Lens culinaris/Lens orientalis, Lens lamottei/Lens odemensis, Lens ervoides, and Lens nigricans. A weak correlation was observed between geographical origin and genetic relationship, except for some accessions of L. culinaris and L. ervoides. Genetic distance matrices revealed a comparable level of variation within the gene pools of L. culinaris (Nei’s coefficient 0.01468–0.71163), L. ervoides (Nei’s coefficient 0.01807–0.71877), and L. nigricans (Nei’s coefficient 0.02188–1.2219). In order to understand any genic differences at species/subspecies level, allele frequencies were calculated from a subset of 263 lentil accessions. Among all cultivated and wild lentil species, L. nigricans exhibited the greatest allelic differentiation across the genome compared to all other species/subspecies. Major differences were observed on six genomic regions with the largest being on Chromosome 1 (c. 1 Mbp). These results indicate that L. nigricans is the most distantly related to L. culinaris and additional structural variations are likely to be identified from genome sequencing studies. This would provide further insights into evolutionary relationships between cultivated and wild lentil germplasm, for germplasm improvement and introgression.Intensive breeding of cultivated lentil has resulted in a relatively narrow genetic base, which limits the options to increase crop productivity through selection. Assessment of genetic diversity in the wild gene pool of lentil, as well as characterization of useful and novel alleles/genes that can be introgressed into elite germplasm, presents new opportunities and pathways for germplasm enhancement, followed by successful crop improvement. In the current study, a lentil collection consisting of 467 wild and cultivated accessions that originated from 10 diverse geographical regions was assessed, to understand genetic relationships among different lentil species/subspecies. A total of 422,101 high-confidence SNP markers were identified against the reference lentil genome (cv. CDC Redberry). Phylogenetic analysis clustered the germplasm collection into four groups, namely,  Lens is a self-pollinating, diploid (2n = 2x = 14), cool season legume with a large genome size of c. 4 Gbp , India is still the world’s largest importer of lentil1,2.The genus c. 4 Gbp . With reears ago . Lentil ears ago . Lentil Lens for a range of agronomic and phenological traits  and secondary  gene pools was found to be higher compared to the species from tertiary (L. ervoides) and quaternary (L. nigricans) gene pools  are widely used variant calling programs to accurately and consistently identify genome variants  and cultivated species (L. culinaris – 304) were obtained from the AGG, Horsham, VIC, Australia. The geographical origins of these accessions varied widely with the majority being from Australia and the Middle East. The remaining accessions were derived from North and South America, Africa, Mediterranean, and European and Asian countries, with the number of accessions from each geographical origin varying from 2 to 240  following the manufacturer’s instructions. Using a NanoDrop UV-Visible spectrophotometer , the concentration and quality of RNA was confirmed at the wavelength ratios of A260/230 and A260/280 nm. The integrity of extracted RNA samples was evaluated using TapeStation 2200 platform with RNA ScreenTape System , following the manufacturer’s guidelines. RNA-Seq libraries were prepared using the Agilent SureSelect Strand Specific RNA Library Preparation Kit and evaluated on TapeStation 2200 platform with D1000 ScreenTape System . Each RNA-seq library was paired-end (2 × 151 bp) sequenced using Illumina Hiseq 3000 Sequencing platforms .Q ≤ 30). Reads with three consecutive unassigned nucleotides (N) were also trimmed and finally any reads shorter than 50 bp in length were removed from the final set. The remaining high-quality trimmed sequence reads were aligned to the lentil reference genome sequence of cultivar CDC Redberry (version 1.2)4 using two alignment pipelines, TopHat2 (version 2.1.1)   and STARn 2.1.1) . The numn 2.1.1) .Lens sp. – 2, L. lamottei – 1, L. odemensis – 22, L. nigricans – 24, L. ervoides – 57, L. orientalis – 57, and L. culinaris – 304), variant calling was performed separately for each species. The number of SNPs obtained from each of the mapping and variant calling methods were compared to identify the best analysis pipeline   and GATKpipeline . The STAGenetic diversity analysis was performed using the abovementioned filtered SNP data from all accessions. Genetic distances for each lentil accession were calculated using Nei’s method within the StAMPP package . A phyloL. odemensis – 22, L. nigricans – 24, L. ervoides – 57, L. orientalis – 57, and L. culinaris – 103) was chosen based on the sample numbers that belonged to each species/subspecies to capture a comparable representation from each group. Variant calling and filtering were again performed using pipelines and parameters discussed above, to generate the final output in VCF format. The allele frequencies for each SNP position were calculated using VCFtools-freq output option  file from CDC Redberry. Based on the exon–intron positions specified in the GFF file, the sequences from the corresponding genes were extracted from the CDC Redberry genome (version 1.2) fasta file using BEDtools (version 2.26.0) . The extThe total number of SNPs or variants identified for all species/subspecies was 3- to 10-fold higher when STAR aligner was used, as compared to TopHat2 , which iQ ≥ 30; 1,414,996 SNPs), and minor allele frequencies (MAF = 0.05) that resulted in a final set of 422,101 high-quality SNP markers , leaving a set of 12,522,726 SNPs. This subset of SNP markers was further filtered for percentage of missing data (50%), quality score  that was originally classified as L. lamottei actually clustered within L. odemensis. In this study, the two lentil accessions (32601 and BGRC-025688), classified as Lens sp., were clustered in L. culinaris and L. orientalis gene pools, respectively. Moreover, 22 accessions out of 467 lentil accessions were classified in clusters that did not correlate to the passport data obtained from AGG. This was more visible in L. orientalis, L. odemensis, L. ervoides, and L. nigricans clusters (L. culinaris accessions (304) with majority being originated from Australia, there was no bias observed in terms of clustering. This was further confirmed by regenerating the phylogenetic tree after removing 201 of the 304 L. culinaris accessions   .L. ervoides (ILWL042) and L. culinaris (10H063L-12HS3003) accessions with Nei’s coefficient value of 0.92295. The two L. orientalis accessions ILWL211 and ILWL069, both originating from the Former Soviet Union, exhibited the lowest genetic distance . Further analysis was performed to understand the variation in genetic distances between and within lentil species/subspecies (L. culinaris exhibited higher genetic distance to L. ervoides (0.33642–0.89817) and L. nigricans (0.31141–0.89375) compared to L. orientalis (0.12622–0.13927) and L. odemensis (0.27913–0.31805). Further analysis revealed wide genetic distance variations within the species/subspecies level .Based on genetic distance calculations, the most divergent pair was observed between bspecies . L. culiL. culinaris, some lentil accessions originating from Canada  showed wide genetic distances to some of the Australian  and Middle East Asian accessions , with Nei’s coefficient value between 0.60 and 0.70. Similar results were also observed within L. ervoides, where distant genetic relationships were observed within two Mediterranean lentil accessions ILWL042 and HOFFMAN-42 . ILWL042 also showed wide genetic distances to multiple lentil accessions originated from Middle East Asia, Russia, and Central Europe  were clustered separately from the Middle East Asian L. ervoides accessions . L. culinaris lines from Australia, North America, Asia, and Mediterranean regions were also clustered separately . In order to explore major variations at the exome levels, we focused on regions with physical distance greater than 1.0 kb  and Pisum sativum (field pea). Potential candidate genes identified are summarized in Potential candidate genes associated with variable genomic regions were identified using the GFF file from CDC Redberry genome as well as BLASTN analysis in NCBI . A totalThe current study has identified taxonomic relationships among cultivated and wild lentil gene pools using SNPs identified from transcriptome sequencing. After comparing different SNP detection pipelines, the STAR aligner-GATK-HC pipeline was chosen as the preferred method for SNP calling due to higher number of SNPs detected compared to other pipelines tested. Similar results have been reported in other studies where the efficiency of SNP and indel detection from both whole genome and exome-captured sequence data was found to be higher in GATK-HC as compared to SAMtools-mpileup . To accuLens species into four major groups, L. culinaris/L. orientalis, L. odemensis/L. lamottei, L. ervoides, and L. nigricans, which is consistent with results from L. orientalis and L. culinaris separately, which is not consistent with the results of Genetic diversity analysis clustered the six Lens accessions for future crossing strategies in order to introgress novel alleles.A weak correlation was observed between clusters and geographical origins, with some exceptions, which is in line with some previous studies . The reaL. ervoides and L. nigricans) indicate that direct hybridization between cultivated and wild lentil accessions that belong to tertiary and quaternary gene pools may be challenging. As an alternative, the use of an intermediate relative as a bridge species may be helpful in transferring alleles/genes that belong to distant gene pools. Attempts have been made in L. nigricans (quaternary gene pool) to transfer resistant genes for ascochyta blight and anthracnose diseases into L. culinaris (primary gene pool) via L. ervoides (tertiary gene pool) as a bridge species  have reported genomic rearrangements such as substitutions, insertion, and deletions  have shown better response to metal  applications, as compared to the cultivated rice (Oryza sativa). These wild species have been used for breeding purposes to enhance the metal tolerance in cultivated rice varieties. In addition, L. odemensis, L. ervoides, and L. nigricans as compared to L. culinaris  . Furtherulinaris , which iulinaris . The canL. culinaris/L. orientalis, L. odemensis/L. lamottei, L. ervoides, and L. nigricans. Wild and cultivated lentil accessions showed a weak correlation to their geographical origins. Therefore, the selection of parents for breeding purposes can be better selected by genetic distance boundaries within and between lentil species/subspecies. Allele frequency-based analysis also confirmed that L. nigricans was distantly related to L. culinaris. The identified native and exotic lentil accessions with wide genetic distances and specific agronomical traits can be used in widening the Australian lentil germplasm for breeding purposes.Assessment of genetic diversity among a global collection of wild and cultivated lentil accessions was performed using a set of genome-wide distributed SNP markers. The genetic diversity analysis revealed a clear grouping of lentil germplasm into four major clusters/gene pools, PRJNA625627, https://www.ncbi.nlm.nih.gov/sra/PRJNA625627.The datasets generated for this study can be found in NCBI SRA accession SB prepared the plant materials and performed the sequencing library preparation. RD performed the data analysis. SK, NC, and KS provided overall project leadership. RD, NC, SK, and KS all assisted in drafting the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aspergillus fumigatus. To raise awareness and emphasize diagnostic challenges, we present a case of mixed bIFIs in a child with acute lymphoblastic leukemia.Breakthrough invasive fungal infections (bIFIs) are an area of concern in the scarcity of new antifungals. The mixed form of bIFIs is a rare phenomenon but could be potentially a troublesome challenge when caused by azole-resistant strains or non-Escherichia coli bloodstream infection and pulmonary invasive fungal infection with Aspergillus fumigatus (early-type bIFIs) while receiving antifungal prophylaxis. Shortly after pulmonary involvement, his condition aggravated by abnormal focal movement, loss of consciousness and seizure. Cerebral aspergillosis with Aspergillus niger diagnosed after brain tissue biopsy. The patient finally died despite 108-day antifungal therapy.A newly diagnosed 18-month-old boy with acute lymphoblastic leukemia was complicated with prolonged severe neutropenia after induction chemotherapy. He experienced repeated episodes of fever due to extended-spectrum beta-lactamase-producing Aspergillus identification at the species level in invasive fungal infections with multiple site involvement in the patients on antifungal prophylaxis.Mixed bIFIs is a rare condition with high morbidity and mortality in the patients receiving immunosuppressants for hematological malignancies. This case highlights the clinical importance of  Cerebral aspergillosis usually occurs secondary to fungemia after inhaling the fungal spores, proliferating and invading the pulmonary alveolar arteries, after the direct invasion from adjacent structures (sinuses), iatrogenic/penetrating trauma, medical surgery, and contamination of indwelling catheters . Multiple brain abscesses could be developed by different mechanisms such as direct angioinvasion, thrombosis, and infarction, mycotic aneurysm, or even intracranial hemorrhage –6. The iBreakthrough invasive fungal infection (bIFI) defined as a new fungal infection in the patients receiving therapeutic or prophylactic antifungal agents. Concomitant fungal pneumonia with central nervous system (CNS) infection is a rare occurrence in patients with hematological malignancies, especially those on anti-fungal (AF) prophylaxis. In this report, a mixed pulmonary and CNS aspergillosis reported in a pediatric patient with acute lymphoblastic leukemia (ALL) and we discuss bIFIs during antifungal (AF) prophylaxis.Aspergillus antigen) was requested (twice per week) for early detection of invasive aspergillosis (IA). Fever developed on the12th day post-admission. Complete sepsis workup was done and meropenem was initiated. Plain X-ray revealed no new parenchymal involvement. Peripheral and central catheter blood cultures performed for the patient using an automated blood culture system . Both cultures were positive for extended-spectrum beta-lactamase-producing Escherichia coli with similar antibiotic susceptibility patterns . Lock-therapy for catheter bloodstream infection with ciprofloxacin performed . Control blood cultures became negative after 5 days of targeted antibiotic treatment. The chest X-ray was normal at that time , platelet count: 10000/mcL (150–400 /microliter) and, white blood cell count (WBC): 106000 cells/mcL ] were detected. B cell ALL diagnosed following bone marrow aspiration/biopsy. Induction chemotherapy started with vincristine, PEG-asparaginase, and daunorubicin. Prophylactic trimethoprim/sulfamethoxazole was initiated simultaneously with his chemotherapy. About 9 days later, his absolute neutrophilic count dropped rapidly  when he was afebrile. Acute phase reactants were within normal limits . Prophylactic liposomal amphotericin B  started, and the galactomannan test  difficile toxin gene by polymerase chain reaction (PCR) after culture for C. difficile. Quantitative PCR for Cytomegalovirus and Mannan test requested in addition to routine indirect mycological  and GM tests. The serum GM test was positive during the second investigation   scheduled. The result of BAL culture was positive for ny) Fig. . The minA few days later, the patient’s condition deteriorated suddenly by abnormal focal movement in his right upper limb, loss of consciousness , and seizure. He transferred to the pediatric intensive care unit. The cerebrospinal fluid (CSF) analysis revealed total cell count 15 without WBC, protein 164 mg/dl, Lactate dehydrogenase 65 U/L , and sugar 48 mg/dl. Brain CT scan showed extensive left hemisphere intracerebral hemorrhage  and revealed that the isolated species is Aspergillus niger. It deposited in the GenBank database with accession number MT561886. The MIC for amphotericin B, caspofungin, voriconazole, posaconazole, and itraconazole in isolated A. niger were 8 mg/l, 0.032 mg/l, 0.5 mg/l, 0.032 mg/l, and 0. 25 mg/l. The patient stayed in the hospital for several weeks without any changes in his clinical status and died due to sudden cardiac arrest associated with severe CNS Aspergillus infection, renal impairment, and septic shock 142 days after admission and 108 days after AF therapy.Brain biopsy performed because of persistent fever despite extensive medical and surgical treatment. KOH revealed septate hyphae Fig. . Tissue Patients with hematological malignancies are at increased risk of various infections, including IFIs. The risk is higher among those receiving chemotherapy for acute myeloid leukemia, myelodysplastic syndrome, ALL, patients during induction-remission chemotherapy, graft versus host disease, and corticosteroid therapy. AF prophylaxis recommended in high-risk patients in the presence of prolonged severe neutropenia (ANC < 500 cells/μL > 7 days) . Even wiMucormycosis is considered a common cause of bIFIs in those on azole prophylaxis in some reports, other mold infections such as Aspergillus and non-Aspergillus species are also well-known as etiologic agents [Aspergillus spp. [Aspergillus spp. (A. terreus), Fusarium spp., Scedosporium spp. and even Mucorales have been reported [There are some risk factors for developing bIFIs like the occurrence of IFIs due to resistant pathogens, development of resistance in previously susceptible fungi, inadequate absorption , abnormal metabolism (CYP2C19 heterogenicity), ineffective distribution, drug interactions, and conditions that perpetuate the infection . Although c agents . The proc agents –24. The lus spp. . Data rereported .The emergence of azole-resistant pathogens warrants careful adherence to the stewardship programs in high-risk settings such as hematology/oncology wards , 27. TheEarly diagnosis and antifungal therapy are vitally crucial for the best outcome of the patient. The treatment of infections caused by fungi may differ, so it is essential to confirm the genus by culture, PCR, and sequencing. Both microscopic examination and culture are insensitive, and therapy should not withhold in the absence of such confirmation. Non-invasive methods like serum biomarkers (GM and beta-D-glucan assays) are established for the diagnosis of IA . MoleculAspergillus species isolated from the patient. Diagnosis of A. niger is difficult because Aspergillus section Nigri is a pigmented fungus  and morphologically is very similar to A. niger. Simões and coworkers by colony morphologic characterizing, microscopic examination, and spectral mass analyses were reported species of Aspergillus section Nigri including Aspergillus aculeatus, Aspergillus brasiliensis, Aspergillus carbonarius, Aspergillus ellipticus, Aspergillus ibericus, Aspergillus japonicas, Aspergillus lacticoffeatus, Aspergillus niger, Aspergillus phoenicis, Aspergillus sclerotioniger, Aspergillus tubingensis, Aspergillus uvarum, and Aspergillus vadensis [Aspergillus section Nigri are rare and more of these were categorized in A. niger infections. Gautier and coworker reported, from 85 A. niger isolated from respiratory samples, 40 species were diagnosed as A. tubingensis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, infected patients suffered from different types of chronic respiratory failure [Aspergillus niger identified by beta-tubulin gene and sequencing in this case.In this study, two vadensis . The inf failure . TherefoA. niger documented in the present case report warrants special consideration in high-risk patients receiving amphotericin B for prophylaxis. Thus, given the different AF susceptibility patterns, identification of the Aspergillus at the species level is suggested [In our case, although both isolates showed low MIC to all tested Azoles, high MIC to amphotericin B was found. Amphotericin resistant uggested , 33 and CNS infections may happen as an occult asymptomatic extra-pulmonary involvement during the diagnostic evaluation of febrile neutropenic patients or symptomatic form, which usually developed after a few weeks (median of 2 weeks) of pulmonary manifestation (range: 5–283 days) . In our Aspergillus spp. was the most prevalent type of identified mixed mold infection in 27 patients retrospectively studied. The most common reported combinations were A. fumigatus/A. terreus, and A. terreus/A. niger, with the mortality rate in such infections being as high as 70% [A. fumigatus/ A. niger in children with vital organ involvement. The definitive diagnosis of CNS aspergillosis requires a high index of suspicion and an aggressive approach. We present herein a case of mixed A. fumigatus/ A. niger infection with fatal outcome in a patient with ALL. Given the different AF susceptibility patterns, identification of the Aspergillus at the species level should considered in invasive fungal infections with multiple site involvement. In line with the current ESCMID-ECMM-ERS guideline, consideration of different AF classes for the treatment of bIFIs in the patients receiving amphotericin B warranted.Data regarding mixed mold infections are somewhat lack in the literature. Based on the most recent report by Magira et al., h as 70% . Mixed m


Listeria spp. are naturally ubiquitous, with L. monocytogenes particularly being ranked as important foodborne disease-causing microorganisms. This study aimed to evaluate the incidence and determine the antimicrobial resistance (AMR) profiles of multidrug-resistant Listeria spp. (MDRL) isolated from different environmental samples (river and irrigation water) in the Sarah Baartman District Municipality (SBDM), Eastern Cape Province (ECP), South Africa. Molecular identification and characterization were carried out using polymerase chain reaction (PCR) and isolates that exhibited phenotypic resistance were further screened for relevant antimicrobial-resistant genes (ARGs). Findings revealed a total of 124 presumptive Listeria isolates; 69 were molecularly confirmed Listeria species. Out of the confirmed species, 41 isolates (59%) were classified as L. monocytogenes while 9 (13%) were classified as L. welshimeri. All Listeria spp. exhibited phenotypic resistance against ampicillin, penicillin, and trimethoprim-sulphamethoxazole and further screening revealed ARGs in the following proportions: sulI (71%), TEMbla (66%), tetA (63%), and CITbla (33%). Results confirmed the occurrence of ARGs among Listeria inhabiting surface waters of ECP. The present study indicates that the river water samples collected from SBDM are highly contaminated with MDRL, hence, constituting a potential health risk.The prevalence of bacteria with multidrug-resistance (MDR) is a significant threat to public health globally.  Listeria is made up of a group of small rod-shaped, non-spore-forming, facultative anaerobic microorganisms that belong to the family Listeriaceae. These bacterial species are motile and capable of thriving at low temperatures and in harsh conditions [Listeria contains 20 major species including L. welshimeri, L. grayi, L. marthii, L. seeligeri, L. ivanovii, L. innocua, and L. monocytogenes and these microbes are present within various environmental niches including soil, vegetables, water, wastewater, and animal feces [Listeria species in different environmental sources such as soil [Listeria in wastewater treatment plants (WWTPs) suggesting that these pathogens can thrive even after chlorination [Listeria spp., L. ivanovii and L. monocytogenes are both considered as relevant animal and human pathogens. However, L. monocytogenes is regarded as the third most common foodborne pathogen causing infections in individuals with higher risk, for instance, elderly and immune-compromised people, as well as pregnant women [Genus nditions ,2. The gal feces . Many st as soil ,5,6, whi as soil , livesto as soil ,9,10,11.rination ,13,14,15nt women . These int women .L. monocytogenes is a transitory resident of the intestinal tract in humans, with about 10% of the world’s population reported to be carriers of the microorganisms without experiencing any apparent symptoms [Listeria spp. harboring antibiotic resistance genes (ARGs) have been detected in agricultural samples [L. monocytogenes (ARLM) has also been reported subsequently [The World Health Organization (WHO) ranked listeriosis among the deadliest foodborne diseases with a 20% to 30% mortality rate . L. monosymptoms ,20. Accosymptoms , listeri samples  and the equently ,23,24.Listeria spp. (MDRL) have rarely been reported, antibiotic-resistant Listeria (ARL) was first reported from a French patient diagnosed with meningoencephalitis in 1988. A recent study disclosed isolates exhibiting phenotypic resistance against streptomycin, chloramphenicol, tetracycline, and erythromycin [Although multidrug-resistant hromycin . Other shromycin ,26. Thushromycin .L. monocytogenes infection in the Republic of Korea. The outbreak was linked to commercially bought enoki mushrooms and was responsible for 36 reported cases, 31 hospitalizations, and 4 fatalities [L. monocytogenes in water sources, particularly those used for irrigation and consumption, may lead to the carry-over of potential pathogens from the water source to fresh produce. Upon consumption of contaminated food or drinking water, infectious doses of the bacterium may manifest in either febrile listerial gastroenteritis (non-invasive listeriosis) or invasive listeriosis associated with the aforementioned symptoms [Listeria spp. recovered from the aquatic milieu in South Africa. This is one of the few studies within the Eastern Cape Province (ECP) that aimed to evaluate the occurrence, as well as delineate the antibiogram profiles of Listeria spp. isolates recovered from river water and irrigation water sources collected from the Sarah Baartman District Municipality (SBDM) in South Africa.In 2011, the Centers for Disease Control and Prevention (CDC) reported an outbreak of listeriosis that was associated with contaminated cantaloupe melons in Colorado, USA which led to 147 cases, out of which 142 were hospitalized, causing 33 fatalities . Recentltalities . A reportalities . Polony,talities ,31. Humatalities . Therefosymptoms . PresentThis study was conducted in the SBDM  situated in the ECP, South Africa. The sampling sites were the Great Fish River and the Bloukrans River. These rivers flow south and are tributaries of the Kowie River situated near Grahamstown. The Bloukrans River serves different purposes such as irrigation water for nearby farms and also has other functions for livestock farms. The river also serves as a receiving watershed for final effluent discharge from the district’s WWTP. Water samples were collected downstream from the Bloukrans River and farm samples  from some farms in the SBDM. Six samples were collected on a once-off regime in the month of September. Water samples were collected accordingly in duplicates by means of sterile bottles (1-L) and conveyed in an ice-box to the laboratory for bacteriological analysis in no more than six hours of collection.Listeria spp., each membrane filter was aseptically picked with sterilized forceps and transferred onto the surface of Chromogenic Listeria agar (ISO) Base  mixed with one vial of OCLA (ISO) selective supplement and OCLA (ISO) differential supplement . This followed the standards as described previously by Ottaviani and Agosti (ALOA) in ISO 11290–1:1997 used for enumerating Listeria spp. including L. monocytogenes [Listeria spp. on the ISO Base. The data were converted to log10 CFU per 100 mL [For bacteriological analysis, the standard filtration method was followed as described by Olaniran et al.  with modytogenes . The medr 100 mL .Listeria spp. by inserting the membrane filter into 90 mL of Tryptic Soy Broth (TSB) and afterward incubated aerobically at 37 °C for 16–18 h . Following incubation, the second enrichment was done by aseptically transferring 1 mL of the enriched TSB suspension onto 9 mL of Fraser Broth , vortexed, and then incubated at 37 °C for a period of 24 h [Listeria agar (LCA) , thereafter supplemented using OCLA (ISO) selective supplement with OCLA (ISO) differential supplement, following the standards as defined by Ottaviani and Agosti (ALOA) in ISO 11290–1:1997 and incubated aerobically for 24 h at 37 °C. After incubation, presumptive Listeria isolates  were subsequently purified onto nutrient agar (NA)  and incubation conditions of 37 °C for a period of 24 h were followed. Pure colonies were further inoculated in sterile nutrient broth (NB) following an incubation period of 18–24 h at 37 °C. The overnight culture was stored in 25% glycerol stock and kept in a freezer at −80 °C for further analysis.Due to the slight turbidity of the river and irrigation water samples collected, enrichment was carried out to increase the detection of  of 24 h . From thThe boiling method was employed to extract bacterial DNA following the methods described by Maugeri et al. . The preListeria isolates were molecularly confirmed using the PCR technique. Listeria species, their respective PCR product sizes, and PCR conditions.The presumptive Listeria species were put through AST using 15 panels of antibiotics as described by the Kirby–Bauer disc diffusion technique. The results were interpreted accordingly following the Clinical and Laboratory Standards Institute [Staphylococcus spp. were referred to in this study because the interpretative criteria for Listeria spp. are unavailable in the CLSI guidelines [All the identified nstitute  guidelinidelines .Listeria species (ARLS) was generated based on isolates that exhibited phenotypic antibiotic resistance (AR) to 3 or more antimicrobial agents as described by Krumperman [a” denotes the sum of test antibiotics the isolates displayed resistance to; “b” denotes the total sum of antimicrobial agents used.The MARPs pattern of antimicrobial-resistant umperman , MARI waKlebsiella pnuemoniae carbapenemase (KPC), Verona integon-encoded metallo-beta-lactamase (VIM) and active-on-imipenem (IMI) genes. A total of 14 ARGs were screened using the PCR technique. The PCR profiling of antimicrobial resistance (AMR) determinants of ARLS involved the screening of 10 antimicrobial-resistant genes (ARGs) encoding for relevant β-lactamases following the method previously described by Dallenne et al. . Table 2Listeria spp. obtained from the river samples collected from Bloukrans River access point one (BRT) and Bloukrans River access point two (BRD) ranged from 3.49–3.88 log10 CFU/100mL. The standard plate count from Duncan Farm irrigation-water (DFW) samples ranged from 0–3.68 log10 CFU/100mL. Therefore, the dissemination of presumptive Listeria spp. was higher for the BRD sampling site in the SBDM, as compared to the other sampling sites.The results showed that the standard plate count for Listeria isolates were recovered from the water samples analyzed. The results revealed that 55.7% (69/124) harbored the prs gene for the confirmation of Listeria spp.  against other test antibiotics including penicillin, cephalothin, ciprofloxacin, levofloxacin, trimethoprim-sulphamethoxazole, nalidixic acid, chloramphenicol, tetracycline, erythromycin, vancomycin, clindamycin, and oxacillin. Among the 15 antimicrobial agents, only amikacin and gentamicin had activity against L. monocytogenes with a low resistance percentage of 39% and 32%, respectively whereas no L. welshimeri isolates exhibited resistance against amikacin. However, L. welshimeri exhibited a resistance of 33% against gentamicin.All confirmed Listeria spp. were generated and are summarized in L. monocytogenes show four MAR patterns against the antibiotics (L. monocytogenes was the AP-PG-KF-CIP-LEV-TS-NA-C-T-ERY-VA-CD-OXA pattern with 41.5% (17/41) of the isolates exhibiting the resistance pattern. The MARI values of all L. monocytogenes ranged between 0.87 and 1  revealed that among the ARGs responsible for sulfonamide resistance, only sulI was detected in 71% (29/41) of L. monocytogenes and 67% (6/9) of L. welshimeri. Tetracyclines tetA was greatly abundant in L. welshimeri by 78% (7/9) and 63% (26/41) of L. monocyotgenes harbored tetA gene; tetB, tetC, and tetM were not detected amongst the Listeria spp. Furthermore, resistance gene catII was present in very low levels, with resistance proportions of 7% (3/41) amongst L. monocytogenes and no phenicol-resistance gene was detected amongst L. welshimeri isolates. Among the screened ESBL determinants, TEMbla was the most dominant resistance genes with L. monocyotgenes isolates displaying great TEMbla presence of 66% (27/41) compared to L. welshimeri with 44% (4/9); OXA-1bla followed with 17% (7/41) and 22% (2/9) amongst L. monocytogenes and L. welshimeri isolates, respectively. L. welshimeri displayed 33% (3/9) CITbla resistance. sulI gene and tetA gene, respectively.PCR screening of ARGs of multidrug-resistant Listeria spp. harboring ARGs. The standard plate count of the presumptive Listeria isolates recovered from the selected sample sites was high and this is in accordance with the findings reported by Bilung et al. [Listeria spp. in the irrigation water samples.The emergence of MDRL poses a serious public health threat to humanity. In this study, our findings provide evidence that the environmental waters of the ECP are probable reservoirs of highly pathogenic g et al.  and Iwu g et al. . The Soug et al. ,49,50, tL. monocytogenes present in river and irrigation waters. In the United States, L. monocytogenes was reported in 43% (604/1405) [Listeria spp. confirming the occurrence of the microorganisms in soil and surface water samples. In this study, the percentage occurrences (PO) observed was 55.7% for Listeria species and 59.4% for L. monocytogenes, while the PO of L. welshimeri (13%) was much lower. Similarly, other studies reported Listeria spp. with PO of 64% [L. monocytogenes (14%) recovered from irrigation water in ECP, South Africa.Globally, several studies have also investigated the prevalence of 04/1405)  and 31% 04/1405)  of water04/1405) . The stu04/1405)  carried O of 64%  reportedListeria spp. in this study revealed that 86.7% of the isolates displayed MDR against the test antibiotics. In contrast, previous studies reported high susceptibility to penicillin, ampicillin, and tetracycline exhibited by L. monocytogenes recovered from pork and slaughterhouses in Brazil [L. monocytogenes observed in this study agrees with another study of Aras and Ardıç [L. monocytogenes isolated from turkey and meat samples with resistance percentage of 75% and 66.7% to ampicillin and penicillin, respectively. In contrast, the findings of this study revealed resistance exhibited by L. monocytogenes against the beta-lactams ampicillin, penicillin, and cephalothin. Similarly, Olaniran et al. [Listeria spp. isolated from treated wastewater (WW) final effluents and receiving watersheds.The patterns of phenotypic resistance exhibited by n Brazil , respectnd Ardıç , which rn et al.  reportedListeria spp. in this study exhibited varied susceptibility to amikacin and gentamicin (aminoglycoside class). Likewise, our finding is corroborated by the study of Bilung et al. [Listeria isolates that were recovered from environmental samples collected from farms in Malaysia. Generally, Listeria spp. have been reported to be susceptible to a wide range of antibiotics. However, many studies have reported Listeria spp. exhibiting phenotypic resistance against antibiotics used in the treatment of listeriosis such as tetracycline, chloramphenicol, and fluoroquinolones [The g et al. , who revinolones ,17,57.L. monocytogenes and L. welshimeri isolates displaying four and two MARP patterns, respectively. Contrary to studies by Arslan and Özdemir [Listeria spp. recovered from homemade white cheese and also food processing environments, respectively, were reported to exhibit single-drug resistance, MDR against a range of 13 to 15 test antibiotics was observed in the current study. Evaluation of the MAR index of ARLS revealed a MAR index that varied from 0.8 to 1  ARGs  were detected. Our findings revealed that among the ARGs detected, sulI (which confers AR against sulphonamides) was the most dominant amongst the Listeria spp., particularly L. monocytogenes. Another dominant ARG was tetA, which confers resistance against tetracycline, followed by TEMbla among L. monocytogenes (66%) and L. welshimeri (44%) isolates (L. monocytogenes recovered from dairy milk farms. Among the ARGs reported included tetA (32%), and sulI (16%). However, other tetracycline resistance genes screened were not found to be harboured by L. monocytogenes [The detection of ARGs harbored by c health . Furtherplasmids ,17. The isolates . Similarytogenes .Listeria spp. that exhibited phenotypic resistance did not harbor ARGs. For instance, all L. monocytogenes and L. welshimeri exhibited phenotypic resistance against chloramphenicol. However, only 7% of L. monocytogenes harbored catII gene. A low percentage occurrence against aminoglycosides was observed; however, the screened Listeria spp. did not harbor ARG. Other studies of Davis and Jackson [Listeria isolates exhibiting phenotypic resistance against aminoglycosides but did not harbor ARGs.In this study, most of the confirmed  Jackson  and Morv Jackson  also repListeria spp. and this is among the few studies reporting the detection of ESBLs encoding genes harbored by Listeria spp. recovered from the aquatic environment in South Africa.There is insufficient information concerning the occurrence of ESBLs encoding genes harbored by Listeria spp. in river water and irrigation water samples. All the confirmed Listeria spp. exhibited multidrug resistance (MDR) and harbored more than one ARG. The occurrence of MDR strains of L. monocytogenes in the water samples indicates that the bacteria are most likely to be transferred to other ecosystems. The ARGs harbored by the Listeria spp. are most likely to be transferred to other bacterial species within the aquatic environment. Therefore, this poses a significant public health risk and environmental crisis that involves the spread of ARB. Therefore, more comprehensive monitoring of the nature of acquisition and spread of ARGs among Listeria spp. is needed particularly in the agroecosystem. The final effluent from WWTPs should also be appropriately depurated before discharge into receiving water bodies.The findings of this current study revealed the presence of


Crazy-paving patterns are rarely reported as radiological manifestations of pulmonary cryptococcosis.Herein, we presented a very rare case of a crazy-paving pattern as a radiological manifestation of pulmonary cryptococcosis in a patient with primary ciliary dyskinesia. The diagnosis of pulmonary cryptococcosis and primary ciliary dyskinesia was ultimately confirmed by bronchoscopic biopsy, fungus culture, whole exome sequencing of blood, etc. The patient received flucytosine  and amphotericin B  during hospitalization and sequential therapy with voriconazole  after discharge. He recovered during follow-up.We concluded that pulmonary cryptococcosis should be considered a possible cause of crazy-paving patterns in chest CT scans. Pulmonary cryptococcosis is a common opportunistic infectious disease . The cra9/L, and the percentage of neutrophils was 88.3%. The level of procalcitonin was 0.21 g/ml, and the PaO2/FiO2 ratio was 115 mmHg during noninvasive mechanical ventilation. The carcinoembryonic antigen level was 14.7 ng/ml, and the CD4 T cell count was 48 cells/µl. The test for human immunodeficiency virus infection was negative. Anti-nuclear antibodies, anti-extracted nuclear antigen (ENA) autoantibodies, myositis-specific antibodies, and anti-neutrophil cytoplasmic antibodies (ANCAs) were all negative. Chest HRCT  was essentially normal. The pressure of the CSF was 145 mm H2O. The CSF was colorless and transparent in appearance, and no nucleated cells, red blood cells or pus cells were found. Biochemical tests of the CSF included trace protein: 0.43 g/L, chlorine: 119 mmol/L, and glucose: 4.58 mmol/L. The ink stain of the CSF was negative. Magnetic resonance imaging (MRI) of the head presented normal results. Based on the above results, the anti-infective treatments were changed to fluconazole  and piperacillin-tazobactam .After 5 days of antibiotic treatment, his symptoms did not improve. As a result, further examinations were performed. The test of serum cryptococcal antigen was positive, and the titer of cryptococcal antigen was greater than 1:2560. The patient underwent bronchoscopy, and bronchoalveolar lavage fluid (BALF) from the right upper lobe was obtained. The BALF was clear and bright in appearance. Periodic acid-Schiff (PAS) staining showed a small number of suspected positive protein-like bodies. The alcian blue staining result was negative. Next-generation sequencing (NGS) of the bronchoalveolar lavage fluid showed 2/FiO2 ratio did not increase significantly. A repeated chest CT scan was performed  severe pulmonary cryptococcosis; (2) type I respiratory failure; and (3) primary ciliary dyskinesia. Patient treatments mainly included infection control and respiratory support. The antifungal therapies were also adjusted to flucytosine  and amphotericin B  according to the results of drug sensitivity analysis. Two weeks later, the treatment was changed to voriconazole . Additionally, the noninvasive ventilator was gradually changed to a high-flow nasal cannula (HFNC). Finally, the patient was discharged after significant improvements in oxygenation.Two months after discharge, the patient underwent follow-up. The chest HRCT scan showed significant improvement Fig. f–h: the Mycobacterium tuberculosis and current severe Cryptococcus neoformans pneumonia, we suspected that the patient might be immunocompromised. This speculation was finally verified by electron microscopic examination and whole exome sequencing of the blood.We reported the first case of a crazy-paving pattern as a radiological manifestation of pulmonary cryptococcosis. Detailed etiological examination ruled out infections from viruses, pneumocystis or other pathogens. The bronchoscopy results were inconsistent with pulmonary alveolar proteinosis. Considering previous infection with Cryptococcus, and the growth of the pathogen in a number of susceptible populations [Cryptococcus causing infection is via inhalation. Spores of Cryptococcus may reach the lower airways and pulmonary alveoli. Respiratory defenses such as mucociliary transport do not prevent these spores from reaching the distal alveoli. [A crazy-paving pattern was first used to describe the imaging characteristics of pulmonary alveolar proteinosis. Currently, this manifestation can be identified in pulmonary edema, nonspecific interstitial pneumonia, pulmonary hemorrhage, sarcoidosis, viral infection, peripheral T-cell lymphoma, invasive mucinous adenocarcinoma, etc. –6. Cryptulations . The mosalveoli.  The centalveoli. . It shoualveoli. . For chealveoli. .Primary ciliary dyskinesia is a rare ciliopathic genetic disorder that causes defects in cilia action. DNAH5 is a main ciliary dyskinesia gene, and mutations of DNAH5 have been identified in 15 % of primary ciliary dyskinesia patients . The hetinterest to disclosure.


Staphylococcus aureus (S. aureus) is known as one of the most important hospital pathogens responsible for a wide range of infections. Limited data concerning the prevalence of nasal carriage of S. aureus and its molecular characteristics are available in Zabol province, Iran. Therefore, the aim of the present study was to determine the prevalence of nasal carriage of S. aureus and its molecular characteristics isolated from health care workers (HCWs).S. aureus (MRSA). PCR amplification method was used for the detection of following genes; sea, seb, sec, sed, see, tst, eta, etb, lukF-PV, lukS-PV and SCCmec types.Totally, 251 nasal swabs were collected from HCWs at a referral hospital, from March to September 2017. Disk diffusion method was used to identify Methicillin-Resistant S. aureus carriers, which 14  isolates were MRSA. The most prevalent detected genes were sea and tst, with 22.6% and 9.7%, respectively. The most prevalent SCCmec type was SCCmec type IV (28.6%).Of 251 collected swabs, 31 (12.4%) cases were identified as S. aureus at hospital.We found that the prevalence of MRSA nasal carriage is at high level and must be considered as a significant health care problem at the investigated hospital. Strict implementation of infection-control policies and rational use of antibiotics are the main pillars for controlling the spread of  Staphylococcus aureus (S. aureus) is recognized as one of the most important community-acquired and hospital-associated pathogens that is responsible for a wide range of infections including blood stream infections, soft tissue infections, pneumoniae and urinary tract infections [S. aureus colonizes various sites of the body; however, the anterior nares are the main ecological niches for S. aureus and it has been reported that 20-30% of individuals are persistent carriers of S. aureus and around 30% are transient carriers [S. aureus can evade host immune system defense barriers by producing different enzymes and toxins. In fact, a strong correlation between toxins and disease has been reported. The main S. aureus toxins have been divided into following three groups; 1) pore-forming toxins , 2) exfoliative toxins (ETs) and 3) superantigens toxins  [S. aureus, however, it was gradually replaced with other beta-lactam antibiotics due to the emergence of beta-lactamase producing strains of S. aureus [S. aureus (MRSA) to methicillin resulted in reducing the efficacy of antibiotic. In fact, MRSA isolates harbor mecA gene responsible for production of modified penicillin-binding-protein (PBP) called PBP2a with low affinity for beta-lactam antibiotics [mecA) is located on a genetic element called staphylococcal cassette chromosome (SCC). Based on genetic elements and composition, two types of MRSA have been identified, community acquired methicillin-resistant S. aureus (CA-MRSA) and hospital acquired methicillin-resistant S. aureus (HA-MRSA). Five main types of SCCmec have been characterized. Types I, II and III are the most prevalent types in HA-MRSA, whereas types IV and V are the most often associated with CA-MRSA [S. aureus, play an important role in the transmission of S. aureus infections within and between wards [S. aureus infections for hospital screening of HCWs and obtain knowledge about virulence factors, genetic diversity can provide useful information for adapting infection control programs. Therefore, the aim of this study was to determine the prevalence of nasal carriage of S. aureus and its molecular characteristics isolated among HCWs at a referral hospital in Zabol province, southeast of Iran.fections . In huma. aureus ,5. Methiibiotics ,5. This  CA-MRSA ,5. Healten wards . To reduStudy design and setting: this cross-sectional study was carried out from March to September 2017 at Amir-al-momenin hospital affiliated to Zabol University of Medical Sciences, Iran. This hospital is the only referral hospital in the region with 224 beds, serving about four hundred thousand people.Sample collection and bacterial identification: we collected samples from staffs who were consent to participate in the study. Participants were excluded if they had history of antibiotics consumption in three weeks prior to sample collections. Sterile cotton swab was used to collect sample from nasal cavity of participated HCWs. For all participants, sex and job were recorded. Nasal swabs were immediately inoculated onto Mannitol Salt Agar . After 24-48 hours incubation at 37oC, S. aureus were identified by common standard microbiological tests including catalase, Gram staining, DNase and coagulase [Antimicrobial susceptibility testing: to identify MRSA isolates, disk diffusion method  was used according to Clinical Laboratory Standard Institute (CLSI) guidelines [mecA-specific primers and PCR [idelines . All iso and PCR .Molecular identification of SCCmec types by multiplex-PCR: bacterial genomic DNAs were extracted according to previously described methods [mecA positive isolates were subjected to SCCmec typing by using previously designed primers and conditions [ methods . All mecnditions .Molecular detection of virulence genes: the PCR amplifications were carried out on extracted DNAs for the detection of following genes; sea, seb, sec, sed, see, tst, eta, etb, lukF-PV and lukS-PV [ lukS-PV ,12. All Statistical analysis: data entry and statistical analysis were performed using SPSS version 16  software and Chi-square or exact Fisher´s tests. P < 0.05 was considered statistically significant.Ethical considerations: this study was approved by the Ethics committee of Zabol University of Medical Sciences, Zabol, Iran (Code: zbmu.1.REC.1396.34).S. aureus carriers including nurses 15 , laboratory staffs five , midwives four , operating room technicians three , nurse assistants three  and physicians one . Out of 31 isolated S. aureus 14  isolates were MRSA. As shown in S. aureus (MSSA) were sea positive (p =0.8). The prevalence of tst among MRSA and MSSA isolates were 14.3% and 5.8%, respectively (p = 0.4). Other virulent genes were not detected. The results of SCCmec typing showed that 28.6% and 7.1% of MRSA harbored SCCmec type IV and I, respectively. The rest of the isolates were untypable.In this study, 251 nasal swabs were totally collected from HCWs. Of 251 taken swabs, 180 (71.7%) and 71 (28.3%) ones belonged to female and male, respectively. The most percentage of samples were taken from nurses with 59.3% (149), followed by midwives with 14.4% (36), operating room technicians with 12% (30), laboratory staffs with 9.6% (24), nurse assistants with 4.4% (11) and physicians with 0.3% (1) of total samples. Among these samples, 31 (12.4%) cases were S. aureus is one of the most important nosocomial pathogens and is the leading cause of life-threating infections such as bacteremia, pleuropulmonary, infective endocarditis and soft tissue infections [S. aureus at hospitals is usually occurred by direct skin-to-skin contact or indirect contact via contaminated medical devices and surfaces [S. aureus nasal carriage among HCWs in Zabol province has not been determined and this is the first report. In this study, nasal carriage of S. aureus was identified in 12.4% of studied HCWs. This prevalence is lower than previous reports from different provinces of Iran. For example, a study conducted by Askarian et al. in Shiraz province, southwest of Iran, showed that 31% of studied HCWs were S. aureus carriers [S. aureus among HCWs was 19% and 37%, respectively [et al. revealed that the average mean prevalence of HCWs carriers in different provinces of Iran was 22.7% [S. aureus are dependent on different factors, such as age, alcoholism, chronic disease, as well as variations in sample size, identification methods and infection control measures [et al. (21.9%) and Dulon et al. (0.2% -14.5%) in Nepal and European countries, respectively [S. aureus was investigated. Our results revealed that the most prevalent virulence factor was enterotoxin A (22.6%) followed by toxic shock toxin (9.7%) and enterotoxin C (3.2%). Other virulence genes such as seb, sed, see, eta and etb were not detected. These results are in agreement with other reports from Malaya and Spain [etb was more prevalent in MRSA and eta was mainly prevalent in MSSA [et al. revealed that some clonal types of S. aureus with as specific genotypes are mostly prevalent in severe infections and are contributed to poor patients' outcomes [fections . The trasurfaces . Regardicarriers . Furtherectively . A comprectively ,18. Moreectively . There a [et al. .9% and Dnd Spain . We did  in MSSA . This di in MSSA ,25,26. FS. aureus nasal carriage at the investigated hospital is lower than most provinces of Iran. However, the prevalence of MRSA is alarmingly high and must be taken into consideration for designing infection control programs. This high rate nasal MRSA carriage among HCWs of investigated hospital can be attributed to misuse of antibiotics, poor compliance to hand hygiene, and ineffective implementation of infection control rules. Our results revealed that in order to prevent infection by S. aureus with considerable virulence factors at the investigated hospital constant monitoring and appropriate eradication of nasal carriage must be performed.We found that the prevalence of Staphylococcus aureus strains are considered a serious public health concern worldwide, causing different kind of infections including blood stream infections, wound infections and pneumoniae;Staphylococcus aureus (MRSA) is identified as a hospital-acquired pathogen that due to production of different virulence factors and emergence of several antibiotic resistance mechanisms are associated with high mortality and morbidity in hospitals.Methicillin-resistant Staphylococcus aureus strains in Zabol hospital is alarmingly high;The prevalence of methicillin-resistant S. aureus strains;Molecular analysis revealed that sea and tst were the most prevalent virulence factors in identified Staffs of investigated hospital could be potential sources of MRSA, therefore elimination of strains from nasal carriage can hamper spread of MRSA infections in Zabol hospital.The authors declare no competing interests.


Rhus (sumac) has received a significant amount of study from phylogenetic and biogeographic perspectives, but genomic studies in this genus are lacking. Rhus integrifolia and R. ovata are two shrubby species of high ecological importance in the southwestern USA and Mexico, where they occupy coastal scrub and chaparral habitats. They hybridize frequently, representing a fascinating system in which to investigate the opposing effects of hybridization and divergent selection, yet are poorly characterized from a genomic perspective. In this study, complete plastid genomes were sequenced for one accession of R. integrifolia and one each of R. ovata from California and Arizona. Sequence variation among these three accessions was characterized, and PCR primers potentially useful in phylogeographic studies were designed. Phylogenomic analyses were conducted based on a robustly supported phylogenetic framework based on 52 complete plastomes across the order Sapindales. Repeat content, rather than the size of the inverted repeat, had a stronger relative association with total plastome length across Sapindales when analyzed with phylogenetic least squares regression. Variation at the inverted repeat boundary within Rhus was striking, resulting in major shifts and independent gene losses. Specifically, rps19 was lost independently in the R. integrifolia-ovata complex and in R. chinensis, with a further loss of rps22 and a major contraction of the inverted repeat in two accessions of the latter. Rhus represents a promising novel system to study plastome structural variation of photosynthetic angiosperms at and below the species level.Plastid genomes (plastomes) represent rich sources of information for phylogenomics, from higher-level studies to below the species level. The genus  The plastid genomes (plastomes) of green plants have been used as phylogenetic tools and studied from an evolutionary standpoint for decades . Across Rhus L.  has received significant attention in phylogenetic and biogeographic studies  and numerous ecologically and economically important trees and shrubs. Within the family Anacardiaceae R. Br. (with ~700 accepted species) the genus  studies . Despitephina L. . InteresRhus integrifolia (Nutt.) Benth. & Hook. f. ex W.H. Brewer & S. Watson and R. ovata S. Watson are two shrubby tree species native to the southwestern USA, Baja California, and outlying islands (R. integrifolia and “sugarbush” for R. ovata)  . Further. ovata) .Rhus, R. integrifolia and R. ovata are sister species , and two individuals of R. ovata  was used to assess read quality before and after trimming and filtering.Voucher specimens were collected for one individual of protocol , modifieprotocol . After p 320c CA . FASTQC Rhus ovata plastid rbcL (GenBank MF963245) as a starting seed. Initial assemblies revealed coverage depth estimates > 1,000×, so each read pool was down-sampled to approximately 200× using a custom UNIX command before assembly. Total read pools were mapped back to assembled circular chromosomes under stringent parameters in Geneious v.10 (http://www.geneious.com/), allowing no gaps and no mismatches, in order to check the accuracy of assemblies and calculate coverage depth. The inverted repeat boundaries were verified via continuous paired-end read coverage and the self-dotplot function in Geneious. FASTA files of each chromosome were then rearranged into the same syntenic orientation, with the Large Single Copy (LSC), Inverted Repeat B (IRb), Small Single Copy (SSC), and Inverted Repeat A (IRa), respectively.Plastomes were assembled into complete circular chromosomes with the Perl script ‘NOVOPlasty v.3.8’  specifyiAnacardium occidentale L. GenBank # KY635877; Mangifera indica L. KX871231; Pistacia weinmaniifolia J. Poiss. ex Franch. MF630953; Spondias mombin L. KY828469; Toxicodendron vernicifluum (Stokes) F.A. Barkley MK419151; Rhus chinensisMF351625 and Citrus aurantiifolia (Christm.) Swingle KJ865401). Annotations were examined in Geneious to identify mis-annotated regions or problems with predicted translations, and then were adjusted in the NCBI Sequin software (https://www.ncbi.nlm.nih.gov/Sequin). Annotated plastomes were submitted to NCBI GenBank under accession numbers MT024991–MT024993 , quantifying the numbers of forward-forward, forward-compliment, forward-reverse, and palindromic repeats with motif lengths >20 bp. A Hamming distance of 3 and e-value cutoff of 10−3 were specified during the search. Perfect tandem repeat content for motifs from 2 to 50 bp were quantified with the Phobos plugin for Geneious 10 . Information content per locus (here termed “IC”) for each spacer, intron, or protein-coding region yielding variation was calculated as: IC = ((# SNP + # Indels)/locus length) × 100. These were ranked by locus length, and length vs. IC were plotted for each locus to identify highly variable regions within the typical target range for PCR amplification and Sanger sequencing with a single primer pair . Primers were designed using the Primer3  plugin for Geneious. Additional information on sequence variation among Rhus accessions was calculated in DnaSP v.6. , and two outgroup taxa: Bretschneidera sinensis Hemsl. GenBank # NC_037753  and Shorea pachyphylla Ridl. ex Symington MK841940 . In order to make the analyses computationally tractable, a single member of each available genus was included representing seven of the nine families of Sapindales (Biebersteiniaceae and Kirkiaceae had no publicly available complete plastomes). Multiple species of some genera were included due to notable plastome length variation, including: Commiphora Jacq. (Burseraceae Kunth); Rhus (Anacardiaceae); Acer L. and Dipteronia Oliv. (Sapindaceae Juss.); and Entandrophragma C. DC. (Meliaceae Juss.). IR annotations were added to plastomes missing this information in GENEIOUS via the self-dotplot function, and the second IR copy was removed from all accessions (IRa). The fifty-four complete plastomes (minus IRa) were aligned with progressiveMAUVE as above. The number of syntenic, locally collinear blocks (LCB) was calculated and subjected to a pairwise “double cut and join” (DCJ) analysis in MAUVE to estimate and visualize the number of genomic rearrangements and major inversions.A representative sample of species was chosen from complete, annotated plastid genomes of Sapindales to investigate plastid genomic structural variation across the order. This included 52 completely sequenced and annotated plastomes  for a total of 209,579 bp in aligned length, including both coding and non-coding regions. RAxML  was usedhttps://irscope.shinyapps.io/irapp/), and outputs a JPEG illustration of IR boundary dynamics.To investigate the relationship between and total plastome length, IR length, and total tandem repeat content, all accessions were compared via phylogenetic least-squares regression (PGLS), which accounts for phylogenetic signal in trait covariance among species , using tRhus ranged from 160,141 bp (R. integrifolia) to 160,262 bp (R. ovata from CA) , and an average pairwise nucleotide diversity (π) of 0.00055 ± 0.00016. The number of nucleotide substitutions in pairwise comparisons ranged from 127 between Californian and Arizonan R. ovata to 208 between Californian R. ovata and R. integrifolia (ndhC-trnVUAC spacer that varied in copy number between the three Rhus accessions (ATT TTT TTT ATT ATT AAT TAT T), with one unit in R. integrifolia, two units in the Arizonan accession of R. ovata, and three in the Californian accession of R. ovata.Assembled plastome sequences for from CA) ; File S1grifolia . All thrgrifolia ; File S2grifolia . Of partRhus accessions (Rhus chinensis (GenBank accessions MF351625 and KX447140) displayed a major contraction of the IR relative to R. integrifolia, R. ovata, R. typhina, and another accession of R. chinensis (MG267385), with the IR being some 10 KB smaller in the former R. chinensis accessions. In the two accessions of R. chinensis with a reduced IR, four genes normally found in the IR are instead found in the LSC region: rpl2, rpl23, trnICAU, and ycf2.Overall plastome structure and synteny did not differ among all three newly sequenced cessions . InteresR. integrifolia and R. ovata, and these are predominantly found in the LSC region  were weakly supported as sister to one another (BS = 72), and together sister to R. integrifolia. The Rhus integrifolia-ovata clade grouped as sister to all other Rhus accessions. Rhus was grouped as sister to Pistacia, Toxicodendron, Anacardium L. + Mangifera L., Sclerocarya Hochst., and Spondias L., respectively.The concatenated matrix of 54 taxa and 209,573 aligned bp had 26,989 variable positions, excluding gaps, with 16,421 parsimony informative sites, 10,568 singleton sites, and 87,070 sites including at least one inferred gap . Fig. 4 p = 0.0017), while there was no signal in IR length  or repeat content  based on analysis of 54 plastomes. Phylogenetic least squares regression revealed no association between IR length and total plastome length , but a significant, albeit weak association between repeat content and total plastome length . A model including both IR length and repeat content as explanatory variables yielded similar results , wherein repeat content was significantly associated with total plastome length (p = 0.0002) and IR length was not (p = 0.2189). Many members of Sapindaceae display relatively smaller total plastome lengths ), it can be inferred that rps19 was lost at least twice within Rhus, based on the current sampling.A closer analysis of IR boundaries within unctions . The moshinensis . The IR C region . This isRhus integrifolia-R. ovata species complex were sequenced, annotated, and analyzed in a comparative context with several other complete plastomes from the order Sapindales. Patterns of nucleotide polymorphism and indel variation were characterized among the three newly generated Rhus plastomes, providing genomic resources for population-level and phylogeographic study of these ecologically and economically important species. Additionally, phylogenetic analysis of 52 complete plastomes in order Sapindales provides resolution and robust branch support among most families within the order, allowing a well-supported basis for phylogenomic comparison of plastid genome evolution. The most striking finding was the dynamic evolution of the inverted repeat boundary in the genus Rhus, which was evident based on limited sampling of only seven accessions representing four species of this genus. This included independent losses of the rps19 gene in R. ovata-R. integrifolia complex and in R. chinensis, with an additional 9.8 KB contraction of the IR within two of three individuals of R. chinensis. These findings suggest that the genus Rhus may have experienced several such events, providing a potentially novel, dynamic model system in which to study plastome structural changes in angiosperms at and below the species level.Three complete plastid genomes of the Rhus accessions are somewhat typical for angiosperms  Garcke  and 613–621 tandem repeats of motif length 2–20 bp  Garcke , Macadam& Betche , QuercusCarruth. , Utricul& Girard , Capsicunnuum L. , Holcogltr. spp. . Repetit 2–20 bp . These r regions . FurtherR probes .Nitraria L. and Peganum L.), which were not included in the aforementioned study.Several previous studies have addressed phylogenetic relationships among families of the order Sapindales, or have included representatives of Sapindales in larger studies of angiosperm relationships . HoweverrbcL, atpB, and the trnL-trnF spacer. While most of the deep branch support values in that study were moderate to high, the placement of Biebersteiniaceae and Nitrariaceae among other members of Sapindales received no support. Additionally, there was moderate support for Meliaceae as sister to Simaroubaceae, whereas in the current study and in that of Missing from the current study are representatives of two sapindalean families, Biebersteiniaceae and Kirkiaceae. Anacardium occidentale, Rhus chinensis; Asarum; Boundary shifts of the IR in plastid genomes are hypothesized to be one of the primary drivers of overall plastid genome size variation . HoweverRhus, based on comparison of complete plastomes across the order Sapindales.Perhaps the most striking finding in the current study is the dynamic nature of evolution of the IR boundary in Rhus, it is evident that IR boundaries are variable at and below the species levels  region of the plastome is the most conserved in terms of sequence variation  relatives levels . Thus, iR. integrifolia-ovata (rps19) and R. chinensis . Plastome evolution was investigated in a robustly supported phylogenomic framework, revealing striking variation within Rhus in terms of dynamics of the LSC-IR boundary, with multiple, independent instances of gene loss. Rhus presents a promising novel system in which to study the dynamics of plastome structural variation in photosynthetic angiosperms at or below the species level, and continued representation of plastomes in this genus is likely to reveal additional changes.Three complete plastid genomes were generated for two ecologically important North American shrub species in the genus 10.7717/peerj.9315/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.9315/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.9315/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.9315/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.9315/supp-5Supplemental Information 5Click here for additional data file.


Gossypium spp.) is the most important world-wide fiber crop but salt stress limits cotton production in coastal and other areas. Growth regulation factors (GRFs) play regulatory roles in response to salt stress, but their roles have not been studied in cotton under salt stress.Cotton  were largely retained in allopolyploid cotton, with subsequent gene expansion in G. barbadense relative to G. hirsutum. Most G. hirsutum GRF (GhGRF) genes are preferentially expressed in young and growing tissues. To explore their possible role in salt stress, we used qRT-PCR to study expression responses to NaCl treatment, showing that five GhGRF genes were down-regulated in leaves. RNA-seq experiments showed that seven GhGRF genes exhibited decreased expression in leaves under NaCl treatment, three of which  were identified by both RNA-seq and qRT-PCR. We also identified six and three GRF genes that exhibit decreased expression under salt stress in G. arboreum and G. barbadense, respectively. Consistent with its lack of leaf withering or yellowing under the salt treatment conditions, G. arboreum had better salt tolerance than G. hirsutum and G. barbadense. Our results suggest that GRF genes are involved in salt stress responses in Gossypium.We identified 19 GRF genes in In summary, we identified candidate GRF genes that were involved in salt stress responses in cotton. Gossypium) is the most important fiber crop in the world. While the genus itself contains more than 50 species, cultivated cottons are composed of four independently domesticated species, among which the Upland cotton (G. hirsutum L.) is the most prominent cultivar, accounting for 95% of global cotton fiber output [Cotton , incc stress , 24, reqve genes .Gossypium. Multiple high-quality genome sequences are available for several species, including two diploid species, i.e., G. raimondii (D5) [G. arboreum  [G. hirsutum (AD1) [G. barbadense (AD2) [Gossypium and their potential roles as salt stress-related genes. Here, we report our genome-wide analysis of the GRF transcription factor genes in four important cotton species, reporting a modestly sized family  that are largely conserved during evolution and polyploidization. We further explore the role of GRF genes in response to salt stress. We identified three G. hirsutum GRF genes, six G. arboreum GRF genes, and three G. barbadense GRF genes that exhibit decreased expression under salt stress, respectively. Compared to the tetraploid species, diploid cotton G. arboreum was more salt tolerant. These candidate GRF genes may be useful in future molecular breeding of salt-tolerant cotton species.Recent advances in cotton genomics have produced the resources necessary to characterize the GRF gene family in dii (D5) , 26 and tivated) , 28, andum (AD1) –31 and Gse (AD2) . These rG. hirsutum genome database with these two conserved domains (PF08879 for WRC domain and PF08880 for QLQ domain) identified 34 putative GRF-encoding genes, here designated GhGRF1A–GhGRF17A (A homoeologs) and GhGRF1D–GhGRF17D (D homoeologs), where the A and D suffix in the gene name indicates the genome of origin. These 34 GhGRF genes are dispersed over 20 of the 26 G. hirsutum chromosomes, with most, but not all, homoeologs conserved  for the recovered GhGRF genes contain predicted introns ; however, intron structure among homoeologous pairs can exhibit variation in intron number (1 to 4) and length. Three of the homoeologous gene pairs did exhibit divergence in structure between homoeologs, namely GhGRF9A vs GhGRF9D, GhGRF16A vs GhGRF16D and GhGRF17A vs GhGRF17D. In two of the three cases , the acquisition of a splice site in the A-homoeolog led to an additional, large intron. For GhGRF17A/D, however, the D-homoeolog exhibits loss of splicing for the first intron, resulting in read-through transcription. Characterization of parental (in the diploids) gene structure for these three homoeologs suggests that this structural variation represents inherited, parental divergence. Phylogenetic analysis of the GRF gene family combined with intron/exon structure characterization naturally divides the family into six groups, here designated A-F, containing 3 to 12 genes  with 9 Arabidopsis thaliana GRFs for phylogenetic analysis , with each homoeolog (At or Dt) sister to their respective parental copies.Overall, the expected diploid-polyploid topology is reflected in the tree for each set of orthologous/homoeologous genes, indicating general preservation during diploid divergence and through polyploid evolution. That is, the number of GRF genes in GhGRF13 is missing from polyploid genome, as is both copy of GaGRF11/GrGRF13 (one from each subgenome). In clade D, genes related to AtGRF8 exhibit duplication in G. arboreum only, while the A-homoeolog from either polyploid species was not recovered from the genome sequence. This may indicate a duplication in G. arboreum after divergence from the polyploid ancestor coupled with loss in the A-subgenome of the polyploid. The evolution leading to clade E is less straightforward in that it is composed of eight genes, including one from each diploid progenitor and three from each tetraploid species. The phylogenetic topology, however, does not suggest a simple duplication of one homoeologous copy; rather, the homoeologous copies of GhGRF8 are placed sister first to each other and then to G. arboreum only. The remaining GRF from G. hirsutum, GhGRF7D, is sister to the only G. raimondii GRF of this clade and has no inferred homoeologous AT copy. Given this pattern of relationships and that G. raimondii has one additional GRF , it seems possible that GhGRF7D represents a uniquely inherited GRF present in the D ancestor only and that the G. raimondii ortholog of GhGRF8D was lost after polyploidization. Alternatively, this pattern could reflect a non-syntenic duplication of GhGRF8D, followed by conversion of the original GhGRF8D into an A-like copy. It bears noting, however, that these observed deviations could be due to errors in the genome sequences; however, the general preservation of GRF copies in the expected relationships suggests general robustness of the analysis. Clade G contains 11 GRF genes, one from G. raimondii and the other ten from G. barbadense, also indicative of substantial duplication in G. barbadense. These new genes belongs to one family and might specially originate in G. raimondii and the tetraploid progenitor of G. barbadense or undergone the homoeolog loss in G. arboreum and G. hirsutum.Although the GRF family exhibits general preservation, a few deviations were noted. For example, Clade C exhibits evidence of homoeolog loss; that is, the D copy of GRF gene was identified as putatively encoding a GRF protein (in Chlamydomonas reinhardtii only); however, all land plant species surveyed recovered a minimum of five putative GRF genes in Physcomitrella patens and nearly double were found in angiosperms . Among angiosperms, GRF copy number varied from the minimum of 8, in Amborella trichopoda, to 48 copies in the tetraploid Brassica napus. Four monocot species were included, where between 9 (in rice) to 27 (in maize) copies were detected. Notably, this high copy number in maize is slightly more than double the copy number in sorghum, likely reflective of the duplicated history of maize. GRF copy numbers in the eudicot species surveyed varied even more, from 12 putative GRF genes in Carica papaya to 48 in Brassica napus, notably another polyploid species genome.We further evaluated the general preservation of GRF genes in plants using 28 representative species and the same search criteria  and G. arboreum (model A-genome ancestor), respectively, dispersed among 10 of the 13 chromosomes , G. barbadense has gained 9 additional predicted copies through duplication  was asGhGRF genes to leaf physiology, we first evaluated their expression under normal conditions in 12 different cotton tissues, including root, stem, leaf, cotyledon, hypocotyl, petal, stamen, pistil, sepals, receptacle, ovules (0 dpa) and fiber (6 dpa), using both qPCR and existing RNA-seq data; homoeologs were not distinguished in the qPCR analysis. Most GhGRF genes exhibited different expression patterns among the various tissues; however, certain generalities were observed. Several tissues exhibited distinct paralog preference in our qPCR survey. For example, six of the examined GhGRF genes  during the primary elongation stage of cotton fiber development (6 dpa). Transcripts of GhGRF1, GhGRF2, GhGRF3 and GhGRF4 were most abundant in stem and root, whereas GhGRF16 and GhGRF17 were preferentially expressed in cotyledon. Over half of the GhGRF genes showed the relatively high level of transcripts in leaves.To evaluate the contribution of these nd fiber  dpa, usind fiber  dpa, usind fiber  dpa, usiG. hirsutum genome sequencing project [GhGRF14, which exhibited the highest expression in pistil by the RNA-Seq data, but showed higher expression in ovule (0 dpa) and fiber (6 dpa) using qPCR  data , whereas treatment with 500 mM NaCl resulted in leaf yellowing and withering  also exhibited significantly decreased expression levels (≥2-fold change) at four time-points during NaCl stress in the available RNA-seq data  exhibited downregulation in either G. hirsutum or G. arboreum. Of the three genes downregulated in G. barbadense, expression changes of GbGRF17 was significant for the 200 mM and 500 mM NaCl at three time-points  post-treatment. These results demonstrate that (1) G. arboreum has better salt tolerance than both G. hirsutum and G. barbadense, congruent with the previous observations [We also observed the phenotypic changes of PCR Fig. . Phenotyves Fig. a, downrenes Fig. b was nevaCl Fig. c; howevenes Fig. d exhibitrvations , 48, 49,Arabidopsis thaliana [Zea mays [Brassica napus [Brassica rapa [Oryza sativa [Brachypodium distachyon [Solanum lycopersicum [G. hirsutum, and show that these genes are generally conserved among the diploid progenitors and the cultivated allotetraploid cottons. Analysis of the GRF genes in an additional cotton species, as well as representatives of diverse plant lineages, show that the family is somewhat labile but the most remarkable changes in copy number are attributable to retention after genome duplication. In tetraploid cottons, the gene copy number is nearly additive in G. hirsutum, but contains 25% more paralogs in the related  G. barbadense genome. Phylogenetic analysis indicated that these additive genes belongs to one family and specially originate in G. barbadense. Further research is required to discern what functional relevance these additional copies have.The GRF transcription factor family of genes has been investigated in a number of plant species, including thaliana , 50–53, Zea mays , Brassicca napus , Brassicica rapa , Oryza sa sativa –58, Bracstachyon  and Solapersicum . The fampersicum  and a plpersicum , 58. TheGhGRF genes are variably expressed in different cotton tissues with particularly high expression in ovules. The decreased expression of several GhGRF genes in response to salt stress treatments implies their function in the regulations of growth and development under the abiotic stress conditions. Together, our results provide data to facilitate the functional identification of the GRF genes in cotton plant growth, development and stress tolerance.We systematically characterized cotton GRF family genes using bioinformatic and phylogenetic approaches and gene expression analyses. We analyzed gene structures, chromosomal locations, intron-exon organizations, phylogenetic relationships and expression profile patterns in different cotton tissues and under salt stress condition to predict their possible biological functions. G. raimondii [G. arboreum [G. hirsutum [G. barbadense [Gossypium genome databases, i.e., G. raimondii, G. arboreum, and G. hirsutum, to identify putative GRF genes (GhGRFs) that contain the relevant conserved protein domains. Genes were considered candidate GRFs if they harbored WRC and QLQ domains within the N-terminal [GRF gene sequences from Arabidopsis thaliana (AtGRFs) were retrieved from the TAIR database [Arabidopsis gene was verified using the SMART conserved domain search tool [We downloaded the genome sequences of four cotton species from the CottonGen database , includiaimondii , G. arboarboreum , G. hirshirsutum , and G. rbadense . To idenrbadense  profile rbadense  using determinal . Previoudatabase  for phylrch tool  and Pfamrch tool . The samGhGRFs were extracted from the genome annotation gff3 file [GhGRFs was visualized using MapChart [GhGRF proteins were determined using the ProtParam tool [Chromosomal locations for each of the above identified ff3 file . ChromosMapChart , and theMapChart . The numram tool .Arabidopsis were aligned using MAFFT with the G-INS-i algorithm [Complete protein-coding sequences for each of these genes from all three cotton species and lgorithm . Phylogelgorithm  by samplG. hirsutum seed, root, stem, leaf, torus, petal, stamen, ovary, calyx, ovule  and fiber  were downloaded from the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA248163) [G. hirsutum genome [GhUBQ7 (accession number: DQ116441), which is detected at relatively constant levels across different cotton samples [G. hirsutum leaf RNA-Seq data (in FPKM) for each GhGRF gene under salt stress  were retrieved from the ccNET database [p-value of less than 0.05. Multiple Experiment Viewer (MeV) [Raw RNA-Seq data for A248163) , represem genome  via HISAm genome , 73. Ream genome  and the  samples . Potenti samples . Gene exdatabase . Genes wer (MeV)  was usedG. hirsutum, G. arboreum, and G. barbadense. For G. hirsutum, seeds of G. hirsutum cv. R15 [To generate new expression information via qRT-PCR, we grew representatives of  cv. R15 , were geG. arboreum cv. Shixiya1 and G. barbadense H7124 were grown for qRT-PCR of salt-exposed leaf tissue timepoints only. For this experiment, seeds of G. arboreum cv. Shixiya1 were provided by Prof. Tianzhen Zhang and G. barbadense H7124 seeds were provided from the Esquel Group. These two Gossypium species were planted in the Damao field in Sanya, Hainan Province, China. For salt treatment, 50-day old plants were sprayed with 200 and 500 mM NaCl solution after surfactant (Triton X-100) treatment. Leaves from salt-treated plants were collected at 0 (control), 1, 3, and 6 h post-NaCl treatment as above.Similarly, G. hirsutum histone-3  and GhUBQ7 (accession number: DQ116441) genes were used as internal references, and the relative amount of amplified product was calculated following the 2-∆∆Ct method [G. hirsutum samples, relative expression levels among different organs were normalized by calibrating with the root sample from that plant. The root sample was washed with DEPC sterile water three times before extracting the RNA.Total RNAs from cotton tissues were extracted using the RNAprep pure plant kit  according to the manufacturer’s protocol. The resulting RNAs were treated with DNase I prior to synthesizing cDNA with oligo (dT) primers and M-MLV Reverse Transcriptase (Invitrogen); these products were diluted 5-fold before use. For quantitative real-time PCR (qRT-PCR), Primer5 software was used to design gene-specific forward and reverse primers  protein sequencesAdditional file 4: Figure S2. Chromosome distribution of GaGRF (Gossypium arboreum GRF) and GrGRF (Gossypium raimondii GRF) genes.Additional file 5: Figure S3. Expression patterns of GRF genes in G. hirsutum using RNA-seq data. The RNA-seq data expression profiles were from Zhang et al., (2015) and ccNET database [database . FPKM re