking status, PD type and disease location, were not associated with CD-related intestinal resection (p > 0.05). Furthermore, AOP patients with complex perianal fistulas were more likely to undergo CD-related intestinal resection  had increased risk for CD-related intestinal resection  but not in POP patients .From 2008.1 to 2017.12, the proportion of patients receiving IFX treatment increased in both the POP and AOP groups Fig. . The occThe presence of PD in CD patients usually indicates increasing hospitalization, progression to more complicated disease phenotypes, and high rate of intestinal resection in some studies about Western patients , 23, 24.Previous studies have shown that approximately one-quarter of CD patients are diagnosed in childhood . PaediatMoreover, we found that PCD patients with POP had earlier use of IFX and a higher cumulative risk of IFX treatment. In the past decade, IFX was the only available biological agent for Chinese CD patients . Due to The presence of PD speeds up the complexity of the natural history of CD, accelerates the development of luminal stenosis, and increases the risk of intestinal resection in some studies of the pre-biologic era , 23, 24.In addition, smoking has been reported as a definite factor associated with recurrence after surgery and a poor response to medical therapy in CD patients . Thus, tOne of the strengths of our study is the inclusion of all kinds of PCD, leading to a reduction in selection bias. The different clinical characteristics and outcomes of PCD patients with POP and AOP were directly compared in this study. We focused on the influence of PD onset age on the rate of intestinal resection in CD patients, and found that there was a higher risk of intestinal resection in PCD patients who developed PD at adults. These findings should alert physicians to pay close attention to these patients to ensure that they receive intensive treatment and close luminal monitoring to achieve better outcomes in clinical practice.However, there are some limitations to our study. The first limitation is the retrospective and single-centre design. Secondly, all of the PCD patients included in our study were hospitalized patients, who may have had relatively serious manifestations. Lastly, some CD patients developed PDs in an early time without more detailed records of PSIs. We failed to provide a more specific description of PSIs. So, we can’t analyse the influence of different PSIs types on the perianal outcome, which was still needed to be further studied and confirmed.In summary, POP and AOP play different roles in pathogenesis and progression of CD. POP showed more complicated perianal lesions than AOP. The clinical impact of PD on the luminal damage of CD patients may be more prominent in patients with AOP than those with POP. Early diagnosis and IFX treatment, as well as closely endoscopic evaluation in CD patients with AOP may therefore allow for earlier identification of potential CD-related luminal damage to reduce the incidence of intestinal resection.


The rise of Multidrug-resistant organisms (MDROs) poses a considerable burden on the healthcare systems, particularly in low-middle income countries like Pakistan. There is a scarcity of data on the carriage of MDRO particularly in the pediatrics population therefore, we aimed to determine MDRO carriage in pediatric patients at the time of admission to a tertiary care hospital in Karachi, Pakistan, and to identify the risk factors associated with it.Extended Spectrum Beta-Lactamase (ESBL) producers, Carbapenem Resistant Enterobacteriaceae (CRE), Vancomycin Resistant Enterococci (VRE), Methicillin Resistant Staphylococcus aureus (MRSA), Multidrug-resistant (MDR) Acinetobacter species and MDR Pseudomonas aeruginosa), nasal swabs and rectal swabs or stool samples were cultured on specific media within 72 h of hospitalization. Data was collected on a predesigned structured questionnaire on demographics, prior use of antibiotics for > 48 h in the last 6 months, history of vaccination in last 6 months, exposure to health care facility regardless of the time of exposure, ICU stay for > 72 h, and about the prior use of medical devices  in last 1 year. Statistical analysis was performed by Standard statistical software.A cross-sectional study conducted at the pediatric department of Aga Khan University Hospital (AKUH) from May to September 2019 on 347 children aged 1–18 years. For identification of MDRO  collected were positive for MDRO. The most isolated species were ESBL Escherichia coli 174/222 (78.3%) followed by ESBL Enterobacter species 37/222 (16.7%) and ESBL Klebsiella pneumoniae 35/222 (15.8%). On univariate analysis, none of the risk factors showed statistically significant association with MDRO carriage.Out of 347 participants, 237 (68.3%) were found to be MDRO carriers. Forty nine nasal swabs from 346 children (14.2%) showed growth of MRSA. The majority of the stool/rectal swabs (Overall, a high prevalence of MDRO carriage was identified among admitted pediatric patients. Implementation of systematic screening may help to identify true burden of MDROs carriage in the health care settings. The rapidly emerging multidrug-resistant organisms (MDROs) associated infections with meager and scarce availability of better therapeutic options are among the prioritized global health concerns . Based oStaphylococcus aureus, Enterococci species, members of family Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter species [The spectrum of hospital acquired multidrug bacteria include  species . Althoug species . Besides species , 6.P. aeruginosa [Enterobacteriaceae strains [vancomycin resistant enterococci infections in hospitalized children also increased from 53 per million in 1997 to 120 per million in 2012.Over the past two decades, the strains of MDROs have quadrupled particularly among children and cause serious bacterial infections (SBIs) including bacteremia, meningitis, bone and joint infections, urinary tract infections (UTI), gastroenteritis, and skin or soft tissue infections , 8. Natiruginosa  and a si strains , 12. TheEnterobacteriaceae [Streptococcus pneumoniae was reported 5% among healthy Thai children [Acquisition of these drug resistant bacteria in infants is either through the mother or from the community . A Reporeriaceae . Colonizchildren .The colonization locations include body sites normally inhabited by indigenous human microbiota or in chronic wounds, renal calculi, devitalized bones, and indwelling foreign bodies . Left unThere are studies regarding MDROs associated infections in Pakistani adults –22, but We conducted a cross-sectional study including all pediatric patients aged 1–18 years admitted to the pediatric ward at the Aga Khan University Hospital (AKUH), Pakistan, from May to September 2019. Participants were screened for identification of MDRO carriers including MRSA, VRE, CRE and ESBL-producing gram negative bacteria within 72 h of admittance. Patients with a documented history of culture proven MDRO infection or carriage during the previous 12 months of admission, diagnosed cases of primary or secondary immunodeficiency, and those who refused to participate in the study were excluded.This study was approved by the Ethical Review Committee (ERC) of AKUH (2019–0653-2209). All methods including enrolment of patients, data collection, microbiological and statistical analysis were in accordance with the guidelines and regulations of institutional ERC. The sample size was calculated by OpenEpi. Assuming a rate of MDRO carriers in the general population as 30%, 5% bound on the error, 5% significance level, the maximum sample size was 323. Sample size was also calculated for associated risk factors such as prior use of antibiotics, prior hospitalization, ICU stay etc. by taking frequency of these factors among non-MDRO carriers between 30% and 50%, 80% power, 5% significance level, and ratio of-MDRO carriers to non-MDRO carriers as 1:2, with a prevalence ratio (PR) of 2.00 and 5% attrition, the sample size estimated was at least 323.Enterobacteriaceae, carbapenem resistant Enterobacteriaceae, vancomycin resistant enterococci, MDR Acinetobacter species, and MDR P. aeruginosa within 72 h of hospitalization for the detection of these pathogens. Patient was identified as MDRO carrier if a  was positive, and the patient was without systemic infection caused by these organisms.Data was collected aftertaking consent from parents or guardians of patients, on demographics, prior use of antibiotics for > 48 h in the last 6 months, history of vaccination in last 6 months, exposure to health care facility regardless of the time of exposure, ICU stay for more than 72 h, and about the prior use of medical devices  in last 1 year. We cultured the nasal swab for MRSA, and either rectal swab or stool specimen for ESBL enterococci were confirmed as E. faecalis or E. faecium by biochemical test and API strep kit test. These isolates were confirmed as vancomycin resistant by vancomycin disc on SB-Mueller-Hinton agar (SBMHA) plate and isolated on 6mcg/ml vancomycin plate with negative and positive control.For the detection of VRE, rectal swabs/stool specimens were streaked directly onto selective agar plates and incubated aerobically for 24 h. Isolated colonies of Swabs were also inoculated into a broth medium containing 6 mcg/ml vancomycin (Enterococcosel). Broth with color change from amber to black after 24–48 h incubation at 37 °C was then sub-cultured on blood agar and selective VRE media and incubated overnight at 37 °C. For further confirmation of isolated colonies API strep kit test and vancomycin disc on Mueller Hinton Agar (MHA) media was performed.2 incubator. A Swab was also inoculated into 2.5% NaCl BHI (Brain Heart Infusion) broth and after incubation at 37 °C for 24 h; it is inoculated onto colistin nalidixic acid agar (CNA), MSA, and SBA plates. The plates were incubated at 37 °C for 24 h. Morphologically resembling S. aureus was confirmed by tube coagulase (for secreted extracellular coagulase), DNase agar, and phenolphthalein phosphate agar. Broth or saline suspension of colonies confirmed as S. aureus was prepared and adjusted to achieve turbidity of 0.5 McFarland standards. Sensitivity testing carried out by disc diffusion method against cefoxitin 30 mcg disc susceptibility breakpoints for cefoxitin against S. aureus and was interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines.For identification of MRSA, nasal swabs plated directly onto sheep blood agar (SBA) and mannitol salt agar (MSA) and incubated aerobically for 24 h in a COEnterobacteriaceae was performed by disc diffusion method by using combination of clavulanic acid (10 μg) and ceftazidime (30 μg). Both discs were placed on Muller Hinton agar plates which were earlier swabbed by respective culture and incubated for 24 h at 37 °C. More than 5 mm increase in the zone diameter for ceftazidime-clavulanic acid was considered positive ESBL production. Phenotypic disc diffusion method with Imipenem disc (10 μg) on Muller Hinton agar plates and incubated for 24 h at 37 °C were used for detection of carbapenem resistance in Enterobacteriaceae (zone size ≤19 mm), Acinetobacter species (zone size ≤18 mm), and P. aeruginosa (Zone size ≤15 mm). Identification of XDR S. Typhi was done using conventional biochemical methods i.e., TSI, SIM agar, and confirmed with the help of serotyping  and API 20E (Biomerieux).Rectal swabs and stool specimens were inoculated into BHI broth and after overnight incubation at 35 ± 2 °C with ambient air, vortexed, and subcultured 100 μl onto MacConkey and CNA agar plate with vancomycin disc placed on 1st streak of CNA. Followed by overnight incubation, all gram negative bacilli were identified by conventional biochemical identification method that includes oxidase test, triple sugar iron (TSI) agar, Sulphide indole motility (SIM) agar, citrate agar, urea agar, Pseudomonas agar P, and Pseudomonas agar F. Identification of ESBL producing p-value ≤0.25 was considered significant. The results are reported as crude PR with 95% confidence intervals.Statistical analysis was performed on Stata version 12.0. Descriptive statistics were reported as frequencies with percentages for categorical variables like gender, prior use of antibiotics, prior hospitalization, etc., and mean ± SD was reported for age (years). Univariate analysis was performed by Cox proportional hazards algorithm, . The common underlying diagnosis of the participants were 48.7% infections followed by 17.3% gastrointestinal and 12.7% respiratory illnesses (Table E. coli 174/222 (78.3%) followed by ESBL Enterobacter species 37/222 (16.7%) and ESBL K. pneumoniae 35/222 (15.8%)  fulfilled the eligibility criteria. 350 (66.3%) consented to participate and were enrolled , ESBL enterobacter species (16.7%), and ESBL K. pneumoniae (15.8%). These observed percentages were also higher than those previously reported from other countries such as Guinea-Bissau (ESBL E. coli 47.7% and K. pneumoniae 15.8%), Madagascar (ESBL E. coli and K. pneumoniae 36.9%), Lebanon (ESBL Enterobacteriaceae 24.9%), and Thailand (ESBL E. coli 67.5% and K. pneumoniae 19.4%) [In this study, we did a systematic screening of Pakistani children to assess MDRO carriage amongst them. The overall MDRO carriage in our study was found to be very high i.e. 68.3% as compared to China where overall MDRO prevalence is 36% . The proe 19.4%) –28.Streptococcus pneumoniae (S. pneumoniae) were significantly associated with carriage of resistant S. pneumoniae [The risk factors reportedly associated with MDRO carriage include age, increased usage of antibiotics , prior eeumoniae . Anothereumoniae . On the eumoniae . Similareumoniae , 32.Poor hygiene and sanitary conditions likely contribute as a factor for the high percentage of MDRO carrier in our region. Our findings also raise the concern of asymptomatic carriage of MDRO in the community. This increases the risk of dissemination of resistance genes by human-to-human transmission or by contamination of the environment . AlthougOur study is not free of limitations. The current data from a single-center study may not reflect the prevalence of MDRO in Pakistan as a whole. Therefore, a multicenter study is needed to compare different healthcare facilities across urban and rural settings of our region to get a better understanding of the situation. As this study was observational, there might be a recall bias which may explain the lack of a significant association between prior antibiotic use and MDRO carriage. Furthermore, the narrow time frame of this study restricted how much the effect of climatic conditions on MDRO carriage could be researched.Regardless of all these limitations, the current study is of great importance as to the best of our knowledge it is the first study of our region that presents data on MDRO carriage in pediatric patients.High prevalence of MDRO carriers in children poses a great public health concern, especially in developing countries like Pakistan where the health care system is already over-burdened. Our findings call for the systematic screening of patients at the time of admission as well as in communities to identify asymptomatic carriers who act as disseminators, in order to find out true burden of MDROs. There should be more targeted epidemiologic and genotypic studies to provide insights on MDRO acquisition and carriage in children.


Senescence represents the last stage of flower development. Phosphorylation is the key posttranslational modification that regulates protein functions, and kinases may be more required than phosphatases during plant growth and development. However, little is known about global phosphorylation changes during flower senescence.In this work, we quantitatively investigated the petunia phosphoproteome following ethylene or air treatment. In total, 2170 phosphosites in 1184 protein groups were identified, among which 2059 sites in 1124 proteins were quantified. To our surprise, treatment with ethylene resulted in 697 downregulated and only 117 upregulated phosphosites using a 1.5-fold threshold (FDR < 0.05), which showed that ethylene negatively regulates global phosphorylation levels and that phosphorylation of many proteins was not necessary during flower senescence. Phosphoproteome analysis showed that ethylene regulates ethylene and ABA signalling transduction pathways via phosphorylation levels. One of the major targets of ethylene-induced dephosphorylation is the plant mRNA splicing machinery, and ethylene treatment increases the number of alternative splicing events of precursor RNAs in petunia corollas.Protein dephosphorylation could play an important role in ethylene-induced senescence, and ethylene treatment increased the number of AS precursor RNAs in petunia corollas.The online version contains supplementary material available at 10.1186/s12870-021-03286-x. Petunia hybrida) flower life and changed the transcript profile of flowers [Senescence represents the last stage of flower development , 2 and i flowers . Differe flowers , 14.Phosphorylation is a highly dynamic and reversible PTM that plays an important role in plant growth and development, and it is involved in many signalling events and controlled by the activity of a large number of kinases (phosphorylation) and phosphatases (dephosphorylation) . GeneralGlycine max SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (GmSARK) and its Arabidopsis homologue AtSARK regulate leaf senescence through synergistic actions of ethylene and auxin [Oryza sativa) [Several key kinases showed a key role in modulating leaf senescence. ENHANCED DISEASE RESISTANCE1 protein kinase negatively regulates ethylene-induced senescence in an ETHYLENE INSENSITIVE2 (EIN2)-dependent manner , 19. ARAnd auxin . The ACT sativa) .Dephosphorylation by protein phosphatases functions as a balancing switch to reverse the effects of phosphorylation by protein kinases. Protein phosphatases also play an important role in the regulation of leaf senescence. In Arabidopsis, the PP2C family protein phosphatase SENESCENCE-ASSOCIATED GENE113 (SAG113) is involved in the control of water loss during leaf senescence . The proPetunia has become a model plant for flower senescence research  because Here, parallel to the proteome and ubiquitylome response to ethylene treatment in petunia corollas previously reported by our group , an iTRA− 1 ethylene for 16 h, the tandem mass spectra of proteome data of petunia corollas were searched against the petunia genome (https://solgenomics.net/organism/Petunia_axillaris/genome), and these tandem mass spectra were also previously searched against these sequences of the transcriptome (SRA accession: SRP077541) that was previously constructed by our group to analyse the petunia proteome [P < 0.05), and a high degree of repeatability was observed . The corollas were treated with air or 2 μl l− 1 ethylene for 16 h and used for the phosphorylation analysis. To increase the accuracy of phosphorylation, three biological replicates were analysed for each sample. Altogether, 2170 phosphosites in 1184 protein groups were identified , Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein domain and subcellular localization analyses  from corollas treated with and without ethylene were assigned GO annotations. In the cellular component category Fig. , both thIn the biological process analysis, several of the downregulated phosphoproteins were highly enriched in protein modification processes, translational initiation and phosphorus metabolic process regulation. Proteins with upregulated phosphosites were highly enriched in transmembrane transport and sucrose metabolic processes Fig. , indicatThe molecular function analysis shows that proteins with catalytic activity and binding were enriched in proteins with downregulated phosphosites, which is consistent with the impact of ethylene treatment on protein modification and transcription regulation described above Fig. . ProteinThe KEGG pathway analysis of the quantitatively changed proteins undergoing phosphorylation showed that the spliceosome pathway was enriched in proteins with downregulated sites. The starch and sucrose metabolism pathway was enriched in proteins with upregulated sites Fig. . These rFor the protein domain analysis, we observed that proteins containing an RNA recognition motif domain, nucleotide-binding alpha-beta plait domain, ABC transporter-like, ABC-2 type transporter, Sec16, central conserved domain, calreticulin/calnexin, initiation factor eIF-4 gamma, zinc finger, and histidine kinase domain were enriched in proteins with downregulated phosphosites. Proteins containing cation-transporting P-type ATPase, P-type ATPase, transmembrane domain, P-type ATPase, sucrose synthase, actin cross-linking, glycosyl transferase of family 1, and transmembrane domain of amino acid transporter were enriched in upregulated quantiles  and 7 pThr motifs  were identified in our petunia dataset at different abundances  were upregulated ; 69 phosphosites on 43 kinases were downregulated and only 10 sites on 9 kinases were upregulated (Supplementary data Table ACS sequences were recovered when the Arabidopsis AtACS1 sequence was used as a query in BLAST searches of the Petunia axillaris draft genome sequence v1.6.2 (https://solgenomics.net/organism/Petunia_axillaris/genome)  oxidase (ACO) and ACC synthase (ACS) are two key enzymes that catalyse the last steps of the ethylene biosynthetic pathway. Eleven The phosphosites of 3 ethylene receptors, PhETR2  (subfamilies 2), were identified and significantly downregulated by more than 2.00-fold by ethylene in vivo. One phosphosite of the raf-like kinase PhCTR1  was identified. Senen phosphosites in petunia ethylene-insensitive protein 2  were identified, and the phosphorylation levels of six sites  were significantly decreased  after ethylene exposure  and the auxin efflux transport protein PIN  decreased after ethylene treatment  has not been reported in plants. SR and hnRNP proteins are alternative splicing regulators. Phosphorylation regulation of SR proteins plays an important role in spliceosome assembly and in regulating SR protein recycling in the cell . In thisIn addition to the arginine/serine-rich protein mentioned above, another eight phosphoproteins involved in RNA transport were identified , alternative exon ends , approximate MIR , multi-IR , approximate IR , intron retention , approximate MSKIP , multi-exon SKIP , approximate SKIP , skipped exon , alternative 3′ last exon  and alternative 5′ first exon . The results showed that the ethylene treatment increased the total number of all types of alternative splicing of mRNA  system , 64. In Previous studies suggested that ethylene treatment promote ethylene autocatalysis and accelerated flower senescence in petunia and carnation . In addiIn this study, a global analysis of phosphoproteome regulation by ethylene and further insights into the dynamics of individual phosphosites were provided. Our results revealed the negative regulation of global phosphorylation by ethylene and showed that protein dephosphorylation may play an important role in ethylene-induced corolla senescence in petunia. Our results revealed the important role of phosphorylation in hormone signal transduction and transport, ion transmembrane transport and remobilization of essential nutrients during ethylene-induced senescence. For the first time, our results show that the spliceosome pathway was enriched in proteins with downregulated sites, indicating the involvement of phosphorylation in RNA metabolism during ethylene-induced senescence. The provided data set may serve as an important resource for the functional analysis of phosphorylation of serine, threonine and tyrosine and facilitate a better understanding of the senescence process in this model petunia.The seeds of petunia ‘Mitchell’ were acquired from the lab of Manzhu Bao . Petunia ‘Mitchell’ plants were grown under greenhouse conditions (22–25 °C) . To prev− 1 ethylene (treatment group) and air (control group) for 16 h. Then, corollas from 8 to 10 flowers were collected, frozen in liquid nitrogen, and stored at − 80 °C for the following experiments. Three biological replicates were performed.Petunia flowers were treated with ethylene according to our previously defined protocols . FlowersPetunia hybrida was ground in liquid nitrogen, and then the cell powder was transferred to a centrifuge tube and sonicated three times on ice in lysis buffer with a high-intensity ultrasonic processor (Scientz). Petunia corollas were ground in liquid nitrogen, and then the cell powder was transferred to a centrifuge tube and sonicated three times on ice in lysis buffer  with a high-intensity ultrasonic processor (Scientz). The protein was precipitated with 15% TCA at − 20 °C for 2 h. The supernatant was discarded after centrifugation for 10 min at 4 °C. The remaining precipitate was washed with cold acetone 3 times. The protein was redissolved in buffer . Three biological replicates were performed.Protein extraction was performed according to the method of Guo et al. . The cor− 1. During the first digestion overnight, trypsin was added at a mass ratio of 1:50 trypsin to protein, and in the second 4-h digestion, the mass ratio of trypsin to protein was 1:100. Approximately 100 μg protein was digested with trypsin for each sample.Trypsin digestion was performed according to the method of Guo et al. . The proTMT labelling and HPLC fractionation were performed according to the method of Guo et al. .To enrich phosphopeptides, the peptide mixtures were incubated with a vibrating suspension of IMAC microspheres. The IMAC microspheres with enriched phosphopeptides were collected, and the supernatant was removed. The IMAC microspheres were washed with 50% ACN/6% TFA and 30% ACN/0.1% TFA sequentially to remove nonspecifically adsorbed peptides. Elution buffer was added, and the enriched phosphopeptides were eluted with vibration to elute the enriched phosphopeptides from the IMAC microspheres. The supernatant containing phosphopeptides was collected and lyophilized for LC-MS/MS analysis.LC-MS/MS analysis was performed according to the method of Guo et al. . Three bhttps://solgenomics.net/organism/Petunia_axillaris/genome). Trypsin/P was specified as a cleavage enzyme, allowing up to 2 missed cleavages, 4 modifications per peptide and 5 charges. Phosphorylation on serine, threonine and tyrosine was specified as a fixed modification.The resulting MS/MS data were processed using the MaxQuant search engine (v.1.5.2.8) . Tandem Bioinformatic analysis was performed according to previously described protocols , 69. Thep value < 0.05 was considered significant.Proteins were classified by gene ontology (GO) annotation into three categories: biological process, cellular compartment and molecular function. For each category, a two-tailed Fisher’s exact test was employed to test the enrichment of the differentially modified protein against all identified proteins. The GO with a corrected http://www.kegg.jp/kegg/mapper.html) was used to identify enriched pathways by a two-tailed Fisher’s exact test to test the enrichment of the differentially modified protein against all identified proteins (81). The pathway with a corrected p value < 0.05 was considered significant. These pathways were classified into hierarchical categories according to the KEGG website.The KEGG database  was used for the statistics of the number of genes with alternative splicing.An RNA-seq analysis was conducted using StringTie v.1.3.3 . The altSpecific primers (Supplemental Table Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.Additional file 7.Additional file 8.Additional file 9.Additional file 10.Additional file 11.Additional file 12.


The evaluation of PET response was based on EORTC criteria. Standard uptake value (SUV) statistics , together with 148 textural features, were extracted from each lesion. Indices that are robust against contour variability (ICC test) were used as independent variables to logistically model tumor response. LASSO analysis was used for variable selection.Patients with operable  or locally advanced  BC were enrolled. All patients underwent chemotherapy (six cycles every 3 weeks). Surgery was performed within 4 weeks of the end of NCT. The MD Anderson Residual Cancer Burden calculator was used to evaluate the pathological response. max and the textural feature index IVH_VolumeIntFract_90 as the most useful to classify PET response, and a combination of PET response, ID range, and ID_Coefficient of Variation as the most useful to classify pathological response.Twenty patients were included in the study. Lesions from 15 patients were evaluable and analyzed: 9 with pathological complete response (pCR) and 6 with pathological partial response (pPR). Concordance between PET response and histological examination was found in 13/15 patients. LASSO logistic modelling identified a combination of SUVOur study suggests the potential usefulness of FLT-PET for early monitoring of response to NCT. A model based on PET radiomic characteristics could have good discriminatory capacity of early response before the end of treatment. Neoadjuvant chemotherapy (NCT) followed by surgery represents the standard strategy in the treatment of locally advanced breast cancer, obtaining an objective response rate of around 70% and a complete pathological response rate of up to 30% –3.It is well known that the pathological response to NCT represents an important prognostic factor in this setting –6. HowevAt present, there is no unanimous agreement about the best imaging method for the early assessment of response to therapy. Morphology-based imaging methods are generally used and interpreted by Response Evaluation Criteria in Solid Tumors criteria (RECIST v1.1) . However18F-labeled fluorodeoxyglucose (FDG) is the most widely used method to monitor response to therapy in BC. Although 18F-FDG is a sensitive tracer, it is not highly tumor-specific as it also accumulates in activated macrophages and other inflammatory cells, with a consequent increase in potential false-positive responses  has been proposed as an indicator of proliferation  are generally used to measure uptake inside a region. The potential utility of 18F-FLT PET/CT images as an early indicator of treatment response, in terms of SUVmax, in patients undergoing NCT was previously demonstrated by Crippa et al. . Semi-quantitative SUV statistics  or locally advanced  human epidermal growth factor receptor 2-positive (HER2-positive) BC taking part in multicenter phase II trial at our cancer (IRST IRCCS).ClinicalTrials.gov NCT02488564).All patients gave written consent to participate in the study, which was approved by the Local Research and Ethics Committee  plus docetaxel  plus trastuzumab plus metformin   or chest wall (T4 tumors).After surgery, if deemed suitable by the investigator, patients were treated with adjuvant anthracycline/paclitaxel or with the cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) scheme. Herceptin was administered every 3 weeks for 1 year.18F-FLT was produced by Advanced Accelerator Applications (AAA) with a radiochemical purity and specific activity >95% and >1 Ci/μmol, respectively.18F-FLT-PET/CT scan a maximum of 2 weeks before the start of NCT  and immediately before the second cycle of chemotherapy (FLT2)  equipped with 18 rings (92.7 cm diameter), each containing 672 BGO crystals , 152 mm axial FOV dimension, combined with a four-slice CT system. Step-and-shoot mode was used for whole-body image acquisition. The second scanner was a Biograph mCT Flow 64-4R PET/CT  equipped with four rings (84.2 cm diameter) of 48 detector blocks, each containing 13 × 13 LSO crystals , 221 mm axial FOV dimension, combined with a 64-slice CT system. Continuous table motion mode was used for whole-body image acquisition . Both po18F-FLT: a CT was performed from the brain to the pelvis immediately before the PET scan, with a multidetector spiral CT scanner . Whole-body PET scan was performed, covering an area identical to that covered by the CT. PET data were reconstructed into a 128 × 128 matrix , using the OSEM reconstruction algorithm. Corrections were applied for attenuation, scatter, random coincidences, isotope decay, and dead time. Fused PET and CT images were subsequently generated.Image acquisition started 1 h after intravenous injection of All volumes of interest (VOIs) encompassing the lesions were manually contoured on both FLT1 and FLT2 CT images by an expert nuclear medicine radiologist (LF) blinded to patient outcome. A second senior nuclear medicine radiologist (FM) supervised each contour. The MimVista software  was used for PET/CT image rigid registration and lesion delineation.In order to test the robustness of textural features against contour variability, four contours  were considered.PET image and structure set were then extrapolated in DICOM format for textural feature image analysis.max, SUVmean, SUVpeak, respectively) , 28 morphological features, and 95 second or higher orders   and IBSIThe PET response to therapy was determined according to EORTC 1999 criteria  between At the end of the chemotherapy course, all patients underwent surgery Table 1.i.e. FLT1); 2) it reflects the longitudinal nature of the study; and 3) the use of percentage changes in place of absolute values allows the comparison of results obtained with different scanners.Continuous data corresponding to SUV statistics and the 148 texture features were considered as percentage changes between FLT1 values and FLT2 values with respect to FLT1: (FLT1−FLT2)/FLT1*100. This has a threefold purpose: 1) it reflects the reasoning of the clinician who is accustomed to evaluating changes from baseline  was computed between the four contours of each lesion obtained with expansion and contraction. This analysis was performed on SUV statistics and texture features and also on their percentage changes. The two-way random effects model  was usedThe accordance between PET response and MD Anderson criteria was tested with the Fisher exact test.i.e., Least Absolute Shrinkage and Selection Operator L1 penalized regression)  or elastic-net (i.e. a compromise between the previous two) was motivated by the need to sensibly reduce the number of covariates in the model due to complexity. The ability of SUV statistics and textural features to describe the PET response was first investigated. Then, the possibility of describing the pathological therapy response (MD Anderson index) based on SUV statistics, textural features, and PET response was evaluated. With respect to model calculation, complete response (CR and pCR) was considered equal to 1, while partial response (PR and pPR) were considered equal to 0.Both the radiological PET response and the pathological treatment response were logistically modeled. LASSO variable selection was used to identify the variables most capable of classifying response correctly (ression) . A leaveStatistical analyses were carried out with R 4.0.4  and packOf the 20 patients selected for the study, three did not have available histology data (one patient refused surgery and two patients were not operated because of systemic disease progression). Two patients did not undergo the second FLT PET/CT scan and were therefore not included in the analysis. Thus, a total of 15 patients were evaluable. Median age at baseline was 54 years with a 10.5 year interquartile range Table 1.On the basis of MD Anderson criteria, there was a complete absence of neoplastic disease (pCR) classified as RCB-0 in eight patients and RCB-I in one patient. The remaining six patients showed a partial remission (pPR) classified as RBC-II in five patients and RCB-III in one patient.max value of 44%.On the basis of EORTC 1999 criteria, a comparison of PET images revealed a complete response (CR) to treatment in seven patients and a partial response (PR) in eight patients, with an average reduction in the SUVComparative analysis of the results showed that PET images were consistent with the subsequent histological examination in 13 (87%) patients (seven with pCR and six with pPR) Table 2.A discrepancy was observed between PET results and histological findings in two patients Table 2,Among 3 SUV statistics and 148 textural features, all 3 SUV statistics and 39 textural features were classified as robust against contour delineation Table 3.max, the model selects IVH_VolumeIntFract_90 as an important texture feature for classification. The selected textural feature stands for “Volume at Intensity Fraction 90%” [IBSI feature classification: BC2M  showed that an early PET with FLT, performed after the first cycle of therapy, was able to classify the pathological response in 100% of cases. It is worthy of note that, despite the limited number of patients analyzed, treatment modality and schedules were homogeneous.The present study, carried out in a population treated with the same NCT schedule  and high percentage change in FLT uptake (60%) were predictive of improved disease-free-survival . They also found that pre-therapy FLT uptake was not a significant predictor of outcome and did not correlate with disease-free-survival. Ulrich et al.  was a more robust index than SUVmax value for uptake characterization. The present study, and the results reported by Baiocco et al.  and MRI (relative change in largest tumor diameter) together in a multivariate analysis combined with breast cancer subtypes, the area under the curve (AUC) was 0.90 (95% CI: 0.83–0.96) (Data in the literature have shown that magnetic resonance imaging (MRI), alone or associated with FDG PET, could represent a non-invasive technique for monitoring response to NCT and for assessing residual disease. In particular, in a single-center study of 93 patients with breast cancer treated with NCT, Pengel et al.  reported83–0.96) . The AUC83–0.96) . The assi.e. slice thickness, voxel dimension). It is known that, especially for textural features, these factors may influence the statistical analysis and reduce the robustness of extracted textural feature indices  can be found in the article/The studies involving human participants were reviewed and approved by Comitato Etico della Romagna, Eudract number: 2014-002602-20; Protocol Code: IRST174.09. The patients/participants provided their written informed consent to participate in this study.Conceptualization: LF, FM, IA, MB, and AS. Methodology: IA, MB, and EL. Formal analysis and investigation: IA, EL, MB, LF, FM, EM, and SA. Writing—original draft preparation: MB, IA, LF, FM, and EL. Writing—review and editing: GP, FM, and SA. Supervision: AS, GP, AF, FM. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pisum sativum L.). To untangle the causal agents of the pea root rot complex and determine the role of the plant genotype in shaping its own detrimental or beneficial microbiome, fungal and oomycete root rot pathogens, as well as previously identified beneficials, i.e., arbuscular mycorrhizal fungi (AMF) and Clonostachys rosea, were qPCR quantified in diseased roots of eight differently resistant pea genotypes grown in four agricultural soils under controlled conditions. We found that soil and pea genotype significantly determined the microbial compositions in diseased pea roots. Despite significant genotype x soil interactions and distinct soil-dependent pathogen complexes, our data revealed key microbial taxa that were associated with plant fitness. Our study indicates the potential of fungal and oomycete markers for plant health and serves as a precedent for other complex plant pathosystems. Such microbial markers can be used to complement plant phenotype- and genotype-based selection strategies to improve disease resistance in one of the world’s most important pulse crops of the world.Plant health is recognised as a key element to ensure global food security. While plant breeding has substantially improved crop resistance against individual pathogens, it showed limited success for diseases caused by the interaction of multiple pathogens such as root rot in pea ( Breeding for disease resistance defends crops against plant pathogens and contributes to sustainable agriculture. Breeding disease-resistant crops face complex challenges at all scales, from molecular and cellular to the field and regional systems . A paradPlant-associated microbiota plays a key role in plant health, and the plant genotype, in turn, can shape the composition of plant-associated microbial communities . Two semResearch is only at the beginning of understanding the factors that direct plants in the complex interactions with the associated microbiome and still little is known about how to steer beneficial associations between plants and microbes. Although challenging, it has been postulated to take this complexity into account early in the resistance breeding process . For thePisum sativum L.) is the most widely grown pulse in the temperate zones  . Co-infeathogens . Similarathogens . Recentlathogens . In lineA. euteiches, F. avenaceum, F. oxysporum, F. redolens, and F. solani. Remarkably, A. euteiches could not be isolated from roots by means of traditional culturing methods, despite its frequent detection by PCR. Through qPCR, it was shown that A. euteiches facilitates root colonisation of Fusarium species eventually leading to increased root rot  genotypes that were selected based on a previous study on root rot resistance. These genotypes showed contrasting levels of resistance to a PRRC present in naturally infested field soil (Kirchlindach) (The experiment involved eight peas (lindach) . The prelindach) .The eight pea genotypes were grown in soil collected from four agricultural field sites showing different levels of PRRC infestation, which were Soil from Feldbach , Kirchlindach , Puch , and Neu-Eichenberg  . Sieved 2O-bleach solution  for 10 min. Finally, seeds were thoroughly rinsed in ddH2O and soaked for 2 h. Seven seeds per genotype were planted in a 2:1 (v:v) mixture of soil and sterilised sand  in plastic pots (600 ml). Pots were arranged in a randomised complete block design with the factors “soil” (four levels) and “genotype” (eight levels) in four replications. Each experimental unit was set up as a pair of two pots containing untreated soil and sterilised soil, respectively. The four replications were sown on four consecutive days and harvested over 4 days in the same order. Plants were grown under controlled conditions in the growth chamber for 29 days. A 16/8 light/dark cycle was applied, providing a photosynthetically active photon flux density of 200 μmol m–2 s–1 over the waveband 400–700 nm. Plants were watered with tap water every 72 h by flooding the pots 4 cm high for 30 min. The growth chamber means temperature over the course of the experiment was 20°C, relative humidity 85%. Pots were inspected on a daily basis for seedling emergence and plants were thinned out to reach a maximum of five plants per pot.Pea seeds were surface-sterilised in 70% ethanol for 30 s followed by a 1:1 (v:v) ddHRel.) was calculated by dividing the biomass of the untreated soil treatment by the biomass of the corresponding sterile control treatment of the same genotype in each replication.The plants were removed from the pots 29 days after sowing, and roots were washed under running tap water. A root rot index  was att® Plant DNA DS 96 Kit  according to the instructions of the manufacturer. DNA concentrations were measured photospectrometrically and samples were normalised to a DNA concentration of 50 ng μl–1. DNA extractions and subsequent qPCR analyses were done on a per pot basis  in two technical replications. The average between both technical replicates was used for all statistical analyses.Previously published qPCR assays were used to quantify ten microbial taxa in the roots of plants grown in the non-sterile treatment . Microbi3 to 100 pg μl–1). To approximate the ratio between target DNA and plant DNA, the serial dilutions were established in diluted plant DNA (50 ng μl–1), extracted from axenically grown  pea seedlings (cv. “Respect”). For each target microbial taxon, DNA was extracted in the same way as the plant material from a patch of mycelium (∼7 cm2) of 10-day-old cultures grown on potato dextrose agar in the dark at room temperature (isolates used in this study are listed in 6 to 102 copies μl–1) of transformed plasmids containing an AMF 18S rDNA sequence. Two replicate reactions were run on a Rotor-Gene Q Thermocycler  for each of the two extracted DNA samples resulting in a total of four technical replicates per pot. The 13 μl qPCR reactions contained 1.5 μl of template DNA, 6.5 μl of KAPA FAST qPCR master mix , primers, and probe, where necessary. PCR programs consisted of an initial denaturation step for 5 min at 95°C, followed by 40 cycles of 10 s at 95°C, 30 s at the assay-specific temperature  of the Bray-Curtis distances between samples was used to explore structural similarities between the microbial composition of the four soils and eight genotypes. Permutational multivariate analysis of variance (PERMANOVA) was used to test differences in the microbial composition among the different factor levels, i.e., “soil,” “genotype,” and their interaction. To assess if the groups of resistant and susceptible genotypes have different microbial communities, the analysis was also performed with the factor “resistance level” (two levels) replacing the factor “genotype” in the model. The associations between the 10 qPCR variables and each NMDS ordination were determined by calculating the goodness-of-fit statistic ge vegan .mvabund  smooth terms retained. Partial r2 for each retained variable in the final model was estimated by calculating the difference in the overall r2 of the final model and the final model without the variable in question. GAMs were calculated for the three infested soils individually and the three soils together. In addition, Spearman correlation between SDWRel. and microbial quantities were calculated within the three infested soils. To test the hypothesis of different quantities of the ten microbial taxa between the groups of resistant and susceptible pea genotypes, Wilcoxon rank-sum test was calculated for the three soils together.Generalised additive models (GAM) were fit using the R package mgcv  to exploRel . The interaction between “soil” and “genotype” was not significant . Mean (SD) SDWRel. was 1.06 (0.43) in the F soil. SDWRel. was significantly lower in the three infested soils (Rel. . SDWRel. was significantly lower for susceptible genotypes in the three infested soils . To analyse the growth performance of the pea genotypes on infested soil further, ANOVA was performed for the three infested soils. This revealed again significant effects of “soil”  and “genotype” . The interaction between these two factors was not significant . Therefore, post hoc analysis was calculated for genotypic means over the three infested soils, revealing significant differences for SDWRel. between the pea genotypes, with genotype S91 being the most resistant and C2 the most susceptible  and 0.91 (0.71/1.17), respectively (–1 (median of 3 pg rct–1).On all ten qPCR assays, average efficiencies and ectively . In the Aphanomyces euteiches and F. solani were the most abundant pathogens in diseased roots, distinguishing the healthy F soil from the three infested soils (SD) of 609 (324) pg rct–1, the diseased roots from P soil showed the highest A. euteiches concentrations compared with 359 (304), 299 (136), and 7 (13) pg rct–1 for the N, K, and F soil, respectively. DNA concentrations of F. solani were highest in the K soil [666 (578) pg rct–1] compared with the P [637 (674) pg rct–1], N [254 (273) pg rct–1], and F [44 (67) pg rct–1] soil. F. oxysporum and R. solani were quantified at intermediate levels: F. oxysporum was present in all soil-genotype combinations, however, considerably lower in the P soil [22 (35) pg rct–1] compared with the three other soils. R. solani showed considerably higher levels in the N soil [64 (77) pg rct–1] than the other soils. In the K soil, F. oxysporum was more present in roots from genotypes that had high overall pathogen loads . F. avenaceum was almost exclusively detected in samples grown in the P soil [3 (6) pg rct–1] and F. redolens in the N soil [7 (7) pg rct–1]. P. ultimum, on the other hand, showed the highest concentrations in the roots from the F soil [32 (33) pg rct–1].ed soils . With a en loads . D. pinoThere was a tendency that resistant pea genotypes had lower total pathogen amounts in the roots than susceptible genotypes . GenotypC. rosea was detected at low levels in samples from the F, P, and N soils  and at intermediate levels in samples from the K soil [29 (34) pg rct–1], generally uniformly present over all genotypes  “soil,” “genotype,” and “soil × genotype” effects for the composition of ten microbial taxa in diseased pea roots, with 41, 14, and 15% of the variance in the microbial composition explained, respectively. When running the PERMANOVA with the factor “resistance level” instead of “genotype,” “resistance level” and the “soil × resistance level” interaction explained 10 and 7% of the variance, respectively. For each of the three infested soils, factors “genotype” or “resistance level” was significant, with the factor “genotype” (“resistance level”) explaining 59 (39), 46 (21), and 33% (18%) of the variance for K, P, and N, respectively. A. euteiches, F. solani, and F. oxysporum appeared as the main contributors of genotype separations with high (r2 > 0.3) and significant (P < 0.05) correlations with the first two dimensions of the ordinations of each of the three infested soils  of the composition of the ten microbial species between individual samples . Over thed soils . AMF shoF. solani and F. oxysporum consistently showed high connectivity (no. of edges). Additionally, AMF, A. euteiches, and F. redolens showed high connectivity in the K, P, and N soil, respectively. AMF showed negative associations with pathogenic species in K and P soils; the negative correlations between AMF and F. solani or R. solani were consistent across both soils. C. rosea showed positive associations with pathogenic taxa, consistently with F. solani in each soil. The network over the three infested soils corroborated the positive association between F. solani and F. oxysporum. These two species showed the highest connectivity  among all taxa. AMF showed negative correlations with A. euteiches, F. oxysporum, F. solani, and R. solani.Networks of conditional dependencies for each of the three infested soils individually showed that the majority of significant correlations between pathogenic species was positive . In eachRel. and the abundance of microbial taxa in the diseased roots revealed a distinct model for each of the three infested soils  reveals tolerance, i.e., enduring infection . R. solani is frequent in pea fields in the US, Canada, or China . For instance, genotypes C1 and G78, while tolerant, showed medium to high overall pathogen levels. Since pea root rot is caused by microbial dysbiosis in the pea rhizosphere as a result of legume intensive crop rotations  can be found in the article/PH, MM, and BS conceived the study. LW, MK, MM, and PH designed the experiment, in consultation with BS. LW and MK conducted the experimental work. LW analysed the data. LW and PH wrote the manuscript with contributions of MM and BS. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Candida species that are more resistant to antifungal drugs are increasing in these patients. In this manuscript, we reported a case of fatal colitis after small bowel transplantation induces by multidrug-resistant (MDR) Candida glabrata. Case Presentation. A 52-year-old man has undergone an extensive small bowel resection with the length of the remaining bowel which was less than 40 cm who became a candidate for transplantation. Four months after transplantation, the patient experienced severe bloody diarrhea with abdominal distension. Ileoscopy and colonoscopy did not show neither pathological change and rejection nor cytomegalovirus (CMV) infection posttransplantation. Abdomen computed tomography showed diffuse moderate small bowel wall thickening. After detection of budding yeast in the stool samples, stool culture was positive for Candida, DNA was extracted, and ITS1-5.8s-ITS2 region of the fungal agent was amplified. Sequencing analysis of PCR and antifungal susceptibility testing revealed that this isolate was multidrug-resistant C. glabrata. Besides, there was no evidence for other pathogens known to cause infection in various laboratory tests. Immediate antifungal treatments with caspofungin remained unsuccessful, and on the eighteenth day of admission, the patient expires with septic shock.  Small bowel transplantation is a potential option for patients with intestinal-failure, and the incidences of infections caused by  These findings highlight the challenging management of candidiasis in patients with small bowel transplantation. Infectious diseases due to MDR organisms have emerged as a vital clinical problem in this patient population. Candida spp. are the most common type of fungal infections among SOT recipients except for lung transplant recipients in which Aspergillus is more prevalent [Candida albicans has been the most common species isolated from IFIs, there is a steady increase in fungal infections caused by non-albicans Candida species [albicans Candida species, C. glabrata is the most common non-albicans isolate. The emergence of multidrug-resistant Candida glabrata, during prolonged and more broad-spectrum exposure to antifungal agents have created a therapeutic challenge [C. glabrata.Infections remain a major cause of morbidity and mortality among solid organ transplant (SOT) recipients. The bacterial followed by viral and fungal infections are the predominant infections which following in SOT . Althougrevalent , 8. Wher species . Among thallenge . In thisPneumocystis jiroveci and Cytomegalovirus (CMV) prophylaxis, respectively, for the first 6 months after ISTx. The patient was discharged from the hospital after 1 month, and then, outpatient visits were performed in the transplant clinic by transplant surgeons, weekly. Four months after transplantation, the patient experienced severe bloody diarrhea with abdominal distension for three consecutive days. Then, the patient came to the Emergency Rooms (ERs) by ambulance in the hypovolemic shock state with drowsiness. Vital signs on arrival were the temperature of 38°C, blood pressure of 82/57 mmHg, pulse rate of 101/min, and respiratory rate of 24/min . He received total parenteral nutrition (TPN) for 18 months and then received a small bowel transplant from donor (14 years brain-dead) with ABO and HLA typing compatible. The patient received methylprednisolone  and thymoglobulin  as induction immunosuppressant and then received mycophenolate mofetil, tacrolimus, and prednisolone as maintenance immunosuppressive regimen. Piperacillin-tazobactam (4.5 g q8h for 3 days), vancomycin (1 g q12h for 3 days), and caspofungin (50 mg daily for 2 weeks) started after transplantation as prophylactic antibacterial and antifungal, respectively. He received trimethoprim-sulfamethoxazole and valganciclovir for f 24/min .Clostridium difficile infection (CDI), detection of fecal toxin A/B by enzyme immunoassay  and culture  were done. All blood, urine, and sputum cultures were negative. Metronidazole plus oral vancomycin were added to his antibiotic regimen to cover suspicious CDI. After detection of budding yeast in the stool samples, caspofungin was started, and more evaluation regarding fungal infection, especially Candida spp., was done as below. Stool culture was positive for C. glabrata with antifungal susceptibility profiles which are showed in The patient was resuscitated with 2 liters of normal saline and transferred to the intensive care unit (ICU) for further evaluation and treatment. On the second day of admission, the patient was intubated, all immunosuppressive was discontinued, inotropes with broad-spectrum antibiotics were started, and sepsis workups were done. The patient underwent diagnostic ileoscopy and colonoscopy. Tissue biopsies did not show neither pathological change and rejection nor CMV organ involvement. Abdomen computed tomography showed diffuse moderate small bowel wall thickening. Stool examination and cultures were analyzed for evaluating infectious causes. Due to colonoscopy features which illustrated diffuse scattered whitish cobblestoning picture  with susCandida CHROMagar  and was plated evenly with a sterile swab. After incubation at 37°C for 48 h in ambient air, the Candida colonies were counted and classified as C. glabrata, according to the color of the colonies. It is notable that a colony count ≥ 105 CFU/ml stool was classified as “Candida overgrowth,” according to Krause et al. [The stool sample was diluted 1 : 10 with saline, and 100 microliters of dilution was transferred onto a e et al. . In addiCandida sp. isolated from the stool sample was performed for the identification of fungal agents. DNA extraction was performed by the boiling lysis method. Single Candida colony from a pure fresh Sabouraud dextrose agar  plate was picked and inoculated into 200 μl of sterile Milli-Q water and kept for 10 min in a heat block  at 100°C. The extracted DNA after incubation at 100°C was kept in a −20°C deep freezer for 10 min and then centrifuged at 10,000 rpm for 5 min. The extracted DNA was stored at -20°C for PCR assay. Amplification of the ITS1-5.8S-ITS2 region was done by universal primers ITS1 (5′-TCC GTA GGT GAA CCT GCG 92G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) at the annealing temperature of 56°C. The amplification was done for 35 cycles of 98°C for 30 s and annealing temperatures of 60°C and 72°C both for 30 s. This was followed by a final extension of 72°C for 5 min. The nucleic acid sequences were compared with the database at the GenBank database using the BLAST sequence search tool. The comparative DNA sequence analysis by nucleotide Basic Local Alignment Search Tool (BLAST) revealed that the amplified sequence was identified as C. glabrata. Molecular identification was consistent with culture methods.Molecular evaluation of the C. glabrata isolate was seeded on the plate containing antifungal drugs, incubated at 37°C for 24 h, and minimum inhibitory concentrations (MICs) were determined by visual examination based on clinical breakpoints or epidemiological cutoff values, which differ across species and the antifungal used. Candida parapsilosis (ATCC 22 019) and C. krusei (ATCC 6258) were used as references for quality control. Antifungal susceptibility testing found that the C. glabrata sample was resistant to FLZ, VRZ, and ITZ, likely resistant to AMB and CAS (The broth microdilution method (CLSI M27-A3/S4) was used for susceptibility testing of our isolate to the following antifungal drugs: fluconazole (FLZ), itraconazole (ITZ), amphotericin B (AMB), caspofungin (CAS) , and voriconazole .  and CAS .Candida spp. is the most common cause of infection among intestinal transplant patients, which has a role of non-C. albicans spp., including C. glabrata which is higher than other species of Candida genus. To the best of our knowledge, this study is the first case report of severe fetal colitis by C. glabrata after ISTx [Pseudallescheria boydii infection [Candida is not similar to each other, so that in our patient who was infected with C. glabrata in line with Praneenararat study [C. tropicalis agent, dysentery and fever were the early symptoms, while according to the Arfa et al. study [C. krusei, fever, and abdominal pain symptoms did not occur, and colitis with diarrhea was the only clinical symptom. In the current case, in addition to the mentioned symptoms, the colonoscopic view was very similar to that of CDI. In SOT transplant recipients, antifungal prophylaxis is usually administered at least for 4 weeks, until anastomosis has entirely healed and resolution of risk factors. Prophylaxis strategies are increasingly used in immunocompromised patients due to the potentially devastating effect of invasive candidiasis in terms of morbidity and mortality. The ideal agent is unclear, but FLZ, CAS, and AMB drugs are logical options [Candida spp. [β-glucan and disrupt in cell wall integrity, are the first line antifungal therapy against C. glabrata infections, as this species has low susceptibility to azole drugs, hereditary [C. glabrata significantly increased. The increasing numeral of C. glabrata clinical isolates reported showing decreased susceptibility for echinocandins is an emergent concern. According to the previous studies, rates of CAS resistance among C. glabrata clinical isolates range from <10% [C. glabrata to the CAS is exposure to low CAS concentrations so that C. glabrata is able to colonize and survive in internal parts of the human body, such as the abdomen [β-D-glucan synthase, such as ibrexafungerp, which has shown potential effectiveness against ECH-resistant C. glabrata isolates [Candida species and, on the other hand, according to the drug resistance pattern reported in this study, it is necessary to reconsider the use of FLZ as part of the prophylaxis regimen.Invasive fungal infections are a major problem in SOT recipients. Overall, the most common fungal infection in SOT is candidiasis, followed by aspergillosis and cryptococcosis , 12, 13.ter ISTx , 13. In ter ISTx . Therefonfection . It is nat study  that repl. study , colitis options –20. Receida spp. . Echinocreditary . Importareditary , 23–25 arom <10%  to as hirom <10% . Based o abdomen , the per abdomen , the gas abdomen , or the  abdomen , due to isolates , or rezaisolates , could hC. glabrata agent as the most reported MDR Candida spp. in a patient undergoing a small bowel transplant by conventional and molecular analysis. In this study, the triazoles, polyene, and echinocandin classes of antifungal drugs appear to be inactive against C. glabrata with high MICs. Further attention is recommended to control fungal pathogens during organ transplantation.In summary, we presented a case of candidiasis with


Mild cognitive impairment (MCI) is a kind of non-functional cognitive decline between normal aging and dementia. With the increase of individual age, the quality of cognitive function has become a more and more important topic. The study of gene loci in patients with MCI is essential for the prevention of dementia. In this study, we evaluate the gene polymorphism in Chinese Han patients with MCI by propensity score matching (PSM) and comparing them to healthy control (HC) subjects.Four hundred seventeen patients with mild cognitive impairment and 508 healthy people were included. The two groups were matched by applying one-to-one PSM, and the matching tolerance was set to 0.002. The matching covariates included gender,age,occupation,marital status,living mode. Then, a case-control associated analysis was conducted to analyze the genotype and allele frequencies of single nucleotide polymorphisms (SNPs) in the MCI group and the control group.P > 0.05). The allele frequency of bridging integrator 1(BIN1) rs7561528 showed minimal association with MCI in the Han Chinese population (P = 0.01). Compared with the healthy control (HC) group, A allele frequency of MCI group patients was significantly decreased. The genotype frequency of BIN1 rs6733839 showed minimal association with MCI in the recessive model (P = 0.03). The genotype frequency of rs7561528 showed minimal association with MCI in the codominant, dominant, overdominant, and log-additive model (P < 0.05). The genotype frequencies of StAR-related lipid transfer domain 6 (STARD6) rs10164112 showed nominal association with MCI in the codominant, dominant, and log-additive model (P < 0.05). Unfortunately, the significant differences did not survive Benjamini-Hochberg false discovery rate correction (adjusted P > 0.05). The patients with SPI1 rs1057233 may be the protective factor of MCI , and patients with APOE rs10164112 may be a risk factor for MCI .Three hundred eleven cases were successfully matched in each group, and there was no statistical difference on all the matching variables, gender, age, occupation, marital status, living mode between two groups after the match (BIN1 gene, and rs1057233 loci in the SPI1 gene may be associated with the MCI in Chinese Han population. APOE gene was the risk factor of MCI, but further verification in a large sample population is still needed.The polymorphisms of rs7561528, rs6733839 loci in the The online version contains supplementary material available at 10.1186/s12888-022-03756-y. Mild cognitive impairment (MCI) is a cognitive impairment mode between normal aging and dementia, which is characteristics of cognitive or some mild memory impairment . In geneBIN1) gene, located on chromosome 2, is a member of the BAR family and participates in physiological processes such as cell endocytosis and actin activation [BIN1 gene may be the pathogenic gene of AD. Some studies confirmed that the polymorphism of different BIN1 loci genes is related to the pathogenesis of AD [Some genetic changes may contribute to dementia in some older people. Bridging Integrator Protein-1 (tivation . In receis of AD –9.SPI1 (Recombinant Human Spi-1 Proto-Oncogene) encodes PU.1, a transcription factor that occupied a major position in myeloid cells’ development and function. The heritability of AD was abundant in the PU.1 cistrome, suggesting the presence of a myeloid PU.1 target gene network in AD. Huang et al.  that expAPOE gene is related to the incidence of AD and MCI, but the abnormal risk accounts for only 20%, suggesting that other genes are involved in the pathogenesis of AD [APOE ε 4 allele raised the risk of developing AD [The Apolipoprotein E (APOE) is the main one of the apolipoproteins in plasma and is also involved in the nervous system’s growth and repair. Several studies have confirmed that the is of AD , 12. Furoping AD . Howeveroping AD . At presBIN1, STARD6, RIN3, APOE, PICALM, SPI1, BZRAP1-AS, PFDN1/HBEGF, TMP21, MTHFR, TMEM106B, MC1R, CENPO, PVRL2 and KL genes to reveal the correlation between the polymorphisms and AD risk.Therefore, to better understand the relationship between these genes, we conducted a study that examined the distribution of SNPsof We performed a multicentric and prospective study. This study included 417 MCI patients and 508 healthy controls (HC) who were recruited between 2018 and 2020, who were all Han nationality over 50 years old in Wuxi City, Jiangsu Province, and its surrounding counties and communities. According to the revised Peterson criteria, MCI patients were diagnosed . The subThe exclusion criteria were as follows: 1) People under the age of 50; 2) those with symptoms of psychosis or congenital mental retardation, history of head injury, severe endocrine diseases, severe cardiopulmonary, severe infectious diseases; 3) those who abuse alcohol or other substances. After a detailed description to the subjects or their representatives, written informed consents were obtained. In addition, the data on general demographics, such as age, sex, lifestyle, marital status, and occupation, were also surveyed.ftp://ftp.ncbi.nlm.nih.gov/hapmap/). The criterion for selecting SNPs is that the minor allele frequency (MAF) ≥0.05 and r2 ≥ 0.8 in Beijing’s Han Chinese population (HCB). We selected eighteen SNPs for genotyping were screened out for analysis, specific information for each gene is given in Table 5 in RIN3 and were selected due to their strong association with AD [Firstly, we selected SNPs in the public HapMap database . Tiangen DNA isolation kit was used to extract genomic DNA from venous blood. The selected SNPs were genotyped by TaqMan SNP Genotyping Assay and ABI PRISM 7900 sequence detection system equipped with SDS2.1 software. In order to quality monitoring, a blind method was used to perform genotype analysis on participants’ status. Ten percent of the samples were genotyped once more, showed a coincidence rate of 99.2%. Two independent researchers scored the genotype data doubly. Deviation from the expectation of Hardy-Weinberg equilibrium (HWE) was evaluated in this queue.Rubin and Rosenbaum first proposed PSM in 1983 to eliminate the impact of confounding factors on the retrospective study results . Propenshttp://analysis.bio-x.cn/myAnalysis.php). We apply the Benjamini-Hochberg false discovery rate correction to account for multiple tests. SNPStats (https://www.snpstats.net/start.htm) was mainly used to assess the connection between SNPs and MCI risk under five genetic models . Then, a Logistic regression analysis was performed with SPSS 24.0.We assessed HWE, the genotype frequency measurements and the allele frequency with SHEsis software  were enrolled, including 168 females. The average age was 66.05 ± 6.21 years . The A allele frequency was 58 (9.6%) in the MCI group and 86 (14.3%) in the healthy control (HC) group. The distribution frequencies of AA, AG, and GG genotypes at rs7561528 loci in the MCI group were 5 (1.7%), 48 (15.8%), and 250 (82.5%), respectively. In HC group, they were 7 (2.3%), 72 (23.9%) and 222 (73.8%), respectively. When we compared the three genotypes’ distribution frequency between these two groups, we find a significant difference . Moreover, the T allele frequency at rs10164112 polymorphism in STARD6 was higher in MCI cases than in controls, and the difference was statistically significant . However, no difference remains significant after Benjamini-Hochberg’s false discovery rate correction (adjusted P > 0.05). Comparing genotype and allele frequencies remaining of the SNPs across the overall sample of MCI patients and controls showed no significant differences. Specific information on each SNP is given in Table 5 in the Except for BIN1 gene rs6733839 and MCI. (CC-CT vs. TT). Similarly, the genotype frequency of BIN1 rs7561528, the distribution frequencies of the codominant model (GG vs. AG), dominant model (GG vs. AG-AA), overdominant model (GG-AA vs. AG), and there was a nominally significant difference between the two groups with respect to log-additive model , codominant model (CC vs. TT), log-additive model. However, when applying Benjamini-Hochberg to correct the false discovery rate, the association is not significant (P > 0.05 after correction).Under five-inheritance models, the age and sex factors of MCI patients and controls were analyzed by unconditional Logistic regression. In the recessive model, there was a minimal association between the genotype frequency of SPI1 was the protective factor for MCI  and rs10164112 in STARD6 was the risk factor for MCI . In our study, the presence or absence of carrying APOEε4 was not statistically associated with the occurrence of MCI (P > 0.05).The results of binary logistic regression are shown in Table BIN1), also known as amphibian protein 2. It has been reported that its expression level in the brain tissue of patients with AD is increased [The bridging integrator 1  . Raj et 28 locus . This po28 locus . Harold  with AD . Signifi with AD . Similar with AD . Previous7561528 . Meanwhied to AD . Based oNLRP3 and Caspase-1 gene deletions can interfere with AD’s progression and improve cognitive function by blocking the formation of NLRP3/Caspase--1 inflammatory body [SPI1 may amplify the genetic variation of other AD-related myeloid genes and regulate neuroprotective or neurotoxic microglial phenotype equilibrium. Huang et al. found that SPI1 rs1057233 and its labeled SNPs may regulate AD risk through changes in SPI1 expression and may represent potential disease sites [After phagocytosis of amyloid-beta (Aβ), microglia initiate the activation of NALP3 inflammatory bodies and then activate caspase-1, which leads to the release of interleukin 1β (IL-1β) and promotes the occurrence of the inflammatory response . NLRP3 iory body . The traory body . As a trse sites , body mase sites , and prose sites , indicatNew research suggests that neurosteroids such as diethylstilbestrol may be a new treatment for AD, indicating that lipid metabolism occupied a significant position in AD , 42. SomSTARD6 rs10164112 was significantly related to AD in the Korean population [APOEε4 allele, resulting in an increased danger of AD [Furthermore, STARD6 appeared in the hippocampus formation in rats, and its level was increased after pilocarpine-induced hippocampal neuron injury of rats . The mulpulation . Yin et er of AD . AlthougAPOE ε4 may be a risk element for AD, and APOE ε2 may be a protective factor for AD [APOE gene between the two groups, which may be related to the fact that populations from different regions may have genetic heterogeneity of MCI.At present, some researchers have reported that r for AD –48. HoweSPI1 and BIN1 variation may be the potential targets for MCI treatment and supported that STARD6 contributes to the risk of MCI. These results are helpful to understand the relationship among the pathogenesis, clinical diagnosis, and the SNPs of MCI in the Han population of southeastern China and provide directions for future research.In summary, the present study demonstrated that BIN1, were also found to be significantly associated with the occurrence of MCI in the Han Chinese population, but our study did not include all relevant SNPs [Several limitations of this study should be taken into consideration and discussed. Firstly, this is a cross-sectional study, so it didn’t consider the order of exposure and the timing of outcome and the causal relationship between exposure and outcome. Secondly, it is worth noting that age and occupation were significant differences between the MCI patients’ controls before PSM. Our study eliminated these variables to reflect the role of genes, but previous studies have confirmed the influence of age and gender on MCI. As a disease affected by many factors, other variables such as marriage, nutritional and mental status should be added when collecting clinical data of MCI. Some other genes, such as rs744373 in ant SNPs . Therefoant SNPs . In addiAdditional file 1: Supplementary Table 5. Detailed information of SNPs in fourteen GWAS-linked genesa.Additional file 2: Fig. 1 OR (95%CI) forest map of the effect of gene polymorphism on MCI.


Phaseolus lunatus, is closely related to common bean and is high in fiber and protein, with a low glycemic index. Lima bean is widely grown in the state of Delaware, where late summer and early fall weather are conducive to pod production. The same weather conditions also promote diseases such as pod rot and downy mildew, the latter of which has caused previous epidemics. A better understanding of the genes underlying resistance to this and other pathogens is needed to keep this industry thriving in the region. Our current study sought to sequence, assemble, and annotate a commercially available cultivar called Bridgeton, which could then serve as a reference genome, a basis of comparison to other Phaseolus taxa, and a resource for the identification of potential resistance genes. Combined efforts of sequencing, linkage, and comparative analysis resulted in a 623 Mb annotated assembly for lima bean, as well as a better understanding of an evolutionarily dynamic resistance locus in legumes.Lima bean,  Phaseolus lunatus L.) was independently domesticated in Mesoamerica and the Andes  to map a resistance locus effective against the predominant field race F of P. phaseoli. To our knowledge, no other resistance loci have been mapped in lima bean. Collinearity analysis with the P. vulgaris (common bean) genome revealed homology with a resistance gene (R-gene) dense region containing different subtypes of nucleotide-binding site leucine-rich repeat (NLR) genes , was chosen to self-propagate seed for the reference genome. Progeny of Bridgeton-DES4 was grown in continuous darkness to generate etiolated tissue for DNA extraction. Separate extractions were performed on tissue from individual plants using the Qiagen Maxi DNA extraction kit, followed by a DNA purification  and subjected to library construction according to their protocols. Five size fractions were selected ranging from 470 bp to 10 kb to construct sequencing libraries following the manufacturer’s protocols . The TruSeq DNA Sample Preparation Kit version 2 with no PCR amplification (PCR-free) was used to make replicate paired-end libraries for the 470 and 800 bp size fractions. The Nextera MP Sample Preparation Kit was used to make mate-pair (MP) libraries with 2–5, 5–7, and 7–10 kb jumps. The 470-bp libraries were sequenced as 2 × 265 nucleotides on the Hiseq2500 v2 in rapid mode. The 800-bp libraries and part of the three MP libraries were sequenced as 2 × 160 bp nucleotides on the HiSeq2500 (v4 Illumina chemistry) while the remainder of the MP libraries were also sequenced as 2 × 150 bp on the HiSeq4000. A total of 246 Gb sequencing data . All library construction and sequencing were performed at Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign.™ software platform . This is a De Bruijn graph-based assembler, designed to efficiently extract the underlying information in the raw reads to solve the complexity of the DeBruijn graph due to genome polyploidy, heterozygosity, and repetitiveness. This task is accomplished using accurate-reads-based traveling in the graph that iteratively connects consecutive phased contigs over local repeats to generate long phased scaffolds  were removed. The PE 450 bp 2 × 265 bp libraries overlapping reads were merged with minimal required overlap of 10 bp to create the stitched reads.Error correction: Following preprocessing, merged PE reads were scanned to detect and filter reads with putative sequencing error .Contigs assembly: The first step of the assembly consists of building a De Bruijn graph (kmer = 127 bp) of contigs from all of the PE and MP reads. Next, PE reads were used to find reliable paths in the graph between contigs to resolve repeats and extend the contigs.Scaffolds assembly: Contigs were linked into scaffolds with PE and MP information, estimating gaps between the contigs according to the expected distance of PE and MP links.Fill Gaps: A final gap-filling step used PE and MP links and De Bruijn graph information to detect a unique path connecting the gap edges.2 progeny from a cross between cultivars Cypress and Jackson Wonder. 192-plex sequencing libraries were constructed following the protocol by Csp6I/MspI and Csp6I/TaqαI pairs of restriction enzymes. Samples included the F2 progeny along with replicate samples of the parents and F1 plants as well as a negative control with no DNA. Separate libraries were constructed for Csp6I/MspI and Csp6I/TaqαI. Sequencing was performed at the University of Delaware’s Sequencing and Genotyping Center on a Hiseq2500 run in rapid mode at 1 × 151 bp.Linkage and synteny mapping approaches were used to cluster and order the assembled scaffolds into draft pseudomolecules. Linkage mapping was performed using genotyping-by-sequencing (GBS) data on 163 Fhttps://github.com/UD-CBCB/RedRep). First, FASTQ files from two lanes of sequencing were merged for the corresponding libraries. Following quality control and barcode deconvolution, FASTQ files from the same barcode were merged and then mapped to the NRGene sequence assembly. Variants were typed using HaplotypeCaller, and the genotype matrix was filtered as follows. VCFtools . First, while retaining softmasked sequences in the reference genome identified by analysis with RepeatMasker, LAST , substitution and gap frequencies were determined with last-train (run settings: –revsym –matsym –gapsym -E0.05 -C2) and lastal alignment was performed . Alignments between repeat sequences were discarded with last-postmask and a python script was used to retain only the top two LAST matches per query sequence. The resulting output was used to run fragmentmap and assembler routines of Chromosomer. Finally, LAST was used to align the Chromosomer assembled P. lunatus genome to the P. vulgaris reference genome (run settings: -m50 -E0.05 -C2).Chromosomer (P. phaseoli) was determined for 384 F2:3 families of the Cypress  X Jackson Wonder (susceptible) cross . Plants were grown in a greenhouse humidity chamber and inoculated at emergence as described by 2:3 progeny was used as an estimate of the corresponding F2 parent phenotype for QTL analysis. QTL IciMapping software v 4.1.0.0  using RepeatMasker (P. lunatus genome), generated from pods (12 libraries), leaves (1 library), and flowers (1 library). We used MagicBlast at NCBI to map these reads to the predicted genes. The resulting SAM file was converted to BAM format and indexed, followed by samtools idxstats to identify predicted genes with either no transcript coverage or those that were covered over their full length. For the remaining genes with partial coverage, bedtools genomecov was used to calculate the fraction of transcript coverage across each gene. In addition, using BLASTp , the predicted proteins for P. lunatus were compared to the P. vulgaris reference proteome .Annotation of the predicted genes was undertaken with multiple tools. First, BLASTp searches  were performed against a custom BLAST database that included all Viridiplantae sequences from the NCBI nr database  chromosomes of P. lunatus  comprised of 898 markers among 41 scaffolds  that together captured 49% of the sequence space. In every case, markers within a given scaffold mapped to a single linkage group. However, within LGs 1, 3, and 5, markers were ordered on the genetic map such that sections of different scaffolds interleaved with other scaffolds (Supplementary Table S2).P. lunatus scaffolds, which were distributed among all 11 P. vulgaris chromosomes (Supplementary File S3). These corresponded to many different scaffolds in the P. lunatus assembly (∼230 Mb or 37% of the assembled genome) than those anchored to the genetic map. There were 16 scaffolds anchored by both maps which comprised ∼105 Mb. Together, nearly 70% of the sequence space was ordered by genetic mapping or synteny analysis, but with only 25% intersection, these separate maps could not be well integrated. Additional work will be required to construct chromosome-scale pseudomolecules. Nevertheless, as described below, combining all of the map data helped to identify a homologous section of the P. lunatus and P. vulgaris genomes associated with variation in disease resistance.Synteny analysis with the common bean genome (PlPp_LG9.1) that explained ∼50% of the phenotypic variation in partial resistance to P. phaseoli (see BIP directory in Supplementary File S2), a slow downy-mildewing phenotype. Previously, with a different population, P. phaseoli. Anchoring the marker sequences from QTL and BSA mapping onto our genome assembly showed that both of these resistance loci reside at the same region of the genome, indicating they are linked or that PlPp_LG9.1 is a weak allele of the race-specific resistance gene.We mapped a QTL on LG 9 . The approximate length of P. lunatus scaffold sequences between the flanking markers was 1.3 Mb, smaller than P. vulgaris by ∼175 kb.Markers present in scaffold 25,456 were associated by both QTL and BSA mapping. Two additional scaffolds contained BSA-associated markers, but these scaffolds were absent from the linkage map used for QTL analysis . Synteny mapping helped to delimit the physical section of the genome containing both resistance loci, but this required a lowering the standard sensitivity threshold for chromosomer . Based on these results, 15 scaffolds spanned the QTL and BSA-associated loci with flanking markers in scaffolds 3610 and 25,456 (see Supplementary File S4 fragment map). Consistent with previous findings (P. lunatus. Inflated gene counts can result from assembly fragmentation (a single gene sequence spread across multiple contigs) and failure to join distant exons together in a single transcript . As is typical of draft genome assemblies, this is likely an overestimation of the true gene number in P. lunatus to protein annotations for P. vulgaris, which diverged from P. lunatus ∼4 mya . Using public transcriptome data for lima bean, we found that the vast majority of P. vulgaris homologs had transcript evidence in P. lunatus  than those with hits (mean of 514 aa). However, 1356 had hits to other Viridiplantae species, and another subset of 2363 had hits to non-Viridiplantae entries in the nr database that were predominantly bacterial sequences. There were 903 hits to Enterobacteriaceae, which are the dominant members of many phyllosphere bacterial communities . The P. vulgaris genome has three particularly large clusters containing some 40 R-genes at the ends of chromosomes 4, 10, and 11 , but six of these, all of which were located on scaffold 974, contained a coiled-coil domain (Supplementary File S8).One of our objectives in sequencing the lima bean genome was to catalog R-genes commonly associated with disease resistance in plants. The majority of these are NLR genes defined by two major domains: a centrally located nucleotide-binding site domain, which has ATPase activity (NB-ARC) and a C-terminus leucine-rich repeat domain (LRR) , but addP. lunatus cultivar that was foundational to lima bean production in the east coast. De novo protein predictions showing high similarity to 96% of the encoded genes for P. vulgaris (common bean) corresponded to 32,505 genes in lima bean, most of which were supported by transcriptome data . Raw read data from the individual libraries at the Sequence Read Archive include: SRX9040258, SRX9040257, SRX9040256, SRX9040255, SRX9040254, SRX9040253, SRX9040252, SRX9040251. Supplementary material is available at figshare: https://doi.org/10.25387/g3.14398910. This Supplementary material includes eight files: a repeat masked version of the genome assembly (Supplementary File S1); the linkage map and QTL mapping results from IciMapping (Supplementary File S2); synteny maps from Chromosomer (Supplementary Files S3 and S4); all genes predicted by Augustus (Supplementary File S5); proteins of P. lunatus with a match to a P. vulgaris protein (Supplementary File S6); transcript coverage for P. lunatus predicted genes (Supplementary File S7); and P.lunatus R-gene annotations and comparison with P. vulgaris (Supplementary File S8).The genome sequence data for this studyis available under the NCBI BioProject PRJNA647124, for BioSample SAMN15394833 (