MicroRNA (miRNA) plays an important role in plant development regulation. Hickory is an economically important plant in which the amount of flowering determines its production.Here, 51 conserved miRNAs, which belong to 16 families and 195 novel miRNAs were identified in hickory genome. For each conserved miRNA family, we used sequences from hickory and other plants to construct a phylogenetic tree, which shows that each family has members in hickory. Some of the conserved miRNA families  have more members in hickory than in other plants because of gene expansion. MiR166 exhibited tandem duplication with three copies being observed. Many members of these conserved miRNA families were detected in hickory flowers, and the expression patterns of target genes were opposite to those of the related miRNAs, indicating that miRNAs may have important functions in floral regulation of hickory.Arabidopsis, grape, and poplar.Taken together, a comprehensive analysis was conducted to identify miRNAs produced in hickory flower organs, demonstrating functional conservation and diversity of miRNA families among hickory, The online version of this article (doi: 10.1186/s12870-017-1180-6) contains supplementary material, which is available to authorized users. DCL1) protein, forming a miRNA/miRNA* complex, which is divided into miRNA and miRNA* after transporting to the cytoplasm [Arabidopsis [MicroRNAs (miRNAs) are classified as small non-coding RNA molecules, whose length range from 18 to 24 nucleotides, acting as central regulators of gene expression [ytoplasm . Plant dytoplasm . Studiesbidopsis –11.Caenorhabditis elegans [In 1993, the first miRNA (lin-4) was discovered and identified in  elegans . In 2001 elegans , and man elegans . High-th elegans . The exp elegans .The evolution and function of miRNAs are revealed by miRNA-based studies from a phylogenetic perspective. Evolutionary conservation enables the identification of miRNAs for which homologs have previously been reported in other species . miRNA gCarya cathayensis), an important economic woody plant, is mainly distributed in Zhejiang and Anhui provinces of China. Hickory has a long juvenile phase, and females and males present different flowering times, imposing a constraint on breeding and stable annual production. The morphological turning point from vegetative to reproductive stage appears in late March, when male floral buds dehisce and pollen releases. Huang et al., (2007) have observed five distinct stages for hickory female floral development including the floral undifferentiated stage, flower bud differentiation and the stage of formation and development of the inflorescence, of the female involucre, and of the pistil [Hickory   and April Flower (AF) (flower bud differentiation) were further analysed. Many miRNAs were obtained and compared with homologous sequences from the genomes of other plants. A comprehensive phylogenetic analysis were also conducted for hickory miRNA family members and their homologs from three other land plant species.−1, with a mean of −56.27 kcal.mol−1. The largest family was the miR167 family consisting of six members, followed by miR156 family, which included five members  with SOAP software, and their secondary structures were also predicted. 51 conserved mature miRNAs of 16 families and their precursor sequences were identified in hickory genome. The minimal folding energy (MFE) of the precursor sequences ranged from −26.1 to −99.62 kcal.mol complex –24. We o complex .Table 1T−1, ranging from −18.7 to −128.5 kcal.mol−1  to further analyze the expression of 15 conserved miRNAs and 12 novel miRNA candidates in different tissues and also different female floral development stages   in grape, eight (miR167a-h) in poplar and six in hickory. The phylogenetic results showed that miR167 was partitioned into four major clades and that hickory miR167 was included in every clade  was previously described and validated as a miR166 target in Arabidopsis [HD-ZIPIII transcripts in early flowers (A2), with similar accumulation patterns in the leaves and stems , and reduced accumulation was observed in the leaves and flowers, indicating that HD-ZIPIII is strongly down-regulated along with the flower development in hickory. This observation is in agreement with the functional role of this transcription factor in regulating hickory flower development [HD-ZIPIII expression is inversely correlated with the expression pattern of miR166  genes during phase transition in Arabidopsis [LOM genes, which encode GRAS family members known to maintain meristem cell polarity, are involved in the regulation of SPL activity [LOM genes preferentially accumulated in early flowers (A2), which showed higher expression levels than mature flowers (A3)   Fig. , whereasA3) Fig. . This exA3) Fig. , suggestExpression analysis indicated that miRNA targets exhibited distinct expression patterns during flower development. We also determined that several miRNAs regulated their targets in the same manner. For example, the targets of cca-miR27, cca-miR38, miR166b and miR171a were all down-regulated to different extents (A2-A3) in the flowering stage Fig. , showingArabidopsis floral transcriptome indicated that many genes encoding transcription factors were preferentially expressed during early flower development compared with other stages [In recent years, a large number of conserved miRNAs and species-specific miRNAs have been identified using high-throughput sequencing . This mer stages . These er stages . OverallArabidopsis, grape and poplar. Most of the mature miRNA sequences exhibited high sequence identity and conservation , A2 stage , A3 stage (flower bud differentiation), and A4 stage (flower bud formation).http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) and Rfam databases (http://www.sanger.ac.uk/Software/Rfam). The conserved miRNAs were subsequently annotated via alignment with miRBase 21.0 (http://www.mirbase.org/index.shtml), allowing two mismatches. The remaining unknown sRNAs were analyzed to predict novel miRNAs using Mireap (http://sourceforge.net/projects/mirea p/). According to recently published criteria, novel miRNAs were screened [http://www.tbi.univie.ac.at/*ivo/RNA).The Raw data were generated from MF and AF sRNA libraries of female flowers . Clean Rscreened . The mapArabidopsis, grape and poplar were obtained from miRBase, Release 21 (http://www.mirbase.org/index.shtml). ClustalW was used to generate multiple alignments of nucleotide acid sequences and MEGA5 was used to generate phylogenetic analyses using the ML method [http://weblogo.berkeley.edu/logo.cgi, accessed 25 Feb. 2015).Precursor and mature sequences of miRNAs from L method . ML phylA modified CTAB method was used to extract total RNA from hickory tissues . The tot2O. All cDNA samples were assayed in triplicate. The primers employed for these assays are listed in Additional file −△△Ct method [A comparative qRT-PCR expression analysis for different tissues  and also for different female floral development stages  were performed. As previous described , miRNAs t method .Least-square means (LS means) and analysis of variance  were performed using Microsoft Office Excel 2007.Additional file 1:Table S1. Cloning of hickory miRNA precursors primers. Table S2. Real-Time PCR of hickory miRNA primers. Table S3. Real-Time PCR of hickory miRNA targets primers and target sequencses. Table S4. Hickory miRNA sequences. Table S5. Conserved and Novel different expression miRNAs identified in Carya cathayensis. (XLS 259 kb)Additional file 2:Figure S1. Conserved and Novel miRNA nucleotide bias at each position. a, Represent conserved miRNA nucleotide bias. b, Represent novel miRNA nucleotide bias. Figure S2. Real-Time PCR analysis of miRNA expression. Real-time PCR was performed with different flower timing. Each bar represents an average of three independent reactions, including both biological and technical replicates. Error bars indicate SD. Figure S3. Real-Time PCR analysis of miRNA expression. Real-time PCR was performed with different tissues. Each bar represents an average of three independent reactions, including both biological and technical replicates. Error bars indicate SD. Figure S4. The maximum likelihood (ML) phylogenetic tree reconstruction using precursor miRNA family sequences from Arabidopsis (ath), grape (vvi), poplar (ptc), and hickort (cca). MUSCLE alignment and ML were used for tree generation. Figure S5. Sequence logo showing a consensus sequence generated from the multiple alignments of miRNA families from four different plant species. a, The miR160, miR162, miR168, miR319, miR390, miR398 and miR408 represent highly conserved miRNAs. b, The miR172 and miR397 represent moderately conserved miRNAs. c, The miR169 represent lowly conserved miRNAs. Figure S6. RT-PCR analysis of miRNA and its targets expression. RT-PCR was performed with different tissues. Each bar represents an average of three independent reactions, including both biological and technical replicates. Error bars indicate SD.(DOC 3078 kb)


Lepidosaurs, a group including rhynchocephalians and squamates, are one of the major clades of extant vertebrates. Although there has been extensive phylogenetic work on this clade, its interrelationships are a matter of debate. Morphological and molecular data suggest very different relationships within squamates. Despite this, relatively few studies have assessed the utility of other types of data for inferring squamate phylogeny.We used developmental sequences of 20 events in 29 species of lepidosaurs. These sequences were analysed using event-pairing and continuous analysis. They were transformed into cladistic characters and analysed in TNT. Ancestral state reconstructions were performed on two main phylogenetic hypotheses of squamates .Cladistic analyses conducted using characters generated by these methods do not resemble any previously published phylogeny. Ancestral state reconstructions are equally consistent with both morphological and molecular hypotheses of squamate phylogeny. Only several inferred heterochronic events are common to all methods and phylogenies.Results of the cladistic analyses, and the fact that reconstructions of heterochronic events show more similarities between certain methods rather than phylogenetic hypotheses, suggest that phylogenetic signal is at best weak in the studied developmental events. Possibly the developmental sequences analysed here evolve too quickly to recover deep divergences within Squamata. Sphenodon punctatus), the only extant rhynchocephalian—and thus the only reasonable proximal outgroup to Squamata in phylogenetic analyses  are one of the most species-rich extant tetrapod lineages . Howeveranalyses . Despiteanalyses , the debanalyses .Developmental data may be useful for phylogenetic inference e.g.,  but theySphenodon was used as the outgroup.Developmental sequences of 20 characters in 21 species representing most major squamate lineages  and 2 weAnother set of cladistic characters was created using the event-pairing method . Comparihttps://www.anbg.gov.au/cpbr/tools/templetontest.tnt). Four replications were conducted: using either ordered or unordered characters; and employing continuous or event-paired characters.With these cladistic characters and files with both molecular and morphological topology in memory, Templeton test  was perfSphenodon was pruned from the tree, and reconstruction was made for the ancestral squamate and compared to the values of terminal taxa.Reconstruction of ancestral states was performed in Mesquite v. 3.2 . DevelopEvent-pair synapomorphies were mapped on both topologies using synapomorphy mapping in TNT. These synapomorphies were subjected to event-pair cracking, following the procedure described in detail by Liolaemus , the strict consensus tree of which is almost completely unresolved. This analysis failed to recover clades of very closely related species such as iolaemus . When allogenies .Furcifer and Varanus indicus are in trichotomy with the clade including all other squamates. This clade is divided into a group containing seven species of iguanians, gekkotan Strophurus, snake Thamnophis, scincoid Mabuya and lacertiform Zootoca, and the second group to which all other squamates belong . In the strict consensus tree s belong . Analysiquamates .Mapping of continuous characters indicates slight differences in tree length between morphological and molecular topologies. With all branches being assigned equal length (=1), the former is 1.49630768 steps long and the latter—1.51610078. With the fossil-calibrated tree, the morphological topology is 0.19257679 steps long and molecular—0.17638729. Mapping of unordered event-paired characters gives the molecular topology a length of 250 steps and the morphological—252 steps. With ordered characters, the molecular topology is 322 steps long, while the morphological is 327 steps long.p > 0.05 in all cases).Neither replication of the Templeton test detected any statistically significant differences between morphological and molecular phylogenies under both present continuous and event-paired character datasets . However, at least some of these groups are represented by only a few members , so these apomorphies may in fact diagnose less inclusive clades .Inferred heterochronic events show more consistency between given methods than between phylogenies . Only a few of these events are common to all methods and phylogenies –14.Varanus) and one scincoid. Members of other important clades, like Amphisbaenia and Dibamidae, were not included. Some of these groups only recently were studied in terms of development . This was also found in similar studies . This mant e.g., . Moreovent e.g., . This maThamnophis and Mabuya that was sister to all other squamates. This resembles the morphological topology, where iguanians are sister group to all other squamates . However, if all studied taxa are extant , the long branches would result in wider confidence intervals and thus ancestral state reconstructions for deep nodes of the phylogenetic tree would be less certain . IntegraIn the continuous analyses , values of all developmental events of the tuatara are located within the confidence interval of the ancestral squamate. This suggests that present data are equally consistent with either hypothesis of squamate phylogeny cf. .In this study only two major phylogenetic hypotheses of squamates were used. It is not beyond imagination that neither of these phylogenies is fully correct. For example, in the analysis combining morphological and molecular data conducted by Cladistic analyses conducted using characters generated by event-pairing and continuous analysis do not resemble any previously published phylogeny. Ancestral state reconstructions are equally consistent with both morphological and molecular hypotheses of squamate phylogeny. Results of the cladistic analyses, and the fact that reconstructions of heterochronic events show more similarities between certain methods than phylogenetic hypotheses, suggest that phylogenetic signal is at best weak in the studied developmental events.10.7717/peerj.3262/supp-1Data S1Click here for additional data file.10.7717/peerj.3262/supp-2Data S2Click here for additional data file.10.7717/peerj.3262/supp-3Data S3Click here for additional data file.10.7717/peerj.3262/supp-4Data S4Click here for additional data file.10.7717/peerj.3262/supp-5Data S5Click here for additional data file.10.7717/peerj.3262/supp-6Data S6Click here for additional data file.10.7717/peerj.3262/supp-7Table S1Molecular phylogeny, ordered characters.Click here for additional data file.10.7717/peerj.3262/supp-8Table S2Click here for additional data file.10.7717/peerj.3262/supp-9Table S3Molecular phylogeny, unordered characters.Click here for additional data file.10.7717/peerj.3262/supp-10Table S4Click here for additional data file.10.7717/peerj.3262/supp-11Table S5Morphological phylogeny, ordered characters.Click here for additional data file.10.7717/peerj.3262/supp-12Table S6Click here for additional data file.10.7717/peerj.3262/supp-13Table S7Morphological phylogeny, unordered characters.Click here for additional data file.10.7717/peerj.3262/supp-14Table S8Click here for additional data file.10.7717/peerj.3262/supp-15Supplemental Information 15Click here for additional data file.


Medicago sativa complex includes both diploid (2n = 2× = 16) and tetraploid (2n = 2× = 32) subspecies. The tetraploid M. ssp. sativa was regarded as having a simple autopolyploid origin from diploid ssp. caerulea, whereas the autopolyploid origin of tetraploid ssp. falcata from diploid form ssp. falcata is still in doubt. In this study, detailed comparative cytogenetic analysis between diploid to tetraploid species, as well as genomic affinity across different species in the M. sativa complex, were conducted based on comparative mapping of 11 repeated DNA sequences and two rDNA sequences by a fluorescence in situ hybridization (FISH) technique.Polyploidy plays an important role in the adaptation and speciation of plants. The alteration of karyotype is a significant event during polyploidy formation. The caerulea were highly similar to those in tetraploid subspecies sativa. Distinctly different FISH patterns were first observed in diploid ssp. falcata, with only centromeric hybridizations using centromeric and multiple region repeats and a few subtelomeric hybridizations using subtelomeric repeats. Tetraploid subspecies falcata was unexpectedly found to possess a highly variable karyotype, which agreed with neither diploid ssp. falcata nor ssp. sativa. Reconstruction of chromosome-doubling process of diploid ssp. caerulea showed that chromosome changes have occurred during polyploidization process.FISH patterns of the repeats in diploid subspecies caerulea is the direct progenitor of tetraploid subspecies sativa. And autotetraploid ssp. sativa has been suggested to undergo a partial diploidization by the progressive accumulation of chromosome structural rearrangements during evolution. However, the tetraploid subspecies falcata is far from a simple autopolyploid from diploid subspecies falcata although no obvious morphological change was observed between these two subspecies.The comparative cytogenetic results provide reliable evidence that diploid subspecies The online version of this article (doi:10.1186/s12862-017-0951-x) contains supplementary material, which is available to authorized users. Nicotiana [Tragopogon [Gossypium [Brassica [Nicotiana, intergenomic translocations have been detected in natural N. tabacum genotypes and this translocation was considered to be significant in tobacco fertility [Lathyrus sativus [Arabidopsis thaliana [Polyploidy is very common in plant evolution. It plays an important role in adaptation and speciation of plants . Accordiicotiana , 4, Tragagopogon , Gossypiossypium , 7 and BBrassica , 9. In Nertility . Compare sativus  and Arabthaliana .Medicago sativa complex includes both diploid (2n = 2× = 16) and tetraploid (2n = 2× = 32) subspecies [M. sativa ssp. sativa L., an important world forage legume, and diploid subspecies M. sativa ssp. caerulea (Less. ex Ledeb.) Schmalh. have a similar morphology with violet flowers and coiled pods [M. sativa ssp. falcata (L.) Arcang. comprises both diploid and tetraploid forms which differ morphologically from the previous two taxa by having conspicuous yellow flowers and straight to sickle-shaped pods [sativa and ssp. falcata have been considered to be autotetraploidy due to appearance of quadrivalents at meiosis and tetrasomic inheritance [caerulea and ssp. falcata in the complex were hypothesized to be the direct progenitor of tetraploid ssp. sativa and ssp. falcata, respectively [M. prostrata may have introgression into the tetraploid ssp. falcata in past. Therefore, the Medicago complex is an interesting model for polyploidy evolutionary study especially for autopolyploid [The bspecies . Tetraplled pods , 14. Subped pods , 14. Witped pods . Tetrapleritance . Two dipectively , 14. Howolyploid .falcata, ssp. caerulea and tetraploid ssp. sativa have been analyzed by C-banding and N-banding techniques [caerulea had similar heterochromatin distribution with tetraploid ssp. sativa: constitutive heterochromatic was distributed mainly around the centromeres, telomere and interstitial region of short arms of the chromosomes and partly presented at the interstitial region of long arms of chromosomes [falcata except centromere regions [falcata than that had been discovered in diploid ssp. falcata [Heterochromatin distributions of diploid ssp. chniques –24. Compomosomes –24. On t regions , 21, 25. falcata .sativa. In this study, we present an in-depth comparative molecular cytogenetic analysis between diploid and tetraploid subspecies in Medicago sativa complex using repetitive sequences and FISH. Chromosome changes will be described in detail in evolution process of autotetraploidy ssp. sativa. The relationship of tetraploid and diploid ssp. falcata will be discussed.Compared with the traditional banding techniques, fluorescence in situ hybridization (FISH), a valuable molecular cytogenetic tool, can display the molecular information on the chromosome more directly, more accurately, and more stably , 27. It caerulea, four diploid ssp. falcata, and six tetraploid ssp. falcata samples were used as materials in this study. Accessions beginning with ‘PI’ were obtained from the National Plant Germplasm System (NPGS) of the United States Department of Agriculture (USDA). Two tetraploid ssp. falcata accessions, XiaNH-072X-824 and Lizj0944, were acquired from the China Germplasm Bank of Wild Species. Accession 2–6 was collected from a wild population in Xinjiang, China. A list of materials with ploidy levels and origins is given in Table Four diploid ssp. v/v) for 4 h at room temperature. Each root tip was squashed in a drop of 45% acetic acid. Finally, the slides were stored at −80 °C before use.Root tips with a length of 1–2 cm were harvested from germinated seeds or growing plants and pretreated in ice-cold water at 4 °C for 20–24 h. Root tips were then fixed in ethanol:glacial acetic acid  were centromeric or pericentromeric, three  were subtelomeric, and three  produced multiple hybridization signals in alfalfa chromosomes [Eleven tandemly repetitive DNA sequences developed in alfalfa by Yu et al.  were useomosomes . We alsoomosomes . The plaFISH procedure was based on Mukai’s description  with mincaerulea accession PI 464715 was conducted by FISH  were co-localized on one end of 12–13 chromosomes : on chromosome 2, 3, 5, 6, 7, 8 and one of chromosome 1; clone 68: on all chromosomes except one of chromosome 7; and clone 87: on all chromosomes except chromosome 7.The physical mapping results revealed E180 produced the greatest number of information of hybridization signals on each chromosome. Thus, double-target FISH between each repetitive sequence and E180 were carried out. Consequently, we used E180 FISH patterns, previous double-target FISH results, and chromosome arm ratios as references to allocate each sequence to a particular chromosome  were also used in cytogenetic analysis. Because 18S–26S rDNA, 5S rDNA, E180, MsCR-3, and MsTR-1 repeats showed a strong ability to distinguish chromosomes according to the results of chromosome allocation, two FISH cocktails—one consisting of 18S–26S rDNA, 5S rDNA, and E180  were co-localized on only one end of one chromosome : on one of chromosome 4; and clone 68 and clone 87: all chromosomes.To further characterize the chromosomes of ssp. caerulea were applied to diploid ssp. falcata PI 631808 and three other accessions  to develop a standard molecular karyotype  to develop a standard molecular karyotype  and ssp. sativa (Zhongmu No. 1). Moreover, the signal numbers of each probe were nearly twice between ssp. caerulea (PI 464715) and ssp. sativa (Zhongmu No. 1). Cocktail FISH results revealed the chromosome distributions of repeat sequences were highly conserved in four ssp. caerulea accessions and four ssp. sativa accessions, respectively  and tetraploid ssp. falcata (XiaNH-072X-824). However, signal distributions of E180, clone 68, and clone 87 probes were more abundant in tetraploid ssp. falcata than in diploid ssp. falcata. The signal numbers of MsCR-1, MsCR-2, MsCR-4, MsCR-5, E180, clone 68, and clone 87 probes were nearly twice between diploid ssp. falcata (PI 631808) and tetraploid ssp. falcata (XiaNH-072X-824). However, the signals of MsCR-3, MsTR-1, clone 65, and clone 74 probes was only located on one chromosome in diploid ssp. falcata rather than on many chromosomes in tetraploid ssp. falcata. Moreover, the cocktail FISH results revealed highly variable karyotypes across different tetraploid ssp. falcata accessions. Thus, chromosome collinearity analysis between diploid and tetraploid ssp. falcata could not be conducted as did between ssp. caerulea and ssp. sativa.The comparative results of chromosome distributions of each repeat sequence Table  showed tMedicago sativa ssp. caerulea and diploid ssp. falcata are sympatrically distributed, with naturally occurring hybrids recorded between them [M. sativa ssp. caerulea and diploid ssp. falcata has been well described by analyses of both C- and N-banded chromosomes [falcata possess only centromeric bands. In contrast, all chromosomes of ssp. caerulea have a centromeric band and a telomeric band in the short arm; in addition, most of the chromosomes of this subspecies have interstitial bands in the short arm, with a few chromosomes featuring prominent interstitial bands in the long arm.een them , 14, 36.een them . In addiomosomes , 21. C- caerulea and diploid ssp. falcata, centromeric bands were revealed to be a heterogeneous mix of MsCR-1, MsCR-2, MsCR-3, MsCR-4, MsCR-5, clone 68, clone 87, and E180 sequences, along with a few 5S rDNA sites. The interstitial bands comprised E180 sequences along with 18S–26S rDNA and 5S rDNA sites, and the subtelomeric bands were represented by MsTR-1, clone 68, clone 87, and E180.C- and N-bands reflect constitutive heterochromatic DNA in chromosomes . Our molcaerulea and diploid ssp. falcata as revealed by FISH were similar to those uncovered by C- or N-banding. The repetitive sequences were physically mapped onto centromeric, subtelomeric, or interstitial regions in ssp. caerulea, whereas the mapped sequences were mainly on centromeric regions in diploid ssp. falcata. Furthermore, conspicuous differences in distribution patterns were observed between ssp. caerulea and diploid ssp. falcata, even though the repetitive sequences detected in centromeric regions of both species displayed similar levels of heterogeneity. Unlike ssp. caerulea, more than half of the chromosomes of ssp. falcata contained E180 sequences in centromeric regions. In addition, centromeric sequences of MsCR-3 were detected on one or no chromosomes of diploid ssp. falcata, whereas they were found on 3–5 pairs of chromosomes in ssp. caerulea.Chromosomal differences between ssp. caerulea and diploid ssp. falcata has been previously revealed by nuclear markers [M. sativa species complex based on chloroplast DNA sequence analysis supports the recognition of ssp. caerulea and diploid ssp. falcata as distinct taxa [caerulea and diploid ssp. falcata and supports their taxonomic differentiation at the chromosome level.Genetic differentiation between ssp.  markers –39. Morenct taxa . Our stucaerulea is postulated to have given rise to tetraploid ssp. sativa  [caerulea support tetraploid alfalfa as an autotetraploid derived from diploid ssp. caerulea [sativa from diploid ssp. caerulea [caerulea to tetraploid alfalfa was reconstructed according to similar FISH patterns. The results strongly supported the simple autotetraploid origin of ssp. sativa from diploid ssp. caerulea.Violet flowered diploid ssp. alfalfa) , 16, 17.caerulea , 23. Seqcaerulea . In our caerulea. Elimination of repetitive DNA was detected in pericentromeric regions of chromosome 6′ and chromosome 16′ of tetraploid ssp. sativa. Increase of repetitive DNA was detected in pericentromeric regions of chromosome 9′ of tetraploid ssp. sativa. Chromosome deletion was postulated to occur in the long arm of chromosome 12′ of tetraploid ssp. sativa. Pericentric inversions were postulated to occur in chromosome 14′ and chromosome 15′ of tetraploid ssp. sativa. Furthermore, significant diversification was recognized in four groups of homologous chromosome of tetraploid ssp. sativa including chromosome 7′ and chromosome 8′, chromosome 11′ and chromosome 12′, chromosome 13′ and chromosome 14′ and chromosome 15′ and chromosome 16′. Thus, we concluded that autotetraploid alfalfa had undergone a partial diploidization by the progressive accumulation of chromosome structural rearrangements during evolution. A previous study of pachytene karyotype reported that at least four groups of the tetraploid chromosomes appear sufficiently alike to be able to form quadrivalents in ssp. sativa, and three of these were seen to form quadrivalents at pachytene [sativa [sativa. Partial diploidization of homologous chromosome groups in tetraploid M. sativa found in our study should be an explanation for the low quadrivalent frequencies at meiotic.It is generally believed that polyploid plants may have unstable genomes in a long term due to a genome-wide gene redundancy . Ma and achytene . Subsequ [sativa . It was  [sativa . This chfalcata have been traditionally treated as a single species, M. sativa ssp. falcata. Diploid ssp. falcata and tetraploid ssp. falcata have been recognized as diploid and tetraploid cytotypes on the basis of chromosome counting and morphology. Diploid ssp. falcata is hypothesized to be the ancestor of autoploid tetraploid ssp. falcata [falcata [falcata was expected to be similar to that of diploid ssp. falcata. Thus, the results of a preliminary study of six accessions of tetraploid ssp. falcata were surprising. Most of the plants possessed chromosomes that had C-bands in addition to normal centromeric bands [caerulea. Even though the studied accessions had yellow flowers with sickle-shaped pods, the accessions were speculated to be the product of hybridization with ssp. sativa [falcata accessions used in our study—three acquired from the NPGS USDA germplasm bank and three collected in situ in China on the basis of morphological identification—also showed highly variable molecular karyotypes. We found that hybridization sites of MsTR-1 and E180, which frequently produce subtelomeric and interstitial bands, respectively, in C-banding analyses, were highly variable across different individuals of tetraploid ssp. falcata. Along with the results of C-banding analysis [falcata.Diploid and tetraploid forms of ssp.  falcata , 14. As [falcata , 21. Becic bands . Highly . sativa . Similaranalysis , our resfalcata were found to possess very different chloroplast haplotypes. The most common haplotype of tetraploid ssp. falcata was shared with M. prostrata rather than diploid ssp. falcata, suggesting past introgression from M. prostrata into the polyploid. The evolutionary trajectory of ssp. falcata does not appear to have involved a simple autopolyploid origin as seen in ssp. sativa [falcata, was uncovered in our study. Although information on the chromosomal distribution of E180 in M. prostrata is not available, multiple E180 hybridization signals have been detected in species of section Medicago, such as M. glutinosa, M. hemicycla, and M. polychroa [falcata could have been introduced from M. prostrata. Our data indicate that the origin of tetraploid ssp. falcata from diploid ssp. falcata is far from simple. Elucidation of the evolutionary history of ssp. falcata will require a large amount of additional data.In an earlier analysis of chloroplast DNA, morphologically identical diploid and tetraploid cytotypes of ssp. . sativa . A high olychroa . This ficaerulea is the direct progenitor of tetraploid subspecies sativa. And autotetraploid ssp. sativa has been suggested to undergo a partial diploidization by the progressive accumulation of chromosome structural rearrangements during evolution. However, the tetraploid subspecies falcata is far from a simple autopolyploid from diploid subspecies falcata although no obvious morphological change was observed between these two subspecies.The comparative cytogenetic results provide reliable evidence that diploid subspecies


Borrelia miyamotoi, is a relapsing fever spirochete vectored by the same species of Ixodes ticks that carry the causative agents of Lyme disease in the US, Europe, and Asia. Symptoms caused by infection with B. miyamotoi are similar to a relapsing fever infection. However, B. miyamotoi has adapted to different vectors and reservoirs, which could result in unique physiology, including immune evasion mechanisms. Lyme Borrelia utilize a combination of Ixodes-produced inhibitors and native proteins  to inhibit complement, while some relapsing fever spirochetes use C4b-binding protein and likely Ornithodoros-produced inhibitors. To evade the humoral response, Borrelia utilize antigenic variation of either outer surface proteins (Osps) and the Vmp-like sequences (Vls) system (Lyme borreliae) or variable membrane proteins . B. miyamotoi possesses putative FHBPs and antigenic variation of Vmps has been demonstrated. This review summarizes and compares the common mechanisms utilized by Lyme and relapsing fever spirochetes, as well as the current state of understanding immune evasion by B. miyamotoi.The emerging pathogen,  Ixodes, Dermacentor, Amblyomma, Ornithodoros)  transmitted by the bite of certain species of hard and soft shell ticks from four genera (hodoros) –3.Borrelia miyamotoi, are currently uncharacterized. Herein, we review some mechanisms Lyme and relapsing fever Borrelia utilize to inhibit and evade host complement and humoral immune responses and relate these to mechanisms that might be used by B. miyamotoi.Intense research efforts are occurring worldwide in an attempt to understand, detect, control, treat, and eradicate these pathogens and their diseases. One step toward preventing and treating infectious diseases is to understand how pathogens evade host defenses to establish infection. Pathogenic Lyme and relapsing fever borreliae establish infection through one or more of the following mechanisms: physical barriers , migration to immunoprivileged sites, and hijacking host processes . The mechanisms utilized by the emerging relapsing fever pathogen, Borrelia form the B. burgdorferi sensu lato complex. Of these 20 species, at least five are classified as causative agents of Lyme disease   though the most common species are I. scapularis and I. pacificus in the US and I. ricinus and I. persulcatus in Europe and Asia.Approximately 20 closely related pathogenic and non-pathogenic species of riensis) –10. LymeOrnithodoros ticks  carry and transmit relapsing fever spirochetes. Several Borrelia spp. cause relapsing fever but B. hermsii, B. turicatae, B. crocidurae, B. hispanica, B. duttonii are more commonly encountered.Species of Ixodes transmit spirochetes of the B. burgdorferi s.l. complex and Ornithodoros transmit relapsing fever borreliae, there are exceptions. B. recurrentis is a louse-borne relapsing fever spirochete endemic mainly to sub-Saharan Africa. B. theileri causes bovine borreliosis and is transmitted by Rhipicephalus microplus, a hard shell tick that parasitizes livestock  Table . While ian arise , 15. EryBorrelia miyamotoi s.s. strains were first isolated and cultured in Japan in 1995 from I. persulcatus and the blood of Apodemus argenteus   . Since tmisphere –84. B. m reasons –92.B. miyamotoi spirochetes  with B. miyamotoi s.l. LB-2001 (US strain) had been unsuccessful leaving severe combined immune deficient (SCID) mice as the only available animal model , Europe , and Asia  . A. AB. miyulcatus) , 109.B. miyamotoi can result in recurrent fever and be self-resolving, similar to other relapsing fever infections  , 107. Onfections , 110. ThB. miyamotoi infection is dramatically different in immunocompromised patients, specifically those treated for non-Hodgkin’s lymphoma (NHL) with rituximab. Two patients treated with rituximab for NHL, one from the US . Instead, both patients exhibited neurological symptoms . A third patient from Germany, also treated with rituximab for NHL, developed Lyme neuroborreliosis-like symptoms   .via the membrane attack complex   Figure  112)..via the Borrelia-specific IgM is produced by a subset of B cells during infection and plays a crucial role in clearing Borrelia  or IgM, binding a bacterial antigen. Importantly, recent studies have shown Borrelia –121. TheThe lectin pathway is very similar to the classical pathway, differing only in the initiation steps. The lectin pathway is typically initiated through mannose-binding lectins, a group of pattern recognition receptors (PRRs) on host cells, binding specific sets of carbohydrates on foreign cells . The lectin and classical pathways converge at the cleavage of C2 and C4 by different mechanisms. In the lectin pathway, C4 and C2 cleavage occurs through mannose-binding lectin-associated serine proteases .2O), which is thought to occur continuously at low levels. The pathway is propagated through interactions with bacterial antigens or a lack of host surface markers   .2O), is cleaved by factor D into Ba and Bb resulting in C3(H2O)Bb, the fluid-phase C3 convertase (cleaves C3 to C3a and C3b). C3b binds the bacterial cell surface where it complexes with additional factor B. Factor D again cleaves factor B, which results in the second, predominant and cell-bound C3 convertase (C3bBb). This cell-bound C3 convertase is stabilized by properdin (C3bBbP). Binding of additional C3b to C3 convertase results in the formation of C5 convertase (C3bBbC3b), which cleaves C5 and initiates the formation of the MAC as described above.Factor B, after binding C3, p43, BBK32, BGA66, BGA71, CD59-like protein] ; B. bavariensis (formerly B. garinii OspA serotype 4) is intermediately resistant. To the best of our knowledge, similar comparisons of multiple strains and species have not been published for relapsing fever Borrelia, though complement resistance is not universal among relapsing fever species among the strains observed. Resistance to complement is important for the transmission, survival, and dissemination of some Borrelia spp. in mammalian and rodent hosts and reservoirs  . FH consectively –135. Theectively –140. FH ectively . For comectively , 142.Borrelia complement inactivation, and complement resistance is correlated with binding FH  has regulatory roles in all three pathways, though is the major regulator of the classical and lectin pathways. C4BP facilitates inactivation of C4b  and fluid-phase C3b  by binding C4b, displacing C2a, and facilitating FI-mediated inactivation of C3 and C5 convertases .Borrelia spp. bind human and various animal C4BP  do not have to be highly resistant to complement in immunoprivileged sites, such as the central nervous system. Finding neurotropic species strongly bind C4BP and very weakly bind FH and FHL-1, while species that are not neurotropic bind C4BP but preferentially bind FH and/or FHL-1 supports this hypothesis  was later reported to be highly but not completely sensitive to complement , this protein has never been identified though several known borrelial proteins can and have been ruled out based on molecular weight   . Given tIxodes spp. with more being identified and characterized , I. ricinus anticomplement (Irac I), Irac II, and Ixac-B1 through -5 (I. ricinus anticomplement). Proteins in this family are similar in function to Salp20  is a lipocalin that binds to and prevents cleavage of C5 ] proteins, while relapsing fever Borrelia possess Vmps   –203. Somroteins) .Borrelia proteins. For comprehensive reviews of Borrelia Osps, see Ref.  and one expression locus, vlsE. All of the identified vls cassettes are located on the same plasmid (lp28-1) in close proximity to but in the opposite direction of vlsE. Expression at vlsE occurs through the random recombination of segments of multiple vls cassettes rather than recombination of an entire, single vls cassette. Thus, recombination events result in thousands of unique VlsE variants, all approximately 36 kDa.In contrast, the Vls system can change the expressed surface antigen u Figure . Antigenresponse –236. TheB. hermsii has approximately 60 unique and promoterless vmp cassettes  scattered throughout its genome and one promoter-driven vmp expression locus . The remaining spirochetes consist of a small population expressing different cassettes. Since the host has not raised a strong antibody response to these non-dominantly expressed Vmps, this minority population of spirochetes can proliferate to high concentrations until an antibody response is mounted and the majority of spirochetes are once again cleared. This cycle of vmp conversion, peaking spirochete loads, and antibody-mediated clearing repeats a minimum of two times resulting in the characteristic symptoms of a relapsing fever illness.The majority of spirochetes are cleared from the host through specific anti-Vmp IgM antibodies raised against the predominantly expressed Vmps, which results in a significant decrease in spirochete load ; however, McDowell et al. were unable to demonstrate FH-binding  with different enzootic cycles and different co-pathogens compared to relapsing fever spirochetes. We should not assume B. miyamotoi utilizes the same set of mechanisms as other relapsing fever spirochetes. B. miyamotoi may use a combination of relapsing fever and Lyme Borrelia mechanisms as well as completely novel mechanisms.Even though B. miyamotoi has not been demonstrated. As discussed above, IgM is key in clearing relapsing fever infections. During B. hermsii infections, IgM targets FhbA and other surface proteins , recurrent fever, and spirochetemia characteristic of relapsing fever. However, additional symptoms characteristic of relapsing fever have not been demonstrated, namely rapid symptom onset with a crisis event suggesting B. miyamotoi infection is not synonymous with relapsing fever and is rather a relapsing fever-like illness (B. miyamotoi compared to the vast majority of relapsing fever spirochetes.Infection with  illness . This shB. miyamotoi utilizes remain undiscovered. Hindered by the lack of a robust animal model, the relatively long-standing inability to culture in vitro, and being unaware of its pathogenicity, our understanding of B. miyamotoi is still in its infancy. However, we are making large strides forward with recent advances in culture techniques, animals models, physicians actively considering B. miyamotoi infection, as well as a growing wealth of epidemiological data that will allow us to clarify the details of infection, genetics, and physiology of this emerging pathogen.The ability to evade the immune response is important for any successful pathogen but many of the mechanisms All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.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.


Bifidobacterium (B.) animalis ssp. lactis HN019 (HN019) supplementation on cellular immune activity in healthy elderly subjects. We conducted a systematic review of Medline and Embase for controlled trials that reported polymorphonuclear (PMN) cell phagocytic capacity or natural killer (NK) cell tumoricidal activity following B. lactis HN019 consumption in the elderly. A random effects meta-analysis was performed with standardized mean difference (SMD) and 95% confidence interval between probiotic and control groups for each outcome. A total of four clinical trials were included in this analysis. B. lactis HN019 supplementation was highly efficacious in increasing PMN phagocytic capacity with an SMD of 0.74  and moderately efficacious in increasing NK cell tumoricidal activity with an SMD of 0.43 . The main limitations of this research were the small number of included studies, short-term follow-up, and assessment of a single probiotic strain. In conclusion, daily consumption of B. lactis HN019 enhances NK cell and PMN function in healthy elderly adults.Elderly people have increased susceptibility to infections and cancer that are associated with decline in cellular immune function. The objective of this work was to determine the efficacy of  Globally, elderly people represent the fastest growing population. Older individuals have typically weaker immune responses to vaccination and elevated risk for infections, certain autoimmune diseases, and cancer . Many ofBacteroidetes spp. [Clostridium difficile associated diarrhea [Gut microbiota plays an important role in immunosenescence and is influenced by physiological aging process, lifestyle, and diet ,12,13,14diarrhea , hospitadiarrhea , antibiodiarrhea , and fradiarrhea . Thus, tB. longum strains induced changes in the bifidobacteria population, which correlated with tumor necrosis factor (TNF)-α and interleukin (IL)-10 levels in plasma [Lactobacillus (L.) casei and L. plantarum strains improved influenza virus vaccination responses in the elderly [Bifidobacterium (B.) animalis ssp. lactis HN019. The objective of this systematic review and meta-analysis of controlled studies was to evaluate the effect of B. lactis HN019 versus non-probiotic control on cellular innate immune activity of healthy elderly subjects.It has been suggested that probiotic bacteria could have potential for improving immune system function in the elderly . Probiotn plasma , and Lac elderly ,23. Alth elderly , systemaB. lactis HN019 consumption in reportedly healthy, elderly (≥60 years) adults. No date restrictions were applied to the searches. The details of the Medline search strategy are listed in The study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . MedlineOne reviewer selected studies for inclusion in the review. Articles were then independently assessed by a second reviewer, who confirmed eligibility. Titles and abstracts were initially screened to exclude non-English manuscripts, review articles, commentaries, letters, case reports, animal studies, and obvious irrelevant studies. Full-texts of the remaining articles were retrieved and reviewed.Data were independently extracted from eligible articles by two reviewers. Data extraction discrepancies between the reviewers were resolved by consensus. The types of data recorded in the standardized data extraction forms included general manuscript information, subject characteristics, study characteristics, PMN phagocytic capacity, and NK cell tumoricidal activity. Potential sources of bias were assessed by evaluating randomization, blinding, type of control, and main outcome definitions among studies.I2 statistic if at least 10 studies were included in the meta-analysis. p-values were two-sided with a significance level <0.05. Statistical analyses were performed using Comprehensive Meta-analysis version 2.2 .A random effects meta-analysis model was developed based on the a priori assumption that treatment effects would be heterogeneous among studies. For each outcome, a pooled standardized mean difference (SMD) and 95% confidence interval were calculated. For reference, SMD values of 0.2, 0.5, 0.8, and 1.0 are defined as small, medium, large, and very large effect sizes, respectively . Forest The initial database search retrieved 82 titles and abstracts; hand searching relevant bibliographies identified three additional records. After screening records for inclusion criteria, 74 records were deemed irrelevant and 11 full text articles were reviewed for eligibility. Ultimately, four studies ,27,28,29B. lactis HN019 relative to control, one study [B. lactis HN019. The daily dosages of B. lactis HN019 ranged from 5 × 109 to 3 × 1011 colony forming units (cfu) and the treatment durations ranged from 3 to 6 weeks , representing a large treatment effect in favor of B. lactis HN019 . Furthermore, each study reported statistically significant improvements in PMN phagocytic capacity with is HN019 . Heterogp = 0.02), representing a statistically significant moderate treatment effect in favor of B. lactis HN019 . Although no individual study reported statistically significant differences between the groups, the pooled SMD for NK cell tumoricidal activity was 0.43  on phagocytic capacity. The meta-analysis of the trials showed that HN019 had a large treatment effect (p < 0.001) on phagocytic capacity of PMN cells  treatment effect for improving NK-cell activity in the elderly  Takeda and colleagues showed that L. casei Shirota may elevate NK cell activity after 4 weeks when compared to placebo [L. casei Shirota was associated with a significant increase in NK cell activity relative to baseline, however the difference was not significant when compared with placebo [L. casei Shirota [Effect of other probiotics than HN019 studies have also been investigated on NK cell activity. A combination of NK cells . In cont feeding  and L. gactivity . Altoget placebo ; (ii) th placebo ; and (ii Shirota . In summB. lactis HN019 on specific cellular immune parameters seems relatively clear there were still limitations inherent in the findings of this meta-analysis, including a potential for bias due to relatively small study populations. In addition, the study outcomes may have been affected by other factors such as the length of intervention, the formula, and the supplementation dose. In the studies included in this meta-analysis, the B. lactis HN019 supplementation was given to healthy elderly adulty for 3–6 weeks suggesting that relatively short term consumption of B. lactis HN019 has an impact on immune cell function in blood; however, it remains unknown if the stimulatory effect would have been maintained in longer term consumption [B. lactis HN019 doses were between 5 × 1010 and 3 × 1011 cfu/day in PMN studies and 5 × 109–5 × 1010 cfu/day in NK cell studies, suggesting that HN019 is efficacious with a range of doses typically used in dietary supplements. In all the B. lactis HN019 clinical trials low-fat milk was used as a delivery vehicle, and as such, no firm conclusions on the role of the vehicle on these immune outcomes can be drawn. A further consideration relates to type of control. The study by Arunachalam et al. [Although the impact of sumption . The daim et al.  used a pm et al. ,27,29 utB. lactis HN019 enhances PMN phagocytic capacity and NK cell tumoricidal activity in healthy elderly adults. As the health of the elderly was not assessed in the original studies, the correlation between B. lactis HN019 mediated PMN and NK cell activity improvement and resistance to infection and diseases remains to be confirmed in future trials.The findings from this meta-analysis suggest that daily short-term consumption of probiotic


Rehmannia is a non-parasitic genus in Orobanchaceae including six species mainly distributed in central and north China. Its phylogenetic position and infrageneric relationships remain uncertain due to potential hybridization and polyploidization. In this study, we sequenced and compared the complete chloroplast genomes of six Rehmannia species using Illumina sequencing technology to elucidate the interspecific variations. Rehmannia plastomes exhibited typical quadripartite and circular structures with good synteny of gene order. The complete genomes ranged from 153,622 bp to 154,055 bp in length, including 133 genes encoding 88 proteins, 37 tRNAs, and 8 rRNAs. Three genes  have potentially experienced positive selection. Plastome size variation of Rehmannia was mainly ascribed to the expansion and contraction of the border regions between the inverted repeat (IR) region and the single-copy (SC) regions. Despite of the conserved structure in Rehmannia plastomes, sequence variations provide useful phylogenetic information. Phylogenetic trees of 23 Lamiales species reconstructed with the complete plastomes suggested that Rehmannia was monophyletic and sister to the clade of Lindenbergia and the parasitic taxa in Orobanchaceae. The interspecific relationships within Rehmannia were completely different with the previous studies. In future, population phylogenomic works based on plastomes are urgently needed to clarify the evolutionary history of Rehmannia. Rehmannia Libosch. ex Fisch. et Mey. is a small genus consisting of six species, among which five  are endemic to China, while Rehmannia glutinosa, a famous and valuable species in Chinese traditional medicine, extends its distribution range from North China to Korea and Japan [Rehmannia has been debated for years. It was traditionally placed in Scrophulariaceae based on morphological traits. Recently, molecular evidence indicated that Scrophulariaceae was polyphyletic [Rehmannia was then transferred to Plantaginaceae [Rehmannia, interspecific relationships within the genus are also unsolved. Despite of the differences in some flower traits, the two tetraploid species R. glutinosa and R. solanifolia share identical chloroplast and nuclear haplotypes [R. piasezkii and R. elata should also be considered one species [R. chingii was the basal taxon of the genus [R. piasezkii was confirmed as the sister group to the remaining taxa within the genus by nuclear data [nd Japan . The sysphyletic . Rehmannginaceae  and lateginaceae ,5 or treginaceae . Besidesplotypes ,8, infer species ,9,10. Moear data ,8.Rehmannia partly lies in the lack of sufficiently effective data. Traditional morphological classification based on limited selected characters is often deeply affected by environmental and developmental factors of samples. For example, bracteoles absence or presence, considered as the critical trait to discriminate R. piasezkii from R. chingii, are not species-specific and variable among intraspecific individuals [Rehmannia [The controversy in systematic position and interspecific relationships of ividuals . Althougehmannia ,7,8, phyMost chloroplast (cp) genomes have a typical quadripartite structure with a pair of inverted repeats (IRs) separated by a large single-copy region (LSC) and a small single-copy region (SSC), and the genome size ranged from 120 to 160 Kb in length . PreviouRehmannia species to verify the familial placement and evaluate the interspecific variation within the genus. These analyses will not only improve our understanding of the evolutionary mechanism of Rehmannia plastome and but also aid to clarify the ambiguous phylogenetic position of Rehmannia.In this study, we sequenced, assembled, and characterized the plastomes of six Rehmannia species were collected for DNA extraction.All samples used in the study were transplanted from their native habitats and cultivated in the greenhouse of Northwest University. No specific permits are required for sampling . HealthyR. glutinosa were isolated using Percoll gradient centrifugation method [R. glutinosa with Illumina MiSeq platform at Sangon Biotech Co. . A paired-end (PE) library with 265-bp insert size was constructed. Total genomic DNAs of other five Rehmannia species  were extracted with simplified CTAB protocol [The organelle-enriched DNAs of n method  and CTABprotocol . A paireR. glutinosa were trimmed to remove the potential low quality bases. Chloroplast genome was assembled using Velvet Assembler version 1.2.07 [http://www.herbalgenomics.org/cpgavas) and the gene homologies were confirmed by comparing with the NCBI’s non-redundant (Nr) protein database, Cluster of Orthologous Group (COG), CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml), PFAM (http://xfam.org), SWISS-PROT (http://web.expasy.org/docs/swiss-prot_guideline.html), and TREMBL (http://www.bioinfo.pte.hu/more/TrEMBL.htm) databases. The raw reads of five other Rehmannia species were quality-trimmed using CLC Genomics Workbench v7.5  with default parameters. Reference-guided assembly was then performed to reconstruct the chloroplast genomes with the program MITObim v1.7 [R. glutinosa as the reference. The cpDNA annotation was conducted using the program GENEIOUS R8 , and used the plastome of R. glutinosa as the reference, coupled with manual adjustment for start/stop codons and for intron/exon borders. Transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), and coding sequences were further confirmed, and in some cases, manually adjusted after BLAST searches. The circle maps of six Rehmannia plastomes were obtained using Organellar Genome DRAW software  [rpoC2) were validated by PCR amplification and Sanger sequencing (Raw reads of n 1.2.07  and SPAdn 1.2.07 . Gaps ann 1.2.07 . The annbim v1.7  using R..mpg.de) . Ambiguoquencing .Rehmannia plastomes were carried out using MAFFT version 7.017 [p-distance) was calculated by MEGA 6.0 [http://bibiserv.techfak.uni-bielefeld.de/reputer/manual.html) with a minimal size of 30 bp and >90% identity  between the two repeats. Gap size between palindromic repeats was restricted to a maximal length of 3 kb. Overlapping repeats were merged into one repeat motif whenever possible. Tandem Repeats Finder [http://tandem.bu.edu/trf/trf.html) was used to identify tandem repeats in the six Rehmannia plastomes with default settings. A given region in the genome was designated as only one repeat type, and tandem repeat was prior to dispersed repeat if one repeat motif could be identified as both tandem and dispersed repeats.Multiple alignments of six on 7.017 . Full alon 7.017 . GeneticMEGA 6.0 . The perMEGA 6.0 . DispersMEGA 6.0  , were computed with codeml tool of PAML package .Rehmannia and 17 representatives of Lamiales species, including six Lamiaceae species, five Orobanchaceae species, and five species from other families , five other Rehmannia species produced large data for each species from 25,724,095 (R. chingii) to 31,171,142 (R. henryi) clean reads (126 bp in average reads length). All paired-end reads were mapped to the reference plastome of R. glutinosa with the mean coverage of 119.6× to 141.8×  (Illumina paired-end sequencing generated 1 Gb raw reads for o 141.8× . Gaps weo 141.8× . All sixX636161) , Table 2Rehmannia ranged in size from 153,622 bp (R. glutinosa) to 154,055 bp (R. chingii). All of them exhibited a typical quadripartite structure consisting of a pair of IRs  separated by the LSC  and SSC  regions  had one intron, while three genes  contained two introns  or gene duplication  and subsequently considered as tRNA or gene similarity repeats [Rehmannia except R. glutinosa with dispersal repeats as the most common. The majority of repeats ranged in size from 30 bp to 44 bp (R. henryi and R. solanifolia. Dispersed repeats had a wider size range (from 30 to 126 bp) than other repeat types. Palindromic repeat, accounting for 41% of total repeats, was the most common, followed by dispersed (39%), tandem (14%), and tRNA or gene similarity (6%) types  and intergenic spacers (43.8%)  types C. A mino (43.8%) D. Rehmannia cpDNAs were plotted using the program mVISTA with R. glutinosa as a reference. The whole aligned sequences showed high similarities with only a few regions below 90%, suggesting that Rehmannia plastomes were rather conserved (psbA-ndhJ) region was identified -psbA, trnS(GCU)-trnG(UCC), psbZ-trnG(GCC), psaA-ycf3, trnT(UGU)-trnL(UAA), cemA-petA, rps12-clpP, nhdD-psaC, and ndhG-ndhL  versus synonymous (Ks) substitutions were calculated for 79 protein-coding genes, generating 241 pairwise valid combinations  located in the LSC region had Ka/Ks ratios above 1.0, which might indicate positive selection  from the six Rehmannia plastomes and 17 published plastomes were used to reconstruct phylogenetic relationships with O. europaea as an outgroup , psaA-ycf3 and trnS(GCU)-trnG(UCC) gene. Lamiales has the highest diversification rates among angiosperms [Rehmannia might be ascribed to the increased evolutionary rates. The presence of repeats in plastomes, especially in intergenic spacer regions, has been reported in all published angiosperm lineages. Compared with other angiosperm species, the number of repeats in spairing ,44,45. Hiosperms . Thus thRehmannia plastomes were conserved in most of regions with sequence identities above 90%. As expected, the noncoding regions exhibited higher divergent levels than the coding regions, and the single copy regions had higher variation than the IR regions. The rpoC2 gene is an exception with lower sequence identity due to various indels, as also reported in grasses [trnS(GGA)-trnP(GAA)) region was identified, which is inconsonant to other herb plastomes [Rehmannia species.Sequences of  grasses . One divlastomes ,48. We alastomes ,50. AddiaccD, rpoA and rpoC2) were under positive selection, of which, rpoA and rpoC2 were reported in Annonaceae [accD gene encodes a plastid-coded subunit of heteromeric acetyl-CoA carboxylase (ACCase), a key enzyme involved in fatty acid biosynthesis in plants [rpoA and rpoC2 encode α and β” subunit of plastid-encoded plastid RNA polymerase (PEP), respectively, a key protein responsible for most photosynthetic gene expression [Rehmannia might have divergent habitats and adapt to different light intensity (unpublished data). These genes experienced natural selection might play an important role in the evolution and divergence of Rehmannia.Our analysis indicated that three genes , and sister to the clade of the parasitic Orobanchaceae species and Lindenbergia philippehsis rather than a genus of Scrophulariaceae. Chloroplast fragments analyses also suggested that Rehmannia, together with Triaenophora, represented the branch sister to Orobanchaceae s.l (including Lindenbergia), or a new familial clade [Rehmannia-Orobanchaceae clade in Lamiales remains uncertain because of the contradictive results of this clade and its related taxa of Paulowniaceae and Phrymaceae from cp fragments data [Rehmannia-Orobanchaceae is still unresolved in our study. But familial relationships of Lamiales taxa in this study were definite and identical to those verified by the rapid evolving cp gene fragments [al clade . The phynts data ,38. For ragments ,54,55.R. elata-R. piasezkii, R. glutinosa-R. solanifolia, had completely identical sequences, respectively and could be treated as two species [R. chingii [R. piasezkii [Rehmannia plastomes were inconsistent with any of these works, inferring all Rehmannia taxa as separate species (Rehmannia species. These comparative genomic analyses will not only provide insights into the chloroplast genome evolution of Rehmannia, but also offer valuable genetic markers for population phylogenomic study of Rehmannia and its close lineages.Recent studies using chloroplast or nuclear gene fragments indicated that two pairs of species groups,  species ,9,10. R. chingii  or R. piiasezkii ,8 were c species . This mi


Echinococcus granulosus (sensu lato) (s.l.) and Echinococcus vogeli are causative agents of chronic zoonotic diseases such as cystic and polycystic echinococcosis, respectively. In Brazil, polycystic echinococcosis has a restricted geographical distribution in the North Region, while cystic echinococcosis is observed in the South Region. Domestic dogs (Canis familiaris) fed with raw viscera represent a risk factor for E. granulosus (s.l.) infection in the South Region. Although this practice is frequent, it remains unclear whether domestic dogs are infected with E. vogeli in the state of Acre, located in the Amazon basin in the North Region of Brazil. The aim of this study was to investigate this gap in the polycystic echinococcosis epidemiology.n = 14; Rio Branco, n = 06; Bujari, n = 06; Xapuri, n = 30; and Epitaciolândia, n = 09) located in the state of Acre, northern Brazil. The samples were screened for parasites by copro-PCR using the cox1 gene associated with automated sequencing.Sixty-five fecal samples were collected from the ground in five municipalities  (s.s.) (G1) in a sample from Rio Branco.Echinococcus taxa from fecal samples of dogs in northern Brazil. The present study is the first molecular record of E. vogeli in domestic dogs found in the state of Acre, reinforcing their role as a source of infection for humans. Because E. granulosus (s.s.) (G1) was detected for the first time in the North Region, from the epidemiological standpoint this finding is highly relevant, because it expands the known geographical distribution, which was previously restricted to the South Region of Brazil.These findings indicate that molecular assays are useful in typing  Canis familiaris) fulfill various specialized activities, such as companionship, herding and hunting [The domestication of canids has evolved into phenotypic changes, docility and adaptation to human-dominated environments, which has led to a lifestyle integrated into modern human societies . During  hunting . Neverth hunting .Echinococcus spp. often grouped based on morphological similarities of their larval stages (metacestodes), as well as molecular-based analyses (metacestode and adult tapeworms) [Echinococcus spp. are trophically transmitted between carnivores that harbor adult tapeworms in the small intestine (definitive hosts) and livestock and rodents, where metacestodes develop in internal organs such as the liver and lungs (intermediate hosts) [For example, echinococcosis is a neglected chronic zoonotic disease caused by peworms) , 6. Echie hosts) , 8. Wilde hosts) .Echinococcus spp. is largely dependent on the presence of competent hosts to transmit each of them. Echinococcus granulosus (sensu lato) (s.l.) causes cystic echinococcosis (CE) and has a wide geographical distribution in Southern Cone countries of South America [E. granulosus (s.l.) is maintained by the dog-sheep, where herding dogs are often fed with raw livestock offal [E. granulosus (sensu stricto) (s.s.) (G1) (ovine strain) is the most common among canids and humans in South America [Echinococcus vogeli causes polycystic echinococcosis (PE). It is restricted to the Neotropical Region and affects areas where people hunt pacas (Cuniculus paca) and live in close contact with domestic/hunting dogs [E. vogeli sylvatic cycle is based on a specific predator–prey relationship between the bush dog (Speothos venaticus), an indigenous canid in South America, and pacas [E. vogeli in the North Region of Brazil.The geographical distribution of  America , affecti America . In Brazck offal  in the Sck offal . Indeed, America . Echinocing dogs –17. The nd pacas , 18. Witnd pacas  and are nd pacas . Despitend pacas , it remaE. granulosus (s.l.), mitochondrial cytochrome c oxidase subunit 1 (cox1) gene-based primers [E. granulosus (s.l.) in canids [Over the last four decades, molecular assays have been proposed, including  primers , 22, to  primers –25. Furtn canids –29.Echinococcus sp. DNA in feces of dogs from the state of Acre, using copro-PCR and subsequent sequencing of the mitochondrial gene cytochrome c oxidase subunit 1 (cox1).This study aimed to investigate The epidemiological survey was conducted in the state of Acre (Brazil) in August 2014 and June-July 2015 on rubber plantations and settlements in the municipalities of Rio Branco, Bujari, Xapuri and Epitaciolândia  and were regularly dewormed. Each fecal sample was transferred to a 50 ml tube containing 70% ethanol. The study also included 14 fecal samples from dogs living within Antimary State Forest located in the municipality of Sena Madureira. These samples were collected in 2013 and stored at the National Reference Service in Hydatidosis of the Laboratory of Helminth Parasites of Vertebrates of the IOC/Fiocruz.c oxidase subunit 1 (cox1) mitochondrial gene as previously described [The macroscopic examination of tapeworm proglottids was performed. Feces were spontaneously sedimented  and the escribed . The copTM . The nucleotide sequences obtained were aligned using the ClustalW method of the MEGA v6.0 software [The amplicons resulting from PCR were purified using the illustra™GFX™ PCR DNA kit , following the manufacturer’s instructions. Both DNA strands were sequenced using the same PCR primers and the PrimTM ABI BigDye Terminator Cycle sequencing kit , according to the manufacturer’s instructions. The analysis of the newly-generated sequences was performed with and automated DNA sequencer , of the RPT01A subunit for automated sequencing - IOC/Fiocruz Technology Platforms Network. The obtained nucleotide sequences were analyzed, aligned and edited by version 4.9 of the program Sequenchersoftware .cox1 was carried out with BLAST program . Phylogenetic analyses were based on alignment obtained from ClustalW of thirteen 366-bp sequences and carried out with MEGA v6.0 software [www.barcodeoflife.org/content/resources/standards-and-guidelines). To determine the robustness of the tree, bootstrap analysis of 1000 replicates was applied. The pairwise distances with the same nucleotide substitution model were calculated with MEGA v6.0 software [E. granulosus (s.s.) (G1) (U50464 [E. granulosus (s.s.) (G2) (M84662 [E. granulosus (s.s.) (G3) ; E. gra (M84662 ); E. gra ; E. equ (M84664 ); E. ort (M84665 ); and E. .n = 30; Sena Madureira, n = 14; Epitaciolândia, n = 09; Bujari, n = 6; Rio Branco, n = 6). The pacas hunting for subsistence was a common practice for at least one family member in 33 rural properties, as well as the habits of feeding domestic dogs with raw entrails of pacas. Cattle and sheep coexisted in 7 properties and dogs had access to the carcasses of livestock. The owners of all of the properties visited admitted never having given anthelmintics to their domestic dogs.Sixty-five fecal samples were collected in peridomestic habitats  was from a settlement in Sena Madureira, where dogs (n = 3) were fed with raw viscera from hunted animals, including pacas. The second (DOG RB-AC-BR) was from a farm in Rio Branco, where dogs (n = 4) had access to the carcasses of livestock (cattle and sheep).There was no finding of dogs having passed E. vogeli  and the sample from Rio Branco (DOG RB-AC-BR) indicated 100% similarity with 17 sequences of E. granulosus (s.s.) (G1). The nucleotide sequences of E. granulosus (s.s.) (G1) and E. vogeli obtained in this study were deposited in the NCBI database under accession numbers KX527915 and KX527916, respectively.The results of the automated sequencing showed that the DNA detected in the sample from Sena Madureira (DOG SM-AC-BR) indicated 99% similarity with 3 sequences of E. vogeli and the sample DOG RB-AC-BR with E. granulosus (s.s.) (G1). The pairwise distance between our samples was 0.094 (variance 0.017). The pairwise distances between DOG SM-AC-BR and E. vogeli  was 0.003 (variance 0.003) and between DOG RB-AC-BR and E. granulosus (s.s.) (G1)  was 0. Higher distances between DOG RB-AC-BR were also observed comparing this element to other sequences used for phylogenetic characterization: 0.094 (variance 0.018) for E. ortleppi (M84665); 0.088 (variance 0.016) for E. equinus (M84664); 0.008 (variance 0.005) for E. granulosus (s.s.) (G2) (M84662); and 0.006 (variance 0.004) for E. granulosus (s.s.) (G3) .Phylogenetic trees based on Neighbor-Joining algorithm with Kimura 2-parameter model confirmed the species identification  in rural communities, it is important to evaluate the risk of transmission of helminthic zoonoses from dogs to humans . AlthougE. vogeli has not yet been investigated. Over the past decade, the epidemiology of echinococcosis has achieved significant advances based on molecular assays [E. granulosus (s.l.) in canids [In northern Brazil, polycystic echinococcosis has been reported in bush dogs , 18, pacr assays , 25, incn canids –29.Echinococcus proglottids in feces. Secondly, copro-PCR associated with automated sequencing allowed the identification of E. vogeli and E. granulosus (s.l.), consistent with epidemiological studies of canids in China [E. vogeli-positive sample was collected from the municipality of Sena Madureira, where clinical cases and seroreactivity to polycystic echinococcosis have been described in urban and rural areas [Our parasitological and molecular findings give rise to some important points. First, there was no finding of dogs having passed in China  and Iranin China . Thirdlyal areas , 49. Foual areas , 14, 50.E. granulosus (s.l.) [E. granulosus (s.s.) (G1) from known endemic areas in southern Brazil [In agreement with previous investigations about s (s.l.) , 12, 51,s (s.l.) . In Acres (s.l.) . From thn Brazil , 53, 54.E. vogeli infection, reinforcing the role of domestic dogs as sources of infection for humans in the municipality of Sena Madureira, where human polycystic echinococcosis has been previously reported [E. granulosus (s.s.) (G1) was detected for the first time in the North Region, from the epidemiological standpoint this finding is highly relevant because it expands the known geographical distribution, which was previously restricted to the South of Brazil.To the best of our knowledge, this article contains the first report of molecularly detected reported , 45. GivThe strengths of our findings should be interpreted in light of limitations. In the state of Acre, during the rainy season (from October to May) the condition of unpaved roads is poor. As a consequence, field studies were only carried out in the dry season. In urban areas, sympatric domestic dogs have restricted space and movements, depending on care and food given by their owners. Management of domestic dogs in rural landscapes is difficult because they often are allowed to roam freely and the size of local dog population is unknown. Although the owners reported they owned one or more dogs, we were unable to collect feces directly after being deposited by the dogs because they were absent during our visit or defecated elsewhere.Echinococcus species. To the best of our knowledge, this is the first report of E. vogeli and E. granulosus (s.s.) (G1) confirmed in domestic dogs from the North Region of Brazil. Further studies are needed to evaluate the extent of canine infection and the formulation of echinococcosis monitoring, considering the circulation scenario of both etiological agents.The environmental contamination with domestic dog feces is a risk factor for the transmission of helminthic zoonoses in Acre. Molecular diagnosis through copro-PCR and automated sequencing were suitable for the identification of


Plasmodium falciparum parasites resistant to ART and partner drugs are spreading, which threatens malaria control efforts. Rodent malaria species are useful models for understanding antimalarial resistance, in particular genetic variants responsible for cross resistance to different compounds.The current first line drugs for treating uncomplicated malaria are artemisinin (ART) combination therapies. However, Plasmodium berghei RC strain (PbRC) is described as resistant to different antimalarials, including chloroquine (CQ) and ART. In an attempt to identify the genetic basis for the antimalarial resistance trait in PbRC, its genome was sequenced and compared with five other previously sequenced P. berghei strains.The PbRC is eight-fold less sensitive to the ART derivative artesunate than the reference strain PbANKA. The genome of PbRC is markedly different from other strains, and 6,974 single nucleotide variants private to PbRC were identified. Among these PbRC private variants, non-synonymous changes were identified in genes known to modulate antimalarial sensitivity in rodent malaria species, including notably the ubiquitin carboxyl-terminal hydrolase 1 gene. However, no variants were found in some genes with strong evidence of association with ART resistance in P. falciparum such as K13 propeller protein.We found that PbRC provide insight into P. berghei genome diversity and genetic factors that could modulate CQ and ART resistance in Plasmodium spp.The variants identified in  Plasmodium falciparum parasites could undermine all recent advances in reducing the disease burden. Laboratory models of antimalarial resistance are needed to develop new drugs effective against parasites resistant to currently available antimalarials, and to understand the molecular mechanisms of resistance.The incidence of malaria is declining around the world, and efforts are being directed toward elimination of this disease in many endemic areas . HoweverPlasmodium chabaudi rodent malaria parasites from laboratory selection were isolated in Plasmodium yoelii rodent malaria parasites display impaired hemozoin production and elevated level of glutathione (GSH), suggestive of a common mechanism of resistance against the two drugs (Plasmodium berghei. The P. berghei RC strain (PbRC) was obtained by laboratory selection with CQ, and is defective for production of hemozoin , a part of BEI Resources, NIAID, NIH: Plasmodium berghei RC, MRA-404, deposited by W. Peters and B.L. Robinson; Plasmodium berghei ANKA 507m6cl1, MRA-867 deposited by C.J. Janse and A.P. Waters. Artesunate was a gift from Dafra Pharma, Turnhout, Belgium. Other reagents, unless otherwise noted, were purchased from Sigma Aldrich .Female BALB/c mice of about 6 to 10 weeks old were purchased from the National Laboratory Animal Center, Mahidol University, Thailand. The following parasites were obtained from the Malaria Research and Reference Reagent Resource Center  using primers listed in Selected genomic regions with candidate variants in the PbRC parasite was passed through a CF11  column to remove white blood cells. Cells were harvested by centrifugation and parasites were liberated from red cells by lysis of the red blood cell membrane with 0.2% saponin. Parasites were washed twice with phosphate buffered saline , resuspended in lysis buffer  and incubated at 37 °C overnight. Proteins were precipitated by the addition of NaCl to 1.5 M, centrifuged and discarded. Isopropanol was added to the supernatant to precipitate nucleic acids. The nucleic acid pellet was dried and resuspended in TE buffer . RNA was removed by digestion with RNAseA. Phenol chloroform extraction and ethanol precipitation of DNA were performed. The integrity of genomic DNA was checked by agarose gel electrophoresis. Genomic DNA was submitted to the Chulalongkorn Medical Research Center, Bangkok, Thailand for genome sequencing. Genomic DNA was sheared by sonication and DNA fragments <1 kb were gel-purified. Sequencing libraries were constructed from sheared genomic DNA using a TruSeq kit . About 2 × 150 bp reads were obtained using a MiSeq instrument (Illumina). The raw data are deposited in the NCBI Sequence Read Archive, accession number PRJNA277169.Parasitized blood obtained from a single mouse infected with the P. berghei ANKA version 3 reference genome  was performed using the ipPCA tool , implemeCopy number variants (CNVs) were identified from the whole-genome sequencing data using the cn.MOPS tool implemented in R . The seqPbRC parasite is reported as CQ and ART resistant  of 2.8 (s.e. = 0.4) and 23.1 (s.e. = 7.3) mg/kg in PbANKA and PbRC, respectively; The esistant . We testPbRC genomic DNA was performed. Sequence reads were obtained from 17,801,263 clusters, and 80.5% of the preprocessed reads could be mapped to the PbANKA v3 reference genome. Genomic sequence data of previously sequenced strains K173, NK65NY, NK65E, SP11_RLL and SP11_A , PBANKA_041290 , PBANKA_1115300  and PBANKA_1137800 .A previous analysis of  species . Howeverng sites . The meang sites . Howeverng sites . At the ng sites . No specPbRC located within genes were examined to identify putative causal variants of the drug resistant trait in this strain. To our knowledge, the only prior evidence of PbRC genetic variants compared with the PbANKA reference is an Ile to Lys substitution at residue 413 (I413K) of the gamma glutamylcysteine synthetase (Pbγ-gcs) gene  gene . We conf to PbRC . A previRC  gene modulate CQ and ART resistance in laboratory-selected drug resistant parasites (PbRC were found in the homologous Pbubp1 gene. Mutations in the CQ resistance transporter (crt) gene modulate CQ resistance in P. falciparum (PbRC was found in the P. berghei homologue (Pbcrt). Mutation of the P. falciparum multidrug resistance associated protein 1 (mdr1) gene modulates sensitivity to several antimalarials, including CQ and ART  , a gene mutated in the P. chabaudi CQ- and ART-resistant AS-ART strain , mutations of which confer reduced ART sensitivity in P. falciparum (We searched for candidate drug resistance variants in genes known to modulate CQ and/or ART drug resistance in other arasites . Five nolciparum . A non-s and ART . A non-s(PfPI3K) . PbRC muT strain  and K13 lciparum .PbRC is defective for hemoglobin catabolism and hemozoin formation (PbRC harbored private variants in genes encoding proteins known to function in catabolism of hemoglobin (PbRC was found only in the dipeptidyl aminopeptidase 3 gene (Pbdpap3)  .PbRC private variants in other genes may be responsible for the traits associated with this strain. PbRC does not produce gametocytes  are prevalent in all strains except PbSP11_A, and PbRC has the greatest number of large deletions.Finally, drug resistance in s common . CNVs aming data . All of P. berghei genomes revealed that the majority of SNVs are private to each strain, in which PbRC is most diverged strain of all. Moreover, the genome-wide pattern of negative Tajima’s D score  are defective for oocyst production , are coookinete . The beroduction . The GAPoduction . The higPbRC makes it difficult to pinpoint causal variants of its drug-resistant phenotype. Moreover, the N strain progenitor from which PbRC was derived by CQ selection was not available to us, and forward genetic mapping is not possible since PbRC does not produce gametocytes ; hence, it is difficult to predict if this mutation affects PbPI3K protein function.Genome sequencing of the CQ- and ART-resistant clone AS-ART isolated in n AS-ART . AP2-μ gn AS-ART . In thisy to ART ; howevertination . PbRC haPfcrt gene modulate CQ sensitivity in P. falciparum, and a V42F variant was found in the homologous gene (Pbcrt) of PbRC , one plasmepsin (plasmepsin IV) and one hemoglobin digestive protein (HDP)  . No vari berghei , and so PbRC private variants with other phenotypic traits  in the vicinity of PBANKA_1443800.1 . Mapped reads from P. berghei RC are shown in the tracks labeled RC.final.bam.Coverage and RC.final.bam. Genomic variants among the six P. berghei strains before and after filtering are shown in the tracks labeled raw_variants and filtered_variants, respectively.Integrative Genomics Viewer screenshot showing Click here for additional data file.


Thelohanellus from India have been described according to morphometric characteristics. Nevertheless, molecular data on these histozoic myxosporean parasites of freshwater fish are scarce. In the present study, molecular characterizations of Thelohanellus qadrii infecting the secondary gill epithelium of Indian major carp Catla catla  and its phylogenetic relationship is reported. The sub-adult cultured catla were observed to have low to moderate gill myxosporean infections. The morphometry of mature spores was in compliance with original descriptions of T. qadrii. Based on the analysis of 18S rRNA gene, phylogenetic clusters which were established according to a consensus sequence, illustrated the taxonomic placement of a series of myxobolids. The DNA sequence homogeneity of T. qadrii (KF170928) with other Thelohanllus spp. ranged from 78% to 95% and formed a dichotomy with cyprinid gill lamellae infecting T. toyamai (HQ338729). Distance matrix results indicated a high genetic diversity among myxosporeans. The present report is the first on the molecular and phylogenetic characterizations of T. qadrii.Myxosporean taxonomy which is traditionally based on the morphology of the myxospore stage, is in a state of flux given new insights provided by the expanding dataset of DNA sequences. To date, more than 40 species of  Thelohanellus Kudo, 1933 are typically histozotic (rarely coelozoic) bivalvulidan myxosporean parasites of freshwater fish with a high diversity of infection sites  fT. qadrii. Earlier, we characterized T. caudatus (KC865607) infecting the caudal fin of L. rohita . According to Eszterbauer [Thelohanellus qadrii clustered with cyprinid gill infecting myxosporeans with 89-95% homogeneity. The phylogenetic tree placed T. qadrii within the freshwater clade. It formed a dichotomy with the closely related T. toyamai (HQ338729) that infected the gill lamellae of common carp, Cyprinus carpio from the USA with maximum node support. In addition, T. qadrii exhibited maximum DNA sequence homogeneity (95%) with T. toyamai (HQ338729). Our previously characterized fin infecting T. caudatus (KC865607) exhibited 78% homogeneity with T. qadrii. Furthermore, freshwater and marine clades (Myxidium incurvatum (DQ377708) and Kudoa iwatai (AY514038)) were distinctly separated within the lineage Myxosporea. Other representatives of the Myxobolidae clade such as Ceratomyxa, Henneguya, Kudoa, Myxidium and Myxobolus were also distinctly different from the gill-infecting Thelohanellus spp. and clustered separately. The out-group B. plumatellae (AY074915) of the Malacosporea class was distinctly and phylogenetically clustered as a separate lineage with Myxosporea. The wide range observed in the evolutionary pair-wise distances between T. qadrii and other myxosporeans (0.00-0.56) including the gill infecting Myxobolus carnaticus (0.23) and the fin infecting T. caudatus (0.25-0.43) from India possibly indicated the high genetic diversity of myxosporeans.This is the first report regarding the molecular and phylogenetic characterization of . rohita . In thisudatus KC65607 infMyxosporeans are best known for the diseases they cause in commercially important fish hosts. With the huge global expansion of freshwater aquaculture, several myxosporeans have been recognized or elevated in status as important pathogens , 6. Sincassumed. Although no mortality was noticed, such infection may cause substantial production loss. Further studies are warranted to establish this pathogenicity and its role in production loss.