Leptospira spp. is the etiological agent of leptospirosis. The high diversity among Leptospira species provides an array to look for important mediators involved in pathogenesis. Toxin-antitoxin (TA) systems represent an important survival mechanism on stress conditions. vapBC modules have been found in nearly one thousand genomes corresponding to about 40% of known TAs. In the present study, we investigated TA profiles of some strains of Leptospira using a TA database and compared them through protein alignment of VapC toxin sequences among Leptospira spp. genomes. Our analysis identified significant differences in the number of putative vapBC modules distributed in pathogenic, saprophytic, and intermediate strains: four in L. interrogans, three in L. borgpetersenii, eight in L. biflexa, and 15 in L. licerasiae. The VapC toxins show low identity among amino acid sequences within the species. Some VapC toxins appear to be exclusively conserved in unique species, others appear to be conserved among pathogenic or saprophytic strains, and some appear to be distributed randomly. The data shown here indicate that these modules evolved in a very complex manner, which highlights the strong need to identify and characterize new TAs as well as to understand their regulation networks and the possible roles of TA systems in pathogenic bacteria.Pathogenic  Leptospira. It is a zoonotic disease affecting humans and a wide range of animals worldwide with significant impact. The genus Leptospira comprises saprophytic and pathogenic species  and were named based on their spiral shape. They are mobile and measure 6 to 20 μm in length by 0.1 μm in diameter [Leptospirosis is caused by pathogenic spirochetes of the genus diameter .Leptospira includes at least 21 species arranged in three groups: pathogenic, intermediate pathogenic, and non-pathogenic or saprophytic [L. interrogans, L. kirschneri, and L. noguchii. Group II intermediate pathogens grow better in culture and cause predominantly mild self-resolving illnesses without fatal complications. Group III saprophytic Leptospira are free-living environmental microorganisms [Based on 16S rRNA phylogeny, DNA-DNA hybridization, pathogenicity, virulence, and in vitro growth characteristics, the genus rophytic ,5, whichrophytic . Group Irganisms .L. interrogans [Mycobacterium tuberculosis [Escherichia coli [Haemophilus influenzae [Salmonella enterica [Toxin-antitoxin (TA) systems represent an important mechanism of bacteria survival during stress conditions, such as starvation or antibiotic pressure. TA operons are widely distributed among bacteria and are characterized by a pair of genes encoding for a stable toxin and an unstable antitoxin. The antitoxin acts as an antagonistic regulator that prevents the toxin from exerting its toxicity, except when some environmental conditions determine a decrease in antitoxin concentration, exposing the cell to toxic effects, leading to a reversible cessation of growth ,7. TA syerrogans , Mycobacrculosis ,11, Eschhia coli , Haemophfluenzae , and Salenterica , they paBorrelia burgdorferi, Treponema pallidum, and other obligated host associated bacteria [Experimental data of toxin-antitoxin and in silico analysis on prokaryotic chromosomes have shown the widespread presence of TA modules among bacteria with few exceptions, like the spirochetes bacteria ,16,17,18bacteria ,20, withbacteria .http://bioinfo-mml.sjtu.edu.cn/TADB/) [Leptospira interrogans [Salmonella enterica [Shigella flexneri [Haemophilus influenzae [Mycobacterium tuberculosis [M. tuberculosis cleave 23S rRNA at the sarcin-ricin loop (SRL) [Type II TA modules are grouped into different families according to the toxin structure and protein sequence similarity ,22. VapBn/TADB/) . They arn/TADB/) ,24, whicn/TADB/) . The Hicn/TADB/) . Toxins n/TADB/) , and alsn/TADB/) . Most oferrogans , Salmoneenterica , Shigellflexneri , and Haefluenzae . Other srculosis . Additioop (SRL) ,33.Leptospira species makes their studies very complex and challenging, providing a rich ground to look for specific mediators with importance for bacterial virulence and pathogenesis. In this work, we have investigated the diversity among toxin-antitoxin type II systems among Leptospira species, with a focus on the toxin of the VapBC family. We searched for and compared the putative TA operons of the whole sequenced genomes of pathogenic L. interrogans and L. borgpetersenii, intermediate-pathogenic L. licerasiae, and saprophytic L. biflexa strains within 20 Leptospira ssp., classified according to the pathogenicity phylogenetic tree [The substantial progress of genomic and proteomic studies has allowed for high-throughput studies of leptospiral proteins aimed mainly at the identification of potential antigens for vaccine and diagnostic development . Most oftic tree . This exhttp://bioinfo-mml.sjtu.edu.cn/TADB2/) [Leptospira we have used the tool TAfinder (http://202.120.12.133/TAfinder/TAfinder.php) from TADB by entering the Refseq Accession Number of the following bacteria: L. interrogans serovar Copenhageni strain Fiocruz L1-130 (Refseq NC_005823), L. interrogans serovar Lai strain 56601 (Refseq NC_004342), L. borgpetersenii serovar Hardjo-bovis strain JB197 (Refseq NC_008510) and strain L550 (Refseq NC_008508), L. biflexa serovar Patoc strain Patoc 1 (Ames) (Refseq NC_010842) and strain Patoc 1 (Paris) (Refseq NC_010602). The set of TA modules of L. licerasiae serovar Varillal strain VAR010 was obtained from the manuscript of Ricaldi et al. [The analyses of bacterial type II toxin-antitoxin loci were performed using TADB 2.0 database (/TADB2/) . To expli et al. .vapBC operons in the 20 Leptospira spp. comprised in this study, we used the program Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi), specifically the protein-protein BLAST tool (BLASTp), to compare the protein sequences of the putative VapC toxins. The VapC query sequences were obtained directly from the TADB database or through locus identification and numbered according to the order of appearance on each genome (base pair numbering) of L. interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC), L. borgpetersenii serovar Hardjo-bovis strain JBL97 (LBJ), L. licerasiae serovar Varillal strain VAR 010 (LEP1GSC185), and L. biflexa serovar Patoc strain Patoc 1 (Ames) (LBF). The subject taxonomic ID (taxid) of the Leptospira used to browse homologous proteins were: Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 (taxid:267671), L. interrogans serovar Lai str. Lai (taxid:1049911), L. borgpetersenii serovar Hardjo-bovis str. JB197 (taxid:355277), L. borgpetersenii serovar Hardjo-bovis str. L550 (taxid:355276), L. biflexa serovar Patoc strain ‘Patoc 1 (Paris)’ (taxid:456481), L. biflexa serovar Patoc strain ‘Patoc 1 (Ames)’ (taxid:355278), Leptospira (taxid:171), L. interrogans (taxid:173), L. kirschneri (taxid:29507), L. noguchii (taxid:28182), L. borgpetersenii (taxid:174), L. weilii (taxid:28184), L. santarosai (taxid:28183), L. alexanderi (taxid:100053), L. alstoni (taxid:28452), L. kmetyi (taxid:408139), L. wolffii (taxid:409998), L. licerasiae (taxid:447106), L. inadai (taxid:29506), L. fainei (taxid:48782), L. broomii (taxid:301541), L. wolbachii (taxid:29511), L. meyeri (taxid:29508), L. biflexa (taxid:172), L. vanthielii (taxid:293085), L. terpstrae (taxid:293075), and L. yanagawae (taxid:293069).To evaluate the presence of putative Conservation value”  formed from values provided by BLASTp analyses, expressed as a frequency between 0 and 1. For each query protein, the C-value was calculated as follows:i” is the amino acid identity, “p” refers to a positive or similar amino acid, and “c” is the amino acid coverage sequence, given by the ratio between the length of the highest scoring matching sequence and the query length. Arbitrarily, we considered sequences with the result of “p” and “c” > 0.5 as having a considerable degree of “C-value”.In order to easily visualize the conservation between two VapCs sequences, we established the “https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi), which considers the proteic conserved domain and sequence similarity information.Alignment between two amino acid sequences and identity (%) were performed using the tool Global Align from BLAST, and multiple sequences alignment was computed using COBALT (Constraint-based Multiple Alignment Tool)  of TAs of the genomes of parallel .Based on the fact that the toxins of TA modules are the elements responsible for the function and substrate specificity, and also for the family classification of these systems, we have focused this study on the distribution and conservation of the VapC toxins, from the VapBC family, which are known to have poor conservation of the primary sequences but high conservation of the PIN domain structural fold .L. interrogans serovar Copenhageni strain Fiocruz L1-130 has four vapBC loci, all on chromosome I, which encode four toxin-antitoxin modules of the VapBC family. Based on the numerical order of the genes in the chromosome, only VapBC-3 (LIC12660–12659) was experimentally characterized and thus considered a bona fide element [2+ or Mn2+ ions at the catalytic site. The cognate VapB antitoxins partners comprise different types of protein domains  and therefore cannot have their primary structures compared.According to the search conducted on TAfinder, the  element , while tL. interrogans serovar Copenhageni Fiocruz L1-130, the 15 VapCs L. licerasiae serovar Varillal strain VAR010, and the eight VapCs L. biflexa serovar Patoc 1 (Ames)  . The anaL. interrogans and L. borgpetersenii), one intermediate strain (L. licerasiae), and one saprophytic strain (L. biflexa) to compare with sequence database of the Leptospira taxid for each of the species described in the methods section. To enable this comparison, we developed a parameter to indicate “conservation” between sequences named C-value (Cv) (see the methods section), which takes into account three results provided by BLAST analysis: the coverage (“c”), the number of identical amino acids (“i”), and also the number of conserved amino acids designed as positive (“p”), which varies from 0 to 1. We did not use the e-value given by BLAST because it varies according to protein size and it does not consider conservative mutations, which are known to be important to structure and function, as previously indicated for VapC-3 [C-value. Results for L. interrogans serovar Copenhageni are shown in C-values, the results also include the “i”, “p”, and “c” values. It is important to consider that BLAST searches for similar sequences based on a large number of entries from several isolates and that each of the analyzed species have (for the most part) many entries too, and that some of the entries of specific strains do not have the serovar group defined. With that said, it should be noted that when one cell of the L. interrogans or L. noguchii), it is not necessarily present in all members of this species, but in one or some isolates of them.In order to study how the amino acid sequences of VapCs are conserved among leptospiral pathogenicity groups  and to infer functional and evolutionary relationships between sequences, we have carried out an extensive study using the BLAST tool, in which we submitted the identified VapCs sequences of two pathogenic strains  share the same amino-acid sequence, probably stemming from genetic duplication events.Among the 28 TAs identified in the intermediate pathogenic cerasiae , 15 are L. biflexa strain was analyzed . It is interesting to note that among the eight VapCs of L. biflexa, three of them  resemble VapC-3 of L. interrogans Copenhageni, which might suggest some evolutionary link and explain their strong presence among the pathogenic species.When the nonpathogenic analyzed , we obseTaken all together, our results describing the distribution of these TA modules among leptospiral strains of varied phenotypes of pathogenicity showed that VapCs are unequally distributed, with some toxins apparently being randomly disseminated, to others present in unique species or specific pathogenicity groups.Toxin-antitoxin systems have been involved in potentially harmful aspects of an infection, such as antimicrobial resistance, persistence, and biofilm formation, and therefore have been the subject of intensive efforts to elucidate biochemical and functional effects besides their role on the physiology of infection. Nevertheless, due to the high number of types, families, and high divergence among them, all these efforts on the elucidation of biochemical and biological functions are still incipient and remain controversial.L. interrogans serovar Copenhageni strain Fiocruz L1-130 and serovar Lai strain Lai: one VapBC [chpIK and mazEF, found in L. interrogans serovar Lai strain Lai, have been described as extensively distributed and the sequences conserved in different pathogenic Leptospira [mazEF operon (LA1780/1781) was not identified in the L. interrogans serovar Copenhageni strain Fiocruz L1-130 using BLAST analysis, but it is present only in the L. interrogans serovar Copenhageni strain HAIO156 from the 19 isolates available of this species-serovar. Considering the strict curation of Fiocruz L1-130 and Lai genomes, this fact indicates that the function of MazEF in pathogenesis is not essential or, more likely, redundant, and could be replaced by other TAs in these strains.Currently, three type II TAs were described and experimentally characterized in the genome of ne VapBC ,46, one ne VapBC ,47, and ne VapBC  since bone VapBC . Even thptospira , the mazLeptospira has shown that there is considerable genomic plasticity even within the same species, as in the case of the large inversion in chromosome I and an ~54 kb genomic island that differentiates the genomes of L. interrogans serovars Lai and Copenhageni, which share ~99% similarity at amino acid level of ortholog genes [L. borgpetersenii analyzed (JB197 and L550) displayed total inversion of the seven TA sequences along the chromosome, which share exactly the same amino acid sequences (data not shown). Differently, the analysis of the 23 TA modules of the genomes of the two strains of L. biflexa (Ames and Paris) did not show any variation between them, even in their position on the chromosome, which is in agreement with data in the literature describing the high conservation in the genomes within this species [The analysis of the whole genomes of og genes . Our dat species .L. interrogans, L. licerasiae, and L. biflexa strains evaluated in our study might indicate that they act on different substrates producing distinct effects, however, no link between toxin homologs and a specific physiological function has been established [vapBC2ST of Salmonella typhimurium [vapBC-1 of Haemophilus influenzae [vapBC of the thermophilic Sulfolobus solfataricus [L. interrogans Copenhageni, VapBC-3 has been biochemically characterized to act on the lysis of the initiator tRNAfMet and therefore to inhibit translation [The low intra-species amino acid sequence identity among VapCs of the ablished ,51. Addiablished , which whimurium , vapBC-1fluenzae , and vapataricus . In L. inslation , but infLeptospira, the overall set of TA modules characteristic among species living in saprophytic or pathogenic conditions reveals that the L. biflexa has a considerable larger number of TAs then L. interrogans and L. borgpetersenii, which can indicate a relatively large subset of adaptive facilities. However, the intermediate L. licerasiae, whose genomic analysis led to the conclusion of its greater proximity to pathogenic strains than to saprophytic [vapBC specifically. Although the data available to date are very incipient, we could hypothesize that this larger number of modules found in the intermediate pathogenic L. licerasiae, which could also be referred as “intermediate saprophytic” [S. pneumonia [M. tuberculosis [TAs are represented by a highly variable number of modules in several bacteria, as highly variable as the extreme environmental or host differences to which these organisms must adapt to live, suggesting it might contribute to their diversification and evolution. Unsurprisingly, the resulting phenotype of each species is affected by a diversity of features including the whole genomic background, among which the diversity of TA modules can be included and may interfere in the response of individual TA systems during adaptation stresses . In the rophytic , has an ophytic” , would bneumonia  and VapBrculosis ), did noIt is not clear how acquisition by horizontal gene transfer of the set of TA loci influenced the evolution of each bacterial species in its broad set of genes, and whether TAs may play roles in determining phenotypic aspects such as virulence and persistence ,51. It hvapBC operons among Leptospira species, through the analysis of the conservation of their toxic elements, we could outline a rather complex picture indicating that these modules evolved differently. It is remarkable that, out of all VapC sequences of pathogenic species submitted to BLAST analysis, VapC-2 of L. interrogans serovar Copenhageni and VapC-2 of L. borgpetersenii serovar Hardjo-bovis are, at the same time, widely and strictly highly conserved only among pathogenic species. Interestingly, proteomic analysis showed an increase of the antitoxin component from VapBC-2 of L. interrogans serovar Copenhageni during stress induced by antibiotic treatment (Ciprofloxacin) [By providing an overview of the distribution of loxacin) . Thus, tEscherichia coli showed that they are unevenly distributed among E. coli phylogroups and are not a universal feature of the species [Leptospira has failed. Except in the specific cases mentioned above, our data were inconclusive and unable to infer this kind of correlation, most likely due to the high diversity of their amino acid sequences. Moreover, it is very important to consider that protein primary sequences are the groundings for the three-dimensional structure folding, which ultimately is responsible for toxin and antitoxin activities.Even though in recent years we have accumulated an increase in the knowledge about TA systems, information on how they have evolved is still missing. One reason that may help to explain this lack of knowledge is the fact that most TA loci are acquired by horizontal gene transfer ,60 and t species . In paraThe large variability in numbers and the low sequence conservation among VapCs shown here emphasizes the strong need to identify and characterize new TAs as well to understand the regulation nets and roles of TA systems in pathogenic bacteria.


Cuscuta (Convolvulaceae) comprises well-known parasitic plants. Cuscuta species are scientifically valuable, as their life style causes extensive crop damage. Furthermore, dried seeds of C. chinensis are used as a Korean traditional herbal medicine. Despite the importance of Cuscuta species, it is difficult to distinguish these plants by the naked eye. Moreover, plastid sequence information available for Cuscuta species is limited. In this study, we distinguished between C. chinensis and C. japonica using morphological characterisation of reproductive organs and molecular characterisation of chloroplast genomes. The differences in morphological characteristics of reproductive organs such as style, stigma, infrastaminal scale, seed shape and testa ornamentation were useful for distinguishing between C. japonica and C. chinensis. Analysis of chloroplast genomes revealed drastic differences in chloroplast genome length and gene order between the two species. Although both species showed numerous gene losses and genomic rearrangements, chloroplast genomes showed highly similar structure within subgenera. Phylogenetic analysis of Cuscuta chloroplast genomes revealed paraphyletic groups within subgenera Monogynella and Grammica, which is consistent with the APG IV system of classification. Our results provide useful information for the taxonomic, phylogenetic and evolutionary analysis of Cuscuta and accurate identification of herbal medicine.The genus  Cuscuta L. (dodder) is a genus comprising 170–200 species of parasitic vines belonging to the family Convolvulaceae [Cuscuta are clearly distinguishable from those in other Convolvulaceae genera based on the absence of leaves, presence of haustoria and acotyledonous embryo, arrangement of flowers in clusters or short racemes and presence of five-fimbriate scales within the corolla [Cuscuta is nearly cosmopolitan in distribution; however, the majority of the species (~75%) are native to North and South America, and other taxa are found only in Asia and Europe [Cuscuta species because of the drastically reduced or almost absent vegetative organs (leaves and stems) and very small floral organs. Therefore, most studies have focused on the microscopic evaluation of seed [Cuscuta species. These studies focused on only the selected character states and their evolution. However, to date, comprehensive species-based analysis of morphological and molecular characteristics has not been performed.d Europe ,4,5, mord Europe . However of seed , stigma  of seed  and infr of seed  and evolCuscuta, complete chloroplast genomes have been reported for five species, including C. reflexa, C. gronovii, C. exaltata, C. obtusiflora and C. pentagona [C. exaltata, C. obtusiflora and Ipomoea purpurea shows that all ndh genes have been lost in C. exaltata. Despite highly variable nucleotide substitutions, C. obtusiflora has retained the genes involved in photosynthesis and photorespiration, similar to other parasitic plants [C. reflexa and C. gronovii shows the presence of two inversions in the LSC region and one inversion in the SSC region. Furthermore, C. reflexa and C. gronovii exhibit different degrees of parasitism, with reduction in RNA editing [Cuscuta, further investigation is needed for high-resolution phylogenetic analysis as well as Cuscuta species identification because of differences in the life style of different parasitic species, numerous existing species and rapid gene evolution.Chloroplasts play important roles in photosynthesis, carbon fixation and starch and fatty acid biosynthesis ,12. Receentagona ,20,21. Cc plants ,20. Chlo editing . AlthougC. chinensis are used as an important herbal medicine, namely, Cuscutae Semen, which is designated as a medicine in the Korean Herbal Pharmacopoeia [C. chinensis are considered as authentic Cuscutae Semen in Korea [Cuscuta species such as C. japonica, C. pentagona and C. australis are also distributed in Korea [Cuscuta species in Korean herbal markets. Thus, accurate identification of Cuscuta species is important for maintaining a uniform pharmacological effect of the herbal medicine.In Korean traditional medicine, dried seeds of acopoeia , and is acopoeia ,24,25. Oin Korea . Howeverin Korea ,27. SincC. japonica and C. chinensis and sequenced the chloroplast genomes of both species. Results showed differences in the morphological traits of floral organs and seeds as well as in the complete chloroplast genomes of C. japonica and C. chinensis. Comparison of chloroplast genome sequences of C. japonica, C. chinensis and related species revealed genomic rearrangements. The genus Cuscuta showed highly variable genome structure and gene content among various species. However, subgenera Grammica and Monogynella exhibited conserved chloroplast genome structure. Our results provide valuable data for the accurate identification of Cuscuta species on the basis of morphological and genomic features and in-depth insight into the evolution of species within the Convolvulaceae. Furthermore, this study will help preserve the quality of Cuscutae Semen as a valuable herbal medicine.In this study, we conducted a microscopic analysis of the reproductive organs of Cuscuta species have drastically reduced vegetative organs (stems and leaves) and diverse reproductive organs (flowers and fruits). Thus, morphological characteristics of reproductive organs have been used for the taxonomic and systematic analysis of Cuscuta species. Here, we conducted a microscopic evaluation of the morphology of reproductive organs of C. japonica and C. chinensis.Like other parasitic plants , CuscutaC. japonica . Micromorphological analysis also revealed significant differences between C. japonica and C. chinensis in the fimbriae of infrastaminal scale (IFS) in flowers. The IFS of C. japonica flowers comprised mainly glandular cells  and 18 sections, based on the phylogenetic framework and floral morphology including IFS types. In the present study, we confirmed that IFS type, style number and stigma morphology were useful for the identification of C. japonica (representative of subgenus Monogynella) and C. chinensis (representative of subgenus Grammica). However, there were no major differences in the epidermal cell type of gynoecium  between the two species. This high homoplasy of gynoecium cell patterns may have been affected by convergent evolution, like other floral characteristics [C. japonica (C. chinensis (C. japonica seeds showed rugulated (puzzle-like) testa ornamentation  regions (IRa and IRb) are 16,554 bp and 14,617 bp in C. japonica and C. chinensis, respectively. The complete chloroplast genomes were validated using PCR-based sequencing using sequence-specific primers  and tandem repeats in chloroplast genomes of genetics ,44. A toectively . The numectively . Tandem ectively . NumerouCuscuta, we compared chloroplast genomes of seven Cuscuta species , while the 1.1 kb inversion was present in the SSC region and contained two genes (ccsA and trnL-UAG); these inversions separated Cuscuta species at the subgenus level. Compared with five Cuscuta species, chloroplast genomes of C. japonica and C. reflexa harboured a 2.3 kb inversion  within the LSC region. The chloroplast genome of C. obtusiflora harboured a species-specific inversion in the SSC region comprising the trnL-ycf1 gene. Another inversion, 7.1 kb in length, comprised trnL-UAG, ccsA, psaC, rps15 and ycf1 genes in C. obtusiflora. Chloroplast genomes in the genus Cuscuta exhibited structural vitiations, contained well-conserved syntenic regions which was divided in two subgenera , the IR/SSC border extended into ycf2, resulting in a pseudogene in all three chloroplast genomes, with pseudogenisation of ycf15 in IRa. By contrast, C. chinensis, C. gronovii, C. pentagona and C. obtusiflora (subgenus Grammica) harboured rpl2 in the LSC region. The ycf1 gene was located in the SSC/IRb border region in all Cuscuta species, except C. exaltata. In C. obtusiflora, inversion was detected in the SSC region. The IR contraction was considerably distinct between Monogynella and Grammica. Nonetheless, similar patterns were observed between the two subgenera. The IR regions are more highly conserved than single copy regions (LSC and SSC) because of copy correction by gene conversion in IR regions in other angiosperms chloroplast genomes [Cuscuta plastid genomes underwent more contraction than those in chloroplast genomes of other angiosperms, indicating an independent evolutionary process in response to the environment [C. japonica, C. chinensis and Ipomoea nil. Results showed no positive selection  method. Most nodes showed bootstrap (BS) values of 100% and Bayesian Inference (BI) posterior probabilities (PP) of 1.0, except the node of Ipomoea species . Representative fresh vines of C. japonica (36°58′34.5″ N and 128°56′47.5″ E) and C. chinensis (33°24′29.9″ N and 126°15′09.8″ E) were used for chloroplast genome sequencing. These samples were assigned identification numbers, and voucher specimens were deposited in the Korean Herbarium of Standard Herbal Resources (Index Herbarium code KIOM) at the Korea Institute of Oriental Medicine (KIOM). Plant samples used for morphological, micromorphological and chloroplast genome sequence analysis are listed in Plant material used in this study was collected from natural populations over 3 years (2014–2016). To confirm the consistency of morphological and micromorphological characteristics, at least two sites were compared for each species  and distilled water (1:200) at 37 °C The rehydrated materials were dehydrated in a graded ethanol series, and dried in an automated critical point drier . The dried materials were fixed on aluminum stubs with double-adhesive carbon tape. The stubs were then coated with gold (Au) using an ion-sputtering device . The prepared floral organs and seeds were observed using a low voltage field emission scanning electron microscope  at an accelerating voltage of 15 kV with a working distance of 9–15 mm [For reproductive morphological observations, flowers and seeds were examined using a stereomicroscope  in order to check the fully mature organs. For scanning electron microscopic observations, the dried materials from voucher specimens were rehydrated overnight using the wetting agent Agepon 9–15 mm .C. gronovii (NC009765) and C. reflexa (NC009766). Gaps in sequences were filled using SOAP de novo gap closer, based on aligned paired-end reads [DNA was extracted using DNeasy Plant Maxi kit , according to the manufacturer’s instructions. Illumina short-insert paired-end sequencing libraries were constructed; sequencing was performed using the MiSeq platform . Chloroplast genome sequences were assembled de novo from low-coverage whole genome sequences. Trimmed paired-end reads (Phred score ≥20) were assembled using CLC genome assembler  with default parameters. Principal contigs representing chloroplast genome sequences were retrieved using Nucmer , and alind reads . To valind reads .C. japonica and C. chinensis was performed using DOGMA annotation [Cuscuta chloroplast genomes were obtained using OGDRAW [Cuscuta species investigated in this study  were identified using MAUVE V2.3.1 program [Annotation of genes in the chloroplast genomes of notation . Codons notation . Circulag OGDRAW . GC contg OGDRAW . Local c program . Substit program .C. japonica and C. chinensis were detected using MISA [SSRs in chloroplast genomes of ing MISA , with miing MISA ,65.Capsicum annuum var. glabriusculum and Nicotiana tabacum were used as outgroups. Molecular phylogenetic trees were constructed using aligned sequences of 50 protein-coding genes with MAFFT [A total of 19 chloroplast genomes were used in phylogenetic analysis. Of these, 17 chloroplast genome sequences were downloaded from NCBI . Capsicuth MAFFT , and seqth MAFFT . The besth MAFFT . The GTRth MAFFT  with 100th MAFFT  using thC. japonica and C. chinensis were analysed in this study. The morphology of reproductive organs such as style, stigma, IFS, seed and testa showed considerable differences between C. japonica and C. chinensis. Chloroplast genomes were highly variable with respect to gene content, gene orientation and GC content, and local variations in genome sequence and structure were observed at the subgenus level. Tandem repeats and SSRs were identified with the aim of developing molecular markers for species identification and authentication of herbal medicines. Inversions were detected in LSC and SSC regions at Cuscuta subgenera. Overall, our results will help in the authentication of herbal medicines containing C. japonica and C. chinensis. Analysis of key morphological/micromorphological characteristics and chloroplast genome sequences of C. japonica and C. chinensis provides valuable information for species identification, taxonomic classification and evolutionary analysis of Cuscuta.Morphological characteristics and chloroplast genome sequences of


Astelia pumila (G.Forst.) Gaudich.  is a major element of West Patagonian cushion peat bog vegetation. With the aim to identify appropriate chloroplast markers for the use in a phylogeographic study, the complete chloroplast genomes of five A. pumila accessions from almost the entire geographical range of the species were assembled and screened for variable positions. The chloroplast genome sequence was obtained via a mapping approach, using Eustrephus latifolius (Asparagaceae) as a reference. The chloroplast genome of A. pumila varies in length from 158,215 bp to 158,221 bp, containing a large single copy region of 85,981–85,983 bp, a small single copy region of 18,182–18,186 bp and two inverted repeats of 27,026 bp. Genome annotation predicted a total of 113 genes, including 30 tRNA and four rRNA genes. Sequence comparisons revealed a very low degree of intraspecific genetic variability, as only 37 variable sites —most of them autapomorphies—were found among the five assembled chloroplast genomes. A Maximum Likelihood analysis, based on whole chloroplast genome sequences of several Asparagales accessions representing six of the currently recognized 14 families (sensu APG IV), confirmed the phylogenetic position of A. pumila. The chloroplast genome of A. pumila is the first to be reported for a member of the astelioid clade (14 genera with c. 215 species), a basally branching group within Asparagales. Astelia pumila (G.Forst.) Gaudich. is a dioecious, cushion-forming perennial herb. It is one of the main constituents of so-called Magellanic moorland  and nuclear (NIA-i3) regions. Astelia, but the placement of sect. Micrastelia, containing only A. pumila, remained unresolved (incertae sedis).misphere . They grmisphere . The infAstelia pumila is being used as a study system for the reconstruction of the ice-age history of Magellanic moorland with phylogeographic methods . Research on chloroplast genome evolution in Asparagales has been primarily focused on orchids , except for its northernmost occurrence at Cerro Mirador (40°S) of south-central Chile’s Los Ríos Region.As a non-model organism, for which genomic resources did not exist previously, next-generation sequencing was used to obtain DNA sequence data of five A. pumila specimens, respective library types and collection localities is given in Different library types were prepared: (1) a chloroplast-enriched library obtained via sorting on a BD FACSAria IIu cell sorter  as a reference, using Geneious 8.0.5 . The resulting mapping was curated manually. Chloroplast contigs from de novo assemblies, performed in VelvetOptimizer 2.2.5. . The junctions between the large single copy (LSC) and the small single copy (SSC) regions and the two inverted repeats (IRs) were additionally validated through Sanger sequencing .Removal of duplicate reads, adapter clipping and quality trimming was done in CLC Genomics Workbench (versions 6.5.1–7.5.1), setting the quality threshold to a qlimit of 0.001. To obtain a first chloroplast genome draft, the pooled quality-trimmed reads of all r 2.2.5. , were ushttp://dogma.ccbb.utexas.edu/html/cp_taxa), and cross-checked using GeSeq  was used as reference when employing GeSeq and Geneious for genome annotations. Where necessary, gene boundaries were corrected manually to match start and stop codons. The annotated chloroplast genome sequences were submitted to GenBank (accession numbers MH752980 –MH752984). Chloroplast genome maps were drawn using OGDRAW. Both OGDRAW and GeSeq are available at the MPI-MP CHLOROBOX website (https://chlorobox.mpimp-golm.mpg.de/index.html).Chloroplast genome annotation was done using DOGMA . A phylogenetic tree was constructed using a Maximum Likelihood approach as implemented in RAxML 8.2.0  served as outgroup.The chloroplast genome sequences of the five pecimens  were alipecimens  and screpecimens  based oner sensu, were doer sensu, togetheML 8.2.0 . In a siA. pumila chloroplast genome sequences vary from 158,215 bp to 158,221 bp due to indel variation , and one a 3-bp mutation . There, it was either missing, pseudogenized, or present at different positions, either within the LSC or the IR. In A. pumila, rps19 is found within the IR, close to the LSC-IR boundaries. Located between the rps19 and the psbA genes, there is a partial rpl22 gene, truncated at the LSC-IRA junction. This kind of gene order was classified as Type IIIg by Asparagus densiflorus and Crinum asiaticum, the LSC-IRA junction lies downstream of the rps19 gene and the IRA does not include a partial rpl22 gene. The structural dynamics of the LSC-IR junctions carry a phylogenetic signal, since there is an IR expansion trend in monocots: basally branching groups have generally shorter IRs than derived ones . The remaining variable sites were all autapomorphies, without any phylogenetically informative content. This contrasts with previous studies on intraspecific chloroplast sequence variability in e sites;  and Theoviduals; , in whicA. pumila, it may be speculated that West and Fuegian Patagonia, and the Falkland Islands, have been colonized only recently, probably after the last glacial. Clearly, the sampling in the present study is not adequate to allow for firm conclusions on the Pleistocene history of A. pumila, but such a scenario would fit to the classical biogeographic hypothesis brought forward by Villagrán .Given the non-existence of genetic structuring in illagrán ; 2001, billagrán . In ordeA. pumila is the first to be reported for a member of the astelioid clade of basal Asparagales. This is a major improvement in terms of published chloroplast genomes from that order, as especially orchids and subfamily Agavoideae (Asparagaceae) are very well sampled (A. pumila individuals and RNA-Seq data of one of them—represents a valuable genomic resource, e.g., for the identification of nuclear single copy genes. Such markers may prove useful to ascertain the still unresolved infrageneric placement of sect. Micrastelia, which contains only A. pumila as its single member.The phylogenetic reconstruction, based on whole chloroplast genome sequences, recovered Orchidaceae as basally branching within Asparagales. Asteliaceae was then retrieved as sister to the remaining clades of the order . This to sampled . FurtherA. pumila accessions, sampled from almost the entire distribution range of the species, revealed extremely low levels of sequence variability. The genomic resources generated in the course of the present study may prove useful for future work on Astelia, e.g., for the development of single-copy nuclear markers. These could be employed to ascertain the yet unresolved phylogenetic placement of A. pumila within the genus.The comparison of whole chloroplast genome sequences of five 10.7717/peerj.6244/supp-1Supplemental Information 1Click here for additional data file.


Viola species  and the results of a comparative analysis between these species and the published plastid genome of the congeneric species V. seoulensis. The total genome length of the five Viola species, including the four species analyzed in this study and the species analyzed in the previous study, ranged from 156,507 (V. seoulensis) to 158,162 bp (V. mirabilis). The overall GC contents of the genomes were almost identical (36.2–36.3%). The five Viola plastomes each contained 111 unique genes comprising 77 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. Among the annotated genes, 16 contained one or two introns. Based on the results of a chloroplast genome structure comparison using MAUVE, all five Viola plastomes were almost identical. Additionally, the large single copy (LSC), inverted repeat (IR), and small single copy (SSC) junction regions were conserved among the Viola species. A total of 259 exon, intron, and intergenic spacer (IGS) fragments were compared to verify the divergence hotspot regions. The nucleotide diversity (Pi) values ranged from 0 to 0.7544. The IR region was relatively more conserved than the LSC and SSC regions. The Pi values in ten noncoding regions were relatively high (>0.03). Among these regions, all but rps19-trnH, petG-trnW, rpl16-rps3, and rpl2-rpl23 represent useful molecular markers for phylogenetic studies and will be helpful to resolve the phylogenetic relationships of Viola. The phylogenetic tree, which used 76 protein-coding genes from 21 Malpighiales species and one outgroup species (Averrhoa carambola), revealed that Malpighiales is divided into five clades at the family level: Erythroxylaceae, Chrysobalanaceae, Euphorbiaceae, Salicaceae, and Violaceae. Additionally, Violaceae was monophyletic, with a bootstrap value of 100% and was divided into two subclades.We report the complete chloroplast genomes of four  With the development of next-generation sequencing (NGS) technology, many studies have performed whole chloroplast genome sequencing. These studies have provided much information about plant taxonomy and evolution. The rapidly evolving loci identified by these studies are very important for resolving unclear phylogenetic relationships because they have a higher resolving power than that of traditional molecular markers. Therefore, many studies have focused on finding genic regions among specific families or genera to provide useful information about molecular markers for further studies –6.Viola L. comprises 583–620 species and is distributed mainly in temperate and tropical regions [Violaceae Batch. consists of approximately 22 genera and 1000–1100 species of herbs, shrubs, lianas, and trees –9. The g regions –8,10–11. regions –15.Viola are still unclear among sections and/or species [Viola. Therefore, to correctly evaluate the phylogenetic relationships of Viola, the most suitable molecular markers should be selected via analyses of the sequence variation at each locus.For this reason, although many studies have been carried out, the phylogenetic relationships of  species , 16–19. Viola species discussed in this study, three species  are very rare because they are endangered in Korea. In particular, it is urgent to establish a conservation strategy for V. raddeana because this species only has a single, relatively small population of individuals in Gyeongsangnam-do Province [V. phalacrocarpa Maxim. is not an endangered species, but various taxonomic data are needed because its taxonomic level is ambiguous due to its close relationship to V. seoulensis.Among the four Province . V. phalViola species  and the results of a comparative analysis between these species and the published genome of a congeneric species (V. seoulensis Nakai). The main goal of this study was to provide important information about the most suitable chloroplast molecular markers for further studies to solve unclear phylogenetic relationships of Viola via the calculation of the rate of evolution of each chloroplast genome loci. Furthermore, this study could expand the current understanding of the chloroplast genome characteristics of the genus Viola and provide basic chloroplast phylogenomic data for Violaceae, thus supporting the development of conservation strategies for endangered Violaceae species.Here, we report the whole chloroplast genome sequences of four Viola species in this study, three  are legally protected species. Therefore, samples of these species were collected with permission from the Ministry of Environment in Korea, with the following license numbers: 2015–15 (V. raddeana) and 2015–39 (V. mirabilis and V. websteri).Among the four V. mirabilis, V. raddeana, V. phalacrocarpa, and V. websteri were collected from Hutan-ri in Gangwon-do Province (37°11'09"N 128°22'16"E), Youngdang-ri in Gyeongsangnam-do Province (35°22'15"N 128°54'34"E), Mt. Oeum in Gangwon-do Province (37°35'53"N 127°57'15"E), and Bugok-ri in Gangwon-do province (37°19'36"N 128°03'15"E) in South Korea, respectively. The voucher specimens were deposited in the National Institute of Biological Resources Herbarium (KB) and the Kangwon National University Herbarium (KWNU). The voucher numbers are GEIBVP0000373630 (V. mirabilis), NIBRVP0000454691 (V. raddeana), KWNU91089 , and GEIBVP0000373612 (V. websteri). Total DNA was extracted from approximately 100 mg of fresh leaves using a DNA plant mini kit .Fresh leaf materials of individual Viola species were sequenced to produce 8,920,660–9,244,544 raw reads with a length of 301 bp. These reads were aligned with the reference genome of Viola seoulensis (GenBank accession number: KP749924). A total 542,183 to 667,526 reads were mapped to the reference genome. The genome coverage was estimated using CLC Genomics Workbench v7.0.4 software . The genome coverages of the sequencing data from V. mirabilis, V. phalacrocarpa, V. raddeana, and V. websteri were 1002, 875, 986, and 1073, respectively.Genomic DNA was used for sequencing by an Illumina MiSeq  platform. The DNA of The protein-coding genes, transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs) in the plastid genome were predicted and annotated using Dual Organellar GenoMe Annotator (DOGMA) with the default parameters  and manuViola species, including the four Viola species of this study and the previously published species (V. seoulensis), were compared using MAUVE [The complete chloroplast genomes of five ng MAUVE . The larViola species were detected using Phobos v.3.3.12 (http://www.ruhr-uni-bochum.de/ecoevo/cm/cm_phobos.htm). Repeats were ≥10 bp in length and had three repeat units for mono-, di- tri-, tetra-, penta- and hexanucleotides.The REPuter program  was usedViola were analyzed to identify rapidly evolving molecular markers that can be used in further phylogenetic studies of Viola. Both coding and noncoding region fragments in each plastid genome were extracted separately by applying the “Extract” option of Geneious v7.1.8 . Then, the homologous loci were aligned individually using MAFFT [The five chloroplast genomes of ng MAFFT . To analng MAFFT .Averrhoa carambola L.) was chosen as the outgroup , V. phalacrocarpa (accession no. MH229817), V. raddeana (accession no. MH229818), and V. websteri (accession no. MH229819) have been submitted to GenBank of National Center for Biotechnology Infromation (NCBI). The total length of the chloroplast genomes of the five Viola species, i.e., the four species analyzed in this study and species analyzed in a previous study (V. seoulensis), ranged from 156,507 (V. seoulensis) to 158,111 bp (V. websteri). All five Viola plastid genomes exhibited the typical quadripartite structure, consisting of a pair of IR regions  separated by an LSC region  and an SSC region . Their overall GC contents were almost identical (36.2–36.3%). The chloroplast genomes of the five species contained 111 unique genes comprising 77 protein-coding genes, 30 tRNA genes, and 4 rRNA genes  contained one intron each, and two genes  contained two introns each.The chloroplast genomes of NA genes . Among tViola plastomes were the same  were investigated. The number of tandem repeats for each class is shown in an 50 bp .V. mirabilis, 56 in V. phalacrocarpa, 50 in V. raddeana, 43 in V. seoulensis, and 73 in V. websteri. The most dominant of SSRs were A/T mononucleotides. Only the V. websteri chloroplast genome had all six types of SSRs, and those of the other species had five types of SSRs, excluding the hexanucleotide SSR  were located in the LSC region, one (rpl2-rpl23) was located in the IR region, and one (ndhF-trnL) was located in the SSC region. Additionally, all regions with a high Pi value were IGSs.A total of 259 exon, intron and intergenic spacer (IGS) fragments were compared among the five o 0.7544 . The IR Viola were monophyletic with a bootstrap value of 100% and a sister to Salicaceae. Additionally, Violaceae was divided into two subclades: sect. Viola W. Becker (subsect. Rostratae W. Becker) and sect.Plagiostigma Solid (subsect. Patellares (Boiss.) Rouy & Foucaud and Bilobatae (W. Becker) W. Becker)  .Viola chloroplast genomes was identical, and the sequence identity was also very similar among species in most of the chloroplast regions  to 47 (V. raddeana), and the number of tandem repeats according to type and length showed a slightly difference across each species  showed a very low Pi of 0.02510 or less  were relatively high (>0.03). For the selection of useful phylogenetic markers, however, the gene length and PIC also must be considered. Among the ten regions, four regions  are too short to be used as phylogenetic molecular markers. Therefore, that the other six regions will presumably be very useful for resolving the many unclear phylogenetic relationships of the genus Viola.The results of this study showed that the Pi values of ten noncoding regions  system . HoweverV. seoulensis was not identified, as it formed an unresolved polytomy with V. phalacrocarpa [V. seoulensis because not enough species were included in the analysis, but V. seoulensis was the most closely related to V. phalacrocarpa. An analysis of the chloroplast genomes of the two species in this study revealed that the total genome size of V. phalacrocarpa was 1335 bp longer than that of V. seoulensis, and the LSC, IR, and SSC junctions also largely differed between the two species. Additionally, the Pi between the two species was 2.22%. Therefore, it would be reasonable to recognize the two taxa as independent species rather than classifying them as variants, and we will carry out additional studies including allied species of V. phalacrocarpa and V. seoulensis to clarify their taxonomic positions.In a previous study, the phylogenetic position of crocarpa . Based oViola species , and analyzed these data compared to published congeneric species in genus Viola. Results of this study, six non-coding regions  will presumably be very useful for resolving the many unclear phylogenetic relationships of the genus Viola. Phylogenetic analyses showed that Malpighiales is divided into five clades at the family level. Also, Violaceae and Viola were monophyletic, and was divided into two subclades.We first report of the complete chloroplast genome sequences of four S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Babesia. Most apicomplexan parasites, including Babesia, have a plastid-derived organelle termed an apicoplast, which is involved in critical metabolic pathways such as fatty acid, iron-sulphur, haem and isoprenoid biosynthesis. Apicoplast genomic data can provide significant information for understanding and exploring the biological features, taxonomic and evolutionary relationships of apicomplexan parasites, and identify targets for anti-parasitic drugs. However, there are limited data on the apicoplast genomes of Babesia species infective to small ruminants.Babesiosis is an economically important disease caused by tick-borne apicomplexan protists of the genus Babesia motasi Lintan and Babesia sp. Xinjiang using Illumina technology. The overlapped apicoplast genomic fragments of six ovine Babesia isolates were amplified and sequenced using the Sanger dideoxy chain-termination method. The full-length sequences of the apicoplast genomes were assembled and annotated using bioinformatics software. The gene contents and order of apicoplast genomes obtained in this study were defined and compared with those of other apicomplexan parasites. Phylogenetic trees were constructed on the concatenated amino acid sequences of 13 gene products using MEGA v.6.06.PCR primers were designed based on the previously reported apicoplast genome sequences of Babesia apicoplast genomes consisted of circular DNA. The genome sizes were 29,916–30,846 bp with 78.7–81.0% A + T content, 29–31 open reading frames (ORF) and 23–24 transport RNAs. The ORFs encoded four DNA-directed RNA polymerase subunits , 13 ribosomal proteins, one elongation factor TU (tufA), two ATP-dependent Clp proteases (ClpC) and 7–11 hypothetical proteins. Babesia sp. has three more genes than Babesia motasi . Phylogenetic analysis showed that Babesia sp. is located in a separate clade. Babesia motasi Lintan/Tianzhu and B. motasi Ningxian/Hebei were divided into two subclades.The results showed that the six ovine Babesia isolates infective to small ruminants in China using Sanger sequencing. The data provide useful information confirming the taxonomic relationships of these parasites and identifying targets for anti-apicomplexan parasite drugs.To our knowledge, this study is the first to elucidate the whole apicoplast genomic structural features of six The online version of this article (10.1186/s13071-019-3581-x) contains supplementary material, which is available to authorized users. Plasmodium spp.), toxoplasmosis (Toxoplasma gondii), cryptosporidiosis (Cryptosporidium spp.), cyclosporiasis (Cyclospora cayetanensis), coccidiosis (Eimeria spp.), babesiosis (Babesia spp.), theileriosis (Theileria spp.) and neosporosis (Neospora caninum) , and one chloroplast genomic sequence of Chromera velie (GenBank: HM222967), were used in the phylogenetic analysis  of each genomic sequence were used to deduce their amino acid sequences. The concatenated sequences of 4683 amino acid residues from each species were put into multi-alignment using Clustal W with further manual verification. Subsequently, MEGA v.6.06 (http://www.megasoftware.net/) software was applied to conduct phylogenetic analysis. A bootstrap phylogenetic tree demonstrating the relationship of BspXJ, BspDH, BmLT, BmTZ, BmNX and BmHB to other apicomplexan parasites was created by the maximum likelihood (ML) method or neighbour-joining (NJ) method, using a distance matrix corrected for nucleotide substitutions based on the JTT with Freqs model. In addition, phylogeny of the whole apicoplast nucleotide sequence was constructed by the ML method based on the Kimura 2-parameter model. A bootstrap analysis was used to assess the robustness of the clusters using 1000 replicates.The 18 apicoplast genomic sequences, six from the ovine Babesia isolates were formed of circular DNA, ranging from 29,916 to 30,846 bp in length with a high A + T content of 78.7–81.0% (Table SSU and LSU), 23–24 transfer ribonucleic acids (tRNAs) and five to six ribosomal protein large subunits (rpl), eight to nine ribosomal protein small subunits (rps), four to five subunits of DNA-directed RNA polymerase (rpo), two ATP-dependent Clp proteases , one elongation factor TU (tufA), and seven to eleven hypothetical protein genes (hyp)  and BmLT (KX881915) sequenced using Illumina technology (designated BspXJ-Illumina and BmLT-Illumina) were compared with those obtained in this study using the Sanger dideoxy chain-termination method . The results indicated that the size of the apicoplast genome, the number of tRNA and protein-encoding genes, and the initiation and termination codons used in the encoding genes are different in the two sets of data from Sanger and Illumina (Tables 914 and BChromera velie (as an outgroup). The two approaches gave the same topology. In the amino acid phylogenetic trees, the piroplasms were divided into three groups: classical Babesia infective to ruminants, Theileria and B. microti. The classical Babesia species were separated into three clades: B. motasi, Babesia sp. Xinjiang/Dunhuang and B. bovis/B. orientalis. In addition, B. motasi were further divided into two subclades: B. motasi Lintan/Tianzhu and B. motasi Ningxian/Hebei  from each apicoplast genomic sequence of 18 apicomplexan parasites and the chloroplast genomic sequence of bei Fig. . In the Babesia spp. are tick-transmitted haemoprotozoans that infect various animal species including humans, causing loss of livestock and public health concern in tropical and subtropical regions of southern Europe, Africa, Asia, Australia and the Americas [Babesia isolates were sequenced, assembled, annotated and compared with those of other apicomplexan parasites. The apicoplast genomes are smaller than those of most apicoplexan parasites [P. chabaudi chabaudi [B. microti [Americas , 36. TheAmericas . In the arasites , 37, 44,chabaudi  and B. m microti .rpl23, rpl11 and sufB genes are deficient in the apicoplast genomes of the six ovine Babesia spp., which is similar to B. bovis, B. orientalis, B. microti, T. parva, T. gondii, E. tenella and C. cayetanensis. It is possible that these genes were lost during the genetic evolution of the apicoplast in most apicomplexan parasites [rpl23 and rpl11 genes do not play an important role in the growth and development of these parasites; alternatively, they may be translated at other sites in the apicoplast genome or directly encoded by the nuclear genome. The loss of the sufB gene may have been caused by the gene inversion of cluster 3 during the evolution of Piroplasmida [hyp genes may be one of the causes of the variation in apicoplast genome size. Previous studies have suggested that rearrangements and loss of the genes involved in photosynthesis were thought to be responsible for the formation of apicoplast [sufB, rpl23, rpl11 and hyp), inversion (RNA polymerase), duplication (ClpC and hyp) and restructuring (SSU and LSU) were also important events during the early evolution of Babesia species. These modifications may have caused the apicoplast genomes of BmNX and BmHB to be the smallest among the reported Babesia in cattle and small ruminants.The content and order of genes in the cluster differ among the parasites. The arasites . Therefoplasmida , 25. In icoplast , 25. Furrpl5 and rpl6 genes were present between rpl14 and rps5 genes in the second-generation sequence data of BmLT, while only the rpl6 gene exists in this region in the first-generation sequence data of the four B. motasi isolates. It is possible that these differences involving nucleotide substitutions, insertions and deletions were due to the different sequencing methods and annotation software used. Additionally, settings of a few parameters comprising ORF length, nested ORFs, start and stop codon, and genetic code were used. Therefore, it is recommended to perform first-generation sequencing to verify sequences based on the second-generation sequencing sequence, to annotate genes using two or more annotation software programs and then further improve the data by using BLAST.Comparison of the BspXJ and BmLT apicoplast genome data sequenced by first-generation and second-generation sequencing techniques showed that there were some differences in genome sizes and the numbers of tRNA and protein-encoding genes. For example, in vivo or in vitro propagation) and molecular classification  of the Babesia species infective to small ruminants in China have shown that these parasites are divided into two Babesia species: B. motasi and Babesia sp. There are two further subspecies of B. motasi, named BmLT/TZ and BmNX/HB . These findings are consistent with the Uilenberg inference [Babesia species infective to small ruminants in China. In the ML and NJ trees, the ovine Babesia fell into two groups: Babesia sp. and B. motasi. Babesia motasi was further divided into two small clades, BmLT/TZ and BmHB/NX.Studies on the biological characteristics  infective for small ruminants in China, and that the four B. motasi isolates possibly belong to two subspecies (BmLT/BmTZ and BmNX/BmHB). Further studies are needed to validate the effects of anti-Babesia drugs against DNA replication, transcription, translation and 2-C-methyl-d-erythritol 4-phosphate pathways of the apicoplast.In the present study, we have accomplished the sequencing, assembly and annotation of the apicoplast genomes of six ovine Additional file 1: Table S1. Primers for amplifying apicoplast genomes of six ovine Babesia isolates.Additional file 2: Figure S1. Circular map of the apicoplast genome of Babesia sp. Xinjiang.Additional file 3: Figure S2. Circular map of the apicoplast genome of Babesia sp. Dunhuang.Additional file 4: Figure S3. Circular map of the apicoplast genome of Babesia motasi Lintan.Additional file 5: Figure S4. Circular map of the apicoplast genome of Babesia motasi Tianzhu.Additional file 6: Figure S5. Circular map of the apicoplast genome of Babesia motasi Ningxian.Additional file 7: Figure S6. Circular map of the apicoplast genome of Babesia motasi Hebei.Additional file 8: Table S2. Comparison of BspXJ/BspDH, BmLT/BmTZ and BmNX/BmHB apicoplast genomes.Additional file 9: Figure S7. Whole genome alignment of Babesia obtained in this study with ten genomes from apicomplexan parasites: B. bovis, B. orientalis, B. microti, C. cayetanensis, E. tenella, L. caulleryi, P. chabaudi chabaudi, P. falciparum, T. parva and T. gondii. Comparison was performed using Mauve. The coloured blocks in the first genome are connected by lines to similar blocks in the other genomes. The region of sequence covered by a coloured block is entirely collinear and homologous among the genomes. Each locally collinear block (LCB) is assigned a unique colour and the apicoplast assemblies presented re-arrangements in some species.Additional file 10: Figure S8. Phylogenetic relationships of six ovine Babeisa isolates and other apicomplexan parasites. Phylogeny was inferred with a maximum likelihood analysis of whole nucleotide sequences based on distances calculated with the Kimura 2-parameter model. Chromera velia (HM222967) was used as the outgroup. Bootstrap values > 50% from 1000 replicates are shown on the nodes. Babesia obtained in this study is shown as triangles.


Rubus genus consists of more than 600 species that are distributed globally. Only a few Rubus species, including raspberries and blueberries, have been domesticated. Genetic diversity within and between Rubus species is an important resource for breeding programs. We developed genomic microsatellite markers using an SSR-enriched R. coreanus library to study the diversity of the Rubus species. Microsatellite motifs were discovered in 546 of 646 unique clones, and a dinucleotide repeat was the most frequent (75.3%) type of repeat. From 97 microsatellite loci with reproducible amplicons, we acquired 29 polymorphic microsatellite markers in the Rubus coreanus collection. The transferability values ranged from 59.8% to 84% across six Rubus species, and Rubusparvifolius had the highest transferability value (84%). The average number of alleles and the polymorphism information content were 5.7 and 0.541, respectively, in the R. coreanus collection. The diversity index of R. coreanus was similar to the values reported for other Rubus species. A phylogenetic dendrogram based on SSR profiles revealed that seven Rubus species could be allocated to three groups, and that R. coreanus was genetically close to Rubus crataegifolius (mountain berry). These new microsatellite markers might prove useful in studies of the genetic diversity, population structure, and evolutionary relationships among Rubus species.The  Rubus genus in the Rosaceae family consists of more than 600 species grouped in 12 subgenera. A few of these species, including raspberries, blackberries, dewberries, arctic fruits, and flowering raspberries, have been domesticated and are the focus of breeding programs [Rubus species have the potential to interact with other species belonging to different Rubus subgenera. Cross-fertilization within the Rubus genus implies that wild populations could be useful resources to improve domesticated species [Rubus coreanus, which is distributed throughout Southeast Asia, could be a valuable resource for breeding programs and the biotech industry [The  species ,4. For tindustry ,6.Rubus populations. Studies focusing on the genetic diversity of Rubus species have been conducted using various types of molecular markers, including restriction fragment length polymorphisms, random amplified polymorphic DNA, simple sequence repeats (SSRs), and amplified fragment length polymorphisms (AFLP), in Rubus caucasicus L. [Rubus glaucus [Rubus idaeus [Rubus occidentalis [Rubus species [Rubus idaeus and Rubus caesius hybrids have been surveyed using internal transcribed spacer markers [Rubus species involved AFLP and SSR markers [During the last few decades, molecular markers have been used to estimate the genetic diversity of sicus L. , Rubus g glaucus , Rubus is idaeus ,10, Rubudentalis ,12, and  species ,14,15. R markers . Cross-aRubus species. The objectives of this study were to develop novel genomic SSR markers using a microsatellite-enriched library of R. coreanus and to test their transferability across six other Rubus species. New microsatellite markers with polymorphisms and transferability across species might be valuable tools to evaluate genetic variability and identify genes controlling agronomic traits in the Rubus genus.More reliable molecular markers are needed to enhance genetic analyses of Actinidia arguta at 74.2% [In this study, 684 positive colonies were randomly selected from an SSR-enriched library and sequenced. A total of 358 kb sequences were acquired in 684 clones, and 646 singleton sequences were used to search for SSR motifs. A total of 38 (5.6%) of the 684 clones were duplicates. Microsatellite motifs were discovered in 546 clones. The enrichment efficiency of microsatellites (84.5%) was higher than for groundnut at 68% , Japanesat 74.2% .Most of the microsatellite motifs (75.3%) were dinucleotide repeats, followed by trinucleotide repeats (20.7%) and repeat motifs that were tetranucleotide or greater (4.0%). Among the SSR motifs, AG/CT and CTT/AAG represented the majority of di- and trinucleotide motifs at 57.1% and 6.0%, respectively. This result corresponds to the general SSR motif distribution in dinucleotide motifs ,22. The R. coreanus accessions, and were thus excluded from further analysis.Among the 546 clones containing microsatellite motifs, 263 primer pairs were designed based on flanking SSR regions. The SSR motifs biased over the acquired sequences might affect primer variation. The divergent distribution of DNA fragments in the SSR-enriched library could introduce variation among crops . In totaRubus species  of the microsatellite markers produced at least one amplicon in other Rubus species. A total of 26 (26.8%) of the microsatellite markers had PCR products in all tested samples , followed by Rubus ursinus (79.4%) and R. idaeus (78.4%); in contrast, Rubus crataegifolius had only 59.8% transferability.We applied the 97 microsatellite markers to analyze their transferability to other  species . The res samples . The traR. coreanus accessions, while the remaining markers were monomorphic. These 29 markers were used to create DNA profiles of 48 Rubus accessions . Polymorphism scores for 29 microsatellite markers were calculated for the R. coreanus collection and for other Rubus species (A) varied among loci, and ranged from two to 13  alleles in R. coreanus and from four to 15  alleles in other Rubus species. Within the R. coreanus collection, the mean expected heterozygosity and observed heterozygosity were 0.582 and 0.558, respectively, and the mean polymorphism information content (PIC) was 0.541 . GB-RC-245 had the highest PIC value of 0.863 (NA = 13), followed by 0.826 for GB-RC-091 and GB-RC-247 . Castillo et al. [R. idaeus) and blackberry (Rubus L. hybrids), with an average PIC value of 0.55 in the red raspberry collection. Michael et al. [A = 8.6) using 21 polymorphic Rubus SSR primers in cultivated and wild black raspberry  collections. Our results revealed a similar diversity index in the R. coreanus collection compared to previously reported microsatellite markers in other Rubus species. This implies that the microsatellite markers developed in this study might be useful for the genetic assessment of Rubus species with high transferability.Among the transferable microsatellite markers, we identified 29 polymorphic markers in selected  species . The numo et al.  reportedR. coreanus and the genetic relatedness among seven Rubus species based on SSR profiles were not previously reported, whereas several studies have focused on cultivated Rubus species such as red raspberry [Rubus accessions using 29 microsatellite loci. The Rubus species were divided into three groups  in the third group (G-3). The alleles of R. crataegifolius for the 29 microsatellite loci were similar to those of R. coreanus, although R. crataegifolius had low transferability (59.8%). The first group (G-1) consisted of other mountain berries (R. crataegifolius var. subcuneatus and R. parvifolius), while the second group (G-2) included blackberry (Rubus fruticosus and R. ursinus) and red raspberry. These microsatellite markers will be useful for studies of the genetic diversity, population structure, and evolutionary relationships among Rubus species. Microsatellite analyses of aspberry ,25. In te groups . Rubus cRubus accessions (32 R. coreanus accessions and 16 accessions of other Rubus species) that were conserved at the Bokbunja substation of the Gochang Agricultural Center , and the fragmented DNA was eluted on a 1.5% agarose gel; the fragments ranged in size from 300 to 1500 bp. After adaptor ligation and pre-amplification, the DNA mixture was hybridized with the following biotin-labeled oligonucleotides: (GA)20, (CA)20, (AT)20, (GC)20, (AGC)15, (GGC)15, (AAG)15, (AAC)15, and (AGG)15. The hybridized DNA fragments were recovered with streptavidin-coated magnetic beads . The fragments were cloned by TOPO TA cloning (Invitrogen). Recombinant colonies, identified as white colonies on an LB plate containing ampicillin, were sequenced using an ABI3100 DNA sequencer . SSR Manager [A modified biotin-streptavidin capture method was used to construct an SSR-enriched library of coreanus . Genomic Manager  was usedThe M13-tailed primer method, in which the M13 sequence is attached to the 5'-end of the forward primer, was used to determine the sizes of the amplified products, as described by Schuelke . PCR ampR. coreanus were cross-amplified in other Rubus species, and their transferability was analyzed. PCR and resolving the amplified products were performed as described above. Genotypes of the 48 Rubus accessions were based on the 29 polymorphic microsatellite markers, and these DNA profiles were used to analyze genetic variability and construct a phylogenetic tree. PowerMarker (ver. 3.25) [Rubus species. A genetic distance matrix was created as a function of shared alleles [The 97 microsatellite loci with a reproducible amplicon in r. 3.25)  was used alleles . The con alleles .Rubus species. The genetic diversity index and clustering results show that these markers are informative for genetic assessments of R. coreanus and other Rubus species. These new microsatellite markers can be used for genetic map construction, molecular breeding, and germplasm management of Rubus species.We created 29 new polymorphic microsatellite markers based on the 97 transferable microsatellite markers in the


While DCTs are typically found in deuterostomes, DCEs are present in several protostome phyla, including arthropods and mollusks. The respective DCEs belong to the yellow gene family, previously reported to be taxonomically restricted to insects, bacteria and fungi. Mining genomic and transcriptomic data of metazoans, we updated the distribution of DCE/yellow genes, demonstrating their presence and active expression in most of the lophotrochozoan phyla as well as in copepods (Crustacea). We have traced one intronless DCE/yellow gene through most of the analyzed lophotrochozoan genomes and we could show that it was subjected to genomic diversification in some species, while it is conserved in other species. DCE/yellow was expressed in most phyla, although it showed tissue specific expression patterns. In the parasitic copepod Mytilicola intestinalis DCE/yellow even belonged to the 100 most expressed genes. Both tissue specificity and high expression suggests that diverse functions of this gene family also evolved in other phyla apart from insects.Melanin plays a pivotal role in the cellular processes of several metazoans. The final step of the enzymically-regulated melanin biogenesis is the conversion of dopachrome into dihydroxyindoles, a reaction catalyzed by a class of enzymes called dopachrome tautomerases. We traced  Innate immunity represents an early defense system in vertebrate and invertebrate animals . It invoAchatina fulica infected by nematodes [Roseovarius crassostreae infecting Crassostrea virginica and Vibrio tapetis infecting Ruditapes phlilippinarum [Although melanotic encapsulation is mostly described in arthropods , the preematodes , Roseovappinarum . Here, appinarum , but mosppinarum ,11, a tippinarum ,13,14.o-diphenols, oxidized into quinones and polymerized to obtain melanin [Manduca sexta [Drosophila melanogaster and called yellow. It was reported associated with larval pigmentation defects [yellow gene family shared a conserved major royal jelly protein (MRJP) domain, not all yellow proteins can convert dopachrome into DHI, like D. melanogaster yellow-f2 and Aedes aegypti yellow [yellow proteins are the main component (80%) of the royal jelly [Yellow genes have been reported as a gene family taxonomically restricted to insects, likely originating from horizontal gene transfer (HGT) from bacteria, and functionally diversified by species-specific duplications [yellow-like transcripts found in the venom of a parasitoid wasp modulate host physiology and behavior [yellow-like salivary proteins of pathogen vectors, such as the sand flies Phlebotomus tobbi and P. sergenti, displayed leishmanicidal properties [yellow proteins of the leaf beetle Phaedon cochleariae likely protect larvae from fungal infections [yellow-like genes were identified in fungi and micro-eukaryotes, but their roles remains to be determined [yellow-like genes have been reported in few other species, namely in Brachiostoma floridae (Chordata), in the amoeba Naegleria gruberi and in the copepod Lepeophtheirus salmonis [Melanin originates from tyrosine and related phenolic compounds, which are hydroxylated into  melanin . Althoug melanin ,17, the  melanin , were se melanin , was ini melanin . Nowaday melanin , it is i melanin  and it p melanin . Subsequca sexta . This ge defects  and the  defects . Althougi yellow ,27,28. Ial jelly . In addial jelly , althougal jelly ,31. Yellications . For insbehavior , yellow-operties ,35 and yfections . Other ytermined . Apart fsalmonis .In bivalves, the enzymes involved in the biogenesis of melanin are often grouped into the general class of phenoloxidases, including three distinct enzyme types: tyrosinases (EC 1.14.18.1), catecholases (EC 1.10.3.1) and laccases (EC 1.10.3.2) , whereasyellow genes in genomic resources from a wider taxonomic distribution with a special focus on the lophotrochozoan phyla. By combining genome sequences with transcriptomic data, we could further demonstrate that these genes are functional and probably also underwent functional diversification outside of insects.The steady increase of sequenced genomes provides the opportunity for a comprehensive analysis of dopachrome tautomerase enzymes in metazoans. In this paper we investigated the taxonomic distribution as well as the phylogenetic relationships of DCT and DCE/https://www.ensembl.org/index.html) and of the Ensembl Metazoa v42 (https://metazoa.ensembl.org/index.html) databases were downloaded from the corresponding ftp repositories (accessed in December 2018). To increase the representativeness of poorly supported lophotrochozoan clades, we retrieved the gene models of two gastropods  [Lingula anatina) [Notospermus geniculatus), one phoronid  [Bathymodiolus platifrons, Modiolus philippinarum [Crassostrea gigas [C. virginica, Pinctada fucata [Ruditapes philippinarum [accostrea glomerata [Mizuhopecten yessoensis [Limnoperna fortunei [Scapharca broughtonii [Tigriopus kingsejongensis) [Schmidtea mediterranea) [Branchiostoma floridae [B. belcheri, http://genome.bucm.edu.cn/lancelet/index.php). RNA sequencing data for selected species were downloaded from the NCBI SRA archive and used for de-novo transcriptome reconstruction or for gene expression analysis , allowing a minimal Phred quality of 30. For de-novo transcriptome reconstruction, the cleaned reads were assembled using CLC Genomic Workbench v11.0 , applying a word size of 20; a bubble size of 50 and minimal contig length of 200 nt. The resulting contigs were subjected to open reading frame (ORF) prediction using the transdecoder tool implemented in the Trinity suite [All the predicted proteins of the Ensembl genome browser v95 (is spp.) , one braanatina) , one nemstralis)  and 10 bppinarum , Crassosa fucata , Ruditapppinarum , Saccostlomerata , Mizuhopssoensis  Limnoperfortunei  and Scapughtonii . We alsongensis)  and one erranea) . Lancelefloridae ) and froty suite . The detyellow proteins and the tyrosinase domain (Pfam ID: PF00264) for DCT proteins. These were coupled to blast searches using manually curated fungal, bacterial and insect yellow sequences retrieved from [E-value cut-off of 10−10. The conserved protein domains were identified using HMMer v3.1 [E-value cut-off of 10−5. The presence of a signal peptide region was verified with Signalp v4.0 [transeq tool (https://www.ebi.ac.uk/Tools/st/emboss_transeq/) and the presence of genomic regions corresponding to unannotated genes of interests was verified using HMMer, as described above.The proteins of interest were identified from the protein datasets using archetypical domains, like the MRJP domain (Pfam ID: PF03022) to identify DCE/ved from  or usingMer v3.1 , applyinalp v4.0 . If the yellow). Bayesian phylogenetic analysis was performed using MrBayes v3.2.6 [http://tree.bio.ed.ac.uk/software/figtree/).The cd-hit tool  was useds v3.2.6 . Markov s v3.2.6 . The finC. gigas DCE/yellow gene (EKC25673) and to further investigate its expression levels, we sequenced two RNA libraries prepared from oysters from the Northern Adriatic Sea . In detail, oyster spat deployed in the lagoon of Goro or in a control tank in a local hatchery were sampled in June 2013. Soon after transportation to the laboratory, the oysters were dissected, the gills were placed in Trizol  and stored at −80 °C. Total RNA was extracted following the Trizol producer’s recommendations. RNA quantity and quality were checked using a Nanodrop instrument (ThermoFisher) and an Agilent RNA6000 Nanochip , respectively. One microgram of high-quality RNA per sample was used to construct libraries for Illumina high-throughput sequencing, subsequently carried out with a 2 × 50 read layout in a Hi-Seq2000 instrument . Reads were trimmed as previously described and the clean reads were mapped on the C. gigas genome (ID: GCA_000297895.1) using the large gap mapping tool implemented in CLC , applying 0.9 for both similarity and length parameters and the reads mapped around the oyster yellow locus (genome scaffold1670) were further taken into consideration. In parallel, the cleaned reads from each library were de-novo assembled, the yellow transcripts identified and compared with the genomic locus. Raw reads were deposited at the SRA archive under accession ID PRJNA484693.To confirm the gene annotation of the yellow genes in different tissues and conditions. These samples included 165 C. gigas datasets [Mytilus edulis with the parasitic copepod Mytilicola intestinalis [We used available RNA-seq datasets of selected non-insect metazoan species to compute the expression patterns of DCE/datasets ,59, 61 Rstinalis , as wellyellow proteins of C. gigas, M. intestinalis, D. melanogaster, Octopus bimaculoides and B. belcheri have been modeled by Swiss Model server (https://swissmodel.expasy.org) [D. melanogaster DCE (sequence identity with PDB template 3Q6K: 28.41%) and B. belcheri (sequence identity with PDB template 3Q6K: 24.47%), while the other three models had a lower quality due to the low sequence identity levels (less than 20%) between queries and templates. The D. melanogaster DCE model, covering 84% of the submitted sequence (residues: 27–429), was built based on the target-template alignment using ProMod3 and fragments libraries to remodel insertions and deletions, and has been evaluated by GMQE function  and overall QMEAN  scoring parameter, resulting in 0.60 and –3.28, respectively. The B. belcheri model, covering the 84% of the submitted sequence (residues: 33–388), has GMQE and QMEAN scores of 0.55 and −3.24, respectively. Models of C. gigas M. intestinalis and O. bimaculoides DCE/yellow proteins instead, covered 60% (residues: 122–725), 82% (residues: 29–400) and 82% (residues: 25–387) of the query sequence respectively, and resulted in GMQE <0.6 and of QMEAN <4. Geometrical parameters calculated for the resulting D. melanogaster DCE model , which contribute to determine the global QMEAN, suffer the relatively low resolution of the best template exploited for the modeling (PDBID: 3Q6K).The sequences of DCE/asy.org) . Other hyellow genes. To identify DCT proteins we used a combination of HMMer, blastp and signal peptide searches in order to select only the proper DCTs from all proteins sharing a tyrosinase domain (Capitella teleta), in platyhelminths, as well as in all the analyzed chordates, including uro- and proto-chordates  and other MRJP-domain containing proteins  reported by Ferguson and collaborators, who previously investigated the phylogenetic relationships of this gene family [yellow genes from some lophotrochozoan phyla, copepods and lancelets. Among lophotrochozoans, DCE/yellow genes are present in Mollusca, Brachiopoda and Rotifera, while they are apparently absent in Nematoda, Annelida, Nemertea and Phoronida , it is possible that DCE/yellow genes could be located on genome regions not included in the available assemblies or they could have been missed during gene annotation processes. To test both these hypotheses, we searched for the MRJP domain in Nemertea and Phoronida genomes translated into the six reading frames, as well as in transcriptomic data of these species . Since we failed to retrieve any MRJP-sequence with both methodologies, we deduced that, like for Annelida and Nematoda, the DCE/yellow gene is absent also in the lophotrochozoan phyla of Nemertea and Phoronida , possessed two MRJP domains in their DCE/yellow proteins. Two-domain DCE/yellow sequences were also retrieved from the bivalve transcriptomes of Villosa lienosa and Margaritifera margaritifera (family Unionidae). However, while the first group of bivalve species encoded only the two-domain DCE/yellow gene, the Unionidae species encoded both single- and two-domain genes. We found further support for this peculiar protein structure by back-mapping RNA-seq reads onto the genomic DCE/yellow locus of C. gigas. The even coverage along the whole gene suggests the complete transcription of two consecutive domains without an intron rather than an assembly artefact , a subset of the identified insect hits , 17 yellow-like protein sequences identified from insect’s salivary secretions, 7 MRJP-domain containing proteins of protozoans as well as 2 bacterial hits considered as possible outgroup.To study the phylogenetic relationships DCE/yellow sequences. The insect sequences , with representatives of honey bee, Atta cephalotes, B. terrestris, Nasonia vitripennis and Solenopsis invicta. As previously reported [ellow-x type appeared as the ancestral insect’s yellow, with the other DCE/yellow types probably originating from x-type ancestors by taxon-specific functional diversification and expansions.The Bayesian phylogenetic tree , rooted in types . In factreported , yellow-yellow-x, copepod and cephalopod hits  appeared ancestral, although their phylogenetic position relative to the yellow-x group and other mollusk proteins remained unresolved. Lancelet DCE/yellows (highlighted in green) clustered as outgroup of metazoans, together with a Reticulomyxa filosa hit (Foraminifera), possibly indicating an independent horizontal gene transfer (HGT) event.Similar to yellow are widespread within mollusks and they clustered in several clades according to their taxonomic group. Gastropod DCE/yellow sequences formed two distinct clades, while bivalve hits clustered in three clades. When species had multiple copies of DCE/yellow , these tended to cluster together, possibly indicating recent duplication events. The first bivalve sub-cluster (highlighted in yellow) comprises most of the sequences (33 out of 57) and it included one sequence for almost all the analyzed species. The second and third sub-clusters included sequences of species showing DCE/yellow gene expansions, like M. yessoensis, Mytilidae and Unionidae species. The two-MRJP domains of double-domain DCE/yellow genes of each species clustered into the same clade, for both bivalve and insect proteins, possibly indicating incomplete duplication events.DCE/It is difficult to assess the functional relevance of genes from sequence data alone. One possibility to gain the functional importance is to analyze the structural conservation and transcription patterns of the genes in question.yellow proteins, we build up structural models of C. gigas, M. intestinalis, D. melanogaster, O. bimaculoides and B. belcheri proteins, based on to two similar structures identified in the Protein Data Bank (PDB) database: the salivary protein LJM11 from L. longipalpis (PDB ID: 3Q6K) and the MRJP1 from Apis mellifera. (PDB ID: 5YYL). Due to the low sequence identity (around 20%), only the structural models of D. melanogaster and B. belcheri  produced reliable scores , while only the aspartate residue is conserved in D. melanogaster DCE (Asp-381), since the glutamine residue is substituted by an arginine (Arg-144). DCE/yellow proteins of O. bimaculoides and B. belcheri instead, share only few of these aromatic residues , that is also present in a putative hydrolase protein of the bacterium Anabaena variabilis (Trp-291). Moreover, the A. varaiabilis protein shares the peculiar 6-bladed β-propeller fold  and moderate sequence identity with B. belcheri and O. bimaculoides DCE/yellow proteins (14% with O. bimaculoides and 26% with B. belcheri).To investigate differences and similarities between the structure of DCE/of LJM11 B. In par protein . MoreoveC. gigas DCE/yellow protein, the structures of both MRJP domains were poorly predictable from a structural point of view. In fact, besides the 6-bladed β-propeller fold and a few conserved residues, it was very difficult to find similarities to MRJP1 or LJM11. Even the C-terminal domain, between the two MRJP domains, representing the one with the highest identity with A. mellifera MRJP1, only had a similarity of 24.58%, while the N-terminal domain only showed 23.10% of similarity.For the yellow genes, we found substantial variation in their expression values, ranging from zero to thousands of TPMs  expression values related to one of its three DCE/yellow genes in kidney and liver tissues, four O. bimaculoides values  as well as 18 out of the 20 M. intestinalis values . Other copepod species, like the free-swimming copepod T. kingsejongensis and the ectoparasitic salmon louse , showed lower DCE/yellow expression values when compared with M. intestinalis. Bivalve DCE/yellow genes appeared poorly expressed, with the highest expression levels in gill, mantle and gonad tissues. Finally, the two B. belcheri DCE/yellow genes are expressed at intermediate levels during developmental stages  was downregulating DCE/yellow up to 3.2-fold at 24 h post infection  events. We showed that DCTs are mainly found in deuterostomes and only in a few non-deuterostome organisms, including cephalopods, platyhelminths, and ematodes . As eumeyellow being a likely candidate [yellow family was previously thought to be exclusively found in insects, bacteria and fungi [yellow genes, while cephalopods and lancelets even possess both DCT and DCE/yellow genes (This suggests that the enzymatic function of DCT (dopachrome tautomerase) is performed by a different protein, with DCE/andidate . The DCEnd fungi ,32. Our ow genes .yellow genes were already present in the protostome and deuterostome ancestor and both genes could have experienced extensive gene loss events. DCE/yellow was then lost in deuterostomes, in Malacostraca among arthropods as well as in the lophotrochozoan phyla mentioned above, whereas DCT has been lost in most protostomes, with the exception of cephalopods. The presence of a DCT gene in a single nematode species (C. teleta), as well as the presence of DCE/yellow genes in lancelets can possibly be explained by HGT events. Prokaryote-to-Eukaryotic HGT is considered to be an important element driving eukaryote genome evolution [R. filosa [yellow proteins of lancelets were closely related to the DCE/yellow of R. filosa, possibly indicating an HGT event . CuriouGT event .D-dopachrome tautomerase (D-DT) and its paralogue Macrophage migration inhibitory factor (MIF) also possess a similar enzymatic capacity, although on the non-physiological substrate D-dopachrome [Major Royal Jelly Protein 1 is artificially expressed in mice the production of D-DT is decreased [C. gigas , in Mytiloida species (3–5 genes) and in gastropod species . Although we could trace one intronless DCE/yellow in several lophotrochozoan species, we observed a considerable syntenic conservation only for species of the same family  or loss  of DCE/yellow genes. The analysis of transcriptomic samples suggested phyla-specific functional adaptations, although, given the importance of melanisation in the immune response, it seems conceivable that these genes also may have maintained the dopachrome converting enzymatic activity. Future studies, leveraging on recombinant proteins and/or gene silencing could help unraveling the detailed functions of DCE/yellow in lophotrochozoans.In the present work we provided a wide-ranging taxonomic distribution of dopachrome tautomerase genes in metazoans. While we confirmed that DCT is mostly distributed among deuterostomes, we reported a wide distribution the DCE/