Helianthus is a diverse taxonomic group with approximately 50 species. Most sunflower genomic investigations are devoted to economically valuable species, e.g., H. annuus, while other Helianthus species, especially perennial, are predominantly a blind spot. In the current study, we have assembled the complete mitogenomes of two perennial species: H. grosseserratus  and H. strumosus . We analyzed their sequences and gene profiles in comparison to the available complete mitogenomes of H. annuus. Except for sdh4 and trnA-UGC, both perennial sunflower species had the same gene content and almost identical protein-coding sequences when compared with each other and with annual sunflowers (H. annuus). Common mitochondrial open reading frames (ORFs)  in sunflowers and unique ORFs for H. grosseserratus (orf633) and H. strumosus  were identified. The maintenance of plastid-derived coding sequences in the mitogenomes of both annual and perennial sunflowers and the low frequency of nonsynonymous mutations point at an extremely low variability of mitochondrial DNA (mtDNA) coding sequences in the Helianthus genus.The genus  From a genomic point of view, plants are unique organisms, since their cells have three genetic systems—nuclear, plastid, and mitochondrial—which function independently, but are simultaneously intimately interconnected. Nuclear genomes are of primary interest, while cytoplasmic genomes, especially mitochondrial, are often underestimated . MitochoHelianthus is a diverse taxonomic group with approximately 50 species [Helianthus and Agrestis, and two perennial sections, Ciliares and Divaricati [H. annuus, and in the case of the perennial species, H. tuberosus, while the other Helianthus species, especially perennial, are predominantly a blind spot. To the best of our knowledge, very few studies of perennial sunflower mtDNA are available [H. annuus—are deposited in GenBank. Mitochondrial DNA of cytoplasmic male sterility (CMS) sources, obtained from wild progenitors, may be considered as exceptions—for instance, the MAX1 CMS mitogenome [Helianthus maximiliani Schrad as a maternal form. Since CMS systems are essential for the hybrid seed production of crops [The genus varicati . Most suvailable . Moreovetogenome  was gainof crops , particuof crops , the invH. maximiliani, the two species H. grosseserratus and H. strumosus represent perennial species and belong to the section Divaricati. H. maximiliani and H. grosseserratus are diploid, whereas H. strumosus can be either tetraploid (2n = 4x = 68) or hexaploid (2n = 6x = 102) [Helianthus is relatively good. However, while crossing tetraploid or hexaploid species is possible, there are considerable problems which require the application of biotechnological methods [H. grosseserratus, H. maximiliani and H. strumosus are characterized by a thin pericarp with a reduced sclerenchymatic layer, which represents an interesting trait in breeding for improved hullability [As with  methods . Many su methods  annual a methods . The chalability .H. grosseserratus and H. strumosus. We analyzed their sequences and gene profiles in comparison to the available complete mitogenomes of H. annuus .In the current study, we assembled the complete mitogenomes of two perennial species: Helianthus: H. grosseserratus Martens and H. strumosus L.  were homogenized by mortar and pestle in 20 ml of STE buffer  and then centrifuged using several steps: (1) 500 g for 5 min, picking the supernatant, (2) 4000 g for 5 min, picking the supernatant, (3) 10,000 g for 15 min, discarding the supernatant. The pellet was treated using 10 units of DNAse  for 7 min and then used for DNA isolation. For both samples, the DNA extraction was performed with a PhytoSorb kit , according to the manufacturer’s protocol.The perennial species of mosus L.  were obtwas used . But forH. grosseserratus and 300 cycles for H. strumosus. A total number of 1,349,630 75-bp paired reads and 3,014,579 150-bp paired reads were generated.Next-generation sequencing (NGS) libraries were made with a Nextera XT DNA Library Prep Kit , following the guidelines of Illumina. The quality and quantity of the libraries were evaluated with the Bioanalyzer 2100  and with a Qubit 4 fluorometer . Sequencing was performed on different runs of NextSeq 500  with the NextSeq 500/550 High Output Kit v2.5: 150 cycles for https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Trimmomatic v 0.39 software [H. strumosus mitochondrial genome were also verified by Sanger sequencing. The mitochondrial genomes were annotated with MITOFY [https://www.ncbi.nlm.nih.gov/orffinder). Graphical genome maps were generated using the OGDRAW tool [H. grosseserratus and H. strumosus, were submitted to the NCBI GenBank database .For quality control of the reads, we used FastQC v 0.11.9 . Such difference significantly influenced the complexity of mitogenome assembly. In the case of H. grosseserratus, a relatively small amount (1.3 mln) of short (75 bp) reads were obtained, and the assembly with a k-mer value equal to 65 generated only 14 high coverage (depths > 50) large contigs  which allowed a quick scaffolding. In the case of H. strumous, twice as many elongated reads  were observed, but many smaller contigs , containing nuclear and chloroplast sequences, were present, which in turn increased the complexity of scaffolding. Additional filtering of mitochondrial contigs had to be performed in the case of H. strumosus mitochondrial genome assembly. Thus, for mitogenome assembly, the proportion of mtDNA in a sample is more crucial than the number of reads.First of all, it should be mentioned that different mitochondrial fraction isolation techniques showed significant variations in the amount of mtDNA derived sequences for the two perennial H. grosseserratus and H. strumosus, respectively. Mitochondrial genomes often have a complex architecture, including sub-genomes or multichromosomal organization [For both of the studied sunflower species, master circles of the mitochondrial chromosomes were gained , with lenization ,24. HoweH. annuus allowed the identification of 12 syntenic blocks , as seen in H. annuus mitogenome, there are two large (>12.5 kbp) repeat regions, but in H. grosseserratus, these repeats are reduced to about 4.5 kbp, and in the case of H. strumosus—to about 1.1 kbp. The most prolonged repeat in H. grosseserratus comprises about 9.4 kbp, whereas in H. strumosus it counts only 7.1 kbp , cob (Complex III) genes, cytochrome c oxidase (Complex IV) genes , ATP synthase (Complex V) genes , cytochrome c biogenesis genes , matR, mttB and ribosomal protein genes . In the mitogenomes of the two perennial sunflowers, succinate dehydrogenase (Complex II) genes are only represented by sdh4 in H. strumosus, but in the case of H. grosseserratus, sdh4 is a pseudogene, which has an internal stop codon. In the studied mitogenomes, nine intron-containing genes were identified. Five of the nine genes  belong to mitochondrial complex I, and the remaining three genes were ccmFC, cox2, mttB, and rps3. The trans-spliced introns were observed in nad1, nad2, and nad5, which is consistent with angiosperm plants [nad7 which counted four introns. The introns’ locations and splicing were similar to those observed in the mitochondrial genes of other flowering plants [The mitochondrial gene content was almost the same in the two perennial sunflower species, counting 31–32 protein-coding, three rRNA, 21–22 tRNA genes, and some open reading frames (ORFs). The mtDNA holds most genes that are typical for plant mitogenomes: NADH:ubiquinone oxidoreductase (Complex I) genes , a limited number of differences could be detected . The mosH. grosseserratus mitogenome includes 21 tRNA genes corresponding to 16 amino acids. The same tRNA genes are present in H. strumosus mtDNA, but an additional one (trnA-UGC) allows, in summary, to carry 17 amino acids by the mitogenome of H. strumosus. The in silico prediction of new open reading frames (ORFs) provided a large number of long (more than 300 bp) ORFs in both species. Meanwhile, we annotated only those . Different ORFs—orf284 (H. grosseserratus), orf334 (H. annuus), and orf365 (H. strumosus)—are encoding psaA-like proteins, which have almost identical amino acid sequences at the N-terminus. Chimeric mitochondrial genes, including cox2 fragments, are often linked to male sterility inducing mtDNA of plants [orf188 encoded protein contains 58 amino acids identical to the N-terminus of cytochrome oxidase subunit 2. While the orf316, present in H. annuus and H. strumosus, has 67 amino acids similar to the middle part of the COX2 sequence. The protein encoded by orf188 in H. grosseserratus shares almost the same structure as the cox2-chimeric protein (QFS00065.1), which we identified in ANN2, a CMS line of sunflower in a previous study [orf316 is an ORF common to sunflower mtDNA. Notably, an uncharacterized protein from the cupredoxin superfamily (XP_022040088.1) annotated in the nuclear genome of H. annuus has a sequence equal to the orf316 encoded protein. The largest ORF (orf633) was observed in the H. grosseserratus mitogenome. Most of the orf633 encoded protein (397 aa) is identical to the N-terminus of ATP ATPase subunit 1.Among the discovered ORFs, we observed several interesting patterns. For instance, we discovered the presence of an ORF encoding the chloroplast-like ribosomal protein S11, in both currently studied species and all up-to-date, complete mitochondrial genomes of us study . Accordisdh4 gene, annual and perennial sunflowers share the same protein-coding gene content. The sdh3 and shd4 genes are among the most “unstable” mitochondrial genes, with frequent cases of their transfer to the nucleus, and even related species may have different sets of succinate dehydrogenase genes [H. grosseserratus and H. strumosus, as well as between H. annuus and both perennial species. Only a few SNPs (1–3 per gene) leading to nonsynonymous substitutions occurred in rps4, cob, rpl16, matR, and atp6. Among the detected polymorphisms, only a single transversion (G to C) in the atp6 gene  CMS type, but was absent in PET1 (MG735191.1), PET2 (MG770607.2) CMS lines. Since the CMS lines have cytoplasmic genetic information  initially obtained from wild species [H. petiolaris mitogenomes, and MAX1 as a H. maximiliani mitogenome. This SNP can likely be tracked only in perennial sunflowers, or at least those belonging to the Divaricati section. Sunflower species have unusually diverse karyotypes [Lactuca sativa) mitogenome with two wild lettuce species  revealed identical sequences and rearrangements for L. sativa and L. serriola, but significant differences with the mitogenome of L. saligna [L. serriola is regarded as a wild ancestor of the domesticated lettuce [The mitochondrial genomes of perennial sunflower species are 7–9% smaller than annual sunflower mitogenomes. Meanwhile, except for the se genes . The mittp6 gene  was comm species , PET1 anryotypes  and highryotypes ,33. Withryotypes . Opposit saligna . Such da PET1 MG7191.1, PErps11 gene is excluded from mtDNA in a vast number of eudicots [orf139, encoding a protein that is similar to the chloroplast-like ribosomal protein S11, in the same position (close to cox1 gene) of all up-to-date sunflower mitogenomes, does not seem accidental. The non-coding sequences in plant mitogenomes vary greatly and may rearrange [orf139 has an identical coding sequence in both perennial sunflowers and all up-to-date H. annuus mitogenomes (including CMS lines), we can assume that orf139 plays a vital role in sunflower mitochondria. The functions of the ORFs similar to the psaA gene, are more cryptic. Three ORFs  have a large identical part and have the same position in the mitogenome—between atp1 and ccmFN (much closer to ccmFN). Thus, it is most likely that the chloroplast DNA (cpDNA) insertion entered the mitochondrial genome in a common sunflower ancestor, and then a limited number of mutations resulted in sequence divergence between these ORFs. Notably, cpDNA-derived sequences with partial homology to the psaA gene have been maintained over long periods in the mtDNA of different Brassica species [Many mitochondrial genomes have abounded with foreign DNA acquired by horizontal transfer, especially from plastid genomes . Howevereudicots ,37. The earrange , while tearrange , being uearrange . Since o species .orf188 that we annotated in the H. grosseserratus mitogenome is similar to the previously described cox2-chimeric ORF (QFS00065.1), potentially playing a role in the formation of ANN2 CMS type in sunflower [H. grosseserratus is orf633, which has no analogs, among other described sunflower mitogenomes. The orf633 has a large part of sequence which is identical to the atp1 gene, but its role is unclear. Studies have revealed the association between the CMS phenotype with mutations in atp1 or a decrease in protein abundance [atp1 encoded protein) lacks transmembrane domains, the association between additional rearranged atp1 copy (orf633) and CMS phenotype is equivocal. Mitogenomes of higher plants are enriched with ORFs with an unknown function [Because of high mitochondrial DNA recombination rates, ORFs with partial mitochondrial gene sequences are common in mtDNA . Such chunflower . Neverthbundance . Howeverfunction ,46, whilfunction . Thus, tH. grosseserratus  and H. strumosus . Except for sdh4 and trnA-UGC, both perennial sunflower species had the same gene content and almost identical protein-coding sequences when were compared with each other or with the annual sunflower (H. annuus). Mitochondrial ORFs  common to sunflowers and unique ORFs for H. grosseserratus (orf633) and H. strumosus  were determined. The observed maintenance of plastid-derived DNA coding sequences in the mitogenomes of both annual and perennial sunflowers and the low frequency of nonsynonymous mutations point at an extremely low variability of mtDNA coding sequences in Helianthus genus. The current investigation may be useful for future studies in mitochondrial genomics.The complete master circles of mitogenomes were obtained for


PGC (Progastricsin) gene, is regulated by microRNAs (miRNA), but how PGII levels vary with Helicobacter pylori (H. pylori) infection and miRNAs genotype remains unclear. Methods: Serum levels of PGI and PGII were determined in 80 patients with gastric cancer and persons at risk for gastric cancer , with and without H. pylori infection. As control from the general population, 52 blood donors were added to the analyses. Associations between PGII levels and genetic variants in PGC and miRNA genes in these groups were explored based on H. pylori seropositivity and the risk for gastric cancer. The two-dimensional difference in gel electrophoresis (2D-DIGE) and the NanoString analysis of messenger RNA (mRNAs) from gastric cancer tissue were used to determine the pathways associated with increased PGII levels. Results: PGII levels were significantly higher in patients with gastric cancer, and in those with H. pylori infection, than in other patients or controls. A PGI/PGII ratio ≤ 3 was found better than PGI < 25 ng/mL to identify patients with gastric cancer (15.0% vs. 8.8%). For two genetic variants, namely rs8111742 in miR-Let-7e and rs121224 in miR-365b, there were significant differences in PGII levels between genotype groups among patients with gastric cancer , but not among other study subjects. Moreover, a strict relation between rs9471643 C-allele with H. pylori infection and gastric cancer was underlined. Fold change in gene expression of mRNA isolated from gastric cancer tissue correlated well with polymorphism, H. pylori infection, increased PGII level, and pathway for bacteria cell entry into the host. Conclusions: Serum PGII levels depend in part on an interaction between H. pylori and host miRNA genotypes, which may interfere with the cut-off of PGI/PGII ratio used to identify persons at risk of gastric cancer. Results reported new findings regarding the relation among H. pylori, PGII-related host polymorphism, and genes involved in this interaction in the gastric cancer setting.Background: Pepsinogen (PG) II (PGII) is a serological marker used to estimate the risk of gastric cancer but how PGII expression is regulated is largely unknown. It has been suggested that PGII expression, from the  Helicobacter pylori (H. pylori) infection, GC remains a worrisome disease due to its poor prognosis and the increasing aging of the population [H. pylori causes atrophic gastritis (AG) and intestinal metaplasia (IM), which are preneoplastic conditions to GC according to Correa’s cascade [CDH1 gene are implicated in hereditary diffuse gastric cancer [H. pylori  is the third leading cause of tumor-related death worldwide . Althougpulation ,3. H. py cascade . Another cascade . Finallyc cancer . Surveilc cancer ,8. Seroli , indepei .Information on each patient’s diagnosis was collected. ACAG had been diagnosed both histologically, following the Updated Sydney System  for atrog for 10 min and stored at −80 °C until use. PGI, PGII, and gastrin-17 were measured using an ELISA kit  and all blood samples were collected and process in the morning in fasting patients. The interval for the dosage was obtained by testing 200 blood donors. According to the parametric method, the 95% distribution reference value range is PGI 30–166 ng/mL, PGII 3–15 ng/mL, and G17 2.5–7 pmol/L.Serum was obtained by centrifugation of venous blood at 2600× PGC transcriptional start site, in binding sites for transcription factors [PGC gene by binding to its 3′UTR [PGC gene [Genomic DNA was extracted from whole blood using the EZ1 DNA Blood kit  and genotyped for six genetic variants . These v factors , three Sts 3′UTR , and an PGC gene .SNP genotyping assays were performed using custom TaqMan genotyping assays , allele-specific oligonucleotide probes , and duaIns/del detection was done by amplifying the DNA fragment encompassing the TATA boxes using a fluorochrome (6 FAM)-labeled forward primer and an unlabeled reverse primer  in a reaH. pylori-positive, were analyzed by using the difference in gel electrophoresis (DIGE) of protein expression. Proteins were isolated, labeled with cyanine dyes and separated as reported in one of our previous papers [For the study, the corpus stomach biopsies of 8 patients with GC, 4 cases were s papers . From ths papers . The comTotal RNA from formalin-fixed, paraffin-embedded tissue sections of 13 patients with GC at diagnosis was extracted using an RNeasy DSP FFPE Kit  according to the manufacturer’s protocol. DNase for optimized removal of eventual genomic DNA contamination was used. RNA quality was assessed using the High Sensitivity RNA ScreenTape kit  on the 2200 TapeStation system . RNA quantification was performed using the Qubit™ RNA HS Assay  on the Qubit fluorometer 2.0. instrument . Among these patients, 7 were H. pylori-positive. For each sample, 250 ng of RNA was hybridized with probes of a custom panel . The assH. pylori value, and to determine its sensitivity and specificity.The Shapiro–Wilk test was used to assess the normal distribution of the data. When values were normally distributed, ANOVA was used to compare quantitative variables among groups. The Kruskal–Wallis test was used when the assumption of normality was not confirmed. To test if medians were ordered, the Kruskal–Wallis test was following by the Jonckheere-Terpstra trend test and Conover post-hoc test to assess differences among groups or by Dunn’s test. Chi-square or Fisher’s exact test was used to analyze differences among groups for qualitative data and the chi-square for linear trends was used to assess differences in distributions of categorical variables among independent groups. Univariate and multivariate linear regression was done to identify clinical variables and genotypes associated with serum PGII levels. Receiver operating characteristic (ROC) curve analysis was used to extract a cut-off value PGII levels predictive of a positive IgG to p < 0.0001, chi-square test). More than half of GC cases had H. pylori infection, about one-third of those at risk of GC  were infected, and only 13.5% of controls had the infection.The study used samples and data from blood donors (controls), first-degree relatives of patients with GC (FDR-GC), ACAG patients, patients with high-grade dysplasia, and GC patients, for a total of 270 study subjects . There wH. pylori infection.Among GC patients, 53.7% had a diffuse and 36.2% had an intestinal histological type, and in 71.2% of cases, the disease was located distally . An adva. Regarding PGII (p < 0.001).Compared with PGI levels in controls , median PGI levels were lower in ACAG patients  and in patients with high-grade dysplasia  and higher in FDR-GC  and GC patients  A. Regarding PGII B, the loWe next explored the usefulness of serum marker cut-offs to predict gastric disease or risk of disease . Using aH. pylori IgG seropositivity as the independent variables. Only anti-H. pylori IgG seropositivity was retained as an independent variable associated with serum PGII (p < 0.0001). By receiver operating characteristic (ROC) curve analysis, anti-H. pylori IgG seropositivity was confirmed to be an important variable for serum PGII levels in all study subjects considered together. The area under the ROC curve (AUC) was 0.804  was found for rs9471643, with higher PGII levels in the C/C group. However, after stratification by H. pylori infection on the study group, the rs9471643 genotype did not associate with PGII concentration , although after multiple gene Benjamin correction none of them retained significant value , molecular events in primary and metastatic colorectal carcinoma , and Retinoic acid receptor, and Retinoid X receptor . This analysis identified significant differences in PGII levels in two genotype groups in GC . For rs8H. pylori infection  and rs121224 G-allele (pri-miR-365b) in individuals based on GC disease and nfection B identifanti-H. pylori seropositivity and specific PGII-related polymorphisms.This analysis confirmed the positive trend between an increase in PGII level and increased risk for GC, in particular in subjects having an Identifying people at high risk for GC is a priority to increase the patient’s survival. A reduced PGI/PGII ratio is known to be associated with an increased risk of GC. However, confounding factors of PGII levels may distort the true association of PGII level and risk for GC.H. pylori infection than in those without. Finally, there were significant differences in PGII levels in subgroups of GC patients, and in particular in those with H. pylori infection, according to rs9471643 C -allele and rs81117242 SNPs in miR-Let-7e gene thought to regulate the PGC gene.This study found that patients with GC have higher PGII levels than persons at risk of GC. PGII levels were also significantly higher in GC patients with In our series, a PGI/PGII ratio ≤3 identified more patients with GC than did a cut-off of <25 ng/mL PGI, which better defines atrophy in the corpus. This observation is in line with the highest serum PGII levels being recorded in GC patients compared to individuals at risk for GC  and controls. A high PGII level reduces the PGI/PGII ratio, resulting in more GC patients falling under the PGI/PGII ratio cut-off ≤3. Thus, combining PGII and PGI was the best serological test for GC risk.H. pylori infection in GC cases. A previous in vitro study demonstrated that H. pylori lipopolysaccharide-induced the release of PGII from guinea pig gastric mucosa [H. pylori-induced gastric mucosal damage and risk of GC [H. pylori-induced gastritis, atrophy, metaplasia, dysplasia, and then GC) is the main model of GC development. This model is principally valid for intestinal-type GC, but several studies have found that diffuse-type GC is also associated with H. pylori infection even if it does not follow Correa’s cascade [H. pylori infection is associated with GC of different histological types.High levels of PGII are related to the presence of c mucosa . Other ssk of GC ,34,35,36 cascade ,38,39. OA direct role of PGII in GC development has been hypothesized ,19. PGIIH. pylori infection in gastric diseases and stepwise progression towards GC, and further, they found that infection increased the PG II concentration in the peripheral blood. Another study in a large series of cases reported an increase of GC development in patients with H. pylori infection and high-titer of PG II level, and conversely, a low incidence rate in subjects stratified with low serum levels of both PG II and H. pylori antibodies [Several animal models and epidemiological studies, including a recent study of Yuan et al. , demonsttibodies . OverallThis study found that three SNPs were associated with serum PGII levels.PGC gene has already been reported to play a role in activating the gene’s promoter, leading to an increase in PG II expression and a change in the nuclear transcription factor binding ability (nuclear mobility shift) [The G>C transversion at rs9471643 in the y shift) . The samH. pylori-positivity and patient’s disease , which may explain why a reduction in its expression in a variety of cancers has been associated with poor prognosis and increased epithelial-mesenchymal transition [H. pylori infection than controls [H. pylori infection. These findings indirectly support a role in miR-Let-7e in GC pathogenesis H. pylori-mediated.Another SNP associated with PGII levels was rs8111742 in miR-Let-7e, an endogenous miRNA predicted to bind the 3′-UTR of PGC ,42. Thisansition ,44,45,46ansition . Two stuansition ,49, and controls . Thus, tThe third SNP associated with serum PGII levels was rs121224 in miR-365b. This miRNA suppresses tumor cell growth and invasion of cancers such as breast cancer, hepatocarcinoma, small cell lung cancer, and GC ,52,53,54H. pylori infection. Serum PGII level was higher in GC patients with H. pylori infection than in those without, in all the genotype subgroups. Therefore, the rs121224 polymorphism may regulate PGC expression, and factors directly related to H. pylori infection or produced by GC cells in subjects H. pylori-infected may interfere with miR-365—PGC interactions. Moreover, differentially expressed genes from the rs8111742 and the rs121224 specimen subtypes were both enriched for the entry of bacterium into host cell/cell adhesion molecule binding and positive regulation of DNA binding.We found an association between serum PGII levels and the genotype of rs121224 in miR-365b, limited to GC patients, with high PGII levels in those with the C/C or C/G genotype and low levels in those with G/G. The G/G genotype was previously associated with a better prognosis in GC [H. pylori infection and/or miRNAs that can interact with nuclear DNA to direct localization of genes concomitantly involved in several pathways to control the production of proteins that may be involved also in different cancer types. Thus, a perturbation in miRNAs expression due to genetic modification in pre-miRNA region linking to specific regulatory gene expression may produce the acquisition of new cellular characteristics as a different response to treatment or tumor hypoxia, or directly to PGII expression [In this contest, in recent years in addition to genetic alterations, several cancer cells change in the epigenome have been reported, including methylation gene due to pression ,60.H. pylori infection and atrophy but also polymorphisms in the PGII promoter region, miR-Let-7e, and miR-365b genes. Overall genes-related to polymorphisms in rs9471643, miR-Let-7e, and miR-365b were found associated with the pathways involved in bacteria entry into the host and with genes found associated with increased PGII levels-related to gastric cancer.The PGI/PGII ratio was better than PGI alone to identify patients with GC, due to higher serum levels of PGII in these patients than in the other study groups. Higher serum PGII is not only dependent on H. pylori in combination with PGII-related SNPs in GC carcinogenesis.Further studies are required to elucidate the role of


Cuscuta australis R.Br. and C. chinensis Lam. are known as Cuscutae Semen (CS) and are widely consumed in Northeast Asia; however, the seeds of other species can be misidentified as CS owing to morphological similarities, leading to misuse. In this report, we propose a multilateral strategy combining microscopic techniques with statistical analysis and DNA barcoding using a genus-specific primer to facilitate the identification and authentication of CS. Morphology-based identification using microscopy revealed that the useful diagnostic characteristics included general shape, embryo exudation, hairiness, and testa ornamentation, which were used to develop an effective identification key. In addition, we conducted DNA barcoding-based identification to ensure accurate authentication. A novel DNA barcode primer was produced from the chloroplast rbcL gene by comparative analysis using Cuscuta chloroplast genome sequences, which allowed four Cuscuta species and adulterants to be discriminated completely. Therefore, this investigation overcame the limitations of universal DNA barcodes for Cuscuta species with high variability. We believe that this integrated approach will enable CS to be differentiated from other species, thereby improving its quality control and product safety in medicinal markets.To guarantee the safety and efficacy of herbal medicines, accurate identification and quality evaluation are crucial. The ripe dried seeds of  Medicinal plants and plant-derived medicines are commonly used worldwide in traditional Chinese medicine and are recognized as natural alternatives or supplements to synthetic chemicals from a modern pharmacological perspective ; howeverCuscuta chinensis Lam. (Convolvulaceae) are a herbal medicine known as Cuscutae Semen (CS), or “To-Sa-Ja” in Korean [C. australis R.Br. and C. japonica Choisy as sources of the same herbal medicine [C. chinensis and C. australis as authentic CS, known as “Tu-Si-Zi” in Chinese [According to the Korean Herbal Pharmacopoeia, the dried ripe seeds of n Korean ,16. Howemedicine ,17, whil Chinese ,18,19. T Chinese ; therefoModern pharmacological studies have revealed that CS can act in various parts of the body, including the reproductive ,22 and iPerilla frutescens (L.) Britton var. frutescens and P. frutescens var. crispa (Thunb.) H.Deane  are often found as adulterants in CS due to their morphological similarities [C. australis, C. japonica, and C. pentagona are also found throughout Korea [Unfortunately, seeds with similar morphological traits from southern regions of Korea are misused as CS. For example, seeds from larities ,36,37. Mut Korea ,39.Cuscuta (dodder) species are parasitic vines with reduced or almost absent vegetation and very small floral organs. Due to their peculiar morphology and parasitism, the species have received interest from various fields, including agriculture [iculture , conserviculture , taxonomiculture ,43,44,45iculture ,47,48. HSeeds are commonly used as traditional herbal medicines; however, commercial markets often sell mixed seeds from different species, including adulterants, within a single package, while some authentic medicinal seeds can be difficult to distinguish ,50. TherC. chinensis and congeneric species using DNA barcodes. Together, these data will facilitate the quality control of the valuable medicinal seed, CS.In this study, we aimed to elucidate detailed morphological and micromorphological characteristics of CS and adulterants using microscopic analysis, thereby developing a convenient and effective identification key. In addition, we designed a specific primer to distinguish between Cuscuta, including CS, were ovoid in shape and triangular in cross-sectional shape  and C. japonica having the largest  were on the central to positive side of the PC2 axis  and C. japonica having the largest  ornamentation with isodiametric cell arrangement, a straight and raised anticlinal cell wall, and a concave outer periclinal cell wall (C. japonica seeds displayed type II rugulate (puzzle-like) ornamentation with elongated cell arrangement, a straight and channeled anticlinal cell wall, and a convex outer periclinal cell wall . As the rbcL gene length varied in C. chinensis (1443 bp), C. pentagona (1446 bp), and C. japonica (1497 bp), we aligned their rbcL sequences and found that the region from 190 to 846 bp was relatively variable for species identification, with approximately 89–95% similarity. The flanking region is conserved and hence primers were designed from those regions. Therefore, we selected a 676 bp sequence from this region to design the forward and reverse primers  had good discriminatory efficiency for Cuscuta species.We confirmed the utility of the equences . The CusCuscuta species and two species of P. frutescens, we performed DNA barcode analysis using the nuclear rDNA ITS and rbcL regions. ITS sequences for P. frutescens var. frutescens and var. crispa were downloaded from Genbank . Similarly, the sequence data were obtained for 15 samples among the four Cuscuta species and aligned  parsimony-informative sites in ITS and 102 (15.5%) in rbcL, whereas the ITS region was more variable than the rbcL region . Intra-species variation in the four Cuscuta species was shown 0–3.4% in ITS, but not found in rbcL. Nine ITS haplotypes were detected in C. pentagona and two in C. japonica, indicating ambiguous species identification; however, only one of the four Cuscuta species shared a rbcL haplotype with P. frutescens. Thus, rbcL is more suitable for Cuscuta species identification than ITS, allowing complete discrimination of all four Cuscuta species.To distinguish between the four  aligned . The ITSrbcL sequences of four Cuscuta and one P. frutescens species using the maximum likelihood (ML) and Bayesian inference (BI) methods. All species clustered into monophyletic groups; however, C. australis and C. pentagona were more closely related to the other species  values, whereas the other only contained C. japonica. Phylogenetic analysis using ITS indicated an ambiguous node in both ML and BI trees  in Korea have a very similar external seed morphology [Cuscuta seeds shared the same hilum position and that, although C. australis seeds were somewhat dark and blackish brown, their color varied widely  in all Cuscuta seeds except for C. pentagona when placed into boiling water. The seed coat consists of three layers: the outer epidermis, two different palisade layers, and an inner multi-parenchymal layer. In most Cuscuta seeds, the endosperm surrounds the coiled embryo [C. campestris embryo is only embedded in the endosperm in small regions [C. pentagona may display a different internal structure to other Cuscuta species. Further comparative ultrastructural studies are required to evaluate the seed coat stratification and internal structure of seeds from different Cuscuta species.CS are small seeds that are difficult to distinguish from adulterants using the naked eye; in particular, three congeneric species  are easily distinguishable from the seeds of adulterant Perilla species (type III and glandular hairs on hilum). In addition, the two types of testa ornamentation observed were consistent with subgenus classification [C. australis, C. chinensis, and C. pentagona  and C. japonica , suggesting that testa ornamentation may be stable at the subgenus level. Although seed micromorphology was a useful diagnostic characteristic, C. australis and C. chinensis could not be clearly distinguished and were difficult to differentiate using morphology or statistical analysis. Therefore, multilateral approaches including morphology with statistical analysis and DNA barcoding are required to accurately identify medicinal materials [Our previous study suggested that the morphological characteristics of reproductive organs, particularly testa ornamentation, can be used to distinguish between japonica . Recentljaponica ,52 and tjaponica ,62. In tfication ; C. austaterials .C. chinensis and C. australis [C. australis and C. chinensis have significantly different chemical constituents [Cuscuta species and adulterant Perilla species using novel genus-specific DNA barcode primers . Moreover, phylogenetic analysis yielded a phylogeny that was consistent with previous studies [Since 2010, the definition of CS in the Chinese Pharmacopoeia has changed to include both ustralis ; howevertituents . To imprtituents ,56,57; htituents ,65. As stituents ,67. In t studies ,48.C. chinensis) and three congeneric species  were collected from natural populations in Korea during the fruiting season (July 2016 to May 2019). All samples collected in this study were identified based on their macroscopic morphology and microscopic characteristics by two authors (Sungyu Yang and Jun-Ho Song) using relevant literature [P. frutescens var. frutescens and P. frutescens var. crispa were sampled from voucher specimens. The morphological and micromorphological characteristics of mature seeds from all species were analyzed. To ensure consistency, at least two samples were examined for each species. All seed samples were deposited in the Korean Herbarium of Standard Herbal Resources at the Korea Institute of Oriental Medicine (KIOM), Naju, Korea. Detailed information about the plant materials used in this study is summarized in Authentic species of CS  were measured and subjected to optical observation. Seed length, width, and the length/width ratio were measured using a digital vernier caliper . Shape, cross-sectional shape, color, hilum hair, and embryo exudation were observed using a stereomicroscope and images captured using a digital camera . To observe embryo exudation, seeds were placed into boiling water for 10 min. Seed color was determined according to the Royal Horticultural Society Mini-color Chart .To verify whether four quantitative variables  grouped the species, principal component analysis (PCA) was performed using PC-ORD version 5.31 .To fully dry the samples, the seeds were placed in silica gel desiccators for 10 days to remove any moisture. For micromorphological observation, seeds were directly mounted on aluminum stubs using a double sided adhesive conductive carbon disk . The stubs were coated with gold using a sputter coater  and testa were observed using a low voltage field emission scanning electron microscope  at an accelerating voltage of 5–10 kV and a working distance of 10–13 mm. The terminology for seed micromorphology followed that of Barthlott ,73.TM 2X Taq PCR smart mix 1, Solgent, Daegeon, Korea) with 10 pmol of each primer . The ITS region was amplified using ITS1 (TCC GTA GGT GAA CCT GCG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) primers, as described previously [rbcL region in chloroplast genomes  was detected using mVISTA [Cuscuta samples in a 20 μL PCR mixture with 10 pmol of CrbcL primers using a Pro Flex PCR system  with the following parameters: initial denaturation at 95 °C for 2 min; 35 cycles at 95 °C for 50 s, 60 °C for 50 s, and 72 °C for 50 s; final extension at 72 °C for 5 min. The PCR products were separated on a 2% agarose gel for 40 min at 150 V. Each PCR product was isolated using a gel extraction kit (Qiagen), subcloned into a pGEM-T Easy vector , and sequenced using a DNA sequence analyzer . The newly sequenced chloroplast genome sequences in this study were deposited in the NCBI GenBank database under the accession numbers MT982734-MT982740, MT982754-MT982757, MT982776-MT982784, MT982792-982799, MT984385-MT984395, and MT998850-MT998863.DNA was extracted from all seed samples using a DNeasy Plant Mini kit  according to the manufacturer’s instructions. Genomic DNA (20 ng) was amplified in a 20-µL PCR mixture (Solgeviously . The conrbcL sequences of 15 samples from four Cuscuta species, eight Perilla ITS sequences, and seven rbcL sequences from Genbank were obtained and aligned using Multiple Alignment using Fast Fourier Transform. Gaps in the alignment were stripped using BioEdit [The newly sequenced ITS and  BioEdit . DNA SP  BioEdit  was used BioEdit . The GTR BioEdit  and bran BioEdit  with theCuscuta species and two Perilla adulterants species using novel genus-specific DNA barcode primers. Therefore, our morphological and molecular data should allow the accurate identification and quality control of CS.To more accurately identify and authenticate herbal medicines, a multilateral strategy combining macroscopic and microscopic techniques with statistical analysis and DNA barcoding is essential. This is the first comprehensive and integrated study to accurately identify medicinal CS and adulterant seeds. Morphology- and micromorphology-based analyses revealed that general shape, embryo exudation, hairiness, and testa ornamentation are useful characteristics for identifying and authenticating the studied species. Furthermore, we were able to completely discriminate between four


Salmonella enterica causes approximately 1.35 million cases of food borne illnesses annually in the United States. Of these salmonellosis cases, almost half are derived from the consumption of fresh, raw produce. Although epiphytic S. enterica populations naturally decline in the phyllosphere, a subset of phytophagous insects have recently been identified as biological multipliers, consequently facilitating the growth of bacterial populations. We investigated whether tomato leaves with macroscopic feeding damage, caused by infestation of adult Western flower thrips , support higher S. enterica populations. To explore this hypothesis, we assessed S. enterica populations in response to thrips feeding by varying insect density, plant age, and the gender of the insect. As a reference control, direct leaf damage analogous to thrips feeding was also evaluated using directed, hydraulic pressure. In a supplementary set series of experiments, groups of F. occidentalis infested tomato plants were later inoculated with S. enterica to determine how prior insect infestation might influence bacterial survival and persistence. Following an infestation period, leaves visibly damaged by adult F. occidentalis supported significantly higher S. enterica populations and resulted in greater amounts of electrolyte leakage  than leaves lacking visible feeding damage. Plant age did not significantly influence S. enterica populations or estimates of electrolyte leakage, independent of initial infestation. Additionally, the gender of the insect did not uniquely influence S. enterica population dynamics. Finally, applications of aggressive water bombardment resulted in more electrolyte leakage than leaves damaged by F. occidentalis, yet supported comparable S. enterica populations. Together, this study indicates that F. occidentalis feeding is one of the many potential biological mechanisms creating a more habitable environment for S. enterica.The human enteric bacterial pathogen  Salmonella enterica is estimated to cause 1.35 million cases of food-borne illnesses annually . While it is generally perceived that cases of salmonellosis are acquired from consumption of S. enterica-contaminated animal products, cross-contamination of fresh produce has become an overwhelming risk throughout the cultivation process, subsequently resulting in a growing rate of produce-associated salmonellosis over the past decade [S. enterica is responsible for the majority of bacterial foodborne illness and, unlike other bacterial foodborne pathogens, the incidence of outbreaks has not diminished over the last decade. The switch in vehicles from contaminated animal products to fresh produce is posited as the leading reason for the ongoing salmonellosis outbreak occurrence. Identifying the biotic mechanisms that allow enteric human pathogens, such as S. enterica, to persist or grow on fresh produce pre-harvest is the first step in reducing the disease burden and increasing food safety.In the United States alone, the human enteric bacterial pathogen S. enterica [S. enterica populations have been observed to naturally decline on healthy leaves [S. enterica populations must persist or find advantageous niches or partnerships on plants in the field pre-harvest, because salmonellosis outbreaks from consumption of raw produce continue to occur regularly. In addition to agricultural practices acting as bacterial reservoirs [S. enterica given their unique behaviors as they facilitate the dispersal and enhance the persistence of the bacterium [S. enterica, thus promoting proliferation and dispersal of bacteria within poultry associated environments [Macrosteles quadrilineatus) and the Green peach aphid (Myzus persicae) are associated with mechanical transmission of S. enterica to uncontaminated lettuce leaves [S. enterica and disperse the pathogen in honeydew [S. enterica-contaminated plants led to a prolonged persistence of the bacteria when compared to plants without insects [S. enterica persistence by providing a more direct route of entry to the apoplast of plant leaves, protected leaf interior, and/or to damaged cells leaking nutrients.Harsh environmental conditions, such as direct UV radiation or lack of available nutrients, make the phyllosphere of plants an adverse environment for enterica . In facty leaves –6. In spservoirs , insectsacterium . Multiplronments , 10. To e leaves ; intereshoneydew . In addi insects , 11. WhiFrankliniella occidentalis Pergande (Thysanoptera: Thripidae) are considered a highly polyphagous insect species and employ a modified form of piercing-sucking feeding, whereby the stylets do not reach the vascular system of plants to withdraw vascular constituents [F. occidentalis feed by initially puncturing surface mesophyll cells and then sucking out cellular contents. In this study, we tested the hypothesis that areas of leaves with macroscopic feeding damage caused by adult F. occidentalis support greater S. enterica populations than undamaged areas. Additionally, we investigated whether insect containment or open infestation of F. occidentalis would differentially affect the magnitude of leaf damage and S. enterica survival. We also postulated that the gender of F. occidentalis could influence the survivorship of S. enterica¸ similar to the results observed with transmission of Tomato spotted wilt tospovirus [TSWV; F. occidentalis possessed higher transmission efficiencies compared to females. The findings herein elucidate whether insect feeding behaviors, namely feeding damage, locally promotes the persistence of S. enterica populations.Western flower thrips, tituents . Insteadus TSWV; , 14, wheS. enterica serovar Typhimurium 14028s used in this study was grown in lysogeny broth  at 37°C with shaking at 200 rpm.A kanamycin (Kan) resistant strain of F. occidentalis were maintained on green bean pods (Phaseolus vulgaris), where the insects utilize the pods as both an ovipositional substrate and a food source. A starter colony of F. occidentalis was provided by Dr. Thomas L. German, Professor Emeritus, University of Wisconsin, Madison, from a long-term colony maintained in their laboratory. Weekly, green bean pods were sterilized in a 5% Clorox solution for 15 min and rinsed twice with sterile water to remove potential pesticide residue prior to placement in rearing containers. Populations of F. occidentalis were maintained in 0.4 L plastic containers  and held under a constant temperature (27°C) and a 16:8 (Light:Dark) photoperiod. Each container included a sheet of filter paper to prevent moisture from accumulating. Voucher specimens of adult female and male F. occidentalis, obtained from the original colony, have been deposited in the Wisconsin Insect Research Collection, University of Wisconsin-Madison (http://labs.russell.wisc.edu/wirc/).Colonies of F. occidentalis infestation density would alter S. enterica populations and electrolyte leakage, a no choice arena experiment was performed. Solanum lycopersicum  seedlings were cultivated using Professional Growing Mix (Sunshine Redi-earth) in 6” pots held in a growth room maintained at a 16:8 (L:D) photoperiod and 24°C light and 19°C dark conditions for three and five weeks. In a 2X2 factorial design, replicate sets of adult F. occidentalis were transferred onto six separate three and five-week-old tomato plants, at densities of 5 (low density) or 20 (high density) individuals per plant. Each plant was contained in a 15.5 cm (ht) and 10 cm (diameter) Plexiglas tube, fashioned with thrips-proof screening  at one end of the tube to prevent insects from escaping the experimental arena, and infested cages were held at 26°C temperature with a 16:8 (L:D) photoperiod. Seventy-two hours after the initial release of insects into cages, all adult F. occidentalis were removed, and the number of feeding lesion sites were visually assessed and counted. Subsequently, each plant was dip-inoculated in 450 ml of sterile water or a 108 CFU/ml suspension of S. enterica  for one minute. For bacterial inoculum preparation, lysogeny broth was inoculated with bacteria from -80°C freezer stocks and incubated, shaking overnight at 37°C. Bacterial cultures were normalized to an optical density at 600 nm of 0.2 in sterile water. Inoculum populations were verified by enumerating populations following serial dilution, plating on LB-Kan agar (50 μg/ml), and growth overnight at 37°C. Dip-inoculated plants were then placed in a clear, uncovered plastic bin at 27°C temperature under a 16:8 (L:D) photoperiod.To investigate whether plant age together with S. enterica populations, plants were sub-sampled 72 hours after dip-inoculation to determine whether bacterial populations were localized to damaged sites on leaves. We chose to examine S. enterica population dynamics 72 hours post inoculation, as bacteria begin to diverge in population size within the presence or absence of insect infestation upon tomato leaves at this point [To assess is point . SpecifiTo further characterize the magnitude of cellular or leaf damage associated with 72-hour thrips infestation, measurements of electrical conductivity were obtained from a comparable set of three, 10 mm-diameter leaf discs with macroscopic feeding damage, and three visibly undamaged leaf discs from the same leaf on each of three plants. Each group of three leaf discs were individually placed in single wells of a 12-well tissue culture plate containing 4 ml of sterile water. Plates were positioned on a rotating table at 50 rpm for approximately 30 min, acting as a wash step to prevent remnant soil particles from influencing conductivity measurements . SubsequF. occidentalis would similarly influence electrical conductivity estimates and S. enterica populations compared with results observed in the no choice experiments. Briefly, adult populations  of F. occidentalis were released onto tomato seedlings for infestation periods of either three, four, or five weeks, resulting in plants infested with various life stages of the insects and obvious sites with feeding damage (silvering). This release of F. occidentalis onto plants allowed insects to actively move and thereby feed wherever they chose, hence the name ‘free choice experiment’. After the initial free-choice infestation period, 24 plants were randomly selected and removed from the experimental arena at one of the three time points post infestation and were dip inoculated into either a 450 ml aliquot of sterile water, or a 108 CFU/ml suspension of S. enterica (each beaker containing 75 μl of Sil-Wet) for one minute. Bacterial cultures were prepared as described above. Replicate sets of 10 mm-diameter leaf discs were extracted 3 days after dip inoculation from separate leaves on each of 24 biological replicates per time point following infestation. Electrical conductance and S. enterica populations were assessed as described previously.An additional, free choice experiment was performed to assess if S. enterica population dynamics, adult populations of male or female F. occidentalis were provided access to separate plants. Six, 2 cm diameter, thrips-proof, Plexiglas clip cages were attached to the underside of three, middle S. lycopersicum leaflets on opposing leaves. Three clip cages were infested with three individual male or female F. occidentalis, whereas the remaining three clip cages remained empty. Three days post infestation, insects and clip cages were removed, and each plant was subsequently dip inoculated into either a 450 ml aliquot of sterile water, or a 108 CFU/ml suspension of S. enterica (each beaker containing 75 μl of Sil-Wet) for one minute. Bacterial cultures were prepared as described above. Replicate sets of 10 mm-diameter leaf discs were extracted 3 days after dip inoculation from separate leaves on each of the 4 individual plants following infestation by male or female thrips, and plants which remained absent of infestation. Electrical conductance and S. enterica populations were assessed as described previously. Experiments were performed with three biological replicates.To determine whether insect gender influences F. occidentalis) and abiotic cellular damage and resulting S. enterica populations, water pressure was used to inflict direct, physical (abiotic) damage to S. lycopersicum leaves, and served as a reference control. Three leaflets, from five-week-old plants randomly chosen for infestation, were faceted with three clip cages and infested with three adult F. occidentalis, whereas the remaining three clip cages on the opposite leaf remained empty on each plant. Additionally, six empty clip cages were applied to six leaflets between two opposing leaves for plants used as a control. At 72 hours after infestation, an inserted color cup was attached to a single action, siphon feed airbrush set  and filled with sterile water, and a new set of five-week-old plants were randomly selected to receive short duration (5 sec) pulses of water at different pressures of 0.35, 1.41, or 3.78 kg cm-2 and directed to the underside of three middle leaflets. After inducing pressurized water damage, insects and clip cages were removed, and each plant was subsequently dip inoculated into either a 450 ml aliquot of sterile water, or a 108 CFU/ml suspension of S. enterica (each beaker containing 75 μl of Sil-Wet) for one minute. Bacterial cultures were prepared as described above. Replicate sets of 10 mm-diameter leaf discs were extracted three days after dip inoculation from separate leaves on each of the 4 plants per treatment group following infestation. Electrical conductance and S. enterica populations were assessed as described previously. Experiments were performed with three biological replicates. To examine cell membrane viability of areas damaged by F. occidentalis or water pressure application, whole leaves were extracted 72 hours after imposed damage (as previously described) and immediately submerged in 10 mL of 0.25% Evans blue dye and incubated on a rotating table at 80 rpm for 20 minutes [To better understand the interaction between biotic , subjected to different F. occidentalis infestation densities (5 or 20 adult thrips), subjected to male or female infestation, and when damaged by F. occidentalis or water pressure. Student’s t-tests were performed to determine if feeding damage prompted a difference in electrical conductance of S. enterica populations when compared to undamaged plant tissues regardless of their treatment. To analyze the same response, student’s t-tests were also applied to the free-choice experiment. Outliers within each experiment were kept.A one-way, analysis of variance (ANOVA) was used to assess if S. enterica were directly influenced by localized macroscopic damage sites from insect feeding, bacterial population dynamics were assessed on damaged and macroscopically undamaged tomato leaves. In a controlled, no-choice environment, tomato plants from two age groups (3 or 5 weeks-old) were exposed to low (5 insects/cage) or high (20 insects/cage) densities of F. occidentalis to investigate if plant age and insect density influenced S. enterica populations. Within each treatment group, S. enterica populations were 1 log higher on macroscopically damaged leaf discs had twice as many macroscopic damaged areas when compared with plants exposed to lower infestation densities.  between macroscopically damaged leaves when assessing bacterial populations and electrolyte leakage  and higher S. enterica populations (P < 0.0001), when compared with visibly undamaged tissues on infested plants, or plants entirely absent of insects  or higher (3.78 kg cm-2) water pressure, together with leaves possessing F. occidentalis feeding sites, exhibited S. enterica populations more than a log greater than leaves damaged by low water pressure (0.35 kg cm-2) or those without any form of visible damage  resulted in significantly greater electrolyte leakage than undamaged leaves , indicating that the aggressive bombardment of water resulted in a greater amount of cellular damage in comparison to thrips feeding , but resulted in equivalent bacterial populations.Cellular damage induced by insect feeding was hypothesized to be a factor that could enhance  feeding . SurprisS. enterica is traditionally studied in the context of animal hosts, the number of salmonellosis cases attributed to consumption of fresh produce warrants an assessment of environmental factors and how they promote bacterial food-borne outbreaks. On healthy plants, S. enterica lacks the necessary mechanisms to maintain phyllosphere populations on its own [Pseudomonas syringae for instance, produces biosurfactants that increase the rate of diffusion of water across plant cell cuticles subsequently prompting a release of foliar nutrients and making them successful, primary phyllosphere colonists [S. enterica’s inability to persist on its own, ubiquitous environmental stressors such as the presence of insects on plants, has previously been demonstrated to enhance S. enterica survival [S. enterica from tomato fruit a month after initial S. enterica foliar inoculation. This affirms the relevance of assessing S. enterica populations upon tomato leaves in relation to food borne outbreak risks especially in the context of foliar feeding insects [S. enterica, F. occidentalis, and tomato plants, specifically investigating the effects of cellular damage induced by F. occidentalis on S. enterica populations, and further evaluating the influence of insect gender, plant age, and infestation density on bacterial populations.Although  its own , most liolonists . Despitesurvival , 11, and insects . In the S. enterica population dynamics are influenced by insect feeding damage on tomato leaves  or minimizing the impact on plant fitness respectively –33. EachS. enterica population. To better understand this interaction, we imposed varying levels of abiotic physical damage to emulate thrips feeding damage in isolation of other biotic interactions. Results from these experiments indicated that although an aggressive bombardment of water resulted in significantly greater cellular damage beyond that which we observed from F. occidentalis  and greater electrolyte leakage (bottom), regardless of plant age or initial F. occidentalis infestation density. Damaged and undamaged leaf discs were extracted from each three or five-week-old plant with high (20 thrips/cage) or low (5 thrips/cage) infestation densities. Measures of electrical conductance for damaged and undamaged leaf discs were used to evaluate the extent of electrolyte leakage over a six-hour period. Boxplots with different letters within each treatment group indicate a significant difference (P < 0.05), as determined by a student’s t-test. Singular dots represent outlier points.In a no-choice experimental arena, damaged leaf tissue exhibited higher (TIF)Click here for additional data file.S2 FigF. occidentalis feeding for 72 hours, or an application of low (0.35 kg cm-2), medium (1.41 kg cm-2), or high (3.78 kg cm-2) water bombardment for five seconds. The blue dot on the drawn leaflet was the location where water pressure or contained thrips damage was applied. Whole leaves were extracted after imposed damage, and immediately dyed to visualize cell membrane viability.Five-week-old tomato leaflets were subjected to (TIF)Click here for additional data file.


Gekko and Hemidactylus. Here, we used RADseq to identify sex-specific markers in two species of Burmese bent-toed geckos. We uncovered XX/XY sex chromosomes in Cyrtodactylus chaunghanakwaensis and ZZ/ZW sex chromosomes in Cyrtodactylus pharbaungensis. This is the third instance of intrageneric turnover of sex chromosomes in geckos. Additionally, Cyrtodactylus are closely related to another genus with intrageneric turnover, Hemidactylus. Together, these data suggest that sex chromosome turnover may be common in this clade, setting them apart as exceptionally diverse in a group already known for diverse sex determination systems.Lizards and snakes (squamates) are known for their varied sex determining systems, and gecko lizards are especially diverse, having evolved sex chromosomes independently multiple times. While sex chromosomes frequently turnover among gecko genera, intrageneric turnovers are known only from  Hemidactylus and Gekko include species with both XX/XY and ZZ/ZW sex chromosomes [Squamate reptiles (lizards and snakes) have evolved a number of different sex-determining systems ,5,6,7,8,omosomes ,10,11. TCyrtodactylus) are the most species rich gecko genus with >300 described species distributed throughout South and Southeast Asia and Oceania [Cyrtodactylus pubisulcus, whose ZZ/ZW sex chromosomes were identified through traditional cytogenetics [Cyrtodactylus karyotypes have failed to reveal heteromorphic, or visually distinct, sex chromosomes [Cyrtodactylus sex chromosomes is a worthwhile endeavor because Cyrtodactylus is closely related to the aforementioned Hemidactylus, a genus with both XX/XY and ZZ/ZW species [Hemidactylus may extend to Cyrtodactylus.Bent-toed geckos , we foubisulcus , these rCyrtodactylus chaunghanakwaensis [C. pharbaungensis [C. pharbaungensis and ten females and nine males of C. chaunghanakwaensis using the QIAGEN DNeasy Blood and Tissue Kit. We prepared RADseq libraries using a modified protocol from Etter et al. [Sbf1 restriction enzyme , and ligated individually barcoded P1 adapters to each sample. We pooled samples into multiple libraries, sonicated, and size selected for 200–500 bp fragments using magnetic beads in a PEG/NaCl buffer [kwaensis  and C. pungensis  are rangr et al. ,24. Briel buffer . We thenWe analyzed the RADseq data using a previously described bioinformatic pipeline . We demuC. pharbaungensis using nine females and ten males and ten markers for C. chaunghanakwaensis using ten females and nine males. After PCR, markers were visualized on a 1% agarose gel to determine whether they amplified in a sex-specific pattern.We validated a subset of the “confirmed” sex-specific markers with PCR. DNA primers  were desHemidactylus turcicus transcriptome [H. turcicus transcripts to genes in the chicken  genome [C. pharbaungensis and C. chaunghanakwaensis have sex chromosome systems syntenic with each other or other amniote species. Alternatively, if each species has a different sex chromosome linkage group, then we can conclude they have different sex chromosomes, even if there is no difference in heterogamety.We queried sex-specified RAD markers against an assembled criptome  using BLcriptome  in Genei) genome ,33, as i) genome ,37,38,39Hemidactylus and Cyrtodactylus species to combine our new results with sex chromosome data from the literature. We accessed ND2 and adjacent tRNAs for 239 Cyrtodactylus species, 61 Hemidactylus species, and 11 outgroup species from GenBank . Export and collection permits were issued by Mr. Win Naing Thaw of the Ministry of Natural Resources and Environmental Conservation Forest Department.C. pharbaungensis included eleven males and four females—barcode errors resulted in several of the female samples not being used for subsequent analysis. We identified 153,555 RAD loci with two or fewer alleles. Of these, 652 markers were female-specific and none were male-specific. We checked the sex-specific markers against the reads from the opposite sex and retained 646 “confirmed” female-specific RAD markers. We designed PCR primers for ten markers and validated two as sex-specific , these genera form an ideal clade for studying the evolutionary drivers of rapid sex chromosome turnover and the early evolution of sex chromosomes.Because the vast majority of  studied A there iery year ,56. HoweC. pharbaungensis is the first amniote shown to have sex chromosomes syntenic with chicken chromosome ten. Synteny information is vital for identifying sex chromosome turnovers, particularly cis-turnovers where a novel sex chromosome system replaces an ancestral system with the same heterogamety, for example, a novel ZZ/ZW system replacing the ancestral ZZ/ZW system or a novel XX/XY system replacing the ancestral XX/XY system. Since both C. pharbaungensis and C. pubisulcus have female heterogamety synteny data could allow us to distinguish whether these are the same or different ZZ/ZW systems. Unfortunately, karyotype data used to identify the heteromorphic sex chromosomes of C. pubisulcus do not provide any synteny information [C. pharbaungensis and C. pubisulcus do not share homologous sex chromosomes, then all three Cyrtodactylus species studied thus far have independently derived sex chromosome systems. Alternatively, if C. pharbaungensis and C. pubisulcus have the same sex linkage group, then at least one turnover occurred in Cyrtodactylus, in which an ancestral ZZ/ZW system was maintained in both C. pharbaungensis and C. pubisulcus but transitioned to an XX/XY system in C. chaunghanakwaensis.Sex chromosomes evolve from autosomes and some autosomes may be more likely to make this change than others ,57. Howeormation . If futuCyrtodactylus RAD markers directly to chicken did not yield useful results, but an alternative approach using the H. turcicus transcriptome as an intermediate step was successful in C. pharbaungensis. Using one or more intermediate steps for BLAST searches has been used previously to identify the sex chromosome linkage group in a frog species with limited genomic resources. In that study, Rana arvalis sex-linked markers were iteratively mapped to a Rana temporaria draft genome. Those R. temporaria fragments were mapped to the Nanorana parkeri genome. Finally, the matching N. parkeri scaffolds were mapped to the highly contiguous Xenopus tropicalis genome [Mapping sex-specific s genome . The incC. chaunghanakwaensis and the ZZ/ZW sex chromosomes of C. pharbaungensis. These results contribute to the ever-increasing diversity of gecko sex chromosome systems. Although squamate lizards are known for a diversity of sex chromosome systems, they tend to have conserved systems within suborders or families, including scincids [Here, we uncovered the XX/XY sex chromosomes of scincids , lacertiscincids , varanidscincids ,62, pleuscincids  (but seescincids ), and cascincids ,64. In c


Although sturgeon production by aquaculture has increased worldwide, a major factor limiting its expansion are infectious diseases, although few data about viral diseases are available however. This review provides a rapid overview of viral agents detected and described to date. Following a general introduction on viral diseases are four sections arranged by virus classification: sturgeon nucleocytoplasmic large DNA viruses, herpesviruses, white sturgeon adenovirus 1, and other viruses. Molecular diagnosis is currently the best tool to detect viral diseases, since cell culture isolation is not yet applicable for the detection of most sturgeon viruses. Acipenseridae family is linked chiefly to caviar production [Sturgeon are known to be more resistant than other species to certain diseases in nature since they are anadromous species . The ecooduction . Over thoduction ,4. The ioduction . Breedinoduction ,7,8, butoduction ,10, viroAcipenseridae have also been reported. Nonetheless, large gaps in viral biology and epidemiology remain. This review presents an overview of viral agents found in sturgeon and described in the literature. The life cycle of viruses is beyond the scope of this review. The aim is to provide a concise summary of the current knowledge on viral diseases and diagnostic analytical techniques.Intensive sturgeon farming is a relatively recent development, as is the study of viral diseases in sturgeon. Several viral agents are known to infect sturgeon  and caseIridoviridae family; however, only frog virus 3 (FV3) is officially recognized as a member of Iridoviridae [Mimiviridae [Mimiviridae, they will be first viruses in this family capable of infecting vertebrate organisms [Sturgeon Nucleocytoplasmic Large DNA Viruses (sNCLDV) represent the most numerous and heterogeneous group of viral agents causing disease in sturgeon. The current nomenclature can lead to error in taxonomy . Most vioviridae . The othiviridae . This farganisms .Acipenseridae species and for intensive aquaculture [Acipenser transmontanus) juveniles from several northern California farms were reported [-The different sizes of sturgeon positive for the virus suggest a possible vertical transmission and adults as WSIV carriers ,46,47;-Acipenseridae family can also be susceptible  [Not only white sturgeon, but other species belonging to the vescens) ;-Intensive breeding conditions  are predisposing factors for disease onset ;-WSIV is considered an endemism of several American basins , which also poses a problem for stocking programs ,14,19.White sturgeon iridovirus (WSIV) is currently the sNCLDV for which more information is available; it poses a considerable problem for the conservation of endangered aculture . The ICTreported . Diagnosreported . Other mreported ,13,19. WA. ruthenus, A. baerii, A. gueldenstaedtii, A. oxyrinchus, A. sturio) and hybrids sampled between 2010 and 2014. In addition, positive cases were reported for Europe, signaling an expansion of the distribution area of WSIV [With regard to the host spectrum, a recent work highlighted WSIV DNA in various sturgeon species  and the shovelnose sturgeon (S. platorynchus) [Missouri River sturgeon iridovirus (MRSIV) is closely related to WSIV and is uniquely associated with two endemic rynchus) . The virrynchus) . Althougrynchus)  and vulnrynchus)  species,Outbreaks in hatchery-reared juveniles of both species occurred in 1999. Symptoms included lethargy, emaciation and skin and fins inflammation; the mortality rate of several shovelnose sturgeon batches was 100%. Although virions have been detected by electron microscopy in epithelial cells, MRSIV has not been isolated on cell monolayer . Rapid vA. brevirostrum). The only published report in the literature is by La Patra and co-authors [Shortnose Sturgeon Virus (SNSV) is another pathogen affecting shortnose sturgeon (-authors  and desc-authors .A. transmontanus) bred in British Columbia, Canada. The sturgeon (8–10 cm length) presented chronic inappetence, lethargy and lack of reactivity; mortality was reported, although the rate was not specified. Diagnosis was based on electron microscopy [The one report concerning British Columbia white sturgeon virus (BCWSV) dates from 2001 in white sturgeon (croscopy . Availabcroscopy .A. fulvescens) that occurred in Manitoba, Canada, in 2009 and 2010 [Namao virus (NV) has been recognized as the causative agent of a mortality outbreak involving hatchery reared juvenile’s lake sturgeon  in Northern Europe dates from the 1990s [A. gueldenstaedtii, A. baerii, A. naccarii, Huso huso) [Acipenseridae. Moreover, discrepancies can also be found in a single species , in size and symptoms: anorexia, lethargy and erratic swimming. Externally, skin ulcers and gills with abundant mucus can be noted, but no specific clinical signs. The virosis appears to affect juveniles more severely and the mortality rate is extremely variable (from 30% to 100%). Moreover, co-infection with opportunistic pathogens or environmental bacteria  can occur [A. stellatus, A. ruthenus) [Research into sNCLDV has increased only recently in Europe. Pending official nomenclature, the causative agent of mortality events in European sturgeon has been named acipenser iridovirus-European (AcIV-E) . An inithe 1990s ; the fouso huso) ,22. Ciulso huso) ; despiteso huso) . Althougan occur ,22. Despan occur ,56,57. Nan occur  and otheuthenus) .Unlike most cases described by American authors, AcIV-E was diagnosed primarily with PCR assays. Histological analysis was also performed, while it was not possible to isolate the virus on cell monolayers using several cell lines  ,22.Mimiviridae. The exception is Frog Virus 3 (FV3), the only member of the Iridoviridae family currently known to cause infection in sturgeon. FV3 is officially recognized by the ICTV as the type species of the genus Ranavirus  [Gasterosteus aculeatus) has also been reported [Acipenseridae family referred to a mortality outbreak in pallid sturgeon juveniles  reared in Missouri (USA). In this epizootic event the cumulative mortality was 95%. The causative agent was identified as FV3 based on histology, electron microscopy and biomolecular assays [Several sNCLDV are named “Iridovirus”, although they are actually more similar to virinae) . Sturgeoreported . The casr assays . No otheAlloherpesviridae family, are known fish pathogens [Acipenseridae have been described. Only one of them is officially recognized by the ICTV: acipenserid herpesvirus 2 (AciHV-2), which is included in the genus Ictalurivirus [Alloherpesviridae have also been reported [Herpesviruses, specifically the species belonging to the athogens ,60,61. Turivirus . Non-stureported ,35.A. transmontanus) juveniles [Acipenserid herpesvirus 1 (AciHV-1) was the first herpesvirus to be identified in sturgeon. The first case was reported in 1989 in California-bred white sturgeon  [A. transmontanus) ovarian fluid: the fish were farmed in California and showed no clinical signs [A. brevirostrum, A. fulvescens) were reported [S. albus) and shovelnose sturgeon (S. platorynchus) [A. baerii) juveniles occurred in Russia. The same causative agent, called Siberian sturgeon herpesvirus (SbSHV), also affected Bester juveniles (A. ruthenus × H. huso) and Siberian sturgeon adults [Acipenserid herpesvirus 2 (AciHV-2) was isolated few years after sturgeon herpesvirus AciHV-1 was detected. AciHV-2 is currently the only sturgeon herpesvirus officially named by the ICTV and added to the virales) . AciHV-2al signs . The viral signs . Subsequreported ,28,29,31rynchus) . AciHV-2n adults . Experimn adults ,63, whicAciHV-2 diagnosis has been carried out on several cell lines  that are useful for the isolation and observation of cytopathic effects  ,33. CellAcipenseridae. The two documented cases concern Atlantic sturgeon (A. oxyrhynchus), Russian sturgeon (A. gueldenstaedtii) [A. ruthenus x H. huso) [Although not specifically a pathogen of sturgeon, Cyprinid Herpesvirus-3 (CyHV-3) or Koi Herpesvirus (KHV) has been reported in taedtii)  and a BeH. huso) . Since oH. huso) . These rGadus morhua, Limanda limanda, Pagrus major, Anguilla japonica) [Although adenoviruses don’t seem to be a problem for sturgeon farming, they have been identified only in sturgeon. Adeno-like particles have, in fact, been seen with electron microscopy in different species (aponica) ,68,69,70aponica) .A. transmontanus) juveniles from California [Ichtadenovirus, of which WSAdV-1 is the type species [The first report dates from 1984 and describes adeno-like particles observed in 0.5 g farmed white sturgeon  was susceptible to IHN infection producing neutralizing antibodies [Infectious hematopoietic necrosis (IHN) is caused by a  disease . A studytibodies . The OIEtibodies . Nonethetibodies .Rhabdoviridae family. Vicenova and co-authors reported a case of SVCV in Siberian sturgeon (A. baerii) from a Czech farm [Another disease is spring viremia of carp (SVC), caused by a virus of the ech farm . The stuA. gueldenstaedtii) was reported in Greece [Among the riboviruses, betanodavirus is the causative agent of viral nervous necrosis (VNN) or viral encephaloretinopathy. The virus is a known fish pathogen, particularly in marine species . A case n Greece . The stuA. baerii), papova-like virus infection in white sturgeon (A. transmontanus) and a reovirus of the Aquareoviridae family in Chinese sturgeon (A. sinensis) have been reported [Acipenseridae reported in China.Finally, infectious pancreatic necrosis (IPN) in Siberian sturgeon  . In suspIn conclusion, to obtain information on viral diseases in sturgeon, the first step is the diagnostic process implementation. The development of sturgeon cell lines may be useful for the isolation of many viruses as possible. If isolation is not possible, specific PCRs are the most efficient tool for establishing a rapid diagnosis. To achieve this, close collaboration between breeders and researchers is needed. The study of sturgeon viruses must find ways of preventing and treating these diseases, in order to reduce farming losses and not affect stocking programs.


Glycosyltransferases (GTs), a large class of carbohydrate-active enzymes, adds glycosyl moieties to various substrates to generate multiple bioactive compounds, including natural products with pharmaceutical or agrochemical values. Here, we first collected comprehensive information on GTs, including amino acid sequences, coding region sequences, available tertiary structures, protein classification families, catalytic reactions and metabolic pathways. Then, we developed sequence search and molecular docking processes for GTs, resulting in a GTs database (GTDB). In the present study, 520 179 GTs from approximately 21 647 species that involved in 394 kinds of different reactions were deposited in GTDB. GTDB has the following useful features: (i) text search is provided for retrieving the complete details of a query by combining multiple identifiers and data sources; (ii) a convenient browser allows users to browse data by different classifications and download data in batches; (iii) BLAST is offered for searching against pre-defined sequences, which can facilitate the annotation of the biological functions of query GTs; and lastly, (iv) GTdock using AutoDock Vina performs docking simulations of several GTs with the same single acceptor and displays the results based on 3Dmol.js allowing easy view of models. Arabidopsis thaliana, AtGLCAT14A can modify beta-1,6-linked galactan and beta-1,3-linked galactan present in type II arabinogalactan (Glycosyltransferases (GTs) are an important group of enzymes that can catalyze the transfer of activated sugar residues onto a wide range of carbohydrates or non-carbohydrate acceptors, generating a remarkable amount of structural diversity in biological systems . These egalactan . In humagalactan . At presgalactan , but fungalactan . It appegalactan .http://www.cazy.org) depicts families of enzymes that degrade, modify or create glycosidic bonds, including GTs, glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, auxiliary activities and carbohydrate-binding modules (https://www.kegg.jp/kegg/glycan/) (https://www.plantcyc.org) (http://bcb.unl.edu/dbCAN_seq/index.php)  (http://csdb.glycoscience.ru/gt.html)  ( modules . The CAZglycan/) , a resoucyc.org) , dbCAN-sdex.php) , the Ricx.shtml)  and CSDBgt.html)  concentrin silico, these results are labeled with corresponding bioinformatics methods. Thus, GTDB provides search methods via different data sources  to meet users’ distinct demands. It also supplies batch download service for annotated data grouped by several characteristic sets. In addition, GTDB is free and available at https://www.biosino.org/gtdb/.Considering the above points, we therefore constructed the GTs database (GTDB), which has combined a variety of contents from well-known public databases including CAZy, UniProt , KEGG 1 and Metahttp://www.cazy.org/) database, and protein names were kept in the original style in this database that uses a combination of trivial names, gene identifications and locus tags. Gene information on GTs, including gene symbol and gene ID, was integrated using data from NCBI Gene (https://www.ncbi.nlm.nih.gov/gene/) (https://www.ncbi.nlm.nih.gov/protein/) and Nucleotide (https://www.ncbi.nlm.nih.gov/nuccore), respectively. The species information, namely taxonomy ID, came from NCBI Taxonomy (https://www.ncbi.nlm.nih.gov/taxonomy/) (https://www.ncbi.nlm.nih.gov/pubmed/) (https://www.uniprot.org/). The 3D structure information, including experimental methods used, resolution, ligand and specific references, was acquired from the RCSB Protein Data Bank (http://www.rcsb.org/) (https://metacyc.org/). All protein sequences from MetaCyc act as a reference dataset when we applied DIAMOND. EC numbers of the GTs in GTDB were mostly from BRENDA (https://www.brenda-enzymes.org) (http://pfam.xfam.org)  . The proxonomy/) . The infpubmed/) . We thensb.org/) . Pathwaymes.org) , which ifam.org) , which ifam.org) .By means of the precomputed eggNOG database clusters and phylogenies analysis, eggNOG-mapper can annotate large sets of proteins via fast orthology assignments . Here, 5The high-throughput program DIAMOND can align DNA reads or protein sequences against a protein reference database with high speed and high sensitivity . It was s of GTs .We developed BLAST and GTdock tools based on known GTs in GTDB. BLAST was carried out via NCBI-BLAST 2.7.1, with default expect threshold of 10 and a maximum of 250 aligned sequences . The entTo predict the possible molecular interactions between GTs and a given glycosyl-acceptor, we developed GTdock tools. To use this function, users should first acquire structure files of macromolecular proteins and small molecular acceptors. For small molecular acceptors, users can select from any of 44 confirmed glycosyl-acceptors provided by UniProt or upload a custom acceptor in SDF format. For macromolecular proteins, the maximum number of structural files allowed is 10. Candidate proteins for docking should only be those with identities >40% and an expected value less than 0.01 in a BLAST comparison with all GTs in GTDB. Finally, Autodock Vina performs molecular docking, where the parameter of each protein center was calculated using the central position of the corresponding molecule and the box dimensions in x, y, z were all set to 40. Once the above analysis was finished, the docking results for each GT with highest interactive score are automatically e-mailed to users.http://3dmol.csb.pitt.edu/) (http://echarts.baidu.com/index.html). In addition, we encapsulated Blast and GTdock services as independent docker applications (https://www.docker.com/), which enabled easier deployment and quick updates.GTDB merges diverse and heterogeneous datasets coming from distinct communities and deposits them in MongoDB after Extract-Transform-Load processes. Compared with traditional relational database management systems, MongoDB provides more flexible ways for necessary future expansion. GTDB was developed using java SpringBoot and integrated a couple of enhanced utilities for providing visual presentations of GTs data. For instance, 3D structures of GTs are represented in 3Dmol.js (tt.edu/) , and a sGTDB was built to offer an integrated resource that incorporates GT sequences and annotations across multiple species, and a user-friendly web interface that allows data retrieval using different types of identifiers, searching against pre-organized protein sequences by BLAST and performing molecular docking of one acceptor with some GTs via GTdock .GTDB collects a large number of sets of GT data that in total consist of 520 179 GTs with 554 892 protein sequences, approximately 21 647 species, 33 720 genes, 394 full EC numbers, 4788 gene ontology (GO) terms and 10 271 references. It covers 105 GT families according to the CAZy database (version 17 April 2019), where GT0 indicates non-classified sequences and GT2 harbors the most abundant number of GTs (159521). In terms of annotated content, 8.9% of GTs have GO terms from third-party database and 35.3% GTs’ GO terms were further predicted in GTDB. ‘Transferase activity (GO:0016740),’ which is one of the main characteristics of GTs, accounts for 44.1% and 89.5% in the two previously mentioned sources, respectively. For KEGG orthology (KO) annotations, 3.2% of GTs have KO from knowledge-based resources, and 68.3% GTs’ KO were widely annotated in GTDB. Notably, there are five KOs—k02563, k05366, k00688, k07011 and k03814—that are all associated strongly with carbohydrate transport and metabolism, making up a large proportion in the total number of KOs from different methods. For domain prediction, 9.3% of GTs’ Pfam data were extracted from UniProt and 79.6% of these domains were extended predictions. All relevant data can be viewed on the ‘Statistics’ page of GTDB.Keyword search and sequence search were both implemented in GTDB, where keyword search consists of three accesses. First, users can input a GT name, protein Genbank accession, or UniProt accession on the top query bar using pre-defined search criteria. Second, users can click on any GT name directly in a word cloud on ‘Home’ page to start a quick investigation of interested GTs. Lastly, an advanced search on the ‘Search’ page allows more sophisticated searching, where various query items are pre-classified into several categories, including protein information , gene information (gene ID and gene symbol), taxonomy (taxonomy ID or organism), tissue, functions  and reaction information . Queries of ‘function’ and ‘reaction’ provide the label of the data source (third-party database or predictions in GTDB), which all assist the users to address specific search concerns accurately. An example of advanced search result is shown in Ureaplasma urealyticum) (Genbank accession: QDI63889.1) is new for GTDB because it was newly created on 9 July 2019 in the NCBI Protein resource. While submitting QDI63889.1 as a query sequence and choosing ‘All’ as the target database, a sorted table of results with links of each aligned entry is generated. As the best alignment results from this search (NP_078340.1 identities = 93% expect = 0)  may belong to GT2 family and has a catalytic mechanism of inverting. The BLAST results are shown in Sequence searches using BLAST can be helpful for analyzing new or similar GTs in GTDB. For instance, GT (ect = 0)  indicateConsidering that GT structure can provide suitable catalytic sites and the special microenvironment for glycosylation reactions, we included binding mode prediction software in GTDB. AutoDock Vina has the advantages of both high speed and high accuracy in molecular docking, so we integrated Autodock Vina and BLAST to build the GTdock pipeline. GTdock can fulfill molecular docking with an acceptor and several GTs at one time and display those results based on 3Dmol.js that provides an interactive interface to view the binding modes.Saccharomyces cerevisiae S288c, UGT51 can catalyze its natural substrate—ergosterol—with very high activity. Researchers have found that UGT51 can also catalyze protopanaxadiol (PPD) with ~13% of the conversion ratio as compared with ergosterol. They then reported mutant M7_1 (S801A/L802A/V804A/K812A/E816K/S849A/N892D), which had an 1800-fold activity improvement for the unnatural substrate PPD  manually based on publications. We will also improve the GTdock tool to achieve more accurate docking results by integrating an improved center finding algorithm. To conclude, GTDB will facilitate further identification of GTs and understanding of the vital roles that GTs play in glycobiology, synthetic biology, drug design and development.Database online.C.Z. performed the analyses, data collection and most web design and drafted the paper. Q.X. set up the database and participated in web design and S.H. and W.Y. constructed the GTdock tool pipeline and participated the website design. R.C. contributed to data collection. P.W. took part in the database building. Y.L., X.Y. and Q.W. edited the final manuscript. G.Z. conceived the study and edited the final manuscript. All authors read and approved the final manuscript.supplymentary_file_baaa047Click here for additional data file.