Saccharomyces cerevisiae to determine the impact of the latest generation of red FPs on their binding partner. We used a yeast assay based on the aggregation and toxicity of misfolded polyQ expansion proteins linked to Huntington’s disease. Since polyQ aggregation and toxicity are highly dependent on the sequences flanking the polyQ region, polyQ expansions provide an ideal tool to assess the impact of FPs on their fusion partners. We found that unlike what is observed for green FP variants, yemRFP and yFusionRed-tagged polyQ expansions  show reduced toxicity.  However, polyQ expansions tagged with the bright synthetically engineered ymScarlet displayed severe polyQ toxicity. Our data indicate that ymScarlet might have significant advantages over the previous generation of red FPs for use in fluorescent fusions in yeast.Development of fluorescent proteins (FPs) enabled researchers to visualize protein localization and trafficking in living cells and organisms. The extended palette of available FPs allows simultaneous detection of multiple fluorescent fusion proteins. Importantly, FPs are originally derived from different organisms from jelly fish to corals and each FP displays its own biophysical properties. Among these properties, the tendency of FPs to oligomerize inherently affects the behavior of its fusion partner. Here we employed the budding yeast Aqueaora victoria  from the jellyfishictoria , allowintramers . While dtramers .ex1) resulting in chorea and cognitive defects in patients (ex1 in yeast results in severe polyQ aggregation and growth defect (We recently established a method to rapidly compare the behavior of FPs against a monomeric variant of superfolder GFP (msfGFP) in yeast  4 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail). Equal amount of proteins were spotted on a nitrocellulose membrane. Membranes were blocked for 30 min in PBS-0.05%Tween at room temperature were and then processed for immunoblot. Membranes were probed with anti-FLAG primary antibody  overnight at 4°C and subsequently with a secondary anti-mouse fluorescent antibody  for 1h at room temperature and imaged using a Bio-Rad ChemiDoc MP imaging system. Membranes were then stripped using the Gene Bio-Application stripping buffer and reprobed with an anti-Pgk1 primary antibody (Thermo 22C5D8) using the same secondary antibody. InUnder the different experimental conditions, cells were diluted 10x in growth media and plated in Lab-tek (Thermo Inc.) imaging chambers and processed for fluorescence microscopy. Images inCell were cultured with appropriate media and processed for flow cytometry using a BD Bioscience FACS Celesta flow cytometer equipped with a 561 Yellow laser for imaging of RFPs. Data were analyzed using the BD FACS Diva software. All conditions were performed in triplicates, 20,000 cells were analyzed and median fluorescence intensities were calculated. No gates were applied.A one way ANOVA followed by a multiple comparison test (Tukey’s or Dunnett’s according to figure legends) was used to determine statistical significance between the different experimental conditions inTo analyze the performance of the three different RFPs in yeast, we first generated codon optimized versions of both FusionRed and mScarlet  . CentromRaw data behind Figures 1, 2, 3 and 4Click here for additional data file.Copyright: © 2018 Albakri MB et al.2018Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).ex1 carrying either 25Q  or 68Q (HD-associated)  . 25Q con fusion . Interes fusion . Based o fusion . In addi fusion . The cau fusion . MoreoveThe data referenced by this article are under copyright with the following copyright statement: Copyright: © 2018 Albakri MB et al.Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).https://doi.org/10.5256/f1000research.15829.d225385 (F1000Research: Dataset 1. Raw data behindBoth p415 GPD-yFusionRed and p415 GPD-ymScarlet are available from addgene (#111916/11917). All plasmids are available upon request from the corresponding author. I have read the revised version and I am happy with the changes the authors have made and I now recommend the work be indexed.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have no further comments to make.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this manuscript Albakri Maram et al. demonstrated superior performance of the mScarlet red fluorescent protein over the FusionRed protein in the Huntington’s disease related yeast assay. First, authors showed the higher brightness of non-fused mScarlet protein vs FusionRed during constitutive expression in yeast. Albakri Maram et al. then made fusions of the mScarlet and FusionRed proteins with first exon of the Huntingtin protein (Httex1) carrying FLAG-tag and either non-pathological 25 repeats of glutamines or Huntington’s disease-associated 68 repeats of glutamines. Authors expressed these fusions in yeast and found that in opposite to the FusionRed protein, mScarlet displayed right cytotoxicity and polyQ aggregation. Overall, mScarlet red fluorescent protein can be used as a right tag in polyQ yeast assay for the Huntington’s disease studies. The strategy suggested in this paper may be used researchers for characterization of the properties of other newly developed fluorescent markers. Major points:  1) Results and discussion section, page 6, right column. Analyzing protein expression level on dot blot authors revealed lower levels of yFusionRed protein as compared to other RFPs and based on this fact they suggested its increased degradation in yeast cells . These findings suggest that lower expression level of yFusionRed protein in yeast my prevent FusionRed protein from toxicity and formation of aggregates, which could be observed at higher FusionRed protein concentrations only. So we cannot say unambiguously about the real reasons of the absence of toxicity and aggregates formation in the case of FLAG-68Q-yFusionRed fusion. Lower expression level of yFusionRed also may masks its tendency to oligomerize inherently and prevent from correct comparison in this respect with other fluorescent proteins. Minor points:  1) Introduction section, page 3, left column, last sentence, please, replace misprint “…and aggregation assays to explore to effects of…” with “…and aggregation assays to explore the effects of…”. 2) Methods section, page 3, right column, Yeast strains and culture conditions section, authors use term W303A. However according to provided reference it should be named as W303-1A. Please, correct. 3) Methods section, page 3, right column, DNA constructs section, authors mention that they do not use proline rich domain. Please, add explanation of the reason of this. 4) Methods section, page 3, right column, Dot blot section, please, replace misprint “…PSMF…” with “…PMSF…”. 5) Methods section, page 3, right column, Dot blot section, please, replace misprint “…at room temperature were then processed…” with “…at room temperature and then processed …”. 6) Methods section, page 3, right column, Fluorescent microscopy section and elsewhere in the text, please, replace misprint “…Fluorescent microscopy…” with “…Fluorescence microscopy…”. 7) Methods section, page 4, Table 1. I would recommend to change the abbreviations of the constructs in order to mention Httex1 and avoid 25Q abbreviation which can be mistakenly interpreted as a substitution of the residue in position 25 with Q, i.e. for example, replace “p415 Gal1-FLAG-25Q-ymsfGFP” with “p415 Gal1-FLAG-Httex1-(Q)25-ymsfGFP”. 8) Methods section, page 5, Figure 2, panel A. I would recommend replace “Httex1” with “Httex1(Q)25/(Q)68”. 9) Methods section, page 5, Figure 2, panel A and elsewhere in the text. In accordance to the 6th point I would recommend replace “25Q” and “Q68” with “(Q)25” and “(Q)68”. 10) Methods section, page 5, Legend to the Figure 1. Please, add explanation that panel (A) represents a “Scheme of fluorescent proteins expression vectors …”. Also, please, be consistent in the same respect for the panel (C). 11) Methods section, page 5, legend to the Figure 2. Please, add explanation that panel (A) represents a “Scheme of fluorescent proteins expression system …”, panel (B) – “Image of yeast colonies …” and panel (C) – “Dot blots of …”. 12) Results and discussion section, page 6, left column and elsewhere in the text and figure legends, please, replace misprint “Fluorescent intensities…” with “…Fluorescence intensities…”.  13) Results and discussion section, page 6, left column, authors mention that “…yFusionRed was significantly dimmer than yemRFP”. Below they write about the strongest fluorescent signal for ymScarlet. Please, provide these comparisons in more quantitative and scientific manner, i.e. mention in how many fold yFusionRed and ymScarlet RFPs were dimmer and brighter than yemRFP, respectively.  14) Results and discussion section, page 6, left column, please, replace misprint “… in yeast can differed from …” with “… in yeast can be different from …”. 15) Results and discussion section, page 6, left column, please, replace misprint “… of mScarlet compared to other RFs…” with “…of mScarlet compared to other RFs …”. 16) Results and discussion section, page 6, right column. Authors use 68 repeats of Q however in their previous paper of the year 2017 they utilized 72 Q repeats. Please, include in the text explanation why did you choose 68 not 72 repeats. It is important for the comparison of the data. 17) Results and discussion section, page 6, right column. Authors write that “…yFusionRed displayed no significant toxicity…”. The term “significant” suggests statistical analysis, could you, please, provide which statistic criterion was used in this case, e.g. a two-tailed student t-test or Mann-Whitney or someone else? Now Statistical analysis section describes Figure1D data only. Overall, if the points raised above will be adequately addressed, indexing in F1000Research is appropriate.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We thank the reviewer for helpful comments. We have addressed concerns in the new version of the manuscript. See below for the authors’ response to specific points raised during the review process. Major points:  1) Results and discussion section, page 6, right column. Analyzing protein expression level on dot blot authors revealed lower levels of yFusionRed protein as compared to other RFPs and based on this fact they suggested its increased degradation in yeast cells . These findings suggest that lower expression level of yFusionRed protein in yeast my prevent FusionRed protein from toxicity and formation of aggregates, which could be observed at higher FusionRed protein concentrations only. So we cannot say unambiguously about the real reasons of the absence of toxicity and aggregates formation in the case of FLAG-68Q-yFusionRed fusion. Lower expression level of yFusionRed also may masks its tendency to oligomerize inherently and prevent from correct comparison in this respect with other fluorescent proteins.We agree with the reviewer on this point. We have now performed additional experiments where polyQ fusions are expressed using multicopy plasmids (2µ) were cells can express up to 50 copies of the constructs. Interestingly, under these conditions, 72Q-yFusionred can indeed aggregate and induce toxicity, albeit to lower extant than ymsfGFP-tagged polyQ counterparts. Therefore it is reasonable to hypothesize that lower expression of yFusionRed (maybe due to increased protein turnover) is responsible for decreased toxicity. Further experiments beyond the scope of this research note will be required to explain the relative lower expression of FusionRed in yeast. Minor points:  1) Introduction section, page 3, left column, last sentence, please, replace misprint “…and aggregation assays to explore to effects of…” with “…and aggregation assays to explore the effects of…”.We have modified accordingly. 2) Methods section, page 3, right column, Yeast strains and culture conditions section, authors use term W303A. However according to provided reference it should be named as W303-1A. Please, correct.We have modified accordingly. 3) Methods section, page 3, right column, DNA constructs section, authors mention that they do not use proline rich domain. Please, add explanation of the reason of this.We added the rationale for the use of the Δ proline constructs which is described in Duennwald et al., 2006 PNAS. 4) Methods section, page 3, right column, Dot blot section, please, replace misprint “…PSMF…” with “…PMSF…”.We have modified accordingly. 5) Methods section, page 3, right column, Dot blot section, please, replace misprint “…at room temperature were then processed…” with “…at room temperature and then processed …”.We have modified accordingly. 6) Methods section, page 3, right column, Fluorescent microscopy section and elsewhere in the text, please, replace misprint “…Fluorescent microscopy…” with “…Fluorescence microscopy…”.We have modified accordingly. 7) Methods section, page 4, Table 1. I would recommend to change the abbreviations of the constructs in order to mention Httex1 and avoid 25Q abbreviation which can be mistakenly interpreted as a substitution of the residue in position 25 with Q, i.e. for example, replace “p415 Gal1-FLAG-25Q-ymsfGFP” with “p415 Gal1-FLAG-Httex1-(Q)25-ymsfGFP”. 8) Methods section, page 5, Figure 2, panel A. I would recommend replace “Httex1” with “Httex1(Q)25/(Q)68”. 9) Methods section, page 5, Figure 2, panel A and elsewhere in the text. In accordance to the 6th point I would recommend replace “25Q” and “Q68” with “(Q)25” and “(Q)68”.We thank the reviewer for the suggestion but the authors believe that to be consistent with the literature in the field, the current nomenclature should be used. 10) Methods section, page 5, Legend to the Figure 1. Please, add explanation that panel (A) represents a “Scheme of fluorescent proteins expression vectors …”. Also, please, be consistent in the same respect for the panel (C).We have modified accordingly. 11) Methods section, page 5, legend to the Figure 2. Please, add explanation that panel (A) represents a “Scheme of fluorescent proteins expression system …”, panel (B) – “Image of yeast colonies …” and panel (C) – “Dot blots of …”. 12) Results and discussion section, page 6, left column and elsewhere in the text and figure legends, please, replace misprint “Fluorescent intensities…” with “…Fluorescence intensities…”. We have modified accordingly. 13) Results and discussion section, page 6, left column, authors mention that “…yFusionRed was significantly dimmer than yemRFP”. Below they write about the strongest fluorescent signal for ymScarlet. Please, provide these comparisons in more quantitative and scientific manner, i.e. mention in how many fold yFusionRed and ymScarlet RFPs were dimmer and brighter than yemRFP, respectively. We added values in the text. 14) Results and discussion section, page 6, left column, please, replace misprint “… in yeast can differed from …” with “… in yeast can be different from …”.We have modified accordingly. 15) Results and discussion section, page 6, left column, please, replace misprint “… of mScarlet compared to other RFs…” with “…of mScarlet compared to other RFs …”.We have modified accordingly. 16) Results and discussion section, page 6, right column. Authors use 68 repeats of Q however in their previous paper of the year 2017 they utilized 72 Q repeats. Please, include in the text explanation why did you choose 68 not 72 repeats. It is important for the comparison of the data.The polyQ repeats are known to be unstable. Sequencing of the DNA revealed that our 72Q original plasmid has mutated into 68Q. This change does not impact the toxicity of the fusion. We added a note in the material and methods section. 17) Results and discussion section, page 6, right column. Authors write that “…yFusionRed displayed no significant toxicity…”. The term “significant” suggests statistical analysis, could you, please, provide which statistic criterion was used in this case, e.g. a two-tailed student t-test or Mann-Whitney or someone else? Now Statistical analysis section describes Figure1D data only.Statistical analysis was performed on the growth curve data and described in the updated figure legends.  This study reports the impact of three different red fluorescent protein (RFP) tags on the expression and toxicity patterns of Httex1 in yeast by benchmarking them against the green fluorescent protein tagged Httex1 as the established model system in the literature. The authors reveal some differences in their effects on Httex1 toxicity and these results are likely to be of interest to those in the Httex1-polyQ field. Overall, the manuscript is well-written and figures are clearly presented. It should be noted that the phenomena reported here is a direct extension of a broader study of other fluorescent protein fusions previously reported by the same group (and has been been referenced).The authors state that the median fluorescent intensities if Fig 1 “reveals yFusionRed was significantly dimmer than yemRFP …  ymScarlet displayed the strongest fluorescent signal”. As written, this implies the median fluorescence intensity provides a measure of the relative brightness of individual fluorophores. However, without quantification of the amount of protein expressed this difference in median fluorescence could simply reflect differences in total fluorophore abundance. We are not familiar with yeast transfection methods - but certainly in mammalian transfection experiments cells will invariably have a massive (many fold) variation in expression levels within populations and between different constructs including a notable population of cells that have no expression. If this is the case in yeast, then the interpretation of difference in brightness is not sufficiently supported by the data. This point needs clarification.Following from the above point, it is difficult to ascertain whether the differences in expression, toxicity and aggregate extents of the various fluorescent protein tagged Htt constructs  arises from the same issues. To us this would be a more likely (and simpler) explanation than what the authors explain as the protein constructs having differences in inherent toxicity (molecule per molecule). Further clarification of this issue is required.   The level of reproducibility should be indicated by including at least 3 independent replicates for the dot blots and growth assays .  Also, the dot blots should be quantified with the appropriate statistical methods. In Fig 1D, the authors report a significant difference in the median fluorescent intensities using a Student’s t-test to compare only two of the RFPs examined. It is suggested that an ANOVA would be more appropriate because there are more than two groups of interest. Based on the data provided, our ANOVA reports a higher degree of statistical significance for the comparison currently reported  and also supports a significant difference between ymFusionRed and ymScarlet (***).   Major queries:This sentence in the abstract has an unclear meaning as it implies ymScarlet is inherently affected by polyQ: “We found that unlike yemRFP and yFusionRed, the synthetically engineered ymScarlet displayed severe polyQ toxicity and aggregation similar to what is observed for green FP variants.”  Thus the sentence should be revised.Additional details for the Dot blot method  would enhance reproducibilityInclude dimension for scale bar in Figure 2D Minor suggestions:We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. We would like to thank the reviewers for their helpful comments. Below is our point-by-point response addressing the concerns raised during the review process. The authors state that the median fluorescent intensities if Fig 1 “reveals yFusionRed was significantly dimmer than yemRFP …  ymScarlet displayed the strongest fluorescent signal”. As written, this implies the median fluorescence intensity provides a measure of the relative brightness of individual fluorophores. However, without quantification of the amount of protein expressed this difference in median fluorescence could simply reflect differences in total fluorophore abundance. We are not familiar with yeast transfection methods - but certainly in mammalian transfection experiments cells will invariably have a massive (many fold) variation in expression levels within populations and between different constructs including a notable population of cells that have no expression. If this is the case in yeast, then the interpretation of difference in brightness is not sufficiently supported by the data. This point needs clarification.Yeast transformations are similar to E. coli transformations that the reviewers must be familiar with. All cells in the population are expressing the contructs since the cells are kept under constant selection. Since all constructs are expressed under the same promoter in a low copy centromeric vector (1-2 copies per cell), there is no reason to believe that some reason yFusionRed cells do not express fusion at the mRNA level. Quantification of the relative protein levels is problematic since anti-RFP antibodies could recognize mScarlet and yFusionred with different affinities. Addition of an additional epitope tag could affect behavior of the FPs. That said, we have used the polyQ fusions to show that yFusionRed protein levels are decreased using dot blot (see #3) and this appears to mask its ability to aggregate and consequently generate a toxic phenotype .Following from the above point, it is difficult to ascertain whether the differences in expression, toxicity and aggregate extents of the various fluorescent protein tagged Htt constructs  arises from the same issues. To us this would be a more likely (and simpler) explanation than what the authors explain as the protein constructs having differences in inherent toxicity (molecule per molecule). Further clarification of this issue is required.  We agree with the reviewer on this point. We have now performed experiments where polyQ fusions are expressed using multicopy plasmids (2µ) were cells can express up to 50 copies of the constructs. Interestingly, under these conditions, 68Q-yFusionred can indeed aggregate and induce toxicity, albeit to lower extant than ymsfGFP-tagged polyQ counterparts. Therefore it is reasonable to hypothesize that lower expression of yFusionRed (maybe due to increased protein turnover or mRNA level) is responsible for decreased toxicity. Further experiments beyond the scope of this research note will be required to explain the relative lower expression of FusionRed in yeast.The level of reproducibility should be indicated by including at least 3 independent replicates for the dot blots and growth assays .  Also, the dot blots should be quantified with the appropriate statistical methods.We have now added quantification of the dot blots and perfrom quantitative growth assays in liquid media to complement the spot assays on agar plates. In Fig 1D, the authors report a significant difference in the median fluorescent intensities using a Student’s t-test to compare only two of the RFPs examined. It is suggested that an ANOVA would be more appropriate because there are more than two groups of interest. Based on the data provided, our ANOVA reports a higher degree of statistical significance for the comparison currently reported  and also supports a significant difference between ymFusionRed and ymScarlet (***).We thank the reviewer and have now added appropriate statistical analysis. Major queries:   This sentence in the abstract has an unclear meaning as it implies ymScarlet is inherently affected by polyQ: “We found that unlike yemRFP and yFusionRed, the synthetically engineered ymScarlet displayed severe polyQ toxicity and aggregation similar to what is observed for green FP variants.”  Thus the sentence should be revised.We have modified accordingly.Additional details for the Dot blot method  would enhance reproducibilityWe have added additional information.Include dimension for scale bar in Figure 2D.We have added the information.  Minor suggestions:


P311 was first identified by the group of  Neuronal protein 3.1 (P311) is a small intracellular protein from an unknown family, whose transcript was first identified by Figure 1), while lung and kidney present a similar fibrotic process . This pathological process has been described in several organs, but is most intensely studied in liver, kidney, and lung. Hepatic stellate cells, renal mesangial cells, and resident lung fibroblasts, respectively, are the organ-specific resident cells that transdifferentiate to myofibroblasts and contribute to the scarring . P311 does not seem to be specific for one or the other migration mechanism. Fibroblasts overexpressing P311 can migrate in both fashions, depending on the culture dish coating or cytokine treatment, such as TGFβ1  by protein kinase C δ, ε, and ζ, resulting in a short-lived P311 protein and no migration. β1 integrin signaling on the other hand stimulates its dephosphorylation and consequently cell migration. Upon plating U118 cells on a motility-activating substrate Ser59’s phosphorylation is reduced, P311 is not degraded and indirectly stimulates ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activity . Additioas TGFβ1 . Correspd slower .Figure 3). Filamin A was shown to interact with integrin β1, present in the cell membrane, to activate TGFβ1 and regulate cell motility . Further research regarding the role of P311’s interaction with these two proteins and the potential effect on circular dorsal ruffle formation still needs to be done . While low P311 mRNA levels can be observed in glioma cell lines with a high expression of Met-HGF/SF, a tumor suppressive function for P311 was excluded since P311 overexpression in U118 glioma cells did not interfere with tumor growth in vivo. This suggested that diminished P311 expression is the result of a more transformed and tumorigenic phenotype. A similar observation was made for terminally differentiated human cortical neuronal cells (HCN-1A and HCN-2) where nerve growth factor exposure decreased P311 expression . Moreover, facial neurons that were transfected in vivo with P311 cDNA regenerated almost three times as fast as non-transfected facial neurons. This could suggest that P311 can interfere with Rho signaling by inducing p21waf1 expression through a thus far unknown mechanism , resulted in less proliferation due to less matrix metalloproteinases (MMP)2 and 9 activity and through direct interaction with P311, DLXIN-1 blocks P311’s invasive function. Finally, P311 is more expressed in regenerating sensory hair cells compared to hair cells that do not renew ] is stored in the matrix and its activated by proteolytic cleavage by MMP-2, MMP-9, or thrombospondin-1 or mechanical cleavage by integrins, upon tissue damage . Active al cells . Apart fin vitro , and reduced phosphorylation of SMAD2/3. As indicated in earlier reports, Tgfβ1 mRNA levels in these mice did not differ between the wild type and P311 null mice, but they present reduced TGFβ1 protein when P311 was absent , and a protein of the translation initiation complex. In addition, P311 forms a complex with the 5′UTRs of Tgfβ1, 2, and 3 by which it recruits Tgfβ mRNA and stimulates its translation . Finally, they also showed that the interaction between P311 and eIF3b occurs on the P311–eIF3b binding motif, which is a highly conserved sequence among different eukaryotes . Moreover, in P311 null mice, the TGFβ1 pathway is clearly less active, which results in hypotensive mice and reduces kidney fibrosis (Table 1). It was demonstrated that P311 could induce a myogenic phenotype in fibroblasts and stimulate its migration . Finally, P311 might have a role in integrin β1 recycling, since P311 interacts with Filamin A and MYH9, while the former is an interconnecting protein between F-actin and integrin β1, the latter is responsible for integrin β1 recycling with F-actin, which is stimulated by the RAC1 pathway . Silencing P311 in vivo or interference with P311 functionality might hamper the fibrogenic process and could allow the tissue to enter the regeneration phase.The question whether P311 is an interesting therapeutic target to be considered for anti-fibrotic therapy still remains unanswered, but circumstantial evidence indicates it might be a good candidate. Tissue fibrosis is mainly characterized by proliferative and contractile resident myofibroblasts and myofibroblast-like cells . A procefibrosis . With rein vivo might randomly interfere with crucial processes for tissue repair. This can be deduced from studies that showed that patients with less P311 have a decreased alveolar repair after emphysema (Table 2). For example, delivery of siRNA loaded in targeted liposomes, as already been shown for hepatic and pancreatic fibrosis (The safety of P311 inhibition can be estimated as relatively high, since P311-deficient mice do not present a detrimental phenotype, indicating that development and homeostasis of the body might not be influenced by P311 inhibition. However, since P311 stimulates cell migration, proliferation, and regeneration it indicates that untargeted delivery of siRNA or a compound that could block P311 function mphysema . Therefomphysema , targetimphysema , but ovefibrosis , or an ufibrosis . Such a in vivo effects of P311 modulation and its consequence to fibrosis (A large body of evidence suggests that P311 is necessary for correct TGFβ1 production and its inhibition might result in a less myofibroblastic phenotype, decreased TGFβ1 protein expression and collagen deposition. Importantly, few studies have explored the fibrosis . Thus, tLS and LvG conceived the work and provided the layout for it. LS and IM drafted the work. LvG critically revised the text and figures and approved the final manuscript for publication.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.


Despite increased use of vaccine in routine immunisation, rotavirus remains a major cause of acute gastroenteritis (AGE) in low-income countries. We describe rotavirus prevalence and hospitalisation in Malawi pre and four years post vaccine introduction; provide updated vaccine effectiveness (VE) estimates; and assess rotavirus vaccine indirect effects.Children under five years of age presenting to a referral hospital in Blantyre with AGE were recruited. Stool samples were tested for rotavirus using Enzyme Immunoassay. The change in rotavirus prevalence was evaluated using Poisson regression. Time series analysis was used to further investigate trends in prevalence over time. VE against rotavirus diarrhoea of any severity was estimated using logistic regression. Indirect effects were estimated by evaluating rotavirus prevalence in unvaccinated children over time, and by comparing observed reductions in incidence of rotavirus hospitalisation to those expected based on vaccine coverage and trial efficacy estimates.2320 children were included. Prevalence of rotavirus in hospitalised infants (<12 months) with AGE decreased from 69/139(49.64%) prior to vaccine introduction to 197/607(32.45%) post-vaccine introduction . Prevalence in children aged 12–23 months demonstrated a less substantial decline: 15/37(40.54%) pre- and 122/352(34.66%) post-vaccine introduction . Adjusted VE was 61.89%(95% CI 28.04–79.82), but lower in children aged 12–23 months (31.69% [95% CI −139.03 to 80.48]). In hospitalised infants with rotavirus disease, the observed overall effect of the vaccine was 9% greater than expected according to vaccine coverage and efficacy estimates. Rotavirus prevalence among unvaccinated infants declined post-vaccine introduction (RR 0.70[95% CI 0.55–0.80]).Following rotavirus vaccine introduction in Malawi, prevalence of rotavirus in hospitalised children with AGE has declined significantly, with some evidence of an indirect effect in infants. Despite this, rotavirus remains an important cause of severe diarrhoea in Malawian children, particularly in the second year of life. We have previously published VE estimates and population impact 3 years after vaccine introduction 2This study was conducted at Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi. QECH is the only government hospital providing free inpatient care to the estimated 151,328 under-5 population of Blantyre City Our surveillance platform has been described previously in detail 2.1Stool samples were batch-tested weekly for rotavirus antigen using enzyme immunoassay . HIV testing of mothers and children was conducted using two sequential EIA rapid tests , or HIV DNA PCR for infants under one year of age 2.22.2.12 tests. Student’s t or rank sum tests were used to compare independent means or medians, respectively.Continuous normally distributed data were described using mean and standard deviation (SD), or median and interquartile range otherwise. Differences in independent categorical covariates were assessed using χ2.2.2To evaluate yearly differences in rotavirus prevalence since vaccine introduction, we analysed the surveillance platform as a cohort and used Poisson regression models with robust standard errors 2.2.3We used time series analysis to describe trend and seasonality in the prevalence of rotavirus in diarrhoeal stools over time. We applied a locally weighted smoother (defined as (1/8) ∗ [1 ∗ x(t − 2) + 2 ∗   x(t − 1) + 2 ∗ x(t) + 2 ∗ x(t + 1) + 1 ∗ x(t + 2)]) to mean monthly proportion of rotavirus-positive stools to define seasonality and a second locally weighted smoother (defined as (1/24) ∗ [1 ∗ x(t − 6) + 2 ∗ x(t-5) + 2 ∗ x(t − 4) + 2 ∗ x(t − 3) + 2 ∗ x(t − 2) + 2 ∗ x(t − 1) + 2 ∗ x(t) + 2 ∗ x(t + 1) + 2 ∗ x(t + 2) + 2 ∗ x(t + 3) + 2 ∗ x(t + 4) + 2 ∗ x(t + 5) + 1 ∗ x(t + 6)], where x(t) = percentage rotavirus-positive stools per month) to the same to define secular trend. Trend in rotavirus prevalence over time was then assessed in a linear model.2.2.4Vaccine effectiveness (VE) was estimated from the nested case-control study as (1-Odds Ratio for 2 vs 0-dose vaccine receipt) among rotavirus EIA-positive rotavirus cases against EIA test-negative gastroenteritis controls, defined as described above, using unconditional logistic regression, and reported as a percentage. Estimates were adjusted for age and for secular and seasonal fluctuations using indicator variables for year and month of admission.2.2.5i.Incidence  was estimated using projected population estimates for Blantyre city, based on the proportion of infants at the national level.ii.The expected population direct effect  was calculated as vaccine coverage × vaccine efficacy,where both vaccine coverage and efficacy were expressed as proportions and using a value of vaccine efficacy of 49.4% for severe disease in infants, 34.5% for all rotavirus gastroenteritis in infants and 17.6% for children with severe disease iii.The observed overall effect was calculated by comparing the semi-annual post-vaccination incidence to incidence for the half-year prior to vaccine introduction (January to June 2012) using:(pre-vaccine incidence – post-vaccine incidence)/pre-vaccine incidence.iv.observed effect – expected effectThe estimated indirect effect was calculated as Poisson regression models with robust standard errors were used to identify any change in the prevalence of rotavirus-positive gastroenteritis among unvaccinated admitted infants (<12 months of age) and children (12–59 months of age) following introduction of rotavirus vaccine using all available data. We then estimated the expected overall reduction in incidence of rotavirus gastroenteritis among admitted infants based on the direct effect of vaccination alone and compared this to the observed overall reduction. We made the assumption that any additional observed reduction in incidence may be attributed to the indirect effects of the vaccine For comparison purposes and to look for evidence of secular trends in diarrhoeal admissions to QECH, the incidence rate of hospitalised rotavirus-negative gastroenteritis was estimated using the denominators described above to estimate child-years at risk, and change over time was evaluated using incidence rate ratios.Analyses were restricted to those children with stool samples collected. All analyses except VE estimates were restricted to admitted children. For analysis of trend in rotavirus prevalence and VE estimates, children were categorised into <12 month or 12–23 month age groups. For assessment of vaccine indirect effects, children up to 59 months of age were included. Analyses were conducted using Stata 12.1 .2.3Ethical approval was obtained from the National Health Sciences Research Committee, Lilongwe, Malawi (867), and by the University of Liverpool Research Ethics Committee (000490).3Stool samples were collected from a total of 2320 children  between 1st January 2012 and 30 June 2016. Of these, 1318  were eligible for both doses of rotavirus vaccine, and 1130  had documented evidence of vaccine status. Characteristics of the population are shown in 3.12p<0.001). Linear regression showed a significant negative trend in prevalence of rotavirus over time in infants  and in children aged 12–23 months , where the regression coefficient represents the percentage change in rotavirus positivity per month.In the four years since vaccine introduction, the relative risk of rotavirus among children admitted with diarrhoeal disease has consistently declined ; however3.2The adjusted VE for two doses of vaccine, across all ages and disease severity was 61.89%  , whilst 3.3Among unvaccinated infants with gastroenteritis, rotavirus prevalence declined from 117/221 (52.94%) in the 10 months prior to vaccine introduction to 65/184 (35.33%) in the 14 months following vaccine introduction  . This anIn contrast to the incidence rate of rotavirus-positive hospitalisations, the incidence of hospitalisation for rotavirus-negative gastroenteritis increased over time. The incidence rate of hospitalisation with rotavirus-negative gastroenteritis among infants was 479 per 100,000 child-years at risk prior to rotavirus vaccine introduction and 655 per 100,000 child-years at risk subsequently . For children, the incidence rate for hospitalisation with rotavirus-negative gastroenteritis was 77 per 100,000 child-years pre- and 136 per 100,000 child-years post-rotavirus-vaccine introduction .4Four years after programmatic rotavirus vaccine implementation in Malawi, we continue to demonstrate VE against rotavirus disease of all severity , and a consistent decline in the prevalence of rotavirus in children presenting to hospital with gastroenteritis. There is some evidence of a reduction in disease among unvaccinated infants, possibly attributable to indirect vaccine effects. However, children aged 12–23 months demonstrate a less pronounced reduction in disease and a lower point estimate for VE compared to those under 12 months of age, and overall there remains a significant residual burden of disease with rotavirus associated with just over 1 in 4 gastroenteritis admissions.Gavi support has allowed LMICs such as Malawi to successfully introduce rotavirus vaccination. However, in the long-term immunisation programmes must be locally funded. Robust vaccine effectiveness estimates, demonstration of sustained impact, and evaluation of indirect effects are essential to allow national governments to evaluate the cost-effectiveness of vaccines. A major strength of our study is its basis in a long-standing surveillance system, within which a case-control study to evaluate vaccine effectiveness has been nested. As vaccine coverage increases, and as it becomes increasingly difficult to identify unvaccinated children for the case-control study, on-going surveillance will allow evaluation of incidence over time. The high vaccine effectiveness seen in infants with severe disease (83.24%  is striking. Although this result should be interpreted within the context of moderately wide confidence limits, it is consistent with our previously reported estimates for Malawi and with recent estimates for RV5 from Rwanda Effectiveness point estimates in children aged 12–23 months appear lower than those in infants. The confidence bounds around these estimates are wide, and it is possible that this simply reflects uncertainty in point estimates as a result of fewer AGE episodes in the older age group, but the finding is corroborated by the relative risk of rotavirus gastroenteritis compared to test-negative controls, which has not decreased in this age group, and the proportion of rotavirus cases occurring in children 12–23 months of age, which has increased from 18% to 38% following vaccine introduction. This lower VE in the second year of life is consistent with observations in Colombia and Brazil Indirect effects of rotavirus vaccine have been observed in high-income countries The absence of observed indirect effects in these groups is contrary to findings from higher income settings and from Rwanda These findings could however also represent under-ascertainment by this study. In particular, our measurement of indirect effects only captures the effect on hospitalised cases which are a small proportion of the community burden of rotavirus disease, and the observed increase in incidence of hospitalisation with rotavirus-negative diarrhoeal disease following vaccine introduction could bias estimation of vaccine impact based on hospitalised cases and estimation of indirect effects derived from this towards the null. Increasingly high vaccine coverage limits further analysis of indirect effects, and mathematical modelling may be required to investigate the presence and extent of such effects in greater depth.It is not clear why we observed an increase in incidence of test-negative diarrhoeal cases following vaccine introduction. This could represent secular changes in health-care seeking behaviour or admission patterns with time, but also may reflect the short period of enhanced surveillance before this point, which limits assessment of trends in rotavirus prevalence prior to vaccine introduction. Prevalence estimates can be affected by secular changes in denominators, and ongoing surveillance is required to evaluate the long term impact of vaccine in our setting.5Our data demonstrate substantial and consistent reductions in rotavirus prevalence in hospitalised children four years since vaccine introduction and robust vaccine effectiveness against all-severity rotavirus disease in infants reinforcing the importance of rotavirus vaccine in reducing the burden of disease in low-income countries. Additionally our data suggest the presence of population indirect effect in infants, although not in other age groups. Although factors other than vaccine introduction may also contribute to overall disease reduction, our analysis was not designed to formally account for these. We are however, unaware of major health system or socioeconomic or nutritional changes in Malawi over the period of our observation that might have also contributed to reductions in disease incidence. Importantly, our data also show the persistent burden of rotavirus, particularly in children in the second year of life. Given the ongoing prevalence of rotavirus disease in this population, strategies to further improve the effectiveness of current vaccines maybe required to maximise protection for all children against rotavirus disease.


Pure red cell aplasia (PRCA) is a rare syndrome that only affects the erythroid lineage. It is defined by a normocytic, normochromic anemia with a marked reticulocytopenia and severe reduction or absence of erythroid precursors in the bone marrow. Treatment of primary, idiopathic PRCA is immunosuppressive therapy. Although it is rare, isolated cytogenetic abnormalities can be seen in PRCA, and abnormal karyotype is associated with poor response to immunosuppressive therapy and poor prognosis. We describe a 77-year-old male with primary, idiopathic PRCA and a deletion of chromosome 20q, del(20q), in the bone marrow cells. He was successfully treated with immunosuppressive therapy and became transfusion-independent. The same cytogenetic abnormality has also been described in a few other reports; taken together, these observations suggest that del(20q) may represent a recurrent cytogenetic abnormality in PRCA. Our case report clearly illustrates that even patients with primary PRCA and an abnormal karyotype can respond to immunosuppression and become transfusion-independent. Pure red cell aplasia (PRCA) is a rare syndrome that solely affects erythroid lineage. It is defined by a normocytic, normochromic anemia with a marked reticulocytopenia and severe reduction or absence of erythroid precursors from the bone marrow , 2. The The primary form of PRCA is considered to be an autoimmune disease with immune-mediated inhibition of the differentiation and maturation of erythroid precursors –3. On th12/L (0.03–0.1), thrombocytes 445 × 109/L (145–348), and total leukocytes 6.8 × 109/L (3.5–11.0). The peripheral blood differential count showed neutrophils, 4.8 × 109/L (1.7–8.2), lymphocytes, 0.9 × 109/L (0.7–5.3), monocytes, 0.7 × 109/L (0.04–1.30), eosinophils, 0.4 × 109/L (0.0–0.7), and basophils, <0.1 × 109/L (0.0–0.3). Thus, the patient had a normocytic, normochromic anemia with low reticulocyte counts but no evidence for a general bone marrow failure.A 77-year-old man was undergoing follow-up at his primary hospital due to chronic kidney disease stage 4. In addition, he had abnormal levels of liver and pancreas serum markers of unknown etiology. His medical history included hypertension, hypercholesterolemia, Barrett's esophagus, and stenting of the left carotid artery due to a transient ischemic attack. During routine follow-up, blood tests revealed a progressive normocytic, normochromic anemia. The patient did not respond to the initial treatment with iron supplements and erythropoietin injections. There was a gradual progression until the blood tests showed hemoglobin (Hb) 6.0 g/dL , mean corpuscular volume (MCV) 101 fL (82–98), reticulocytes <0.010 × 10A bone marrow biopsy showed total absence of erythropoiesis with normal megakaryocytes and normal granulocytopoiesis with large amounts of iron in the bone marrow . This waFurther investigations included serological tests for B19 parvovirus, Epstein–Barr virus (EBV), cytomegalovirus (CMV), hepatitis A, hepatitis B, hepatitis C, and human immunodeficiency virus (HIV), but there was no evidence for any of these infections being the cause of the hematological disease. A computed tomography (CT) scan of the chest showed no signs of thymoma, and flowcytometric analyses of the bone marrow did not detect any evidence for monoclonal B-cell or T-cell populations. Karyotyping of bone marrow cells revealed a deletion at the long arm of chromosome 20 and the karyotype 46,XY,del(20) (q11)[6]/46,XY[14] .The findings justified the diagnosis of PRCA with the chromosomal abnormality del(20q). Previous studies suggest treatment with cyclosporine A to be superior to corticosteroids and cyclophosphamide with regard to response rate and relapse-free survival , 7. ThisTapering of corticosteroids was initiated after six weeks and was initially without any complications . HemogloIn this case report, we present a PRCA patient with a deletion of chromosome 20q in the bone marrow cells, and no evidence was found for concomitant hematologic disorders or other disorders commonly associated with PRCA  , 2. The Although the associations with other conditions and differences in the responsiveness to various therapeutic strategies suggest a diverse pathophysiology of PRCA , 7, 8, tOur patient had an isolated deletion of chromosome 20q which has only been reported in a few cases of PRCA. Kurtin et al. reported two such PRCA cases with no other hematologic disorders, and they postulated an association between this karyotypic abnormality and PRCA . Wang etL3MBTL1 polycomb tumor suppressor protein; this has been shown to cause replicative stress and genomic instability in vitro [L3MBTL1 represses human-induced pluripotent stem cells (iPSCs) for hematopoietic differentiation and enhances commitment toward the erythroid lineage [L3MBTL1, it is not known which of the lost genes are involved in the development of MDS and other myeloid disorders [The del20q) abnormality is associated with dyserythropoiesis and dysmegakaryocytopoiesis and is thought to represent an early aberration in hematologic malignancies due to the loss of the in vitro , 10, 13. lineage . Del(20q lineage , 13, 15  lineage , whereas lineage . However lineage . The abn lineage . Besidesq abnormaisorders , 21, 22.PRCA is a very diverse and rare disease with no standardized therapeutic strategy. The treatment is mainly based on experience and case reports . The firMonoclonal antibodies such as alemtuzumab and rituximab are other alternatives commonly used for patients with refractory PRCA. Varying results have been reported for these drugs , 27, 28,PRCA with abnormal karyotype seems to be associated with poor response to immunosuppressive therapy and consequently poor prognosis. Lacy et al.  reportedTo conclude, PRCA is a rare disease, and cytogenetic abnormalities are detected for a subset of these patients. The role of such abnormalities in the pathogenesis of PRCA remains elusive. However, even patients with cytogenetic abnormalities may respond to immunosuppressive treatment and become transfusion-independent. For this reason, detection of an abnormal karyotype should not exclude immunosuppression as the first-line treatment.


Wolbachia influence several aspects of the fitness and evolution of their terrestrial hosts, but few analogous partnerships have been found in marine systems. We report here the genome, phylogenetics, and biogeography of a ubiquitous and novel Rickettsiales species that primarily associates with marine organisms. We previously showed that this bacterium was found in scleractinian corals, responds to nutrient exposure, and is associated with reduced host growth and increased mortality. This bacterium, like other Rickettsiales, has a reduced genome indicative of a parasitic lifestyle. Phylogenetic analysis places this Rickettsiales within a new genus we define as “Candidatus Aquarickettsia.” Using data from the Earth Microbiome Project and SRA databases, we also demonstrate that members of “Ca. Aquarickettsia” are found globally in dozens of invertebrate lineages. The coral-associated “Candidatus A. rohweri” is the first finished genome in this new clade. “Ca. A. rohweri” lacks genes to synthesize most sugars and amino acids but possesses several genes linked to pathogenicity including Tlc, an antiporter that exchanges host ATP for ADP, and a complete Type IV secretion system. Despite its inability to metabolize nitrogen, “Ca. A. rohweri” possesses the NtrY-NtrX two-component system involved in sensing and responding to extracellular nitrogen. Given these data, along with visualization of the parasite in host tissues, we hypothesize that “Ca. A. rohweri” reduces coral health by consuming host nutrients and energy, thus weakening and eventually killing host cells. Last, we hypothesize that nutrient enrichment, which is increasingly common on coral reefs, encourages unrestricted growth of “Ca. A. rohweri” in its host by providing abundant N-rich metabolites to be scavenged.Bacterial symbionts are integral to the health and homeostasis of invertebrate hosts. Notably, members of the Rickettsiales genus  Symbioses are common in marine systems, and the stability of these relationships among hosts and symbionts are often essential to each member’s health . Yet symAcropora cervicornis, identified a Rickettsiales-annotated OTU that increased in relative abundance from 11.4 to 87.9% of the total coral bacterial community after exposure to elevated inorganic nutrients [Our recent study of the endangered Caribbean staghorn coral, utrients . Furtherutrients . We alsoutrients .Rickettsia)-like organisms (RLOs) [The order Rickettsiales is one of several microbial groups being investigated as a disease agent of corals, but the high prevalence of these bacteria in both visually healthy and diseased corals renders their etiological role unclear , 11. Rics (RLOs) , 12, buts (RLOs)  found thRickettsiaceae, Anaplasmataceae, and “Candidatus Midichloriaceae” [Rickettsiaceae consists of bacteria that induce spotted fever rickettsiosis in humans [“Ca. Midichloriaceae” have been shown to infect protists and the mitochondria of metazoans [Anaplasmataceae consists of four genera: Anaplasma, Ehrlichia, Neorickettsia, and Wolbachia [Members of the order Rickettsiales are typically obligate intracellular parasites, often associated with invertebrate vectors and sometimes pathogenic to humans. They have highly reduced genomes, averaging 1.1–1.5 Mb –17, withriaceae” , 20–22. n humans , 23, whietazoans –26. The Candidatus Aquarickettsia rohweri,” which is named for the association of members of this genus with aquatic organisms and Dr. Forest Rohwer, whose group first described this taxon in corals. This study places “Ca. A. rohweri” in the family “Ca. Midichloriaceae”, and assesses genome features related to its host dependency and potential virulence mechanisms. The presence of genes for secretion and metabolite acquisition as well as genes involved in sensing extracellular nutrient concentrations suggests that “Ca. Aquarickettsia” exploits its host for necessary nutrients, energy, and amino acids. Last, we report that members of “Ca. Aquarickettsia” are found not only in corals but also in other aquatic organisms, including sea anemones, sponges, hydrozoans, placozoans, ctenophores, and protists worldwide.We report here the genome assembly, phylogenetics, and biogeography of the Rickettsiales OTU identified in our previous studies. For this organism, we propose the novel genus and species “A. cervicornis in the field. In brief, we obtained 40 fragments of A. cervicornis from the Coral Restoration Foundation’s coral nursery in Key Largo, Florida. Fragments were transported by boat to Pickles Reef, Florida, and secured to mounting structures using All Fix Marine Epoxy®. Corals were allowed to acclimate for 1 week before 20 fragments were exposed to 70 g of Osmocote 19-6-12 Smart-Release® Plant fertilizer in mesh bags that were attached ~5 cm below corals. The remaining 20 fragments were maintained under ambient conditions at the site to serve as unenriched controls. Enrichment was maintained for 4 weeks with fertilizer bags replaced after 2 weeks. Shaver et al. [Following the methods of Shaver et al. , we perfr et al.  demonstrSymbiodiniaceae abundance, and tissue components  were assessed using a previously established numerical system [Due to mortality or breakage, 34 coral fragments of the initial 40 were sampled for histological analysis. Fragments were placed in 50-mL centrifuge tubes filled with a formaldehyde-based fixative composed of 1:4 parts Z-Fix Concentrate  and Instant Ocean® seawater. Centrifuge tubes were capped, tightly sealed using Parafilm, and shipped to the Histology Laboratory at George Mason University for analysis using methods from Miller et al. . and Pril system . Image aA. cervicornis specimens from the high-nutrient treatments were sectioned onto glass slides at a thickness of 10–15 μm. Sections were deparaffinized 2× for 10 min in xylene, 2× for 10 min in 95% ethanol, rinsed once in distilled water, and air dried. FISH with end-labeled oligonucleotide probes was performed in a humidity chamber in hybridization buffer  with 40% formamide for 2–4 h. After hybridization, the slides were incubated at 48 °C in wash buffer  for 20–40 min, depending on the hybridization incubation period. The wash buffer was rinsed away with distilled water, and slides were air dried and mounted in VectaShield . Slides were visualized on a Nikon Eclipse E800 epifluorescence microscope. All probes, including the general bacterial probe suite  that together target most known bacteria, the bacterial probe negative control (EUB338-NEG), and the sequence-specific probes designed in this study , were used at a final concentration of 10 ng/μl in hybridization buffer.The protocol for fluorescence in situ hybridization (FISH) was adapted from a previously described method . Three pCandidatus Aquarickettsia rohweri” 16S rRNA gene sequence obtained from the draft genome for use, as confirmation of their presence in A. cervicornis specimens. In order to ensure specificity, the probe was designed to target a hypervariable region of the 16S rRNA, and for efficiency, it targets a region of extremely high accessibility on the 16S rRNA molecule [http://rdp.cme.msu.edu/probematch/search.jsp) using 100% sequence match suggests that the probe only matches 16S sequences previously assigned to Rickettsia species or unclassified alphaproteobacteria in the database. A single-mismatch probe, RICKNEG (5′-CCTCCAATACTCCATTGG-3′), was designed as a negative control to confirm probe specificity and matches 0 bacterial sequences in the RDP database. Optimal formamide concentration (40%) for the specific probe was empirically determined by using the highest formamide concentration that simultaneously exhibited presence of mixed EUB338 binding and RICK binding, and absence of RICKNEG binding.A suite of probes  targeting most bacteria 16S rRNA sequences  was used for FISH , 30. Promolecule . The speAcropora cervicornis fragment from ref. [An rom ref.  with a rAcropora digitifera [A. cervicornis), and the algal symbiont Symbiodiniaceae spp[Trichoplax adhaerens were retained due to previous observations that genomic scaffolds of this organism contained sequences from Rickettsial endosymbionts that may have been horizontally transferred to the placozoan host [Symbiodiniaceae spp. that had erroneously been retained. CheckM [Rickettsiaceae in the marker gene database. The program Pseudo-Finder [The filtering and trimming of low-quality sequences and removal of adaptor sequences were conducted with FQtrim  and gene coordinates generated by Prodigal [Prokka  was usedProdigal   [Trichoplax sp. H2 [Hoilungia hongkongenis [2, 10 mM each dNTP, 5 µM each primer, 1 µ Taq polymerase. The PCR program included 3 -min pre-heating (95 °C) followed by 35 cycles of 30 s heating (95 °C), 30 s annealing (63 °C), and 1 min 30 s extension (72 °C). After the cycles, a final extension step of 3 min (72 °C) completed the PCR. The Rickettsial 16S was amplified from DNA isolations of either newly isolated single individuals  , Trichopx sp. H2 , and Hoikongenis . To amplsee ref. ) or fromsee ref. ). For deCa. A. rohweri” and closely related sequences from “Ca. Midichloriaceae.”The full-length 16S rRNA region of the genome was identified using BLASTn searches against 16S rRNA regions from other Rickettsiales species from Greengenes Version 13.8)  as a par.8  as a bpcomp and tracecomp to monitor maximum discrepancy in clade support (maxdiff), the effective sample size (effsize), and relative difference in posterior mean estimates (rel_diff) as summary statistics of the model. The appropriate number of samples to discard as “burnin” was independently determined first by visual inspection of parameter trace plots, and then by optimizing convergence criteria. For amino acid analyses, the maxdiff statistic was 0, while for Dayhoff6 recoded data the statistic was 0.046. The minimum effective sample size was >50 (>100 for Dayhoff6) and the maximum rel_diff statistic was <0.3 (0.1 for Dayhoff6). Node support was evaluated using posterior probabilities.A concatenated marker protein-based phylogeny was generated using a previously curated set of 92 orthologous, single-copy marker genes present in all bacteria  Supplem. HmmsearCa. A. rohweri” and other OTUs within the genus “Ca. Aquarickettsia” in the Qiita database. The Rickettsiales OTU from the sample selected for “Ca. A. rohweri” genome assembly was determined by Shaver et al. [Ca. A. rohweri” (Greengenes ID 150441). Many of the categories were, however, either ambiguous , redundant , or miscategorized . For clarity, we manually re-annotated these into 23 categories: by compiling the available metadata files, we were able to differentiate samples into more explanatory biomes ” and “reef sediment”). A map of the distribution of “Ca. A. rohweri” in corals was generated by plotting the sample collection coordinates and host genus, available as part of the EMP metadata . Analyses through Redbiom were confirmed by analysis using the tool IMNGS [The Earth Microbiome Project (EMP) tool Redbiom  was usedr et al.  to matchr et al.  OTU 1504ol IMNGS  to queryA. cervicornis  designed in this study is sequence specific: at 40% formamide, RICK hybridizes to cells in the clusters but no signal from RICKNEG is apparent , but places “Ca. Midichloriaceae” as the sister group to both Anaplasmataceae and Rickettsiaceae. Strikingly, “Ca. A. rohweri” was part of a distinct clade along with other uncultured and uncharacterized bacteria associated with marine invertebrates. This distinct clade, present in other phylogenies [Gorgonia ventalina, the stony corals Orbicella annularis, Orbicella faveolata, and Acropora cervicornis, and the sponge Cymbastela concentrica. While most members of this group were identified in marine hosts, several species were identified in freshwater organisms, including the ciliate Euplotes woodruffi and freshwater cnidarian Hydra oligactis. “Ca. A. rohweri” shared species-level homology with multiple 16S rRNA sequences from endosymbionts of Trichoplax adhaerens, as well as with sequences from both sponge and coral hosts , and encodes two unique gene clusters  as well as 324 unique genes (not assigned to any orthogroup). While 262 of the unique genes were hypotheticals, 14 of the 62 annotated unique genes (Supplementary Table Wolbachia on NCBI (NC_010981 and NC_010981). The other unique cluster consists of hypothetical genes and ankyrin repeats. Ankyrin repeats, along with tetratricopeptide repeats , are eukaryotic-like repeat domains which were observed in the genome of “Ca. J. acanthamoeba” and may be involved in symbiont–host interactions [Based on analysis of orthologous gene sets using OrthoFinder 2.2.3 Fig. , “Candidractions .Fig. 4OrPelagibacter ubique HTCC1062 and Rhodobacter sphaeroides, more genes mapped to the categories “Translation” , “Replication and Repair” (9% vs. 4% and 4%), “Intracellular Trafficking and Secretion” (4% vs. 2% and 1%), and “Energy Production and Conversion” (10% vs. 8% and 7%), while fewer genes mapped to the categories pertaining to amino acid, nucleotide, and inorganic ion metabolism and transport  numbers matching to coding sequences. The KEGG pathways with the greatest numbers of KOs found were: ribosome (47 KOs), oxidative phosphorylation (38), carbon metabolism (34), two-component system (29), biosynthesis of amino acids (24), aminoacyl-tRNA biosynthesis (24), purine metabolism (22), and pyrimidine metabolism (22). Clusters of Orthologous Groups of proteins (COG)  functionrvh Type IV secretion system  are present to interconvert Gly to Ser and Thr  have inhr Fig. S, the orgr growth . Lys, AsCa. A. rohweri” potentially can produce ATP through oxidative phosphorylation coupled to electron transport and driven by Glu oxidation, it also possesses an ATP/ADP symporter . Tlc1 allows for the import of ATP in exchange for ADP, effectively sapping the host of energy ; only Tlc1 functions as an ATP transporter, while Tlc4 and Tlc5 import host ribonucleotides [Ca. A. rohweri” encodes only one copy, with 51% amino acid identity to the Tlc1 gene of “Ca. J. acanthamoeba”.The cell membrane of Rickettsiales spp. has been shown to be permeable to host NAD+ . Althougrgy Fig. . Rickettbacteria , 82. Wolleotides . “Ca. A.Ca. A. rohweri” possess a functional pyruvate dehydrogenase complex (PDC) and tricarboxylic acid (TCA) cycle, there is no evidence of functional glycolysis/gluconeogenesis  [While “sis Fig. . Rickettsis Fig. . Unlike ne (SAM) . The prene (SAM)  confirmsCandidatus A. rohweri” lacks any complete pathways for nitrogen metabolism , which are utilized for other purposes. Despite an inability to metabolize nitrogen, “Ca. A. rohweri” does possess the NtrY-NtrX two-component system for regulating nitrate metabolism  known as Rickettsiales vir homolog (rvh), which compared with canonical T4SSs lacks a homolog of virB5, the gene encoding the minor pilus subunit [Ca. M. mitochondrii,” which has one rvh T4SS [Ca. J. acanthamoeba,” which has three T4SS clusters, only one of which is related to rvh [Ca. A. rohweri” has annotations for all necessary components of the rvh T4SS , while VirB11 was the most conserved at 76.8% amino acid identity (to “Ca. J. acanthamoeba”) , seawater, and freshwater, and marine organisms such as corals, sponges, the ctenophore Mnemiopsis, and kelp  database; in all, our Redbiom analysis represents an evaluation of a total of 173,714 16S V4 Greengenes Illumina 100 nt samples , whereas 494 (38.2%) had OTUs within “Ca. Aquarickettsia.” As metadata for coral samples in the EMP database included sampling coordinates and host identity, we had the capability to determine how “Ca. A. rohweri” was distributed globally and in which coral species it was identified  had OTUs identified as “Ca. A. rohweri” was identified , the Red Sea, the Caribbean, and Australia.When we mapped the distribution of EMP sponge samples (primarily from the Sponge Microbiome Project ) in whicied Fig. , we founCa. A. rohweri” using IMNGS at 97% and 99% similarity thresholds  and 1855 coral metagenomes queried, “Ca. A. rohweri” was only found in 21 samples. This is likely due to our use of the full-length 16S rRNA sequence for this analysis, which is more informative than an 100 -bp fragment.To supplement results from analysis of the EMP database, we queried the SRA database with the full-length 16S rRNA sequence of “Ca. Aquarickettsia” broadly associates with corals and with many members of the non-bilaterian metazoan phyla , as well as the even more ancient protists. This broad host range is not uncommon in Rickettsiales: many species of this family  are able to switch hosts, infecting both vertebrate and invertebrate hosts while still maintaining virulence [Together these data suggest that the proposed genus “irulence , 100.Ca. A. rohweri,” a novel bacterium in a new genus of Rickettsiales that is found globally distributed in corals and sponges and also associates with protists and placozoans. As its proposed name suggests, the genus “Ca. Aquarickettsia” is primarily associated with marine-dwelling organisms. Based on the metabolic capabilities of “Ca. A. rohweri,” we postulate that this group of parasitic organisms plays a role in coral disease development. Previously, we reported evidence that inorganic nutrient exposure increased “Ca. A. rohweri” populations, reduced coral growth [Ca. A. rohweri” from commensal to parasitic in coral hosts (Fig. Ca. A. rohweri” detects nutrient enrichment through NtrY-NtrX, triggering other functions such as cell growth and division or the upregulation of transporters to acquire more metabolites from the host. As nutrient enrichment also allows the coral host and algal symbiont to produce more N-rich amino acids and proteins, surplus resources could easily be scavenged by the parasite. Last, we hypothesize that in nutrient-replete conditions, the presence of surplus nitrogen metabolites encourages the proliferation of this parasite to levels that may overwhelm host metabolic capabilities (Fig. We characterized the genome and metabolic capabilities of “l growth , and incl growth . Based osts Fig. . We hypoies Fig. .Fig. 8MoCa. A rohweri” is globally associated with many coral hosts, and possesses the genomic capacity to parasitize the coral holobiont for amino acids and ATP. We also previously showed that in response to nutrient enrichment, these parasites proliferate and reduce coral growth. Thus through shifts in its population dynamics “Ca. A rohweri” may leave the host more susceptible to other opportunistic pathogens leading to disease. Alternatively, “Ca. A rohweri” may serve as a primary pathogen, although this still remains speculative. As nutrient pollution increasingly affects reefs, we suspect that parasites within the proposed genus will proliferate and may contribute to heightened vulnerability to disease and mortality.Rickettsiales have been implicated in coral disease for more than 25 years. However, their presence in seemingly healthy corals has led to doubt that they are the primary pathogen responsible for white-band disease. We show here that “Supplementary TablesSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5Supplementary Figure S6Supplementary Figure S7Supplementary Figure S8Supplementary Figure S9Supplementary Figure S10Supplementary Figure S11Supplementary Figure S12


The protocol is simple, does not require any additive such as acid, base, ligand, etc., and encompasses a broad substrate scope for primary, secondary and heterocyclic amines. Moreover, the acetylation and propanylation of amines also can be achieved with good to excellent yield by this strategy.The MoS And the amide framework is widely applied in medicines10, natural products11, and functional materials  and (NH4)2MoO4·4H2O (12.5 mol%) were used as catalyst, an excellent yield of 99% and 97% can be obtained, respectively (entries 2 and 3). Afterward, other metal salts, such as Fe(OAc)2·4H2O, Mn(OAc)3·2H2O, Cu(OAc)2, and Ni(OAc)2·4H2O were investigated as catalysts. Satisfactory yields could also be achieved, but none of these catalyst worked better than MoS2. Therefore, MoS2 was selected as the optimal catalyst (entries 5–8). This reaction can be completed within 18 hours . Decreasing the reaction temperature (entry 14) proved to be unfavorable to this transformation. Additionally, the presence of air was harmful to this reaction (entry 15). Finally, this reaction was conducted by using stoichiometric amounts of DMF (1.5 equiv.) as the reagent in another solvent, but only poor yield was obtained (entries 16–17).Initially, the transamidation of tetrahydroisoquinoline and DMF catalyzed by MoSion Fig. . Various2a–f). This reaction was tolerable for various functional groups such as methoxy, bromide, and nitro. Notably, the electron-withdrawing group is unfavorable to this reaction (2d), and a lower yield would be found. In the case of heterocyclic amines, this transformation proceeded well and the transamidation products were obtained with excellent yields (2g–h). Other secondary amines, either circular or linear amines, underwent this reaction smoothly with high yields in most cases (2i–o). Remarkably, compound 2o which containing trifluoromethyl group, a marker for fluoxetine51, could be synthesized by this strategy with good yield (78%). However, the substrate with obvious steric hindrance group, such as ethyl (1j) on nitrogen was suffered from a reduced yield. This transamidation strategy was also applicable to primary amines, and good results as well as secondary amines were achieved (2p–v). Particularly, substrate containing hydroxyl group also converted to desired product with good yield (2 u). The transamidation reaction and the corresponding product are valuable in organic synthesis reaction. For example, the natural product homoveratrylamine (1x) can be modified with formyl group by this strategy, and the corresponding product 2x could be converted to a series of natural compounds such as pseudopalmatine, 8-oxopseudopalmatine, and ilicifoline B52. However, when aromatic amines such as aniline, tetrahydroquinoline and tetrahydroindole were used as substrates, only trace amount of desired products could be detected.With the optimized condition in hand, various amines, including primary, secondary and heterocyclic amines, were tested for this reaction Fig. . The resN-methylformamide as formyl sources (entries 1–2). However, in the case of using sterically hindered amide such as N,N-diethylformamide as substrate, the yield would be reduced seriously (entry 3). Delightedly, acetamide and propionamide could be applied as carbonyl sources, and the corresponding N-acetylation and N-propionylation reaction were achieved with excellent yields (entries 4–5). These results further confirmed the transamidation strategy developed by us has a broad scope and is valuable. Moreover, in the case of aromatic amides used as carbonyl sources, a yield of 30~40% still could be found (entries 6–7).After the expanded substrate scope of amines, we then considered other appropriate carbonyl sources except DMF Fig. . The resN-acetylation and N-propionylation reaction were expanded .To verification the practicability of this strategy, the ded Fig. . Either 2g using only 3 mol% MoS2 as catalyst was proceeded  is generated. And then, the sterically congested intermediate (I) is disintegrated with a proton transfer, and intermediate II is formed. Finally, a ligand exchange reaction occurs between intermediate II and DMF to release the target molecule (T.M.).Based on the radical blocking experiments and previous reported metal-catalyzed transamidation reactionssed Fig. . First, 2-catalyzed transamidation reaction using amides as carbonyl sources was reported. The advantages of this reaction are the readily available and inexpensive metal applied as catalyst, cheap amides applied as carbonyl source, scalable, broad scopes, free of any additives such as acid, base, ligand, and etc.In summary, an efficient MoS1H, 13C and 19F NMR spectra were recorded on 400, 100, 377 MHz NMR spectrometer using CDCl3 as solvent unless otherwise stated. HRMS were made by means of ESI. Melting points were measured on micro melting point apparatus and uncorrected. Unless otherwise noted, all reagents were weighed and handled in the air, and all reactions were carried out in a sealed tube under an atmosphere of argon. Unless otherwise noted, all reagents were purchased from reagent company, and used without further purifications. Notably, the powder MoS2 were used in this work.The prepared thin-layer chromatography (Prep TLC) was performed for product purification using Sorbent Silica Gel 60 F254 TLC plates and visualized with ultraviolet light. IR spectra were recorded on a new Fourier transform infrared spectroscopy. 2  in DMF (1.0 mL) was stirred in a sealed tube under an atmosphere of argon at 150 °C for 18 h. After being cooled to room temperature, the reaction mixture was filtered, washed with ethyl acetate (20 mL). Afterward, the solution was added 10 mL water and extracted with ethyl acetate (3 × 15 mL), and then the combined organic layers were dried with Na2SO4. The solvent was evaporated under vacuum and the crude product was purified by Prep TLC on silica gel with petroleum ether and ethyl acetate to obtain the pure product.A typical experimental procedure for transamidation was conducted as follows: A solution of amine (0.2 mmol), MoSSupplementary Information (traceless)


Pyrus spp.) is an economically important temperate fruit tree worldwide. In the past decade, significant progress has been made in pear molecular genetics based on DNA research, but the number of molecular markers is still quite limited, which hardly satisfies the increasing needs of geneticists and breeders.Pear . A total of 534 primer pairs were synthesized and preliminarily screened in four pear cultivars, and of these, 332 primer pairs were selected as clear, stable, and polymorphic SSR markers. Eighteen polymorphic SSR markers were randomly selected from the 332 polymorphic SSR markers in order to perform a further analysis of the genetic diversity among 44 pear cultivars. The 14 European pears and their hybrid materials were clustered into one group (European pear group); 29 Asian pear cultivars were clustered into one group (Asian pear group); and the Zangli pear cultivar ‘Deqinli’ from Yunnan Province, China, was grouped in an independent group, which suggested that the cultivar ‘Deqinli’ is a distinct and valuable germplasm resource. The population structure analysis partitioned the 44 cultivars into two populations, Pop 1 and Pop 2. Pop 2 was further divided into two subpopulations. Results from the population structure analysis were generally consistent with the results from the UPGMA cluster analysis.In this study, a total of 156,396 simple sequence repeat (SSR) loci were identified from a genome sequence of The results of the present study showed that the use of next-generating sequencing to develop SSR markers is fast and effective, and the developed SSR markers can be utilized by researchers and breeders for future pear improvement.The online version of this article (10.1186/s12864-018-4822-7) contains supplementary material, which is available to authorized users. Pyrus spp., 2n = 2× = 34) is an economically important temperate fruit tree worldwide. The genus Pyrus belongs to the subtribe Malinae of the tribe Maleae in the subfamily Amygdaloideae of the family Rosaceae [Pear  and other genomics studies –5.Microsatellites, or simple sequence repeats (SSRs), are tandemly repeated units of 1–6 nucleotide sequence motifs flanked by unique sequences . Given tPyrus pyrifolia Nakai) for the first time by using an enriched genomic library, RAHM (random amplified hybridization microsatellites) and 5′ anchored PCR methods. Fernández-Fernández et al. developed 19 microsatellite primer pairs from the genomic DNA of European pear (Pyrus communis L.) [Currently, several efforts have also been made to develop SSR markers in pears –26. Yamaunis L.) . Yamamotunis L.) . Fan et unis L.) . Chen etunis L.)  developeunis L.) . There aunis L.) , 25, 26.Pyrus bretschneideri Rehd was completely sequenced [Although a great deal of effort has been made for the development of SSRs based on the genome sequence , 15, 18 equenced , the numA total of 156,396 SSR motifs were identified within the scaffold sequences. Of the total SSRs identified, mono-nucleotide repeat motifs  were predominant, followed by di-nucleotide repeat motifs , tri-nucleotide repeat motifs , tetra-nucleotide repeat motifs , penta-nucleotide repeat motifs  and hexa-nucleotide repeat motifs . For the mono-, di- and tri-nucleotide repeat motifs, the frequency is as high as 97.36% in total. The most abundant mono-nucleotide motif was A/T, accounting for 95.94% of mono-nucleotide motif repeats. In di-nucleotide repeats, the most frequent motif was AG/CT (42.34%), followed by AT/TA (40.36%). Of the tri-nucleotide repeats, AAG/CTT and AAT/ATT were the most abundant, accounting for 27.61 and 26.45%, respectively. The number of each major SSR type identified is summarized in Additional file Further comparison of the number of SSRs with different repeat motifs revealed that the number of SSRs with shorter motifs was much higher than that with longer motifs. In all repeat types, the number of SSRs exceeded 10,000 only for the mono-nucleotide repeat motifs with the repeat numbers of 12, 13 and 14. The number of SSRs for the mono-, di-, tri-, and tetra-nucleotide repeat motif types were more than 1000 when the repeat numbers was within 28, 17, 7 and 5, respectively, while the number of SSRs was not more than 500 for the penta- and hexa-nucleotide repeat motifs even with the least number of repeats , among the 156,396 SSR loci identified, there are 120,469 SSR loci that were located within or near the 28,819 genes. Among them, the number of SSRs in the intergenic region was 62,984 (40.27%), which was the highest, followed by the number of SSRs located in the intron of the gene . There are 23,953 (15.32%) SSRs in total located within the 1 KB area upstream of a transcription start site, within the 1 KB area downstream of a transcription start site, or within a 1 KB area upstream of a gene while downstream of another gene. There are 5052 SSRs in total in the region of 5’ UTR, or in the region of 3’ UTR. Only 2135 SSRs were located in the exon region of coding genes  database, and 24,333 genes were annotated within the database. These genes were also aligned by BLASTx to the protein database KEGG, and 4835 genes were annotated within the KEGG database. These genes were enriched into 128 pathways of 6 KEGG A classes and 21 KEGG B classes. With the 6 KEGG A classes, 417 genes were enriched into Cellular Processes, 385 genes were enriched into Environmental Information Processing, 1990 genes were enriched into Genetic Information Processing, 69 genes were enriched into Human Diseases, 4674 genes were enriched into Metabolism, and 217 genes were enriched into Organismal Systems  and classified further using GO terms. After acquiring a GO annotation for every gene, we used WEGO to perform a GO functional classification for all the genes. Based on the GO annotation, 15,808 (54.85%) genes of pear were assigned to 112,706 GO-term annotations. These annotations were summarized into 48 GO-terms of the three major categories. Of them, biological process  comprised the majority of the GO annotations, followed by cellular component  and molecular function . Genes were mostly enriched in the terms of metabolic process (10257), cellular process (10158), cell (9421), cell part (9405) and binding (9088)  primer pairs were designed from the 156,396 identified SSRs. After deletion of the SSR loci with more than one identical primer, a total of 101,694 SSR loci  from Yunnan Province, China, and the offspring  of the Yunnan sand pear cultivar ‘Huobali’. Cultivar ‘Shuihongxiao’ of white pear and cultivar ‘Honghuaguan’ of Pyrus ussuriensis from Hebei Province, China are also clustered in this group. The second subgroup is composed of ‘Korla pear’, ‘Pingguoli’ and the descendants of ‘Pingguoli’, such as ‘Hongxiangsu’, ‘Yuluxiang’, ‘Xinli No.7’ and ‘Red Zaosu’, which can be called the Chinese white pear (P. bretschneideri) subgroup. The third subgroup is composed of ‘Wuxiangli’ and other 6 varieties of Pyrus ussuriensis  phenotypes of 43 peach varieties, and they considered that PpYUC11 might be responsible for the stony hard phenotype in peach. In other research, Kang et al. [Psy1/Unknown), which was generated by joining exons from Psy1 and an unknown gene and resulted in the yellow-fruited phenotype of tomato accession PI 114490, might be caused by an SSR with 19 A/T repeats in the downstream sequence of the Psy1 gene with Psy1/Unknown. A recent study shows that intron DNA sequences constitute a previously unrecognized type of downstream regulatory element for genes transcribed by RNA polymerase II and play an important role in determining the site of transcription initiation [SSRs can be divided into genomic SSRs and genic SSRs, and genomic SSRs are usually derived from SSR-enriched genomic libraries or random genomic sequences, while genic SSRs are derived from coding regions of the transcriptome or EST sequences , 26, 29.g et al.  reporteditiation . ClearlyThe functions of genic SSRs can often be inferred by performing a homology comparison of gene-containing genic SSRs, which is one of the reasons researchers think genic SSRs are more useful than genomic SSRs , 30. WitPyrus. Yue et al. [Pyrus accessions with 28 genic SSR markers and identified 9.43 alleles per locus and an average PIC value of 0.585. Liu et al. [Pyrus communis L.) using 134 core SSR markers and showed that the average number of alleles per SSR locus was 5.45. Erfani et al. [P. pyrifolia cultivars detected a mean of 4.93 alleles per locus for 134 SSR markers varying from 3 to 9. Brini et al. [Pyrus accessions (0.26 to 0.91). Erfani-Moghadam and Zarei [In the present study, the 18 SSR loci distinguished the 44 pear individuals with an average of 10.56 alleles per locus and an average PIC value of 0.7808, indicating a high level of polymorphism. Comparison of our results with previous findings of the SSR-based studies in Pear revealed that the average number of alleles per locus and PIC values recorded in the present study were compatible or higher than other SSR-based studies in e et al.  evaluateu et al.  studied i et al.  studied i et al.  in the si et al.  reportedi et al.  found PIi et al.  reportednd Zarei , respectP. pyrifolia white pear group [P. ussuriensis cultivars were considered to have originated from a hybrid between wild P. ussuriensis and P. bretschneideri [P. ussuriensis cultivars were sometimes clustered alone [P. ussuriensis cultivar ‘Honghuaguan’ was not clustered with the P. ussuriensis cultivars in subgroup III, but first with the white pear cultivar ‘Shuihongxiao’ and then clustered with the 15 sand pear cultivars as subgroup I of Asian pear.In the present study, we used the UPGMA method to cluster the 44 pear varieties, and the European pears and Asian pears were clustered into different groups. A similar distinct clustering pattern has also been reported, and the Asian pear and European pear were completely separated from each other in the phylogenetic tree , 47 and ar group . In the hneideri , so P. ued alone  and someed alone , 50. In Huobali is a group of local cultivars in Yunnan Province, China. ‘Wanshuhuobali’, ‘Xiangyunhuobali’ and ‘Xiangyunzhongshuhuobali’ are closely related cultivars to each other, all of which were clustered in subgroup I of Asian pear. ‘Mantianhong’, ‘Hongsucui’ and ‘Meirensu’, the descendants of their male parent Huobali , were also clustered together with these three cultivars. Also in subgroup II, ‘Hongxiangsu’, ‘Xinli No.7’ and ‘Yuluxiang’, all of which were offspring of ‘Korla pear’, were clustered together with ‘Korla pear’. Also grouped with these breeds are ‘Xinli No.7’ , ‘RedZaosu’ (bud mutation of ‘Zaosu’) and ‘Pingguoli’ . That these relatives were clustered together showed that the clustering results in this study were relatively reliable.P. ussuriensis cultivars, but the European pears were not included in the study [Pyrus, and at the same time, can help us make use of these resources.Zangli pear, a variety of landrace, distributed in the higher altitude junction region of Tibet, Yuannan and Sichuan Provinces in southwestern China, is a kind of semi-wild or semi-cultivated type, with hardy characteristics and resistance to a hypoxic environment. This type of pear is a potential germplasm resource available for resistance breeding in pear; for example, Zangli varieties ‘Deqinli’ pear, distributed in Deqin County of the Diqing Tibetan Autonomous Prefecture in Yunnan Province, China, have morphological characteristics of both Oriental and European pear, but its genetic relationship is closer to European pear, with smaller and leathery leaves. In the present study, the results of a cluster analysis of 44 pear cultivars with newly developed SSR markers also showed that the phylogenic status of ‘Deqinli’ in the dendrogram was rather special, indicating that it was closer to European pear. Another study also showed that the relationship between Zangli and sand pear cultivars from Sichuan, Yunnan and Guizhou Provinces was closer than that between Zangli and he study . There ahe study , indicatIn this study, we developed 101,694 genomic SSR markers from the reference genome sequences of pear. Among them, a total of 38,377 SSR marker loci were 2–6 base repeats, which is the largest number of SSR markers developed from a single development in pear by far. These SSR markers were also marked with the relative position information of both the physical location and the linkage groups, and the adjacent genes of the SSR loci were also annotated, which can provide better help for related research work and the developed SSR markers can be utilized by researchers and breeders for future pear improvement.A total of 44 accessions, planted in the Pear Germplasm Resources Garden in Zhengzhou, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, were used in the present study Tables  and 2. XGenomic DNA was extracted using the modified CTAB (cetyltrimethy-lammonium bromide) method. The DNA quality and concentration were determined by electrophoresis in 1% agarose gel and NanoDrop1000 spectrophotometry . DNA were diluted with sterilized ultrapure water, normalized to 50 ng/μL, and stored at − 20 °C until use.ftp://ftp.ncbi.nlm.nih.gov/genomes/Pyrus_x_bretschneideri/CHR_Un/ [Scaffold sequences in a compressed file “225117_ref_Pbr_v1.0_chrUn.fa” for the genome of ‘Dangshansuli’ pear were obtained through /CHR_Un/ . SSR mot/CHR_Un/ . A minimftp://ftp.ncbi.nlm.nih.gov/genomes/Pyrus_x_bretschneideri/GFF/) using the ANNOVAR software [https://blast.ncbi.nlm.nih.gov/Blast.cgi) with an E-value threshold of 1e-5 to the NCBI non-redundant protein (Nr) database (http://www.ncbi.nlm.nih.gov) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg). Protein functional annotations could then be obtained according to the best alignment results. The GO annotation of genes was analyzed by the Blast2GO software (version 2.6.0+) [We carried out the SSR annotation from the reference genome with the Generic Feature Format 3 (GFF3) (software . To anno 2.6.0+) , and the 2.6.0+) .Pyrus bretschneideri), followed by the repeated motif type, a linkage group number and a consecutive number.Based on SSR motifs, the Primer 3 software  was used2+ plus), 0.1 μL of Taq DNA polymerase solution (5 unit/μL), 1.6 μL of 25 mM dNTPs, 0.6 μL (10 μM) of each primer solution and 14.1 μL of sterilized ddH2O. The PCR reagents  were purchased from TaKaRa Bio Engineering Co., Ltd. , and the primers were synthesized by GENEWIZ Bioengineering Ltd. . Amplification was programmed as 5 min at 94 °C for initial denaturation; 30 cycles consisting of 30 s at 94 °C for denaturation, 30 s at the annealing temperature (Ta) for annealing, 45 s at 72 °C for extension; and finally, a 10-min extension step at 72 °C. The PCR products were loaded for electrophoresis in an 8% polyacrylamide gel.A total of 534 primers were randomly selected from the newly designed primers to first validate for polymerase chain reaction (PCR) and SSR polymorphisms among four pear accessions . The PCR reaction was carried out in a reaction mixture with a volume of 20 μL comprised of 1.0 μL (50 ng) of template DNA, 2.0 μL of 10 × PCR buffer . The PCR products were identified on an ABI 3130 genetic analyzer . Determination of the fragment sizes and data collection were done using the GeneMapper 4.0 software .The genotyping data of the accessions in the 18 SSR loci selected in the initial screening were then used to evaluate the genetic diversity of the 44 pear accessions. To estimate the genetic diversity among the pear accessions, some parameters were calculated using the software POPGENE version 1.32 , includiAdditional file 1:Figure S1. Percentages of different motifs among mono-(a), di- (b) and tri- (c) nucleotide repeats in the ‘Dangshansuli’ pear genome. (PDF 183 kb)Additional file 2:Table S1. Frequency of different SSR repeat motifs. (XLSX 9 kb)Additional file 3:Table S2. Frequency distribution of different SSR motifs. (XLSX 9 kb)Additional file 4:Table S3. Type and number of repeat motifs. (XLSX 10 kb)Additional file 5:Table S4. Statistics of SSRs in different categories. (XLSX 9 kb)Additional file 6:Table S5. Number of different types of SSR motifs in different LGs. (XLSX 10 kb)Additional file 7:Table S6. Information of new designed 101, 694 SSR primers, including information of the primer sequences, annealing temperature (Ta), repeat motifs, target size, linkage groups, and positions in genetic and physical maps of pear. (XLSX 10192 kb)Additional file 8:Table S7. Information of 534 SSR primers, including information of the primer sequences, annealing temperature (Tm), repeat motifs, target size, linkage groups, and positions in genetic and physical maps of pear. (XLSX 72 kb)Additional file 9:Table S8. Information of 332 good SSR primers, including information of the primer sequences, annealing temperature (Tm), repeat motifs, target size. (XLSX 36 kb)Additional file 10:Figure S2. Amplified fragments of Pb3L11N5758 and Pb3L4N5130 SSR loci from five pear varieties. (PDF 188 kb)Additional file 11:Table S9. Polymorphism information of 18 primers in 44 pear cultivars. (XLSX 10 kb)Additional file 12:Table S10. Similarity coefficient among 44 pear cultivars. (XLSX 18 kb)