Euonymus hamiltonianus and its relatives (Celastraceae family) are used for ornamental and medicinal purposes. However, species identification in Euonymus is difficult due to their morphological diversity. Using plastid genome (plastome) data, we attempt to reveal phylogenetic relationship among Euonymus species and develop useful markers for molecular identification. We assembled the plastome and nuclear ribosomal DNA (nrDNA) sequences from five Euonymus lines collected from South Korea: three Euonymus hamiltonianus accessions, E. europaeus, and E. japonicus. We conducted an in-depth comparative analysis using ten plastomes, including other publicly available plastome data for this genus. The genome structures, gene contents, and gene orders were similar in all Euonymus plastomes in this study. Analysis of nucleotide diversity revealed six divergence hotspots in their plastomes. We identified 339 single nucleotide polymorphisms and 293 insertion or deletions among the four E. hamiltonianus plastomes, pointing to abundant diversity even within the same species. Among 77 commonly shared genes, 9 and 33 were identified as conserved genes in the genus Euonymus and E. hamiltonianus, respectively. Phylogenetic analysis based on plastome and nrDNA sequences revealed the overall consensus and relationships between plastomes and nrDNAs. Finally, we developed six barcoding markers and successfully applied them to 31 E. hamiltonianus lines collected from South Korea. Our findings provide the molecular basis for the classification and molecular taxonomic criteria for the genus Euonymus (at least in Korea), which should aid in more objective classification within this genus. Moreover, the newly developed markers will be useful for understanding the species delimitation of E. hamiltonianus and closely related species. Euonymus hamiltonianus Wall., a plant belonging to the Celastraceae family, is widely distributed from Northern India to Far East Asia (http://www.plantsoftheworldonline.org). E. hamiltonianus are valuable ornamental plants due to their beautiful shapes and colors. However, the morphological variations lead to ambiguous delimitation of this species. The uncertainty of species delimitation is not only the case for E. hamiltonianus but also for other closely related species. Fruit morphology of Euonymus was used to divide them into five sections, but molecular phylogeny of Euonymus was poorly supported in the previous study [us study .Euonymus [matK, rbcL, and nuclear ribosomal internal transcribed spacer (nrITS) regions have been utilized to construct the phylogenies of Celastraceae [Euonymus have been conducted on individual species [Euonymus plastomes was recently conducted, no in-depth analysis of one species with its relatives has been performed [E. hamiltonianus and its relatives, it is important to conduct in-depth comparative analysis using various resources.Although Celastraceae is a large family containing 96 genera and 1,350 species, few studies have been conducted for the genus Euonymus –4. Univestraceae . Neverth species . Even thPlastomes have been widely employed for phylogenetic studies and DNA barcoding in various plants due to their uniparental inheritance  in most land plants . UnipareNuclear ribosomal DNAs such as 45S rDNA and 5S rDNA exist in a tandem repeat array of hundreds to thousands of copies and are genetically conserved due to concerted evolution ,22. HoweEuonymus plants by de novo assembly of low-coverage whole-genome shotgun sequencing (dnaLCW) [Euonymus plastomes using data from NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The newly discovered genetic features of E. hamiltonianus and its relatives advance our understanding of the molecular identification and plastome evolution of these species.In this study, we assembled the complete plastomes and nrDNA (45S and 5S) sequences of five (dnaLCW) ,28. We aE. hamiltonianus, one E. europaeus, and one E. japonicus were collected from various sources including wild and commercial samples  libraries. The libraries were sequenced using the Illumina MiSeq platform at Phyzen . This analysis yielded 0.82–1.20 Gbp of sequencing data per sample.Thirty-one  samples . The colde novo assembly using the CLC assembly cell . Among assembled contigs, only contigs with similarity to the reference plastome  were extracted by MUMmer and BLASTZ [https://chlorobox.mpimp-golm.mpg.de/geseq) and Artemis [All newly assembled plastomes and 45S rDNA were assembled using the dnaLCW method ,28. In sd BLASTZ ,31. Fina Artemis ,33.E. hamiltonianus, KY921875.1) were extracted. The start and end position of each 45S rDNA subunit  were determined using RNAmmer and the reference sequence [Assembly of 45S rDNAs was performed in a similar manner following the pipeline used for plastome assembly. Only contigs with sequence similarity to the reference 45S rDNA . After completing the assembly of 5S rDNA, the IGS region was assembled by mapping and elongation. Elongation of the IGS region was repeated until the mapped reads met the next 5S rDNA unit. Finally, the complete 5S rDNA unit with IGS sequences was confirmed via NCBI BLAST [Assembly of 5S rDNA was performed by reference mapping. Briefly, the draft sequence of 5S rDNA was assembled by mapping raw reads to the reference sequence of BI BLAST .Euonymus plastome sequences were obtained from NCBI GenBank (E. hamiltonianus (NC_037518.1) as a reference sequence with default parameters [https://irscope.shinyapps.io/irapp/) [In addition to the five newly assembled plastomes, five other previously reported  GenBank . The simrameters ,37. Stru/irapp/) .Euonymus plastomes were aligned using the PRANK aligner with the +F option [http://circos.ca/) [To check the variants in whole plastomes, the ten F option –41. The cos.ca/) . Pi valucos.ca/) .Euonymus plastomes and one Catha edulis plastome (GenBank accession No.: KT861471) were used for comparative analysis. Among the 88 protein-coding genes, the sequences of 77 genes were collected because they were nonredundant and shared by the 11 individuals. The dN/dS analysis was performed on these common genes using a branch-site model  of codeml in the paml version 3.14 package [Ten  package . A likelThe 77 protein-coding gene sequences were independently aligned using PRANK aligner with the +F and translate option, and then were concatenated as a single supermatrix ,40. The https://mafft.cbrc.jp/alignment/software/) [To reconstruct the 45S rDNA phylogeny, species in ftware/) . The subftware/) ,47.E. hamiltonianus individuals. Three SNP markers were developed into dominant markers, while three InDel markers were codominant. All marker sets were subjected to in silico validation with NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) [Euonymus samples. The following were the PCR amplification conditions: Initial denaturation: 94°C for 5 minutes, denaturation: 94°C for 20 seconds, annealing: 58°C for 20 seconds, extension: 72°C for 20 seconds, final extensions: 72°C for 7 minutes. Denaturation, annealing and extension were conducted 35 cycles. After that, the PCR amplicon was validated by 3% agarose gel with gel-electrophoresis. E. japonicus (section Ilicifolii) was excluded from this validation, because the species was phylogenetically distant from E. hamiltonianus and E. europaeus (sect. Euonymus). Grouping of each accession was conducted with PowerMarker v3.25 by the NJ method with 1,000 bootstrap replicates [Three single nucleotide polymorphisms (SNPs) and three insertion or deletions (InDels) markers were developed based on the plastome variations among four -blast/) . The newplicates . Finallyplicates .Euonymus lines. By adding plastome sequences from NCBI, a total of ten complete plastomes, six 45S rDNA sequences, and six 5S rDNA sequences were compared in downstream analyses.We assembled complete plastomes, 45S nrDNA, and 5S nrDNA sequences from five Euonymus plastomes ranged from 157,263 bp to 157,702 bp in size, with a quadripartite structure that is typical of plastomes in most plant species   . By contEuonymus plastomes. Hundreds of SNPs (71–270) and InDels (70–213) were identified among the four E. hamiltonianus lines  . Among the polymorphic sites, 1,767 sites (1.15%) were parsimony informative sites, while the remaining 810 sites (0.53%) were singleton sites. The pi value was calculated as 0.00695 throughout the plastome with sliding window methods . The individual windows had pi values ranging from 0 to 0.02993. The highest pi value was observed in the rbcL–accD region (0.02993). Six intergenic regions  had the higher pi values than the other regions (> 0.02). All six highly diverged regions were located in SC regions: five of these were located in the LSC region, and only one (ndhH–ycf1a) was located in the SSC region.To identify divergence hotspot regions within the i value) . We idenEuonymus plastomes, we estimated the ratio of non-synonymous (dN) to synonymous (dS) substitution rates (dN/dS). Based on dN/dS ratios, no gene was estimated to be under positive selection (>1.0), which was similar to most seed plants [psbI, psaI, psbJ, psbF, psbT, psbN, and ycf3) and two ribosomal protein genes (rpl36 and rps7). In addition, 15 genes  contained only synonymous substitution sites. In other words, these 24 genes had conserved protein-coding sequences in the Euonymus plastomes. Among the four E. hamiltonianus plastomes, 51 genes had conserved protein-coding sequences, 33 of which had no nucleotide variations.To investigate sequence divergence of protein-coding genes in the d plants –54. HoweE. japonicus-specific SNPs and 17 SNPs in E. hamiltonianus and E. europaeus. Among the 112 SNPs identified, 5, 3, 34, and 70 were detected in 18S, 5.8S, 26S, and ITS regions, respectively  than in other species (343 to 347 bp) with abundant SNPs . The BI and ML trees showed almost identical topologies  was sister to the three other E. hamiltonianus accessions. However, the phylogenetic relationship inferred from nrDNA sequences was inconsistent with the results inferred from plastome sequences (E. hamiltonianus accessions (Hantaek and Jeju) and the two other E. hamiltonianus accessions (Hongcheon and ‘Snow’) formed independent subclades, with sister relationships  differed from those of E. hamiltonianus and E. europaeus by having a shorter IGS (139 bp) with lower sequence homology. Such variations in the IGS sequence have been reported for other plants [Nicotiana tabacum and Solanum species, respectively [The complete 5S rDNA sequences showed high similarity among the  species . Howeverr plants –60. For ectively ,60.Euonymus may have undergone a specific evolutionary event. Further research will be needed to elucidate the genomic characteristics and evolutionary history of the nuclear genomes of Euonymus species.Considering its sequence characteristics, the 5S rDNA tandem repeat array in Euonymus lines could also be divided into two groups. We predicted a divergence event in the Euonymus plastomes based on the results of phylogenetic analysis and differences in IR junctions. The Euonymus plastomes could be divided into two types based on the location of the rps19 gene. The plastomes of E. hamiltonianus, E. europaeus, and E. szechuanensis had shorter IR regions, and the rps19 gene was located in the LSC region  had higher posterior probabilities than the other genes, with a significant p-value (< 0.05)  . Both gesubunits ,69. Prodsubunits . The losy plants ,71,72. Ipressure . The dN Euonymus species and performed comparative analysis. The plastome structures in the Euonymus genus were quite similar. However, the divergence of the Euonymus plastomes were revealed by comparing IR junctions and calculating pi values. We also confirmed the sequences and structures of nrDNAs in Euonymus. Phylogenetic analysis revealed possible cytonuclear discordance between the plastid and nuclear genomes, which we used to infer the times of IR expansion/contraction. The six molecular markers developed in this study will be useful for exploring genetic diversity of E. hamiltonianus distributed in South Korea. Further studies might help confirm the putative cytonuclear discordance between the organelle and nuclear genomes, as well as the divergence of the ndhE gene in E. hamiltonianus through large-scale data analysis.In this study, we documented the complete plastomes of S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S7 Fig(DOCX)Click here for additional data file.S8 Fig(DOCX)Click here for additional data file.S9 Fig(DOCX)Click here for additional data file.S10 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Purpose: Observational research has indicated the presence of a causal relationship between sleep disturbances and low back pain (LBP). However, the link may have been biased by confounding factors. The purpose of this study was to examine the potential causal association of insomnia and daytime sleepiness with LBP by using mendelian randomization (MR).Methods: Genome-wide association study (GWAS) summary statistics of insomnia were obtained from a large-scale GWAS meta-analysis  or UK Biobank alone . The summary statistics of daytime sleepiness were from UK Biobank  and LBP were provided by the FinnGen Release 6  or UK Biobank . Linkage disequilibrium score (LDSC) regression and bidirectional MR analysis was employed to estimate genetic correlation and causal relationship. In the MR analysis, the inverse variance weighted method (IVW) was utilized as the main analysis procedure, while MR-Egger, Weighted median and Robust adjusted profile score (RAPS) were utilized for supplementary analyses.Results: LDSC analysis showed that LBP were significantly genetically correlated with insomnia  and daytime sleepiness . The MR analysis revealed that genetically predicted insomnia was significantly associated with an increased risk of LBP . However, the reverse causality was not confirmed. No evidence was identified supporting causality of daytime sleepiness and LBP.Conclusion: This study demonstrates a putative causal link of insomnia on LBP and a null causal effect of LBP on insomnia. Furthermore, a causal link between daytime sleepiness and LBP were not reported. This finding may stimulate new strategies for patient management in clinical practice, benefiting public health. Low back pain (LBP) is one of the most common patient complaints in orthopedic outpatient departments and imposes a high burden on individuals and society. According to The Global Burden of Disease study 2013, chronic LBP is the most frequent cause of disability worldwide . The preInsomnia, a frequently occurring sleep disorder characterized by trouble falling and/or staying asleep, is commonly reported by LBP patients, with a prevalence of at least 50% . A cohorTraditional observational studies are inevitably biased since they are subject to many confounding factors. On the other hand, large randomized trials, which can provide the best evidence for causation, are expensive and time-consuming. Mendelian randomization (MR) provides an ingenious method for inferring the causal nature of the exposure-outcome relationship by using instrumental variables (IVs), such as single nucleotide polymorphisms (SNPs) . Due to Previous MR studies have successfully confirmed that sleep traits contributed to the risk of several diseases including psychiatric disorder , cardiovvia the exposure (Assumptions Ⅲ)  . Robust N = 1,331,010 individuals from UK Biobank and 23andMe) GWAS meta-analysis of insomnia from individuals of European ancestry  (Genetic correlations (rg) of insomnia, daytime sleepiness with LBP were estimated using Linkage disequilibrium score regression (LDSC) software package (ik/ldsc) .-statistics markedly greater than 10 usually indicates a small instrument bias  developed by Burgess and coworkers  and the lowest for changes in daytime sleepiness   .r2 < 0.001). Unless otherwise specified, significance threshold p of < 5 × 10−8 and the absence of linkage disequilibrium (LD)  were selected in our MR analysis. Under the same conditions, 38 SNPs as IVs of daytime sleepiness were obtained. In the reverse direction study, SNPs were identified to associate with LBP at a broadened threshold (p < 5 × 10−6)  . We extrInitially, 4 SNPs of 158 lead SNPs were not found in the summary statistics for LBP . Subsequently, nine SNPs  were removed for being palindromic with intermediate allele frequencies. For the remaining 145 SNPs, MR-PRESSO test detected seven potential outliers . Finally, 138 SNPs as IVs of insomnia were selected to perform the MR analysis.p = 1.69e-16] (p = 2.98e-05), RAPS  except for MR-Egger method  (p = 0.435) and Cochran’s Q test did not detect significant heterogeneity in both MR-Egger and IVW methods (p > 0.05) , 1.186–1.318; .69e-16] . This st= 0.179) . The MR- > 0.05) . The cau > 0.05) . Based o > 0.05) . Further > 0.05) . The meap = 0.636), and the other methods were concordant with this result (p = 0.135). Cochran’s Q test did not report any heterogeneity (p > 0.05)  was not found in the summary statistics for LBP and one SNP (rs2048522) was removed for being palindromic. In the MR-PRESSO test, no outliers were detected. Finally, 36 SNPs as IVs of daytime sleepiness remained in this part of the analysis. No genetic effect of daytime sleepiness on the risk of LBP was found by IVW method  . Supplemp = 0.573; MR-Egger: OR = 0.989, CI: 0.971–1.007, p = 0.235; Weighted median: OR = 1.00, CI: 0.986–1.014, p = 0.996; RAPS: OR = 1.00, CI: 0.993–1.012, p = 0.663)  were found as a potential outlier by MR-PRESSO analysis. All MR methods did not support significant causal effect of LBP on insomnia  . The sca= 0.663) . The forp = 0.235). The Weighted median, MR-Egger and RAPS estimations provided supporting evidence , while there was no evidence of a reverse causality. No association was found between daytime sleepiness and LBP. No significant heterogeneity and pleiotropy were found in the replication practice. Specific information could be found in Summary statistics of LBP from UK biobank were included for replication purposes. In the analysis, genetically determined insomnia was positive associated with the risk of LBP  and LBP by MR methods, extending the relevant studies. Second, potential confounders and other biases were removed by genetic variants. Third, potential horizontal pleiotropy was detected and corrected by the MR-PRESSO method.Some inevitable limitations should also be mentioned. Firstly, our study cannot evaluate the influence of within-population structures resulting from the between-sex difference in the prevalence of insomnia . SecondlIn summary, the results of this study reveal a deleterious effect of genetically predicted insomnia on LBP and a null causal effect between daytime sleepiness and LBP. Nonetheless, large-scale longitudinal studies, as well as in-depth mechanistic studies, need to be done. Importantly, the MR result implies that attention should be paid in clinical practice to comorbid insomnia and chronic LBP. A better understanding of the relationship between these two conditions will contribute to pain prevention and improvement in global public health.


Diaphorina citri, the vector of the citrus greening disease pathogen ‘Candidatus Liberibacter asiaticus’, has a reduced immune system in comparison to the non-hemipteran insects, including those of Lepidoptera, Dipterans, and Hymenoptera. Other hemipterans, such as Acyrthosiphon pisum, and Rhodnius prolixus, possess a partial or absent immune deficiency (IMD) pathway. D. citri does not have the IMD pathway, but has a complete Toll system that protects them from Gram-positive bacteria. Here we characterized the Toll signaling system genes in D. citri. In addition, we studied the expression profile of Toll genes in different life stages. Discovering this system and its dynamic expression may help in designing an innovative strategy by which we can control this insect vector.Diaphorina citri is the main vector of citrus greening disease, also known as Huanglongbing (HLB). Currently, mitigating HLB depends on the control of D. citri using insecticides. To design innovative control strategies, we should investigate various biological aspects of D. citri at the molecular level. Herein we explored the Toll signaling system-related proteins in D. citri using in silico analyzes. Additionally, the transcripts of the identified genes were determined in all life stages from eggs to adults. Our findings reveal that D. citri genome possesses Toll signaling pathway-related genes similar to the insect model, Drosophila melanogaster, with slight differences. These genes include cact, TI, Myd88, Dif/DI, pll, tub, and spz encoding Cactus, Toll, Myeloid differentiation factor 88, Dorsal related immunity factor/Dorsal, Pelle, Tube, and Spaetzle, respectively. Unlike D. melanogaster, in D. citri Dorsal, immunity factor and Dorsal are the same protein. In addition, in D. citri, Pelle protein possesses a kinase domain, which is absent in Pelle of D. melanogaster. Gene expression analysis showed the transcript for cact, TI, Myd88, pll, tub, and spz are maximum in adults, suggesting the immunity increases with maturity. Instead, Dif/DI transcripts were maximal in eggs and adults and minimal in nymphal stages, indicating its role in embryonic development. The overall findings will help in designing pioneering control strategies of D. citri based on repressing its immunity by RNAi or CRISPR and combining that with biological control.The Asian citrus psyllid,  Candidatus Liberibacter asiaticus’ is a Gram-negative bacterium, not available in culture, and phloem-limited [Ca. L. asiaticus’ is transmitted by Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) in a circulative–propagative manner [D. citri, and thus, to reduce the spread of ‘Ca. L. asiaticus’ [D. citri populations [D. citri populations, especially if overwintering adult populations were targeted during tree dormancy [Citrus greening disease, also known as Huanglongbing (HLB), is one of the most destructive citrus diseases worldwide . It thre-limited ,9,10. ‘Ce manner ,11. All e manner . Currentiaticus’ ,14. Appliaticus’ . Additioulations ,17,18. Tdormancy ,20,21. TIn general, insects lack adaptive immune systems , but theD. citri has a reduced immune system compared to the non-hemipteran insects including those of Lepidoptera, Diptera, and Hymenoptera [Acyrthosiphon pisum Harris, Rhodnius prolixus Stal, and Siphonaptera, Pediculus humanus Linnaeus possess a partial or absent IMD pathway [D. citri has no IMD pathway but it has a complete Toll system that improves its survival after infection with Gram-positive bacteria [D. citri and some other insects, such as A. pisum, the insects compensate this deficiency by using bacterial endosymbionts. It is reported that Wolbachia symbiont activate the immune system in mosquitoes and cause resistance against the pathogens [Candidatus Profftella armatura’ in D. citri produces a cytotoxic polyketide toxin, diaphorin, and act as a potential defensive symbiont against pathogens [Ca. L. asiaticus’-infected D. citri [D. citri using the Drosophila melanogaster Toll pathway genes and protein sequences as inquiries in BLAST. We used bioinformatic techniques to identify Toll signaling pathway-related proteins and predict their three-dimensional structure, active domains, and binding-receptor profiles. We performed RT-PCR to study the gene expression profiles for all predicted Toll genes in D. citri developmental stages from egg to adult.enoptera ,45,46,47 pathway ,49,50. Dbacteria . Becauseathogens . ‘Candidathogens ,54. It wAll experiments were performed at the laboratories and facilities of the Citrus Research and Education Center (CREC), Institute of Food and Agriculture Sciences (IFAS), University of Florida, Lake Alfred, Florida.D. citri, was originally collected in 2014 from citrus groves in Polk County, Florida, USA. Colonies of D. citri were continuously reared on Citrus macrophylla and Murraya koenigii (curry leaf) in secured growth rooms  at the Citrus Research and Education Center (CREC), University of Florida, Lake Alfred, Florida. The adults of D. citri and samples of Citrus macrophylla leaves were tested randomly every month for the presence of ‘Ca. L. asiaticus’ by polymerase chain reaction (PCR) assays described by Tatineni et al. (2008) [Ca. L. asiaticus’-free.The Asina citrus, psyllid, . (2008)  to keep D. citri that resembled the query amino acid sequence of known Toll pathway genes of the model insect, Drosophila melanogaster. In brief, the protein sequences of Toll pathway-related genes Cactus  [Dif gene; AAA28465.1) [TI gene; AAA28941.1) [Pll gene; AAA28750.1) [tub gene; P22812.4) [Di gene; NP_724054.1) [Myd88 gene; AAL56570.1) [Spz NP_733194.1) [D. melanogaster, were blasted against D. citri using protein–protein BLAST (BLASTp 2.11.0+) [https://www.ncbi.nlm.nih.gov/gene/, 15 April 2021), and “Diaphorina citri OGS v2.0 proteins” and “Diaphorina citri OGS v2.0 CDS” Blast database available in the Citrus Greening Solutions website  [D. citri.The protein–protein BLAST (BLASTp) algorithm from the basic local alignment search tool (BLAST) was performed to identify sequences of Toll signaling pathway-related genes in 85908.1) , Dorsal-28465.1) , Toll (T28941.1) , Pelle (28750.1) , Tube (t24054.1) , Myeloid56570.1) , and Spä33194.1) , from D.2.11.0+) ,62, baseil 2021) , using cil 2021) . Myd88 aD. melanogaster in the NCBI BLASTp search were retrieved and aligned using the constraint-based multiple alignment tool (NCBI COBALT)  with default parameters. The highly conserved and low-level conserved areas of each sequence were identified with different colors.For each protein, top-matched amino acid (AA) sequences from different insect families showing high similarity with Tube, Pelle and Dorsal proteins from D. melanogaster and D. citri and insects belonging to several families that showed high similarity (over 25%) and had good coverage were selected for the analyzes. Multiple alignment of AA sequences was performed using CLUSTALW. The phylogenetic analysis of protein sequences was conducted using MEGA X software [Pelle, Tube, and Dorsal proteins from software . The evosoftware , and thesoftware . Initialhttps://swissmodel.expasy.org/, accessed on 14 April 2021). The PDB structure of each protein was generated by submitting the protein FASTA sequences to the website. The template was selected based on quality of the resulting models, as estimated by quaternary structure quality estimate (QSQE), global model quality estimate (GMQE), coverage, sequence identity, and sequence similarity [https://www.cgl.ucsf.edu/chimera/, accessed on 15 May 2021) [The protein structure homology modeling, structure-based function annotation, creation of three-dimensional (3D) structure, and prediction of the membrane topology of the predicted sequence were conducted using Swiss-model web software (milarity . The UCSay 2021)  was usedD. citri eggs and stages, including five nymphal instars (1st to 5th), teneral, and mature adults. Teneral is the newly emerged adult, it is pale and soft-bodied, and it lasts a few hours before it gets harder and darker (mature). Eggs were collected using forceps and nymphs were transferred to petri dishes using a fine camel hairbrush. Nymphs were examined under a stereoscope to classify them into instars based on size and morphological features. Teneral and mature adults were collected using an insect aspirator and sex was determined under the stereoscope. Total RNAs were extracted from different stages of D. citri using the RNase mini kit . The quality and quantity of extracted RNA was measured by the Nanodrop 1000 spectrophotometer . The gene expression was measured by quantitative RT-PCR as described by Nehela et al. (2018) [alpha-tubulin were used as internal control genes [D. citri under biotic stresses. Both internal control genes gave very similar results, however, we used actin for normalization. The gene expression for each biological sample was performed in triplicate. Primers for the Toll signaling pathway-related genes  . The houol genes  for traned genes  were useproducts .D. citri stages, followed by post hoc pairwise comparisons using the Tukey–Kramer honestly significant difference test . Simple linear regression (SLR) analysis was performed to model the gene expression (as a dependent variable). The fitted regression line is expressed as a significant equation, as determined by the F test (p < 0.05). Both coefficients of determination (R2) and adjusted coefficient of determination (R2adj) were also obtained. Further, due to the observed nonlinear phenomena, data were fitted with a second-degree polynomial regression model (quadratic model) to understand the curvilinear relationship between life stage (as an independent variable) and gene expression (as a dependent variable). Polynomial regression models, the 95% confident curves for the estimated regression, quadratic equation, R2, R2adj, and p-value based on the F test (p < 0.05) were also obtained. JMP statistical software  was used for all statistical analyzes listed above.The analysis of variance technique (ANOVA) was used for statistical comparison of the gene expression among different D. citri OGS v2 protein dataset website  because we did not find any similarity in the BLASTp search using NCBI  search resulted in the identification of Toll-signaling pathway proteins, including Cactus, Toll, Pelle, Tube, Dorsal, Dorsal- immunity-related factor based on the NCBI database, while Spaetzle and MyD88 were identified based on the ing NCBI . InteresD. citri  and other insect families. The multiple protein sequence alignment using ClustalW and phylogenetic trees analysis showed that DcPelle was phylogenetically closer (72%) to the query sequence DmPelle and Pelle in other insects  begin with glutamine and end with aspartate (in 34 out of 35 insects). ScanProsite server predicted the presence of different functional motifs in Pelle protein in all insects, including the presence of protein kinase domain and conserved protein sequence (200 AA) in the protein kinase domain (D. citri and D. melanogaster (DmPelle (AAA28750.1) was aligned with the sequences of the top matched protein from the  insects A. Furthee domain B. InterPe domain C. The conogaster C. In brinogaster C.For the homology modeling, we used Swiss modeling to compare the crystallographic three-dimensional (3D) structure of DcPelle, (XP_026680533.1) and DmPelle (AAA28750.1). The 3D structures of DcPelle and DmPelle were predicted using the crystal structure of human IRAK1  in the Protein Data Bank (PDB ID: 6bfn.2. A), as the template for structural modeling. Based on the target and template chain sequence alignment, about 64% of amino acid residues of DmPelle (SER 197 to VAL 499) were modeled with template protein  with reliable global model quality estimation (GMQE = 0.30) and good qualitative model energy analysis  of DcPelle were modeled with the target protein  with good GMQE of 0.2 and reliable QMEAN of −2.93 D. The prD. citri  and 17 other insects , Halyomorpha halys (XP_014275275.1), Tribolium castaneum (XP_008198084.1), and Nilaparvata lugens (XP_039275902.1). However, the Tube protein from D. citri was phylogenetically far from the DmTube query, with less than 50% similarity indicating diversity of Tube between the insect model and D. citri. Multiple protein sequence alignment using COBALT analysis showed that all predicted Tube protein sequences have high homology with the D. citri Tube protein (XP_008485884.1). COBALT analysis shows that around 150 amino acids were conserved among D. citri and the other insects in these analyzes (DmTube (P22812.4) was aligned with the sequences of the top-matched protein sequences from the  insects A. Multipanalyzes A. HMMER D. melanogaster having death domain (IPR000488), and death-like domain (IPR011029) as a superfamily , Tube, death domain (IPR029397), death-like domain superfamily (IPR011029), protein kinase-like domain superfamily (IPR011009), serine/threonine-protein kinase (IPR008271) as the active site, protein kinase, ATP binding site (IPR017441), and unintegrated proteins, such as Interleukin-1 receptor-associated kinase (PTHR24419), AGAP003062-PA (PTHR24419:SF23), transferase domain 1 (G3DSA:1.10.510.10), phosphorylase kinase; domain 1 (G3DSA:3.30.200.20) and erfamily B. Among nogaster C.DmTube and DcTube separately. Three-dimensional structure of the complex between the death domains of Pelle and Tube (death domain of Tube) with Protein Bank Data ID: 1d2z.1.B and 2.00 Å resolution was selected as template for structural modeling of DmTube. The 3D structure for the DcTube protein was built after alignment of the target sequence and the template structure of interleukin-1 receptor-associated kinase 4 (IRAK4 in complex with inhibitor) with PDP ID: 6tia. 2. A and 2.52 Å regulations. In DmTube, approximately 32% of amino acid residues (SER 24 to GLU 172) were modeled with template protein . The predicted protein model had a reliable global model quality estimation (GMQE = 0.22) and good qualitative model energy analysis (QMEAN Z -score = −0.37)  structure for DcTube was predicted using the Swiss-model server. Among all templates predicted by the Swiss-model, there was not a common template for DcTube and DmTube. Therefore, the most accurate template based on sequence identity and QMEAN z-scores was selected for = −0.37) A. The pr= −0.37) A,B with = −0.37) C.In the predicted monomer DcTube, 67% of amino acid residues (residues GLU 150 to ASN 454) were modeled with the template protein  with a GMQE of 0.42 and an acceptable QMEAN score of −1.35 D. In theD. citri. The BLASTp search of D. melanogaster produced significant similarities to the embryonic polarity protein Dorsal-like isoform X1 (XP_017305228.1), embryonic polarity protein Dorsal-like isoform X2 (XP_026675878.1) and embryonic polarity protein Dorsal-like isoform X3 (XP_017305229.1). The availability of Dorsal protein sequences from different insect families allowed us to refine the phylogenetic analysis of Dorsal protein sequences across species. Multiple alignment of top-matched amino acid sequence in BLASTp search using ClustalW resulted in the construction of a phylogenetic tree (Diuraphis noxia (XP_015370635.1), Myzus persicae (XP_022170154.1), Acyrthosiphon pisum (XP_001949498.2), Aphis gossypii (XP_027846205.1), and Rhopalosiphum maidis (XP_026817157.1) belonging to the Hemiptera. However, DcDorsal and DmDorsal fell into two different clades, showing divergence in Dorsal protein between these two insects  as the query in NCBI resulted in the identification of the Dorsal protein in tic tree A. The ph insects A. Multip insects A. Furthe insects B.Annotation of the Dorsal protein sequence using InterPro scan in DcDorsal (NP_724054.1) and DmDorsal (XP_026675878.1) showed that they share NF-kappa-B/Dorsal (IPR000451), Rel homology domain (RHD), DNA-binding domain (IPR011539), IPT domain (IPR002909), Rel homology dimerization domain (IPR032397), p53-like transcription factor, DNA-binding (IPR008967), immunoglobulin E-set (IPR014756), immunoglobulin-like fold (IPR013783), DNA-binding domain superfamily (IPR037059), and Rel homology domain as a conserved site (IPR030492) C. AlthouThe crystallographic three-dimensional (3D) structure of DcDorsal and DmDorsal was predicted using Swiss modling. The 3D structure of DmDorsal and DcDorsal was predicted using the protein transcription factor NF-κB P65 (a novel DNA recognition mode by NF-κB P65 homodimer) in the Protein Data Bank (PDB ID: 1ram.1. D) as the template for structural modeling. Based on target and template chain sequence alignment, about 28% of amino acid residues of DmDorsal (PRO 47 to PRO 326) were modeled with the template protein (PDB ID: 1ram.1. D)  with valid global model quality estimation (GMQE = 0.16) and good qualitative model energy analysis  of DcDorsal were modeled with the target protein (PDB ID: 1ram.1. D)  with good GMQE of 0.23 and reliable QMEAN of −3.02 D. SimilaDif/DI, Pll, tub, cact, TI, Myd88, and spz were analyzed at eight developmental stages of D. citri eggs, first, second, third, fourth, and fifth nymphal instars and teneral and mature adults, both females and males. DcDif/DI genes were found to be expressed throughout developmental stages, with the strongest level of expression in eggs and teneral, as well as mature adults both male and female  in the fat body in response to infection [The Toll signaling system is required for embryonic Dorsal–ventral pattern formation, and for innate immunity against pathogens in adult insects. The Gram-positive bacterial or fungal infection activates the Toll pathway signaling and stimulates protease cascade enzymes, which finally cleave the Spaetzle protein of the host. This cleaved Spaetzle binds to one of the Toll receptors, and Spaetzle-mediated activation leads to the interaction of three cytoplasmic proteins, Pelle, Tube, and MYD88 via the death domain, underneath the cell membrane. Subsequently, the signaling complex of Pelle/Tube/MyD88 leads to the phosphorylation and proteosome degradation of cytoplasmic components of NF-κB proteins Dorsal/Dif and IκB protein Cactus. The degradation of IκB protein Cactus following the Toll pathway activation causes their release and the nuclear location of the Dif and Dorsal, and expression of antimicrobial peptides (AMPs) nfection ,60,75.Dif/di. Upon the infection and signal induction, Cactus degradation occurs and causes the translocation of Dif/di to the nucleus, which activates the expression of antimicrobial peptides [Spaetzle protein consists of a C-terminal and the pro-domain region that is synthesized and secreted as an inactive form of pro-protein and processed to the functional form of C-106 by serin protease cascades. Tube and MyD88 function as protein adaptors when the receptor is active. The TIR interleukin-1 receptor domain of Toll binds to the domain of MyD88, but Myd88 and Pelle do not contact each other directly; instead, two separate death domains on the surface of the adaptor protein, Tube, connect the MyD88 and Pelle. In fact, Tube, as an adaptor, engages the Pelle kinase at the formed complex with Toll and functions upstream of Pelle and downstream of Tube. Pelle is a serine/threonine kinase that contains the C-terminal catalytic domain, and also contains an N-terminal death domain. It is shown that Pelle, because of its kinase activity, causes autophosphorylation, induces phosphorylation, and subsequently degradation of the inhibitor Cactus in two separate N-terminal motifs ,77. Tubepeptides .D. melanogaster [The Toll/Cactus signaling pathway plays an important role in regulating density and proliferation of hemocytes during larval development of nogaster . The NF-nogaster .D. citri. Our InterPro-based analysis showed that the Pelle protein has kinase and death domains. The analyzes suggest that Pelle protein in both D. citri and D. melanogaster function similarly. The Tube protein in D. citri possesses kinase domain that is absent in D. melanogaster Tube protein, indicating that it is remarkably similar in overall structure to Pelle in D. melanogaster and IRAK4 in mammalian cells. In addition, the presence of a death domain in the Tube protein of D. citri and D. melanogaster does not require their association with a protein kinase family protein, as death domains are observed in many proteins lacking catalytic function or a protein kinase domain, and this result agrees with findings by Towb et al. [Based on the above, we focused on three main genes of Toll pathway signaling, including Pelle, Tube, and Dorsal for further analyzes in b et al. . The IRAb et al. . Kinase b et al. . Our resb et al. .D. melanogaster against the D. citri, both genes hit the same protein of embryonic polarity protein Dorsal-like. Dif/dI and NF-κB are similar homologues that are translocated in fat body cell nuclei upon Toll pathway activation by pathogens, but they have distinct functions. dI plays an important role in early embryo development, while Dif is is involved in the adult immune system [Dif genes are absent in D. citri and it have only a Dorsal gene (dI). This result is consistent with Berni et al. (2014) [D. melanogaster, and is not present in the genome of Rhodnius prolixus and other insects.In our protein–protein BLAST search of the Dif/dI protein sequence from e system . Our in . (2014)  in whichD. citri during all developmental stages. Our result show that dI is expressed higher in eggs and female teneral, as well as in adults, indicating that the female delivers the majority of DcdI mRNAs required for embryonic developmental. In fact, RNAi of dI in the early stages of Rhodnius prolixus decreased the mRNAs level up to 90% and caused misplaced anterior embryonic and severe defects in embryonic development, suggesting embryo patterning is controlled by dI maternally [In the current study, we investigated the expression dynamics of Toll pathway genes with the goal of better understanding their roles in the development and physiology of ternally . In additernally  showed tMyd88, tub, pll, Ti, and Spz genes followed similar expression patterns and were upregulated during developmental stages from eggs to the adult stages, and reached the maximum expression in late nymphal stages and adult. Our results show that cact was expressed at similar levels, but the lowest expression level was found in eggs. This result was in agreement with gene expression in Tribolium castaneum, in which most of the genes were upregulated during developmental stages from larva to adult and reached the highest expression in the pupal and adult stages [T. castaneum cact, Dif, Myd88, tub, and pll genes had the highest expression level in eggs compared to the developmental larval stages. Similar to our results, cact gene expression in T. castaneum was similar in all developmental stages but was lowest in the fifth instar.On the other hand, t stages . In contD. citri, we identified the Toll system signaling pathway-related genes. Comprehensive in silico analyzes and gene expression assays were performed. Our overall findings suggest that D. citri possesses an active Toll signaling pathway with the major proteins; however, some unique differences from most insects were found, such as the presence of a kinase domain in Pelle protein, and that the Dorsal immunity factor and Dorsal are most likely the same protein. The gene expression patterns indicate that the innate immunity increases with maturity of D. citri, as the higher expression of genes is in adults. Taking all these findings together, D. citri. This work opens a new avenue to designing innovative strategies for controlling D. citri. For example, silencing Toll signaling-related genes may increase D. citri’s susceptibility to pathogens that are used for biological control. Combining RNA interference or CRISPR with biological control applications could be an efficient control strategy for mitigating HLB.To elucidate the mechanisms regulating the innate immunity in


Cryptosporidium species are zoonotic protozoan parasites responsible for gastroenteritis in various animals and humans. The diagnosis of Cryptosporidium presents many challenges. This research attempted to match the diagnostic efficiency of the modified Ziehl–Neelsen technique (mZN), immunochromatographic assays (IC), and enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium in faecal samples of cattle in Kuwait. In addition, polymerase chain reaction (PCR) was utilised to determine the predominant species infecting cattle in Kuwait and correlating the detected species with the results of different diagnostic tests used, the presence or absence of clinical signs, and the age group of the infected cattle.Cryptosporidium positive samples were 23%, 15.25%, and 14% using IC, ELISA, and mZN. IC had the highest sensitivity (74.07%), and mZN had the highest specificity (98.29%) using a composite reference standard (CRS) as a gold standard. The rapid IC test results in high false-positive results of cryptosporidiosis, whereas using mZN alone is insufficient to declare a negative faecal sample. Only 74.5% (35/47) of Cryptosporidium-positive samples by the three assays could be amplified by PCR. This study was the first to genotype Cryptosporidium in Kuwait. Cryptosporidium parvum (n = 26) was the dominant species detected from cattle samples, followed by C. andersoni (n = 6), C. bovis (n = 2), and C. raynae (n = 1). The findings showed a statistically relevant relationship between diarrhoea and the detection of Cryptosporidium spp. in faecal samples of cattle . Pre-weaned calves were the most vulnerable age group to Cryptosporidium spp. infection .Of 400 analysed faecal samples, Cryptosporidium infection in faecal samples, antigen detection or PCR methods combined with one of the microscopy techniques should be used. Cryptosporidium parvum was the prepoderant Cryptosporidium spp. recovered from cattle samples in Kuwait followed by C. andersoni. Cryptosporidium parvum is a significant risk factor for diarrhoea in pre-weaned calves. However, further study is needed as many other causes of diarrhoea in calves must be ruled out before a diagnosis of Cryptosporidium diarrhoea can be made.For screening of The online version contains supplementary material available at 10.1186/s12917-022-03435-w. Cryptosporidium species, the enteric apicomplexan protozoan parasite, can infect various hosts, including humans, livestock and poultry. However, cattle are the most significant host from a veterinary and public health perspective [Cryptosporidium spp. have already been recorded in cattle, only four spp.; Cryptosporidium parvum, C. bovis, C. ryanae, and C. andersoni are the most frequently found [C. parvum is the only spp. linked to clinical illness in neonatal calves, with older animals (> 6 weeks) demonstrating asymptomatic oocyst shedding [Cryptosporidium bovis and C. ryanae are the predominant species in post-weaned calves [C. bovis and C. ryanae; although, diarrhoea was reported in native calves in Nigeria, presumably due to C. bovis and C. ryanae infection [Cryptosporidium andersoni is more commonly found in adults than in young cattle. Cryptosporidium andersoni infections are clinically associated with weight gain impairment and reduced milk yield in adult cows [spective . Howeverly found –5. Nevershedding . Cryptosd calves –5. Therenimals >  weeks deult cows , 7.Cryptosporidium spp. in clinical cases, the specimen typically contains considerable oocysts count and a high concentration of the parasite antigen, even though methods with low sensitivity grant a positive result. Whereas specimens with few oocysts may necessitate an epidemiological investigation to detect asymptomatic carriers, using an initial screening method then a confirmatory test, for example, molecular or microscopic techniques, can boost reliance in the diagnosis [Cryptosporidium spp. during diarrhoea epidemics in calves can help perform timely interventions, reduce economic losses, and improve animal welfare [Diagnosis of iagnosis . Further welfare .Cryptosporidium DNA (PCR). Conventional microscopy is time-consuming, laborious, and needs expert microscopists to identify oocysts accurately. Simultaneously ELISA and PCR are costly and require well-equipped laboratories and skilled technicians. Although IC is a rapid test and easy to perform and interpret but may have many false-positive results [Several diagnostic assays were applied to detect Cryptosporidiosis in various hosts. They include 1- conventional , 2- antigens-detection tests (IC and ELISA), and 3- detection of  results , 11.Many diagnostic circumstances lack a gold standard, and it can be argued that what is popularly known as a gold standard may not be a proper one. As a result, numerous approaches to assessing diagnostic tests in the lack of a gold standard have been established , 13. ForCryptosporidium oocysts. Genotyping of Cryptosporidium species was applied. In addition, correlating the detected species with the results of different diagnostic tests used, the presence or absence of clinical signs, and the age group of the infected cattle were also studied.All assays used commonly in Kuwait for detecting clinical and asymptomatic cryptosporidiosis in cattle have not been evaluated. Thus, the objectives of this study were to assess routinely used laboratory tests such as microscopic examination of mZN stained faecal smears, IC, and ELISA. Given that there is no gold standard assay for diagnosis of Cryptosporidiosis , we applThe total number of cattle on the visited farms was 9365. Rectal faecal samples were collected randomly from 400 cattle: 175 pre-weaned, 49 post-weaned, and 176 adults.Cryptosporidium oocysts and antigens revealed that the Cryptosporidium positive samples were 23%, 15.25%, and 14% using IC, ELISA, and mZN, respectively , 4 (1.0%), and 73 (18.25%), respectively. In addition, coinfections of rotavirus with Cryptosporidium spp. and E. coli with Cryptosporidium spp. were detected in 2.25% (9/400) and 4.5% (18/400), respectively. Mixed infections with Cryptosporidium spp., rotavirus, and E. coli were detected in two of 400 examined faecal samples (0.5%).Examination of 400 cattle faecal samples for Cryptosporidium in cattle faeces was 47.22%, 74.07%, and 48.00%, respectively. In contrast, the diagnostic specificity was 98.29%, 89.97%, and 95.67% for mZN, IC, and ELISA, respectively. The agreement between IC and the pseudo-gold standard was substantial (κ = 0.61). On the other hand, a moderate agreement was reported for both mZN and ELISA compared to the pseudo-gold standard . Table The diagnostic sensitivity of mZN, IC, and ELISA to detect Cryptosporidium with the PCR results and the genotype identified in 47 faecal samples was reported in Table Cryptosporidium species. Thirty-two of the 35 positive PCR samples gave positive result by IC (91.4%), 28 samples were positive by mZN (80.0%), and 24 samples were positive by ELISA (68.6%). Cryptosporidium parvum was detected in 26 samples; 15 samples were positive by mZN, IC and ELISA, six were positive by IC and ELISA, and five were positive by mZN and IC. Cryptosporidium andersoni was detected in six samples; two were positive by both mZN and IC, whereas two were positive by both mZN, and ELISA. The other two samples were positive only by one test, mZN or IC (Table A comparison between the three diagnostic tests used to detect Cryptosporidium spp. showed that C. parvum was recorded in 46.8% (22/47) diarrheic pre-weaned cattle and four adult cattle (8.5%) with normal faecal consistency (Table C. andersoni was detected in four adult cattle (8.5%) with normal faecal consistency, one pre-weaned cattle with normal faecal consistency, and one diarrheic post-weaned cattle. C. bovis was detected in two pre-weaned cattle with normal faecal consistency, and one C. ryanae was diagnosed in post-weaned cattle with normal faecal consistency . Pre-weaned calves were the age group most likely to contract Cryptosporidium spp. infection .Statistical correlation between faecal consistency, age group, and different genotypes of ryptosporidium, forming 4 groups . Among the same species of Cryptosporidium, subgroups were formed like in the C. andersoni group. This indicates genetic variation within the genus Cryptosporidium  is routinely used to detect Cryptosporidium infection in faecal samples using various laboratory techniques [The detection and diagnosis of chniques . Becausechniques .Cryptosporidium oocysts in faecal samples of various animals using a variety of gold standards such as cumulative positivity [Cryptosporidium oocysts. Identifying Cryptosporidium species is significant because different species have different epidemiology, clinical manifestations, and sequelae [Previous studies evaluated different diagnostic techniques applied to identify sitivity  and latesitivity . Howeversitivity  considersitivity . Additiositivity . Furthersequelae .Cryptosporidium in cattle faeces was studied. The results revealed that IC had the highest sensitivity, and mZN had the highest specificity using a CRS as a gold standard. The sensitivity of the three tests ranged from low in mZN and ELISA  to moderate in the IC test (74%), whereas the specificity of the three tests was high (90% or higher). The low sensitivity of the three tests may be due to the low intensity of infection rate in the studied cattle population, and the present study was a cross-sectional study where only one faecal sample was collected from each animal. However, repeated examination of more than one faecal sample on three consecutive days enhances the detection of Cryptosporidium oocysts [In the present study, the performance of the three tests  used to detect  oocysts .Cryptosporidium in stools of diarrheic children using PCR as a gold standard. mZN revealed higher specificity than ELISA and rapid strip, while ELISA showed the highest sensitivity [Cryptosporidium in diarrheic animals , ELISA (40.9%) showed higher sensitivity than IC (22.7%), while IC (100%) showed higher specificity than ELISA (78.9%) [C. parvum in diarrheic calves using cumulative positivity as a gold standard. They found that the three kits had high sensitivity and specificity .Ezzaty Mirhashemi et al.  found thsitivity . In anot (78.9%) . Papini  (78.9%)  studied Cryptosporidium in faecal samples have been reported. These differences are highly dependent on infecting species and the concentration of oocysts in the faecal sample [Different sensitivities and specificities of diagnostic tests used to diagnose l sample , 23.Cryptosporidium spp. in faeces is highly specific and less sensitive [The diagnostic performance of mZN revealed a high PPV and NPV; such a higher value may be because mZN confirms the diagnosis by tracing the parasites. It was reported that the microscopic method for detection of ensitive  and can ensitive .Cryptosporidium antigens (copro-antigens) in faecal samples using ELISA and IC assays is widely used. However, ELISA and IC detection limits ranged from 3 × 105 to 106 oocysts per ml [Detecting s per ml , 26, whis per ml , 27. TheCryptosporidium spp. were controversial. One possible explanation is that not all commercially produced antibodies recognize all Cryptosporidium spp. oocyst antigens of individual species. Hence, immunological testing cannot detect every copro-antigen found in the more than 30 species and genotypes identified until 2016 [In the present study, the results of immunological tests used to detect different til 2016 .Cryptosporidium-positive samples recovered by the three assays could be identified by PCR. This could be attributable to different factors, including the low PPV of IC and ELISA or PCR inhibition. Cryptosporidium parvum was the most common species detected from cattle samples in Kuwait, with only a few samples containing C. andersoni (n = 6), C. bovis (n = 2), and C. raynae (n = 1). There have been few studies of Cryptosporidium molecular diagnosis in the Middle East. In terms of Cryptosporidium spp. distribution, our results differ from those of previous studies. Cryptosporidium parvum was the only discovered species in Tunisia and Syria [C. parvum and other Cryptosporidium spp. infecting cattle farms. Cryptosporidium parvum was recorded as the most predominant species, with C. andersoni in Iran [C. raynae in Turkey [C. bovis and C. raynae in Egypt [C. ryanae, C. andersoni, and C. bovis in Sudan [Cryptosporidium identified in Jordan was C. ryanae, then C. parvum and C. andersoni [C. bovis was the most detected spp. in cattle, then C. ryanae and C. parvum [Cryptosporidium spp. that infect cattle are distributed differently over the world [Only 74.5% 35/47) of nd Syria –30. In c in Iran , C. raynn Turkey , C. boviin Egypt , and C. in Sudan . The mos/47 of nd. parvum . The fouynae n = . There hC. andersoni was detected in six samples from four adult cattle, one post-weaned and one pre-weaned. All positive animals were from the same farm. Only one animal had diarrhoea (post-weaned), also was positive for both rotavirus and E. coli by rapid IC test. Another positive animal was a cow with a history of decreased milk production and severely emaciated. Cryptosporidium andersoni was identified in adult cattle more than in young cattle [C. andersoni have been linked to decreased weight gain and milk production in adult cows [C. bovis was reported in two healthy pre-weaned calves, and C. ryanae was identified from one healthy post-weaned animal. Previous studies reported that neither C. bovis nor C. ryanae have been implicated in clinical disease in cattle of any age group [In this study, g cattle . In addiult cows . Whereasge group , 38–40.C. parvum was the prepoderant species detected from cattle in Kuwait. It was identified in four adult cattle without signs of diarrhoea, and 22 pre-weaned calves suffered from diarrhoea. C. parvum is a widely endemic pathogen that causes self-limiting gastroenteritis in pre-weaned calves suffered from profuse watery diarrhoea as a common symptom and can be fatal in severe cases [Escherichia coli, and Salmonella spp.) can cause neonatal diarrhoea [In the present research, re cases , 42. Muliarrhoea . In contCryptosporidium spp. were clustered into four groups  in the phylogenetic tree. In addition, subgroups of Cryptosporidium were formed among the same species. This indicates genetic variation within the genus Cryptosporidium [The detected poridium .Cryptosporidium infection in faecal samples, antigen detection or PCR methods combined with one of the microscopy techniques should be used. Cryptosporidium parvum is a significant risk factor for diarrhoea in pre-weaned calves. However, further study is needed as many other causes of diarrhoea in calves must be ruled out before a diagnosis of Cryptosporidium diarrhoea can be made.The antigen detection test results in false-positive results of cryptosporidiosis, whereas using mZN alone is insufficient to declare a negative faecal sample. As a result, for screening of Cryptosporidium infections in cattle with or without clinical symptoms. The farms were chosen without being aware of the level of Cryptosporidium infestation. Twenty-two dairy cattle farms were visited once in the Sulaibiya area.Between October 2014 to September 2015, a 1-year cross-sectional study was carried out to assess the diagnostic performance of several tests used to identify n = 127) and non-diarrhoeic (n = 273).Four hundred cattle were randomly selected to examine their faecal samples. Five to ten grams of faeces were collected from the rectum or immediately after defecation and kept in a sterile capped cup. The faecal samples were categorised according to their consistency into diarrhoeic  was utilised to discover Cryptosporidium antigens. The plate is coated with the anti-Cryptosporidium-specific antibody. The methodology, and interpretation of the results were performed according to the manufacturer’s guidelines. Samples were stored at -20◦C without preservatives and the test was applied within a month from the day of collection. Faecal samples were diluted and added to the coated wells, then the plate was incubated for 1 h at 21◦ ± 3◦C. Next, the conjugated anti-Cryptosporidium monoclonal antibody was appended and reincubated. Finally, tetramethylbenzidine (TMB) was visualized the reaction as showed in Fig. A sandwich, double wells ELISA kit  was used to detect Cryptosporidium species. Criteria of selection of samples for PCR test was mainly those positive by two or more methods except four samples were positive using one test . The samples preserved in potassium dichromate were centrifugated twice in distilled water. DNA was extracted from all specimens (frozen and preserved) using the FastDNA SPIN kit for soil .Forty-seven samples were selected for PCR testing and molecular typing of Cryptosporidium molecularly differentiated by RFLP of the secondary PCR products using SspI and MboII  rRNA gene was applied . The prioII Fig.  as previCryptosporidium spp. were aligned with publicly available sequences to determine the relationship between Cryptosporidium species using ClustalX . The 18S rRNA sequences of C. andersoni isolated from cattle in China (KC580754), C. parvum; strain Bovine C. parvum genotype (BOH6) isolated from calf in Ohio (AF093490), C. bovis; Bovis 2622 isolated from cattle (AY120911), C. ryanae RYanaeS6293a1 isolated from calves in China (HQ009807), and C. parvum_2_Sheep_Kuwait isolated from sheep in Kuwait [The 18S rRNA gene sequences of the recovered hio AF0930, C. bovCryptosporidium. The animal was classified as Cryptosporidium spp.-infected if either one of the reference tests, excluding the index test, were positive. For instance, to evaluate IC (index test), results of mZN and ELISA were used to build the pseudo-gold standard. The diagnostic performances and the 95% confidence interval of the three tests was computed using MdCalc® Statistical software. Using QuickCalcs, GraphPad Software®, kappa (κ) test agreement was computed and assessed. The Chi-square test (χ2) was used to evaluate the correlation between the different Cryptosporidium spp. diagnosed by PCR with the presence or absence of diarrhoea, and age group in the examined 47 faecal samples.A composite reference standard was applied to build a pseudo-gold standard for evaluating mZN, IC, and ELISA used to detect Additional file1. Nested PCR and RF LP of restriction enzymes SspI and MboII


Dalbergia L.f. is a pantropical genus consisting of 269 species of trees, shrubs, and woody lianas. This genus is listed in CITES Appendices because of illegal logging and trafficking driven by the high economic value of its heartwood. Some species are also used medicinally. Species identification of Dalbergia timber and herbs is challenging but essential for CITES implementation. Molecular methods had been developed for some timber species, mostly from Madagascar and Southeast Asia, but medicinal species in south China were usually not included in those studies. Here, we sequenced and assembled the chloroplast genomes of five Dalbergia species native to Hong Kong, four of which are medicinal plants. Our aim is to find potential genetic markers for the identification of medicinal Dalbergia species based on divergence hotspots detected in chloroplast genomes after comparative and phylogenetic analysis. Dalbergia chloroplast genomes displayed the typical quadripartite structure, with the 50 kb inversion found in most Papilionoideae lineages. Their sizes and gene content are well conserved. Phylogenetic tree of Dalbergia chloroplast genomes showed an overall topology similar to that of ITS sequences. Four divergence hotspots (trnL(UAA)-trnT(UGU), ndhG-ndhI, ycf1a and ycf1b) were identified and candidate markers for identification of several Dalbergia species were suggested. Dalbergia L.f. is a pantropical genus with 269 accepted species of trees, shrubs, and woody lianas according to Plants of the World Online [Dalbergia is of high economic value. The heartwood of some of the Dalbergia species are known as rosewood, or Hongmu in Chinese. Because of its distinctive color and durability, rosewood is a precious timber for making high-end furniture, valuable carvings, and musical instruments. Fifteen Dalbergia species have been listed as authentic sources of rosewood in National Standard for Hongmu (GB/T 18107-2017) by the Standardization Administration of China [Dalbergia timber, the whole genus Dalbergia was included in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix II in 2016, except for Dalbergia nigra (Brazilian rosewood) which was already listed in Appendix I.d Online  and the d Online  in the td Online . It belod Online . Membersof China . The higDalbergia species are medicinal plants. According to the Medicinal Plant Names Services provided by Royal Botanic Gardens, Kew [Dalbergia species have been cited in medicinal sources, i.e. pharmacopoeias and reference collections, all over the world. Heartwood of Dalbergia odorifera is a Chinese materia medica listed in the Chinese Pharmacopoeia [D. odorifera, D. assamica, D. benthamii, D. hainanensis, D, hancei, D. hupeana, D. millettii, D. mimosoides, D. obtusifolia, D. rimosa, D. sissoo, and D. yunnanensis have all been listed as sources of medicinal materials in Zhonghuabencao, a reference collection of Chinese medicinal herbs [D. odorifera (listed in the Chinese Pharmacopoeia) and D. hancei (listed in the Standards of Zhuang Materia Medica of Guangxi Zhuang Autonomous Region), these medicinal materials have not been listed in any official standards in China. There is no official guidance for morphological identification and no validated methods for quality control of these herbal materials.Besides, some ens, Kew , 57 Dalbacopoeia . In addial herbs . Except Dalbergia. Various molecular techniques have been employed for the identification of Dalbergia species, such as random amplified polymorphic DNAs [rbcL+matK+ITS [ITS2+trnH-psbA [rbcL+matK+trnL [Dalbergia species involved in the analyses.It is also not easy to distinguish between different timber species, especially in the form of finished products. A reliable identification method must be in place to support law enforcement. Traditional wood identification relies on wood anatomy, based on both macroscopic and microscopic characteristics. Online databases and recognition tools, such as the InsideWood database  and CITEhic DNAs , simple hic DNAs , and DNAhic DNAs ,18,19,20matK+ITS ,17, ITS2Dalbergia L.f. is a genus with pantropical distribution whose complete infrageneric classification is difficult [A Synopsis of the Dalbergieae, a tribe of the Leguminosae in 1860 [Dalbergia in The species of Dalbergia of southeastern Asia [Sissoa and Amerimnon), five sections  and 24 groups, based on floral morphologies. In 1989, André de Carvalho published his treatment on Brazilian Dalbergia [Dalbergia, Triptolemae, Selenolobium, Pseudecastaphyllum, and Ecastaohyllum) based on the morphological evidence of reproductive organs [ITS sequences do not match these treatments completely [Dalbergia.ifficult . The fir in 1860 . Based oern Asia . He clasalbergia ,24. He ce organs . Howevermpletely . Hence, Stipa species) [Ilex species [Fritillaria species [Hedyotis species [Dalbergia species and identified eight divergence hotspots after comparing the nine sequences [Dalbergia species have been published, but those of medicinally used Dalbergia species in South China are still unavailable. In this study, we have assembled the complete chloroplast genomes of five Dalbergia species native to Hong Kong. All but one species, D. candenatensis, are medicinal plants. A total of 46 complete cp genomes from the 26 Dalbergia species available, including all but one medicinally used Dalbergia species in China, have been analyzed for divergence hotspots identification and study of phylogenetic relationship. To check if the divergence hotspots are also present in the mitochondrial genome, which may hamper the use of those hotspots for identification, the newly assembled cp genomes were compared against the complete mitochondrial genome of D. odorifera by BLAST to identify potential mitochondrial plastid DNAs. Finally, the divergence hotspots and internal transcribed spacer 2 (ITS2), a popular DNA barcode, were evaluated for their potential for species discrimination.Complete chloroplast (cp) genomes have been proposed to be super-barcodes that provide higher discriminatory power than conventional barcodes . By compspecies) , Ilex sp species , Fritill species , and Hed species . In 2019equences . ChloropDalbergia species and characteristics of the cp genomes are listed in D. benthamii is the largest in size at 156,638 bp. The smallest genome belongs to D. assamica, with a genome size of 155,835 bp. All cp genomes demonstrated the quadripartite structure typical of angiosperms, with a large single copy (LSC) ranging from 85,253 bp to 85,767 bp, inverted repeats (IRs) between 25,671 bp and 25,742 bp in size, and a small single copy (SSC) ranging from 18,978 bp to 19,427 bp. The GC content is between 36.02% and 36.19%, similar to the value previously reported [The Illumina NovaSeq 6000 system produced 16,579,278 to 19,675,290 clean, paired-end reads per species. Complete cp genomes were assembled by de novo assembly. Clean, paired-end reads were mapped to the assembled contigs for validation. The cp genomes obtained had mean coverage ranging from 154.29 X to 829.73 X . Figure reported .Dalbergia cp genomes ranged from 111 to 114, with 75 protein-coding genes, 2–3 hypothetical proteins, 30–32 tRNAs, and 4 rRNAs. All the genes were categorized according to the gene functions and shown in Dalbergia species studied. The trnG-UCC gene is absent in the genome of D. benthamii but is present in the other four genomes. The ycf68 gene is only present in D. hancei and D. millettii. The number of duplicated genes in the IR regions ranged from 18 to 19, depending on the presence/absence of ycf68. Among the duplicated genes, there are seven tRNA genes, four rRNA genes, five protein-coding genes and two to three hypothetical genes . Sixteen genes that harbor intron(s) were found, with 13 genes carrying one intron and three genes, pafI, rps12, and clpP1, containing two introns.When only single copies of duplicated genes are counted, the total number of genes in the assembled Dalbergia cp genomes , followed by the SSC region with 35–38 SSRs (18.32–19.59%) and the IR region with 5–6 SSRs (2.62–3.35%). However, if we take the length of each region into account, the SSC region has a higher SSR density (number of SSRs per nucleotide) (0.001824–0.001978) than LSC (0.001546–0.001712)  was detected exclusively in D. millettii. Same as other Dalbergia cp genomes previously reported [Dalbergia cp genomes are AT-rich. There are 125–134 A/T mononucleotide repeats and 35–42 AT/AT dinucleotide repeats but only 5–8 mononucleotide C/G repeats and 3 AG/CT dinucleotide repeats  . Mononuc.001712) b. Pentane) 0.0018–0.001978 repeats c. An AATlgorithm d. Among aponicus , Phaseols luteus .Dalbergia species is quite conserved , with 232 bp of the gene located in the SSC region. For the other four species, their ycf1 pseudogene is shorter in length (467–468 bp) and the first nucleotide of the SSC region is the last base of the stop codon. The ndhF gene is located in the SSC region of the junction. The ndhF gene of D. hancei is truncated with a length of only 1479 bp and it is 782 bp away from the junction of IRb/SSC. For the other four Dalbergia species, their ndhF gene is intact with a length of 2249–2273 bp. They are either just spanning across the junction, with 2–14 bp of the gene at the IRb region , or a mere 2 bp away from the border at the SSC region (D. candenatensis). The full-length ycf1 gene spans across the SSC/IRa junction in all five species.Looking into the gene arrangement and border regions, it was found that the gene arrangement of the cp genomes of the five onserved . There ip ≥ 0.03) were identified as divergence hotspots , followed by the trnL-trnT intergenic spacer (p = 0.035), ndhG-ndhI intergenic spacer (p = 0.032), and ycf1b (p = 0.032).In the sliding window analysis, four regions with nucleotide diversity values , identified as MTPTs, accounting for 9.98–10.51% of the cp genomes. More than half of the MTPTs (28–33) belonged to transfer RNA and ribosomal RNA. The rest were protein-coding MTPTs (11–12) and non-coding MTPTs from intergenic spacer regions (2–5 only). The majority of the MTPTs (30–32) are from the IR regions, probably because of its prevalence in ribosomal RNA genes. The protein-coding MTPTs are from ndhB, ycf1, ycf2, atpA, atpB, aptE, and rps12. The MTPTs containing the ndhB gene are the longest, with a length of 2493 bp. The four divergence hotspots have not been identified as MTPTs.Inter-organelle DNA transfer and mitochondrial plastid DNAs (MTPTs) were first reported in maize in 1982 ,35. Sincequences . In ordeDalbergia cp genomes available on NCBI GenBank, except for the few dubious ones stated in Methods. The condensed maximum likelihood tree is shown in D. hancei and D. millettii obtained in this study are in the same clade as D. mimosoides and D. hancei. D. assamica was grouped into a small paraphyletic clade together with D. hypeana, D. balansae, and D. hainanensis, which is in line with previous studies using ITS sequences [rbcL+matK+ITS sequences [D. benthamii and D. candenatensis were grouped into a small clade distinct from all other Dalbergia species. This is in agreement with the study of Hartvig et al., but different from the study using ITS sequences [D. benthamii and D. candenatensis were in different clades, Clade V and Clade III.The five newly assembled cp genomes were analyzed together with all equences  and rbcLequences . D. bentequences , in whicDalbergia species analyzed, the intraspecific and interspecific variations of the Dalbergia species cannot be fully represented. We could only attempt to evaluate whether the hotspots could give monophyletic clades for species of interest and would be worthy of further investigations. Neighbor-joining trees were built for the four divergence hotspots (D. cultrata and D. bariensis. The trnL(UAA)-trnT(UGU) and ycf1b gave monophyletic clades for D. cochinchinensis. In addition, the trnL(UAA)-trnT(UGU) formed monophyletic clade for D. hancei, while the ndhG-ndhI intergenic spacer gave monophyletic clades for D. yunnanensis and D. hancei. The ycf1b is the only hotspot that could produce monophyletic clade for D. odorifera. Since this evaluation is not applicable to species with only one cp genome analyzed, we also looked into the discriminatory power of ITS2 sequences using ITS sequences with voucher specimen number in GenBank. In D. retusa, D. sissoo, D. trichocarpa, D. hancei, D. millettii, D. dyeriana, D. melanoxylon, D. cochinchinensis, D. nigra, D. obtusifolia, D. miscolobium, D. candenatensis, D. pinnata, D. nigrescens, and D. stevensonii. These regions could be potential candidate markers for identifying the corresponding species.As there are only 1–5 cp genomes for each of the 26 hotspots . All fouDalbergia cp genomes available in GenBank, the five newly assembled cp genomes displayed the quadripartite structure typical of angiosperms, as well as the 50 kb inversion commonly found in most taxa of Papilionoideae [trnS genes (trnS-GCU and trnS-GGA) [Dalbergia cp genomes do not possess the ~36 kb inversion specific to core Genistoid, which is embedded within the 50 kb inversion. In the cp genomes of the Dalbergia species, and those of other species in Dalbergieae tribe , there was a base substitution in the 29 bp fragment of trnS-GCU. This might have prevented the inversion from happening. All Dalbergia cp genomes have lost the infA gene and rpl22 gene, which is common in all legumes [rps16 gene, which has been reported to be lost in different legume taxa [Dalbergia cp genomes, except for Dalbergia nigra because of a unique InDel . However, when we extracted the exon sequences of ycf68 from the alignment for translation, we found that most Dalbergia species have the same amino acid sequences for this gene  [ycf68 is not a protein-coding gene [Extensive structural rearrangements, gene loss and mutations have occurred in chloroplast genomes of different subfamilies of Fabaceae, which is therefore regarded as a “model system for understanding chloroplast genome evolution” . Same asonoideae . In cp grnS-GGA) ,42. Dalb legumes . The rpsume taxa ,42,44, wue InDel . The ycfhis gene . There itic tree . The ycf15901.1) ,46. Howeing gene .Dalbergia cp genomes is generally similar to the one obtained with ITS sequences [D. candenatensis, which was in Clade III in the study with ITS sequence. The large clade contains two big subclades, corresponding to Clade IV and Clade V assigned by Vatanparast et al. The major discrepancy is on D. benthamii, which was grouped into the small clade with D. candenatensis in our study but assigned to Clade V by Vatanparast et al. In another study using rbcL+matK+ITS [D. benthamii and D. candenatensis were also in one small subclade, which was also sister to a large subclade containing most Dalbergia species in their study. It was also noticed that the two cp genomes of D. hancei were not clustered together. One was clustered with D. millettii (OM328092) and together they were sister to D. mimosoides (MN714221) and the other cp genome of D. hancei (OM328090). Our results showed the close phylogenetic relationship between D. hancei and D. millettii, which was also supported by the phylogenetic tree of ITS2 sequences  and D. oliveri (0.0036)  . This shequences . Using rmatK+ITS , Hartvigt et al.  and of tdes MN7141 and theto Prain . Our resdes MN7141 and theDalbergia cp genomes. These four hotspots had also been detected in the previous study, which identified eight hotspots in total [Dalbergia species. While the entire genus Dalbergia is listed in CITES Appendices, we are particularly interested in medicinal Dalbergia species, such as D. odorifera (listed in the Chinese Pharmacopoeia) and D. hancei (listed in the Standards of Zhuang Materia Medica of Guangxi Zhuang Autonomous Region), as well as D. assamica and D. millettii, which are used as folk medicine in South China. Our results suggest several candidate markers, ycf1b for D. odorifera, trnL(UAA)-trnT(UGU), ndhG-ndhI and ITS2 for D. hancei, and ITS2 for D. millettii. For D. nigra, the only Dalbergia species listed in CITES Appendix I, ITS2 appears to be a good candidate. Unfortunately, the sequence data currently available is still insufficient for thorough evaluation of the discriminatory power of candidate markers. For one thing, GenBank has currently only collected cp genomes of 26 Dalbergia species. For barcode sequences that are more abundant in GenBank, there are only 96 Dalbergia species with ITS sequences available and voucher specimen numbers deposited. Even for D. nigra, there are only four ITS sequences provided by two research groups. Dalbergia is a genus containing 269 species that are widespread and require protection. Concerted effort from the scientific community is needed to enrich the genomic data of this genus. Further experiments are still needed for the generation and validation of short genetic markers (~100–200 bp) for developing an identification method applicable to timber samples and processed medicinal materials.In this study, we identified four divergence hotspots among in total . The redDalbergia assamica and Dalbergia candenatensis were collected with the assistance of the Hong Kong Herbarium, Agriculture, Fisheries, and Conservation Department of the Government of the Hong Kong Special Administrative Region. Specimens of Dalbergia benthamii, Dalbergia hancei, and Dalbergia millettii were collected by members of the Shiu-Ying Hu Herbarium with a field collection permit in Hong Kong in 2019–2020. All collections are permitted and legal in Hong Kong.Specimens of Plant samples were identified by Dr. David Tai-Wai Lau, curator of the Shiu-Ying Hu Herbarium of the Chinese University of Hong Kong. Detailed information is listed in Total genomic DNA was extracted from 0.1 g dried leaves according to the spin column DNA extraction protocol for plant-derived Chinese materia medica (Annex A2 of GCMTI RD-5:2020) of the Government Chinese Medicines Testing Institute, Hong Kong SAR . Qualityhttp://en.novogene.com/, accessed on 23 March 2022) on the NovaSeq 6000 platform . Approximately 3.0 Gb of raw data were generated.Paired-end libraries with 150 bp insert were constructed from total genomic DNA. Sequencing was performed by Novogene Bioinformatic Technology Co. Ltd.  . Mapped contigs were selected and assembled into complete cp genomes. Gaps between the contigs, if any, were amplified and sequenced using specific primers. Newly obtained chloroplast genomes were annotated on the GeSeq platform  [D. odorifera (NC_049008.1) and D. martinii (NC_049049.1) as reference. OrganellarGenomeDRAW  [CLC Assembly Cell package v5.1.1  was used to perform quality trimming, mapping, and de novo assembly. Poor-quality reads with a Phred score below 33 were removed to obtain clean filtered reads, which were assembled into contigs with a CLC assembler CLC Assembly Cell package. Gaps were filled using GapCloser in SOAPdenovo v3.23 and contigs were re-ordered by NUCmer 3.0. Contigs were then mapped to the reference genome, fera NC_0008.1. Mach 2022)  was usedhttps://webblast.ipk-gatersleben.de/misa/index.php?action=1, accessed on 23 March 2022) [https://bibiserv.cebitec.uni-bielefeld.de/reputer, accessed on 23 March 2022) [MIcroSAtellite identification tools, MISA (ch 2022)  and REPuch 2022)  were usehttps://genome.lbl.gov/vista/mvista/submit.shtml, accessed on 23 March 2022) [D. hancei (OM328090) was selected as reference. For better visualization of junction sites of the boundary regions, the online program IRscope  was adopted [Visualization of genome structure of newly obtained cp genomes was performed with mVISTA software (ch 2022) . The Shuch 2022)  and the  adopted . The diaD. hainanensis was also deleted because of suspected misidentification [https://mafft.cbrc.jp/alignment/server/, accessed on 23 March 2022) [D. odorifera (MT009405.1) and D. oliveri (NC_053827.1), were further removed as they are apparently different from other sequences of the same species. Sliding window analysis was performed using DnaSP v6.12.03 software [To identify divergence hotspots, all cp genome sequences available in GenBank (as of 4 January 2022) were downloaded. Accessions selected as reference sequences of GenBank were removed to avoid duplication while the reference sequences were retained. Accessions NC_036961.1 of fication . The remch 2022) . Two seqsoftware . The winD. odorifera (NC_049008.1) with the mitochondrial genome sequence of D. odorifera (NW441235.1) using BLASTn [Potential MTPTs, mitochondrial sequences of plastid origin arising from horizontal inter-organelle DNA transfer, were identified by comparing the newly assembled cp genome sequences and the NCBI refseq of g BLASTn  with theDalbergia species, a maximum likelihood (ML) tree was constructed based on the complete cp genomes using MEGA X software [Glycine max (NC_007942.1) was added as an outgroup. Pairwise genetic distance was also computed with MEGA X [To infer the phylogenetic relationships of the software . The genh MEGA X .Pterocarpus indicus (MT249115.1) was added as the outgroup. If the divergence hotspot could produce an NJ tree with a monophyletic clade containing all analyzed sequences of a certain species, we would regard the divergence hotspot as a potential marker for distinguishing that species from other congeneric species.Sequences of the four divergence hotspots were extracted from the alignment of cp genomes to construct their own neighbor-joining (NJ) trees using MEGA X software  based onITS2) for evaluation, the assembled contigs were mapped to an ITS sequence of D. odorifera (KY489987.1). We then re-mapped the clean filtered reads to the contig obtained to ensure the coverage. The ITS sequences obtained were then aligned with ITS sequences downloaded from GenBank for building a neighbor-joining tree. Only accessions annotated with a voucher number were downloaded.To obtain the sequences of internal transcribed spacer 2 (Dalbergia species native to Hong Kong. The chloroplast genomes displayed the typical quadripartite structure and the 50 kb inversion common for most Papilionoideae lineages. Size and gene content among all Dalbergia chloroplast genomes are quite conserved, and an abundance of SSRs have been observed. Four divergence hotspots were identified from a phylogenetic analysis containing 46 chloroplast genomes of 26 Dalbergia species. Candidate markers for identification of several medicinally used Dalbergia species were suggested based on phylogenetic trees of individual divergence hotspots and ITS2 sequences. Our work provides the foundation to further enrich the DNA barcode and genomic data of this genus, as well as for the development and validation of short genetic markers for use in timber samples.In this study, we have sequenced and assembled the chloroplast genomes of five


Previous studies on plant long noncoding RNAs (lncRNAs) lacked consistency and suffered from many factors like heterogeneous data sources and experimental protocols, different plant tissues, inconsistent bioinformatics pipelines, etc. For example, the sequencing of RNAs with poly(A) tails excluded a large portion of lncRNAs without poly(A), and use of regular RNA-sequencing technique did not distinguish transcripts’ direction for lncRNAs. The current study was designed to systematically discover and analyze lncRNAs across eight evolutionarily representative plant species, using strand-specific  and whole transcriptome sequencing (RiboMinus) technique.IPS) was found so far to be the only plant lncRNA group with conserved motifs, which may play important roles in the adaptation of terrestrial life during migration from aquatic to terrestrial. Most highly and specially expressed lncRNAs formed co-expression network with coding genes, and their functions were believed to be closely related to their co-expression genes.A total of 39,945 lncRNAs  were identified, which showed molecular features of lncRNAs that are consistent across divergent plant species but different from those of mRNA. Further, transposable elements (TEs) were found to play key roles in the origination of lncRNA, as significantly large number of lncRNAs were found to contain TEs in gene body and promoter region, and transcription of many lncRNAs was driven by TE promoters. The lncRNA sequences were divergent even in closely related species, and most plant lncRNAs were genus/species-specific, amid rapid turnover in evolution. Evaluated with PhastCons scores, plant lncRNAs showed similar conservation level to that of intergenic sequences, suggesting that most lincRNAs were young and with short evolutionary age. INDUCED BY PHOSPHATE STARVATION (The study revealed novel features and complexity of lncRNAs in plants through systematic analysis, providing important insights into the origination and evolution of plant lncRNAs.The online version contains supplementary material available at 10.1186/s12864-022-08602-9. Long noncoding RNAs (lncRNAs) were found to account for a large part of the whole transcriptome in plants. Most of lncRNAs identified in plants were lincRNAs, which were transcribed from intergenic regions. Another lncRNAs transcribed from intragenic regions were lncNATs, which were antisense to coding genes. Both types of lncRNAs have been identified in many plant species, among which a few were found to play important roles in plant development and stress resistance –4. BeneflncRNA-314 was originated from a full-length LTR retrotransposon inserting into downstream of a promoter in the ancestral tomato genome [Arabidopsis, lincRNA11195 contained a LTR retrotransposon and was activated under abiotic stresses [To date, important question about how plants lncRNAs originated and evolved remains largely un-answered. Several ways for the origin of lncRNAs have been documented, among which transposable elements (TEs) are an important cause. TE was thought as an important factor to drive the transcription of lncRNAs , 16. In stresses . In addiBrassicaceae and Cleomaceae [LncRNAs had a fast evolutionary rate and a high degree of sequence diversity in animals –20. Someeomaceae , 23. Beseomaceae . A comprThe current study was designed to comprehensively discover and analyze lncRNAs in multiple plant species across the evolutionary landscape using strand-specific and whole transcriptome sequencing data. We employed eight plant species from the primitive to higher taxa in plant, which were representatives at key position of plant evolution. Using RiboMinus and strand-specific RNA-sequencing techniques in combination with consistent plant tissues, we have expanded the lncRNA landscape, including long noncoding natural antisense RNAs (lncNATs) and lncRNAs without poly(A). Our results showed that both lincRNAs and lncNATs were widely distributed in plants. TEs played important roles in the origination and transcription of lncRNAs. The molecular features of lncRNAs were found to be conserved in plants, but sequences were non-conserved even between evolutionarily close species. Most highly and specially expressing lncRNAs participated in the co-expressional network with coding genes, which may function as regulators in the network.Chlamydomonas reinhardtii (C. reinhardtii), Selaginella moellendorffii (S. moellendorffii), Zea mays (maize), Oryza sativa subsp. Japonica (rice), Arabidopsis lyrata (A. lyrata), Arabidopsis thaliana , Populus trichocarpa (poplar) and Solanum lycopersicum (tomato)  Fig. . These sto) Fig. : Table 1C. reinhardtii and S. moellendorffii, we used all available data. Considering the batch effects coming from PCR artifacts, sequence depth and gene expressional abundance, etc. among different data sources, we eliminated PCR artifacts and low-quality sequencing data by pre-processing and mapping high-quality reads to genome in our procedure. We required the mapped data size for each tissue to be above 10X genomic coverage depth in order to retain transcripts with low-level expression  data for multiple plant tissues, either collected from public database or sequenced by ourselves when they are not available  were found, and their distribution in each species were listed in “Additional file A total of 39,945 lncRNAs were obtained in all the eight species, for which the transcriptional orientation of each lncRNA was also confirmed. LncRNAs identified in this study were categorized into two types: lncRNA in intergenic region  and lncRNA antisense to one or more exons of coding genes lncNAT: 14,595) . Here we searched lncNATs using strand specific RNA-seq data, and found that lncNATs dispersed widely in plant Fig. . LncNATstii Fig. . There aThe characteristics of lncRNAs, such as transcript length, AT content, exon number, and so on, were explored and compared to coding genes in individual plant species , 26, 27.p-value < 2.2e-16, t-test)  , compared to the ratio of mRNAs from 65.3% (rice) to 92.6% (C. reinhardtii)  of coding genes, while which was higher than intergenic sequences Fig. D. This fTEs were widely existed in eukaryotic genomes and acted as key factors for gene and genome evolution , 32. InsTEs were classified into two types: Class I TEs or retrotransposons, and Class II TEs or DNA transposons. They are both present in the plant genome. However, we found that Class II TEs favored transcribed regions, including lncNAT and lincRNA, whereas Class I TEs favored regions of non-transcribed sequences Fig. B. The raTEs play important roles on the origination of lncRNAs. Benefitting from the strand-specific sequencing data, we can confirm the positions of promoters. We found about 38 ± 21% lincRNAs had promoters originating from TEs, which was higher than the percentage of coding genes (29 ± 20%) and lncNATs (27 ± 21%) , and expressed in anther, pistil, leaf, root, stem and seed, but especially high in pistil  homeostasis in plants, including AtIPS1 and AtIPS2/At4 in Arabidopsis, OsIPS1 and OsIPS2 in rice, and TPSI1 in tomato [IPS contained conserved 24-nt nucleotides motif in several species [IPS in algae may be explained by their lack of vascular tissues, whereas IPS was expressed in vascular tissues when lacking of Pi. IPS first appeared in S. moellendorffii, a representative of primitive vascular plant   Fig. A. Similap-value < 2.2e-16)  score in all of the plant species, which returned a score between 0 and 1 . JS scorA. lyrata.The expressional patterns of lncRNAs evolved rapidly in plant, which formed sharp contrast to the coding genes. We defined tissue-specific (TS) transcripts as transcript with JS score > = 0.9. We found TS lncRNAs and TS mRNAs were distributed to tissues differently in the same species Fig. C. Also, A. thaliana, while lncRNAs were significantly different among tissues  from A. thaliana to construct WGCNA network. A total of 178 lincRNAs, 555 lncNATs and 20,729 coding genes were selected to construct the network. In this network, lncRNAs and coding genes were clustered into 24 modules according to their expressional characteristics  tails by oligo(dT) selection, which would exclude a large part of lncRNAs without poly(A) . In adding genes , 52, we A. thaliana, rice and maize, the proportion of TE-related lincRNAs in total lincRNAs were reported to range from 22.9 to 51.5% [In plants, insertion of TEs is one of the important mechanisms for lncRNA origin , 53. We to 51.5% , similarto 51.5% . Our resSome lncRNAs were found to be conserved in animals with the species were distantly separated in evolution , 55. TheIPS was a lncRNA participating in Pi regulation in several plant species, and was induced expressing in the vascular tissues of root and shoot [IPS crossed all tracheophyte , and then cultivated in phytotron respectively. Leaves, stems, roots and flowers of A. thaliana and A. lyrata were collected from mature plants. Siliques of A. thaliana were collected one week after flowering, and seeds were collected when siliques were full but epidermis were still green. The seedlings of poplar were provided by Cuiting Wang, CAS Center for Excellence in Molecular Plant Sciences. Leaves, stems, roots and shoot tips of poplar were collected from seedlings with height of ~ 1 m. The mature plants of rice (Japonica) were obtained from the lab of Zuhua He, CAS Center for Excellence in Molecular Plant Sciences (http://sippe.ac.cn/zuhuahe/). Leaves and stems of rice were collected before heading period. Samples of S. moellendorffii were described in our previous study [The seeds of us study .Our dataset contained more than 700G read pairs from eight plant species, of which 490G were previously published data and 210G were generated by RNA sequencing , and the quality threshold was set as 20. Reads shorter than 50 bp or with ambiguous nucleotides were removed after quality checking. Genomes and gene sets of the eight plant species were downloaded from public databases, and the versions of them were shown in “Additional file .210, andAll clean reads were mapped to genome using bwa (version 0.7.17-r1188) . SamtoolA. thaliana, A. lyrata, rice and maize were downloaded from SRA database, and their accession numbers were listed in “Additional file ChIP-seq data from Whole genomic repeats and TEs of the eight plant species in this study were identified using RepeatMasker 3.3.0 with parameters setting as -xsmall, and the RepBase20.02 were used as reference library. TE-lncRNAs were defined as lncRNAs with exon sequence overlapping at least 5 bp to TE sequences.Lastz (version 1.02.00) was used for inter-species alignments, then TBA (version v12) was used for final integration to obtain genome-wide alignment files for all eight species . PhastCoA. thaliana [Arabidopsis thaliana” [The WGCNA co-expressional network was constructed with R package WGCNA (version 1.68) using the FPKMs from all the 12 tissues of selected genes in thaliana . GO enrihaliana” .Additional file 1.Additional file 2. RNA-seq datasets.Additional file 3. lncRNA-gff3.Additional file 4: Figure S1. (A) Expressional levels of lncNATs and their antisense coding genes. The expressional levels were evaluated by log (FPKM+1). (B) Expressional levels of lncNAT, antisense coding genes, and coding genes without lncNATs expressing at their antisense strand. Figure S2. (A) Ratio of splicing and alternative splicing transcripts. (B) Base distribution of splicing acceptor and donor for lncRNAs and mRNAs. Figure S3. Expressional levels of lncRNA Osat_00007032 in rice. Figure S4. The distribution of PhastCons score in lncRNAs and coding genes. (A) The distribution of PhastCons score in lncRNAs, intergenic sequences and coding sequences of coding genes. (B) Percentage of lncRNAs and mRNAs with different PhastCons scores in total lncRNAs and mRNAs, respectively. Figure S5. Expressional patterns of lncRNAs and mRNAs in divergent species. (A) Expressional levels of lncRNAs and mRNAs in each species, and the numbers of y-axes were calculated by log10(FPKM). (B) Tissue-specificity of lncRNAs and mRNAs in each species with multiple tissues, and the y-axes stands for JS score.Additional file 5. ChIP-seq datasets.