O-acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein–protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS.The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and  Organization of proteins and especially enzymes in multiprotein assemblies responds to many functional and structural requirements, from substrate channeling to regulation, from spatial co-localization to stabilization of poorly stable conformational states ,2,3. ProCysteine metabolism  , is intiMycobacterium tuberculosis: O-acetylserine sulfhydrylase isozymes A and B (CysK/CysM) [O-acetylserine (OAS) and stabilized by bisulfide [Haemophilus influenzae [E. coli [Salmonella enterica serovar Typhimurium [Only four of these enzymes have been the target of significant medicinal chemistry efforts, either in enteric bacteria or in sK/CysM) ,37,40,41sK/CysM) , and sersK/CysM) . AlthougsK/CysM) ,29, the sK/CysM) ,40. One sK/CysM) ,50,51,52sK/CysM) ,49,53. SsK/CysM) ,55,56. Oisulfide ,58, a prisulfide . Despitefluenzae ,51,53, E[E. coli ,61, Salmhimurium ,62, and himurium ,65,66,67himurium ,50,68. Ihimurium .E. coli [Recently, Hayes and co-workers discovered that CysK is the permissive factor that activates a bacterial tRNase, the C-terminal region of Contact-dependent growth inhibition A protein (CdiA-CT) toxin in an uropathogenic strain of E. coli . The autE. coli  and funcE. coli  studies d of about 5 nM and are apparently not in competition with each other [CysK/toxin and CS complexes have roughly the same Kch other . DespiteSalmonella [We believe that understanding the structure and the regulatory role of CS is of particular relevance in the development of potential antibiotics, in light of the observation that inhibition of one enzyme does not only affect the activity of the main target but has also “pleiotropic” effects due to complex perturbation, most of which are, at the moment, unpredictable. For example, high-affinity inhibitors of CysK might potentially lead to complex dissociation. Indeed, it has been reported that CysE mutants, deficient in CS formation, induce cysteine auxotrophy in lmonella . Furtherlmonella ,72, its lmonella ,73. To oProtein painting is a recently developed technique ,75 that http://www.ebi.ac.uk/pdbsum/ [2. On the CysK sequence, four lysine residues  appear to participate in the interface with the toxin and should, in principle, be inaccessible to dyes  allowed to dyes .K221 cannot be recognized by trypsin because of the presence of a proline residue at its C-terminus; conversely, K118, K121, and K226 are three out of 34 potential trypsin cleavage sites. K226, which is protected by dye labelling from trypsin digestion in CysK , becomes buried at the toxin interface upon complex formation. The corresponding peptide is indeed identified in the complex A.K118 and K121 are not covered by the dyes on the uncomplexed CysK , but, whPrevious works by Hayes’s group and ours demonstrated that the CysK/CdiA-CT complex shares many mechanistic features with CS, among which the occupation of the CysK active site ,25. ThusBinding of CysE to CysK can be followed by fluorescence emission spectroscopy since CysK active site occupation by CysE modifies the fluorescence emission properties of the pyridoxal 5′-phosphate (PLP) cofactor ,39. The Arabidopsis thaliana CysK identified residues K217, H221, and K222  as essential for CS formation [S. Typhimurium have been isolated in the crystal: an unligated, open form [The identification as a PPI hot spot of K226, that was also identified at the CysK/toxin interface, further supports the highly similar binding mode of the two CysK binding partners. Interestingly, a mutagenesis study on ormation . In addipen form ; a substpen form ; and an pen form . In a prpen form . The trapen form . This mog, the maximum dimension Dmax, forward scattering I(0), etc.) are reported in g < 10.SAXS data were collected to further characterize the quaternary structure of the CS complex in solution and compare it with the protein painting results. In order to validate all structures involved in the CS complex, CysK and CysE alone were characterized with SAXS. An overview of the solution scattering data and the corresponding PDDF is presented in Interestingly, both CysK and CysE give solution scattering profiles that are consistent with the known crystal structures ,84. The g = 6.606 ± 0.003 nm and Dmax = 22.0 nm, and showed an elongated S-shaped envelope (as obtained by DAMMIF) that was compatible with the proposed model sketched in the bottom panel of g and a Dmax of 18.5 nm, which is not supported by the experimental data shown in -1. The information obtained by protein painting was used to define the residues involved in CysK–CysE interaction: K226 on CysK and R242 on CysE. This is part of the conserved PARIV sequence in the last strand of the α-helix that is solvent exposed and thus available for the interaction with CysK. Three different constrained distances, namely 1.0, 1.5, and 2.0 nm, were set between the CA atoms of residues K226 (CysK) and R242 (CysE). Fit was not satisfactory with 1.0 nm  and 2.0 nm distance , but 1.5 nm resulted in a good fit for each of the three independent solutions obtained . The resulting models fitted well into the ab initio real-space envelope, giving a reasonable overlap with the SAXS pattern and the q-space data regularized with GNOM  analysis gave structural parameters of Rith GNOM A,BThe S-shape of the complex also indicates that the three potential binding sites available to CysK on each protomer of CysE are not equivalent, but that, once CysK binds to one protomer, its binding site on the other protomer is defined. This observation suggests the existence of an allosteric communication between the two trimers within the hexamer. In the model, the CysK active site points towards the C-terminus of CysE but is still solvent accessible A, likelyUnless otherwise specified, reagents were purchased from Sigma Aldrich  and used as received.E. coli, cloned in pET21P and pSH21p vectors, respectively, were over-expressed in the bacterial strains BL21(DE3) and BL21(DE3) TunerTM. Cells were grown at 37 °C in Luria Bertani medium and induced in the presence of 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). CysK and CysE were then purified and the tag was removed from CysE as previously described [−1·cm−1, calculated by the alkali denaturation method [−1·cm−1. The purity of the enzymes assessed by SDS-PAGE was estimated higher than 95%. The specific activity of CysK, determined with the discontinuous assay of Gaitonde [L-Ser, 0.25 mM AcCoA, and 7 nM enzyme (monomer), was 83 U/mg, in agreement with previously-reported kinetic data [CysK and CysE from escribed , with min method . CysE coGaitonde  in the ptic data .E. coli BL21(DE3) TunerTM and was purified as CdiA-CT:CdiI-His6 complex, as previously described in [−1·cm−1.CdiA-CT was expressed in ribed in . CdiA-CTAbsorption measurements were carried out at 20.0 ± 0.5 °C using a Varian  CARY400 spectrophotometer. All spectra were corrected for buffer contributions. Fluorescence emission spectra were collected using a FluoroMax-3 fluorometer  at 20 ± 0.5 °C, equilibrating samples for 5 min prior to spectra acquisition. CysE/CdiA-CT stoichiometric binding to CysK was monitored by measuring PLP fluorescence emission at 500 nm following excitation at 412 nm (see ) 39,50,. All speThe oligomeric state of CysE and CysE/CysK complex in native conditions was determined on an analytical HPLC-SEC Superdex 200 increase 3.2/300 column  in PBS in the presence of 1 mM tris(2-carboxyethyl)phosphine (TCEP). A calibration curve was obtained by running five commercial standards for size exclusion chromatography  and the home-made standard glyceraldehyde 3-phosphate dehydrogenase .w/w for CysK alone and CysK in CS and 1:3.1 for CysK in complex with CdiA-CT. The reactions were stopped with trifluoroacetic acid (TFA) at 0.1% final concentration and peptides mixtures were desalted using 10 μL Pierce® C18 Tips .All protein preparations, either alone or in complex with binding partners, were incubated for 15 min in a solution containing 5 mM of each of the following molecular paints, in accordance with a published protocol : disodiuv/v acetonitrile and 2.5% v/v TFA, and 0.5 µL of the solution was spotted onto the plate. Each sample was analyzed in triplicate. External calibration was performed using the following mixture of standard peptides: Bradykinin fragment 1–7 (m/z 757.3997), angiotensin II (human) (m/z 1046.5423), P14R (m/z 1533.8582), ACTH fragment 18–39 (human) (m/z 2465.1989), and insulin chain B oxidized (m/z 3494.6513). Calibrations were accepted at the following conditions: 15 ppm mass tolerance and max outlier error, 4 minimum peaks to match, 5 as minimal signal-to-noise ratio (S/N). Resulting MS spectra were submitted to a homemade database search using the Mascot search engine. Methionine oxidation was selected as variable modification and carbamidomethylation of cysteine as fixed modification, two missed cleavages were tolerated, and peptide mass tolerance was set at 100 ppm. To ensure the quality of database searching results, the peak assignment for every peptide was manually checked, considering S/N = 3 as the limit of detection (LOD).Mass spectrometry analyses were carried out using a 4800 Plus MALDI TOF/TOF™ spectrometer  in positive ion reflectron mode combining 400 shots in the mass range 500–3600 Da. MALDI spots were prepared using the dried droplet method. Briefly, 1 µL of sample was mixed with 1 µL of 10 mg/mL α-cyano-4-hydroxycinnamic acid (HCCA) in 75% CysK, CysE, and CS prepared in a stoichiometric ratio were re-purified and buffer exchanged in 20 mM sodium phosphate, 85 mM NaCl, 2 mM EDTA, 10 mM 2-MCE, pH 7.5 using a Superdex 200 10/300 GL . After the chromatographic run, proteins were dialyzed against their storage buffer, which was later used to record the SAX-scattering baseline. Proteins were concentrated by ultrafiltration and their final concentration was determined by Bradford assay. SAXS measurements were performed using freshly-diluted protein samples at concentrations of 1.5, 1.0, and 0.5 mg/mL. Data sets of samples that showed indication of aggregation were merged with the corresponding data collected at lower concentration.−1) by using a freshly-prepared 5 mg/mL BSA solution in 50 mM Hepes pH 7.5. The scattering curves were normalized to the primary beam intensity, corrected for sample transmission, and normalized to absolute scattering units using IGOR Pro . Each set of scattering patterns was carefully checked and the average after a positive control over radiation damage was performed. Radiation damage was not observed on samples presented in this study. GIFT [P/M = 1.5). Bead ab initio modeling was conducted using DAMMIF [Small-angle X-ray scattering: Synchrotron SAXS data of all samples were collected on the Austrian beamline at the Elettra Synchrotron  using a Pilatus3 1M detector system at a sample-detector distance of 1.232 m and at a wavelength λ = 0.154 nm. Measurements were carried out at 10 °C in capillaries of 1.5 mm outer diameter/0.01 mm wall thickness made from borosilicate , enclosed within a custom-made thermostatic compartment connected to an external circulation bath and a thermal probe for temperature control. Raw data were radially averaged and calibrated to absolute units (cmdy. GIFT  was useddy. GIFT , which wg DAMMIF , from thg DAMMIF  from ATSThe CysK and CysE crystallographic model structures were superimposed to the ab initio bead models using SUPCOMB . The mulCysteine plays a central role in bacterial metabolism, in the resistance to oxidative stress, in antibiotic resistance, and in biofilm formation. Therefore, cysteine biosynthesis needs a fine-tuning of the involved enzyme activities. Despite the considerable efforts made towards the elucidation of these processes, the subtle mechanisms governing cysteine homeostasis are still poorly understood. Among these, the regulation of CS assembly, its conformation, and its role in CysE activity modulation are still debated. Cysteine biosynthesis is a putative target for enhancers of antibiotic therapy since cysteine-depleted bacteria exhibit a decreased fitness . In part


Qualea grandiflora, the most widely distributed tree species in the Cerrado, a large Neotropical savanna. We analyzed genetic variation with microsatellite markers in 23 populations  and phenotypic variation of 10 metamer traits  in 36 populations . To evaluate the role of geography, soil, climate, and wind speed in shaping the divergence of genetic and phenotypic traits among populations, we used Generalized Dissimilarity Modelling. We also used multiple regressions to further investigate the contributions of those environmental factors on leaf trait diversity. We found high genetic diversity, which was geographically structured. Geographic distance was the main factor shaping genetic divergence in Qualea grandiflora, reflecting isolation by distance. Genetic structure was more related to past climatic changes than to the current climate. We also found high metamer trait variation, which seemed largely influenced by precipitation, soil bulk density and wind speed during the period of metamer development. The high degree of metamer trait variation seems to be due to both, phenotypic plasticity and local adaptation to different environmental conditions, and may explain the success of the species in occupying all the Cerrado biome.Identifying the environmental factors that shape intraspecific genetic and phenotypic diversity of species can provide insights into the processes that generate and maintain divergence in highly diverse biomes such as the savannas of the Neotropics. Here, we sampled  Understanding the role of environmental factors in shaping genetic and phenotypic intraspecific diversity can provide important insights into the processes that are responsible for the generation and maintenance of biological diversity . In respSpecies with broad geographic distributions in heterogeneous environments constitute good models to investigate the role of environmental factors in shaping genetic and phenotypic diversity. In this context, the largest Neotropical savanna, the Cerrado, provides an ideal opportunity to conduct such research since it comprises one of the most biodiverse and heterogeneous tropical biomes on the world. The Cerrado harbors almost 10,000 plant species, and comprises a landscape marked by phytophysiognomies ranging from grassy fields to dense gallery forests . The bioThe Quaternary climatic oscillations have been recognized as playing a role in shaping the levels and spatial distribution of genetic diversity in Cerrado plant species. Phylogeographical and ecological niche modelling studies of Cerrado species have suggested a common pattern of reduction in species ranges during glacial periods, with retractions mainly in their southern extents. During interglacial periods, with wetter climate and higher temperatures, species expanded their distributions . A few rQualea grandiflora (Vochysiaceae), the most common and widely distributed tree species of the Cerrado   .ca. 20 adult individuals per population, from 23 populations, totaling 418 individuals. The distance between sampled populations ranged from 128.9 to 2,397.2 km .For the genetic study, we collected young leaves of 397.2 km . Quantit2+ during the reaction, 0.25 mM of each dNTP, 1 U Taq polymerase (Phoneutria) and 0.05–0.10 µM of each primer, depending on the primer set used  using a model that includes, simultaneously, the parameters null alleles (N), inbreeding coefficients (FIS), and genotyping failures (B) in a bayesian framework. we performed 500,000 Steps, with 50,000 steps as burn-in and keeping every 100th step for each population using INEST version 2.2  in the total sample by Bayesian analyses using the software STRUCTURE version 2.3.3  replicates and burn-in of 100,000 replicates, assuming an admixture model with correlated alleles between populations. The most probable K explaining the data was determined from average likelihood values ]) across runs for each K, as suggested by K statistic was calculated according to K from STRUCTURE can be sensitive to the presence of isolation by distance (IBD), we also inferred genetic clustering considering the geographic localization of the populations, with the software TESS 2.3.1  of the second node from distal branches of different parts of the crown of 744 individuals from 36 populations . All samples were collected in the rainy season and the distance between sampled populations ranged from 5.33 to 2,067 km (2), leaf length , leaf width  and leaf left-side and right-side width (in cm), using the software Image J. All samples were put in paper bags and dried in an oven at 70 °C for 72 h for determination of dry mass of each part of the metamer, separately . From these data we estimated the following metamer traits: Internode Mass Ratio (IMR), Petiole Mass Ratio (PMR), Leaf Mass Ratio (LMR), Specific Leaf Area (SLA), Leaf Area Ratio (LAR), Leaf Length per Leaf Width (LLW), Leaf Fluctuating Asymmetry (LFA) and Metamer Fluctuating Asymmetry (MFA). The traits were selected due their physiological importance. Metamer mass (MM), IMR and PMR indicate the biomass investment in biomechanical and hydraulic support , with the R package ‘gdm’ , retaining eight bioclimatic variables, wind speed, and six soil variables as predictors (Q. grandiflora is an anemochoric species (2) and the soil maps were at a resolution of 250 m, we used the function resample from the ‘raster’ package in R (P < 0.05).The climate variables and wind speed for each population were extracted from WorldClim  and soiledictors  and geneedictors  in the Cedictors . For theedictors , since Q species . For the species . Since tage in R  to make In addition to GDM, in order to investigate the association between each metamer trait and the environmental variables, we performed a multiple regression analysis using the mean of the traits of each population as the response variable. We used a stepwise model selection routine from the function ‘step’ implemented in the ‘stats’ package in R  in orderRST and pairwise Mahalanobis distance matrices.Finally, to test for an association between genetic and metamer traits divergence, we performed a multiple regression on distance matrices with the function ‘mrm’ from ‘ecodist’ package in R , using pNA) varying from 6.3 to 10.9, allelic richness (AR) varying from 5.1 to 7.7, observed heterozygosity (HO) and expected heterozygosity (HE) ranging from 0.42 to 0.77 and 0.69 to 0.85, respectively , Corinto (COR), Campo Grande (CQG), and CHG showed higher HE  and peripheral northeastern population PRI. The F(nfb) was significant (P < 0.05) in seven populations, ranging from 0.029 (PIR) to 0.189 (FUR) ] and geographic distances , indicating IBD. The test performed with the program localDiff showed that genetic differentiation between populations seems to remain constant across sampled area since the stationary hypothesis was not rejected (P > 0.05). Structure inferences, both from STRUCTURE and TESS analyses resulted in K = 5 genetic groups, geographically structured  and the northern group the lowest value (12.0)  were significant and remained in the model (P < 0.05). The GDM final model was then composed by only these two variables, which explained 23.1% of the total variance. Geographic distance was the most important variable in the model (I-spline = 0.31), showing its strong influence in the genetic divergence among populations and corroborating the Mantel test. Mean diurnal temperature range had lower influence on the genetic divergence among populations (I-spline = 0.06)  in addition to geographic distance. So, the final model to describe the metamer trait dissimilarity in Q. grandiflora includes soil bulk density , wind speed (I-spline = 0.22) and geographic distance (I-spline = 0.25), which explains 24.9% of the total metamer trait variation among populations .Leaf area (LA) and metamer mass (MM) were associated with variables that provide higher plant productivity. They were positively associated with precipitation of the warmest quarter (Bio 18) and temperature of wettest quarter (Bio8), but negatively associated with wind speed  appeared to be the main determinant of genetic divergence among populations, although IBE also had a limited effect. Soil bulk density and wind speed during the period of metamer development were important drivers of metamer trait divergence among populations along with geographic distance. Although soil factors explained the variation in several metamer traits, climate (mainly precipitation) was related to all metamer traits.Our study of the most widely distributed tree species in the Cerrado biome, Q. grandiflora populations was corroborated by the Mantel test that found a strong correlation between genetic and geographic distances (r = 0.833). In addition, our results also suggested that differences in the mean diurnal temperature range of sampled sites also contribute towards explaining the genetic differences among populations. Although geographic distance has been emphasized as the most common factor limiting gene flow among populations, recent landscape genetic studies suggest that this factor is just one of many possible causes of genetic divergence and isolation and that environmental heterogeneity may also play an important role in this process . Edaphic factors have been pointed out as mainly responsible for promoting distinct floristic composition even under similar climate conditions in the Cerrado biome  was affected by precipitation in the quarters with highest rainfall during the year, and was also higher in populations located in areas with less wind. A trade-off seems to exist between the investment in leaf mass (LMR) and the investment in internode mass (IMR) in Q. grandiflora, since they were affected by the same environmental variables, but in opposite directions. Internode mass is important for biomechanical leaf sheet support and the hydraulic pathway  in  pathway . In Q. gn plants .Soil characteristics show even more complex relationships with the metamer traits than climate variables. Bulk soil density was positively related to SLA, LMR and LAR, but negatively related to IMR. These relationships of metamer traits with soil bulk density need to be better studied, since they are apparently contradict theoretically expectations. It is known that high soil bulk density affects plant growth . The infQ. grandiflora, since the GDM analysis revealed an association with the overall metamer trait divergence among populations and the multiple regression showed its association with four specific metamer traits. Populations in environments with higher wind speed had smaller LA, MM and LMR, and higher IMR. Our results are concordant with some studies that reported reductions in LA and SLA and an increase in leaf thickness as morphological responses of plants to higher wind speeds  showing the highest genetic diversity. Moreover, the populations located in this central portion showed high admixture levels. These results are consistent with the colonization history of Q. grandiflora, which revealed that the central portion of the Cerrado core was the species’ refuge during the cold ages of the Quaternary Period  likely plays an important role in promoting divergence in metamer traits. The high variation in metamer traits, which our data suggest are likely due to genetic variation and phenotypic plasticity and can explain the ability of the species to occupy the entire Cerrado, including savanna enclaves in the Amazon. In addition, our data suggest that if considerable areas of the Cerrado are preserved and connected to each other, the high genetic and leaf trait diversity found in this species may allow Q. grandiflora to cope with current and future climate change.In summary, our results demonstrate that geographic distance and past climate change shaped the genetic diversity and structure of Microsatellite genotypes and leaf trait datasets generated and analysed in this study can be found in the Figshare repository with DOI: 10.6084/m9.figshare.11106881. Leaf trait dataset was also submitted to TRY Plant Trait Database. All other datasets for this study are included in the article/JL-F and ML planned and designed the research, provided financial resources for the research and collected the population samples. RB and TP performed the laboratory work. RB, TP, AM, VE, and RS analyzed the data. RB, TP, JL-F, and ML interpreted the data. RB, TP, JL-F, and ML wrote the manuscript and all authors reviewed the final version. RB and TP should be considered joint first authors. The authors JL-F and ML should be considered joint senior authors.This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).The authors declare that the research was conducted in the absence of any personal, professional or financial relationship that could constitute a conflict of interest.


In most smallholder dairy programmes, farmers are not fully benefitting from the genetic potential of their dairy cows. This is in part due to the mismatch between the available genotypes and the environment, including management, in which the animals perform. With sparse performance and pedigree records in smallholder dairy farms, the true degree of baseline genetic variability and breed composition is not known and hence rendering any genetic improvement initiative difficult to implement. Using the Girinka programme of Rwanda as an exemplar, the current study was aimed at better understanding the genetic diversity and population structure of dairy cattle in the smallholder dairy farm set up. Further, the association between farmer self-reported cow genotypes and genetically determined genotypes was investigated. The average heterozygosity estimates were highest (0.38 ± 0.13) for Rwandan dairy cattle and lowest for Gir and N’Dama . Systematic characterization of the genetic variation and diversity available may inform the formulation of sustainable improvement strategies such as targeting and matching the genotype of cows to productivity goals and farmer profile and hence reducing the negative impact of genotype by environment interaction. Smallholder dairying has the potential to drive people out of poverty, provide sustainable livelihoods and enhance household food and nutritional security. In different countries in Sub-Saharan Africa, a variety of dairy development initiatives are being implemented either by national governments or Non-Governmental Organisations (NGOs) . An examAll procedures performed in the study involving human participants and the protocol for animal hair sample collection were reviewed and approved by the Ethics Committee of the University of Rwanda’s Research and Postgraduate Studies (RPGS) Unit and the National Institute of Statics Rwanda (NISR) based on the guidelines provided by the Rwanda National Ethics Committee and in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Animal handling was done by knowledgeable personnel to ensure maximum comfort and minimal injury at all stages of the research.This study was conducted as a survey that combined social economic data, data on indictor traits for cow productivity, biological data in terms of animal hair samples. All the numerators were properly trained to conduct the survey and all standard biosecurity and institutional safety procedures were adhered to under the supervision of the expert from the University of Rwanda. A total of 1564 smallholder dairy farmers from the South and North provinces of Rwanda were interviewed. The respondents were beneficiaries of the Girinka programme. Socio-economic and productivity data that were collected included information on gender issues, production systems, access to relevant dairy production inputs such as fodder, water, labour, and animal health services. Animal hair samples were collected from the tail switch, taking care to avoid faecal contamination, following a protocol provided by the International Livestock Research Institute (ILRI). A total of 2717 cows were sampled from smallholder dairy farms consisting of 1492 samples from the North province and 12245 samples from the South province. Due to budget limitations a total of 150 random samples were selected from each of the provinces and shipped for genotyping. Samples were heat treated at 70°C for 2 h in preparation for shipping and genetic analysis. Of the 300 submitted samples, genotyping results were obtained from 299 samples. The rest of the samples have been safely stored in a biorepository at ILRI for future use. Results from the socio-economic survey are beyond the scope of the current paper.n = 28 samples), Holstein (n = 63), Norwegian Red (n = 17), Jersey (n = 36), and Guernsey (n = 21) breeds. To capture genetic signatures representative of African cattle, an African taurine breed (N’Dama (n = 24)) and two indicine breeds, the East African Shorthorn Zebu (EASZ) (n = 50) and Gir (n = 30) were also included in the analysis.A panel of genotypes from commercial international taurine dairy breeds was used as a reference for breed composition assignment. These included Friesian  using the Geneseek Genomic Profiler (GGP) High Density (HD) SNP array consisting of 150,000 SNPs, while SNPs for the reference breeds had been genotyped with the Illumina HD Bovine (777K SNPs) array. The SNPs in GGP array were optimised for use in dairy cattle having the most informative SNPs from Illumina Bovine 50k and 770k chips and additional variants known to have a large effect on disease susceptibility and performance. Genotype data quality control and cheques were carried out using PLINK v 1.9  and inclFIS-values was set to zero because such inbreeding coefficient estimates reflects sampling error  were estimated using PLINK. The distribution of MAF in each subpopulation  was represented as the proportion of all the SNPs used in the analysis and subsequently grouped into five classes as follows: , , , , and . The results were plotted for comparison between subpopulations using R . The obsng error .Principal component analysis (PCA) was used to describe the genetic structure of the crossbred cattle population using PLINK  by way oIn order to understand the relationships between the populations, the Euclidean distance between populations was evaluated using dartR package  in R. A Table 1). Ten percent of the farmers received pure Holstein-Friesian cattle while 6% farmers received Jersey cattle. Other farmers received local Zebu (20%). Quite a substantial proportion (18%) of farmers did not know the genotype of the cow that they received. From the genetic analysis, the majority (87%) of the cows was determined as cross-breeds between exotic dairy breeds such as Holstein Friesian, Jersey and Ayrshire; and local zebu type of animals. The rest were either pure exotic breeds (7%) or local zebu breeds (6%). There was 46.2% agreement and 29.4% disagreement between the farmer-reported genotypes and the genetically determined genotypes. The rest of the animals (24.4%) had their owners reporting that they did not know the genotype at all. The majority of the farmers received the animals as either calves (66%) or growing heifers (24%).Farmer-self reported information showed that the predominant genotype (45%) used for milk production in the Girinka programme was the cross between Holstein-Friesian and Zebu  and African taurine (N’Dama) breeds had the highest proportion of SNPs with the low MAF category  compared to European taurine (ET) breeds. The Rwandan crossbred cattle had relatively high proportion of SNPs with high MAF . The observed heterozygosity estimates for the study populations are illustrated in Table 2. The average heterozygosity estimates were high for the Rwanda cattle (0.38 ± 0.13) and lowest for Gir and N’Dama . Heterozygosity estimates for European taurine breeds used as references ranged between 0.30 ± 0.19 and 0.37 ± 0.12 for Jersey and Holstein breeds, respectively.The distributions of average minor allele frequencies for all populations under study  are shown in Table 2). The values obtained were not significantly different from zero.The study populations showed low detectable levels of inbreeding for both Rwanda cattle and the reference samples  from the indicine breeds. The Rwandan samples dispersed intermediate between EASZ and the Taurine breeds. A significant number of the Rwandan samples dispersed close to the N’Dama breed, suggesting a significant contribution of the breed in some of the animals in the population.The first principal coordinate vector accounted for 12% of the total variation and separated European taurine breeds from non-European breeds as shown in Figure 4. Each animal is represented by a vertical line divided into K coloured segments representing the estimated fraction belonging to each cluster. Short vertical lines at the bottom of each horizontal bar delimit individuals of different populations. Reference breeds are labelled as Guernsey (GN), Norwegian Red (NR), Friesian (FR), Holstein (HO), Jersey (JE), N’Dama (ND), East African Shorthorn Zebu (ZB) and Gir (GI). Based on visual inspection of the admixture plot, scrutiny of the separate CV error plots and the PCoA plots, K = 8 represented the most appropriate population number for the dataset. Importantly, increasing K above 8 did not reveal any detectable population substructure and the breed clusters remained the same. Based on results obtained with K = 8, most animals were crosses of Holstein-Friesian breeds which contributed on average 58.3% of the total genes in the crossbred animals. The predicted absolute exotic breed gene content in the crossbred cattle ranged from 12 to 100%  and African taurine (N’Dama) breeds compared to European taurine (ET) breeds. This result is consistent with the design of the genotyping array used which targets e breeds . This ase breeds . This ise breeds . The hige breeds . The relThe results also showed minimal differences in inbreeding coefficient estimates between European taurine and the Rwanda population. Given the huge admixture observed for the Rwanda population, this was expected. To accurately assess population structure of the study populations, we chose the PCoA method to assess dissimilarity between populations. The PCoA plot illustrates the wide range of genetic composition and breed contribution. The Rwanda cattle in the Girinka programme are not only highly admixed but also mainly crosses of Holstein Friesian, African taurine (N’Dama) and the East African Zebu. The dispersion pattern observed in this study reflects the practised indiscriminate crossbreeding, where farmer’s continually upgrade their animals to high exotic levels in a bid to increase productivity. ADMIXTURE results agree with the PCoA results and demonstrate the wide range of breed types that constitute the Rwanda Girinka cattle. The dominance of Holstein-Friesian breeds over other cattle breeds reflects the goals for the Girinka programme, in terms of maximising milk yields.Farmers’ ability to identify the genotype of their animals was limited. This implies that farmers either have poor knowledge of dairy breeds or the animals are not performing as expected. Based on the phenotypic performance of their animals, farmers may not have been convinced that the breed that they were told they would receive is the one they have when it does not perform at the level that the farmers expected. This could be in terms of both underperforming as well as over performing. This mismatch in terms of the breed that the farmers has and what they believe they have also reflect on poor pedigree record keeping and poor access to breed choices. Currently, there are no large farms that would provide large numbers of suitable animals, when needed. A scheme for appropriate sire selection and animal identification ought to be instituted across east Africa. In the meantime, handlers of the Girinka programme need to start instituting a breed composition profiling campaign after they purchase the animals so that they can match animals to specific farmer production systems. Farmers with the capacity to provide the right inputs such as animal feed, proper health management and have access to markets should receive the animals with the highest taurine composition, while those farmers with low capacity to provide inputs, ought to receive animals with a composition consistent with their production system. To ensure that the Girinka programme fulfils its goal, farmer education on dairy best practises and with consideration to cow genetic diversity must precede farmer acquisition of the cattle. This will ensure that farmers are well prepared with regard to the demands of rearing dairy cattle and have the requisite knowledge and inputs. The low dairy productivity reported in different countries in Sub-Saharan Africa reflects the inappropriateness of the breed allocation programmes and also general lack of proper preparatory work done prior to breed allocation.This study has demonstrated that a substantial number of farmers in the Girinka programme did not know the real breed of their cow. This would be a major bottleneck in any efforts for breed improvement. The application of high density SNP markers can be used in smallholder production settings to inform decision making and offer insightful options in breed development and distribution among smallholder farmers. Such information is vital in developing future breed sourcing strategies and development efforts among governments and NGOs targeting smallholder farmers. Further, the diversity of breed types used and the wide admixture spread presents the Rwandan dairy population with the opportunity for in-depth studies to identify the appropriate breed types and admixture level for different production systems.https://doi.org/10.6084/m9.figshare.7046768. Requests for the reference genotypes must be made directly to the owners of this data, as indicated.The data supporting the conclusions of this manuscript has been uploaded to Figshare by the authors at MC, TD, and OM conceived and designed the study. TD and OK oversaw the data and sample collection. FM and EC conducted the data analysis. MC, FM, TD, OK, EC, and OM wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Scophthalmus maximus) is a marine flatfish of high commercial value living on the European continental shelf. While wild populations are declining, aquaculture is flourishing in southern Europe. We evaluated the genetic structure of turbot throughout its natural distribution range  by analyzing allele frequency data from 755 single nucleotide polymorphism discovered and genotyped by double‐digest RAD sequencing. The species was structured into four main regions: Baltic Sea, Atlantic Ocean, Adriatic Sea, and Black Sea, with subtle differentiation apparent at the distribution margins of the Atlantic region. Genetic diversity and effective population size estimates were highest in the Atlantic populations, the area of greatest occurrence, while turbot from other regions showed lower levels, reflecting geographical isolation and reduced abundance. Divergent selection was detected within and between the Atlantic Ocean and Baltic Sea regions, and also when comparing these two regions with the Black Sea. Evidence of parallel evolution was detected between the two low salinity regions, the Baltic and Black seas. Correlation between genetic and environmental variation indicated that temperature and salinity were probably the main environmental drivers of selection. Mining around the four genomic regions consistently inferred to be under selection identified candidate genes related to osmoregulation, growth, and resistance to diseases. The new insights are useful for the management of turbot fisheries and aquaculture by providing the baseline for evaluating the consequences of turbot releases from restocking and farming.Unraveling adaptive genetic variation represents, in addition to the estimate of population demographic parameters, a cornerstone for the management of aquatic natural living resources, which, in turn, represent the raw material for breeding programs. The turbot ( Within the Baltic Sea, excluding the transition area with the North Sea, salinity can also reach higher values in the south (≈13 PSU). In the Mediterranean Sea, the salinity is even higher than in the Atlantic Ocean (≈38 PSU), but drops abruptly in the transition to the Black Sea, whose salinity levels resemble the Baltic Sea (≈11 PSU). Contrasting patterns of surface temperature also occur across latitude and between seasons. A north–south cline exists in the Atlantic area , which increases further in the transition to the Mediterranean Sea (≈21°C), especially during summer. A sharp winter versus summer variation is also found within the inner Baltic and Black seas (≈20°C difference). In the latter, turbot was formerly described as a subspecies  and the Baltic Sea  database; The uneven distribution of turbot has been associated with phylogeographic events related to rapid adaptive radiation following the last glaciation and to heterogeneous environmental conditions across its distribution range  technology , and then, a comprehensive catalog of loci was created using the cstacks module (‐n 7) and the full dataset. A correction of SNP calls was made using population data with the rxstacks module, which improves SNP calls and filters unreliable and confounded loci. Finally, the populations module was used to retrieve a first dataset of SNPs in genepop format for doing the final filtering step. Genotyping accuracy was validated using sample replicates from within and among libraries.A ddRAD genotyping‐by‐sequencing (GBS) approach was carried out following the procedure first described by Peterson et al.  with slip > .05) FIS values  genotyped in >80% individuals, (ii) contained a single SNP per RAD‐tag, (iii) had a unique match against the turbot genome , and observed (HO) heterozygosities and percentage of polymorphic loci at 95% (P95: frequency of the most common ≤0.95) and 99% (P99: ≤ .99) were calculated to assess genetic diversity. HE was also calculated for a set of the most polymorphic, potentially more informative, loci, that is, where MAF ≥ 0.05. Departure from Hardy–Weinberg equilibrium (HWE) and significance of FIS values were tested for each population. Analyses were performed using GENEPOP v4.0 , expected  from 1 to 10. Five independent runs were used for each K estimate when the full marker dataset was used, while 10 runs per K were tested for the neutral and outliers marker subsets. The locprior model, which specifies the population of origin of each individual, was also used, as previously suggested for data showing weak structure . Data were transformed using principal component analysis (PCA) and an appropriate number of principal components and discriminant functions retained.Discriminant analyses of principal components (DAPC) were run in the ADEGENET package  between sampling sites were obtained using Google Earth .A Mantel test was performed using GENEPOP v4.0 to test for isolation by distance (IBD). The correlation between p < .05) was corrected for multiple comparisons using the sequential Bonferroni method  was used as an initial threshold but log10 BF >1.3 (p > .95) was also evaluated for comparisons with other methods. BAYESCAN outcomes were plotted using the R functions provided by the program. The FDIST FST method implemented in the LOSITAN software  of 0.01, and the “neutral mean FST” option. ARLEQUIN v3.5 was applied to investigate outlier loci among groups of samples under a hierarchical island model, testing 20,000 simulations, 50 groups, and 100 demes per group. SNP markers with p < .05 or <.01 were considered as outlier loci depending on the analysis.To identify candidate loci subjected to selection, three different algorithms and related software packages were applied as previously suggested  were retained, and additionally, only those that were identified both with LOSITAN and ARLEQUIN at the highest confidence level (CI < 99 and FDR = 0.010 for LOSITAN and p < .010 for ARLEQUIN). Outlier loci for balancing selection are more difficult to detect, and a high rate of false positives is returned with most programs  including the most consistent set of outlier SNPs were mapped to the turbot genome to obtain their genomic position using local BLASTn . These outliers were subsequently anchored to the turbot genetic map (linkage group (LG) and predicted position in cM) to investigate their putative relationship with previously reported outliers  were retrieved from the Environmental Marine Information System (EMIS) database ( http://mcc.jrc.ec.europa.eu/emis/) and the Copernicus database ( http://marine.copernicus.eu/), corresponding with the average along the year of the monthly mean values for each sampling site with a 4‐km resolution for the period comprising 1997–2014.The environmental variables “sea surface temperature” and “sea bottom temperature” , “sea surface salinity” and “sea bottom salinity” , and “primary production”  and the registered environmental variables  was studied by a canonical redundancy analysis (RDA) using the R platform VEGAN software Oksanen, . For eac33.1populations module extracted a first dataset of 5,564 polymorphic loci. A final set of 755 SNPs was obtained after the application of the following filtering steps: (i) 3,656 loci did not pass the cutoff of genotyping coverage (>80%), (ii) 899 loci showed MAF <0.002, (iii) 40 loci showed consistent deviations from HWE across populations (p < .05), suggesting null alleles (FIS > 0) or mixing of reads from different paralogs (FIS < 0), (iv) 71 loci corresponded to the same SNP due to overlapping RAD‐tags, (v) 136 loci co‐mapped in the same RAD‐tag with another SNP, and (vi) 7 SNPs did not match with the turbot genome. Genotyping accuracy was evaluated in this filtered sample using the sample replicates within and among libraries, and an average genotyping error rate of 0.5% was recorded. A total of 25 individuals with very low DNA quality and genotyped for <20% of the SNP panel after filtering were discarded. The final panel of 755 SNP loci was analyzed in 672 individuals to evaluate genetic diversity and structure, identification of outlier loci and landscape analysis.A total of 783,829,288 read‐pairs were retrieved from the sequencing platform, with ~1 million read‐pairs on average per sample. The distribution of reads per sample was rather unbalanced, ranging from 125,563 to 5,842,110, mostly attributed to the low DNA quality of some archived samples, along with the specific methodology employed; that is, samples were combined immediately following adapter ligation  to 0.189 ± 0.154, and P99 to 49.0% ± 2.1% . Likewise, a lower effective population size (Ne) was estimated for these samples, especially for BLS‐N (126) and AD , and most Ne values were higher than 1,000 . Within the ATL region, the lowest Ne values were for IR‐E (264), BB‐SE (166) and SP‐W (411). Significant deviations from HWE, resulting from heterozygote deficit, were only found within the Atlantic/Baltic Sea area at BAS‐N (FIS = 0.162) and BB‐SW , but especially at BAS‐S .On average genetic diversity values across all populations amounted for Na to 1.49 ± 0.50, H3.3FST = 0.090, p < .001) was detected across the whole turbot distribution using the 755 SNP panel, mostly attributable to divergence between the more isolated areas (Black Sea and the Adriatic Sea) and the Atlantic Ocean and the Baltic Sea (Table K) was four as revealed by STRUCTURE , and low, but significant, between the Baltic and the Atlantic region . When applying the most strict criteria  and ARLEQUIN . Among these, eight outliers detected both with LOSITAN and ARLEQUIN, and consistent with environmental variation, were retained  and the eight for balancing selection were removed from the dataset to obtain the most consistent set of 513 SNPs not showing evidence of selection, here designated as the “neutral panel.” Sequences of the 25 most consistent outlier loci  were located in the turbot genome and most of them (23) anchored to the genetic map (Table  and the ADGRL2 (adhesion G protein‐coupled receptor L2), a gene related to exocytosis regulation , a gene related to fertilization , a gene strongly up‐regulated during osteoblast differentiation in humans , KRT8 (keratin 8), KRT18 (keratin18); Grady et al., LG2: Outlier 6850_51 mapped to an intronic region of MTMR7 (myotubularin‐related protein 7), a gene related to lipid and protein metabolism , a candidate gene for VHSV resistance , MICALL1 (MICAL like 1), and PATL1 , a gene which plays an important role in macrophage differentiation , a poorly characterized gene with conserved immunoglobulin‐like domains, related to neuronal modulation and associated with growth traits in cattle , a candidate gene related to growth and A. salmonicida resistance , a gene related to osmoregulation  and outlier (25 loci) SNP datasets to compare patterns of genetic differentiation related to demographic parameters and selection Table . A thirdFST = 0.003, p < .050), ATL versus BLS , and BAS versus BLS . The global differentiation pattern was similar to that observed when using the whole marker dataset .Levels of genetic differentiation estimated for the neutral panel were lower than estimated from the full SNP dataset Table b and S5, Figures b. Ne wasr = .07648, p = .297).When only ATL samples were examined, DAPC with neutral markers yielded similar results as the full panel indicating that turbot from Norway and Spanish coasts were slightly differentiated from the other samples Figure c. NevertFST markedly increased between ATL versus BAS  and ATL versus BLS , but lower than that computed from the full SNP panel (FST = 0.169), suggestive of stabilizing selection acting on some of these outlier loci in both regions. Genetic structure resolved by only the six ATL‐BAS divergent outlier loci showed that both BAS and NOR were distinct from the remaining ATL samples, as three main clusters were identified both with STRUCTURE  developed with a genotyping‐by‐sequencing (GBS) strategy, namely ddRAD , which suggests some caution for statistical bias when analyzing the whole dataset. However, this sampling reflects quite well turbot distribution, which is common in the Atlantic Ocean and very scarce in the Mediterranean Sea, and therefore, the analysis should take this fact into consideration. Anyway, we split our analysis also by region and made all meaningful comparisons between regions to obtain the most comprehensive view of turbot structure.E: 0.095) was much lower than previously reported , mainly due to the high number of samples used to identify polymorphic loci in our study (697) and the low MAF cutoff used (0.002). This resulted in the detection of a large fraction of loci with very rare alleles compared to previous studies  approaching to previous results  was similar to the current study (FST = 0.090), especially considering that the allozyme study did not include the Black Sea. In our work, turbot samples were consistently separated into four main genetic regions: Atlantic, Baltic Sea, Black Sea, and Adriatic Sea. However, it should be kept in mind that turbot is essentially a northeastern Atlantic species with relict populations in the Mediterranean Sea, such as in the Gulf of Lion and the northern Adriatic Sea. Its isolated occurrence in the Black Sea can be attributed to the more suitable environmental conditions compared to the Mediterranean Sea for a temperate cold‐water species. Hence, genetic differentiation between the sampled Atlantic and Baltic turbot was much lower (FST = 0.007) than between the Atlantic and either Adriatic (FST = 0.170) or Black Sea (FST = 0.240) samples.Given the general lack of physical barriers in the sea, marine fish, such as turbot, with wide distribution ranges, high fecundity, pelagic eggs, and larvae, are expected to be at best weakly structured across large areas. The only study exploring genetic structure in turbot covering a roughly similar distribution range was based on allozyme marker data , a substructure previously reported based on other SNP and microsatellite genotypes  between the Atlantic Ocean and the Baltic Sea . Differentiation due to selection (and genetic drift) may be favored by limited gene flow related to differences in salinity tolerance. It is known that Atlantic turbot eggs do not survive at the lower salinities of the Baltic Sea , and further, Norman, Ferguson, and Danzmann , which contains three genes related to osmoregulation . This genomic region at LG1, which includes several outlier loci, growth and VHS resistance QTLs, and growth and salinity tolerance candidate genes, seems relevant to explain the genetic structure of turbot.Interestingly, the outlier 5397_68, located very close to 2372_27 at LG1 (<100 kb distant), showed signals of stabilizing selection in the BAS‐BLS comparison. The marker SNP 5397_68 is located in the vicinity (~100–400 kb distant) of genes related to growth . As mentioned above, two of these markers (7033_88 and 5397_68) are linked to several growth‐related loci  has been identified as an important factor in the evolution of some marine species, for example, related to coral reef fishes , indicating that demographic and historical factors have contributed to shaping population structure of turbot across its natural distribution. The information reported here also provides for the first time new insights on turbot adaptation, especially in the Black Sea, and suggested parallel evolution between areas with similar environmental conditions. Both strong neutral evolutionary forces and adaptive selection appear to be acting simultaneously on geographically isolated populations in the natural distribution of turbot. However, subtle neutral differentiation and local adaptations might also be occurring within regions. Candidate outlier loci, mostly anchored to the turbot genome, and especially at specific regions of LG1 and LG9, showed a positive correlation with environmental variables related to salinity and temperature.4.3Our results represent useful information for the management of wild stocks, and they can be valuable for breeding programs of farmed turbot. An improved definition of management units considering both demography and adaptation to environmental variation along the whole distribution range can now be delineated, allowing the future definition of adaptive management units (AMU, Bernatchez et al., None declared. Click here for additional data file. Click here for additional data file.


Gentiana radix is used in traditional Chinese medicine and has functions of clearing heat and drying dampness, as well as purging liver and gallbladder fire. A highly sensitive and effective strategy for rapid screening and identification of target constituents has been developed by using ultra high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap) in crude and wine-processed Gentiana radix. Based on the accurate mass measurement (<5 ppm), retention times, and MS fragmentation ions, 52 constituents were unambiguously or tentatively characterized from Gentiana radix, including 21 iridoids, 11 flavonoids, 19 xanthones, and a triterpenoid. This study demonstrated that the established method could be a rapid, effective analytical tool for screening and characterization of compounds in the complex systems of Gentiana radix. By comparing the structure and peak areas of chemical constituents in crude and wine-processed Gentiana radix, we found that some compounds in crude and wine-processed Gentiana radix were significantly different. Gentiana radix is the dried root and rhizome of Gentiana manshurica Kitag., Gentiana radix Bge., Gentiana triflora Pall. and Gentiana rigescens Franch. +, m/z 193 [M + H − H2O − H2O]+, suggesting the presence of two hydroxyl groups, m/z 211 was identified as the base peak. In addition, all of them produced [M + H − CH3OH]+ and [M + H − CH3OH − CO]+ions at m/z 197 and m/z 169, suggested the presence of carbomethoxy [m/z 179 [M + H − H2O − CH3OH]+, m/z 151 [M + H − H2O − CH3OH − CO]+, m/z 161 [M + H − H2O − H2O − CH3OH]+, m/z 133 [M + H − H2O − H2O − CH3OH − CO]+. By comparison with the reference standard, compound 1 was predicatively annotated as loganetin.Compound 1 produced the [M + H]omethoxy . It yiel+ ion at m/z 391.12317 and 391.12280 . In the MS2 spectrum, m/z 229 [M + H − Glc]+ was identified as the base peak. Other product ions like m/z 373 [M + H − H2O]+, m/z 211 [M + H − H2O − Glc]+ and m/z 193 [M + H − H2O – Glc − H2O]+. The ion at m/z 125 [M − 266]+ was yielded by RDA cleavage fragmentation at 5-position of the O-ring and 7-position of the C-ring. By comparison with the literature data, compound 2 was tentatively identified as eustomoside.Compound 2 showed the [M + H]+ ion at m/z 405.13898 and 405.13864 . It generated a serial of ions at m/z 387 [M + H − H2O]+, m/z 373 [M + H − CH3OH]+, m/z 345 [M + H − CH3OH − CO]+, m/z 243 [M + H − Glc]+, m/z 225 [M + H − Glc − H2O]+, m/z 211 [M + H − Glc − CH3OH]+, m/z 193 [M + H − Glc − CH3OH − H2O]+, m/z 175 [M + H − Glc − CH3OH − H2O − H2O]+ and m/z 165 [M + H − Glc − H2O − CH3OH − CO]+. Thus, compound 4 was tentatively determined as methyl (1S)-1-(d-glucopyranosyloxy)-6,7-dihydroxy-7-methyl-1,6,7,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate.Compound 4 showed the [M + H]− at m/z 375.13019 and m/z 375.13022 (mass error = 4.310 ppm and mass error = 4.390 ppm) (C16H23O10), respectively. Both of them generated the same ESI-MS2 ions at m/z 213 [M − H − Glc]−, m/z 195 [M − H − Glc − H2O]−, m/z 169 [M − H − Glc − CO2]−, 151 [M − H − Glc − H2O − CO2]− and m/z 125 [M − H − Glc − 2CO2]− [n of compound 5 is shown in Compound 5 yielded its quasi-molecular ions [M − H]− 2CO2]− . The pro+ at m/z 385.14673 and m/z 385.14664 . It generated a serial of ions at m/z 367 [M + Na − H2O]+, m/z 357 [M + Na − CO]+, m/z 339 [M + Na − CO − H2O]+, m/z 355 [M + Na − HCOH]+, m/z 223 [M + Na − Glc]+ and m/z 205 [M + H − Glc − H2O]+. By comparison with the reference standard, compound 6 was predicatively annotated as 1-O-d-Glucopyransylampexine.Compound 6 showed the [M + Na]− ions at m/z 581.17352 and 581.17328 , 419.11990 and 419.11984 , and 403.12506 and 403.12488 , respectively. All of them generated [M − H]− and [M − H − H2O]− ion at m/z 535, m/z 373, m/z 357, m/z 517, m/z 355, m/z 339, respectively. Compounds 8 and 14 also generated the [M − H − Glc − H2O − CH2]− ion at m/z 341, m/z 179. Compounds 14 and 21 also generated the [M − H − Glc]−, [M − H − Glc − H2O]− and [M − H − neutral molecule]− (RDA) ion at m/z 211, m/z 195, m/z 193, m/z 177, m/z 151, m/z 141, m/z 125. The MS2 ions at m/z 193 (C10H11O5) were generated by cleavage fragmentation from 1-position of quasi-molecular ions in compounds 8 and 14. Compound 8 generated [M − H + HCOOH − H2O]−, [M − H + HCOOH − H2O − H2O]− and [M − H − H2O − H2O]− ions at m/z 563, m/z 545 and m/z 499. The fragmentation pathways were consistent with deduced of compounds 8, 14 and 21, which further proved the validity of the results .The [M − H]− ion produced the ions at m/z 371, m/z 345 and m/z 209 in the MS2 spectrum, which originated from the neutral loss of H2O, CO2 and a glucose. In addition, the molecular ion also produced the minor ion at m/z 191 [M − H − Glc − H2O − H2O]− and m/z 163 [M − H − Glc − H2O − H2O − CO]−. Thus, compound 11 was tentatively determined as secologanol.Compound 11 generated [M − H]+ ions at m/z 405.13895 and m/z 405.13858 , m/z 407.15421 and m/z 407.15466 . Its MS2 spectrum produced ions at m/z 387, m/z 369, m/z 389 and m/z 371, which involved the loss of one and two molecules of H2O, respectively. In addition, compounds 12 and 45 also produced a serial of ions at m/z 243 and 245 [M + H − Glc]+, m/z 225 and 227 [M + H − Glc − H2O]+, m/z 193 and 195 [M + H − Glc − H2O − CH3OH]+, m/z 183 and 185 [M + H − Glc − CH3OH − CO]+, m/z 165 and 167 [M + H − Glc − H2O − CH3OH − CO]+ [3OH]+ ions at m/z 211. So, compounds 12 and 45 were tentatively determined as kingiside and morroniside.Compounds 12 and 45 produced the [M + H]H − CO]+ . Compoun− ions at m/z 563.16193 and m/z 563.16144 , m/z 401.10907and m/z 401.10892 , respectively. Both of their deprotonated molecular ions produced [M − H]−, [M − H − Glc]−, [M − H − Glc − CO]−, [M − H − Glc − CO − H2O]− and [Glu − H]− ions at m/z 517, m/z 355, m/z 355, m/z 193, m/z 327, m/z 165, m/z 309, m/z 149, m/z 341, and m/z 179, respectively −, m/z 165 [M − H − Glc − Glc − CO]−, m/z 323 [M − H − Glc − H2O − CH2]− and m/z 305 [M − H − Glc − H2O − CH2 − H2O]−. So, compound 15 was tentatively ascertained as 6′-O-β-d-glucosyl gentiopicroside, compared with the tR values and mass spectra with the reference standard, compound 20 was tentatively ascertained as gentiopicroside.Compounds 15 and 20 generated their [M − H + HCOOH]ectively . In addi− ion at m/z 435.15146 , respectively. Compound 22 could generate ions at m/z 389 [M − H]−, m/z 371 [M − H − H2O]−, m/z 227 [M − H − Glc]−, m/z 209 [M − H − Glc − H2O]− and m/z 127 . So, compound 22 was tentatively ascertained as loganin.Compound 22 generated their [M − H + HCOOH]− ions at m/z 655.18951 and 655.18927 , m/z 797.21667 and 797.21637 , m/z 875.22638 and 875.22614 , respectively. The ions at m/z 315 (C15H19O10) were generated by α-cleavage fragmentation of [M − H]− ion and loss a neutral molecule . Both of their deprotonated molecular ions produced [M − H − H2O]− and [M − H − Glc]− ions at m/z 637, m/z 779, m/z 857, m/z 493, m/z 635, m/z 713. Compounds 26 and 51 also generated the [M − H − R1′]−, ions at m/z 577, m/z 357. And compounds 50 and 51 generated the [M – H − CH3OH − CO]− and [M − H − CH3OH − CO − Glc]− ions at m/z 815, m/z 737, m/z 653, m/z 575. Compounds 26 and 50 generated the [M − H− Glc − H2O]− ions at m/z 475, m/z 617 [m/z 195 [M − H − R1′ − Glc]−. Compound 50 produced [M − H − 2H2O]−, [M − H − CH3OH]−, [M − H − 2CH3OH − 2CO]−, [M − H − 3CH3OH − 3CO]−, [M − H − CH3OH − Glc]−, [M − H − 2CH3OH − 2CO − Glc]− and [M − H − 3CH3OH − 3CO − Glc]− ions at m/z 761, m/z 755, m/z 677, m/z 617, m/z 593, m/z 617, m/z 475, m/z 515, m/z 455. Compound 51 produced [M − H − R2′]−, [M − H − R2′− CH3OH − CO]− [M − H − R2′ − CH3OH − CO − Glc]− and [M − H − R2′ − R1′]− ions at m/z 739, m/z 679, m/z 517, m/z 441. Compounds 26, 50 and 51 were predicatively annotated as gentiotrifloroside, rindoside, macrophylloside A.Compounds 26, 50, and 51 generated their [M − H] m/z 617 . In addi+ ion at m/z 567.20477 . It generated a series of ions at m/z 549 [M + H − H2O]+, m/z 471 [M + H − H2O − CH3OH − CO]+, m/z 453 [M + H − 2H2O − CH3OH−CO]+, m/z 453 [M + H − 3H2O − CH3OH − CO]+, m/z 229 [M + H – Glc − C10H8O3]+ and m/z 211 [M + H − Glc − C10H8O3 − H2O]+. Therefore, it was tentatively identified as 2′-feruloy loganin.Compound 31 produced its [M + H]+ ion at m/z 401.14362 and m/z 401.14392. It generated a serial of ions at m/z 383 [M + H − H2O]+, m/z 365 [M + H − 2H2O]+, m/z 341 [M + H − CH3OH − CO]+, m/z 197 [M + H − C8H12O6]+, m/z 179 [M + H − C8H12O6 − H2O]+, m/z 151 [M + H − C8H12O6 − H2O − CO]+and m/z 127 . Therefore, compound 41 was tentatively annotated as 3′-acetyl swerside.Compound 41 generated its [M + H]+ ion at m/z 579.26117 . The [M + Na]+ ion produced the ions at m/z 561 and m/z 543 in the MS2 spectrum, which involved the loss of one and two molecules of H2O. In addition, it produced m/z 417 [M + Na − Glc]+, m/z 399 [M + Na − Glc − H2O]+, m/z 255 [M + Na − 2Glc]+, m/z 237 [M + Na − 2Glc − H2O]+ and m/z 383 [M + Na − Glc − CO − CH4]+. Thus, compound 48 was tentatively determined as gentianaside.Compound 48 generated [M + Na]− ion at m/z 841.26135(mass error = 1.356 ppm) (C34H49O24 ). The molecular ions yield [M − H]−, [M – H − Glc]−, [M − H − 2Glc]−, [M − H − 2Glc − C2H4]−, [M − H − 2Glc − C2H4 − 4H2O]− and [M − H − 3Glc − C2H4]−, m/z 841, m/z 679, m/z 517, m/z 489, m/z 477 and m/z 337. According to the literature data, compound 49 was tentatively annotated as scabran G4.Compound 49 produced the [M − H]− ion at m/z 781.22137 and m/z 781.22119 . The ions at m/z 619 and m/z 601 were yielded by neutral loss one molecule of glucosyl and glucose, respectively. Moreover, the molecular ion generated ions at m/z 655 [M − H − C6H6O3]−, m/z 493 [M − H − C6H6O3 − Glc]− and m/z 339 [M − H − C6H6O3 − 2Glc − H2O]−. According to the literature data, compound 52 was ascertained as depressoside.Compound 52 generated [M − H]− ions at m/z 609.14783 and 609.14764 , m/z 593.15302 and 593.15277 , m/z 431.09903 , m/z 653.17395 and m/z 653.17328 , respectively. All of them produced [M − H − H2O]− ions at m/z 591, m/z 575, m/z 413 and m/z 635, respectively. Compounds 16, 17 and 30 also generated the [M − H − 2H2O]−, [M − H − C3H6O3]− and [M − H − C4H8O4]− − ions at m/z 447, m/z 431, m/z 491. Compounds 16 and 17 generated the [M − H − Glc − C4H8O4]− ions at m/z 327, m/z 311. Moreover, compounds 16 generated ions at m/z 429 [M − H − Glc − H2O]− and m/z 411 [M − H − Glc − 2H2O]−.Compound 30 ion generated ions at m/z 281 [M − H − C5H10O5]− and m/z 283 [M − H − Glc − C4H8O4 − CO]−. In addition, compound 38 ion generated ions at m/z 477 [M − H − Glc − CH2]−, m/z 357 [M − H − Glc − C4H8O4 − CO]− and m/z 315 [M − H − Glc − C4H8O4 − CO − 2CH2]− − ions at m/z 801.21179 and 801.21118 , m/z 639.15826 and 639.15808401.10892 , respectively. Both of them produced [M − H]− and [M − H + HCOOH − Glc]− ions at m/z 755, m/z 593, m/z 639 and m/z 477. Compound 25 generated a serial of ions at m/z 621 [M − H + HCOOH − Glc − H2O]−, m/z 477 [M − H + HCOOH − 2Glc]−, m/z 681 [M − H + HCOOH − C4H8O4]− and m/z 635 [M − H − C4H8O4]− −, [M − H − C4H8O4]− and [M − H − C5H10O5]− at m/z 431, m/z 357, m/z 311 and m/z 281, respectively. Therefore, according to the fragmentation pathways and literature data, compounds 25 and 28 were isovitexin-2′′-4′-O-B-d-glucosyle, isovitexin-2′′-O-B-d-glucosyle.Compounds 25 and 28 produced their [M − H + HCOOH]C4H8O4]− . Compoun+ ions at m/z 449.10727 and 449.10684 , m/z 493.13306 and 493.13287 , respectively. Both of them generated [M + H − H2O]+ and ions [M + H − 2H2O]+ at m/z 431, m/z 475, m/z 413 and m/z 457, respectively. Compound 27 could generate a series of ions at m/z 395 [M + H − 3H2O]+, m/z 329 [M + H − C4H8O4]+ and m/z 299 [M + H − C5H10O5]+ +, [M + H − C5H10O5 − HCHO]+ and [M + H − C5H10O5 − C10H10O3]+ respectively. By comparing with the literature data, compounds 27 and 36 were tentatively annotated as isoorientin and 6-C-B-d-glucospranosyltricine (isopyrenine).Compounds 27 and 36 yielded their [M + H]5H10O5]+ ,51. Comp3COOH]− ions at m/z 683.18146  and m/z 521.12724 . Both of the deprotonated molecular ions yield [M − H + CH3COOH − H2O]− at m/z 503 and m/z 665. Compound 39 produced their [M − H + CH3COOH − 2H2O]−, [M − H − CH2]−, [M − H − Glc − CO]−, [M − H − Glc − C5H10O5]− and [M − H − Glc − C5H10O5 − CH2]− ions at m/z 647, m/z 609, m/z 433, m/z 311 and m/z 297 [3COOH − CO2]−, [M − H − C4H8O4]−, [M − H − C5H10O5 − CH2]− and [M − H − C5H10O5 − C9H8O2]− ions at m/z 477, m/z 341, m/z 297 and m/z 163. According to the literature data, compounds 39 and 46 were tentatively annotated as ioscoparine-7-O-B-d-glucosyle and isoscoparine.Compounds 39 and 46 produced their [M − H + CH3COOH]− ion at m/z 1009.24139 . It generated a serial of ions at m/z 991 [M − H + CH3COOH − H2O]−, m/z 973 [M − H + CH3COOH − 2H2O]−, m/z 949 [M − H]−, m/z 931 [M − H − H2O]−, m/z 847 [M − H + CH3COOH − Glc]−, m/z 846 [M − H + CH3COOH − Glc − H]−, m/z 489 [M − H − C15H16O9 − C4H8O4]− and m/z 489 [M − H + CH3COOH − C15H16O9 − C5H10O5]−. According to the literature data, compound 42 was ascertained as 4′-O-B-d-glucospranosyl-2′′-O--2′′-isoorientin.Compound 42 produced the −, m/z 253[M − H − C5H10O5]− and m/z 179 [Glu − H]− [C-β-d-glucospranosyl-glucopyranosyl-1-hydroxy-7-methoxyxanthone.Compound 3 showed [M − H + HCOOH]− ion at m/z 451.14664 , m/z 377.08716 and 377.08698 . Both of the deprotonated molecular ions at m/z 405 and 331 [M − H]−. Compound 7 generated a series of ions m/z 433 [M − H + HCOOH − H2O]−, m/z 415 [M − H + HCOOH − 2H2O]−, m/z 243 [M − H − Glc]− and m/z 269 [M − H − C7H4O3]−. Compound 24 generated ions m/z 347 [M − H + HCOOH − HCHO]−, m/z 197 [M − H − C8H6O2]−, m/z 119 [M − H − C9H10O4]−, m/z 153 [M − H + HCOOH − HCHO − C8H6O2]− and m/z 179 [M − H − C7H6O2 − HCHO]− − HCHO]− . Therefo3COOH]− ions at m/z 375.10927 , m/z 523.14722 and 523.14703 . Both of the deprotonated molecular ion yield [M − H + CH3COOH − C9H8O4]− at m/z 195 and at m/z 343. Compound 9 produced a serial of ions at m/z 357 [M – H + CH3COOH − H2O]−, m/z 287 [M − H − CO]−, m/z 255 [M − H − 2HCHO]−, m/z 179 [M − H − C8H8O2]−, m/z 151 [M − H − C9H8O3]− and m/z 121 [M − H − C9H8O3 − HCHO]−. Compound 29 generated [M − H + CH3COOH − 2H2O]−, [M − H + CH3COOH − 4H2O]−, [M − H + CH3COOH − Glc]−, [M − H + CH3COOH − C8H8O3]−, [M − H + CH3COOH − C9H8O3]−, [M − H − C9H8O3]−, [M − H – Glc − 2HCOH]− and [M − H – Glc − C8H8O2]− at m/z 487, m/z 451, m/z 361, m/z 371, m/z 359, m/z 299, m/z 241 and m/z 165, respectively. According to the literature data, compounds 9 and 29 were tentatively determined as tetramethoxy-1,3,7,8-xanthone and glucosyl-1-decussatin.Compounds 9 and 29 produced − ion at m/z 534 and m/z 549. Compounds 13 and 54 also generated the [M − H − H2O]− and [M − H − 2H2O]− ions at m/z 431, m/z 577, m/z 413 and m/z 559. Compound 10 generated [M − H − 2HCHO]−, [M − H − CO − 2H2O]−, [M − H − CO − 3H2O]−, [M − H − CO − Primeverosyl]− and [M − H − HCHO − Primeverosyl]−, ions at m/z 521, m/z 517, m/z 499, m/z 249 and m/z 247. Compound 13 generated the [M − H − CO − H2O]−, [M − H − C7H6O2]−, [M − H − C8H6O4]− and [M − H − Glc − CO − H2O]−, ions at m/z 403, m/z 327, m/z 283 and m/z 241. Moreover, compound 54 generated ions at m/z 495 [M − H − CO − 4H2O]−, m/z 477 [M − H − CO − 5H2O]−, m/z 417 [M − H − CO − C5H8O5]−, m/z 415 [M − H − C9H8O4]− and m/z 279 [M − H − C5H8O4 − C8H8O2]−. Therefore, combined with bibliography data and fragmentation pathways, these three compounds were tentatively identified as gentiabavaroside, glucosyl-1-gentiacaulein, primeverosyl-1-decussatin.Compounds 10, 13 and 54 generated their [M − H]− ion at m/z 481.09833 . The [M − H + HCOOH]− ion of compound 18 produced the aglycone ion at m/z 273 in the MS2 spectrum, which originated from the neutral loss of an glucose moiety (162 Da). It also generated a series of ions at m/z 435 [M − H]−, m/z 445 [M − H + HCOOH − 2H2O]−, m/z 403 [M − H − CH2 − H2O]−, m/z 359 [M − H − C7H6O2 ]− and m/z 241 [M − H − Glc − 2H2O]−. Therefore, it was tentatively identified as glucosyl-8-swertianin.Compound 18 produced its [M − H + HCOOH]3COOH]− ion at m/z 481.09711 . It generated a serial of ions at m/z 463 [M − H + CH3COOH − H2O]−, m/z 445 [M − H + CH3COOH − 2H2O]−, m/z 435 [M − H + CH3COOH − H2O − CO]−, m/z 403 [M − H − H2O]−, m/z 361 M − H + CH3COOH − C4H8O4]−, m/z 301 [M − H − C4H8O4]−, m/z 273 [M − H − C4H8O4 − CO]− and m/z 179 [M − H − C4H8O4 − C7H4O4]−. The fragmentation pathway was consistent with deduced of compound 19, which further proved the validity of the results . It generated [M − H + CH3COOH − CH3]−, [M − H + CH3COOH − H2O]−, [M − H + CH3COOH − HCHO]−, [M − H + CH3COOH − C9H8O4]−, [M − H − C7H6O3]− and [M − H − C7H6O3 − H2O − CO2]− ions at m/z 362, m/z 359, m/z 347, m/z 197, m/z 179 and m/z 153. Compound 23 was ascertained as 2-methylcorymbiferin.Compound 23 produced the [M − H + CH4]+ ions at m/z 570.18396 , m/z 570.18060 and 570.18390 , m/z 570.18390 and 570.18054 , so they were isomers. They all produced the [M + H − CO]+ and ions at m/z 525. Besides, compounds 32 and 33 gave [M + H − CO − H2O]+ ion at m/z 507. Compounds 32 and 44 could generate [M + H]+ ion at m/z 553. In addition, compound 32 could generate [M − C7H6O2 − C5H8O4 − CH3]+, [M + H − C7H6O − C5H8O4]+ and [M + H − C5H8O5 − C8H6O3]+ at m/z 283, m/z 287 and m/z 253. Compound 33 generated [M + H − CO − 2H2O]+, [M + H − C5H8O4 − C7H6O3]+ and [M + H − C7H6O3 − C5H8O4]+ at m/z 489, m/z 283 and m/z 265. Compound 44 generated [M + H − C5H8O4 − C7H4O2]+, [M + H − C5H8O4 − C6H4O2 − CO]+, [M + H − C11H19O9]+, [M + H − C11H19O9 − CO]+ and [M + H − C5H8O4 − C7H4O2 − CO]+ at m/z 283, m/z 265, m/z 259, m/z 229 and m/z 219. Therefore, compounds 32, 33 and 44 were tentatively ascertained as gentioside, 1-hydroxy-2-methoxy-7-O-primeveroylxanthone and 7-hydroxy-2-methoxy-1-O-primeveroyl xanthone.Compounds 32, 33 and 44 gave their [M + NH+ ions at m/z 298.99619  and m/z 298.99518 . Compounds 34 and 35 produced a serial of ions at m/z 299 [M + K]+, m/z 271 [M + K − CO]+, m/z 255 [M + K − CO − H2O]+ and m/z 155 [M + K − C6H8O2 − 2H2O]+ and m/z 136 [M − C6H4O3 + H]+ [Compounds 34 and 35 produced [M + K]O3 + H]+ . Accordi4]+ ions at m/z 586.17798 and 586.17841 , m/z 444.15210 . Both of them produced [M + H]+ and [M + H − CO]+ ions at m/z 569, m/z 427, m/z 541 and m/z 399. Compound 37 showed [M + H − H2O]+, [M + H − HCHO]+, [M + H − C5H8O4 − C7H6O3]+ and [M + H − C5H8O4 − C8H6O4 − CO]+ ions at m/z 551, m/z 539, m/z 299 and m/z 243. Compound 43 generated [M + NH4 − H2O]+, [M + H − CO − H2O]+. [M + H − Glc − CO]+ and [M + H − Glc − CO − H2O]+ at m/z 426, m/z 381, m/z 237 and m/z 219. Compounds 37 and 43 were tentatively determined as gentiakochianoside and 1,3,4-trihydroxy-8-β-d-glucospranosyl-5,6,7,8-tetrahydroxanthone.Compounds 37 and 43 gave their −, m/z 475 [M − H + HCOOH − 2H2O]−, m/z 467 [M − H + HCOOH − CO2]−, m/z 465 [M − H]−, m/z 349 [M − H + HCOOH − Glc]−, m/z 305 [M − H + HCOOH − Glc − CO2]−, m/z 297 [M − H − H2O − C8H6O3]− and m/z 153 [M − H − Glc − C8H6O3]−. By comparing with the literature data, compound 40 was tentatively identified as 1-O-glucosyl corymbiferi.Compound 40 showed [M − H + HCOOH]+ ion at m/z 443.09613 . It produced the [M + Na − H2O]+, [M + Na − 2H2O]+and [M + Na − 3H2O]+ ions at m/z 425, m/z 407 and m/z 389. The [M + Na]+ ion produced the ions at m/z 281, m/z 263 and m/z 247 in the MS2 spectrum, which originated from the neutral loss of glucose, H2O and CH4. Therefore, compound 47 was tentatively deduced as 8-O-glucosyl bellidifolin.Compound 47 generated its [M + Na]− ion at m/z 455.35406 and 455.35413 (mass error = 2.784 and 2.812 ppm) (C30H47O3). The molecular ions yield [M − H − H2O]−, [M − H − CO2]−, [M − H − HCOOH]−, [M − H − C15H26O]−, [M − H − C15H22O2]−, [M − H − C15H26O − CO2]− and [M − H − C15H26O − CO2 − CH2]− at m/z 437, m/z 411, m/z 409, m/z 233, m/z 221, m/z 189 and m/z 175. According to the literature data, compound 53 was tentatively deduced as oleanolic acid.Compound 53 produced the [M − H]Gentiana radix was purchased from Anguo herbs market  and was identified as the dry root and rhizome of Gentiana scabra Bge. by professor Li Feng of Liaoning University of Traditional Chinese Medicine. Wine-processed Gentiana radix is made by reference to the Chinese pharmacopoeia (2015). Gentiopicroside, swertiamarin, sweroside, loganin, isovitexin, and mangiferin were purchased from Jiangsu Yongjian Pharmaceutical Technology CO., Ltd. . Formic acid, methanol and acetonitrile (HPLC grade) were purchased from Fisher Scientific . Ultrapure water was purchased from Hangzhou Wahaha Group Co., Ltd. . Yellow rice wine was purchased from Zhejiang Tapai Shaoxing Liquor Co., Ltd. . Preparation of wine-processed Gentiana radix: After diluting the yellow rice wine with water, mix it with raw Gentiana radix. (raw Gentiana radix: yellow rice wine: water = 10:1:1.5). After moistening for 30 min, stir it in a stir-frying container and keep it stir-frying for 4 min at 100 °C, stirring 100 times per minute. When the raw Gentiana radix is dark yellow on the surface and slightly fragrant with wine, take it out and cool it, resulting in wine-processed Gentiana radix.Gentiana radix were weighed precisely (0.50 g); the powders were placed into 20 mL of methanol and the quality was recorded, and then ultrasonically extracted at room temperature for 0.5 h. The extracts were cooled to room temperature and filtered to a 25-mL volumetric flask, then methanol supplemented to 25 mL. Methanol extracts were filtered through a 0.22-µm membrane for analysis. All of the solutions were stored at 4 °C and brought to room temperature before analysis.The standard solutions of gentiopicroside, swertiamarin, sweroside, loganin, isovitexin, and mangiferinwere prepared in methanol at appropriate concentrations. The powders of crude and wine-processed v/v) formic acid and acetonitrile (B). A gradient program was adopted as follows: 0–2 min, 2% B; 2–20 min, 2–22% B; 20–24 min, 22–80%; 24–26min, 80% B; 26–27 min, 80–2% B; 27–30 min, 2% B. The flow rate was set as 0.3 mL/min.UHPLC analysis was performed on DIONEX Ultimate 3000 UHPLC system  with a binary pump and an autosampler. Samples were separated on a BEH C18  at room temperature. The mobile phase consisted of 0.1%  was connected to the UHPLC system via an electrospray ionization (ESI) interface. The effluent was introduced into the ESI source in a post-column splitting ratio of 1:4. The analysis was performed in both negative and positive ion mode with a mass range of m/z 100–1500. The optimized ESI parameters in negative ion mode were set as follows: capillary temperature of 350 °C; sheath gas (nitrogen) flow of 30 arb.; auxiliary gas (nitrogen) flow of 10 arb.; source voltage of 3.0 kV; capillary voltage of −35 V; tube lens voltage of −110 V. The capillary voltage was 25 V, source voltage of 4.0 kV and tube lens voltage was 110 V in positive ion mode; and other parameters were same as those of negative ion mode. The resolution of the orbitrap mass analyzer was set at 30,000. The isolation width was 2 amu, and the normalized collision energy (CE) was set to 35%. Collision-induced dissociation (CID) was conducted in LTQ with an activation q of 0.25 and activation time of 30 ms. All instruments were controlled by the Xcalibur data system, and the data acquisition was carried out by analyst software Xcalibur (version 2.1) from Thermo Electron Corp .The analysis was performed in both negative and positive ion mode with a mass range of n was established for systematically characterizing constituents in crude and wine-processed Gentiana radix. By this method, the structure of constituents could be identified and the peak area of constituents could be determined. The wine-processing would change the structure of chemical constituents of Gentiana scabra, which resulted in partial loss and abscission of unstable chemical groups , and carbon–carbon double bond would be displaced or broken in the structure of some constituents.In this study, an effective and sensitive analytical method by UHPLC-LTQ-Orbitrap-MSGentiana radix, which were 2-O-β-d-glucospranosyl-1,6-dihydroxyxanthone, tetramethoxy-1,3,7,8-xanthone, glucosyl-8-swertianin, mangiferin, 2′-feruloy loganin, isoscoparine-7-O-B-d-glucosyle, 4′-O-B-d-glucospranosyl-2′′-O--2′-isoorientin, 8-O-glucosyl bellidifolin, gentianaside, scabran G4. And 8 characteristic constituents in wine-processed Gentiana radix, which are gentiabavaroside, glucosyl-1-gentiacaulein, gentioside, desmethylfoidilin, norswertianin, 1,3,4-trihydroxy-8-β-d-glucospranosyl-5,6,7,8-tetrahydroxanthone, isoscoparine, primeverosyl-1-decussatin.The results showed that there were 10 characteristic components in the crude Gentiana radix are the same, by comparing the peak area of their chemical constituents, it can be seen that the contents of some chemical constituents change in Gentiana radix after being wine-processed. Among the constituents, 2-C-β-d-glucospranosyl-glucopyranosyl-1-hydroxy-7-methoxyxanthone, methyl (1S)-1-(d-glucopyranosyloxy)-6,7-dihydroxy-7-methyl-1,6,7,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate, glucosyl-1-decussatin, gentiakochianoside, 1-O-glucosyl corymbiferin, macrophylloside A and oleanolic acid increased significantly after being wine-processed. While 2-methylcorymbiferin, 1-hydroxy-2,3,4,7-tetramethoxy xanthone, 1-hydroxy-2-methoxy-7-O-primeveroylxanthone and morroniside decreased significantly after being wine-processed.Since the sampling amounts of crude and wine-processed Gentiana radix, which showed wine-processing can change the structure and contents of some constituents in Gentiana radix. The result of this paper can be used to explain the processing principles of Gentiana radix to some extent.By comparing the structure and peak area of the constituents, we could see that there are differences between crude and wine-processed


Moreover, the proposed polymerization reaction can be finished completely within a very short time, which is useful for large-scale manufacturing. Impressively, the developed Fe1−xS/NS-MCS catalyst demonstrates outstanding ORR catalytic activity in terms of a more positive onset and half-wave potential as well, as much a better methanol tolerance and stability, in comparison with that of Pt/C benchmarked catalyst. The remarkable ORR electrocatalytic properties are strongly associated with the favorable characteristic spherical N, the S co-doped porous graphitic carbon nanoskeleton incorporated with the Fe1−xS nanoparticle-encapsulation structure.The unique micro/mesoporous spherical nanostructure composed of non-noble metal nanoparticles encapsulated within a heteroatom-doped carbon matrix provides great advantages for constructing advanced non-precious oxygen reduction (ORR) electrocatalysts. Herein, a promising oxygen electrocatalyst comprising iron sulfide (Fe Importantly, the proposed polymerization reaction can be finished completely within a very short time (7 min), which makes it useful for large-scale manufacturing. Moreover, together with the advantages of the characteristically spherical N, S co-doped a porous graphitic carbon nanoskeleton incorporated with the Fe1−xS nanoparticle-encapsulation structure; the resulting Fe1−xS/NS-CS demonstrated an outstanding ORR catalytic activity in terms of a more positive onset and half-wave potential, as well as much better methanol tolerance and stability, in comparison with that of the Pt/C benchmarked catalyst.Being mindful of the above ideas, this paper proposes a facile and fast strategy to fabricate iron sulfide (Fe2 protection with a ramp rate of 1°C min−1 for 1 h to form Fe-containing N-doped carbon spheres (Fe/N-CS). Finally, 0.06 g of the Fe/N-CS sample and 10 g of thiourea are placed in the center and front end of tube furnace, respectively, and they are then heated to 850°C for 1 h with a heating rate of 10°C min−1 and an argon flow of 100 sccm. After that, the Fe1−xS/NS-CS catalyst is obtained.Typically, 2.81 g of melamine is added into 5.6 mL of formaldehyde with constant stirring until it forms a homogeneous transparent solution at 65°C. Meanwhile, 0.04 g of AFC and 0.56 g of poly  are dissolved completely in 80 mL of deionized water to form a uniform orange liquid. Then the two solutions are stirred at 60°C to mix homogeneously. Subsequently, 1.3 mL of acetic acid is injected into the above mixed liquid with continuous stirring at 60°C for 7 min to produce the Fe containing nitrogen-rich carbon polymer spheres (Fe-NCPS). Next, 0.35 g of Fe-NCPS sample is annealed at 600°C under N3O4/N-CS control material is also synthesized by a similar way, just replacing thiourea with urea in the process of synthesis. Additionally, the N-CS control catalyst is obtained through a similar way without adding AFC and replacing thiourea with urea.For comparison, the NS-CS control sample is prepared in a similar way without the addition of AFC. Moreover, the Fe1−xS/NS-CS catalyst was fabricated via a facile three-step method, including polyreaction, pyrolysis, and a high-temperature vulcanization step, as schematically displayed in 2 protection to form Fe-containing N-doped carbon spheres (Fe/N-CS). Finally, the obtained Fe/N-CS samples were vulcanized into iron sulfide/nitrogen and a sulfur co-doped carbon sphere (Fe1−xS/NS-CS) by the pyrolysis of thiourea.The Fe1−xS/NS-CS. It can be seen from 1−xS (JCPDS: 22-1120). In 1−xS (JCPDS: 22-1120), respectively. The result suggests that the iron species in Fe-NCPS samples are transformed into Fe1−xS after the pyrolysis and high-temperature vulcanization step. The accurate microstructures of Fe1−xS/NS-CS are further investigated by HRTEM. Compared to the solid carbon spheres of Fe-NCPS, the Fe1−xS/NS-CS sample not only shows the porous structure, but also displays a large number of Fe1−xS nanoparticles (~18 nm) embedded in the sphere  plane of graphitic carbon. Additionally, the interplanar lattice spacing of 0.260 nm observed in 1−xS. Peculiarly, such a nanoparticle-encapsulation geometric confinement structure can not only effectively suppress the oxidation, agglomeration, and dissolution of Fe1−xS nanoparticles during the ORR catalysis process, but can also activate the interfacial contact between neighboring graphitic carbon layers and Fe1−xS nanoparticles, thereby enhancing the ORR electrocatalytic activity and durability  method. It is noteworthy that the Fe1−xS/NS-CS catalyst displays the type II adsorption isotherms with a typical type H4 hysteresis loop  due to the air contact  is more positive compared to N-CS (0.660 V), NS-CS (0.706 V), and Fe3O4/N-CS (0.759 V), suggesting pronounced ORR catalytic activity of Fe1−xS/NS-CS material. The corresponding linear sweep voltammetrys (LSVs) curves were recorded to further evaluate the excellent ORR catalytic performance of Fe1−xS/NS-CS (0) of the Fe1−xS/NS-CS catalyst was 0.989 V, which was more positive than that of Pt/C catalyst (0.973 V) and much more positive than that of N-CS (0.833 V), NS-CS (0.895 V), and Fe3O4/NS-CS (0.967 V) , which even exceeded the benchmarked 20% Pt/C (0.831 V) and other Fe-based related electrocatalysts previously reported , highlighting the similar ORR kinetic processes of Fe1−xS/NS-CS as commercial Pt/C. The excellent ORR electrocatalytic activity of Fe1−xS/NS-CS is further confirmed by the much higher kinetic current density JK, as shown in JK of Fe1−xS/NS-CS (13.83 mA cm−2) was much higher than that of N-CS (0.48 mA cm−2), NS-CS (0.94 mA cm−2), and Fe3O4/NS-CS (4.25 mA cm−2) at the potential of 0.8 V. To deeply elucidate the ORR pathway and kinetics, the LSVs of N-CS, NS-CS, Fe3O4/N-CS, Fe1−xS/NS-CS, and 20% Pt/C at different rotation rates  were recorded  of N-CS, NS-CS, Fe3O4/N-CS, Fe1−xS/NS-CS, and 20% Pt/C. The average values of n at the potential range from 0.55 to 0.70 V for the N-CS, NS-CS, Fe3O4/N-CS, Fe1−xS/NS-CS, and 20% Pt/C were 2.84, 3.18, 3.63, 3.99, and 3.98, respectively, revealing a dominant four-electron ORR catalytic pathway under the electrocatalyst of Fe1−xS/NS-CS.Motivated by the characteristic spherical nitrogen and sulfur co-doped graphitic carbon nanoskeleton incorporated with the Feptimized . For com3O4/N-CS , and comxS/NS-CS . Remarka0.967 V) . Moreovereported . Figure recorded . Meanwhi1−xS/NS-CS sample displayed an efficient ORR activity and pathway, which can be ascribed to several factors. Firstly, the graphitic carbon matrixes contribute to excellent electrical conductivity and stability, thus leading to good electrochemical performances , which is more positive than that of NS-CS and Fe3O4/N-CS  nanoparticles were embedded into a nitrogen and sulfur co-doped carbon sphere (Fe1−xS/NS-CS), has been successfully explored through a simple and fast polymerization between MMA and AFC as well as a subsequent high-temperature vulcanization process. Compared to the commercial Pt/C catalyst, the resulting Fe1−xS/NS-CS demonstrated a superior ORR catalytic performance and methanol tolerance together with much better stability. Therein, the moderate N and S co-doping, the graphitic carbon nanoskeletons with large surface areas and abundant porous architectures, formed Fe1−xS catalytic active substance, and the possible synergetic interaction between Fe1−xS nanoparticles and the protective N and S co-doped graphitic carbon layer further improve the outstanding electrocatalytic properties of Fe1−xS/NS-CS. In consideration of the simple and fast synthetic preparation, the strategy proposed here can potentially be employed for the synthesis of other non-noble metal-based catalysts in large-scale industrial production.In summary, an excellent ORR catalyst, in which iron sulfide (FeAll datasets generated for this study are included in the article/All authors contributed to the writing of the manuscript and have given approval to 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.