Boswellia sacra (Burseraceae), a keystone endemic species, is famous for the production of fragrant oleo-gum resin. However, the genetic make-up especially the genomic information about chloroplast is still unknown. Here, we described for the first time the chloroplast (cp) genome of B. sacra. The complete cp sequence revealed a circular genome of 160,543 bp size with 37.61% GC content. The cp genome is a typical quadripartite chloroplast structure with inverted repeats  separated by small single copy  and large single copy  regions. De novo assembly and annotation showed the presence of 114 unique genes with 83 protein-coding regions. The phylogenetic analysis revealed that the B. sacra cp genome is closely related to the cp genome of Azadirachta indica and Citrus sinensis, while most of the syntenic differences were found in the non-coding regions. The pairwise distance among 76 shared genes of B. sacra and A. indica was highest for atpA, rpl2, rps12 and ycf1. The cp genome of B. sacra reveals a novel genome, which could be used for further studied to understand its diversity, taxonomy and phylogeny. Most of these assembled genomes are associated with economically important crop plants [A major distinguishing organelle of plant cells is the chloroplast, which was suggested to have originated from cyanobacteria through endosymbiosis –3. The mp plants . Chloropp plants . The tecp plants . Thus, uEucommia ulmoides [Poplus cathayana [Quercus spinosa [Acacia ligulata [Pinus armandii [Cocos nucifera [Citrus aurantiifolia [Musa acuminata [Chloroplasts contain circular DNA with nearly 130 genes ranging in size from 72–217 kb –11. Chloulmoides , Poplus  spinosa , Acacia ligulata , Pinus aarmandii , Cocos ntiifolia , Musa accuminata , Norway cuminata , etc. Thcuminata –13.Boswellia sacra; Burseraceae) [Boswellia species, and B. sacra is an endemic species growing only in Dhofar region of Oman [B. sacra using ITS and ISSR analysis, where detailed genomic and conservation studies were suggested. Therefore, it is high time to further understand this keystone species. Hence, in the current study it was aimed to elucidate the complete chloroplast genome of B. sacra and determine the phylogenetic relationship of this tree with related species.To understand the genome structure of chloroplasts, we have investigated the economically and culturally important frankincense-producing tree (eraceae) . There a of Oman –26. The  of Oman , whereas of Oman . In resp of Oman . The cry of Oman . In addi of Oman . The ess of Oman –32. Ther of Oman  has workThe study site was managed by the Museum of the Frankincense Land . The samples were collected from the Natural Park of Frankincense Tree, which is part of the Land of Frankincense sites, inscribed in the UNESCO’s World Cultural and Natural Heritage List. A permission was obtained from the Museum of the Frankincense Land to use the leaf part of the tree resources for research purposes. The trees used for sampling were treated ethically, and our study did not harm the local environment.Wadi Dawkah, Dhofar-Oman  is a completely arid desert region with small sandstone hills. The annual mean temperature of the sampling area is ~35°C, and the annual rainfall is approximately 40 mm . The temBoswellia sacra were washed with sterile distilled water to remove sand particles and soil debris. The chloroplast DNA was extracted by using a modified protocol of Shi et al. [de novo assembled and mapped with the reference genome [de novo assembled into contigs using Mimicking Intelligent Read Assembly (MIRA 4.0) [Azadirachta indica (accession number: NC 023792) through Map Reads to Reference Tool in CLC Genomics Workbench (version 8.5). The order of contigs was resolute according to the reference genomes. The gaps (7 to 150 bp) between the de novo contigs were replaced with consensus sequences of raw reads, which were later, re-mapped to the reference genomes. Any additional gaps were either filled manually by mapping tools in CLC Genomics Workbench (version 8.5) or using GapCloser (http://soap.genomics.org.cn/index.html).The fresh leaves from i et al. . To prepe genome . The gapIRA 4.0) . Second,de novo assembled B. sacra cp genome sequence and Azadirachta indica cp genome sequence, as a reference. In addition, full alignments with annotations were visualized using the VISTA viewer [Genome annotation was performed by using Dual Organellar GenoMe Annotator (DOGMA) , tRNAscaA viewer .http://pgrc.ipk-gatersleben.de/misa/) was used to analyze the types and positions of various simple sequence repeats (SSRs) in B. sacra. The results of this analysis were also correlated with the reference cp genome of A. indica according to the methods of Su et al. [B. sacra cp genome were identified using Tandem Repeats Finder version 4.07 b [MISA  polymorphism of shared genes were calculated using DnaSP 5.10.01 [https://www.biostars.org/p/119214/) based on single-nucleotide polymorphism (SNP) definition (a variation in a single nucleotide that occurs at a specific position in the genome) was employed to call SNPs. In addition, the SNPs in coding regions were classified in two ways: synonymous and nonsynonymous as shown by Guo et al. [B. sacra and Azadirachta indica was performed on MEGA 7.0 [Complete cp genomes as well as a separate partition using only 76 shared genes were employed to analyze the average pairwise sequence divergence for mparison . These rmparison  with themparison . The ind 5.10.01 . A custoo et al. . The numMEGA 7.0 .Boswellia sacra cp genome was performed using Create Alignment and Create Tree tools in CLC genomics workbench version 8.5  [Vitis venifera was used as an outgroup. The accession numbers are provided in Phylogenetic analysis of the -Cantor) . A totalBoswellia sacra. A total of 373,248 reads  were de novo assembled showing at total of 703x cp genome coverage. The chloroplast genome sequence quality was re-confirmed through repeating library preparation and sequencing of the extracted cpDNA from B. sacra tree. This result was sufficient to produce a single consensus sequence representing the complete chloroplast genome of Boswellia sacra. The de novo assembled genome was referenced with already reported cp genomes of Sapindales such as Citrus sinensis and Azadirachta indica. The gaps between the de novo contigs were replaced with consensus sequences of the raw reads and re-mapped to the reference genome. The sequences of the chloroplast genomes were deposited in GenBank (accession numbers: KT934315).Genome sequencing and assembly of Ion Torrent PGM sequencing produced 286.8 Mb of data for chloroplast genome of B. sacra was almost similar to Azadirachta indica  and Citrus sinensis . However, the size was almost 380 bp higher than closer relative in Order Sapindales i.e. Citrus aurantifolia . In case of other arid-land trees, B. sacra size was higher than Acacia ligulata  as shown in the previous studies of cp genomes of these plants [The complete chloroplast genome was 160,543 bp in size, comprising a large single copy (LSC) region of 88,055 bp and a small single copy (SSC) region of 18,962 bp. In addition, a pair of inverted repeats (IRa and IRb) of 26,763 bp each  was alsoe plants , 22, 52.e plants , althouglarge subunit rbcL, two ORF, and other important genes related to ATP-dependent protease/ATP binding subunit (clpP), translational initiation factor (infA), Maturase K (matK), envelope membrane protein (cemA), subunit of acetyl-Co-Acarboxylase (accD), and c-type cytochrome synthesis gene (ccsA) were identified during annotation. The existence of chloroplast-encoded clpP in B. sacra suggested that this gene is indispensable for cell survival. Though this needs further verifications, but the presence of clpP indicates a better system to combat severe heat and drought stress in B. sacra, whereas Cicer arietinum, Lathyrus sativus, and Pisum sativum have lost an intron in both clpP1 and rps12 [clpP gene has also been identified in Acacia ligulata [Trifolium subterraneum [Camellia sp. [Wollemia nobilis [Most of the essential photosynthesis-related genes were identified in the cp genome . Twenty nd rps12 . The clpligulata , Trifolierraneum , Camellillia sp. , Actinidllia sp.  and Woll nobilis .B. sacra and A. indica cp genomes are presented in B. sacra cp genome had approximately 37.61% GC content, which is higher than that of A. indica (37.5%). In the B. sacra cp genome, the intergenic regions, introns, and genic regions account for ca. 33.5%, 9.6%, and 57.5%, respectively, as shown in Tables Indels (3.96%). The sizes of the LSC and SSC regions were somehow different between these two cp genomes, primarily reflecting the large index in each region. The 10 most divergent intergenic regions included the spacer between trnH—GUG-psbA , accD—psaI , atpH—atpI , psbZ—trnG-UCC , trnK—UUU-rps16 , petN—psbM , ycf4—cemA , ccsA—ndhD , trnR-UCU—atpA  and trnL-UAG—ccsA . These findings suggest that a deletion might have occurred in this region in A. indica after the divergence of A. indica and B. sacra.The general characteristics of the indels, the cp genome of B. sacra was highly conserved, however, in total 25 loci were identified with most variable regions. ycf1 was highly variable region showing a 4354 indel sites, followed by atpA with 124 indel sites. The average indel length for ycf1 was 174 bp, followed by rpl14 with deletion of 25.5 bp. On the other hand, the indel diversity per site was highest for rpl2 , followed by rps11 (0.301).In case of for rpl2 . We alsoed genes . Of thesand rps8 . The higtrnH/psbA intergenic region has been studied as a barcode of differentiation at the species level in the plastids of inland plants, such as Araucaria [psbA, which encodes photosystem II protein D1, and trnH-GUG, which functions in histidine translation [trnH/psbA intergenic region in B. sacra. Yap et al. [Agathis dammara compared with Wollemia nobili. Previous detailed BLAST analyses have shown that this Indel is present in nineteen Araucaria species, including W. nobili [psbA gene improves drought tolerance in tobacco plant. The occurrence of psbA in B. sacra suggests a strong ability to counteract oxidative stress, as this tree frequently encounters low-water conditions, however, this still needs further in-depth investigations.The raucaria –59. Thisnslation –13. The p et al.  suggeste. nobili . In addi. nobili  demonstrB. sacra and A. indica. These regions showed homology in genome organization between the two species, although the sequences are inverted relative to each other. The LCBs of protein coding, tRNA, and rRNA genes showed a similar synteny for both cp genomes. Similarly, the two cp genomes were run global alignment through VISTA to understand the comparative genomic organization [petN and psbM; accD and psal; ycf4 and cemA; rpl32 and trnL; atpH and atpl). The most divergent in coding regions were trnl, matK, clpP, rpl33, accD, rps3, ndh , ycf3 and ycf1  of the total sequence , 31.21% (T/U), 19.49% (C), and 18.71% (G) in the cp genomes. The transition/transversion rate ratios are k1 = 2.149 (purines) and k2 = 2.072 (pyrimidines). The overall transition/transversion bias is R = 0.996. These shows that B. sacra, A. indica and Citrus sinensis species with long generation times, and both exhibit little changes in the structural organization of the chloroplast genomes. This could further reveal how these species evolved at low rates. Similar perspectives were shown for the chloroplast genomes of five Quercus species [In case of single-nucleotide polymorphism (SNP) (a variation in a single nucleotide that occurs at a specific position in the genome), maximum likelihood analysis of natural selection codon-by-codon, was performed to estimate the number of inferred synonymous (s) and nonsynonymous (n) substitutions which are presented along with the numbers of sites that are estimated to be synonymous (S) and non-synonymous (N) . These end dN-dS . However species . The est species –68.orf56 and ycf68 genes of three tree species viz. B. sacra, A. indica and C. sinensis , 2 species from Melianthaceae (Francoa sonchifolia and Theorbroma cacao), 3 species from Malvaceae , 15 species from Brassicaceae , one species from Caricaceae (Carica papaya) and one species from Rutaceae (Citrus sinensis).To obtain insight into the position of outgroup . We condA. indica species from Maliaceae. The neighbor-joining phylogenetic tree resolved 33 nodes, with strong bootstrap support of 55–100 . Krishnan et al. [A. indica and Citrus sinensis form a homogenous group (100%), validating the conventional taxonomic classification of A. indica. Thus, the results of the phylogenetic analysis of the cp genome of both species showed the formation of a distinct cladogram with B. sacra, revealing new insights to understand the gene structure of these three plants.The phylogenetic analysis included some commercially important Sapindales plants and the more relevant f 55–100 . These rn et al.  suggesteB. sacra and A. indica are different, however, the fact that both plants produce oleugum and belong to the same order suggests interesting prospects for broader evolutionary studies of Sapindales [C. sinensis and A. indica (C. sinensis and A. indica. Whereas a sister relationship of B. sacra was well supported (100/1.00). The posterior probabilities (PP) were also 1.0 for B. sacra, C. sinensis and A. indica , B. sacra). A similar phylogenomic reconstruction was also noticed by Wang et al. [The habitats of pindales . This phpindales , suggest. indica . The Bay. indica , suggestg et al. ,75, wherde novo assembly and reference genome analysis, we provided the first completely sequenced chloroplast genome of endemic Boswellia sacra. The genome organization and gene structure is almost typical to that of angiosperms plants. The main difference in the gene composition between the B. sacra and A. indica cp genomes was the protein-coding gene in the trnH-GUG-psbA region. Notably, eight shared SSR regions were identified in the B. sacra and A. indica cp genome comparison. Elucidating these regions could provide additional phylogenetic insights at the taxonomic level, and these results could be used to develop molecular markers [Boswellia chloroplast genome evolution. The current new genomic datasets will enable further exploration of the genetic diversity of Boswellia sacra and the other 18 species of this genus.Using a combination of  markers . Previou markers  suggesteS1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLS)Click here for additional data file.S1 FigB. sacra genome is shown on top as the reference genome. Within each of the alignments, local collinear blocks are represented as blocks of the same color connected with lines. Annotations are shown above and below the LCBs, protein-coding genes are indicated as white boxes, tRNA genes are shown in green, and rRNA genes are shown in red. The lowered position of a box indicates an inverted orientation.The (TIFF)Click here for additional data file.S2 FigGossypium incanum was included as an outgroup to root the tree. A total of 21 complete chloroplast genomes were aligned. In total, 21 nodes were resolved. The position of B. sacra is shown in green circular symbol.The (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.


Burkholderia pseudomallei (a gram-negative bacterium) is the major causative agent for melioidosis, which is prevalent in Singapore, Malaysia, Thailand, Vietnam, and Northern Australia. Melioidosis patients are increasingly being recognized in other parts of the world. The bacteria are intrinsically resistant to many antimicrobial agents, but prolonged treatment, especially with combinations of antibiotics, may be effective. Despite therapy, the overall case fatality rate of septicemia in melioidosis remains significantly high. Intracellular survival of the bacteria within macrophages may progress to chronic infections, and about 10% of patients suffer relapses. In the coming decades, melioidosis will increasingly afflict travelers throughout many global regions. Clinicians managing travelers returning from the subtropics or tropics with severe pneumonia or septicemia should consider acute melioidosis as a differential diagnosis. Patients with open skin wounds, diabetes, or chronic renal disease are at higher risk for melioidosis and should avoid direct contact with soil and standing water in endemic regions. Furthermore, there are fears that B. pseudomallei may be used as a biological weapon. Technological advancements in molecular diagnostics and antibiotic therapy are improving the disease outcomes in endemic areas throughout Asia. Research and development efforts on vaccine candidates against melioidosis are ongoing.This review briefly summarizes the geographical distribution and clinical impact of melioidosis, especially in the tropics.  B. pseudomallei is the causal agent of melioidosis  . Th. Th29]. The prevalence of this infection is 0.2% among the population in Singapore, yet it is related to huge death and relapse rates. An earlier study showed that of 12 patients, 6 had relapsed and 2 died . As justB. pseudomallei, while other cases were confirmed by an indirect hemagglutination antibody titer of 1:16 or greater [The first case of melioidosis was reported in Thailand in 1955 , 3 indig greater ,42.Melioidosis cases have been documented in soldiers stationed in various regions throughout Vietnam. Military personnel serving in Vietnam have been reported with acute and fatal melioidosis ,44  among 3,653 blood cultures from febrile patients admitted to the Centre for Tropical Diseases in Ho Chi Minh City [B. pseudomallei was detected from 73 sites ; however, only 21% of the isolates were of the virulent L-arabinose (ara)-negative variety from 9 different rice fields. The low incidence of melioidosis in the provinces around Ho Chi Minh City can be attributed to the restricted distribution of B. pseudomallei in the soil throughout the area [B. pseudomallei was isolated in 40%. Among patients who died during hospitalization, 32% were infected with B. pseudomallei [Clinical surveillance and environmental sampling in southern Vietnam (1992–1998) showed only 9 inh City . Soil sathe area . There wthe area . Pleuropthe area . Subsequthe area . A more domallei ,57.Melioidosis was first described in Australia (in North Queensland) following a 1949 outbreak in sheep . A melioB. pseudomallei isolates were collected from temperate areas in the Southwest and Western regions of Australia, and molecular typing reveals similar clonality for the past 25 years. Melioidosis is endemic throughout Northern Australia and Queensland, according to an epidemiology report on melioidosis from 2001 and 2002 [The spreading of melioidosis was mainly due to importation of animals from the temperate region of Northern Australia. and 2002 . The annand 2002 ,60. Prevand 2002 .B. pseudomallei infection has been documented in a patient who acquired the infection by aspiration of river water in Philippines during a near-drowning incident [B. pseudomallei infection (1996–2000). One patient was a 56-year-old man working as a ranger at Ken-Ting Farm in southern Taiwan. He had been to Thailand for sightseeing 5 years before [B. pseudomallei on the fifth day following admission. Another patient died by the ninth day, despite intensive care and a broad-spectrum antimicrobial regimen [Melioidosis is an important disease in Taiwan. incident . Nearly s before . The pat regimen . MelioidB. pseudomallei [Several cases have been reported from different regions of India, but only a few medical centers have successfully identified this bacterium –69. For domallei ,71–72, wdomallei ,66. Chrodomallei . In Indidomallei . A previdomallei .In other parts of Asia, infrequent cases of melioidosis from Indonesia were reported in the Dutch literature . HoweverB. pseudomallei has also been detected in the soil and animals in various African countries [Sporadic cases of melioidosis have been reported in Nigeria, Gambia, Kenya, and Uganda; however, the overall situation in Africa is uncertain ,85–87. Sountries ,93. A reountries . The firB. pseudomallei in the lymph node of a pig [Infrequent cases of melioidosis have also been documented in other moist regions, such as Mauritius. The first case was reported in Madagascar in 1936 after isolation of of a pig . Galimanof a pig  previousof a pig , but notof a pig . Howeverof a pig . A recenof a pig .B. pseudomallei from a 10-year-old boy [B. pseudomallei was detected by blood culture [Melioidosis patients with chronic granulomatous infection or diabetes have been reported during the rainy season in Puerto Rico –103. Per-old boy . More re-old boy . The var culture . There a culture , Costa RB. pseudomallei [B. thailandensis and B. pseudomallei [Glanders was eradicated from United States (US) domestic animals in the 1940s. However, a human case of glanders was documented in a Department of Defense laboratory worker (2000). Since 1945, this is the first human case of melioidosis infection reported in the US . In addidomallei . Later, domallei ,116.B. pseudomallei was isolated from 49 patients for the past decade. Almost 33 cases in the UK were diagnosed in patients after visits to Thailand, Bangladesh, and Australia, while 3 patients with cystic fibrosis also acquired the infection in Malaysia, the British Virgin Islands, or Brazil. Twenty-one clinical isolates of B. pseudomallei have been misidentified by European diagnostic laboratories during confirmation.Melioidosis is rare in Europe and is often linked with travel to Southeast Asia. The cases imported into the United Kingdom (UK) most likely occurred in immigrants from prevalent countries. Both human and animal cases have been imported from Bangladesh, Pakistan, India, Indonesia, and Philippines –120. B. B. pseudomallei possesses different types of secretion systems essential for its dissemination and intracellular survival [B. pseudomallei is successfully transmitted from an environmental reservoir to lung epithelial cells via bacterial constituent-like capsules. The type III secretion system-3 (T3SS-3) activates and contributes to vacuolar escape and intracellular motility through BimA-mediated actin polymerization in infected epithelial cells [B. pseudomallei escapes by inducing cell-death (apoptosis), with secondary spread through the lymphatics and migrating macrophages containing the bacterium. The host often develops an adaptive immune response with T cells recruited in response to interferon-gamma (IFN-γ) production, which facilitates cell-mediated immune responses and antibody production after bacterial infection , intraperitoneal (IP), oral, subcutaneous (SC) and inhalation routes of infection [B. pseudomallei infection, and these include capsular polysaccharide, LPS, adhesins, specialized secretion systems , actin-based motility, and myriad secreted factors that enable survival within a host and contribute to the pathogenesis of melioidosis [B. mallei survived for 21 days versus mice exposed to wild-type bacteria that died within 4 days. Pathogenesis included modulating the host ubiquitination pathways, phagosomal killing, and actin–cytoskeleton rearrangement. Furthermore, virulence factors were associated with a host protein (BMAAO728) responsible for the closure of phagosomal membranes. The GABA(A) receptor-associated protein-like I, which is found in mice and humans and is responsible for cellular internalization, escape, evasion, and interaction with host proteins, involves BMAAO728 and BMAA1865/BMAA0553 [Several animal models for studying nfection –130. Thiioidosis ,132. ForBMAA0553 . There wBMAA0553 .B. pseudomallei. Severe pathological symptoms of acute inflammation and necrosis are found in the lungs, liver, and spleen [B. pseudomallei (3 x 105 CFU) was administered by different routes, such as IV, IP, SC, intranasal and orally, in mice, and the severity of infection and pathogenesis was compared. Bacterial loads were measured in various organs—such as the spleen, liver, lungs, lymph nodes, and brain—and blood after 3 days of infection. Bacterial loads did not show any tropism towards lung tissues after intranasal infection by a lethal dose of a highly virulent strain (NCTC 13178). However, the number of bacteria increased in the liver and spleen, but not in the lungs, of C57BL/6 and BALB/c mice after 24 h. Results clearly show systemic spread of B. pseudomallei from the lungs to other organs [6 CFU) after day 2 of infection as a result of innate immunity not controlling infection that ultimately leads to sepsis and death. Pathogenesis caused by B. pseudomallei was studied by infecting mouse macrophages via intraperitoneal injection. Microscopic examination of U-937 macrophages infected with B. pseudomallei proves the occurrence of intracellular bacteria within membrane-bound vacuoles. In vitro study clearly shows that B. pseudomallei can survive and multiply in human phagocytes  lesions and have been related to protease activity. The proteolytic enzymes, as well as a unique lethal exotoxin, are separable entities [B. pseudomallei strains induce severe mortality after IP injection in mice [B. pseudomallei are toxic to both hamsters and mice models. B. pseudomallei is generally able to survive within various cell lines and the process of phagocytosis, as examined from pathological sections. B. pseudomallei have several T3SS-3 that play important roles in bacterial survival and dissemination [B. pseudomallei in the absence of nitric oxide synthase (iNOS) production. This enzyme is essential for controlling the multiplication and survival of B. pseudomallei inside cells. In addition, IFN-γ and IFN-β (a type I interferon) are responsible for initiation of innate immunity against various microbial infections [The development of drug resistance by markable . The celmarkable . The patentities . Further in mice . The toxmination . IFN-γ imination . Septicemination  reportedfections .B. pseudomallei-infected diabetics have impaired IL-12P70 production that results in decrement of IFN-γ [B. pseudomallei to produce IL-12 and, subsequently, IFN-γ in response to certain infections hampers initiation of a proinflammatory response. IFN-γ is essential for inducing phagocytosis  and switching on potent bactericidal effects. A number of reports show that depleted GSH in antigen-presenting cells (APCs) can change T cell maturation into Th2 response. GSH or NAC addition of APCs results in a high level of IL-12 expression through a Th1 response [Various studies clearly demonstrate that almost 50% of melioidosis patients have varying degrees of diabetes mellitus. Additionally, of IFN-γ . IL-12 aof IFN-γ . The cloof IFN-γ . Howeverresponse –153.B. pseudomallei from the sputum, urine, tissues, blood samples, and wound exudates. However, blood cultures have been confirmed negative just before death in the septicemic form of infection. This bacterium presents itself as a wrinkled colony on Ashdown’s selective agar (ASA) [B. cepacia  and B. pseudomallei; thus, further confirmatory tests, such as polymerase chain reaction (PCR), may be needed. Detection of bacteria-specific antibodies from a blood sample is another form of diagnosis. Serological tests revealing high-antibody titers are very useful in the presence of clinical diagnosis. Clinical diagnostic tests include agglutination, indirect hemagglutination, complement fixation, immunofluorescence, and enzyme immunoassays [B. mallei, which is recognized as a causative agent of glanders. Mainly, a direct immunofluorescent antibody test (DIF) is available for the quick diagnosis of melioidosis from patient sputum, pus, and urine. The DIF is more sensitive (73%) and of higher specificity (99%) versus culture of the bacterium from patients (n = 272) with a suspected case of melioidosis [Melioidosis is usually diagnosed by isolation and identification of ar (ASA) , whereasar (ASA) ,156. Thenoassays . Cross-rioidosis . Furtherioidosis , 160–162B. pseudomallei often develops resistance to existing antibiotics [B. pseudomallei. Third-generation antibiotics, including cephalosporins, are not clinically useful in treating melioidosis; however, carbapenems and amoxicillin-clavulanate are used for treatment with broad spectrum effects. Recently, combined therapy with trimethoprim and sulphonamides was noted to decrease bacterial growth [B. pseudomallei [B. pseudomallei develops resistance mechanisms against existing antibiotics due to enzymatic inactivation, target deletion, and efflux from the bacterium caused by chromosomally encoded genes. As a result, excessive production and mutations of the class A PenA β-lactamase can cause resistance to ceftazidime and amoxicillin-clavulanic acid. Deletion of the penicillin-binding protein-3 (PBP-3) leads to drug resistance towards ceftazidime. Similarly, over expression of the BpeEF-OprC drug-efflux pump causes resistance to trimethoprim (TMP) and TMP-sulfamethoxazole (SFZ) antibiotics [ibiotics . Howeveribiotics . The secl growth . Moreovel growth . Fluoroqdomallei , but expibiotics  Fig 4)..B. pseudB. pseudomallei. IFN-γ was recognized as a potent antibacterial force against B. pseudomallei in infected macrophages by using this assay system. Third-generation cephalosporins combined with penicillin and aminoglycosides exert powerful effects [B. pseudomallei and then treated with subtherapeutic concentrations of ceftazidime and liposome-DNA-immune stimulatory complexes experience enhanced survival and completely clear the bacterial load compared to treatment with either therapeutic agent alone [In vitro screening assays have been established for the categorization of cytokines and antimicrobial drugs exerting synergistic activity for preventing intracellular pathogenesis of  effects . Additiont alone . Therefont alone . In addint alone –173. Thent alone . It is vB. pseudomallei to antimicrobial agents has also been scrutinized [B. pseudomallei are variably susceptible to coamoxiclav but resistant to gentamicin and colistin. However, most melioidosis cases can easily be treated with suitable therapies. Early treatment must be initiated immediately after clinical diagnosis. Patients with acute melioidosis may die within hours or days unless prompt treatment is initiated. Long-term treatments with antibiotic combinations are needed for a complete cure. Although bloodstream infections with melioidosis are fatal, other forms of infection are usually nonfatal. Antibiotic therapies for the treatment of acute septicemic melioidosis have varying degrees of effectiveness.The susceptibility of utinized –176 and,utinized . B. pseuB. pseudomallei is commonly susceptible to the following antibiotics: imipenem, penicillin, amoxycillin-clavulanic acid, azlocillin, ceftazidime, ticarcillin-vulanic acid, ceftriaxone, and aztreonam [ztreonam . Based oztreonam . Meropenztreonam . Oral trztreonam . Thus, cztreonam . Mortaliztreonam .B. pseudomallei strains often exceed levels achieved in serum [B. pseudomallei resides [Fluoroquinolones are given for treatment of acute melioidosis; however, this drug is not strongly recommended due to a high relapse rate. In addition, an in vitro study also clearly demonstrated that the minimum inhibitory concentrations (MICs) for some in serum . Ciprofl resides –181. Cip resides .Several earlier reports have demonstrated that different types of genetically modified bacterial vaccines were evaluated to analyze their protective effects –185. In B. pseudomallei genome sequences has greatly aided the progress of vaccine target discovery for the alternative therapeutic and prophylactic strategies [Burkholderia lethal factor-1 (BLF-1) that prevents cells from breaking down protein in an infected host. Another study clearly demonstrated the potential of various DNA molecules from B. pseudomallei with vaccine potential [The accessibility of several rategies . For exaotential .B. pseudomallei pathogenesis. Furthermore, two suitable monoclonal antibodies (mAbs) have been established that target the CPS and LPS to date [B. pseudomallei infection in mice. Although both mAbs confer protection when given singly, the combination treatment provided significantly better protection at low doses [There are two surface-associated antigens, capsular polysaccharide (CPS) and LPS, which are responsible for  to date . The CPSow doses .ilvL, serC, aroB, purN, purM, BPSS1509, lipB, pabB, aroC and bipD in B. pseudomallei vaccines [B. mallei Hcp1), autotransporter protein (BimA), or type III secreted protein (BopA) and ABC transporter protein  reveals that BopA shows the greatest protection (60%–100% efficacy) against B. mallei- and B. pseudomallei-infected mice. Furthermore, serum obtained from the BopA-immunized mice was reactive with type III secreted bacterial recombinant proteins. T cells from vaccinated mice also show increased levels of IFN-γ [Until now, vaccines have not been developed commercially for the prevention of human melioidosis. Several novel vaccines for melioidosis are currently under development and being tested in animals –192. Howvaccines ,194; subvaccines ,195; andvaccines . For insof IFN-γ ,198.B. pseudomallei bipD mutant with dysfunctional T3SS-3 was employed to vaccinate mice. Incomplete defense was obtained in vaccinated mice after challenge with virulent wild-type B. pseudomallei, but inoculation with the purified bip D protein was not protective [+ T cell responses via MHC class I presentation, which is important for protection against intracellular pathogens [Additionally, an attenuated otective –200. Attotective . A signiathogens . In receB. pseudomallei. Synthetic peptides of this antigen were recognized by melioidosis patient sera [Moreover, previous human studies have demonstrated that the properties of oligopeptide-binding protein A (OppA Bp) may play a vital role in the ATP-binding cassette (ABC) transport system, because they can react with convalescent patient sera . Recentlent sera ,205. Thient sera . TherefoMelioidosis continues to pose a potential threat, especially in Southeast Asian countries . HoweverB. pseudomallei vaccine candidates are not available for the prevention of melioidosis. After exposure to the causative organism, combination treatment with co-trimoxazole and doxycycline is recommended. Trovafloxacin and grepafloxacin are effective in animal models. In conclusion, more work and research remains to be carried out to better manage and prevent B. pseudomallei-related diseases that afflict not only humans but also animals.Diabetes, open wounds, lung disease, and immune deficiency are important factors for melioidosis, and patients with those conditions must avoid direct exposure of contaminated clay soil and standing water in prevalent areas. In particular, persons engaged in agricultural activities  must wear boots that can prevent direct infection via the feet and lower limbs. Standard precautions include wearing masks, gloves, and gowns to prevent infection, especially among healthcare workers attending to patients with melioidosis. In addition, clinicians examining travelers with severe pneumonia or septicemia returning from the subtropics or tropics should consider the differential diagnosis of acute melioidosis. Currently, Burkholderia pseudomallei is the causative agent of melioidosis, which is prevalent throughout Southeast Asia.This bacterium is an important bioweapon and bioterrorism risk worldwide.The overall fatality rate of septicemia in melioidosis is very high, and bacteria are intrinsically resistant to many antimicrobial agents.Melioidosis increasingly affects travelers visiting endemic areas, thereby leading to septicemia.Clinicians should consider acute melioidosis as a differential diagnosis.PLoS Negl Trop Dis 2013; 7: e2072.Limmathurotsakul D, Kanoksil M, Wuthiekanun V, Kitphati R, deStavola B, Day NPJ, Sharon J. Peacock SJ. Activities of daily living associated with acquisition of melioidosis in Northeast Thailand: A matched case-control study. N Engl J Med. 2012; 367: 1035–1044.Wiersinga WJ, Currie BJ, Peacock SJ. Melioidosis. Clin Infect Dis 2000; 31: 981–986.Currie BJ, Fisher DA, Howard DM, Burrow JN, Lo D, Selva-Nayagam S. Endemic melioidosis in tropical northern Australia: a 10-year prospective study and review of the literature. Lancet 1989; 2: 697–701.White NJ, Dance DA, Chaowagul W, Wattanagoon Y, Wuthiekanun V, Pitakwatchara N. Halving of mortality of severe melioidosis by ceftazidime. Oxf Med Case Reports. 2016; 6: 113–116.Goel A, Bansal R, Sharma S, Singhal S, Kumar A Chronic melioidosis presenting with multiple abscesses.


Klebsiella pneumoniae is a nosocomial pathogen with clinical importance due to its increasing resistance to carbapenems and colistin. Here, we report the genome sequences of eight colistin-resistant K. pneumoniae strains which might help in understanding the molecular mechanism of the species. The sequence data indicate genomes of ~5.2 to 5.4 Mb, along with several plasmids. Klebsiella pneumoniae is gaining importance due to increased carbapenem and colistin resistance with very few treatment options  from clinical samples. The isolates were observed to be phenotypically resistant to meropenem and colistin by the E-test method.In this study, we present the draft genome sequences of eight De novo assembly of raw data was achieved using AssemblerSPAdes version 5.0.0.0 embedded in the Torrent suite server version 5.0.3. Sequence annotation was done in PATRIC, the bacterial bioinformatics database and analysis resource (http://www.patricbrc.org) (http://rast.nmpdr.org) (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html).To further understand the mechanism behind colistin resistance, whole-genome sequencing was performed using the Ion Torrent PGM platform with 400-bp chemistry. Complete details about the eight genomes are given in https://cge.cbs.dtu.dk//services/MLST) (http://www.cbs.dtu.dk/services) returned multiple antimicrobial resistance genes for most of the antibiotic classes  identified IncFII(K) and IncFIB(pQil) in all isolates in addition to a few other plasmids (Moreover, the sequence types of the isolates were found to be ST-11 for PM565, PM1842, PM1995, and PM138; ST-14 for PM716 and PM1134; and ST-231 for PM5186 and PM1168, as analyzed by the MLST version 1.8 tool ( classes . Interesplasmids .K. pneumoniae isolates were deposited in GenBank/DDBJ under the accession numbers mentioned in The whole-genome sequences of the eight


Salmonella serovars.Many of the bacterial genomic studies exploring evolution processes of the host adaptation focus on the accessory genome describing how the gains and losses of genes can explain the colonization of new habitats. Consequently, we developed a new approach focusing on the coregenome in order to describe the host adaptation of Salmonella enterica subsp. enterica to mammalian- (S. Dublin), multi- (S. Enteritidis), and avian-  hosts.In the present work, we propose bioinformatic tools allowing (i) robust phylogenetic inference based on SNPs and recombination events, (ii) identification of fixed SNPs and InDels distinguishing homoplastic and non-homoplastic coregenome variants, and (iii) gene-ontology enrichment analyses to describe metabolic processes involved in adaptation of S. Dublin diverged from the polyphyletic clade S. Enteritidis which includes the divergent clades S. Pullorum and S. Gallinarum (i). The scripts ‘phyloFixedVar’ and ‘FixedVar’ detected non-synonymous and non-homoplastic fixed variants supporting the phylogenetic reconstruction (ii). The scripts ‘GetGOxML’ and ‘EveryGO’ identified representative metabolic pathways related to host adaptation using the first gene-ontology enrichment analysis based on bacterial coregenome variants (iii).The ‘VARCall’ workflow produced a robust phylogenetic inference confirming that the monophyletic clade Salmonella serovars Dublin , Enteritidis (i.e. multi-hosts), Pullorum (i.e. avian-hosts) and Gallinarum (i.e. avian-hosts) at the coregenome scale. All these polyvalent Bioinformatic tools can be applied on other bacterial genus without additional developments.We propose in the present manuscript a new coregenome approach coupling identification of fixed SNPs and InDels with regards to inferred phylogenetic clades, and gene-ontology enrichment analysis in order to describe the adaptation of The online version of this article (10.1186/s12866-017-1132-1) contains supplementary material, which is available to authorized users. Recent advances in bacterial genomics aim to identify small insertions/deletions (InDels) of the coregenome  in additRepresenting several tens of Bioinformatics tools developed since the beginning of the twenty-first century, the gene-ontology enrichment analysis allows identification of relevant biological processes among large lists of genes . CoverinSalmonella enterica, especially Salmonella enterica subsp. enterica serovars, is the main cause of foodborne gastroenteritis [Salmonella is related to acquired pathogenicity islands (PAIs) called Salmonella pathogenicity islands (SPIs), and genes involved in intestinal phase of infection, colonization of deeper tissues, and expansion in host range, successively [S. enterica subsp. enterica serovars, especially those causing severe human infections (e.g. S. Typhimurium and S. Enteritidis), while the reduction of host range  would mainly be explained by the losses of genes due to large deletions and accumulation of pseudogenes (i.e. start/stop gained codons and/or frameshift mutations) [Salmonella serovars [With around 80 million cases annually, nteritis . Host adessively . Severaltations) . The fewtations) –22 or inserovars , 23–25, serovars .Salmonella and other Enterobacteriaceae (e.g. locus ccm of 7.5 kb in S. Typhimurium) [S. enterica serovars, as Abortusequi, Abortusovis, Choleraesuis, Dublin, Enteritidis, Gallinarum, Pullorum, Sendai, Paratyphi C, and Typhimurium, are known to harbor different virulence plasmids [spvR, spvA, spvB, spvC, and spvD) in the 7.8-kb spv region  which are required for bacterial multiplication in the reticulo-endothelial system [The evolution of serovars, as Agona, Dublin, Hadar, Heidelberg, Javiana, Kentucky, Newport, Saintpaul, Schwarzengrund, Virchow, Weltevreden, Choleraesuis, Enteritidis, Gallinarum, Paratyphi A, Paratyphi B, Paratyphi C, Typhi and Typhimurium, seems to be highly mediated by losses of coding sequences which lead to functional reduction, and horizontal acquisitions of plasmid and phage sequences, hypothetically under regulation of a defense mechanism against exogenous invading sequences, particularly through clustered regularly interspaced short palindromic repeats (CRISPR) . With thimurium) , and manplasmids  heterogel system  and cytol system .S. Enteritis is introduced in the food chain by products from diverse hosts [S. Dublin is mainly due to contaminated cow’s milk and cheese [S. Gallinarum [S. Pullorum [S. Enteritidis into two major clades [S. Enteritidis strain P125109, NC_011294.1) [S. Dublin and S. Enteritidis diverged from a most recent common ancestor (MRCA), and that S. Enteritidis was constituted of two clades , one of them being the origin of the avian-adapted serovars S. Gallinarum and S. Pullorum [Salmonella pathogenicity is strongly associated with the acquisition of SPIs [S. Gallinarum contributes to the colonization of the gastrointestinal tract of chickens, as demonstrated with chickens orally infected by bacterial mutants [S. Enteritidis, but were intact in the mammalian-adapted serovar S. Dublin (i.e. cattle and humans), as well as the avian-adapted serovars S. Gallinarum and S. Pullorum [std.∆, stiC, stfF, safC, stbC, pegC, lpfC, sefD, sefC, sthB, sthA, sthE in S. Gallinarum [ssf and saf operons are highly specific to avian adaptation of S. Gallinarum and S. Pullorum [Known as a common serovar involved in domestically acquired foodborne illness in humans, se hosts . In contd cheese , while Sllinarum  and S. PPullorum  infect ar clades , a recen11294.1)  demonstrPullorum . Focusin of SPIs , especia of SPIs . While T mutants , severalPullorum . Among mPullorum .Salmonella serovars: Enteritidis (i.e. multi-hosts), Dublin , Gallinarum (i.e. avian-hosts) and Pullorum (i.e. avian-hosts) [Here, we propose a novel approach coupling identification of clade specific SNPs and InDels , 2 and an-hosts) , 22, 25.S. Enteritidis (strain P125109) [S. Typhimurium reads against the reference LT2 genome  [After trimming and mapping of paired-end reads, the previously published genome dataset  . This deP125109) , and thi8% max.) .Salmonella genomes was analyzed through the variant calling workflow ‘VARCall’ where high-confidence variants were retained [Salmonella dataset falls inside the boundaries of 0.5–2 InDels / 10 SNPs which have been previously observed in other prokaryotic species  and eukhimurium , e.g. Huhimurium ).S. Enteritidis chromosome  [S. Agona serovar, we also detected 35% of intergenic InDels (i.e. 411/1157) across the 59 studied genomes.We found that 12,031 variants  were intragenic, corresponding to 85% of the 14,086 coregenome variants . As the coding regions represent about 88% of the p < 5.0×10−2, Wilcoxon rank sum or Kolmogorov-Smirnov tests). For instance, the average pairwise distances of S. Enteritidis clade  were significantly different than those calculated for Pullorum , Gallinarum  and Dublin  serovars .Interestingly, the average pairwise distances of SNPs or InDels were significantly different Fig.  between 6 for 1000 computed trees [The phylogenetic inference converged after 200 bootstrap replicates  with a led trees , resultiS. Dublin as a distinct clade with differences of 4945 ± 378 SNPs and 296 ± 17 InDels from the S. Enteritidis clade, as confirmed by our genetic structure analysis [S. Enteritidis described as classic and second clades by Langridge et al. [S. Enteritidis by microarray [The phylogenetic tree Fig.  showed tanalysis  and accoanalysis . Exceptie et al.  Fig. 2)S. Dublincroarray  and SNP-croarray , we suppS. Pullorum and S. Gallinarum are highly specialized toward avian-hosts; they are non-motile members of serogroup D, indistinguishable by serotyping and historically recognized as distinct biotypes considering several criteria: the distinct septocaemic diseases they cause in avian species, biochemical characteristics and multilocus enzyme electrophoresis [S. Pullorum and S. Gallinarum isolates arose from the second clade of S. Enteritidis , while by contrast the S. Pullorum clade displayed a larger significant diversity with regards to SNPs  or InDels . These results confirm at the genetic level the biochemical classification performed by Crichton and Old [phoresis . As prevphoresis , our phydis Fig. . Interesdis Fig. : the S.  and Old .S. Dublin and S. Enteritidis have a most recent common ancestor, and that the clade S. Pullorum/S. Gallinarum diverged from S. Enteritidis. According to Langridge et al. [S. Enteritidis , as well as significantly higher than those of the mammalian-adapted serovar S. Dublin and avian-adapted serovar S. Gallinarum .All together, these observations are in accordance with Langridge et al.  who conce et al. , we alsodis Fig. . In oppoudogenes , we obsedis Fig. , expressSalmonella, the PAIs called SPIs have long been recognized to play a determinant role in the virulence properties of the pathogen [Following lateral transfer of genetic material, recombination events are a common modality of evolution of bacterial genomes . In Salmpathogen . These rpathogen . We perfn = 91) were detected in S. Dublin, the most distant clade of the collection .Surprisingly, most of these recombination events  either of branches in the phylogenetic tree (i.e. ‘PhyloFixedVar’) or between groups defined by the user (i.e. ‘FixedVar’).S. Typhimurium estimating non-synonymous SNPs at 44% [p < 1.0×10−8; Wilcoxon signed rank test) variants . According to a study on s at 44% , most ofS. Dublin, S. Enteritidis, S. Gallinarum and S. Pullorum , divergence of S. Pullorum/Gallinarum from S. Enteritidis , divergences of S. Pullorum  and S. Gallinarum  with each other   and est (SPAdes ), is curAlthough the removal of variants from recombination events must be theoretically performed when the phylogenetic inference assumes  or requires (i.e. Maximum Likelihood) a Markov chain model of nucleotide substitution , we obseS. Dublin, S. Enteritidis, S. Gallinarum and S. Pullorum genomes. Considering all leaves of the phylogenetic inference, the non-homoplastic fixed variants represented 65% of all fixed variants. It should however be mentioned that it is fully possible to use the scripts ‘phyloFixedVar’, ‘FixedVar’, ‘GetGOxML’ and ‘EveryGO’ to select homoplastic variants for studies that would focus on coevolution [Because of our objective to detect clade specific traits related to host adaptation, we decided to exclude the homoplastic variants in the analyses of the volution .The gene-ontology enrichment analysis was applied to synonymous and non-synonymous fixed variants because synonymous variants may be involved in regulation of gene expression or level of protein synthesis even if they do not impact the protein sequence .In order to browse easily between tree branches, genes and their annotations, the xml file produced by the scripts ‘phyloFixedVar’ or ‘FixedVar’ centralizes the annotations of variants including the homoplastic variants for all the combinations of genomes present in the phylogenetic inference. The polyvalent scripts ‘phyloFixedVar’, ‘FixedVar’, ‘GetGOxML’ and ‘EveryGO’ were developed to perform the first gene-ontology enrichment analysis based on bacterial coregenome variants and can be applied to all kind of bacterial genome collection.Salmonella displayed a decreasing in coregenome diversity  may correspond to frameshift insertions of T or TT in S. Enteritidis, or frameshift deletions of T or TT in S. Dublin  are essential reactions during mice infection by S. Typhimurim [S. Typhimurium in mice requires also the twin-arginine translocation system (Tat) which exports across the cytoplasmic membrane numerous cofactors containing virulence factors and anaerobic respiratory chain proteins [astB) (GO:0009015) is indeed the second enzyme of the arginine succinyltransferase pathway involved in arginine catabolism as a sole nitrogen source [The results of the present study highlighted the accumulation of fixed variants in intragenic regions connected to metabolic pathways of amino acids Table . The mosars Fig. . Concernin Table , the glyphimurim . Aerobicproteins   is in fact a key enzyme in the biosynthetic pathway from aspartate to homoserine, which is a common precursor for the synthesis of amino acids methionine, threonine and isoleucine.With regard to the GO-terms of interest for in Table , the accmolality  and aspamolality . Also inS. Dublin , hyperosmotic stress and proline limitation in host compartment is indeed known to lead S. Typhimurium responds to a decrease in the levels of proline-charged tRNAPro by promoting expression of the mgtCBR virulence operon [S. Dublin (GO:0003842) is encoded in the putA gene of S. Typhimurim by a bifunctional membrane-associated dehydrogenase which oxidizes proline to glutamate for use as the sole carbon, nitrogen or energy source [Regarding the proline metabolic and catabolic processes impacted by non-homoplastic fixed variants in y source .alr of S. Typhimurim, also called dal genes, encode alanine racemases which are biosynthesis sources of D-alanine for cell wall formation and also necessary to the catabolism of L-alanine as a source of carbon, energy and nitrogen [S. Pullorum  may consequently be due to his adaptation to this avian alimentation.The genes nitrogen . L-arginnitrogen , the sto+-dependent D-glucose transporter SLGT1), whereas enzymes required for solid food and pinocytotic uptake capacity are produced during weaning (e.g. fructose and starch), and activities of sucrase-isomaltase, maltase-glucoamylase, trehalase and fructose transport (i.e. GLUT-5) increase when adult diet switches from lactose to sucrose and starch. In contrast, the pre- and post-natal periods of birds and chicks are associated with a switch of gastrointestinal functions driven by the transition from a lipid-rich yolk diet inside the egg  to a carbohydrate- and protein-based diet after hatch in young chickens and house sparrows  [Compared to similar sized nonflying mammalian, the avian gastrointestinal tracks present a typical shorter retention time and quantitatively much more important passive adsorption of L-glucose. During ontogeny, the architecture and functioning of mammalian and avian gastrointestinal tracts are closely related with their food diet and intake rate. The gestational phase of mammalian is dominated by production of gastrointestinal enzymes required for digestion and absorption of milk (e.g. amino acid transporters and the Naivities) . Severalivities) .S. Dublin  acid resistance system to prepare for the stresses of host-cell interactions [Further with respect to the GO-terms of interest related to metabolic and catabolism processes of amino acids in in Table , S. Typht pH 2.5 . Salmoneractions .ileS encoding the essential enzyme isoleucine-tRNA ligase (IleRS) were observed in S. Enteritidis under selection pressure induced by the mupirocin, an inhibitor of this enzymatic activity [S. Pullorum  may consequently also be linked to other antibiotics  . For instance, plasmids encoding aerobactin (i.e. iucABCD and iutA) and Sit iron transport operons (i.e. sitABCD), as well as other iron acquisition genes (e.g. iss) play indeed a major role in survival abilities of S. Kentucky (e.g. IncFIB plasmid) and some S. Heidelberg (e.g. pSH163_120 and pSH696_117 plasmids) in the extraintestinal environments of poultry where iron is in limited supply [Concerning the loss or modification of potassium ion transmembrane transporter activity in the avian-adapted serovar 9) Table , the modd supply .Salmonella serovars Dublin , Enteritidis (i.e. multi-hosts), Pullorum (i.e. avian-hosts) and Gallinarum (i.e. avian-hosts) at the coregenome scale. Among the multiple metabolic pathways impacted by fixed variants during host adaptation of Salmonella serovars, our main observation emphasized that glutamate metabolism could play a major role in adaptation of S. Dublin to mammalian-hosts.In conclusion, we proposed the first validated procedure to identify fixed SNPs and InDels according to inferred phylogenetic clades and performed the associated gene-ontology enrichment analysis in order to describe the adaptation of Salmonella strains and was constituted by Langridge et al. [Salmonella serovars S. Enteritidis (i.e. multi-hosts), S. Dublin , S. Gallinarum (i.e. avian hosts) and S. Pullorum (i.e. avian-hosts)  rather than to perform time consuming variant calling across reference genome alignments (UnifiedGenotyper) [We develop currently a new version of the ‘VARCall’ workflow  based on the HaplotypeCaller algorism (GATK) in order to call SNPs and InDels at the same time by local notyper) .Based on the pseudogenomes, the phylogenetic inference was performed with the multi-core architecture of RAxML . FollowiA script called ‘phyloFixedVar’ uses the annotated vcf file from the variant calling analysis and the nwk file from a binary tree. This script was developed in order to detect sensitive and specific variants at each branch of the phylogenetic inference. The variants are defined with a biological point of view either as fixed (i.e. variants with sensitive and specific genotypes at intermediate branches) or transient . The script ‘phyloFixedVar’ (a) identifies all comparisons of genome groups referring to all nodes, (b) selects sensitive and specific variants, and (c) defines if these variants are homoplastic (i.e. convergence of genotypes between independent phylogenetic clades), successively. More precisely, right child leaves are listed with corresponding leaves of left child at each node and reversely, then both comparisons (i.e. right versus left children and reversely) are connected with comparison numbers which are associated to identifiers of single nodes, all together grouped into node labels in a new nwk file (a). On the one hand the sensitive variants are detected as presenting common genotypes into leaves of right or left child of previously listed comparisons, and on the other hand the specific variants are identified as presenting different genotypes in corresponding right or left children (b). With regard to these sensitive and specific variants, the genotypes of all the other genomes are screened in order to tag homoplastic variants (i.e. common genotypes between variants of independent phylogenetic clades) (c). The selected variants are finally written in the xml file with their related annotations  for each node and all comparisons of lists of leaves. Similarly to ‘phyloFixedVar’, another script called ‘FixedVar’ requires a vcf file and lists of genomes IDs that have to be compared. This script was developed in order to detect sensitive and specific variants independently of the phylogenetic inference.http://geneontology.org/page/download-ontology), and the script ‘GOxML’ associates gene identifiers (i.e. NP or WP) from the xml file with GO-terms available in the QuickGO browser (http://www.ebi.ac.uk/GOA) of the UniProt GO annotation program (https://www.ebi.ac.uk/QuickGO/). In addition, the script ‘GOxML’ allows comparison of these GO-terms in order to exclude potential eukaryote GO-terms from the dataset and retains prokaryote GO-terms. Finally, the script ‘GOxML’ integrates the curated GO-terms (i.e. prokaryote GO-terms) and related biological processes to the common xml file in order to centralized the GO-terms and functional annotations of variants . With a view to select intragenic variants (SNPs and InDels), and distinguish between GO-terms from the variants of interest (i.e. tested sample) and all variants (i.e. universe) which are used for the hypergeometric test of the gene-ontology enrichment analysis based on the parent-child approach [p-values of the hypergeometric tests versus the branch levels from the GO-terms of the DAG).With the objective to associate the selected variants to corresponding prokaryotic GO-terms, a driver script called ‘GetGOxML’ invokes the scripts ‘GOSlimer_XML’ and ‘GOxML’, successively Fig. . More prapproach , the driapproach  implemenapproach . Finallyhttp://www.paidb.re.kr) from KonKuk University  [S. Enteritidis .Candidates of PAI-like region overlapping genomic islands (cPAIs) of the Pathogenicity Island Database (h Korea)  were useAdditional file 1:Salmonella enterica subsp. enterica serovars Enteritidis, Pullorum, Gallinarum and Dublin were described by Langridge et al. . (XLSX 15 kb)Genome dataset used in the present study. The genomes of Additional file 2:Salmonella enterica subsp. enterica serovars Dublin, Enteritidis, Pullorum and Gallinarum were described by Langridge et al. . (XLSX 16 kb)Statistical report of the ‘VARCall’ workflow. The serovars Additional file 3:Salmonella enterica subsp. enterica serovars Dublin, Enteritidis, Pullorum and Gallinarum. The variants were identified by the ‘VARCall’ workflow against the reference genome S. Enteritidis . The positions of recombination events detected by Maximum Likelihood and default gamma priors of ClonalFrameML are removed with a script ‘Clonal_VCFilter’ in order to compute phylogenetic inference based on pseudogenomes excluding variants linked to recombination events. The produced pseudogenomes  were inferred with RAxML based on a bootstrap analysis and search for best-scoring Maximum Likelihood tree with General Time-Reversible model of substitution and the secondary structure 16-state model. The color legend corresponds to phylogenetic clustering performed by Langridge et al. . The trees are rooted on the branches of S. Dublin before comparison. The comparison of the tree topologies were performed using the cophylo function of ‘phytools’ R package. (PDF 1733 kb)Phylogenetic inferences performed based on coregenome single nucleotide polymorphisms (SNPs) excluding (A) or including (B) variants from recombination events detected in Additional file 4:Salmonella enterica subsp. enterica serovars Dublin, Enteritidis, Pullorum and Gallinarum. The color legend corresponds to serovars presented by Langridge et al. . The variants were identified by the ‘VARCall’ workflow against the reference genome S. Enteritidis . The produced pseudogenomes  were inferred with RAxML based on a bootstrap analysis and search for best-scoring Maximum Likelihood tree with General Time-Reversible model of substitution and the secondary structure 16-state model. The phylogenetic inference converged after 200 bootstrap replicates with a log likelihood score of −8.106 for 1000 computed trees. The tree is rooted on the branch of S. Dublin. The pseudogenomes and the RAxML inference were used to perform detection of recombination events based on default gamma priors of ClonalFrameML. The number of recombination events is defined closed to white circles which represent recombination events occurred on a branch of the phylogenetic tree. The recombination events with sizes higher than 400 bp are presented. (PDF 752 kb)Phylogenetic inference based on coregenome single nucleotide polymorphisms (SNPs) and recombination events identified in Additional file 5:Salmonella enterica subsp. enterica serovars Dublin, Enteritidis, Pullorum and Gallinarum. The variants were identified by the ‘VARCall’ workflow against the reference genome S. Enteritidis . The produced pseudogenomes  were inferred with RAxML based on a bootstrap analysis and search for best-scoring Maximum Likelihood tree with General Time-Reversible model of substitution and the secondary structure 16-state model. The pseudogenomes and the RAxML inference were used to perform detection of recombination events based on default gamma priors of ClonalFrameML. The recombination events with sizes higher than 400 bp are presented. (XLSX 16 kb)List of recombination events identified in Additional file 6:Salmonella enterica subsp. enterica serovars Enteritidis (n = 33), Pullorum (n = 5), Gallinarum (n = 8) and Dublin (n = 13) diverged. The variant calling analysis was performed with the ‘VARCall’ workflow . The fixed non-homoplastic variants are defined by common genotypes across the considered group of genomes, as well as different genotypes in all the others compared genomes. The fixed homoplastic variants are defined by common genotypes across the considered group of genomes and genomes of independent phylogenetic clades, as well as different genotypes in genomes of the compared child-leaves. The variants were annotated with SnpEff against reference genome S. Enteritidis . (XLSX 13 kb)Phylogenetically relevant single nucleotide polymorphisms (SNPs) and small insertions/deletions (InDels) fixed at phylogenetic branches where genomes of Additional file 7:Salmonella enterica subsp. enterica serovars Dublin versus all the others genomes , Pullorum/Gallinarum versus Enteritidis , Pullorum versus Gallinarum , and Gallinarum versus Pullorum . The variant annotation was performed with SnpEff against reference genome S. Enteritidis . The identification of variants, detection of fixed variants, assignment of GO-terms to variants, and gene-ontology enrichment analysis were performed with the scripts ‘VARCall’, ‘phyloFixedVar’, ‘GetGOxML’, and ‘EveryGO’, respectively. The level, biological process (BP), molecular function (MF), and cellular component (CC) of GO-terms are represented. The p-values of hypergeometric tests were adjusted by Bonferroni correction. (XLSX 327 kb)Gene-ontology (GO) terms of intragenic and non-homoplastic variants (SNPs and InDels) fixed in Additional file 8:Salmonella enterica subsp. enterica serovars Dublin versus all the others genomes , Pullorum/Gallinarum versus Enteritidis , Pullorum versus Gallinarum , and Gallinarum versus Pullorum . Pathogenicity Island Database from KonKuk University  were used to detect Salmonella Pathogenic Islands (SPIs) SPI-1 , SPI-2 , SPI-4 , SPI-5 , SPI-6 , SPI-11 , SPI-12  and PAI III 536  of the reference genome S. Enteritidis . (XLSX 251 kb)Candidates of PAI-like region overlapping genomic islands (cPAIs) impacted by intragenic and non-homoplastic variants (SNPs and InDels) fixed in Additional file 9:Salmonella enterica subsp. enterica serovars Dublin, Enteritidis, Pullorum and Gallinarum. The variants were identified by the workflow ‘VARCall’ against the reference genome S. Enteritidis . The produced pseudogenomes  were inferred with RAxML based on a bootstrap analysis and search for best-scoring Maximum Likelihood tree with General Time-Reversible model of substitution and the secondary structure 16-state model. The pseudogenomes and the RAxML inference were used to perform detection of recombination events based on default gamma priors of ClonalFrameML. The recombination events with sizes higher than 400 bp are presented. Pathogenicity Island Database from KonKuk University  were used to detect Salmonella Pathogenic Islands (SPIs) SPI-1 , SPI-2 , SPI-4 , SPI-5 , SPI-6 , SPI-11 , SPI-12  and PAI III 536  of the reference genome S. Enteritidis . (XLSX 19 kb)Candidates of PAI-like region overlapping genomic islands (cPAIs) impacted recombination events identified in


Staphylococcus lugdunensis is a skin commensal classified as a coagulase-negative Staphylococcus (CoNS). Though CoNS is typically associated with less aggressive clinical disease than Staphylococcus aureus, there is growing awareness that S. lugdunensis may be as virulent as S. aureus. The association between S. lugdunensis and infective endocarditis is well known, but few reports of native-joint disease with this organism exist. We report a case a 28-year-old male with no prior medical problems presenting with native-joint septic arthritis. Cultures grew S. lugdunensis. To our knowledge, this is the fifth case reported in the literature. S. lugdunensis was first described in 1988 [S. lugdunensis has been associated with higher severity and mortality than other coagulase-negative Staphylococcus (CONS) species, presenting with features more similar to disease caused by S. aureus. A case series with native valve infective endocarditis (IE) caused by S. lugdunensis reported mortality as high as 42% and surgical needs for more than 51% of cases [S. lugdunensis have also been reported and may be more common than once thought in community-acquired infections [S. lugdunensis and S. aureus can infect joints via direct inoculation or hematogenous spread. The association between S. lugdunensis and prosthetic joint disease is well established, with reports of infections ranging between 6 weeks and 4 years after implantation [fections . S. lugdantation , 8. TherA 28-year-old male with no past medical history presented to the emergency room with two weeks of right knee pain. He denied any recent sexually transmitted diseases, illicit drug use, or prior episodes of knee pain. He reports no trauma to the knee. Over-the-counter pain medications did not relieve his pain, which progressed and eventually limited his ability to use his joint.S. lugdunensis. Magnetic resonance imaging (MRI) revealed diffuse soft-tissue edema, most prominent in the popliteal fossa around the knee and a large knee joint effusion with synovitis . Imaging revealed mild soft-tissue swelling and a moderate effusion. A diagnostic arthrocentesis was performed, and fluid analysis revealed 28,875 white blood cells per mcL, predominantly neutrophilic. Gram stain revealed Gram-positive cocci in clusters at 1 day of growth. At 48 hours, cultures grew ynovitis . Six synThe patient underwent washout by orthopedic surgery on day one and day three. Vancomycin was given for four days and subsequently changed to clindamycin. The patient completed seven days of parenteral antibiotics and transitioned to oral clindamycin for the remainder of a four-week course. He had full clinical resolution at his third week of therapy.S. lugdunensis is rare. Our review of the English literature found only four cases [S aureus [S. lugdunensis [Native-joint septic arthritis caused by ur cases , 9–11. Our cases , 9–11. Pur cases . One prour cases . The mosS aureus . Howeverdunensis . The incdunensis .S. lugdunensis is classified as a CoNS. It nevertheless retains partial coagulase activity and has many similarities with S. aureus [S. aureus, but performance characteristics of these tests for S. lugdunensis are variable and can lead to false identification of S. aureus [S. lugdunensis may be underappreciated. Samples may be reported as S. aureus, and both pathogens cause similar disease. Both organisms are skin commensals and are associated with soft-tissue infections, bacteremia, and infective endocarditis. Like S aureus, S. lugdunensis can cause severe disease. Infective endocarditis by S. lugdunensis can present with complications such as valvular perforation, myocardial abscesses, embolic stroke, and death [. aureus , 8. Aggl. aureus , 16. Thund death , 4, 11. nd death , 11.S. lugdunensis infections. Three cases reported in the literature underwent joint irrigation, debridement, or other surgical interventions, and one did not specify (There are no randomized controlled data to guide the treatment of bacterial septic arthritis, and there is a paucity of data on  specify  [7, 9–11S. lugdunensis. Clinicians must be aware of the association between S. lugdunensis and severe clinical disease. Lincomycin-based therapy was a successful treatment option for our case. Further studies are needed to compare different therapeutic options.The present case contributes to a small but growing list of cases of native-joint septic arthritis by


NDUFB11, which encodes a structural component of complex I of the mitochondrial respiratory chain (MRC), were recently independently reported to cause histiocytoid cardiomyopathy (histiocytoid CM) and microphthalmia with linear skin defects syndrome (MLS syndrome). Here we report an additional case of histiocytoid CM, which carries a de novo nonsense variant in NDUFB11  identified using whole-exome sequencing (WES) of a family trio. An identical variant has been previously reported in association with MLS syndrome. The case we describe here lacked the diagnostic features of MLS syndrome, but a detailed clinical comparison of the two cases revealed significant phenotypic overlap. Heterozygous variants in HCCS  and COX7B, which, like NDUFB11, encodes a protein of the MRC, have also previously been identified in MLS syndrome including a case with features of both MLS syndrome and histiocytoid CM. However, a systematic review of WES data from previously published histiocytoid CM cases, alongside four additional cases presented here for the first time, did not identify any variants in these genes. We conclude that NDUFB11 variants play a role in the pathogenesis of both histiocytoid CM and MLS and that these disorders are allelic .Variants in  NDUFB11 (OMIM *300403) variants have recently been independently proposed to cause histiocytoid cardiomyopathy  ( 500000)  and micr 500000) . Histioc 500000) . Predomi 500000) . Associa 500000) . Extraca 500000) . Variant 500000) , but thi 500000) ., characterized by unilateral or bilateral microphthalmia and linear skin defects. Skin defects are classically limited to the face and neck along Blaschko's lines, are present from birth, and heal with time, often leaving minimal scarring. Additional clinical features may include neurological and cardiac abnormalities (HCCS (OMIM *300056) (COX7B (OMIM *300885)  , was firmalities . Heteroz*300056) , which e*300885) , which e*300885) . HoweverNDUFB11 )  cardiomyopathy . The aut8*)) see . An iden8*)) see , highligNDUFB11 c.262C > T; p.(Arg88*) and FAM135A c.474C > G; p.(Tyr158*). Sanger sequencing confirmed the presence of both variants in the affected child and their absence in the unaffected parents , with a mitochondrial function in keeping with previous understanding of histiocytoid CM as a mitochondrial disorder. Alternative inheritance models  were considered with no additional strong candidate genes identified. We interrogated four additional unrelated cases of histiocytoid CM for variants in NDUFB11, COX7B, or HCCS, and no putative disease-causing variation was identified. We also reviewed publicly available WES data  and COX7B (OMIM 300885), which are X-linked genes encoding or targeting mitochondrial proteins and are implicated in MLS, are good candidate genes for histiocytoid CM. We note that HCCS nonsense variant ) and extremely skewed X-inactivation (98:2) with classical features of MLS including linear skin defects on her neck, microphthalmia, anophthalmia, and sclerocornea, who died at 4 mo with ventricular tachycardia (VT) and was found to have histiocytoid CM on postmortem examination, further supporting that MLS and histiocytoid CM are allelic disorders. However, no rare variants were found in HCCS or COX7B in seven additional histiocytoid CM cases without NDUFB11 variants ) that was also reported in an additional case with both MLS and histiocytoid CM (Comparisons see  between  variant  show con variant . Previouytoid CM . Around ytoid CM . In the HCCS or NDUFB11 (see NDUFB11 variant (case GHCG) a second nonsense variant was detected in cytochrome b, but only at a frequency of 20% and only in the cardiac tissue, possibly indicating a clonal selection of a somatic variant in the diseased heart. The authors suggest this is one of the potential avenues through which the expressivity of defects in MRC complex 1 activity might be regulated . Only variants that passed standard GATK filters, with genotype quality score >20, coverage >20×, and allelic balance >20, were included in the analysis. We filtered for rare  de novo variants, predicted to be protein altering  in the proband. All inheritance models were considered.The trio (of affected child and both unaffected parents) underwent WES using the Agilent SureSelect system (Human All Exon v4 + UTR kit) with Illumina sequencing . Reads were aligned to the human reference genome (hg19) using Burrows–Wheeler alignment (BWA)  v0.7.5, NDUFB11 were amplified by polymerase chain reaction (PCR), and directly sequenced using the BigDye Terminator Cycle Sequencing Kit and an ABI3500 Genetic Analyzer (Applied Biosystems). Sequences were analyzed using Sequencher (v5.3) software. The coding sequence of all samples was fully covered in both directions.The coding exons and intron–exon boundaries of NDUFB11, F-TCCAGCCATGACTAGAGCTG, R-TCATC TCAGCTCCCCATTCC, and for FAM135A, F-GTTGTACTGCAGCCTTGTAATAAAC, R-CAGCCTGAAGAACCATGACC.The sequencing primers were, for NDUFB11 (SCV000297804.1) and FAM135A (SCV000297805.1) variants have been submitted to ClinVar (htpp://www.ncbi.nlm.nih.gov/clinvar/). We do not have consent from patients to deposit complete sequencing data in a repository.This study received research ethics committee approval  and all subjects (or parents where appropriate) provided written informed consent.We thank the families presented in this report for their participation.G.R., P.J.R.B., S.A.C., and J.S.W. conceived and designed the study; G.R., J.T., F.R., T.H., M.A., R.J.B., A.W., S.W., S.J., and R.W. collected and analyzed data. G.R., P.J.R.B., S.A.C., and J.S.W. interpreted the data and wrote the paper. All authors have reviewed and approved the final manuscript.This work was supported by the Academy of Medical Sciences, Wellcome Trust, British Heart Foundation, Arthritis Research UK, National Institute for Health Research (NIHR) Biomedical Research Unit in Cardiovascular Disease at Royal Brompton and Harefield NHS Foundation Trust and Imperial College London, Medical Research Council (UK), Foundation Leducq, a Health Innovation Challenge Fund (HICF) award from Wellcome Trust and Department of Health, The Lily Foundation, and the UK NHS Highly Specialised Commissioners.The authors have declared no competing interest.Gholson J. LyonBahig M. Shehata


Darevskia rostombekowi and other unisexual species of this genus reproduce normally via true parthenogenesis. Typically, diploid parthenogenetic reptiles exhibit some amount of clonal diversity. However, allozyme data from D. rostombekowi have suggested that this species consists of a single clone. Herein, we test this hypothesis by evaluating variation at three variable microsatellite loci for 42 specimens of D. rostombekowi from four populations in Armenia. Analyses based on single nucleotide polymorphisms of each locus reveal five genotypes or presumptive clones in this species. All individuals are heterozygous at the loci. The major clone occurs in 24 individuals and involves three populations. Four rare clones involve one or several individuals from one or two populations. Most variation owes to parent-specific single nucleotide polymorphisms, which occur as heterozygotes. This result fails to reject the hypothesis of a single hybridization founder event that resulted in the initial formation of one major clone. The other clones appear to have originated via post-formation microsatellite mutations of the major clone.The all-female Caucasian rock lizard  Questions concerning origin and evolution of parthenogenesis in lizards have received considerable attention in recent years –6. ParthIndotyphlops braminus [Sphyrna tiburo [Lepidophyma (L. reticulatum and L. flavimaculatum) [Leposoma percarinatum [Darevskia) or triploid (as in Aspidoscelis) and commonly exhibit high levels of nuclear gene diversity because of fixed heterozygosity at codominant loci [Among vertebrates, true parthenogenesis, where reproduction proceeds without male participation, has been detected in squamate reptiles, especially in lizards  and alsobraminus ,9. It ala tiburo . The Laca tiburo . In mosta tiburo ,13, but a tiburo . Only paculatum)  and the arinatum  did not arinatum , a new sarinatum . Becauseant loci . The morant loci .Cnemidophorus lemniscatus [Heteronotia binoei [Aspidoscelis neomexicana [A. tesselatus [Each parthenogenetic species usually consists of several clonal lineages, possibly caused by either mutations , multiple origins from different pairs of founders, rare new hybridization events, or some level of genetic recombination ,20,21. Cniscatus ,26 and Ha binoei . Aspidosmexicana  and A. tsselatus  exhibit Darevskia is of particular significance because its species have been the subjects of extensive ecological and biogeographical study, and because parthenogenesis has arisen several times within the group [Darevskia includes 24 bisexual species and seven parthenogenetic, diploid species of hybrid origin [Darevskia are in accordance with Parker et al.’s model [D. rostombekowi.The lacertid genus he group . Darevskd origin . Previoud origin –34. Gene’s model , with thDarevskia rostombekowi  and D. raddei . The parental species belong to different clades of Darevskia [Darevskia rostombekowi have a chromosome set of 2n = 38 [D. raddei [D. dahli, D. armeniaca, and D. unisexualis, in which several allozyme clones were found [D. portschinskii occurs in southern Georgia, northern Armenia and western Azerbaijan, and the maternal species D. raddei is distributed in Armenia and adjoining Georgia, western Azerbaijan, and northern/western Iran. Several discrete populations of D. rostombekowi occur in Lori and Tavush provinces of northern Armenia (elevation range 700–1500 m), and one small, isolated high-montane (2000 m) population along the southeastern shore of Lake Sevan (Gegharkunik province), in the vicinity of the village of Zagalu (renamed recently to Tsovak). The Sevan and Vardenis mountain ranges, which rise to more than 3000 m, isolate this population from the nearest populations inhabiting the foothills near the Kura River by Lake Gai-Gel’, Azerbaijan. The mountains are insurmountable geographical barriers for D. rostombekowi. No allozyme analysis exists for this population.g Murphy ) is one arevskia ,24 and tarevskia . Phyloge 2n = 38 , are cha 2n = 38  and exhi. raddei . This ware found . At presD. rostombekowi  originated from one southern Armenian population (Yeghegnadzor) of D. raddei [b; Cytb) from D. rostombekowi at Tsovak indicated a maternal origin from D. raddei at Yeghegnadzor [Cytb, which suggested the existence of a second matriline of D. rostombekowi specific to Tsovak. In addition, interpopulation comparisons of genetic variation in D. rostombekowi, based on multilocus DNA fingerprinting with microsatellite hybridization probes, and RAPD markers for the nuclear genome, indicated significant differences between lizards from northern Armenian and Tsovak [Analysis of mitochondrial DNA (mtDNA) showed that all northern matrilines of Armenian . raddei . Initialegnadzor , but subegnadzor –40 resold Tsovak ,42. Thesd Tsovak , highligD. rostombekowi including variation at three microsatellite-containing loci identifies genotypes of D. rostombekowi from four Armenian populations. We apply a new approach to genotyping the related parthenogenetic species D. dahli [D. rostombekowi—previously presumed to be a monoclonal parthenogenetic species—has several clones. New, the approach reveals parent-specific markers consisting of microsatellites and single nucleotide polymorphisms (SNPs) located out of the microsatellite cluster for each allele of each locus. The SNPs yield direct information about interspecific hybridization founder events, and microsatellite variability provides information concerning possible mutations in the initial hybrid clones that give rise to new genotypes. Interspecies allele comparisons involve homologous microsatellite loci from the parental bisexual lizard species D. raddei and D. portschinskii. All alleles present in D. rostombekowi also occur in the parental species.Our study examines the clonal diversity in D. dahli  and showD. rostombekowi (n = 42) from four populations, D. raddei (n = 65) from 13 populations, and D. portschinskii (n = 27) from two populations in Armenia were analyzed (D. rostombekowi as an Endangered Species by the IUCN (2009) and the species is not protected by CITES. The study was approved by the Ethics Committee of Moscow State University (Permit Number: 24–01) and was carried out in strict accordance with their ethical principles and scientific standards. All specimens were captured with a noose. Blood samples were taken from the tail veins of lizards under chloroform anesthesia, and then these lizards were released. DNA was isolated from lizard blood by using the standard phenol–chloroform extraction method with proteinase K, and resuspended in TE buffer, pH 8.0.DNA samples of analyzed . Sample Three loci, Du215, Du281, and Du323 were PCR-amplified using previously described primer pairs ,44. ThesD. rostombekowi [D. raddei and D. portschinskii were shown in Data on genetic variation of these loci in ombekowi  and on aThe number of alleles, allelic richness, as a measure of allele counts adjusted for sample size, and expected heterozygosity, as a measure of gene diversity, were calculated per locus and per population by using Fstat v.2.9.3.2 , GenePopD. rostombekowi were heterozygous at the three loci and contained two alleles that differed from each other in length and structure of the microsatellite clusters. Two of them, Du281 and Du323, had also single nucleotide variants (SNVs) in fixed positions of the flanking regions , Du215 had five alleles, Du281 had four, and Du323 had two , genotype 1 was most abundant; it was found in three populations. Genotypes 2 and 3 were represented by eight individuals each  in two and one population, respectively. Finally, rare genotypes 4 and 5 were represented by one individual each .D. rostombekowi varied from 0% to 75%  and D. portschinskii (n = 27) were given in D. raddei, and a little more for male D. portschinskii. Du215 and Du281 had heterozygote deficits. That of Du281 also occurred in male D. raddei. Female D. raddei had an excess of heterozygotes at Du323. Deviations from Hardy-Weinberg expectations probably owed to small sample sizes. The heterozygote deficits may have owed to a Wahlund effect due to the 65 individuals of D. raddei coming from 13 localities.Indices of gene diversity in males and females of the parental species D. rostombekowi originated from a single or multiple interspecies hybridization event(s), genotype-specific markers, formed by combinations of parent-specific SNVs of both alleles in Du281 and Du323 were evaluated  and D. portschinskii (n = 27). Sequencing obtained 37 alleles from D. raddei and 38 from D. portschinskii 2 formed Du323(rad)1 in D. raddei and allele Du323(rost)1 occurred in D. portschinskii as Du323(port)5. Similarly, alleles Du215(rost)1–4 were the same as Du215(port)7, 5, 10, 7, respectively, and Du215(rost)5 was the same as Du215(rad)12. Further, Du281(rost)1–3 had the motif (GATA)n, and specifically (GATA)2 GAGAT. The locus also appeared as Du281(rad)3 and Du281(rost)4 with a perfect (GATA)10; these coincided with alleles Du281(port)6 and 7, respectively. Due to the high mutation rate of microsatellite DNAs [D. rostombekowi and its parents. The dendrograms of genetic similarity for Du281 and Du323 loci, for which we found SNV markers, were shown in D. raddei, and others from paternal species D. portschinskii. For example, Du281(rost)1, 2, and 3 showed similarity with all Du281 alleles of D. raddei, and Du281(rost)4 was similar to two Du281 alleles of D. portschinskii. Du323(rost)1 had high similarity with Du323 alleles of D. portschinskii, and Du 323(rost)2 had high similarity with Du323 alleles of D. raddei.Alleles of Du215, Du281, and Du323 in chinskii . Microsaite DNAs , in someD. rostombekowi reject the hypothesis of monoclonality based on allozymes [D. armeniaca, D. dahli and D. unisexualis have more than one allozyme clone [D. dahli, whereas three microsatellite-containing loci detected 11 clones [D. rostombekowi, suggesting that it was formed by one hybridization event. Identical SNVs but different microsatellite markers show that the hybridization event involved very similar female D. raddei and male D. portschinskii in one population.Given that lizards with identical genotypes represent a distinct clone, the several microsatellite clones of llozymes . Howeverllozymes . The obsme clone  and micr1 clones . IdenticD. rostombekowi formed first. Assuming that unisexual species originating from a single hybridization exhibit a widespread and common clone with a few rare clones [D. rostombekowi might be ancestral . However, the finding of a second mitotype of D. rostombekowi specific for the Tsovak population [D. raddei in the hybridization. To examine this possibility, additional genotype-specific markers from more microsatellite loci are necessary.Analysis of mitochondrial DNA shows that pulation  indicateDarevskia exhibit low levels of clonal diversity and a high degree of mtDNA similarity with respect to their parental species. Their recent origins appear to best explain these patterns, while they may have largely different ages. Darevsky [Darevskia originated in the Caucasus after the last glacial maximum and that all species have had the same amount of time to evolve new clones, approximately 5000–7000 years ago. However, a multilocus analysis for the parthenogens D. unisexualis, D. bendimahiensis and D. uzzeli dated their origins to between 200 000 and 70 000 years ago [Darevskia and these predate the last glacial maximum. Different parental lineages hybridized several times in different geographic regions [Cytb data. If this calculation is applicable to lizards, the age of D. rostombekowi would be approximately 200 000 years old (2% divergence) [Parthenogenetic species of Darevsky  suggesteears ago . These r regions . Further regions  providedergence) .Heteronotia binoei (Gekkonidae) [Aspidoscelis tessellatus and A. neomexicanus [D. dahli, D. armeniaca, and D. unisexualis [Darevskia rostombekowi has two times lower clonal diversity than D. dahli [D. rostombekowi may have a more recent origin than D. dahli, or it suffered a severe bottleneck.Electrophoretic studies of proteins and mitochondrial DNA have shown that parthenogenetic lizards tend to be multiclonal. A high level of variability of allozyme loci was discovered in konidae) . Geneticexicanus  and in psexualis . DarevskD. dahli . AlthougD. rostombekowi inhabiting the coast of Lake Sevan constitutes a chromosomal race and/or a clone that emerged because of chromosomal mutations occurring in the course of karyological evolution.Some cytogenetic and molecular characteristics of the genome of hybrid species differ from those of the bisexual species due to disturbance in the segregation of chromosomes. This results in cytogenetic variability, different genomic activity and mutations in some loci ,31,42,61D. rostombekowi. Future studies may determine whether the clonal diversity owes to a single or multiple interspecific hybridization events, and identify the role mutations play in altering the initial clone(s).In summary, our analyses discover multiclonal genetic structure in the parthenogenetic lizard S1 Fig*The number of alleles is shown in square brackets.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Moschus berezovskii, is one of seven musk deer (Moschus spp.) and is distributed in Southwest China. Akin to other musk deer, the forest musk deer has been traditionally and is currently hunted for its musk . Considerable hunting pressure and habitat loss have caused significant population declines. Consequently, the Chinese government commenced captive breeding programs for musk harvesting in the 1950s. However, the prevalence of fatal diseases is considerably restricting population increases. Disease severity and extent are exacerbated by inbreeding and genetic diversity declines in captive musk deer populations. It is essential that knowledge of captive and wild forest musk deer populations' immune system and genome be gained in order to improve their physical and genetic health. We have thus sequenced the whole genome of the forest musk deer, completed the genomic assembly and annotation, and performed preliminary bioinformatic analyses.The forest musk deer, A total of 407 Gb raw reads from whole-genome sequencing were generated using the Illumina HiSeq 4000 platform. The final genome assembly is around 2.72 Gb, with a contig N50 length of 22.6 kb and a scaffold N50 length of 2.85 Mb. We identified 24,352 genes and found that 42.05% of the genome is composed of repetitive elements. We also detected 1,236 olfactory receptor genes. The genome-wide phylogenetic tree indicated that the forest musk deer was within the order Artiodactyla, and it appeared as the sister clade of four members of Bovidae. In total, 576 genes were under positive selection in the forest musk deer lineage.We provide the first genome sequence and gene annotation for the forest musk deer. The availability of these resources will be very useful for the conservation and captive breeding of this endangered and economically important species and for reconstructing the evolutionary history of the order Artiodactyla. Moschus are endemic to Asia. They are currently listed in Appendix II in CITES and in Category I of the State Key Protected Wildlife List of China ) were identified using software MSDB  in the fTo estimate species-specific and shared genes in the forest musk deer in comparison to 10 mammal species, we used orthoMCL  to definWe constructed the phylogenetic trees based on Bayesian inference  and maxiHere, we report the first draft assembly of the forest musk deer genome, a species that is of particular importance to Chinese ecology, biodiversity conservation, economy, and medicine. The availability of the genome and these results will be very useful for the conservation and captive breeding of this endangered and economically important species and for reconstructing the evolutionary history of the order Artiodactyla.GigaScience repository, GigaDB [The DNA sequencing data have been deposited into the NCBI Sequence Read Archive under the ID PRJNA317652. Other supporting data, including the assembled genome, gene annotations, and BUSCO results, are available via the , GigaDB .Figure S1: K-mer (k = 17) distributions in forest musk deer genome.Figure S2: GO comparative analysis and functional classification between forest musk deer, sheep and cattle.Figure S3: Distribution of divergence of each type of TEs in forest musk deer genome. The divergence rate was calculated between the identified TE elements in the genome and the consensus sequence in the TE library used. SINEs: Short interspersed elements. LINEs: Long interspersed elements. LTR: Long terminal repeat retrotransposon.Moschus bweezovskii), cattle (Bos taurus), yak (Bos grunniens), sheep (Ovis aries), Tibetan antelope (Pantholops hodgsonii), alpaca (Vicugna pacos), and pig (Sus scrofa), which representing Artiodactyla; human , horse , and dog , mouse . For each animal, proteins were represented by bars and were classified based on orthoMCL analysis. Single_copy (green) included the common orthologs with the same number of copies in different species; Multi_copy (red) included the common orthologs with different copy numbers in different species; Unique (magenta) included the orthologs that were only in one species; Unclustered genes (yellow) included the genes that could not be clustered into known gene families; Other (blue) included the genes that could be clustered into known gene families, but were not belonged to Single_copy, Multi_copy or Unique.Figure S4: Protein orthology comparison between different genomes. There were forest musk deer , and National Natural Science Foundation of China (NSFC31702032)The authors declare that they have no competing interests.Z.F., X.Z., J.L., W.Q. and B.Y. designed and supervised the project. Z.F., W.L., C.Y., J.J., C.P., J.Y., P.B., Y.S., and K.C. performed the bioinformatics analyses. M.P. revised the manuscript. Z.F. and B.Y. wrote the manuscript.GIGA-D-17-00185_Original_Submission.pdfClick here for additional data file.GIGA-D-17-00185_Revision_1.pdfClick here for additional data file.GIGA-D-17-00185_Revision_2.pdfClick here for additional data file.Response_to_Reviewer_Comments_Original_Submission.pdfClick here for additional data file.Response_to_Reviewer_Comments_Revision_1.pdfClick here for additional data file.Reviewer_1_Report_ -- Aaron Shafer9/13/2017 ReviewedClick here for additional data file.Reviewer_1_Report_(Revision_1) -- Aaron Shafer1/27/2018 ReviewedClick here for additional data file.Reviewer_2_Report_ -- Sylvain Foissac9/19/2017 ReviewedClick here for additional data file.Reviewer_2_Report_(Revision_1) -- Sylvain Foissac1/22/2018 ReviewedClick here for additional data file.Supplemental materialClick here for additional data file.