There is an increased demand for efficient biomarkers for the diagnosis of non-small cell lung cancer (NSCLC). This study aimed to evaluate plasma levels of TrxR activity in a large population to confirm its validity and efficacy in NSCLC diagnosis. Blood samples were obtained from 1922 participants  and 729 sex- and age-matched healthy controls). The plasma levels of TrxR activity in patients with NSCLC (15.66 ± 11.44 U/ml) were significantly higher (P < 0.01) than in patients with BLDs (6.27 ± 3.78 U/ml) or healthy controls (2.05 ± 1.86 U/ml). The critical value of plasma TrxR activity levels for diagnosis of NSCLC was set at 10.18 U/ml, with a sensitivity of 71.6% and a specificity of 91.9%. The combination of NSE, CEA, CA19-9, Cyfra21-1, and TrxR was more effective for NSCLC diagnosis  than was each biomarker individually (P < 0.001). TrxR can also efficiently distinguish the metastatic status of the tumor, and it can further differentiate between various histological differentiations. Together, plasma TrxR activity was identified as a convenient, non-invasive, and efficient biomarker for the diagnosis of NSCLCs, particularly for discriminating between metastatic and non-metastatic tumors, or for histologic differentiation. It is estimated that more than 1.8 million new lung cancer cases and around 1.6 million deaths associated with lung cancer occur annually worldwide, and these incidence and mortality rates are projected to undergo continued rapid growth2. Approximately 80% of lung cancer cases are categorized as being non-small cell lung cancer (NSCLC) based on histologic classification. Compared with other carcinomas, NSCLC carries a far worse prognosis and the overall 5-year survival rate after diagnosis remains below 15%5. Early and appropriate treatments for improving the 5-year survival rates largely depend upon the early and accurate diagnosis of lung cancer6. Unfortunately, delays commonly occur in the clinical diagnosis of lung cancers10. In China, the average diagnostic delay for lung cancer ranges from 2.9 to 8.4 months, and as a consequence 65.3% of the patients are first diagnosed at stage III or IV15.Currently, lung cancer is one of the most common malignancies and the most frequent cause of cancer-related death16. Furthermore, instances of over-diagnosis related to CT scans and the negative influences of radiation exposure have provoked widespread controversy. In addition, some invasive diagnostic strategies, including bronchoscopy and needle biopsy, are not widely utilized due to the associated pain and inconvenience17. Thus, the exploration of novel and efficient diagnostic biomarkers remains an urgent and necessary pursuit.At present, imaging techniques such as computed tomographic (CT) scans and chest X-rays play an important role in the clinical diagnosis of lung cancer. However, these tests have high false-positive rates and usually fail to uncover the hidden or subclinical lesions or small metastases, resulting in their limited application18. However, the applications of these biomarkers remains unsatisfactory owing to the low sensitivity of diagnosis, which remains below 50%21. Hence, a top priority in lung cancer research should be the identification of a non-invasive, non-radiative, convenient, and fast diagnostic approach that offers both high sensitivity and specificity.In recent years, a limited number of tumor-specific proteins have been identified, including neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), cytokeratin fragment 21-1 (Cyfra21-1), and squamous cell carcinoma antigen (SCC-Ag). Some of these biomarkers, such as CEA and Cyfra21-1, have been applied in the clinical diagnosis of NSCLC according to the National Academy of Clinical Biochemistry guidelines27. This protein attracted our attention because it plays a key role in the tumor-related redox process28. Redox imbalance has long been recognized to be a factor in the pathology of NSCLCs30. Previous studies have found that TrxR is highly expressed in NSCLC both in vitro and in vivo, and is involved in regulating redox balance, transcription factor activities, and tumor growth in NSCLC32. In addition, the inhibition of TrxR activity by the TrxR-specific inhibitor Ethaselen resulted in a remarkable antitumor effect on the A549 NSCLC cell line as well as in vivo in NSCLC xenograft mice, suggesting that TrxR may play a crucial role in NSCLC28. It has also been reported that TrxR may be associated with aggressive tumor growth and poor patient outcomes35. More importantly for diagnostic purposes, the secretion of TrxR into the peripheral blood under conditions of oxidative stress has been observed and this secreted TrxR has proven to be a valuable indicator of oxidative stress38. These previous studies thus suggested that TrxR may be a potential and efficient biomarker for the diagnosis of NSCLC.Thioredoxin reductase (TrxR) is a component of several redox-sensitive signaling cascades that mediate specific physiological processes, including those relating to cell survival, maturation, growth, migration, and inhibition of apoptosisIn our previous retrospective investigation, we evaluated a small cohort of patients with NSCLC (43 cases), revealing that plasma levels of TrxR activity were significantly higher in these patients than in healthy controls. In the present study, we further analyzed the diagnostic efficiency of TrxR activity as a plasma biomarker of NSCLC in a large population, and compared TrxR activity levels with different characteristics and features of the recruited patients and controls, including gender, age, body mass index (BMI), smoking history, histological type, tumor-node-metastasis (TNM) stage, metastasis, and histologic differentiation. Then, we further evaluated the levels of NSE, Cyfra21-1, CA19-9, CEA, TrxR and combinations thereof in a large clinical population to improve the efficiency of NSCLC diagnosis for future clinical application.39. Tumor stage was defined according to the 7th IASLC/AJCC staging system41. All samples were randomly separated into a training set and a validation set and matched with respect to age and sex. All patient clinicopathologic information, including age, gender, BMI, pathology, differentiation, TNM stage, and history of smoking is shown in Table Patients with pathologically diagnosed NSCLC (638 cases), BLDs (555 cases), and sex- and age-matched healthy controls (729 cases), as shown in Supplemental Fig. 5 ml samples of preoperative peripheral blood were collected in EDTA and anticoagulant-free tubes. Samples were centrifuged at 3,000 rpm at room temperature for 5 minutes within 2 hours of collection, and the supernatants were collected and tested immediately.42, both DTNB and Trx reduction assays were used to measure TrxR activity in vitro. For the DTNB reduction assay, the assessment of TrxR activity was based on the enzymatic activity of TrxR to catalyze the reduction of 5, 5′-dithiobis (2-nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2-nitrobenzoic acid (TNB2-), which generates a strong yellow color with maximum absorbance at 412 nm43. For the Trx reduction assay, human recombinant Trx was added and incubated with mammalian TrxR, followed by monitoring the initial decrease in absorbance at 340 nm43. In biological samples such as human plasma, only DTNB reduction assay has been widely used to measure the TrxR activity46. All commercially available TrxR activity colorimetric assay kits, including BioVision , Sigma , and Clairvoyance , were designed to determine TrxR activity based on the DTNB reduction. Since several enzymes present in biological samples can reduce DTNB, a specific TrxR inhibitor is used to determine the reduction of DTNB due only to TrxR activity. However, the determination of TrxR activity in biological samples based on human recombinant Trx reduction assay has not been reported or validated in previous studies.According to the previous literature46. Determination and calculation of TrxR activity were performed according to the manufacturer’s instruction and full details are provided in Supplemental Experimental Procedures. The coefficients of variation (CVs) of inter-assay and intra-assay were 4.2–8.1% and 5.9–8.8%, respectively. The detection range of this assay was between 0.5 U/mL-100 U/ml.In this study, TrxR activity was measured by commercially available thioredoxin reductase (TrxR) activity colorimetric assay kits , which was based on DTNB reduction and has been widely applied to determine the TrxR activity in human samples in many previous clinical studiesWe examined four tumor markers [carcinoembryonic antigen (CEA), cancer antigen 19-9 , and cytokeratin 19 fragment  and analyzed using a Cobas® analyzer . All reagents and kits were purchased from Roche diagnostics , and performed according to the manufacturer’s instruction. The upper normal limits for the tumor markers were 13 ng/ml for NSE, 5 ng/ml for CEA, 39 U/ml for CA19-9, and 3.3 ng/ml for Cyfra21-1.Results are described as percentages for categorical variables and as medians  for the continuous variables. Proportions were calculated and compared using the Chi-squared test, while continuous variables between groups were compared by the non-parametric Mann-Whitney test with a Bonferroni correction. The diagnostic efficacy of biomarkers in NSCLC and BLD was evaluated based on the receiver operating characteristic (ROC) curves and the area under the curve (AUC) values. Correlations between plasma TrxR activity and confounding factors such as age, BMI and smoking history were analyzed primarily based on binary logistic regression analysis. Results are expressed as OR (odds ratio) values with the corresponding 95% CI . All statistical analyses were performed using GraphPad Prism 6  and SPSS version 19.0 . The significance threshold was set at 0.05 for two-tailed analyses.This study protocol involving human participants was approved by the Ethics Committee of Renmin Hospital of Wuhan University Cancer Center , Peking University Third Hospital , Zhejiang Cancer Hospital , the First Affiliated Hospital of Nanjing Medical University , Changyi People’s Hospital , Hubei Traditional Chinese Medicine Hospital  and Wuhan first hospital . The methods were carried out in accordance with the approved guidelines. Informed consent was obtained from all patients.A total of 1922 participants were recruited, including 970 in the training cohort  and 952 in the validation cohort , as shown in Supplemental Fig. The plasma levels of TrxR activity (median ± IQR) in patients with NSCLCs, patients with BLDs, and healthy controls were detected and analyzed. In the training set, the plasma levels of TrxR activity in patients with NSCLCs (15.61 ± 11.63 U/ml) were significantly higher  being used to determine the optimal cut-off value of TrxR activity as a means of distinguishing patients with NSCLCs from those with other conditions. Using this approach, the critical value of plasma TrxR activity levels for the diagnosis of NSCLC was set at 10.18 U/ml, based on the ROC curve  of TrxR activity levels (>10.18 U/ml) was 94.99% for NSCLC diagnosis. Furthermore, in the assessment of our validation cohort, TrxR activity also showed great AUC 0.847  relative to any individual biomarker , revealing that smoking status significantly correlated with the severity of lung diseases (P < 0.001). Also, according to linear-by-linear association results, the severity of lung diseases was elevated as the pack-years of lifetime smoking history increased in a linear relationship (P < 0.001). Pearson correlation analysis indicated no correlation between TrxR activity levels and smoking history (pack-years) in any of the groups. We also conducted an ROC analysis in our study according to smoking history and the AUCs of the ROC curves were similar for smokers and non-smokers.In the training cohort, the plasma levels of TrxR activity in the 127 NSCLC patients with metastatic tumors  were significantly higher (P < 0.001) than in those patients with non-metastatic NSCLCs , whereas no other markers were able to effectively distinguish metastasis , while those with poorly-differentiated NSCLCs ranked second  were further elevated and significantly different from that in benign diseases, potentially due to increased oxidative stress and extensive TrxR secretion under tumor growth conditions38. Although there were no differences in activity levels among the three histological types of NSCLCs, partly because of the similar oxidative status of SCCs, ADCs, and LCCs, TrxR activity could effectively and efficiently distinguish these patients from BLDs and healthy controls, making up for the absence of an effective biomarker for diagnosing ADCs and LCCs. In addition, very similar results and identical conclusions were drawn from our validation cohort, including 318 patients with NSCLCs, 272 patients with BLDs, and 362 healthy controls, thus verifying the effectiveness of plasma TrxR activity levels as a means of discriminating among NSCLCs, BLDs and healthy controls.In this study, the detection of plasma TrxR activity levels in 320 patients with NSCLCs, 283 patients with BLDs and 367 healthy controls in a training cohort was conducted, revealing that TrxR activity levels in patients with BLDs (6.24 ± 3.75 U/ml) were significantly higher than in healthy controls (2.04 ± 1.87 U/ml), which may be the result of the cell damage associated with lobar pneumonia, pulmonary TB, chronic bronchitis, COPD, and PIPImportantly, an appropriate sample preparation procedure is essential for the accurate determination of TrxR activity levels in plasma. In this study, we excluded all samples with evidence of hemolysis and lipemia as hemolysis and lipemia have both been shown to skew results. The effect of hemolysis is likely due to additional TrxR release from red blood cells, while lipemia has been shown to lead to a significant interference with the reaction system itself. The construction of reliable diagnostic criteria for TrxR activity levels capable of discriminating between NSCLCs and BLDs is important for future clinical application. By conducting ROC analyses and establishing AUCs for TrxR activity, we confirmed its diagnostic efficiency in discriminating between BLDs and NSCLCs with AUCs of 0.899  and 0.837 , respectively. In addition, we set 4.01 U/ml and 10.18 U/ml as the critical cut-off values for the diagnosis of BLDs and NSCLCs, respectively, based on the maximal Youden Indexes, which appropriately balanced the sensitivity and specificity. Thus, the diagnostic criteria for NSCLCs and BLDs were constructed as shown in Table 17, which may result in the limitation of its clinical application in the diagnosis of NSCLCs . CEA and NSE have been applied as biomarkers to the diagnosis and prognosis of several cancer types, including as a means of monitoring recurrence and evaluating prognosis in NSCLCs57, but these markers only performed moderately well as a means to diagnose NSCLCs in our training cohort . In the present study, we also incorporated the biomarker CA19-9, which has mainly been used for the diagnosis of several types of gastrointestinal cancers58, but found that its sensitivity only reached 18.8%. Compared to these individual biomarkers, TrxR activity appeared to be more effective and efficient for NSCLC diagnosis, with a sensitivity of 71.60% and a specificity of 96.50%. We further established a joint detection model for the diagnosis of NSCLCs, incorporating Cyfra21-1, NSE, CEA, CA19-9, and TrxR activity in our panel. The combination of Cyfra21-1, NSE, CEA and CA19-9 improved diagnostic sensitivity to 53.8% for NSCLCs. After adding TrxR activity into this diagnostic panel, the sensitivity greatly increased to 85.6%. In addition, TrxR activity maintained great diagnostic accuracy for patients with NSCLCs who were negative for recommended routine biomarker screening . These outcomes were further verified in our validation cohort  elevated as the pack-years of lifetime smoking history increased, with a linear relationship between these two factors (P < 0.001). According to a Pearson correlation analysis results, there is no correlation between TrxR activity levels and smoking history (pack-years) in any of the assessed groups.60. In this study, the level of TrxR activity also increased significantly as NSCLCs progressed to a metastatic state. An ROC analysis indicated that the optimal cut-off value for distinguishing between metastatic and non-metastatic patients was 16.00 U/ml, with a sensitivity of 66.14% and a specificity of 78.76%, as shown in Table 45.We also investigated correlations of TrxR activity and various clinical characteristics and features of patients with NSCLCs, such as metastasis, TNM stage, and histologic differentiation. Notably, previous studies have shown that TrxR plays a key role in mediating tumor metastasis61. For example, Chen et al. conducted a restrospective clinical study and identified TrxR as an independent poor prognostic indicator for EGFR wild type and ALK negative NSCLC patients46. Another study performed by Zhou et al. found that almost 90% of NSCLCs patients (138/149) have shown a significant decrease of TrxR level after surgery, suggesting TrxR activity as a promising biomarker during NSCLC surgery and clinical treatment61. In the current research, we mostly focused on the identification of TrxR as an efficient biomarker to distinguish NSCLCs, benign lung diseases (BLDs), and healthy group, indicating its diagnostic value in NSCLC detection. In addition, this is the first study to compare the diagnostic value of TrxR activity with multiple NSCLC biomarkers, including NSE, CA19-9, CEA, and Cyfra21-1, and report the sensitivity and specificity of TrxR in combination with other NSCLCs biomarkers, which was distinct from the previous studies.Several previous studies have suggested TrxR as a potential biomarker in NSCLCsAt present, CT scans have been a recommended approach of diagnosing NSCLC owing to their non-invasive nature. Despite this and other advantages, the contraindications for the use of this diagnostic approach including radiation exposure, high cost, and high false-positive rates must be considered. Compared to CT scans, TrxR activity detection is inexpensive, reproducible, easy  and convenient (requiring <2 ml plasma). Importantly TrxR activity also has a high sensitivity and specificity, making it a more competitive, efficient, effective, and reliable approach for diagnosing NSCLCs. The combination of Cyfra21-1, NSE, CEA, CA19-9, and TrxR, along with imaging and clinicopathologic examination information, may offer a better panel of diagnostic tools for the early and accurate diagnosis of NSCLCs, which will contribute to the prompt and effective treatment of NSCLCs and a reduction in associated mortality. However, as most participants in our study were recruited in central and eastern China, the presence of common confounding variables and comorbid conditions cannot be ignored. Given to the complex causes of NSCLCs and the variance in genomic and molecular signatures as a function of ethnicity, future studies using an independent cohort of patients will be needed. It will also be necessary to investigate the molecular mechanisms and clinical implications of elevated TrxR activity levels in cancer patients. In addition, in this study, we measured TrxR activity levels in plasma, and not in histologic specimen. It is still uncertain whether peripheral TrxR levels reflect similar changes in the corresponding tumor tissue, and so this topic also warrants further exploration.In summary, this is the first large population study to report the clinically diagnostic relevance of TrxR activity as a plasma biomarker for NSCLCs in both training and validation cohorts. Our study showed that plasma TrxR activity is an effective and efficient biomarker of diagnosing NSCLCs, particularly as a means of characteristic discrimination among tumors of different metastatic states and histological differentiation. Hence, we recommend the convenient, economical, relatively non-invasive, and reproducible detection of TrxR activity levels, along with CEA, NSE, and Cyfra21-1, to aid in diagnosis of NSCLC.Supplementary Info


Normalization of cDNA is widely used to improve the coverage of rare transcripts in analysis of transcriptomes employing next-generation sequencing. Recently, long-read technology has been emerging as a powerful tool for sequencing and construction of transcriptomes, especially for complex genomes containing highly similar transcripts and transcript-spliced isoforms. Here, we analyzed the transcriptome of sugarcane, a highly polyploidy plant genome, by PacBio isoform sequencing (Iso-Seq) of two different cDNA library preparations, with and without a normalization step. The results demonstrated that, while the two libraries included many of the same transcripts, many longer transcripts were removed, and many new generally shorter transcripts were detected by normalization. For the same input cDNA and data yield, the normalized library recovered more total transcript isoforms and number of predicted gene families and orthologous groups, resulting in a higher representation for the sugarcane transcriptome, compared to the non-normalized library. The non-normalized library, on the other hand, included a wider transcript length range with more longer transcripts above ∼1.25 kb and more transcript isoforms per gene family and gene ontology terms per transcript. A large proportion of the unique transcripts comprising ∼52% of the normalized library were expressed at a lower level than the unique transcripts from the non-normalized library, across three tissue types tested including leaf, stalk, and root. About 83% of the total 5,348 predicted long noncoding transcripts was derived from the normalized library, of which ∼80% was derived from the lowly expressed fraction. Functional annotation of the unique transcripts suggested that each library enriched different functional transcript fractions. This demonstrated the complementation of the two approaches in obtaining a complete transcriptome of a complex genome at the sequencing depth used in this study. Advances in sequencing technologies in recent years have allowed a great amount of transcriptomic data to be generated within a relatively short time at an affordable price. Short-read technologies have been used for transcriptome sequencing and transcript expression across a broad transcript dynamic range. However, the quality of transcriptomic analyses including transcript profiling and differential expression analysis greatly relies on the quality of the available reference transcriptome . StudiesConventionally, for transcriptome library preparation, a sample comprised of multiple cells isolated from the tissue/organ being studied is normally used for RNA extraction. When sequenced, this protocol tends to work well for medium to highly or ubiquitously expressed transcripts which are normally represented in high copy number and are predominant in the transcriptome. However, transcripts of genes that are expressed at low levels or expressed in a small fraction of the cell types of a tissue and/or at certain phases of development can comprise a small fraction of the total transcripts of an extracted RNA sample. As a result, the predominant transcripts have a higher probability of being sequenced multiple times compared to those transcripts expressed at low levels, which might not be sequenced at all considering the relatively low throughput (at an affordable cost) of most recently available long-read transcriptome sequencing platforms including the PacBio Iso-Seq , the IllWe now report the use of the PacBio Iso-Seq data, generated for the transcriptome of the highly polyploid sugarcane genome in an earlier study , to asseSugarcane materials and DNA extraction were described previously . In brieThe cDNA library preparation followed the PacBio Iso-Seq guidelines on an aliquot of 1 μg pooled RNA, as previously described . In summThe read data processing followed steps previously described  with somSequences of 99% identity and 99% length coverage were collapsed into one single cluster by CD-HIT-EST from the CD-HIT package ver4.6.8  with theArabidopsis genomes using KOBAS ver3.0  ver3.0.2b employing 303 orthologous groups derived from 90 representative eukaryotic species . Coding S ver3.0  with an S ver3.0  and BLASS ver3.0 , respectS ver3.0 . GO termS ver3.0 . FurtherS ver3.0  and analS ver3.0  to disseS ver3.0  by GMAP S ver3.0  with 80%S ver3.0 .Venn diagrams were created using the online Venn tools . StatistThe raw data supporting the conclusions of this study have been previously deposited in NCBI SRA database under BioProject PRJNA356226, accession numbers from SRR5259105 to SRR5259110. The RNA-Seq read data for leaf and root tissues can be downloaded in NCBI Study Accession Number SRP152893. All other relevant data that supports the findings in this study are included within the article and supplementary files.The initial data processing was performed for two datasets obtained from non-normalized and normalized cDNA libraries . Each library had two bins of 0.5–2.5 kb and 2–3.5 kb. The NN dataset has 15,014 high-quality (HQ) and 7,773 low-quality (LQ)-FL transcript isoforms (transcripts) in the 0.5–2.5 kb bin; 11,880 HQ; and 8,309 LQ in the 2–3.5 kb bin. This resulted in the total number of pooled transcript isoforms in the NN dataset being 42,976 sequences. Similarly, the NO dataset had 17,597 HQ- and 8,147 LQ-FL transcript isoforms in the 0.5–2.5 kb bin; 27,703 HQ; and 15,624 LQ in the 2–3.5 kb bin. The total number of transcripts obtained for the normalized library was 69,071. The HQ- and LQ-FL transcripts in each dataset were combined into single files for further analysis. To reduce the redundancy, we first collapsed all sequences of 99% similarity and 99% overlapped length by CD-HIT-EST, which resulted in 131 sequences in the NN dataset and 49 sequences in NO dataset being removed. The sequence summary statistics for each bin in the two datasets are presented in The first question we asked was how many of the total transcripts in the two libraries were common and how many were recovered during the normalization that were not present in the NN library. To get a general idea of the difference between the two datasets, we compared them in two different ways, comparing bins of the same length and comparing total pooled data from each of the libraries. This was done by using the two-directional comparison (CD-HIT-EST-2D) at a sequence identity threshold of 80%. When the two bins of 0.5–2.5 kb were compared, a total of 7,661 transcripts in the NN dataset were found to be similar to 9,981 transcripts in the NO dataset, which accounted for 33.6% and 38.8% of the respective datasets. Similarly, a total of 7,513 transcripts in the 2–3.5 kb bin of the NN dataset were found to be similar to 14,954 transcripts in the 2–3.5 kb bin of the NO dataset, which accounted for 37.2% and 34.5% of the respective datasets. When the pooled datasets representing all the sequences that were captured in both 0.5–2.5 and 2–3.5 kb bins from each library were compared, we identified 20,279 transcripts (47.3% of the NN dataset) and 32,948 (47.7% of the NO dataset) were similar at 80% identity, while 22,566 and 36,074 transcripts were unique to NN and NO datasets, respectively (Saccharum officinarum gene index) , unigenes from transcriptome reported in Cardoso-Silva et al. (2014), and the SUGIT, which was the combined dataset of NN and NO corrected by Illumina short-reads  were extracted from transcripts using TransDecoder ver5.3.0, and the longest ORFs (min 100 aa) were retained for orthologous gene prediction. Consistent with the previous observation, the NN dataset produced 105,605 ORF sequences , while the NO dataset had 117,101 ORF sequences   compared to the NN dataset (7% sequences). We asked whether the normalization recovered more long noncoding (lnc) transcripts since the lnc transcripts are generally shorter and expressed at a lower level than that of coding transcripts . We firsWe validated the expression of the transcript isoforms of the two datasets by mapping RNA-Seq data representing leaf, root tissues , and stalk tissue  against The aim of transcript normalization prior to sequencing was to reduce the abundance of the very highly expressed transcripts, so that the lowly expressed transcripts could be represented and sequenced, and thereby, to increase the representation of the transcriptome. A higher representation of the transcriptome was shown by the higher total transcript isoform number, higher BUSCO completeness, and greater number of gene families and ortholog counts for the NO dataset, compared to the NN dataset, in the previous sections. We tested the hypothesis of whether the NO dataset recovered more lowly expressed transcripts by mapping the RNA-Seq read data from three tissue types  against the two unique fractions of NN and NO datasets identified in The transcripts in the two datasets represented only polyadenylated (polyA) RNAs in the sugarcane transcriptome, since oligo(dT) was used in our RNA extraction protocol. PolyA transcripts could be protein-coding (mRNAs) or noncoding transcripts . We haveSorghum bicolor with an e-value of 1e-5. The number of unique sorghum genes identified were 10,544 and 14,069 genes, respectively, for the NN and NO datasets, totaling 15,749 unique sorghum genes identified  , and 57,699 out of 69,022 transcripts (∼84% of the NO dataset) using KOBAS against the complete genome of entified Table 2.10 . Most of the significantly different GO terms showed a higher percentage in the NN unique dataset, compared to that of the NO unique faction. Since WEGO compared GO distribution using percentage of transcripts within the total annotated transcripts, the difference could be attributed to the lower GO terms per transcript in the NO dataset compared to that in the NN dataset , and probably the distinct functional roles of transcripts from each unique fractions.Next, we looked into the two unique fractions from the NN and NO datasets  identified by CD-HIT-EST-2D by comparing their functional enrichment. Similar to the total sets, there was a higher proportion of un-annotated transcripts in the unique NO compared to that in the NN dataset. About 92%  and ∼84%  for the two unique fractions from NN and NO datasets were mapped against the NCBI NR protein database, of which 18,011 sequences and 24,897 sequences were assigned to 45,352 and 59,136 GO terms, respectively Table 2.Arabidopsis gene IDs obtained in transcript annotation using KOBAS. About 83.2%  of the NN dataset and 75.1%  of the NO dataset were annotated. Using the Arabidopsis gene IDs, we identified 1,499 and 3,925 Arabidopsis genes that were only found of the NN and NO datasets, respectively. We parsed these unique gene sets using the Mercator pipeline and compared their functional annotation. Further functional differences were analyzed by Mercator 4 using et also possessed more transcript isoforms per gene family and GO terms per transcripts. The NO dataset, on the other hand, included more transcript isoforms, of a shorter length, representing a higher transcriptome completeness and more gene families, and had generally lower expression levels and more un-annotated transcripts using the known protein databases. Many of the NO transcripts were found to be long noncoding transcripts, mostly derived from the unique NO fraction, and were expressed at a lower level than protein-coding transcripts.Taken together, the results suggest that the two datasets included different functional transcripts of the studied sugarcane transcriptome, in addition to the common transcripts presented in both libraries. The NN captured a wider length range of transcripts and had a lower number of transcripts resulting from deeper sequencing of highly represented sequences and a higher percentage of sequences that were annotated using the known protein database and GO database. This datas; 40–60% are from 500–2,000 genes; while the rest 20–40% are originally from low/rare transcripts . This vaIn recent years, the third-generation of sequencing technologies has been emerging as a powerful tool to recover the complexity of transcriptomes without the need of assembly and provide more accurate evidence of transcript length, alternative splicing events, and polyadenylation sites. There are a growing number of plant transcriptomes that have been sequenced by long-read technologies, which, in turn, facilitated the reference genome annotation and isoform-level transcript profiling . cDNA noBy using the highly polyploid sugarcane transcriptome to test the impact of the normalization on long-read sequencing by PacBio Iso-Seq, we showed that, besides many of the same transcripts being captured, normalization removed many longer transcripts, while include many new generally shorter transcripts. The normalized library recovered more transcript isoforms  in total compared to the non-normalized library . Of the total transcripts, 93.2% of the non-normalized transcripts and 92.3% of the normalized transcripts were supported by the Illumina short-read data of different tissues including leaf, stalk, and root tissues. Although medium-sized Illumina RNA-Seq data (maximum sample reads were ∼66 million) was used to confirm the expression of transcript, the use of deeper coverage data is likely to confirm more transcripts, especially those lowly expressed in the normalized dataset. Moreover, 75.5% and 70.5% of the transcripts from respective non-normalized and normalized datasets were aligned against the sorghum genome version 3, while 61.5% and 59.3% aligned to the new monoploid sugarcane genome. Transcripts aligned evenly across the sugarcane chromosomes, but mostly on the two arms toward the telomeres on the sorghum chromosomes. The lower percentage of transcripts mapping against the sugarcane monoploid genome could be due to the genome sequences being derived from a French cultivar R570 representing only the sugarcane gene space that covered the sorghum genome . It coulWhen compared, the two libraries had about 47.3% and 47.7 of sequences that were common (at 80% identity), while the rest  were unique to one of the libraries. Similar to the results for the total sets, the unique normalized transcripts exhibited shorter length compared to the unique non-normalized transcripts. Analysis revealed that the unique non-normalized transcripts were expressed at a higher level than the unique normalized transcripts. This suggested that the normalization recovered a proportion of lowly expressed transcripts that were likely to be absent in the non-normalized library. The unique non-normalized transcripts could be those long transcripts that were removed by the normalization due to its bias toward shorter transcripts. Among the unique normalized transcripts, we identified 3,527 long-noncoding transcripts, accounted for ∼9.8% of the unique sequences and ∼79.9% of the total 4,416 long non-coding transcripts found in the normalized library. Only 932 long non-coding transcripts were found in the non-normalized library, of which, 692 sequences  were in the unique fraction. Therefore, the total number of long non-coding transcripts identified in this study were 5,348, more than the total of ∼2,400 sequences reported previously in  using hode novoAS , in which a higher rate of gene discovery was found in the normalized library . It is ne novoAS , IsoCon e novoAS , and isOe novoAS  could beSaccharum spontaneum . "SRA" had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We are grateful to the Australian Agency for International Development (AusAID) for financial support through an Australian Awards Scholarship to NH.Author FB was employed by the company, Sugar Research Australia. All 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.


Background and Objective: Neuroblastoma (NB), the most common pediatric solid tumor apart from brain tumor, is associated with dismal long-term survival. The aim of this study was to identify a gene signature to predict the prognosis of NB patients.Materials and Methods: GSE49710 dataset from the Gene Expression Omnibus (GEO) database was downloaded and differentially expressed genes (DEGs) were analyzed using R package “limma” and SPSS software. The gene ontology (GO) and pathway enrichment analysis were established via DAVID database. Random forest (RF) and risk score model were used to pick out the gene signature in predicting the prognosis of NB patients. Simultaneously, the receiving operating characteristic (ROC) and Kaplan-Meier curve were plotted. GSE45480 and GSE16476 datasets were employed to validate the robustness of the gene signature.Results: A total of 131 DEGs were identified, which were mainly enriched in cancer-related pathways. Four genes  were selected as a gene signature, which was included in the top six important features in RF model, to predict the prognosis in NB patients, its area under the curve (AUC) could reach 0.86, and Cox regression analysis revealed that the 4-gene signature was an independent prognostic factor of overall survival and event-free survival. As well as in GSE16476. Additionally, the robustness of discriminating different groups of the 4-gene signature was verified to have a commendable performance in GSE45480 and GSE49710.Conclusion: The present study identified a gene-signature in predicting the prognosis in NB, which may provide novel prognostic markers, and some of the genes may be as treatment targets according to biological experiments in the future. MYCN, ALK, LMO1, PHOX2B, ARID1A, and ARID1B (Neuroblastoma (NB) is a highly heterogeneous pediatric solid tumor both in clinical and biological characteristics. It is the third most common malignant disease, which takes about 7% of malignant tumors in children less than 14 years, with the incidence of close to 60/100,000 during the first year of life . Howeverd ARID1B . Nonethed ARID1B .MYCN, 11q, mitosis-karyorrhexis index (MKI), histology, and 1p] were more important than age and metastatic pattern for predicting the clinical outcomes, which should be considered for risk stratification in patients aged less than 18 months. Moreover, Typically, patients at stage 4 and stage 4s all have metastatic tumors, but they have remarkably different outcomes. Notably, most patients with stage 4 diseases are associated with high risks and inferior outcomes regardless of multi-modal therapy . In contRandom forest (RF) is among the most important machine learning methods thanks to their relatively good accuracy, robustness, and ease of use. They can be used to rank the importance of features in a regression or classification problem in a natural way. Mean decrease impurity and mean decrease accuracy are employed as criteria for feature selection .MYCN not amplified. Thus, the gene signature was considered as a prognostic marker of NB, and some genes (like ERCC6L) in the signature might serve as the therapeutic targets based on biological experiments in the future.In this paper, we have identified a four-gene signature via RF and risk score model in predicting the prognosis of NB patients via the GEO dataset (GSE49710), the gene signature also had good performance in discriminating other groups of GSE49710, as well as in other two independent datasets. Importantly, the obtained gene signature we picked out could separate NB patients with different outcomes in stage 4 or age less than 18 months, or p-value of < 0.05 was considered as differentially expressed genes (DEGs). Moreover, log-rank test and Cox proportional hazard regression model    term BP ogy (GO)  analysisFirstly, we put the DEGs into the RF model as features, then built the RF model using Scikit-Learn tools. We defined a grid of hyperparameter, and sampled from the grid, performing 10-fold cross-validation with each combination of values. Subsequently, the grid search algorithm outputs the settings that achieved the highest AUC in the validation procedure and the feature importance based on mean decrease impurity . Secondli indicated the coefficient of each gene in multivariate Cox regression analysis with overall survival as the dependent parameter and χi represented the expression value by log2 transformation of each gene.where βSimultaneously, all samples were divided into the high or low-risk group according to the median risk score, and the Kaplan–Meier curves of overall survival and event-free survival were plotted.p-value of < 0.05 was considered statistically significant.All statistical analyzes were carried out using the IBM SPSS version 23 software and R. Bivariate coefficient was calculated through Spearman’s rank correlation. Additionally, Kaplan-Meier method and log-rank test were employed to plot and compare the survival curves. Survival data were assessed through univariate and multivariate Cox regression analyses. A two-tailed MYCN amplification, death from disease and high risk, respectively. In contrast, stage 4s patients with MYCN amplification, high risk and death from disease had occupied less than 4.5%, and those with progression had accounted for 7.5%. In comparison, in the TARGET-NBL dataset, stage 4 patients had taken up more than 80% in death, event, progression and relapse groups, and the proportions were especially high in death and relapse groups . However, stage 4s patients had only occupied no more than 2% (Supplementary Table n = 168) revealed that the age of ≥ 18 months, MYCN amplification, and high risk were associated with inferior outcomes . Results of risk factor analysis in this dataset were consistent with those from related articles  and 5 KEGG pathways were revealed finally Figures , S4. As ERCC6L, AHCY, STK33, and NCAN) were selected as a gene signature in predicting the vital status of NB patients  were closely correlated with overall survival and event-free survival. As shown in Supplementary Figures MYCN amplification, advanced stage, high risk, disease progression, and unfavorable class label groups had high risk scores (p < 0.0001)  , ERCC6L reached 0.799 in predicting the survival status of NB patients, which was the highest. In order to obtain the potential function of ERCC6L in NB development, the gene-encoded protein–protein interaction (PPI) network was constructed by employing the STRING database2, so as to find the co-expression relationship of genes. CytoHubba  with the Clustering Coefficient of > 0.9 were selected, as shown in Figure p-value < 2.2E-16)  were used for evaluation. As shown in Figure MYCN amplification and ALK mutation have been comprehensively studied as the prognostic factors and important treatment targets  signature in predicting the prognosis of NB, with the AUC of as high as 0.86. Simultaneously, the grouping ability of the gene signature was assessed, the results of which revealed that the four-gene signature had good performance in predicting other groups. When predicting the MYCN amplification group, the AUC of the four-gene signature was 0.965, and in another two independent datasets, the AUC in predicting MYCN amplification was higher than 0.95. We guessed there are two reasons: (1) the AUC in predicting MYCN amplification of AHCY alone was 0.946, which could be attributed to the fact that AHCY was directly regulated by MYC proteins  in the untreated stage 4 and stage 4s patients, and utilizing bioinformatic means to intensively analyze the data. We had identified 131 DEGs, and BP in GO term and KEGG functional enrichment analysis showed that the DEGs were mainly enriched in the cancer-related pathways, such as DNA replication, cell division and cell cycle . We pickproteins ; (2) migMYCN amplification, ALK mutation  is a protein-coding gene, also known as PICH, its related pathways include cell cycle and mitosis. Pu et al. had found that higher ERCC6L expression was notably associated with inferior outcomes in breast and kidney cancers, and silencing of ERCC6L would inhibit the growth of breast and kidney cancer cells. Besides, their further studies considered that ERCC6L might affect the cell cycle via RAB31-MAPF-CDK, so as to promote cancer cell proliferation . Typically, the AUC of ERCC6L alone in predicting different groups was higher than 0.75 (p < 0.0001).Of the four genes, is in NB . NCAN hais in NB . ERCC Exferation . Albers feration , which iERCC6L, we have constructed a PPI network and calculated the PCC of ERCC6L with other key genes which may interact with ERCC6L, eight genes had been identified that had a close relationship with it. Most of them were involved in the cell cycle pathway. Importantly, of the eight genes, MAD2L (CCNB1 (BIRC5 (ERCC6L may play an important role in NB, biological experiments are needed for further study of its mechanism.In order to find the potential function of s, MAD2L , CCNB1 (L (CCNB1 , and BIR1 (BIRC5  have beeYL conceived of and directed the project. XZ designed the study, analyzed the data, wrote the manuscript. HL revised the manuscript critically for important intellectual content. YZ collected data and samples. LW reviewed the data. All authors have read 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.


For the treatment of primary canaliculitis, 1,2,3-snip punctoplasty and canalicular curettage are commonly used; however, a recurrence rate of 6.6% to 22% has been reported. Herein, we describe a case of recurrent primary canaliculitis that was completely cured by 4-snip punctoplasty and canalicular curettage.A 53-year-old woman was admitted to our hospital with chief complaints of epiphora, discharge, eyelid flare up, and swelling near the inferior lacrimal punctum in the left eye, which initially presented 6 months earlier.Based on the aforementioned symptoms, the patient was initially diagnosed with bacterial conjunctivitis at a local ophthalmologic clinic and used antibiotic eye drops for 6 months. However, her symptoms did not improve and they worsened at 2 weeks prior to admission. She was subsequently diagnosed with chronic dacryocystitis and referred to our hospital for surgical treatment. Slit lamp examination results showed conjunctival congestion in the inner corner of the left eye, along with eyelid flare up, swelling near the inferior lacrimal punctum, and yellowish discharge and concretion from the lacrimal punctal orifice. Furthermore, punctal regurgitation was not observed in the lacrimal sac compression test. Thus, the patient was diagnosed with primary canaliculitis on the basis of her clinical symptoms and laboratory findings.Based on the diagnosis of primary canaliculitis, 1-snip punctoplasty and canalicular curettage were performed, and the patient was prescribed oral antibiotics for 2 weeks together with topical antibiotics for 4 weeks. Overall, the patient's symptoms improved after surgery, but epiphora and yellowish discharge from the lacrimal punctal orifice developed again 2 months after surgery during outpatient follow-up. Based on the diagnosis of recurrent primary canaliculitis, 4-snip punctoplasty and canalicular curettage were performed, and the patient was prescribed oral antibiotics for 2 weeks together with topical antibiotics for 4 weeks.Over a 6-month follow-up period, the symptoms disappeared completely and no other findings were observed.Four-snip punctoplasty and canalicular curettage are simple clinical procedures that can minimize the recurrence rate of primary canaliculitis. Hence, 4-snip punctoplasty and canalicular curettage should be considered as the 1st-line treatment for primary canaliculitis and recurrent cases. It accounts for approximately 1.4% to 2% of all lacrimal diseases.,3,10 Medical and surgical management are options for treating primary canaliculitis; however, surgical management is considered the definitive treatment.,5,11,12 In cases of recurrence after the initial treatment, conservative treatment should be avoided, and snip punctoplasty with canalicular curettage should be performed.Clinical symptoms include epiphora, swelling of the eyelid or punctum, erythema, pain, and redness. Clinical signs include thickening of the canalicular portion of the eyelid margin, punctal regurgitation of pus and concretions, a pouting erythematous punctum, yellowish discoloration of the canalicular region, and localized hyperemia.,8,11,13 Currently, there are no reports discussing the most appropriate surgical approach to treat recurrent primary canaliculitis; therefore, a more definitive treatment method should be considered to reduce the recurrence rate.At present, 1,2,3-snip punctoplasty and canalicular curettage may be an effective treatment modality for primary canaliculitis; however, previous studies have reported recurrence rates of 6.6% to 22%.In the present case, a patient diagnosed with primary canaliculitis was treated using 1-snip punctoplasty and canalicular curettage; however, symptom recurrence was observed during the postoperative outpatient follow-up. Subsequent treatment with 4-snip punctoplasty and canalicular curettage successfully cured the recurrent primary canaliculitis. Accordingly, we recommend 4-snip punctoplasty and canalicular curettage as an effective treatment method for complete curettage when managing recurrent primary canaliculitis after initial treatment.2A 53-year-old female patient was admitted to our hospital with chief complaints of epiphora, discharge, eyelid flare up, and swelling near the inferior lacrimal punctum in the left eye, all of which had developed 6 months earlier. Based on the aforementioned symptoms, the patient was initially diagnosed with bacterial conjunctivitis at a local ophthalmologic clinic and administered antibiotic eye drops  for 6 months. However, her symptoms did not improve, and they had worsened 2 weeks prior to her admission. Subsequently, she was diagnosed with chronic dacryocystitis at a local ophthalmologic clinic and transferred to our hospital for recommended surgical treatment. The Institutional Review Board/Ethics Committee of Bucheon St Mary's Hospital approved this study. It was performed in accordance with the tenets of the Declaration of Helsinki. Written informed consent was obtained from the patient for publication of this case report and accompanying images.The patient had hypertension , but no other specific underlying disease or history of previous surgery. On admission, her corrected visual acuity in both eyes was 1.0 and the intraocular pressure was normal. Slit lamp examination results showed conjunctival congestion in the inner corner of the left eye, eyelid flare up, swelling near the inferior lacrimal punctum, and yellowish discharge from the punctal orifice  for 2 weeks along with 4 weeks of antibiotic eye drops .One month after the 2nd surgery, a well-formed punctum was observed, and all signs of epiphora, discharge, eyelid flare up, and swelling near the inferior lacrimal punctum in the left eye had disappeared Fig. .There were no findings of recurrence or complications during the subsequent 6-month follow-up period.3–4,8–10Primary canaliculitis is a rare disease that involves infection in the lacrimal canaliculus. It accounts for approximately 1.4% to 2% of all lacrimal diseases; however, it is commonly misdiagnosed due to its clinical signs overlapping with other diseases that occur near the lacrimal apparatus.–7,9 As a result, there are often cases in which the appropriate treatment is not provided in timely manner due to delayed diagnosis.,6–8 In particular, initial treatment is delayed in many cases due to symptoms of epiphora, discharge, redness, and eyelid swelling, being misdiagnosed as chronic conjunctivitis, chalazion, or chronic dacryocystitis.,8,11 However, specific clinical signs, such as pouting of the punctum, yellowish discoloration of the canalicular region, peripunctum hyperemia, expressible punctal discharge and concretion, and negative regurgitation during the lacrimal sac compression test, can help differentiate primary canaliculitis from other diseases that present with similar symptoms.,5,8–10Literature reviews report that the rate of misdiagnosis ranges from 45% to 100%.The patient in the present case was admitted for primary symptoms of epiphora, discharge, eyelid flare up, and swelling near the inferior lacrimal punctum that persisted despite long-term treatment with antibiotics for 6 months. She was ultimately diagnosed with primary canaliculitis based on gross findings of yellowish discoloration of the canalicular region, expressible punctal discharge and concretion, and negative regurgitation during the lacrimal sac compression test; therefore, the appropriate treatment was provided.Actinomyces and Nocardia have been reported as the causative organisms of primary canaliculitis–9,15–17; however, recent reports have revealed that Staphylococcus and Streptococcus species are the most common causative organisms.,10,14,18 In the present case, the bacterial culture test could not identify the exact causative organism, but gram-positive rods were found. Additionally, the histologic examination identified tangled clumps of filamentous organisms, which are findings consistent with a diagnosis of sulfur granules.In most case reports, –9,13,18 In particular, it has been reported that medical treatment can have a recurrence rate of 33%; furthermore, there have been many other reports of failed cases using medical treatment.,6,7,13Treatment options for primary canaliculitis include medical management such as warm compresses, digital massage, topical and systemic antibiotics, antifungals, corticosteroids, and hyperbaric oxygen therapy; or surgical management. Some studies have reported that medical management is somewhat effective for treating primary canaliculitis, but the frequency of recurrence may be high due to the fact that such management cannot completely eradicate the bacterial reservoir.; however, surgical management is still considered the definitive treatment for canaliculitis.,5,11,12A recent report has indicated that conservative, incision-sparing treatment had an 83.3% successful treatment rate and low recurrence rates,4,10,15Surgical management includes punctoplasty with canalicular curettage and canaliculotomy with canalicular curettage. Further, punctoplasty and canalicular curettage can use 1-, 2-, or 3-snip punctoplasty, which has been reported to be an effective treatment for primary canaliculitis.; however, the canaliculus plays an important role in allowing tear flow into the lacrimal sac, and canaliculotomy and canalicular curettage can ultimately cause lacrimal pump dysfunction and epiphora due to scarring or fibrosis of the canaliculus, canalicular luminal narrowing, or canalicular fistula formation. Thus, serious consideration should be given as to whether canaliculotomy should be used for treating primary canaliculitis.,5,7,20Canaliculotomy and canalicular curettage has been reported as a safe and efficacious treatment In the present case, a minimally invasive surgery was planned, and 1-snip punctoplasty and canalicular curettage were performed initially. It was believed that complete canalicular curettage was achieved during the 1st surgery; however, it was found that the patient's symptoms recurred 2 months after that surgery and a 2nd surgery was planned.It has been reported that 1-snip punctoplasty and canalicular curettage is a minimally invasive and effective procedure with a high successful treatment rate of 83.3% and a low complication rate. Therefore, widening the incision size may help reduce the likelihood of leaving concretions or debris behind, which would ultimately increase the success rate of surgery and lower the recurrence rate. In the present case, good treatment outcomes were achieved by performing 4-snip punctoplasty and canalicular curettage with punctal dilation and a wider incision.It is believed that the recurrence may have been caused by incomplete removal of concretions and debris during the 1st surgery. Some concretions or debris may have been left unintentionally due to the small size of the incision.Although 4-snip punctoplasty can be a good alternative for achieving punctal dilation and a wider incision size, it may be more invasive than 1-snip punctoplasty, which may present problems with maintaining anatomical and physiologic functioning of the lacrimal system. Another study has reported that 4-snip punctoplasty does not have a major impact on the function of the punctal sphincter or the pumping action of the canaliculus, and thus, it can be performed safely.,8,11,13 Therefore, to reduce the recurrence rate, 4-snip punctoplasty and canalicular curettage, which allows a more definitive removal of canalicular concretions or debris, should be considered as the 1st-line treatment to reduce the recurrence rate, in addition to the method used for recurrent cases (Fig. While 1,2,3-snip punctoplasty and canalicular curettage may be an effective treatment modality for primary canaliculitis, it is associated with a recurrence rate of 6.6% to 22%.We cannot conclude that 4-snip punctoplasty and canalicular curettage is the best treatment modality for recurrent primary canaliculitis based solely on the treatment outcome in the present case. This is a single case report with surgical outcomes observed over a short follow-up period. In the future, an analysis is required to investigate treatment outcomes in a larger group of patients with observations over a longer follow-up period.Performing 4-snip punctoplasty and canalicular curettage for recurrent primary canaliculitis after 1-snip punctoplasty and canalicular curettage can increase the success rate of surgery and reduce the recurrence rates. 4-Snip punctoplasty and canalicular curettage may also be considered as the 1st-line treatment for primary canaliculitis.Conceptualization: Min Ho Kim, Ho Ra.Resources: Min Ho Kim, Ho Ra.Visualization: Min Ho Kim, Ho Ra.Writing – original draft: Min Ho Kim.Writing – review & editing: Min Ho Kim.


Dear editorUrinary tract infections (UTIs) are among the most common hospital-acquired infections, which are caused by urinary catheterization in most cases , 2. ThesVarious approaches have been proposed for decreasing these infections. Among these approaches, using aseptic methods, limiting the use of catheters to necessary cases, using disposable tools, and training the personnel can be pointed out. One of the most recent methods suggested in this regard is using catheters coated with anti-septic agents, antibiotics, and agents and metals such as silver, silicon, nitrofurazone , 6.In a study in 24 hospitals, using catheters coated with silicone-nitrofurazone compounds led to a slight decrease in UTI rate with odds ratio of 0.68 . In anotFortunately, this kind of catheter has become available to the physicians in Iran. However, since enough experience does not exist regarding their possible harms and benefits in the Iranian population, the researchers of the present study attempted to design a case-control study for evaluating the effects of using urinary catheters coated with gold, silver, and palladium on the rate of infections and mortality due to UTIs in patients hospitalized in the ICU of a teaching hospital in Tehran, Iran.Inclusion criteria were age over 18 years and having a catheter for 2 to 7 days. Presence of any infection or antibiotic consumption at the time of catheterization or development of an infection not related to the catheter during the study, the patient being pregnant or lactating, and having a history of allergy to the metal compounds of the catheter were among the exclusion criteria. Positive urine culture was defined as presence of at least 100000 bacterial colonies in each milliliter.Finally, 314 patients were randomly divided into 2 groups of 157 and catheterized with either regular or metal coated catheters. The 2 groups were similar regarding sex distribution (p = 0.518). Mean age of the patients was 59.80 ± 19.85 years in those with metal coated catheters and 61.69 ± 19.57 years in the regular catheter group (p = 0.396). Throughout the study, 26 (16.6%) patients in the coated catheter group developed symptoms related with UTI due to catheter, 18 (11.4%) of which had positive urine cultures . All the mentioned microorganisms were resistant to fluoroquinolone family, aminoglycosides, penicillin, third and fourth generation cephalosporins, carbapenems and colistin. In the non-coated group, 38 (24.2%) patients developed symptoms related to UTI due to catheterization, 29 (18.5%) of which were reported as positive . All the mentioned microorganisms were resistant to fluoroquinolone family, aminoglycosides, penicillin, third and fourth generation cephalosporins, carbapenems and colistin. There was no statistically significant difference between the 2 groups regarding developing UTI (p = 0.134). Additionally, no significant difference was found between the coated and non-coated groups regarding infection-related mortality (6 (3.8%) patients in the coated groups vs. 11 (7.0%) in the non-coated group; p > 0.05).Based on the findings of the present study, although the group using heavy metal coated urinary catheters had a lower number of infections and mortality cases, this difference was not statistically significant. Therefore, we should be cautious regarding suggesting their use considering the price of these catheters and their availability in hospitals. It seems that more accurate and multicenter studies with controlling other probable confounding factors are required for reaching a final decision regarding the use of this tool.


PeHB001–PeHB115) were identified from moso bamboo (Phyllostachys edulis) and grouped into 13 distinct classes  based on the conserved domains and phylogenetic analysis. The number of members in the different classes ranged from 2 to 24, and they usually varied in terms of exon–intron distribution pattern and length. There were 20 conserved motifs found in 115 PeHBs, with motif 1 being the most common. Gene ontology (GO) analysis showed that PeHBs had diverse molecular functions, with 19 PeHBs being annotated as having xylem development, xylem, and phloem pattern formation functions. Co-expression network analysis showed that 10 of the 19 PeHBs had co-expression correlations, and three members of the KNOX class were hub proteins that interacted with other transcription factors (TFs) such as MYB, bHLH, and OVATE, which were associated with lignin synthesis. Yeast two-hybridization results further proved that PeHB037 (BEL class) interacted with PeHB057 (KNOX class). Transcriptome expression profiling indicated that all PeHBs except PeHB017 were expressed in at least one of the seven tissues of moso bamboo, and 90 PeHBs were expressed in all the tissues. The qRT-PCR results of the 19 PeHBs showed that most of them were upregulated in shoots as the height increased. Moreover, a KNOX binding site was found in the promoters of the key genes involved in lignin synthesis such as Pe4CL, PeC3H, PeCCR, and PeCOMT, which had positive expression correlations with five KNOX genes. Similar results were found in winter bamboo shoots with prolonged storage time, which was consistent with the degree of lignification. These results provide basic data on PeHBs in moso bamboo, which will be helpful for future functional research on PeHBs with positive regulatory roles in the process of lignification.Homeobox (HB) genes play critical roles in regulating various aspects of plant growth and development. However, little is known about HB genes in bamboo. In this study, a total of 115 HB genes ( Then,elopment . In the elopment . Arabidopsis and the peach have shown that AtBP and PpKNOPE1 can interact with the typical KNOX DNA-binding site (TGACAGC) in 4CL, COMT, and LAC to regulate their expression during lignification [The HD-ZIP classes are characterized by a leucine-zipper conserved domain adjacent to the C-terminus of the HD, while the other 10 classes not only contain the HD, but also have their own specific conserved domains. For example, PLINC is distinguished from other zinc finger classes by the substitution of the usually conserved homeodomain residue F49, with a methionine residue and the insertion of one amino acid between helix 1 and helix 2 ,16,17,18fication ,25.Phyllostachys edulis) belongs to the subfamily Bambusoideae of the Poaceae family, with characteristics of fast growth and excellent materiality, it can grow about 20 m in 1.5 months [Moso bamboo  and RGAP 7.0 , respectively [http://bamboo.bamboogdb.org/) [−10. Then, all putative sequences were validated by the Pfam program (http://pfam.xfam.org/) [http://www.expasy.org/tools/) [To identify the members of HB genes in moso bamboo, the previously identified HB genes of ectively ,38, and db.org/) . The seqam.org/)  and aligam.org/)  to remov/tools/) .Arabidopsis, and rice by Clustal X 2.1 [https://www.megasoftware.net/) with the Maximum-likelihood (ML) method and the bootstrap test was replicated 1000 times [http://gsds.cbi.pku.edu.cn) was used to analyze the exon/intron of HB genes obtained from moso bamboo [http://meme-suite.org/tools/meme) with the following parameter settings, which included the distribution of motifs: zero and one per sequence; maximum number of motifs to find: 40; minimum width of motif: 6; and maximum width of motif: 120 [To explore the conservation of obtained sequences, multiple alignments were carried out using the full-length translated protein sequences of HB proteins from moso bamboo, al X 2.1 . Phyloge00 times . The Geno bamboo . The contif: 120 . https://bioinformatics.psb.ugent.be/plaza/) with the software-set parameters [http://bioinformatics.cau.edu.cn/bamboo/cytoscape/network.php) web site [https://cytoscape.org/) [PAL, 4CL, C3H, C4H, HCT, CCR, CCoAOMT, CAD, F5H, COMT, and LAC) related to lignin biosynthesis in moso bamboo were downloaded from PLAZA 4.0 [http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [The proteins encoded by the HB genes of moso bamboo were annotated using the PLAZA 4.0 to assign GO terms (rameters . The Bamweb site  and Cytope.org/)  were useLAZA 4.0 , and thee/html/) .https://matrix2png.msl.ubc.ca/bin/matrix2png.cgi) [To investigate the expression of HB genes in the different tissues and developmental stages of moso bamboo, the transcriptome data generated from leaves, early panicles, advanced panicles, roots, rhizomes, 20-cm shoots and 50-cm shoots were downloaded from the NCBI Short Read Archive (SRA)  and usedpng.cgi) .To examine the expression levels of different HB genes associated with lignin biosynthesis in moso bamboo shoots during the lignification process, the basal parts of shoots with different heights  were collected from the bamboo forest experimental site of Jiangxi Academy of Forestry located in Nanchang, Jiangxi Province, China. In addition, winter bamboo shoots  without any blemishes and disease were harvested and stored in darkness in perforated plastic bags at 25 °C. The basal parts of the first internode of shoots were collected as samples after 0, 3, 6, and 12 d for RNA isolation and lignin content determination. Each sample was three technical replicates with five shoots per replicate, frozen immediately in liquid nitrogen and stored at –80 °C. Meanwhile, formalin–acetic acid–alcohol (FAA) was used to fix the samples collected from the same part, which were stored at 4 °C for further morphology analysis.The total RNA was extracted using the plant total RNA extraction kit  according to the manufacturer’s instructions. The integrity of the total RNA was analyzed on a 1.0 % agarose gel. RNA quantity was determined using a NanoDrop 2000 Spectrophotometer . Total RNA was used for the synthesis of cDNA by a reverse transcription system . PeNTB gene as an internal control [2O. PCR was performed with cycling parameters as follows: 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 10 s. The expression levels were calculated using the 2−ΔΔCT method [Quantitative real-time PCR (qRT–PCR) analysis was performed using the  control . The speT method . Each PCThe winter bamboo samples were dried in an oven at 80 °C, until there was no change in their weight. The samples were ground into a powder with a mortar and pestle, then filtered with a 40-mesh sieve and used for further lignin content measurement using the acetyl bromide method . The datw/v) Toluidine Blue O [The fixed samples were taken out and rinsed with sterile water 3–5 times to remove the FAA fixative on the surface. Then we cut the samples into a pyramid shape and fixed them on the tray with glue. The samples were sectioned using a vibratome  at a thickness of 20 μm. The sections were stained with 0.01%  were selected for further validation by using yeast two-hybridization. To explore the transcriptional activity of the genes, the yeast expression vectors were constructed in pGBKT7 vectors. Primer sequences for the construction of expression vectors were designed as listed in p < 0.05 and ** p < 0.01.IBM SPSS Statistics  was used for statistical analysis, and the mean and standard deviations of three biological replicates are presented. Significant differences are indicated at * PeHB001 to PeHB115 according to their physical locations on the genome scaffolds. The proteins encoded by PeHBs ranged from 197 aa to 542 aa in length, with an average of 357.5 aa. The basic characteristics of PeHBs including isoelectric point (pI), molecular weight (MW), instability index, grand average of hydropathy (GRAVY) value, and subcellular localization are shown in After a series of comprehensive comparison analyses, including the BLAST search, conserved domain analysis, sequence multiple alignment and phylogenetic analysis, a total of 115 HB genes were identified in moso bamboo, which were named Arabidopsis, and rice are shown in To investigate and identify the characteristics of homologous domains in PeHBs, the multiple sequence alignment was carried out. The result demonstrated that the specific domains of 13 identified classes  were found in the sequences of PeHBs, and the representative homeodomain sequences of moso bamboo, Arabidopsis (107), and rice (107) were classified into 14 classes based on the similarity of their HD domain and the presence of specific domains  were grouped into the HD-ZIP class and two (KNOX and BEL) formed the TALE class ,  domains . Most meLE class . The memand rice . HoweverPeHBs   and ‘xylem and phloem pattern formation’ (GO:0010051) is 19; these were then selected for co-expression analysis and expression pattern validation in shoots of the moso bamboo. Gene Ontology (GO) enrichment analysis was performed to characterize the main biological functions of re found . More thess (BP) . The resomponent . MoreovePeHBs annotated as GO:0010089 and GO:0010051 were selected. The co-expression network diagrams were generated basing on the transcriptome data [PeHBs had co-expression correlations. Six gene pairs were found to have positive co-expression with each other, including PeHB07 and PeHB027, PeHB017 and PeHB057, PeHB031 and PeHB109, PeHB042 and PeHB109, PeHB074 and PeHB031, and PeHB074 and PeHB109. The only negative co-expression gene pair was PeHB005 and PeHB095. These results indicated that the co-expressed PeHBs might work together in regulating lignification in the cell wall of moso bamboo.To construct the gene networks involved in lignification, the 19 ome data   of the KNOX class presented in the PPI network were selected, they were cloned and sequenced to confirm their complete ORF, and then constructed in pGBKT7. The AH109 yeast cells containing the recombinant plasmids were tested on the selective media. The results showed that the positive control group (pGBKT7-53) exhibited visible blue colonies with satisfactory growth states on the SD/−Trp/−His/−Leu/X-α-GAL medium, while the transformants of pGBKT7::KNOXs and pGBKT7 were unable to survive , indicatTo investigate the PPI between different PeHBs, the predicted PPI pair of PeHB037 (BEL class) and PeHB057 (KNOX class) was used for further validation by yeast two-hybrid. The ORFs of PeHB037 and PeHB057 were constructed into activation domain and DNA-binding domain plasmid vectors, respectively. As depicted in PeHBs related to the biosynthesis and deposition of the secondary cell wall were selected for further validation using qRT-PCR, with PeNTB as the reference gene. The results demonstrated that the expression levels of most PeHBs increased significantly along with the shoot growth, and reached the highest expression level in 6.7-m shoots  showed an increasing expression trend along with the storage time, and reached the highest level at the end of the storage (12 d). For example, PeHB031, PeHB057, and PeHB095 were upregulated more than 6.5-, 14.8-, and 3.2-fold in winter shoots at 12 d compared to those at 0 d, respectively. The expression levels of PeHB064, PeHB088, and PeHB100 upregulated rapidly and reached the maximum at 3 d, followed by a decline; those of PeHB019 and PeHB070 of the WOX class showed a similar trend of upregulation, with the maximum at 6 d, then a decline at 12 d. The expression of five genes in winter shoots during storage showed a trend of first decreasing and then rising, in which PeHB024 and PeHB104 were first slightly downregulated and then significantly upregulated to reach the maximum at the end of the storage (12 d); PeHB007 and PeHB027 were first significantly downregulated and then upregulated; PeHB005 was the only one that was significantly downregulated. PeHB024 and PeHB104 were downregulated slightly at 3 d, and then increased significantly. To further understand whether Bamboo shoots are the immature culm of the bamboo; the process of lignification occurs with the increase in height and the storage days. The lignification process was mainly characterized by the deposition of lignin in the cell wall, which has been proved in different height shoots . Therefop < 0.05) (PeHBs (14 of 19 PeHBs) in winter shoots  e. The inr shoots . These rage days , which wPe4CL (PH01000809G0020), PeC3H (PH01001724G0360), PeCCR (PH01001334G0240), and PeCOMT (PH01001283G0360), indicating that they might be regulated by KNOX. The qRT-PCR results showed that the expression of Pe4CL, PeC3H, PeCCR, and PeCOMT increased obviously with the process of lignification. In particular, the expression of PeCOMT in 6.7 m shoots was markedly upregulated to more than 53-fold of that in 0.2 m shoots, and that in winter shoots after storage for 12 days was 122-fold of that in the fresh shoots  in winter shoots  was found in the promoters of four lignin synthesis genes, h shoots . These ro shoots  and threr shoots , which pArabidopsis [The HB genes are common in plants, and play important roles in regulating cell differentiation, embryonic shoot meristem formation, embryo patterning, vascular development, floral organogenesis, and fruit ripening ,57. Therbidopsis  and ricebidopsis , but onlbidopsis ,61, so iArabidopsis, and rice (107 each) [Arabidopsis, and rice. No members belonging to the LD class were found in moso bamboo, suggesting that gene deletions may have occurred during its evolution. The members of the BEL, PHD, and SAWADEE classes of moso bamboo were all more numerous than in those classes of Arabidopsis and rice. In particular, the members of the BEL class were almost twice as numerous of those in Arabidopsis and rice, which indicated that gene expansion occurred in the BEL class of moso bamboo, maybe leading to the functional redundancy. The members of the WOX class in most seed plants had been divided into modern/WUS (Wuschel), middle, and old evolutionary branches [Arabidopsis and rice, suggesting that the bamboo WOX class might have a unique pattern of evolution. We first identified 115 HB genes from the genome of moso bamboo, which was more than the number of members of 07 each) ,62. The branches , but thoPeHBs (85%) had fewer than 10 introns, and only 21 PeHBs had more than 10 introns. Half of the members of the PLINC class had no introns, and the remaining genes contained at least one intron. Overall, the number of introns was similar in the same class. For example, the members of the ZIP I class had one or two introns, and those of the ZIP III class contained more introns than the ZIP I, II, and IV classes, but they were relatively conserved, similar to those in grapes [In this study, 94 of 115 n grapes . In genen grapes . In addiPeHBs in different tissues and developmental stages in moso bamboo, it was found that they had different constitutive and tissue specific expression patterns. For examples, some PeHBs such as the members of the HD-ZIP III, SAWADEE, and NDX classes were highly expressed in all tissues, suggesting that they might be involved in various biological processes and play important roles in the growth and development of moso bamboo. By contrast, PeHB017 had a very low expression level in all tissues, suggesting that it may be involved in other biological processes. In addition, the expression levels of some PeHBs in the PHD and HD-ZIP IV classes in roots were significantly lower than those in other tissues, indicating that they may play a smaller role in the root-related growth and development process. Similarly, most members of the BEL class were highly expressed in the leaves and panicles, but relatively low in the other four tissues, especially in the shoots. On the other hand, PeHBs belonging to the PLINC class showed dominant expression in shoots, and were relatively low in the other bamboo tissues. Based on the expression patterns of PeHBs were upregulated except PeHB005, and showed a downregulation in the highest shoots and in the winter shoots at the end of storage. However, the upregulated PeHBs demonstrated different expression patterns. For examples, the expression abundance of eight PeHBs increased continuously along with the increase of lignification degree in different height shoots and the winter shoots during storage, suggesting that these genes play positive regulatory roles with a continuously rising pattern in the process of lignification. Meanwhile, five upregulated genes  showed a similar trend of first rising and then falling with the maximum appearing in 1.0-m or 3.0-m shoots and the winter shoots after 3 d or 6 d storage. PeHB005 was the only one downregulated in both different height shoots and the winter shoots during storage. These results indicated that the lignification process was a complicated regulatory network associated with multiple PeHBs. Under natural conditions, with the increase in shoot height, the lignification degree in the cell wall of shoots was intensified. The lignification degree of winter bamboo shoots increased in a passive state with the prolongation of storage. All 19 PeHB074) and two members of the BEL class  were positively co-expressed in the network, which was supported by the qRT-PCR results of them expressed in the shoots undergoing lignification. In addition, many studies have reported that TFs can regulate the lignin pathway by binding with the key structural genes involved in lignin synthesis [Pe4CL, PeC3H, PeCCR, and PeCOMT, and three PeHBs of the KNOX class showed a positive co-expression correlation with these lignin synthesis genes. The co-expression network of PeHBs, and PPI of PeHBs, as well as the regulatory roles of PeHBs on structural genes indicated that lignification might be a complicated regulatory network in moso bamboo, which needs further experimental verification.The plant lignification process is a complex process of cell differentiation and development, which is driven by the co-expression of many transcription regulators and functional genes ,65,66,67ynthesis . For exaPeHBs identified from moso bamboo were classified into 13 classes, which had different expression patterns in various tissues. Nineteen PeHBs were predicted to be related to lignin biosynthesis and validated by qRT-PCR, in which 10 PeHBs had co-expression correlations, and three members of the KNOX class were hub proteins that interacted with other TFs. PeHB037 interacted with PeHB057 in yeast, which further proved that the PPI prediction was correct. With the increasing degree of lignification, the expression patterns of the 19 PeHBs in different height bamboo shoots were similar to those in winter shoots during storage, and three PeHBs of the KNOX class had a positive correlation with those of four lignin synthesis genes . These results indicated that the involvement of PeHBs in bamboo lignification was a multi-level regulatory network.Lignification is considered to be a key step in timber formation, and some HB genes are regulators of lignin biosynthesis. The 115


Hevea genus of the Euphorbiaceae family, Hevea brasiliensis (Willd. ex Adr.de Juss.) Muell. Arg. is the main commercial source of natural rubber production worldwide. Knowledge of the population structure and linkage disequilibrium (LD) of this species is essential for the efficient organization and exploitation of genetic resources. Here, we obtained single-nucleotide polymorphisms (SNPs) using a genotyping-by-sequencing (GBS) approach and then employed the SNPs for the following objectives: (i) to identify the positions of SNPs on a genetic map of a segregating mapping population, (ii) to evaluate the population structure of a germplasm collection, and (iii) to detect patterns of LD decay among chromosomes for future genetic association studies in rubber tree. A total of 626 genotypes, including both germplasm accessions (368) and individuals from a genetic mapping population (254), were genotyped. A total of 77,660 and 21,283 SNPs were detected by GBS in the germplasm and mapping populations, respectively. The mapping population, which was previously mapped, was constructed with 1,062 markers, among which only 576 SNPs came from GBS, reducing the average interval between two adjacent markers to 4.4 cM. SNPs from GBS genotyping were used for the analysis of genetic structure and LD estimation in the germplasm accessions. Two groups, which largely corresponded to the cultivated and wild populations, were detected using STRUCTURE and via principal coordinate analysis. LD analysis, also using the mapped SNPs, revealed that non-random associations varied along chromosomes, with regions of high LD interspersed with regions of low LD. Considering the length of the genetic map  and the mean LD (0.49 for cultivated and 0.02 for wild populations), a large number of evenly spaced SNPs would be needed to perform genome-wide association studies in rubber tree, and the wilder the genotypes used, the more difficult the mapping saturation.Among rubber tree species, which belong to the  Hevea brasiliensis, or the rubber tree, is an important crop species that produces a high-quality natural rubber in commercially viable quantity, accounting for more than 98% of the total natural rubber production worldwide , perennial, and cross-pollinated tree species with an estimated haploid genome size of 1.47 Gb  and amplified fragment length polymorphisms (AFLPs) , and denHordeum vulgare L.) and wheat (Triticum aestivum) genomes and has been shown to be an effective tool for developing molecular markers for these species  have lowered the cost of DNA sequencing to the point that genotyping-by-sequencing (GBS)  is now f species .Evaluation of the molecular diversity encompassed in rubber tree genetic resources is a prerequisite for their efficient exploitation in breeding and the development of conservation strategies of genetic diversity. LD is the non-random association of alleles at distinct loci in the genome of a sampled population  and is tWith the rise of sequence-based genotyping, precise and accurate estimates of population structure and the LD across the genome are now attainable for the rubber tree. Our goals in this study were to characterize the genome distribution of single-nucleotide polymorphisms (SNPs) in a rubber tree mapping population using GBS technology, to examine population structure, to investigate how LD breakdown relates to chromosomes, and to compare the LD between cultivated and wild populations. For this study, we used accessions selected from the germplasm collection previously analyzed by H. brasiliensis accessions, composed of both wild germplasm and cultivated genotypes. Details of the plant materials can be found in Supplementary Table S1. The other set is an important mapping population of the rubber tree consisting of 252 F1 hybrids, derived from a cross between PR255 × PB217 and comprising three replicates of each parental genotype, which were mapped with 505 markers  prior to this publication  according to the procedures described by the manufacturer. DNA quality parameters and concentrations were measured using a UV-Vis spectrophotometer  and agarose gels.Two sets of samples were selected for this study, and a total of 626 samples were sequenced. One set consisted of 368 EcoT22I, a methylation-sensitive restriction enzyme. The resultant fragments from each sample were directly ligated to a pair of enzyme-specific adapters and combined into pools. PCR amplification was carried out to generate the GBS libraries, which were sequenced on the Illumina HiSeq 2500 platform . The raw data were processed, and SNP calling was performed using TASSEL 5.0  as described by SSEL 5.0 . InitialSSEL 5.0  with BowSSEL 5.0  using thTo perform the analysis, the data were divided into the mapping population and germplasms. SNP calling was performed using the TASSEL 5 GBSv2 pipeline  and filtR package Onemap “suggest_lod”) and a maximum recombination fraction of 0.35.All linkage analyses were performed using OneMap software , versionThe map construction utilized only markers with 0.05 missing data and tested the pattern of allelic segregation for χ2 goodness of fit to the expected Mendelian segregation ratios, and markers with significant segregation distortion were excluded from further analysis. GBS-based SNPs were added to the previous genetic map using the “try.seq” function in OneMap, which determines the best position for a given unpositioned GBS marker in a specific linkage group (LG). Finally, the fractions of recombination were converted to centimorgans (cM) using the Kosambi map function , and theK), ranging from 1 to 10. Each run included 500,000 Markov chain Monte Carlo (MCMC) iterations, among which the first 100,000 iterations (used to monitor whether a chain reached stationarity) were discarded as burn-in. The delta K method was used to identify the number of subgroups in the dataset (Q) of a given accession, the accession was classified as admixed in clusters with a membership of Q < 0.70. Subsequently, genetic distances between pairs of accessions were calculated, and PCoA was performed for the SNPs using the GenAlEx program (version 6.5) . Initially, the structure was analyzed with the software STRUCTURE 2.3.4 . Ten rep dataset . Based oion 6.5) .r2) between each pair of SNPs with the R software and GGT 2.0  between markers on the same chromosome using the following model: y = a + b-c/x  consisted of good barcoded reads. Of these reads, 69,408 (23.42%) were aligned to the mapping population exactly one time and 177,143 (59.78%) more than one time, corresponding to an 83.20%  overall alignment rate. The total rate of alignment to the germplasm was 89.97% , of which 22.59% of the tags  were aligned to the rubber tree reference genome exactly one time  and 67.3A total of 386,180 SNPs were identified in the germplasm, and 76,191 SNPs were detected in the mapping population, of which 350,965 SNPs (germplasm) and 66,453 SNPs (mapping population) were biallelic. After excluding markers showing (1) more than 20% missing data or (2) a MAF ≤ 0.05, the whole dataset was reduced to 77,660 and 21,283 SNPs in the germplasm and mapping populations, respectively. The SNP frequencies were one biallelic SNP every 20.7 kb for the mapping population and every 3.9 kb for the germplasm.Sequence data are deposited under EMBL-EBI accession PRJEB26962.P≤ 0.05), which revealed segregation ratios of 1:2:1 and 1:1. Markers exhibiting statistically significant segregation distortion were excluded from further analysis to obtain accurate genetic linkage maps. The data analyses were performed using SNPs with a maximum of 5% missing data, resulting in a linkage map with 1,062 markers presenting 348 SSR markers (Supplementary Table S2) and 138 SNPs markers genotyped using the Sequenom MassARRAY® platform  and the Fluidigm® platform , developed from de novo transcriptome assemblies .The genetic map was organized according to the numbers obtained from the map previously developed by Rosa et al. (unpublished). Eleven markers were removed after data diagnosis using heat map graphs, thereby permitting the visualization of the recombination fraction and LOD scores from markers, to group the SNPs in the LGs without changing the order of the base map. Only LG18 was further divided into subgroups “A” and “B” , distributed among all the chromosomes. The LGs ranged from 23.8 cM (LG18B) (Supplementary Table S3) with 14 markers to the largest group with 404.6 cM (LG10) with 85 markers (of which 78 were from GBS), and the average interval size between two adjacent markers was 4.4 cM.Thus, a genetic map was generated spanning a cumulative length of 4,693 cM (The maximum gap size was 36.4 cM (LG11), which was maintained from the previous map. Furthermore, some regions could not be sampled using the selected restriction enzyme; in LG16, for example, only two GBS-based markers were added.K values ranging from 1 to 10 showed that the model likelihood increased steeply at K= 2, followed by a drastic decline starting at K = 3, suggesting that the optimal K value was 2. The assignment results for K= 2 showed that some of the sampled individuals exhibited admixtures from two gene pools : group 1 (red bars) mainly consisted of accessions originating from the Mato Grosso and cultivated genotypes, and group 2 (green bars) consisted entirely of wild accessions from Amazonas, Rondônia, Para, and Acre (identified as IS – Ilha Solteira). Twenty-one accessions showing a membership probability  below 0.70 were defined as admixed and were removed from subsequent analyses.The germplasm collection was selected in a prior work by Figure 2). On the first axis, most of the breeding genotypes were separated from the other genotypes. On the second axis, the wild germplasm samples were clustered together, but some accessions from the Amazon were isolated in their own subdivision.As a second analysis of differentiation, we employed PCoA based on a similarity matrix that explained 22.8 and 16.7% of the genetic variation with the first and second PCoA axes, respectively. Plotting the two first PCoA axes separated the germplasm into two clusters, though some overlap was present between the wild germplasms and cultivated genotypes , and pairwise LD estimates were performed within the gene pool of each of these groups. To visualize LD throughout the genome, heat maps were produced based on pairwise 2r estimates, and corresponding p-values were calculated using permutations for all marker pairs . These heat maps were employed to identify variations in LD between the cultivated and wild rubber tree germplasm groups.Based on population genetic structure, accessions could be divided into two distinct groups (cultivated and wild group) (P < 0.05) in the cultivated and wild germplasms, respectively. Of these significant associations, 78 and 64 were intrachromosomal in the cultivated and wild germplasms, respectively, accounting for 0.5 and 0.4% of the total possible intrachromosomal correlations. Among the unlinked loci, the proportions of LD were 0.7 and 3.3% for the cultivated and wild germplasms, respectively. In both clusters, an uneven distribution of LD among the 18 chromosomes was observed.In 16,025 pairwise combinations, we identified 186 (1.2%) and 592 (3.7%) statistically significant associations that were in LD (P < 0.05) was very different between two clusters, as reflected by the mean 2r values of 0.49 and 0.02 obtained for the cultivated and wild germplasms, respectively. The 346 SNPs that were localized in the integrated map were used for the estimation of LD decay among the different LGs. LD decay was estimated across the germplasms and was found to be more pronounced in the wild germplasms, with the range being dependent on the chromosome group . Using a fixed baseline 2rvalue of 0.1 for the wild germplasm, LD decay ranged from 2.3 cM (LG9) to 11.4 cM (LG17). In contrast, in the cultivated accessions, this decay was slower, ranging from 4.3 cM (LG8) to 50 cM (LG17), with average values of 5.6 cM and 19.6 cM for the wild and cultivated germplasms, respectively . The patterns of LD can also be visualized across the genome from the diagonal of the heat maps .The strength of LD  and analyzed the population structure and LD with two different populations of rubber tree, formed from 47 accessions from a breeding program and 300 accessions from a germplasm collection. For this study, we employed SNPs obtained via the GBS approach, which enabled the detection of polymorphisms distributed across the genome.An important breakthrough of the GBS approach is that a reference genome is not necessary for SNP genotyping. However, the availability of a reference genome offers additional benefits, as it allows proper alignment and ordering of the sequenced tags . The firHevea genome   . Based ormplasm) , the resThe frequency of nucleotide substitutions was five times higher in the wild (3.9 kb) than in the cultivated (20.7 kb) germplasm sequences. One explanation for this finding is that selection in cultivated breeding programs acts to reduce diversity and alter allele frequencies in the DNA sequence. Depending on how LD is increased surrounding these loci; the effects of such a selection may not extend sufficiently far to affect the overall genome diversity. Rubber tree breeders have had to develop cultivars that are appropriate for the specific temperature and humidity conditions encountered in different cultivation areas, along with various biotic and abiotic stress resistance factors. High-density SNPs are also common in non-genetic regions where there is no selection pressure, and the abundance of these SNPs will be very useful for future assessments of breeding populations. The high diversity of SNPs is characteristic of outbreeding trees; in other studies examining rubber tree, GBS does not require complete genome sequencing; only a targeted sequencing approach is necessary. Due to its high-throughput efficiency, GBS has been used for SNP identification and mapping in many plant species . In rubbThe linkage map constructed in the present study exhibits a regular marker distribution. However, the cumulative genetic map is significantly longer  than would be expected for maps derived from other mapping populations that have presented a lower resolution; for example, a map of 2,052 cM  was obtaGenetic mapping of the population (PR255 × PB217) was initiated in the first publication by H. brasiliensis has been reported to be highly heterozygous, with 71% of the genome length comprising repeats   , and theDiversity analyses were performed in a previous work that included the entire collection of rubber germplasm, analyzing 1,117 genotypes with 13 microsatellite markers , and shoRecent work using SSR markers has revealed similar results to those obtained using SNPs from GBS. For example, Population genetic structure is a principal factor influencing the generation of false positives and the success of the association or LD mapping . These 3Supplementary Figure S1). LD estimation is possible without the positions of molecular markers along the genome and was indeed performed in this study. However, these positions are crucial if one is interested in determining the decay or extension of LD in relation to the genetic distance, regardless of by chromosome.LD analysis with the mapped SNPs revealed that LD varied along the chromosomes, with regions of high LD being interspersed with regions of low LD  than in the wild germplasm (0.02), corroborating the results of The average The higher LD level detected in the cultivated group may have been influenced by the partly identical-by-descent of these genotypes from a limited number of founders of the breeding programs, with only a few generations between them. Thus, many longer pieces of chromosomes have not had time to undergo disruptions. Domesticated crop cultivars necessarily represent a subset of the genetic variation found in their wild ancestors, and the process of crop domestication is responsible for genetic bottlenecks . AlthougPopulus nigra, in which a decay of 2r with distance in the CAD4 gene was observed at approximately 16 bp  and genomic selection (GS). Genomes with low LD require a high marker density for GWAS or GS; therefore, our SNPs may be valuable for GWAS in rubber tree breeding. Considering the length of the genetic map  and the mean LD observed (0.49 in breeding and 0.02 in wild populations), many evenly spaced SNPs would be necessary to perform GWAS in the rubber tree, and the wilder the genotypes that are used, the more difficult is the saturation of the mapping. However, to obtain a sufficient SNP density throughout the genome and to account for variation in LD along the chromosomes more markers must be genotyped. Our study results provide a valuable resource for further genetic studies involving linkage or association mapping, marker-assisted breeding and Hevea is necessary for such endeavors, and the present study supplies a major contribution to this goal.Furthermore, GWAS of wild germplasm accessions in the future will provide a substantial contribution to dealing with new challenging situations that will arise as a consequence of global climatic changes. Useful QTLs and genes to face these new situations are currently unknown, but the best way to identify them would be through analyses relying on GWAS of large panels of wild genotypes. A precise assessment of the LD pattern across the genome of LMdS, LHBdS, VLG, and AdS designed the study and performed the experiments. LMdS, LHBdS, AC, CdS, CM, ES, JF, and MdM performed the experiments. LMdS, LHBdS, JR, GM, CM, VLG, MdM, PG, and AG analyzed the data. LMdS, LHBdS, and AdS wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.