Rheumatoid arthritis (RA) is a chronic progressive autoimmune disease leading to considerable disability over time. The disease can be characterized by the presence of multiple autoantibodies in the serum and synovial fluid. Microbial dysbiosis is proposed to play a role in the pathogenesis of RA. Increased systemic bacterial exposure leads to elevated levels of antimicrobial response factors (ARFs) in the circulation. In the present study, we tested whether RA patients have increased levels of ARFs by analyzing the levels of multiple ARFs in serum from RA patients and healthy age and sex-matched controls. The levels of soluble CD14 (sCD14), lysozyme, and CXCL16 were significantly elevated in RA patients compared to healthy controls. Lipopolysaccharide binding protein (LBP) levels remained unchanged in RA patients compared to healthy controls. A positive correlation of LBP with rheumatoid factor (RF) was also found in RA subjects. Interestingly, the levels of anti-endotoxin core antibodies (EndoCAb) IgM, total IgM, EndoCAb IgA, and total IgA were significantly elevated in RA patients compared to healthy controls. No significant changes in the levels of EndoCAb IgG and total IgG were observed in RA patients compared to healthy controls. Furthermore, lysozyme and CXCL16 levels were positively correlated with disease severity among RA subjects. Increases in the levels of several ARFs and their correlations with clinical indices suggest systemic microbial exposure in the RA cohort. Modulation of microbial exposure may play an important role in disease pathogenesis in individuals with RA. Rheumatoid arthritis (RA) is a chronic progressive autoimmune disease leading to severe disability. Genetic, environmental, and epigenetic factors instigate the production of autoantibodies and the loss of tissue tolerance in RA –4. ThesePrevotella copri and decrease in Haemophilus spp. compared to healthy controls  are released into the circulation to neutralize microbial products. ARFs are diverse pleiotropic molecules that include cytokines, chemokines, anti-endotoxin core antibodies (EndoCAb), peptides, and proteases , 35. TheIn the present study, we tested whether RA patients have increased levels of ARFs by analyzing the levels of multiple ARFs in serum from RA patients and healthy age- and sex-matched controls. Increased levels of ARFs may indicate an increase in systemic bacterial exposure. The ARFs tested include soluble CD14 (sCD14), lipopolysaccharide-binding protein (LBP), lysozyme, CXCL16, EndoCAb IgG, EndoCAb IgA, and EndoCAb IgM. Our results revealed a marked elevation of several ARFs in RA patients. These significant elevations of ARFs may be clinically relevant since they correlate with clinical indices. Our results point to systemic microbial exposure as a common stimulus in RA, which could perpetuate the disease.Subjects were recruited for the Studies of the Etiology of Rheumatoid Arthritis (SERA), a prospective longitudinal study designed to evaluate the contributions of environmental and genetic factors to the development of RA. Recruitment of RA population has been described in detail previously . Healthy® serum separator tubes  from both RA patients and healthy controls. After clotting, the whole blood collected was centrifuged  and the serum layer was removed. Measurements of rheumatoid factor (RF), high sensitivity C-reactive protein (CRP), and anti-cyclic citrullinated protein antibodies (anti-CCP) in the serum were measured using previously described methodologies  and Hycult Biotech , respectively. EndoCAb IgG, EndoCAb IgA, and EndoCAb IgM were measured using direct ELISA kits procured from Hycult Biotech. CXCL16 was analyzed using a sandwich ELISA kit procured from Thermo Scientific . Lysozyme levels were measured using sandwich ELISA kit procured from MBL . Total IgG, IgA, and IgM were measured using sandwich ELISA kits procured from Invitrogen . To block non-specific antibodies that may interfere with the assay, the samples were diluted in appropriate buffers, which contained 50 μg/ml of HeteroBlock  and kept for 30 minutes before adding into the ELISA plate. All the analyses were performed blinded to case/control and clinical status. In order to maintain the test quality and reproducibility, an internal control was included in all the assays and the coefficient of variation (CV) of replicates was set at ≤ 10%.t-test was used. For correlation analysis, Pearson product-moment correlation coefficient (Pearson's r) analysis was performed. Gender stratification was also done to detect sex-related changes in the levels of ARFs in RA subjects compared to their respective control subjects. For all statistical tests, P < 0.05 was considered to be statistically significant. All the statistical tests were done with GraphPad Prism 7 . Descriptive statistics of all the analyte levels are given in We transformed all the data into base-10 logarithm values for statistical analysis and correlation studies. Square root transformation was employed for disease indices, which included true zeros. For testing statistical significance, the unpaired P = 0.004) compared to healthy controls (P = 0.009). RA females showed a trend toward an increase in sCD14 levels when compared to control females (P = 0.075).Since CD14 acts a co-receptor for LPS, elevated levels of sCD14 are considered to reflect LPS exposure and subsequent monocyte/macrophage activation –44. Intecontrols . Gender P = 0.224)  in RA patients is observed in our study  . A positur study . Howeverr = 0.363, P = 0.041)   . Further= 0.058) . We did P = 0.033) compared to healthy controls (P = 0.118) nor RA females (P = 0.141)   were sigP = 0.0003) compared to healthy controls (P = 0.016) and RA females (P = 0.006) showed a significant increase in the levels of CXCL16 compared to their respective controls , lysozyme , and CXCL16  in both RA patients and in healthy controls  was also observed in both RA subjects and in healthy controls (Pearson's controls . Moreovecontrols .P = 0.001) and EndoCAb IgM (P = 0.011) were elevated in our RA cohort compared to healthy controls (P = 0.007) whereas males showed only a trend toward an increase (P = 0.06) (P = 0.032) compared to control females. RA males did not show any significant difference in EndoCAb IgM levels (P = 0.156) compared to control males  . EndoCAbol males .P = 0.001) and IgM (P = 0.0004) were significantly elevated in RA patients compared to healthy controls, confirming earlier reports (P = 0.096) (P = 0.001) whereas RA males showed a trend toward significant increase (P = 0.293). IgM levels of both RA males (P = 0.038) and RA females (P = 0.003) were elevated compared to respective control subjects  in RA cohort showed that IgA ( reports –64. The = 0.096) . RA femasubjects .P = 0.194), EndoCAb IgA:total IgA (P = 0.697), and EndoCAb IgM:total IgM (P = 0.528) in RA subjects compared to healthy controls . Healthy controls did not show any significant correlation between these factors   and EndoCAb IgA  in both RA subjects and healthy controls  and total IgA levels  in both RA patients and healthy controls  . We founcontrols . Moreovecontrols .r = 0.308, P = 0.032)  and the activity index vs. CXCL16  in RA subjects   . A trendsubjects . Further= 0.019) . Thus, tNo other ARFs showed a significant association with disease activity measures. In addition, we did not find any significant correlation between the levels of anti-CCP antibodies with ARFs in RA patients (data not shown).Systemic exposure to microbial products has been hypothesized to trigger and/or potentiate several autoimmune diseases including RA –67. In rWe observed a significant elevation of sCD14 levels in RA patients compared to healthy controls confirming previous reports –76. sCD1Similar to sCD14, LBP is a critical circulatory molecule involved in endotoxin clearance . We did Levels of lysozyme were significantly elevated in RA patients, confirming previous observations . LysozymWe found that CXCL16 was elevated in our RA subjects compared to healthy controls, confirming earlier results , 102. CXB cells play an important role in the pathogenesis of RA by secreting autoantibodies, presenting antigens and producing cytokines. We observed increased levels of total IgA and total IgM in RA patients compared to healthy controls. Increased levels of total IgA and IgM in RA patients reflects activation of the immune system –63. EndoSimilar to other autoimmune conditions, RA mainly affects females . We obseMultiple studies suggest the potential role of microbes as triggering and/or accelerating factors in autoimmunity , 123–126Elevated levels of ARFs in our study support the role of myeloid cell activation in disease pathogenesis, possibly via systemic microbial exposure in RA cohort . The gasAll datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by University of Colorado's Institutional Review Board (COMIRB#01-675). The patients/participants provided their written informed consent to participate in this study.PA: experimental design and execution, data analysis, and manuscript preparation. RH and NS: experimental design, data analysis and reviewed the manuscript. JS, EB, MF, KD, MD, TM, and VH: substantial contribution by acquiring patient samples and clinical data and manuscript revision and review. All the authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here we consider aspects of the application of biotechnological methods to rapid creation, propagation, and maintenance of plants with improved or new traits in sugar beet breeding. The results of the works carried out in these fields by the Federal State Budgetary Scientific Institution “The A.L. Mazlumov All-Russia Research Institute of Sugar Beet” are reviewed. A close association between morphological and physiological changes in in vitro cultured organs and tissues, on the one hand, and breeding traits, on the other hand, which allows the development of experimental systems for non-amphimictic plant reconstruction is shown. The influence ofin vitro growth conditions on haploid cells of unfertilized sugar beet ovules in the course of obtaining doubled haploid lines with high degree of homozygosity and maintenance of valuable breeding properties is considered. As compared to common inbreeding, this method shortens the time for development of homozygous material from 10–12 to 3–5 years, which is of great importance for speeding-up the breeding process. The results of studies on the culturing of mature sugar beet zygotic embryos based on in vitro selective systems have made it possible to improve the adaptive potential of plants and to provide complex resistance to environmental stress factors. Strict selection under abiotic stress conditions allowed creation of sugar beet isogenic lines with tolerance of drought, salinity, and soil acidity. It is shown that the proposed original design of mass-scale microclonal in vitro reproduction and deposition of elite plants as components of highly productive hybrids can be used to obtain seeds of uniform high-quality breeding material. The technologies developed by biotechnological methods are a topical and innovative direction of inquiry, since the application of these techniques to sugar beet breeding will promote obtaining of competitive hybrids with a set of commercially valuable traits. The combination of biotechnology methods, including tissue culture, and traditional breeding techniques is expected to provide an opportunity to obtain a new starting material to develop domestic varieties and hybrids of new generation with heterosis effect and a wide resistance spectrum persisting across generations. One of the current topical directions in sugar beet breedingis the development of highly productive hybrids on alinear basis. The traditional breeding process involves timeconsumingselection of self-pollinated lines with uniformmorphological traits, economically useful properties , and high quality of seeds. However,the breeding methods in use take much time and labor. Itis determined by the two-year development cycle of plants,inbreeding depression, and the phenomenon of self- andcross-incompatibility, hampering the maintenance of geneticaluniformity in the breeding material. In this connection,biotechnological methods based on in vitro culturingof organs and tissues are promising, as they permit one todevelop and stabilize breeding material and to maintain itsvaluable traits. The application of biotechnologies to thebreeding process will allow reduction and then eliminationof our lagging behind foreign countries in different indicesof agricultural industry.Raise of sugar beet doubled haploids in tissue culturewill produce homozygous lines to obtain hybrids witha set of commercially useful traits. This is an importantstage of sugar beet breeding, which should be finishedand included in the technological process. Use of in vitroselection systems promoting the breeding of plants withhigh resistance to abiotic stresses, which is based on theability of vegetative organisms to develop mechanisms ofprotection from adverse environment factors at the celllevel  is alsopromising in sugar beet industry.In practical sugar beet breeding, the refinement of biotechnologicalmethods for mass reproduction and longmaintenance of valuable forms is one of the main directionsin works with isolated tissue cultures. An important basisof these methods is the unique property of totipotency inplant cells characterizing the whole potential of traits of anindividual, which, when cultivated, is implemented throughdifferent ways of morphogenesis: embryoidogenesis, organogenesis,and histogenesis . In this connection,the morphogenetic competence of cultivated tissue and organs of sugar beet plants can promote a certainway of development, which demands special attention ofresearchers when microcloning the developed material.To carry out research in the above-mentioned directions,a number of international scientific institutions have beeninvolved. In particular, to improve sugar beet germplasm,methods for development of doubled haploids, estimationof resistance to stresses, various molecular methods,and so on are used in the United States. The AgriculturalResearch Service of the United States Department of Agriculture(USDA ARS) supervises all studies . The international interest to haploid plants inthe European Union is confirmed by development of EUGOST Action 851 “Gametic cells and molecular breedingfor crop improvement”. Homozygous lines are used in allbreeding and seed-growing companies in Europe such asStrube, KWS, Syngenta, and Fiorimond Despres, engagedin the development of sugar beet hybrids and seed material.The development of sugar beet haploid and doubledhaploid forms (x/xx) is an integral part of investigationsat the Planta research center. There, basic investigations indifferent fields of biotechnology including working out ofmethods of sugar beet breeding for resistance to stressesare concentrated. Using biotechnologies, sugar beet breedingmaterials were obtained in Germany, Turkey , Poland , Bulgaria, and so on. Technological indicesof the breeding material produced with the help of sugarbeet doubled haploids in Byelorussia, which demonstratesuccess in breeding at (or above) the level of usual hybrids,are of interest. Tests of the doubled haploids showedthat they exceeded the diploid standard in beet root yield(43–45 t/hectares) and sugar content (20.9 and 21.1 %). A long-term study of hybridsinvolving DH lines produced in Bulgaria showedhigh performance as compared to traditional hybrids. Asfor beet root yield, dihaploid hybrids exceeded the controlby 11.4–13.7 %, and the sugar yield was about the standardlevel . These results are ofpractical importance, as they promote the introduction of technologies based on tissue culture methods for rapid raiseof breeding material with valuable traits to be included intobreeding programs.In connection with the above, investigations in the developmentand introduction of biotechnologies for targetedproduction of genetically improved starting material fordomestic sugar beet breeding in order to produce newgenerationpromising hybrids are topical. Success of theseinvestigations can be ensured if taking into account specificfeatures of morphogenetic potentialities of the developmentof certain plant organs tending to intense regeneration andreproduction under in vitro conditions.In the course of long-term investigations at the FederalState Budgetary Scientific Institution “The A.L. MazlumovAll-Russia Research Institute of Sugar Beet”, methods ofin vitro haploid parthenogenesis induction were developed,and optimum conditions of sugar beet explants’ culturewere determined . The choiceof initial plants as donors of explants with regard to phenotypefeatures, including the morphological habitus ofseed-bearing plants, high degree of seed monogermity,and seed setting, is an important issue. Ovules located inbuds in the central ear of the pleiochasium cluster wereprimary explants for in vitro culturing. Cytoembryologicalstudies  indicated that in vitro formationof haploid embryoids occurred at all developmentstages of embryo sac nuclei and cells. It was promoted bypronounced polarity and high ability of haploid cells todevelop. Ovules containing eight haploid nuclei or sevencells  in embryo sacs displayed thegreatest activity in the formation of haploid embryoids.According to the present notion, the switch of the developmentprogram from the gametophyte to sporophyte typecan occur in this time span .Exposure of material to low positive temperatures during2–4 days stimulates this process.The method using alternation of В5 nutrient media differingin consistence proved to be the bottom line in ovuleculturing. High gibberellin concentrations in the liquidnutrient medium caused direct regeneration of haploid embryoidswith embryo root and cotyledon leaves formation.Transplantation to a solid nutrient medium was followedby the formation of continuance shoots. Presence of 6-BAPin a liquid medium induced the formation of callus tissue,whose transplantation to a solid nutrient medium with additionof kinetin (Kn) and 2.4-D induced the developmentof regenerants by hemmorhizogenesis. In this technique,the cultivated explants retained their viability for up to4–6 months and the proliferation and differentiation processeswere activated in female gametophyte nuclei. Thespecialization of the formed cells of plant tissues increased the output of haploid regenerants by up to 18.9 % throughdirect embryoidogenesis and by up to 11.0 % by callusogenesis.Such method accelerated cell differentiation by25–30 % and increased the number of the formed regenerantsby up to 13–19 % .At the next step, morphologically normal haploid regenerantswere selected and transformed to diploids incolchicine-containing nutrient medium. All stages of theprocess were accompanied by selection for cytological,biochemical, and molecular traits . Raise of haploid plants has long been known. Ploidywas most often determined by directly counting chromosomesin a nucleus . Analysis of nDNA by flow cytometry based onfluorescent staining of plant nuclei  provided an opportunity to select regenerants withthe haploid chromosome set (n = 9) rapidly and efficiently,and, after treatment with colchicine, to select doubledhaploids (2n = 18). It should be noted that cell divisionaberrations were often observed after mutagenesis; thisresulted in varying chromosome quantities in regenerantsand occurrence of mixoploid and aneuploid forms, whichwere rejected .Biochemical evaluation revealed different enzyme activitypatterns in experimental specimens. In comparison withinitial diploid forms, haploid plants were characterized bythe protein quantity elevated by 60 % and greater activitiesof peroxidase, glucose-6-phosphate-dehydrogenase, andisocytrate-dehydrogenase. After chromosome doublingin regenerants, these values returned to the control orslightly higher levels . It isconjecturable that the change in enzyme activities in sugarbeet haploid regenerants was due to methylation of DNAgenome sites connected with protein functioning. The revealeddifferences reflected more profound changes causedby cell differentiation during tissue culturing . This issue demands a special comprehensive study.After diploidization of regenerants, the detection ofhereditary changes not expressed phenotypically was ofgreat importance. РСR and RFLP analyses using Hind IIIrestrictase  made it possible toidentify the cytoplasm type from the number of fragmentsafter chromosome doubling. In microclones with normalcytoplasm, one fragment (800 bp) was amplified. In formswith the sterile type of cytoplasm, two digestion products,320 and 480 bp in length, were revealed. Doubled haploidsin which this fragment was not digested by Hind III includedcompletely fertile forms with normal cytoplasm (N) andnuclear genes in the recessive form (rf). In other specimens,polymorphism of fragments was observed, which supposedthe presence of sterile cytoplasm (S) and different combinationsof recessive and dominant alleles of the nuclear genesRf1/rf1 and Rf2/rf2 in the corresponding haploid forms. The identification of cytoplasm type in regenerants at earlysteps of culturing facilitated the development of lines withCMS, being of considerable interest for sugar beet breeding.The last step of in vitro culturing included the cloningof regenerants with doubled ploidy and their subsequentrooting on a nutrient medium with modified hormonalcomposition. The selected microclones with root systemsand well-developed leaf surface were planted to soil undergreenhouse conditions. Keeping air humidity high allowedup to 72 % survival of microclones. Further, plants weregrown for 3 months till obtaining stecklings of 70–100 gin weight, which were then vernalized for 45 days. Atthe beginning of seedstalk formation, the greenhouse airtemperature was raised to 14–20 °C, and constant illuminationwas kept for a month. In this time, intense growthand development of seed-bearing plants were noted. Then,budding and flowering were observed. The heights of plantswere 1.5 meters or more, and the thicknesses of the mainflower-bearing stems were about 3.0 cm. Flowering wassynchronous and intense. The fertility of pollen grainsvaried from 87 to 92 %. In 8–10 weeks, seeds with a highlevel of monogermity were formed.Our study revealed the basic conditions for inducinghaploid parthenogenesis and allowed a three-year cyclefor producing homozygous DH lines to be elaborated. Theobtained seeds of four DH lines became a new startingmaterial, and now they are used in breeding work. Thedevelopment of sugar beet homozygous lines will promoterapid raise of hybrids with a set of economically valuabletraits .As a result of our experiments, a method of in vitro selectionof sugar beet forms was originated in order to developunique breeding material with resistance to adverse environmentalconditions .The method is based on double passage of regenerantsunder severe selective conditions, after which the regenerantssurviving high chloride concentrations constituted66.0–81.7 %. Further passage of salt-tolerant forms on anutrient medium supplemented with a nonionic osmoticcomponent (0.45 M) promoted an improvement of adaptationmechanisms of plant protection under osmotic stressconditions. In this case, the survival rate of genotypesdoubled, and the level of general regenerant adaption toabiotic stress  reached 59.0 %.The passage of the obtained regenerants at elevated acidity(рН 3.5) was accompanied by intense development ofcontinuance shoots, which pointed to a high resistance ofmicroclones, 69.1–87.5 %. The level of general adaptionof regenerants to a more complex stress (drought + acidity)increased tenfold .When developing the method, of great importance werethe experiments that demonstrated that the growth of unspecializedplant tissues  on selective nutrient media with addition ofsea salt (2 %) and sorbitol (0.45 M at рН 4.0) led to almostcomplete degradation of plant material. During primaryselection, the emergence of regenerants with resistancewas observed only in 2–4 % of explants, for which matureembryos were used. This period was accompanied by slowformation of continuance shoots in the survived explants. Itwas connected with retardation of cell extension and divisionprocesses, which resulted in inhibition of regenerantdevelopment . Use of secondaryselection, which, in addition, increased the degree of resistancein the regenerants under severe selection conditions,proved to be efficient.Further mass-scale systemic selection of regenerants accordingto the root length index , whichvaried from 1.0 to 1.2 in the experiments, increased thepercentage of survived tolerant plants to 87.5–89.2 %.The regenerants selected according to root length underconditions that simulated high soil acidity (рН 3.5) anddrought were remarkable for well-developed root systemalong with the uniformity of leaf apparatus. Root formationunder conditions of osmotic stress was less changeablethan leaf development. After the exposure to osmoticcomponents, the rate of root elongation restored quickly.The intense development of the root system under conditionsof osmotic stress was determined by the accelerationof lateral root growth, providing better access to water andnutrients .The revealed features of the morphological developmentof sugar beet regenerants were associated with changes in avariety of biochemical processes that enhanced the work ofoxidative stress enzymes, increased protein synthesis rates, and caused accumulation offree proline .Under conditions of water deficiency, proline increasedosmotic pressure of cell juice, which was indicative of itsosmoregulation function . Underabiotic stress conditions, the accumulation of proline (from36.9 to 79.3 microgram/g) in salt-tolerant sugar beet regenerantswas associated with the genotype. The resistancecoefficient K, determined as the ratio between the quantityof proline at stress and its initial content, was 5.8–10 timesmore in salt-resisting forms than in the control. Furtherpassage of salt-tolerant forms with the presence of sorbitolat concentrations 0.45–0.40 М increased the resistancecoefficient by a factor of 1.5–1.9. During the systemicselection , the overall osmotic resistanceof sugar beet genotypes increased by a factor of7–12. These data allowed proline quantity to be used asan index of sugar beet regenerant adaptation to stress and as an indicator of salt-resistant components .At the final stage, the selected forms with well-developedroot systems were grown under greenhouse conditions toobtain stecklings and then seed-bearing plants.These methods make it possible to carry out selection ofsugar beet forms with resistance to edaphic environmentalfactors to produce isogenic lines within 3–4 years instead ofthe previously required 8–10. It is of great importance forbreeding process when developing new sugar beet hybridstolerant of abiotic stresses.It has been shown that in the course of the development ofstress-resistant isogenic lines cultured sugar beet petioles,or leaves of young shoots, or embryos produce a new organismthrough embryoid formation . The key difference between the emerging embryolikebodies and haploid embryoids is the formation of theformer from diploid somatic cells of plants without participationof sexual gametes. The morphogenesis was basedon the formation of initial cells, having dense cytoplasmand a large nucleus, in epidermis. In the next division, atwo-cell proembryo formed to produce then a sphericalsomatic embryoid. At the next stage, the formation of aheart-shaped embryoid and a shoot with development of astem with cotyledon leaves was observed. Apparently, thestressful situation created by in vitro conditions causedmorphogenesis implementation via somatic embryoidformation. The resulting gametophytic and somatic embryoids,irrespective of their origin, were new organismsin their nascence and were primitives of plant reproductionby analogy with meristematic cells . In this connection, the stage ofembryoid induction is the key step of in vitro micropropagationof the produced sugar beet plant material.It is known that sugar beet is reproduced under naturalconditions solely by seeds and that it has only one backupmode to survive: vegetative propagation by traumaticpartition. This method was widely used in the breedingpractice of the 20th century for vegetative propagation ofbeet roots serving as ancestors of valuable starting materialwith maintenance of their most important traits: highsugar content, productivity, beet root shape, and so on.Now, it has been proven that types, methods, and forms ofseed reproduction can be responsible for the reproductiveability of any plant species in combination with vegetativereproduction . Only those speciesare the most productive in which generative and vegetativereproduction are combined. Therefore, the use of plantpropagation in sugar beet by microcloning may improve itsreproductive ability, seed production, and other valuabletraits and properties.At present, in vitro micropropagation on the basis ofmeristem culture is the main method both for the rapidpropagation of valuable breeding materials, and for theirlong-term maintenance . Byusing microcloning in practical breeding, a variety of initiallines with traits of CMS fixing ability, smooth surface ofa beet root, great quantity of seeds on seed-bearing plantsand so on can be maintained in culture, propagated, andemployed in the breeding process .Studies on using micropropagation and in vitro depositionto improve the morphogenetic potential of hybridcomponents produced according to the three-line breedingdesign were of considerable practical interest. The involvementof tissue culturing in the breeding process providedan increase in the reproductive ability of the developedhybrid components and brought about high-grade sugarbeet seeds .At the first step, elite genotypes were selected from threecomponents of a highly productive hybrid: lines with cytoplasmicmale sterility, a pollinator of O-type with the CMSfixing ability, and a fertile pollinator able to stimulate theheterosis effect in F1. When selecting the genotypes, usewas made of instant diagnostics with regard to morphologicand cytoembryologic traits reflecting developmental stagesof seed-bearing plants, mono-/multigermity, pollen grainfertility, synchrony of flowering, ploidy, etc. For in vitroculture introduction of the selected genotypes, apexes of10–20 floral shoots from the same seed-bearing plant wereused. The main inducers of continuance shoot developmentfrom apical meristem were hormonal componentsof the Gamborg’s and Murashige–Skoog nutrient medium. The presence of growth hormones –6-benzylaminopurine, kinetin, and gibberellin – in thegrowth medium caused the formation of up to eight adventiveshoots per one explant, and the height of the regenerantswas about 60–73 mm. At the end of stabilization, theformed regenerants had deep green leaves, the optimalratio between the petiole and leaf blade, good stooling,and developed apical point. Such microclones successfullyformed many axillary branches and then sufficient numbersof elite CMS, O-type, and multigerm pollinator regenerants.The required number of morphologically developedregenerants of hybrid line components were simultaneouslyin vitro cultivated in a collection of elite clones bydeposition. It allowed long-term maintenance of hybridline component purity at the level of the first generation.The regenerants were cultured without transplantationson hormone-free nutrient medium with high agar contentat weak illumination (500–600 lx), and the 16L:8D lightschedule.The second stage included mass-scale micropropagation,rooting of the regenerants, and raise of hybrid componentstecklings. For mass propagation, the deposited elite regenerants were transplanted to hormonal В5 nutrient medium,which activated meristems and induced the proliferationof lateral shoots. Repeated replanting of shoots providedthe sufficient number of microclones. The greatest numberof regenerants was obtained from monogerm fertile lines(48 shoots per explant on the average), and MS-lines andpollinators gave the least ones .It should be noted that forms with CMS appearedto be very sensitive to changes in culturing conditions anddied more often than fertile genotypes. They were more difficultto stabilize under in vitro conditions and to reproducelater on .The regenerant rooting was associated with the inductionof adventitious roots, achieved by modification of thenutrient medium hormonal composition. Root formationlasted 3–4 weeks. The resulting sugar beet microclones withstrong root systems and well-developed above-ground partswere transferred to nonsterile conditions of soil substrate,where adaptation of the microclones was conducted. Themicroclone survival, growth, and development during theadaptation period were determined by the humidity conditionsfor the microclones. The keeping of high air humidityallowed 80–100 % survival of microclones. The plantsformed in a greenhouse were characterized by uniformityof morphological traits within a line. The developed plantsof hybrid line components were grown under greenhouseconditions till the formation of stecklings. The final importantprocess of this step was the harvesting of stecklingsand their vernalization by storing in refrigerators.At the third stage, breeders used the seed-bearing plantsobtained from stecklings for hybridization under fieldconditions, in separate field plots according to the schemesused to obtain seeds of a simple hybrid and a multigermpollinator of improved quality .The elaboration of biotechnological methods for massscalein vitro reproduction and deposition of sugar beetbreeding materials is innovative. It is of top priority andgreat importance, as it makes it possible to obtain sugarbeet lines with high genetic uniformity and better seedmaterial. The introduction of this method, which speedsup breeding twofold, would add much to the developmentof highly productive hybrids for commercial use and inseed industry, providing maintenance of high indices ofeconomically useful properties of the developed breedingmaterial.In this review, we describe the main biotechnology methodspresently used to obtain and propagate new material forsugar beet breeding. Haploid parthenogenesis is a novelmethod promising for breeding programs. Eliminating thenecessity of multiple self-pollinations in plants, the method halves the time required for the raise of homozygous materialwith valuable traits. Lines of doubled haploids havebeen produced and monogerm seeds of sugar beet hybridcomponents have been obtained on its basis.Studies that have enabled the development of in vitrobreeding principles making sugar beet regenerants moretolerant of great salinization, drought, and soil acidity areof vital importance. The obtained results hold remarkablepromise for breeding, as the approach under considerationallows the development of isogenic lines with high osmoticresistance to adverse environmental factors within threeyears.Mass-scale micropropagation and in vitro depositionof elite hybrid components using the three-line breedingscheme is helpful in sugar beet breeding. This method notonly increases the productive ability of seed-bearing plant,but also produces high-grade sugar beet seeds.Introduction of these technologies into sugar beet breedingand seed industry is a focal and innovative direction ofstudies enabling increase of the homozygosity level in linesand improvement of seed material quality with maintenanceof high performance in the developed hybrids.The authors declare no conflict of interest.Barykina R.P., Veselova T.D., Devyatov A.G., DzhalilovaKh. Kh., Ilyina G.M., Chubatova N.V. The Handbookof Botanic Microtechniques: Fundamentals and Methods.Moscow: Publ. House of Moscow State University, 2004.Available at http://bookre.org/reader?file=1483744&pg=1(in Russian)Bates L.S., Waldren R.P., Teare I.D. Rapid determinationof free proline for water-stress studies. Plant Soil.1973;39:205-207. DOI 10.1007/BF00018060.Batygina T.B. Embryoidogeny is a new type of a vegetativepropagation. In: Batygina T.B. (Ed.). Embryology ofFlowering Plants: Terminology and Concepts. Vol. 3. ReproductiveSystems. St. Petersburg: Mir i Semya Publ.,2000;334-349. Available at https://www.twirpx.com/file/435188/ (in Russian)Batygina T.B., Kruglova N.N., Gorbunova V.Yu., TitovaG.E., Seldimirova O.A. From a Microspore to a Variety.Moscow: Nauka Publ., 2010. (in Russian)Batygina T.B., Vasilyeva V.E. Plant Reproduction. St. Petersburg:St. Petersburg University Publ., 2002. (in Russian)Beckmann J.S., Soller M. Restriction fragment length polymorphismsin genetic improvement: methodologies, mappingand costs. Theor. Appl. Genet. 1983;67:35-43. DOI10.1007/BF00303919.Butenko R.G. Isolated Tissue Culture and Physiology ofPlant Morphogenesis. Moscow: Nauka Publ., 1964. (inRussian)Cherkasova N.N., Zhuzhzhalova T.P. Obtaining osmosis-resistantsugar beet plants under in vitro culture. SakharnayaSvekla = Sugar Beet. 2011;7:22-24. (in Russian)Cherkasova N.N., Zhuzhzhalova T.P. Modeling of in vitrobreeding conditions to develop sugar beet forms resistantto environment stress factors. Sakharnaya Svekla = SugarBeet. 2014;6:16-18. (in Russian)Cherkasova N.N., Zhuzhzhalova T.P., Kolesnikova E.O.Development of sugar beet regenerant plants resistantto a set of stress factors. Sakhar = Sugar. 2018;5:28-30.(in Russian)Dukhovsky P., Yuknis R., Brazaitite A., Zukauskaite I. Plantresponse to integrated impact of natural and anthropogenicstress factors. Fiziologiya Rasteniy = Russ. J. Plant Physiol.2003;50(2):147-154.Gürel S., Gürel E., Kaya Z., Erdal M., Güler E. Effects ofantimitotic agents on haploid plant production from unpollinatedovules of sugar beet (Beta vulgaris L.). Biotechnol.Biotechnol. Equip. 2003;17(2):97-101. DOI10.1080/13102818.2003.10817065.Kikindonov G., Kikindonov Tz., Enchev S. Economical qualitiesof crosses between doubled haploid sugar beet lines.Agric. Sci. Тechnol. 2016;8(2):107-110. DOI 10.15547/ast.2016.02.018.Kilchevsky A.V., Khotyleva L.V. (Eds.). Genetical Basisof Plant Breeding. Vol. 3. Biotechnology in Plant Breeding.Cell Engineering. Minsk: Belarus. Navuka Publ.,2012;203-216. (in Russian)Kolesnikova E.O., Vasilchenko E.N., Cherkasova N.N.Possibilities of using biotechnology methods for sugarbeet in breeding process. In: Scientific Support of Beet-Growing Industry: Proc. of the Int. scientific-practicalconf. dedicated to the 90th anniversary of the Sugar BeetExperimental Research Station . Minsk: Belarus. Navuka Publ., 2018a;40-48.(in Russian)Kolesnikova E.O., Zhuzhzhalova T.P. Microcloning andmaintenance of lines material in sugar beet breeding process.In: Proc. of IV (XII) Int. Botanical Conf. of YoungScientists in St. Petersburg, April 22–28, 2018. St. Petersburg,2018;267-268.Kolesnikova E.O., Zhuzhzhalova T.P., Cherkasova N.N.,Vasilchenko E.N. Рerspective technologies of isolatedtissue culture in sugar beet breeding process. RossiyskayaSelskokhozyaystvennaya Nauka = RussianAgricultural Sciences. 2018b;6:13-17. DOI 10.31857/S250026270001825-2 (in Russian)Kosareva I.A. The study of crops and wild relatives collectionsfor signs of resistance to toxic elements of acid soils.Trudy po Prikladnoy Botanike, Genetike i Selektsii = Proceedingson Applied Botany, Genetics, and Breeding.2012;170:35-45. (in Russian)Kozlovsky V.F., Apasov I.V., Zhuzhzhalova T.P., FedulovaT.P. Main directions and results of sugar beet breedingin USA. Sakharnaya Svekla = Sugar Beet. 2016;8:35-41.(in Russian)Lamaoui M., Jemo M., Datla R., Bekkaoui E. Heat anddrought stresses in crops and approaches for their mitigation.Front. Chem. 2018;6:26. DOI 10.3389/fchem.2018.00026.Nitzsche W., Wenzel G. Haploids in Plant Breeding. Berlin;Hamburg, 1977. Pausheva Z.P. Workshop on Plant Cytology. Moscow:Agropromizdat Publ., 1988. (in Russian)Rahnama A., Munns R., Poustini K., Watt M. A screeningmethod to identify genetic variation in root growth responseto a salinity gradient. J. Exp. Bot. 2011;62;69-77.DOI 10.1093/jxb/erq359.Shabala S., Munns R. Salinity stress: physiological constraintsand adaptive mechanisms. In: Shabala S. (Ed.).Plant Stress Physiology. 2nd edn. CABI, 2017;24-64.Soshnikova T.N., Radyukina N.L., Korolkova D.V., NosovA.V. Proline and functioning of an antioxidant plantsystem and cultivated cells of Thellungiella salsugineaduring oxidative stress. Russ. J. Plant Physiol. 2013;60(1):41-54. DOI 10.1134/S1021443713010093.Tomaszewska-Sowa M. Cytometric analyses of sugarbeet (Beta vulgaris L.) plants regenerated from unfertilizedovules cultured in vitro. Plant Breed. 2010;2:231-235.Tomaszewska-Sowa M. Effect of growth regulators andother components of culture medium on morphogenesis ofsugar beet (Beta vulgaris L.) in unfertilised ovule in vitrocultures. Acta Agrobotanica. 2012;65(4):91-100. DOI10.5586/aa.2012.025.Vanyushin B.F. DNA methylation is an epigenetic regulationof growth and development of plants. In: DevelopmentBiology: Morphogenesis of Reproductive Structuresand a Role of Somatic, Stem Cells in Ontogenesis andEvolution: Proceedings of the Int. conf. dedicated to the50th anniversary of the Laboratory of Embryology andReproductive Biology, Biological Institute of the RussianAcademy of Sciences . Moscow:KMK Association of Scientific Editions. 2010;41-43.(in Russian)Vasilchenko E.N. Induction of haploid partenogeny of sugarbeet in vitro. Sakharnaya Svekla = Sugar Beet. 2016;6:2-4.(in Russian)Vasilchenko E.N., Zhuzhzhalova T.P., Vashchenko T.G.,Kolesnikova E.O. The technology of creating restitutionsugar beet lines. Vestnik VGAU = Bulletin of VoronezhState Agrarian University. 2018;1(56):42-50. DOI10.172338/issn2071-2243.2018.1.56. (in Russian)Zemlyanukhina O.A., Cherkasova N.N., Zhuzhzhalova T.P.,Kalaev V.N. Biochemical and morphological traits of sugarbeet acid-resistant plants-regenerants (Beta vulgaris L.).Vestnik VGU = Bulletin of Voronezh State University.2017;2:59-67. (in Russian)Zemlyanukhina O.A., Vasilchenko E.N., Karpechenko N.A.,Zhuzhzhalova T.P., Karpechenko I.Yu., Kalaev V.N.Molecular-biochemical characters of haploid and dihaploidsugar beet regenerant plants. In: Proc. of the V Congressof CIS physiologists, V Congress of Russia biochemists,Adflim Conferences. ActaNaturae (spec. iss.). 2016;2:59.(in Russian)Zhuzhzhalova T.P., Kolesnikova E.O., Vasilchenko E.N.,Cherkasova N.N. Issues of microcloning use in sugar beetbreeding. Proceedings of the VII International ScientificConference “Breeding and Genetic Science and Education:Pariyskiy readings. March 19–21, 2018. Uman, Ukraine,2018:78-81. (in Russian)Zhuzhzhalova T.P., Podvigina O.A., Znamenskaya V.V.,Vasilchenko E.N., Karpechenko N.A., ZemlyanukhinaO.A. Sugar beet (Beta vulgaris L.) haploid parthenogenesisin vitro: factors and diagnostic characters. SelskokhozyaystvennayaBiologiya = Agricultural Biology.2016;51(5):636-644. DOI 10.15389/agrobiology. 2016.5.636eng.Zhuzhzhalova T.P., Znamenskaya V.V., Oshevnev V.P., GribanovaN.P., Kolesnikova E.O., Vasilchenko E.N., CherkasovaN.N. An innovative method of sugar beet in vitro micropropagation in the breeding process. SakharnayaSvekla = Sugar Beet. 2017;4:12-18. (in Russian)Zhuzhzhalova T.P., Znamenskaya V.V., Podvigina O.A.,Yarmolyuk G.I. Reproductive Biology of Sugar Beet. Voronezh:Sotrudnichestvo Publ., 2006. (in Russian)Znamenskaya V.V. In vitro microcloning as a method ofmaintenance and reproduction of sugar beet lines. In: Encyclopediaof Beta Genus. Biology, Genetics, and Breedingof Beet. Novosibirsk: Sova Publ., 2010;420-437. (inRussian)


Michelia champaca var. champaca is an ornamentally important tree in Magnoliaceae. The paper reported the complete chloroplast genome (cpDNA) of M. champaca var. champaca and its basic annotated information. The size of cpDNA is 160,008 bp, with a typical quadripartite structure of a large single-copy (LSC) region of 88,037 bp and a small single-copy (SSC) region of 18,809 bp separated by a pair identical inverted repeat regions (IRs) of 26,581 bp each. The genome contained 131 genes (113 unique), including 86 protein-coding genes (80 unique), 37 tRNA genes (29 unique), and eight rRNA genes (four unique). Phylogenetic analysis showed that M. champaca var. champaca is affinal to M. baillonii and they form a nomophyletic group with other eight Michelia species. This Michelia clade is sister to the Aromadendron cathcartii clade with high support. All genera mentioned in this analysis are nomophyletic under the system of Magnoliaceae by Sima and Lu. Michelia champaca Linnaeus is the type species of the genus Michelia Linnaeus in the family Magnoliaceae (Sima and Lu champaca Linnaeus and var. pubinervia (Blume) Miquel. The latter variety occurs in China , Cambodia, India, Indonesia, Laos, Malaysia, Myanmar, Nepal, Thailand, and Vietnam , Bangladesh, India, Malaysia, Myanmar, Nepal, Pakistan, and Sri Lanka and widely cultivated as a good ornamental species in Southern China and Southeastern Asia, which is probably originally from India  from the fresh leaves of M. champaca var. champaca Linnaeus collected from Kunming Arboretum, Yunnan Academy of Forestry and Grassland Science, Yunnan Province of China . The sheets of vouchered specimen (Y. K. Sima and S. Y. Chen 99278) are deposited at the herbaria, YAF and YCP.The complete sequence of chloroplast genome of M. champaca var. champaca Linnaeus was sequenced by Illumina HiSeq Sequencing System  and shotgun library was constructed. About 2.0 G pair-end (150 bp) raw sequence data were obtained and the low-quality sequences were filtered through CLC Genomics Workbench v8.0  to get high-quality clean reads. NOVO Plasty software  N. H. Xia and C. Y. Wu (JX280400) served as the reference. The genome was automatically annotated using CpGAVAS . To better determine the phylogenetic position of M. champaca var. champaca Linnaeus, the complete chloroplast genome sequences of other thirty-five species of the subfamily Magnolioideae from NCBI were aligned using MAFFT v. 7  Sargent (KU170538) and Liriodendron tulipifera Linnaeus (DQB99947) were served as the outgroup. The maximum likelihood (ML) tree was reconstructed with RAxML  region of 88,037 bp and a small single-copy (SSC) region of 18,809 bp separated by a pair of identical inverted repeat regions (IRs) of 26,581 bp each. The cp genome contained 131 genes (113 unique), including 86 protein-coding genes (80 unique), 37 tRNA genes (29 unique), and eight rRNA genes (four unique). The result of phylogenetic analysis showed that M. champaca var. champaca Linnaeus is affinal to Michelia baillonii (Pierre) Finet and Gagnepain (MK782763) and they form a nomophyletic group with other eight species of the genus Michelia Linnaeus (Michelia Linnaeus is sister to the clade of Aromadendron cathcartii (J. D. Hooker and Thomson) Sima and S. G. Lu with high support. All genera mentioned in this analysis are nomophyletic under the system of Magnoliaceae by Sima and Lu (The complete cp genome of Linnaeus . This cla and Lu . The stu


Burkholderia cepacia complex (BCC) species is essential for therapy, prognosis assessment and research. The taxonomic status of BCC remains problematic and an improved knowledge about the classification of BCC is in particular needed.Accurate classification of different recA, hisA and MLSA . Using the available whole genome sequences of BCC, we inferred a species tree based on estimated single-copy orthologous genes and demarcated species of BCC using dDDH/ANI clustering.We compared phylogenetic trees of BCC based on 16S rRNA, recA, hisA and MLSA have limited resolutions in the taxonomic study of closely related bacteria such as BCC. Our estimated species tree and dDDH/ANI clustering clearly separated 116 BCC strains into 36 clusters. With the appropriate reclassification of misidentified strains, these clusters corresponded to 22 known species as well as 14 putative novel species.We showed that 16S rRNA, This is the first large-scale and systematic study of the taxonomic status of the BCC and could contribute to further insights into BCC taxonomy. Our study suggested that conjunctive use of core phylogeny based on single-copy orthologous genes, as well as pangenome-based dDDH/ANI clustering would provide a preferable framework for demarcating closely related species.This article was reviewed by Dr. Xianwen Ren. Burkholderia cepacia complex (BCC) is a group of gram-negative bacteria comprising more than 20 valid species names, including B. cepacia, B. multivorans, B. cenocepacia, B. vietnamiensis, B. stabilis, B. ambifaria, B. dolosa, B. anthina, B. pyrrocinia and B. ubonensis, etc.  was obtained using a nonlinear simulation analysis method with the default option of the Curve Fitting Tool implemented in MATLAB R2018a. The two approaches revealed a significant correlation, with an r2 = 0.9947.Additional file 6. Single-marker phylogenies of reclassified 116 BCC genomes. The trees are the same as in Fig. Additional file 7. Species tree and MLSA of 116 reclassified 116 BCC genomes. The trees are the same as in Fig.


Heracleum have shown poor correspondence between observed molecular clusters and established taxonomic classification amongst closely related species. This might reflect both unresolved taxonomy but perhaps also a lack of good genetic markers. This lack of appropriate and cost effective species-specific genetic markers hinders a resolved relationship for the species complex, and this in turn causes profound management challenges for a genus that contains both endemic species, with important ecological roles, and species with an invasive potential. Microsatellites are traditionally considered markers of choice for comprehensive, yet inexpensive, analyses of genetic variation, including examination of population structure, species identity, linkage map construction and cryptic speciation. In this study, we have used double digest restriction site associated DNA sequencing (ddRADseq) to develop microsatellite markers in Heracleum rechingeri. Genomic DNA from three individuals were digested with Sbf1 and Nde1 and size selected for library construction. The size-selected fragments were sequenced on an Ion Torrent sequencer and a total of 54 microsatellite sequences were bioinformatically confirmed. Twenty five loci were then tested for amplification, resulting in 19 of these being successfully amplified across eight species, comprising both the so-called thick-stemmed species , and thin-stemmed species . Both Bayesian and distance-based clustering, and principal coordinate analyses clearly separated these into two groups. Surprisingly, three H. pastinacifolium populations were not separated from populations of the morphologically similar endemic species, H. anisactis, suggesting lack of genetic differentiation. Likewise, high genetic similarity was found between H. persicum and H. rechingeri populations, questioning taxonomic separation at the species level between these taxa. Further analyses are needed to re-evaluate the taxonomic significance of observed morphological variability currently applied to distinguish these sister taxa. Nevertheless, our results represent progress in the effort to develop cost-efficient molecular tools for species discrimination in this genus.Many studies on  Heracleum is a taxonomically complex genus, and previous investigations have found incongruent results between observed molecular clusters, morphological data and taxonomic classifications . For instance, species-specific markers have successfully been applied to discriminate between large and small-glanded individuals of the biaceae) . Likewisanariens . Additio, 22. ForIn recent years, next generation sequencing (NGS) techniques have been widely adopted to identify molecular markers including both microsatellite and single nucleotide polymorphism (SNP) in non-model organisms. This can be done at low cost and in a short time, even without an associated reference genome , 26. FurHeracleum species complex. Specifically we wanted to develop microsatellite markers first, based on the H. rechingeri genome, and subsequently try to utilize these markers in other closely related species of Heracleum. We wanted to test whether thick-stemmed species complex including H. rechingeri and H. persicum and H. gorganicum represent a distinct genetic lineage, and also whether we are able to discriminate endemic thin-stemmed species H. anisactis and H. transcacasicum from the widespread H. pastinacifolium.In this study, we wanted to use ddRADseq to identify and develop microsatellite markers to help resolve the Heracleum rechingeri were collected from a population in Fandoghlu  and immediately placed on silica gel  protocol as described by Vivian-Smith and Sønstebø [Sbf1-HF and Nde1-HF restriction enzymes (New England Biolabs) and ligated to adaptors all in a single unidirectional one-tube reaction [DNA was extracted from the dried tissues using the ENZA SP Plant DNA Kit as specified by the manufacturer’s protocol  and eluted in water. DNA was quantified using the broad range Qubit assay (Thermofisher Scientific) and normalized to a working concentration of 5 ng ulSønstebø  from whireaction . The indreaction . ConsensHeracleum including H. persicum, H. gorganicum and H. antasiaticum, H. anisactis, H. pastinasifolium and H. transcaucasicum and H. lasiopetalum as listed in Heracleum , for each K and the plot was obtained with Structure Harvester [Clustering analysis was conducted based on pairwise genetic distances among populations, Da , using tDa , to evalarvester . For graarvester . This enH. rechingeri individuals. The results revealed that 7886 reads  contained microsatellite motifs with two, three, four, five and six nucleotide repeats, respectively. From these reads, 54 singleton and consensus sequences contained adequate flanking regions next to the microsatellite repeats enabling the design of primers n, and (AC)n constituted 43%, 17%, and 15% of the microsatellite repeats, respectively (Dinucleotide repeats (AT)ectively . In addiH.rech482 consisted of only two invariant alleles throughout all individuals and therefore this locus was excluded from all further analyses. Thus, 26% of loci were polymorphic across all individuals. A total of 17 alleles were observed across polymorphic loci, with an average 3.4 alleles per locus. The number of alleles varied between two (H.rech3_80 and H.rech436) and six (Hrech60_5). All alleles were shared in at least two populations, thus no private allele was found across populations. Because of limited number of samples used in most populations the within-population statistics were not computed.Six loci comprised more than two alleles across all individuals from the studied species . HoweverHeracleum showed that 100% and 94% of markers were successfully amplified in the thick-stem  and thin-stem  species, respectively. Also seven out of 19 loci (37%) were amplified in H. lasiopetalum.Testing the cross-amplification of 19 microsatellite markers on 17 populations from eight different species of Heracleum populations with both model-based Bayesian structure and by distance-based analyses. Principal coordinate and clustering analyses showed that the group of thick-stemmed species including H. persicum, H. rechingeri, H. gorganicum and H. antasiaticum were recovered together as a single cluster n is also an abundant repeat in the taxa studied here. The percentages of total reads containing microsatellite motifs may vary among species and the types of sequencing methodology. A comparison amongst sequences obtained by traditional Sanger sequencing and NGS techniques, based on a review of 71 different studies, showed that NGS was approximately 40 times more effective in microsatellite marker development than Sanger sequencing [Satureja bachtiarica and Heracleum persicum using 454 sequencing with an enrichment step for microsatellite loci, resulted in a discovery of 16% (8371 out of 51051 sequences) and 15% (3904 out of 25951) microsatellite motifs, respectively [Solanum melongena and Cedrus atlantica, has shown that 0.02% (2000 reads) and 8.5% (5714 reads) consisted of microsatellite motifs, respectively [Eleusine coracana and Taxus florinii have shown that 0.15% (10327 reads out of 6810971) and 1% (94851 of 8823053 reads) contain microsatellite motifs, respectively [Previous investigations have reported (GA)e.g. see  for a requencing . In compectively . RADseq ectively . LikewisH. persicum do not form a distinct group compared to the other closely related sister species, including H. rechingeri  and thick-stemmed species (H. rechingeri and H. persicum) are more closely related than what is perhaps expected from their described diagnostic taxonomic criteria. Long sequence reads obtained by ddRADseq provide a valuable tool to develop and deploy novel microsatellite markers, and provide a time and cost-effective approach for a genus that contains both endemic and invasive species.This study demonstrates the utility of microsatellite markers in studying complex species with otherwise weak taxonomic signals. The present analysis suggests that the thin-stemmed species (S1 Table(XLSX)Click here for additional data file.


Plasmodium vivax has been largely neglected over the past century, despite a widespread recognition of its burden across region where it is endemic. The parasite invades reticulocytes, employing the interaction between Plasmodium vivax Duffy binding protein (PvDBP) and human Duffy antigen receptor for chemokines (DARC). However, P. vivax has now been observed in Duffy-negative individuals, presenting a potentially serious public health problem as the majority of African populations are Duffy-negative. Invasion of Duffy-negative reticulocytes is suggested to be through duplication of the PvDBP and a novel protein encoded by P. vivax erythrocyte binding protein (EBP) genes. The emergence and spread of specific P. vivax strains with ability to invade Duffy-negative reticulocytes has, therefore, drawn substantial attention and further complicated the epidemiology and public health implication of vivax malaria. Given the right environment and vectorial capacity for transmission coupled with the parasite’s ability to invade Duffy-negative individuals, P. vivax could increase its epidemiological significance in Africa. In this review, authors present accruing knowledge on the paradigm shift in P. vivax invasion of Duffy-negative reticulocytes against the established mechanism of invading only Duffy-positive individuals and offer a perspective on the epidemiological diagnostic and public health implication in Africa. Plasmodium vivax is the most widespread species of human malaria and more than 3 billion people live in P. vivax-endemic regions [Plasmodium falciparum, P. vivax was believed not to be a major health problem in Africa. However, until recently, P. vivax infections have been widely reported in several African countries and its emergence is not surprising given the presence of a suitable environment, competent vectors and susceptible human hosts [Plasmodium vivax not only differs from P. falciparum in terms of its epidemiology, but also its biology and evolutionary trajectory. These unique features enable P. vivax to survive under a wide range of climatic and ecological settings with the abilities to adapt to both temperate and tropical climates. Other features, including P. vivax ability to form hypnozoites, a stage of which the parasite can remain quiescent from months to years [P. falciparum [Malaria affects one-third of the world’s population and kills hundreds of thousands across the globe . Plasmod regions . Unlike to years , an invalciparum .P. vivax in Africa remains poorly understood, although P. vivax transmission from almost all countries across the continent has been reported from various entomological, serological, community prevalence surveys, including clinical infection data from local residents and travellers returning to malaria-free countries [P. vivax transmission is ongoing throughout Africa [P. vivax in the region. There are increasing reports of P. vivax infections in Duffy-negative populations of Africa [P. vivax has been observed to be less severe in Duffy-negative Africans compared to their Duffy-positive counterparts [P. vivax is poorly adapted to Duffy-negative individuals and may change as the parasite evolves to optimize its ability to infect Duffy-negative populations.The epidemiology of ountries . These rt Africa , 7. Howef Africa  as deterterparts . This maP. vivax among the spectrum of Duffy phenotype populations across Africa and synthesize data accrued on the invasion mechanisms of Duffy-negative erythrocytes and novel parasite ligands and host receptors that enable the parasites to invade reticulocytes. To achieve this, published articles was searched in the following electronic databases: Google Scholar, (www.google.scholar.com) and PubMed (www.pubmed.gov). Search terms included Plasmodium vivax, Duffy OR CD234 OR DARC, parasite ligands, host receptors, molecular epidemiology, invasion, mechanism. No restriction on year of publication was imposed but only articles published in English were considered.Here, authors review the contemporary biology and epidemiology of P. falciparum and P. vivax co-infections or pure P. vivax infections; and, (3) studies were conducted in African or South American populations. Selected studies were entered into Mendeley Reference Manager and the following information was recorded: (1) country where study was conducted; (2) the case prevalence of P. vivax reported; (3) Duffy phenotypes reported; (4) comments on disease pathogenesis, particularly for Duffy-negative infections; (5) proposed mechanisms of infections of Duffy-negative individuals; (6) novel receptor–ligand interactions reported; and, (7) study conclusions.Studies were selected if they met the following criteria: (1) The study involved human participants; (2) infections analysed were either Plasmodium vivax infections persist with low levels of parasitaemia either as mono-infections or co-infections with P. falciparum in co-endemic settings, making it relatively difficult to diagnose with microscopy, which is the gold standard for malaria detection [P. falciparum and sometimes minor species, such as Plasmodium ovale and Plasmodium malariae, but not specifically P. vivax, despite the increasing number of P. vivax reported across Africa and in Duffy-negative individuals. Therefore, P. vivax infections are often misdiagnosed either by mistake or because parasitaemia is too low given that its P. vivax invades reticulocytes that comprise only a small fraction of the circulating erythrocytes [P. vivax infections are beginning to report increasing numbers of these infections in community surveys [P. vivax prevalence in endemic areas across Africa. Moreover, with the advent of whole genome sequencing of Plasmodium spp, genome analysis of P. vivax from clinical sources will uncover the true magnitude of infections among Duffy-positive/negative populations [etection . In mosthrocytes . Owing t surveys . This is surveys , 12 and,ulations .P. vivax to infect human erythrocytes with or without the Duffy antigen to cause blood-stage infections [P. vivax infections in Duffy-negative anaemic children. In another study conducted in Cameroon by Russo et al. [P. vivax-infected Duffy-negative patients presented with fever. In admixture populations, according to Menard et al. [P. vivax, providing an opportunity for this parasite to infect hepatocytes of Duffy-negative people with the selection of novel P. vivax strains with the ability to invade Duffy-negative erythrocytes. Taken together, these data suggest a paradigm shift in the understanding of P. vivax invasion biology and raises more questions than answers. Could the adaptation of vivax to infect Duffy-negative individuals be frequency dependent? If so, what ratio of Duffy phenotypes fuel vivax adaptation to invade Duffy-negatives? There is yet limited evidence which supports the inability of vivax to spread to populations such as those in West Africa where the Duffy-negative allele is fixed. At any rate, given that P. vivax can cause both clinical and sub-clinical infections in Duffy-negative patients, these evolutionary realities are of public health concern, particularly when major investments in interventions against P. falciparum are driving falciparum to elimination but creating a niche for other Plasmodium spp.For many years epidemiologist thought that Duffy-negative individuals were totally resistant to vivax malaria and the complete resistance of Duffy-negatives to vivax malaria was the most striking example of association of a genetic trait with an infectious disease . On the fections , 15, 16.fections , found 2o et al. , 27 P. vP. falciparum is considered the deadliest malaria parasite with most severe clinical outcomes, P. vivax is more widespread and often associated with high levels of morbidity. The overall burden, economic impact and severity associated with vivax malaria has been underestimated until some recent studies showing the association of P. vivax infections with severe malaria and death [P. vivax-associated malaria ranges from asymptomatic parasitaemia and uncomplicated febrile illness to severe and fatal malaria. Other severe clinical manifestations include multi-organ dysfunction with anaemia and thrombocytopaenia [P. vivax is its association with spontaneous abortions, premature and low birth weight in pregnant women. These clinical features have mostly been described for Duffy-positive populations and may vary among Duffy-negative individuals in Africa. In areas where P. falciparum and P. vivax co-exist, while the total malaria burden decreases, the proportion of cases attributable to P. vivax increases [While nd death . The clitopaenia . Anotherncreases .P. vivax, gametocytes appear simultaneously with the asexual schizonts [P. falciparum, gametocytes usually appear 10–14 days after infection. Such a difference in development not only increases the length of time the individual remains infectious, but also increases the likelihood of transmission before the infected individual seeks treatment [P. vivax ability to relapse from dormant liver-stage hypnozoites, from weeks to years after clearance of the primary blood-stage infection is a major obstacle to its control and elimination [P. vivax infection and the growing clinical burden across regions with a spectrum of Duffy phenotypes puts vivax malaria on the spotlight as an important public health issue [P. vivax infection and the public health significance of vivax malaria should no longer be neglected [In chizonts  while inreatment . Moreovemination . The incth issue . Definiteglected .P. vivax in Africa is viewed as a consequence of the lack of expression of the Duffy antigen on red blood cells (RBCs). Exhibiting distinct red cell tropism, malaria parasites use sophisticated invasion mechanisms to identify, penetrate and multiply in vector and human host cells. Plasmodium vivax blood-stage infection is restricted to human reticulocytes, whereas P. falciparum can infect all types of RBCs [Plasmodium species requires multiple interactions between parasite ligands and host receptors, some of which have overlapping and partially redundant roles [P. vivax, the connection between the PvDBP and DARC is a critical pathway, and the only pathway identified so far, for P. vivax invasion of human reticulocytes, in spite of the increasing reports of P. vivax infections in Duffy-negative individuals that rise questions on the specificity of Duffy antigens [P. vivax infection in Duffy-negative populations throughout Africa and South America, it is possible that these infections were historically not detected rather than a recent adaptation of certain P. vivax strains.The sparse distribution of  of RBCs . RBC invnt roles . With thantigens , 29. WitP. vivax had been known to be completely dependent on the interaction between PvDBP and DARC [P. falciparum merozoites that use several erythrocyte receptors  and glycophorin C (GPC) and ligands  for erythrocyte invasion. Although DARC is present on the erythrocyte surface, P. vivax reticulocyte binding proteins (PvRBPs) bind receptors on reticulocytes that are subsequently lost during erythrocyte maturation [P. vivax tropism [P. vivax, but are protected from blood-stage P. vivax infection [The invasion of human reticulocytes by and DARC , unlike turation . This en tropism . Based o tropism . PvRBP2dnfection  indicatiP. vivax merozoites invasion of RBCs. Due to its strong geographic differentiation and association with vivax malaria resistance, DARC is considered as best example to depict positive selection in human genome. It belongs to the G protein-coupled receptor family (a 35–50 kDa glycoprotein) expressed both on the surface of RBCs and endothelial tissue [P. vivax invasion could be occurring to some degree [FY gene main alleles, FY*A (FY*01) and FY*B (FY*02) that are codominantly expressed  define four main phenotypes: Fy(a+b−), Fy(a+b+), Fy(a−b+) and Fy(a−b−). The Duffy-negative phenotype, Fy(a−b−), occurs in two-thirds of African–American Blacks [FY*B  [FY*B in RBCs (thus blocks the RBC invasion by P. vivax), but without affecting FY*B expression in other tissues [DARC is a membrane protein present on the surface of erythrocytes and central to e degree . The DufFY*A FY*0 and FY*Bn Blacks  and is a tissues , 38.TablFy, is characterized by a SNP in a GATA-1 transcription factor binding site associated with the erythrocyte silent (ES) phenotype that has been shown to protect against P. vivax infection and is at near fixation in sub-Saharan Africa and is virtually absent any where else. Indeed, the Duffy antigen exhibits extreme geographic differentiation in Africa, but nearly absent from Asia and Europe. While ES  phenotype impairs promoter activity in erythroid cells by disrupting a binding site for the GATA-1 erythroid transcription factor [P. vivax infections have been observed in Fy*01N.01 homozygotes [P. vivax might be evolving escape-variants able to overcome the protective effect of Fy*01N.01/FY*01N.01 or has evolved a Fy-independent RBC invasion pathway or that the GATA-1 SNP does not abolish Fy expression. Different allelic forms of the Duffy blood group gene could also modify the antigen’s expression level, leading to weak phenotype. For example, two variants c.265C>T (rs34599082) and c.298G>A (rs13962) of glycoprotein Duffy have resulted in weak Fyb expression [FY*02W.02 allele has also shown to weaken the expression of the Fyb antigen [The gene encoding the Duffy blood group, n factor  and confozygotes , which ipression . Another antigen , 42. TheP. vivax merozoites appears to rely heavily on the interaction between PvDBP and DARC [P. vivax clinical isolates collected from Duffy-negative individuals indicates the presence of two and higher copies of the PvDBP gene [P. vivax clinical isolates from Cambodia [P. vivax genomes from over 200 isolates originating from across the world indicated that copy number variation (CNV) at the PvDBP locus is one of the most common CNVs within the P. vivax genome irrespective of the geographical origin of the isolates [P. vivax [P. vivax indicates that several sub-populations are present [Red blood cell invasion by and DARC . Whole-gDBP gene . This obDBP gene . Two difCambodia , EthiopiCambodia  and MadaCambodia . Moreoveisolates . For insisolates . PvDBP CP. vivax . In otheP. vivax . The out present .P. falciparum and P. vivax [P. vivax? For example, expansion of mdr1 has been shown to result in increased anti-malarial resistance, however, it is unlikely that anti-malarial drug pressure plays a role in PvDBP duplication unless the duplication confers an increased growth rate to P. vivax parasites [P. vivax erythrocyte invasion, it is expected that the binding affinity of DARC with high-copies PvDBP could be higher than with single-copy PvDBP parasites. However, no association has been found between PvDBP duplications and the efficiency of parasite to invade reticulocytes with FY*A or FY*B genotypes [Plasmodium species, it is likely that the duplications emerged in the parasites in response to the variation in human Duffy blood group across malaria-endemic settings. Recent studies have shown that PvDBP gene amplification not only facilitates binding to an alternative lower affinity receptor in Duffy-negative reticulocytes [P. vivax to evade host anti-PvDBP humoral immunity [P. vivax [PvDBP certainly allows P. vivax to colonize diverse ecological niches as well as to evade the host immune system.CNVs are common in both P. vivax . What inarasites . Owing tenotypes . Assuminulocytes , 44, butimmunity , reassurP. vivax . The polFY*A allele compared to individuals carrying the FY*B [A molecular epidemiology study conducted in Ethiopia observed that the proportion of parasites with PvDBP amplification was higher in individuals carrying the the FY*B . On the the FY*B . It is nthe FY*B . Such a P. vivax came from travellers who presented with P. vivax infection after returning from countries in Africa where Duffy-negativity is at near fixation [P. vivax can remain as latent infections for months or even years before triggering a relapse infection, it is difficult to confidently identify the source of P. vivax responsible for the blood stage infection in those individuals. In addition, there is yet insufficient data demonstrating the presence of P. vivax in Anopheles mosquitoes collected in areas where the majority of people are Duffy-negative [P. vivax infections in Duffy-negative individuals.The first indirect evidence of invasion of Duffy-negative reticulocytes by fixation , 55. Becnegative . HoweverP. vivax DNA by molecular and serological assays in a Duffy-negative individual’s blood sample could represent pre-erythrocytic stages of the parasites, independent of blood-stage infection [P. vivax within Duffy-negative erythrocytes coupled by confirmation by genotyping has essentially proved Duffy-negative infection [P. vivax infection in a Duffy-negative erythrocyte was reported by Ryan et al., in Kenya in [P. vivax infection within Duffy-negative erythrocyte from patients in Madagascar [P. vivax through mosquito bite followed by the establishment of P. vivax blood-stage infection [P. vivax antibody testings in Duffy-negative populations would reveal the extent of exposure and possible adaption of P. vivax for continuous transmission in these populations. Furthermore, P. vivax infections of Duffy-negative people have also been reported from the American continent using serological, molecular, and microscopic detection tools [P. vivax infections, a recent study of P. vivax patients in the Brazilian Amazon region has shown that the null allele FY*02N.01 and weak allele FY*02W.01 were associated with low parasitaemia and low symptomatology [P. vivax adhesion by the reduction of the Duffy glycoprotein, as supported by earlier studies showing erythrocytes, expressing the FY*/FY*02N.01, have a significant reduction in parasite adhesion when compared to erythrocytes expressing FY*02/FY*02 [FY*02N.01 and FY*02W.01 alleles may have an impact on the reduction of clinical manifestation in malaria, leading to the development of sub-clinical malaria.The detection of nfection . Therefonfection . The firKenya in  followeddagascar . Furthernfection , though on tools , 57. Whimatology . The pre02/FY*02 –61. TherP. vivax in the absence of Duffy-positive receptor is imperative with respect to parasite epidemiology, public health importance and elimination strategies against vivax malaria. To date, the question of whether P. vivax has recently evolved its strategies to infect Duffy-negative erythrocytes and utilized an alternative DARC-independent invasion pathway remains unclear. Characterizing this pathway is important in the development of innovative strategies to prevent spread of P. vivax infections in Africa, and vaccine development. Unfortunately, the inherent challenge of culturing P. vivax in vitro has made studies targeting vivax malaria very difficult [P. vivax in Duffy-negative reticulocytes [Understanding of the mechanisms of RBC invasion by ifficult . The invulocytes .P. vivax is through the interaction between the human Duffy antigen/receptor for chemokines  and the P. vivax EBP2, a new member of the DBP family [P. vivax field isolates and monkey-adapted strains, two possible invasion mechanisms have been proposed. The first mechanism is through duplication of the PvDBP gene observed in multiple P. vivax strains. The other is invasion through a novel PvEBP-driven pathway that contains conserved Duffy-binding-like and C-terminal cysteine-rich domains that can be recognized by the Duffy receptor  [PvRBPs previously examined, PvRBP1a and PvRBP2c bind to Duffy-negative erythrocytes [P. vivax invasion of Duffy-positive cells [PvRBP family, PvRBP2b, was recently shown to be a key ligand involved in reticulocyte recognition and invasion through the transferrin receptor 1 (TfR1) [P. vivax invasion of reticulocytes remain unclear. The membrane protein TfR1 is lost during red blood cell maturation and, therefore, absent in normocytes [DBP engages with the Duffy protein, allowing entry of the merozoite in the red blood cell during invasion in Duffy-positives [P. vivax parasite invasion.One potential parasite ligands involved in Duffy-positive/Duffy-negative reticulocyte invasion is the family of (PvRBPs) . Althoug(PvRBPs) , 65. Of hrocytes . Howeverve cells . The thi1 (TfR1) . Howeverrmocytes . Theoretrmocytes . Althougrmocytes . PvRBP2bositives . It has Plasmodium vivax merozoite surface protein-1 paralog (PvMSP1P) and P. vivax glycosylphosphatidylinositol-anchored micronemal antigen (PvGAMA) were also shown to bind to both Duffy-positive and Duffy-negative RBCs [P. vivax erythrocyte-binding protein  has also been shown to bind moderately to Duffy-negative reticulocytes [ive RBCs –70. Takeulocytes , 71.P. vivax-infected Duffy-negative individual [P. vivax for some time before symptoms emerged. These asymptomatic cases could have escaped the detection by current diagnostic tools and/or regimes in most African countries that primarily target P. falciparum. Plasmodium vivax is now confirmed not to be completely absent from western and central Africa, though the dominance of Duffy-negative individuals in these regions has kept the prevalence of P. vivax low. Moreover, the presence of very low proportion of Duffy-positive individuals could have minimized P. vivax transmission in these regions, though there is no evidence of direct transmission among Duffy-negative individuals and between Duffy-positive and Duffy-negative individuals. Lastly, the contribution of alternative hosts such as apes [P. vivax parasites [P. vivax blood stage malaria [P. vivax within infected individuals must be considered [It is as yet unclear whether a few Duffy molecules present on the surface of the erythrocyte enabling parasites with multiple PvDBP gene copies to invade the cell, or whether the invasion process of Duffy-negative reticulocytes could occur through alternate pathways by non-DBP ligands  . Anotherdividual . This mu as apes , the geoarasites , its redarasites , and thearasites  cannot b malaria , new resnsidered .P. vivax remains an enigma for parasitology and evolution. Since most individuals of African origin are Duffy-negative and do not express the DARC antigen on the surface of their erythrocytes, it is unclear whether the high prevalence of Duffy-negative alleles in African populations results from P. vivax resistance selection [P. vivax among Duffy-negative populations. The public health significance of P. vivax is increasingly apparent in Africa. Duffy-negative reticulocyte infection by P. vivax highlights the risk of emergence and spread of vivax malaria in Africa where it was non-existent before. Apart from the Horn of Africa, Madagascar and Mauritania, P. vivax is grossly overlooked in many parts of the African continent. Given the low parasitaemia associated with P. vivax infections in Duffy-negative individuals, microscopy and RDTs are not sensitive enough to detect. Therefore, the contemporary epidemiology of vivax malaria in Africa is vastly unknown [P. vivax infections and studies that further characterize genomic loci under selection in P. vivax isolates infecting Duffy-negative individuals. Future studies towards a better insight of erythrocyte invasion mechanism could be obtained by comparison of gene expression profiles of parasites infecting Duffy-negative and Duffy-positive people and identifying genes specifically up-regulated in Duffy-negativity infections. The evolution of P. vivax strains able to infect RBCs not expressing Duffy antigen could have important implications for vaccine development. Increasing efforts for continuous surveillance of P. vivax strains capable of invading red cell through Duffy-independent pathway is essential for designing P. vivax-specific vaccine candidates. A better understanding of the epidemiology of P. vivax enables the design of P. vivax-specific control and elimination strategies. Given the presence of an ideal temperature, highly competent vectors for its transmission, and the apparent increase in the host range, P. vivax seems to expand to areas where it was non-existent in the past in Africa. It is imperative to consider how the growing evidence that P. vivax is not restricted to a Duffy-dependent invasion pathway is affecting the African population at risk of vivax malaria.Infection of Duffy-negative individuals by election  or there unknown . This ca


Proteus spp. are commensal gastrointestinal bacteria in many hosts, but information regarding the mutual relationships between these bacteria and their hosts is limited. The tree shrew is an alternative laboratory animal widely used for human disease research. However, little is known about the relationship between Proteus spp. and tree shrews. In this study, the complete genome sequencing method was used to analyse the characteristics of Proteus spp. isolated from tree shrews, and comparative genomic analysis was performed to reveal their relationships. The results showed that 36 Proteus spp. bacteria were isolated, including 34 Proteus mirabilis strains and two Proteus vulgaris strains. The effective rate of sequencing was 93.53%±2.73%, with an average GC content of 39.94%±0.25%. Briefly, 3682.89±90.37, 2771.36±36.01 and 2832.06±42.49 genes were annotated in the NCBI non-redundant nucleotide database (NR), SwissProt database and KEGG database, respectively. The high proportions of macrolide-, vancomycin-, bacitracin-, and tetracycline-resistance profiles of the strains were annotated in the Antibiotic Resistance Genes Database (ARDB). Flagella, lipooligosaccharides, type 1 fimbriae and P fimbriae were the most abundantly annotated virulence factors in the Virulence Factor Database (VFDB). SNP variants indicated high proportions of base transitions (Ts), homozygous mutations (Hom) and non-synonymous mutations (Non-Syn) in Proteus spp. (P<0.05). Phylogenetic analysis of Proteus spp. and other references revealed high genetic diversity for strains isolated from tree shrews, and host specificity of Proteus spp. bacteria was not found. Overall, this study provided important information on characteristics of genome for Proteus spp. isolated from tree shrews. Proteus spp. are common commensal gastrointestinal bacteria in many hosts and belong to the Morganellaceae family [Proteus mirabilis, Proteus vulgaris, Proteus penneri, Proteus hauseri, and unnamed genomospecies 4, 5, and 6 [Proteus spp. is actually a normal, ubiquitous commensal constituent of human flora and it is recognized that the gut is a reservoir for this proteolytic organism. Previous studies have illustrated the Proteus spp. antagonistic or commensal relationships with numerous animals and sometimes found cooperated relations with different animal hosts, acting as animal symbionts [Proteus spp. and animals coexisting with the bacteria in the environment is still unclear. Information regarding the mutual relationships between Proteus spp. bacteria and their natural habitats is currently limited.e family –2. Curre5, and 6 –4. Theseymbionts . HoweverTupaia belangeri) is a new laboratory animal belonging to the family Tupaiidae, order Scandentia, widely distributed in South Asia, Southeast Asia and Southwest China [Tupaia belangeri in China, namely, T. belangeri gaoligongensis, T. belangeri modesta, T. belangeri yaoshanensis, T. belangeri tonquinia, T. belangeri yunalis and T. belangeri chinensis [Proteus spp. was the most isolated bacteria from feces of tree shrew by using culture method [Proteus spp. isolated from tree shrews, and comparative genomic analysis was performed to reveal their relationships.The tree shrew   at 37°C for 24 hours, and finally identified using the Vitek Compact 2 biochemical identification system (bioMérieux). Total genomic DNA of the isolated bacteria was extracted using a bacterial total genomic DNA extraction kit  following the manufacturer’s instructions. All DNA samples were stored at -20°C for complete genome analysis.The us study . All faeBacterial genome sequencing of all isolates was performed by our laboratory on the Illumina MiSeq platform using 2×250 bp paired-end reads. The libraries were built using a Nextera XT DNA Library Prep Kit following the manufacturer’s reference guidelines. Generally, 1 ng genomic DNA of each strain was used. After segment and purification, index PCR was performed to add the Illumina Nextera barcodes using i5 and i7 primers, and then the purification was executed again to remove non-target fragments. Finally, the libraries were normalized, pooled and sequenced using an Illumina MiSeq sequencing system .The raw data were trimmed for quality control, and low-quality (<Q40) reads were filtered by Trimmomatic (version 0.38) . Draft gProteus spp. was analysed by PhiSpy (version 2.3) with default parameters [Clustered regularly interspaced short palindromic repeats (CRISPR) locus screening of bacteria was predicted by CRT (version 1.2), with minRL 19, maxRL 38, minSL 19 and maxSL 48 . The reprameters . The intrameters , and theProteus mirabilis HI4320: NC_010554 and Proteus vulgaris FDAARGOS_366: NZ_CP023965) with the BWA software package [For the detection of single nucleotide polymorphism (SNP) variants relative to the reference, we used clean reads mapped to each reference genome ( package –28. The  package –30. The  package .Proteus spp. in this study with 114 reference Proteus spp. (102 P. mirabilis and 12 P. vulgaris) from different sources and countries  method with 1000 bootstrap replicates to generate a phylogenetic tree. The SNPs of predicted ICEs were called by using the methods mentioned above, and a phylogenetic tree was built by MEGA 6.0 with 18 reference ICE sequences . Phylogeountries . A maximProteus spp. in this study.Statistical analysis was performed using the SPSS software package . Kolmogorov-Smirnov Z, T-test or Kruskal-Wallis H test were used if appropriate. A P value of <0.05 was recognized as statistically significant. The Tajima’s D values were calculated by MEGA 6.0 for www.ebi.ac.uk/ena with Accession Numbers ERS3013925 to ERS3013960 (PRJEB30582). The sequences of predicted ICEs were deposited into the NCBI database with GenBank Accession Numbers from MK460213 to MK460220.The sequence data have been deposited into the European Nucleotide Archive, P. mirabilis and two P. vulgaris strains were isolated in this study, and the details of the strains with host information were shown in Proteus spp. bacteria, and 11,608,128 clean reads were retained after quality control. The average clean Q20 (%) was 91.25±0.99, clean Q30 (%) was 88.35±1.28, and effective rate (%) was 93.53±2.73, as shown in Thirty-four Proteus spp. in this study, with an average number of 133.42±6.31 and a range from 120 to 151 , YNTSP34 (11 loci), YNTSP35 (11 loci), and YNTSP69 (11 loci) . The detP. mirabilis isolates had putative ICEs with T4SS structures and belonged to SXT/R391 family, as shown in PmiYNTSP19), while the shortest was 86,907 (ICEPmiYNTSP40). Phylogenetic analysis revealed four clustered groups, indicated with red, yellow, green and blue colours . A total of 9320.24±2117.44 base transitions (Ts) were obtained for Proteus spp., compared with 2910.35±602.98 base transversions (Tv) , as shown in Proteus spp. with an average of 19.31±6.00 InDels. In general, 33.72±9.76 were insertions, and 31.36±10.51 were deletions. No significant difference was found between the insertions and deletions   . A large= 0.336) . The aveP. mirabilis indicated high genetic diversity for strains isolated from tree shrews because 136 isolates were divided into four clustering groups, indicated with yellow, green, blue and purple colours (P. mirabilis bacteria isolated in this study (red font). Twenty-eight strains were clustered into the purple group. Although ten isolates  were located at an identical branch, others were clustered into different branches of the tree. Most of the reference strains in the purple group were isolated from humans, especially from urinary tract infections, and P. mirabilis from tree shrews showed high similarity with these strains, such as 1150_PMIR, 1134_PMIR, Pm_Oxa48, K670, GN2, 51_PMIR, 418_PMIR and 1091. Furthermore, these reference strains were isolated from different countries without any epidemiological relationship. YNTSP26 was clustered into the blue group, and most strains in this group were also isolated from patients, except P. mirabilis Wood, which was isolated from Lucilia sericata, BC11-24 from swine, and 25933GTA from beef. There were two strains (YNTSP57 and YNTSP24) divided into the green group, and all of the reference isolates were isolated from patients. The last three P. mirabilis bacteria from tree shrews were clustered into the yellow group, and the major sources of strains were from patients as well, except for 11985-2-3 and SAS71, which were environmental strains. We further performed phylogenetic analysis of P. mirabilis isolated from tree shrews independently, and 34 strains were clustered into four groups, as shown in P. vulgaris were separated into three groups, including two strains isolated from tree shrews in this study and 12 references . Phylogenetic analysis of 34 P. mirabilis isolated from tree shrews demonstrated four clustering groups were obtained by NJ method, shown with red, green, blue and yellow colours . However, statistical significance  was identified for all 136 P. mirabilis in this study.Phylogenetic analysis based on ML method of  colours , and eacferences . YNTSP35ferences . The clu colours . In addied above . All theProteus spp., including mammals, birds, amphibians, reptiles, and insects [P. mirabilis and P. vulgaris inhabited in the intestines of western lowland gorillas. Gaastra et al. [P. mirabilis from the faeces and urine of dogs with urinary tract infections, and Kroemer et al. [P. mirabilis bacteria caused urinary tract infections in dogs and cats in European countries. Lowe et al. [P. mirabilis or P. vulgaris strains dominated the pig tonsil, and Kobashi et al. [P. mirabilis from pig faecal samples that was resistant to tetracycline. Other authors emphasized the role of wild birds in the transmission and spread of Proteus spp. bacteria to domestic poultry, cattle, or humans [Proteus spp. with laboratory animals, specifically for the tree shrew. Our results indicated that Proteus spp. strains were the most commonly isolated bacteria from the gut of tree shrews, even though all of these laboratory animals were closed populations without any contact from outside environments.A previous study showed that many wild and domestic animals are hosts of  insects . These b insects  identifia et al.  isolatedr et al.  reportede et al.  revealedi et al.  identifir humans –39. HoweProteus species possess different antibiotic resistant abilities. Previous studies on clinical patients revealed that polymyxins resistance was mediated via lipid A of bacteria, and they also had intrinsic resistant abilities to tetracycline, tigecycline and colistin [P. mirabilis HI4320 reference strain demonstrated tetAJ encoded tetracycline resistance, cat encoded chloramphenicol acetyltransferase, bcr encoded sulfonamide resistance, and multidrug efflux genes mdtG, mdtH, mdtK, mdtL were all contained in this uropathogenic strain [P. mirabilis acquired Salmonella genomic island 1 (SGI1) was reported, and it was resistant to tetracycline, sulfonamides, fluoroquinolones, streptomycin, trimethoprim, chloramphenicol, and β-lactam antibiotics [P. mirabilis strain PM58, an extensively drug-resistant isolate contained the New Delhi metallo-β-lactamase 1 (NDM-1) gene was also reported. P. mirabilis isolate (NO-051/03), a recent sequenced strain from a patient acquired β-lactam, trimethoprim, sulfonamides, and aminoglycosides resistant genes [Proteus spp. strains with these clinical ones, we found similar antimicrobial resistant genes acquired by both Proteus spp. isolates. In our study, ARDB annotations indicated high proportions of macrolide resistance profile. The tetracycline resistance profiles were involved in tetB, tetM, tetJ and otrA genes, and the descriptions of those genes were tetracycline efflux pump and ribosomal protection protein. mdtL and cat genes with chloramphenicol resistance profile were annotated, and they mainly referred to multidrug resistance efflux pump and chloramphenicol acetyltransferase. lsa, carA and tlrC genes annotated with lincosamide, streptogramin, macrolide resistance profile. The penicillin resistance profile with pbp gene was also annotated. In addition, our previous study [Proteus spp. strains possessed NDM-1 gene resistant to Imipenem. All these pieces of evidences indicated that the antimicrobial resistant profiles or genes were similar between tree shrew Proteus spp. isolates with human clinical ones.colistin –41. Wholc strain . Recentlnt genes . Compareus study  revealedProteus spp. bacteria in these hosts. It would be interesting to compare the features of Proteus spp. from clinical isolates and animals, explaining the factors contributing to the different lifestyles of Proteus spp. in various environments [Proteus species are considered potential pathogenic bacteria in human gastrointestinal or urinary tract infections [Proteus spp., especially for the P. mirabilis species. However, the interactions between Proteus spp. and hosts may lead to the pathogenicity of this genus resulting from population expansion. Several virulence factors of this genus have been revealed in previous studies. Proteus spp. possess similar characteristics to other Enterobacteriaceae for lipopolysaccharides (LPS), such as Salmonella spp. and Escherichia coli. The chemical structure of LPS may lead to the adaptation of Proteus spp. bacteria to environmental conditions and enhance their pathogenic abilities [Proteus spp. due to their immunogenic structure. The flagellins activate the inflammatory pathways of hosts and contribute to the stimulation of the host innate immune systems [Proteus spp. infections is adhesion to epithelial cells or surfaces for urinary or gastrointestinal tracts. A previous study [P. mirabilis HI4320, and six types of fimbriae have been characterized thus far, including MR/P fimbriae (mannose-resistant Proteus-like fimbriae), MR/K fimbriae (mannose-resistant Klebsiella-like fimbriae), NAF (non-agglutinating fimbriae), ATF (ambient-temperature fimbriae), PMP (P. mirabilis P-like pili), and PMF (P. mirabilis fimbriae). All of these fimbriae play an important role in the pathogenesis of P. mirabilis infections. In this study, all of the Proteus spp. strains isolated from tree shrews possessed the virulence factors mentioned above, such as LPS, flagella, type 1 fimbriae and P fimbriae. Therefore, the laboratory animal-isolated Proteus spp. strains showed no significant differences from clinical strains, especially in their pathogenicity.The evolutionary relationships of different niches in humans, animals, and plants using proper techniques could reveal the roles of ronments . Proteusfections –46. It ibilities . In addi systems –49. Furtus study  showed 1P. mirabilis from tree shrew had closer genomic relations with human clinical isolates. There was no host specificity of Proteus spp. isolated from human patients, animals or environments. Close similarity could be found between strains from different epidemiological resources, and even within the same host species, such as the tree shrews in this study, and characteristics of high genetic diversity in the isolates were also identified. This unexpected result contradicted previous studies, and several studies have indicated that microorganism specificity to their hosts supports the co-evolution of the host and its microbes [Proteus spp. strains from our results with other references did not reveal host specificity of isolates.Furthermore, phylogenetic analysis based on total SNPs of strains demonstrated that microbes –52. HoweProteus spp., we found the antimicrobial resistance, virulence factors and genomic features of these isolates were similar with human clinical Proteus spp. strains. These results indicated some of the gut bacterial population was somewhat similar between tree shrew and human, specifically for Proteus spp. However, the antimicrobial resistant bacteria and related genes, especially for erythromycin, tetracycline and β-lactamase, were commonly existed in this laboratory animal. This should be paid attention in future clinical research applications.Based on ARDB, VFDB annotations and phylogenetic relation analysis of tree shrew Proteus spp. isolated from tree shrews and compared their characteristics with other references. Overall, the host specificity of isolates was not discovered and Proteus spp. strains were somewhat similar between tree shrew and human. This investigation provided important information on characteristics of genome for Proteus spp. isolated from tree shrews.In this study, we firstly analyzed the genomics of S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S1 Appendix(RAR)Click here for additional data file.S2 Appendix(RAR)Click here for additional data file.