Wild species of cotton are excellent resistance to abiotic stress. Diploid D-genome cotton showed abundant phenotypic diversity and was the putative donor species of allotetraploid cotton which produce the largest textile natural fiber.G. thurberi (D1), G. klotzschianum (D3-k), G. raimondii (D5) and G. trilobum (D8) to reference genome. The numbers of differently expressed genes (DEGs) were significantly higher under cold stress than salt stress. However, 34.1% DEGs under salt stress were overlapped with cold stress in four species. Notably, a potential shared network  was mined out by gene co-expression analysis. A total of 47,180–55,548 unique genes were identified in four diploid species by De novo assembly. Furthermore, 163, 344, 330, and 161 positively selected genes (PSGs) were detected in thurberi, G. klotzschianum, G. raimondii and G. trilobum by evolutionary analysis, respectively, and 9.5–17% PSGs of four species were DEGs in corresponding species under cold or salt stress. What’s more, most of PSGs were enriched GO term related to response to stimulation. G. klotzschianum showed the best tolerance under both cold and salt stress. Interestingly, we found that a RALF-like protein coding gene not only is PSGs of G. klotzschianum, but also belongs to the potential shared network.A total of 41,053 genes were expressed in all samples by mapping RNA-seq Illumina reads of 2+ signal transduction and oxidation-reduction mechanisms. Our work establishes a transcriptomic selection mechanism for altering gene expression of the four diploid D-genome cotton and provides available gene resource underlying multi-abiotic resistant cotton breeding strategy.Our study provided new evidence that gene expression variations of evolution by natural selection were essential drivers of the morphological variations related to environmental adaptation during evolution. Additionally, there exist shared regulated networks under cold and salt stress, such as CaThe online version contains supplementary material available at 10.1186/s12870-020-02726-4. Gossypium) provides the most natural fiber for the manufacture of textiles [G. hirsutum and G. barbadense  are widely planted, and domesticated through a long-time history. Genetic evidence suggests that allotetraploid cotton was formed by diploid A- and D-genome species hybridization events at about 1–2 million years ago [G. thurberi, G. klotzschianum, G. raimondii, and G. trilobum, which distribute four different latitude areas of the Americas [G. thurberi and G. trilobum show the close relationship of phylogeny, even though there is distinctly different latitude of natural distribution. Moreover, G. thurberi a member of the D genome is tolerant of cold temperature [Cotton . We also found a module was negatively correlated with salt and cold stress by WGCNA. G. klotzschianum showed better resistance under cold and salt stress, and 171 common DEGs under cold and salt stress were identified in this species. In summary, gene expression variations were essential drivers of the morphological variations related to environmental adaptation during evolution and there are shared networks that involved in cold and salt stress response, such as signal transduction and oxidation-reduction processes. Our work provides an insightful understanding of expression divergence, conservation, and response to environmental adaptation during evolution by combining protein-coding sequence and gene expression diversity.In our research, to reveal the genetic and expression diversity under cold and salt stress, we perform transcriptomic sequencing of four diploid D-genome species, including  studies . Six speG. thurberi (D1), G. klotzschianum (D3-k), G. raimondii (D5) and G. trilobum (D8) with a rich diversity of morphological characteristics were selected for further analysis. These species show tremendous phenotypic variations in flower color and leaf shape, although these are phylogenetically most closely related to each other  after two stress treatment samples , two repeats for each sample. A total of 40 data sets with 273.01 GB raw data were obtained. The resulting GC content rates of 44.41–46.64% are similar in a different dataset. A total of 273.01 Gb clean data without an adapter, ploy-N, and lower quality reads were obtained after quality control. Every dataset is at least 17.1 million clean reads and more than 89.03% of base Q30. After that, the RNA-sequenced clean reads were then anchored onto the reference genome, G. raimondii. The mapped clean read ranged from 81.37 to 91.37% of the clean reads were uniquely mapped to the reference genome (Table SR2 > 0.7); suggested datasets of other samples are reliable  have a fewer number of SNPs as compared to the other three species. In general, transcriptomes of GD5 samples should have the same genome with a reference genome. Although the transcriptome of GD5 samples and reference genome was very similar to each other (< 1 SNP per Kb of most genes), some SNPs were observed between GD5 and the reference genome, suggesting that there existed in differences in the leave′s transcriptome among plants of the same donor material. A total, 3, 2651 genes were expected to have high functional effects by SNPs in four diploid D-genome kinds of cotton. Go ontology (GO) enrichment analysis showed that a high number of the genes were highly involved in various processes such as the oxidation-reduction, stress response, protein modification, and protein phosphorylation. It has been demonstrated that abiotic stresses have played an important role in driving the transcriptional diversity among the four cotton species  lengths were ranged from 200 to 500 bp. To initiate our evolutionary analysis, we identified the strictly orthologous unigenes among 7 species, including three cultivated  and four wild species. For rigorous analysis, the genomes of allotetraploid species were separate A and D subgenome to identify orthologous transcripts. A total of 47,119 orthologous gene pairs were characterized, and 5312 single-copy transcript pairs of those were used to construct the phylogenic tree of the nine genomes by maximum likelihood methods. Phylogenetic analysis using these unigenes revealed a quite similar topology  denotes either purifying or neutral selection. Therefore, we evaluated the dN, dS, and dN/dS of the identified orthologous single-copy genes among nine genomes. Based on the above-constructed phylogenetic tree, the dN/dS for each orthologous unigene pair was evaluated in the different branches using a free ratio model (model = 1), which allows for a separate dN/dS ratio for each branch. We found that wild diploid cotton  had higher dN/dS ratios than the branch of cultivated cotton . Under natural selected pressure, wild species are showed a fast evolutionary ratio based on adaptive choices  and synonymous (dS) are used to estimate the evolutionary rate and positively selected genes (PSGs) in each species. If dN/dS value is GAD1, GAD, and GA2ces Fig. . PSGs ofces Fig. .We detected expression levels of a total of 41,053 transcripts in at least a single sample. Global expression level distributions of 40 data sets were similar to each other. Twenty-four datasets of leaves without stress treatment were used to detect gene expression divergence and conservation of four species. Samples of GD5C0R1 and GD5C0R2 were as a control group to identify different expression transcripts. Patterns of differential gene expression were characterized by four species  were removed. In conjunction with connection strengths  among 12,110 genes  were identified as hub genes. Ultimately, a total of 425 hub genes (ranging from 1 to 293 within the modules) were detected.Notwithstanding the analysis of gene expression pattern provided insights of gene expression divergence and conservation, co-expression gene networks, constructed by highly connected genes, and is more relevant to the vital biological processes of growth and development, and complex regulatory process of abiotic stress response. To grasp gene co-expression network conservation and divergence, all 40 datasets of leaves were used to construct a co-expression network by weighted gene co-expression network analysis (WGCNA). To enhance the accuracy of the WGCNA analysis, genes with fragments per kilobase of exon model per million reads mapped of less than one FPKM <  were remP < 0.05). Four major modules of highly co-expression genes were most strongly correlated (Pearson’s correlation r > 0.9) with four genomes, respectively. The largest module (turquoise), containing 3128 highly connected genes in GD5, enriched in response to stimulus and immune system process . Seven sample conditions were defined for identified significant modules Fig. b. Share.s Table S. A seconOf particular concern is that co-expression networks related to cold and salt stress, due to four diploid species presented different cold and salt stress resistance. Association analyses between co-expression modules and abiotic stress  revealed that 17 modules correlated with abiotic stress , thus representing suites of interconnected genes underlying the biological process of the abiotic stress response -like was identified in within the module. The homolog gene from the Arabidopsis perhaps could be responsible for the regulation of the plant stress response and or adaptation, growth, and development. Fifteen genes in this module were interconnected with the RALF-like protein coding gene. Except for five uncharacterized genes, ten out of fifteen interconnected genes were associated with the GO term, response to the stimulus, including Gorai.005G234900, Gorai.007G094200, Gorai.N023400, and Gorai.011G238800. The homologs from Arabidopsis have been annotated for four important transcription factors: MYB44, TCP9, TCP12, and GATA8, and are majorly involved in abiotic stress response. Moreover, two xyloglucan endotransglucosylase/hydrolase protein 22 (XTH22)-encoding genes, Gorai.003G052400 and Gorai.009G006400, were observed to be involved in carbohydrate transport and metabolism. Homologs of Gorai.005G094300 in Arabidopsis, EXORDIUM protein-encoding gene, required for cell expansion in leaves, and may be involved in signaling processes that coordinate brassinosteroid (BR) responses to environmental or developmental signals  were used to identify DEGs in four species. The number of DEGs varied from 459 to 3372 among treatments of 8 groups  ps Fig. Sa, indicaGorai.002G073700 encodes the homolog of Arabidopsis NAC72, which binds to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter [Gorai.001G239000 and Gorai.006G017400 encode homologs of MYB-like protein in Arabidopsis that involve in plant defense response [Gorai.006G147500 of skyblue3 module genes is positively selected and is interconnected with the hub gene, RALF-like protein coding gene. It further confirmed the potential regulated network that was identified in the previous analysis. Validation of the key genes through reverse genetics revealed that the VIGS-plant’s ability to adapt to the salt-alkali was highly compromised. The VIGS-plants exhibited wilting and gradual drying of the leaf margin upon exposure to salt stress . And then, those SNPs were applied for the construction of a phylogenetic tree to investigate the relationship of four species. The relationship of the four species is in line with previous studies, and the result further confirmed feasible genomic sequences analysis based on RNA-Seq de novo assembly. Allotetraploid cotton contains seven species. Based on the similar distribution of 45S and 5S rDNA between allotetraploid and diploid species in chromosomes, some researchers thought different allotetraploid species of the cotton genus have different donor species, such as G. thurberi, G. klotzschianum, G. raimondii and G. trilobum [G. raimondii showed most synteny blocks with D-subgenome of G. hirsutum and G. barbadense by comparative genomic analysis, suggested G. raimondii is the possible donor species of G. hirsutum and G. barbadense. So, some researchers thought allotetraploid cotton may be monophyletic. In our study, 5312 single-copy genes were used to construct a phylogenic tree and estimate the evolutionary rate. A-genome and A-subgenomes formed monophyly, and D-genomes and D-subgenomes formed monophyly. Our result support that G. raimondii is donor species of D subgenome of G. hirsutum and G. barbadense. It is consistent with monophyletic evolutionary theory of allotetraploid cotton [In a previous study, some researches focus on the relationship of D-genome diploid cotton, for instance, the relationship of aimondii . Range ftrilobum . But, G.d cotton .Positive selection plays a significant role in the evolution and adaptation of the plants to biotic and abiotic stresses, as gene expression and regulation changes by positive selection have been postulated to be key determinants of the rates of adaptive evolution . Our resWe observed morphological differences between the four species. The different ecosystem of four species leads to the divergence of their morphology. Previous genetic pieces of evidence indicated gene expression alteration is essential to drive phenotypic diversity during evolution . Our resG. klotzschianum is tolerant to cold and salt stress. G. klotzschianum, the natural range is the Galapagos Islands, can adjust itself to the environment. Therefore, we investigated genes in G. klotzschianum under positive selection. The numbers of PSGs are low in G. klotzschianum . Nonetheless, 33 PSGs exhibited differential expression under salt and cold stress, indicating that gene expression alteration caused by natural selection might have played an essential role to improve the environmental adaptation.The morphological analysis found that G. klotzschianum showed astonishing tolerance under cold and salt stress, we speculate a shared network that involved in cold and salt stress response was formed during the adaptive evolution of G. klotzschianum. Thirty-three (~ 10.6%) PSGs were differently expressed under cold or salt stress. In particular, in WGCNA, we found a module that was negatively correlated with salt, cold, and share.T.S conditions. 74% of genes in this module were enriched GO terms related to response to stimuli and 36.3% genes were enrichment GO term related to the signaling process. This result further confirmed our speculation. Simultaneously, we found the stress signaling process is a tentative share regulated network of salt and cold stress response. One hub gene in skyblue3 was found in WGCNA. And, 15 genes with significant correlation to the hub gene were identified. Interesting, Gorai.006G147500 among 15 high connective genes, were found. These 16 genes of the skyblue3 module were potentially regulated networks under cold and salt stress. The researcher presumes that plant cells must be capable of sensing various environmental signals [Gorai.010G168400 in Arabidopsis, encoding AtOSCA3.1 protein, play a role in the hyperosmolarity-gated non-selective cation channel that permeates Ca2+ ions [Gorai.010G168400, showed a higher expression level under salt and cold stress compared to control groups, was a potential sensor that mediates cold and salt stress in cotton, and calcium ion acts as the second messengers in response to abiotic stress [Gorai.009G294400, encoding CBL-interacting protein kinase 18, which involved in Ca2+ signal transduction, was also found that different express under cold and salt stress. Core stress-signaling pathways involve protein kinases related to the yeast SNF1 and mammalian AMPK [Gorai.005G081200 and Gorai.002G103300 which share homology to SNF1/AMPK in their kinase domains.Combining the analysis of the preceding context that  signals , and som signals , G prote signals , and COL signals . Salt an signals . The homa2+ ions . And Gorc stress . Gorai.0ian AMPK . SNF1/AMGorai.004G093200, Gorai.013G025400, Gorai.013G059500 and Gorai.013G176900 were up-regulated under cold and salt stress, correlating with the oxidation-reduction process. PSGs and sky blue-module genes also generated many important candidate genes, such as Gorai.004G227900 and Gorai.002G226600, which are related to keeping the homeostasis of reactive oxygen species. Extensive investigations of these genes under cold and salt stress would help us understand how DEGs regulate homeostasis of reactive oxygen species and signal transduction and their function during cold and salt response, thus helping us to increase yields in cotton.The oxidation-reduction process is also involved in salt and cold stress response by keeping the homeostasis of reactive oxygen species . We founG. showed klotzschianum showed better resistance under cold and salt stress. Compared with the other three species, more PSGs were detected in G. klotzschianum, and 9.6% PSGs were differently expressed under cold or salt stress. In G. klotzschianum, 27.1% DEGs under salt stress were overlapping with that under cold stress and we found the skyblue3 module was significantly negatively correlated with cold, salt, and share.T.S condition groups. Thus, there are share networks involved in cold and salt stress response, such as signal transduction and oxidation-reduction processes. Based on our multiple analyses, a set of candidate genes involved in cold and salt stress response is putatively proposed, providing genetic resources for multi-abiotic resistant cotton breeding.Comparative transcriptome analysis of four diploid D-genome kinds of cotton reveals sequence and gene expression variations. Gene evolution analysis of wild and cultivated kinds of cotton identifies positively selected genes which involve in the domestication and evolution of cotton and estimate the evolutionary rates. In this work, we found that evolutionary selection could drive gene expression alteration to adapt environment and gene expression variation is the primary evolutionary event during the divergence of four D-genome species. The expression pattern analysis found that six profiles showed distinct species-specific characteristics and genes of those profiles were involved in response to stimulation. Thus, gene expression variations were essential drivers of the morphological variations related to environmental adaptation during evolution. More DEGs were identified under cold stress in contrast to DEGs under salt stress, indicated cold stress lead to expression change of more genes. G. thurberi (D1), G. klotzschianum (D3-k), G. raimondii (D5) and G. trilobum (D8). There are 13–14 species in diploid D-genome cotton and they are originally distributed from Southwest Mexico to Arizona, with additional disjunct species distributions in Peru and the Galapagos Islands. Here, G. thurberi, G. klotzschianum, G. raimondii and G. trilobum were obtained from USDA-ARS Southern Agricultural Research Centre in College Station, Texas, USA and currently perennially preserved in the National Wild Cotton Nursery, located in Sanya, Hainan province, in the peoples republic of China and managed by the Institute of Cotton Research, Chinese Academy of Agricultural Sciences (ICR-CAAS). The wild cotton species seeds were obtained from the wild cotton nursery which is under the management of the institute of cotton research, Chinese Academy of Agricultural Sciences, (ICR-CAAS), China. The seeds first germinated at 28 °C, 16 h light/8 h dark cycle, and light intensity of 150 μmol m− 2 s− 1 in 15% water content sands. Three days after germination, the proper plants were potted in soil and placed in a growth room under the same condition. The seedlings were grouped into the three categories, at two simple leaves and one heart-shaped leaf (time point: 0 h) stage. First group put under normal condition , a second group was watered with 300 mM NaCl solution  and the third group put at low temperature . The first group was used to reveal the expression divergence and conservation and leaves of seedling were collected at 0 h, 6 h, and 12 h. Second and third groups were used to reveal regulation under cold and salt stress, and leaves were collected at 12 h. This experiment had two repeats.Four diploid wild species of D-genome cotton were used in this study, including http://www.biomarker.com.cn) provides experimental procedures and commercially performed it.The RNA from the samples was extracted by use of TRlzol Reagent  in three biological replicates as the instructional manual. RNA quality and concentration was verified by use of Agilent 2100 Bioanalyzer . The RNA quality was further checked by NanoDrop 2000 spectrophotometer and electrophoresed on the agarose gel. RNA with quality and purity in the 260/280 ratio of 1.8–2.1, 260/230 ratio ≥ 2.0 were applied for further analysis. The RNA was then transcribed into by use of M-MLV transcriptase kit, obtained from TaKaRa Biotechnology, Dalian, China. Moreover, the mRNAs retrieved by NEBNext Poly (A) mRNA Magnetic Isolation Module . The transcribed cDNAs were applied to construct the cDNA libraries using the NEBNext Ultra RNA Library Prep Kit for Illumina  and NEBNext Multiplex Oligos for Illumina . Briefly, the enriched mRNA was fragmented into RNAs with approximately 200 nt, which were used to synthesize the first-strand cDNA and then the second cDNA. Adaptor ligation procedure was used to analyse the double stranded cDNAs, and the conformed fragments were retrieved by Agencourt AMPure XP beads , and enriched by polymerase chain reaction (PCR) amplification. Sequencing of the constructed cDNA libraries was carried out on a flow cell by use of an Illumina HiSeq™ 2500 sequencing platform. Beijing Biomarker Technologies  [The evaluation of the quality of the various gene expression profiles, the expression levels were analysed by fragments per kilobase of transcript per million fragments mapped (FPKM) . Differes (DEGs) .MQ0 > = 4 and (MQ0/ (1.0*DP)) > 0.1; QUAL < 10; QUAL < 30.0 or QD < 5.0 or HRun > 5, and only those SNPs with distance > 5 were used.Picard - tools v1.41 and samtools v0.1.18 was employed to sort and remove the duplicated reads and integrate the bam alignment per sample. The Samtools software was employed to carry out the Single nucleotide polymorphisms (SNP) calling. The raw vcffiles were filtered with GATK prescribed standard filter method and other variables. The standard and variables were programmed as clusterWindowSize: 10; www.biocloud.net) in this research.The left and the right files from all libraries were pooled into one large left and right.fq file. Transcriptome assembly was established based on the left.fq and right.fq by use of Trinity with parameters set with min_kmer_cov set to 2 by default and all other set to default . ExpressG. hirsutum and G. barbadense) and two diploid kinds of cotton (G. arboreum and G. raimondii) were obtained from Cottongen database (https://www.cottongen.org). The genome of allotetraploid species contains two subgenomes (A- and D-subgenome). To ensure the accuracy and reliability of ortholog identification, two subgenomes of allotetraploid species were separated. Together with our results of transcriptome assembly , a total of nine genomes were used for phylogenetic analysis and A-genome clades were as out-group . Orthofinder [G. thurberi, G. klotzschianum, G. raimondii, and G. trilobum) SNPs using BMK Cloud (www.biocloud.net). The evolutionary rate of each lineage for the nine gnomes was estimated using the codeml program in the PAML [model = 1). We grabbed the dN, dS, and dN/dS from the result of Codeml. We filter the Genes with dS = 0. If the PSGs dN/dS > 1 were reported as PSGs.High-quality draft genomes of two allotetraploid cotton  using BMKCloud (www.biocloud.net).To determine the expression patterns of the genes over time within each species, K-means clustering was used to visualize genes expression pattern . The cotton seedlings were planted in the greenhouse with the temperature set at 26 °C, and 16 h light/8 h dark cycle. A total of 24 seedlings were inoculated for each construct. After 14 days of Agrobacterium inoculation, the VIGS-plants, and non-VIGS plants were then exposed to salt-alkali treatment for 6 days. The samples were then collected for morphological and physiological analysis.The virus induced gene silencing (VIGS) of candidate genes, RALF and FLA, in ocedures , 45. FirAll data generated from this experiment was analysed by using the R (v3.5.0) software.https://www.ncbi.nlm.nih.gov/sra/ PRJNA554555).The RNA-seq data generated in this research work and reported have been deposited in National Center for Biotechnology Information (NCBI) under accession number PRJNA554555  Analysis of the scale-free fit index for various soft-thresholding powers (β). (B) Analysis of the mean connectivity for various soft-thresholding powers.Additional files 9:Figure S2. Heatmap plot of the topological overlap matrix.Additional files 10:Figure S3. Venn diagrams of DEGs; PSGs and genes of skyblue3 module. A-D represents the Venn diagram of GD1, GD3, GD5, and GD8, respectively.


Theileria genus, transmitted by hard ticks and which possesses a complex life cycle. The clinical course of the disease ranges from benign to lethal, but subclinical infections can occur depending on the infecting Theileria species. The main clinical and clinicopathological manifestations of acute disease include fever, lymphadenopathy, anorexia and severe loss of condition, conjunctivitis, and pale mucous membranes that are associated with Theileria-induced immune-mediated haemolytic anaemia and/or non-regenerative anaemia. Additionally, jaundice, increases in hepatic enzymes, and variable leukocyte count changes are seen. Theileria annulata and Theileria parva induce an incomplete transformation of lymphoid and myeloid cell lineages, and these cells possess certain phenotypes of cancer cells. Pathogenic genotypes of Theileria orientalis have been recently associated with severe production losses in Southeast Asia and some parts of Europe. The infection and treatment method (ITM) is currently used in the control and prevention of T. parva infection, and recombinant vaccines are still under evaluation. The use of gene gun immunization against T. parva infection has been recently evaluated. This review, therefore, provides an overview of the clinicopathological and immunopathological profiles of Theileria-infected cattle and focus on DNA vaccines consisting of plasmid DNA with genes of interest, molecular adjuvants, and chitosan as the most promising next-generation vaccine against bovine theileriosis.Theileriosis is a blood piroplasmic disease that adversely affects the livestock industry, especially in tropical and sub-tropical countries. It is caused by haemoprotozoan of the  Theileria species belonging to the phylum Apicomplexa [T. parva) have been recorded per year, leading to the loss of about USD 300 million [Bovine theileriosis is an important tick-borne disease of domesticated cattle in tropical and subtropical countries, caused by several complexa . Some spcomplexa ,3,4,5. Fcomplexa . In Sub- million .Theileria species that infect bovines include T. annulata, T. parva, T. mutans, T. orientalis complex , T. tarurotragi, T. velifera, T. sinensis and Theileria sp. Yokoyama, a newly discovered Theileria species closely related to T. annulata [Theileria species is reported in T. orientalis/sergenti/buffeli group should be considered as a single or separate species individually referred. The phylogenetic analyses of Theileria species are based on 23S ribosomal RNA, 18S ribosomal RNA and the major piroplasm surface protein (MPSP) gene [T. orientalis [T. orientalis complex for all three benign Theileria species in the T. orientalis complex group, while others still refer to them separately, but a consensus on whether it should be considered as a single species or referred to individually is yet to be reached [annulata ,11,12,13SP) gene ,15. The  reached ,17.Theileria species possess unique characteristics that make them different from other apicomplexa such as Toxoplasma and Plasmodium, in that they do not reside inside a parasitophorous vacuole [Theileria sporozoites are free within the host cell cytosol after the rapid dissolution of the surrounding host cell membrane and immediately associate with the host cell microtubules to differentiate into schizonts [Theileria spp. are transmitted by Ixodid ticks of the genera Haemaphysalis, Rhipicephalus, Hyalomma and Amblyomma , is caused by the infection of cattle with buffalo-derived T. parva, formerly known as T. parva lawrenci [T. parva exhibits much greater genotypic and antigenic diversity than the cattle-adapted T. parva . Theileus ticks . Buffalost fever  and buffd cattle . In cont species , the bufd cattle .T. orientalis complex T. buffeli, T. sergenti, and T. orientalis. Besides tick transmission [T. orientalis complex exerts its major effect following the infection of the erythrocytes by the piroplasmic forms and subsequent erythrocyte destruction [T. orientalis exist, which cause reduced growth and production losses in infected cattle and therefore have an economic impact [T. orientalis complex parasites have been identified based on the sequence variations in the MPSP gene, which is conserved in T. orientalis complex piroplasms and is expressed during the intraerythrocytic stage of the protozoa [chitose type I and ikeda type 2) of the 11 genotypes of T. orientalis have been demonstrated to be pathogenic [T. buffeli, while genotype 6 was reclassified with the taxonomic name T. sinensis to reflect its divergence from other benign Theileria species [T. orientalis with different variants is common [T. orientalis was considered to be genotype-related; however, one of the means by which T. orientalis evades the host immune system is that infection usually occurs as a set of mixed infections with more than one of its genotypes [Theileria orientalis double or triple genotype co-infection is very common, and affected cattle seldom show any clinical signs of the disease, thereby making it difficult to diagnose subclinical and mild forms of theileriosis [T. orientalis infections are highly dependent on environmental stress factors, the level of acquired immunity in the affected cattle and herd genetics [The non-schizont-transforming species include emerging haemoprotozoans belonging to the smission , their tsmission ,63. Theitruction . Recent c impact ,64,65. Eprotozoa . Two  have been recorded [Theileria annulata caused a regenerative anaemia with significant decreases in the RBC count, packed cell volume (PCV), haemoglobin (Hgb) concentration, mean corpuscolar haemoglobin concentration (MCHC) with increased mean corpuscular volume (MCV), and marked reticulocytosis in infected Holstein cattle [In recorded ,71,72. Arecorded . Theilern cattle . Decreasn cattle ,76.T. orientalis infections in cattle raised in mountainous areas [T. orientalis infection and they showed a significant more severe regenerative anaemia compared to the MPSP negatives [Theileria species-infected Malaysian cattle [T. buffeli infection [Similar to tropical theileriosis, regenerative anaemia associated with either intravascular or extravascular haemolysis  and signus areas . In Koreegatives . Interesegatives . Macrocyn cattle  and in anfection .Theileria species and strain. In fact, panleukopaenia was a remarkable finding in tropical theileriosis and Omer et al. [T. buffeli infection. Higher levels of parasitaemia in cattle with tropical theileriosis resulted in reduced lymphocyte and monocyte numbers and increased absolute neutrophil numbers [T. annulata-infected Egyptian water buffaloes [T. parva infection with two different strains, fluctuations in leukocyte and granulocyte counts were found, with an early decrease followed by a more severe agranulocytosis just before the animals died [Leukocyte count varied based on infecting r et al.  reportedr et al.  reported numbers . Lymphop numbers . Signifi numbers . Leukopa numbers ,76. Throuffaloes . Variabiuffaloes . Howeveruffaloes  reportedals died .T. annulata-infected cattle includes a prolonged activated partial thromboplastin time and prothrombin time, thrombocytopenia and hyperfibrinogenaemia [The coagulation profile of genaemia .T. annulata-infected water buffaloes. Similarly, sialic acid value increased in T. annulata-infected Holstein cattle as well as adenosine deaminase activity, while total antioxidant capacity dropped [Anaemic cross-bred cows with tropical theileriosis had significant decreased levels of sodium, ionized calcium, total protein and albumin compared to non anaemic cows, and increased concentrations of blood urea nitrogen and creatinine . El-Deeb dropped .T. buffeli [Significant elevations in the serum activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), serum levels of low-density lipoprotein cholesterol (LDL), very-low-density lipoprotein cholesterol (VLDL), β-hydroxybutyrate and non-esterified free fatty acids, and significant decreases in serum albumin, globulin, cholesterol, triglyceride, glucose, calcium and phosphorus levels were notable findings in Egyptian water buffalo tropical theileriosis ,82. Simi buffeli .Urinalysis was performed in 8-day-old newborn female twin Holstein calves with fatal disease and showed trace amounts of proteins in urine, haemoglobinuria and haematuria .Theileria orientalis is also known to induce immune-mediated haemolytic anaemia [T. sergenti is usually shortened as the immune system produces antibodies directed against the parasites as well as against its own erythrocytes [Theileria-infected cattle. Exposure of the phosphatidylserine on the cell surface can induce an antibody response and function as a marker of the phagocytic clearance of RBCs by macrophages [T. sergenti infected RBCs occur without the involvement of antibodies or complement. Shiono et al. [The pathophysiologic mechanism of anaemia in oriental theileriosis is multifarious . Anaemia anaemia . The lifhrocytes ,86. Autorophages . Hagiwarrophages  evidenceo et al. .T. parva strain caused lymphocytolysis in lymph nodes where lymphoproliferation was low and only few schizonts were found. Conversely, anaemia was mild and regenerative in cattle and buffaloes with East Coast fever, and numerous macrophages were present in the lymphoid organs [T. parva merozoites infect erythroid and other haematopoietic precursor cells, resulting in the extensive destruction of haematopoietic cells in bone marrow [Mbassa et al.  reportedd organs . Howevere marrow .Theileria infection. Innate immunity provides a front line of host defense, ensuring rapid but nonspecific defense mechanisms immediately after pathogen invasion in the host. Specific and protective immune response against all parasite stages  is mounted following inoculation by infected ticks [Theileria parasites, natural killer cells (NK), macrophages and γδ T lymphocytes secrete cytokines that modulate the interaction between immune system cells in the host. These interactions promote synergy between macrophages and NK cells and increase the interferon (IFN)-γ level, which activates macrophages and interleukin (IL)-12 for the proliferation and activation of NK cells. The innate response of macrophages and NK cells drives the adaptive response towards T helper 1 responses [Theileria schizont-infected and activated uninfected macrophages have an influence on both the innate and adaptive immune response. Therefore, NK, CD8+ and γδ T cells produce IFN-γ, which acts on macrophages. The macrophages in turn produce nitric oxide that clears both trophozoite and schizont-infected cells. NK cells lyse schizont-infected cells, clearing schizonts while the macrophages undergo apoptosis, as reviewed in [+ T cells) to differentiate, thereby forming a link between innate and adaptive immunity [+ T cells secrete cytokines such as IL-2 and IFN-γ, which are needed for the clonal expansion of the cytotoxic T cells [Theileria by inhibiting the trophozoite-infected cells and macro-schizont infected cells . NevertTheileria annulata infections are usually fatal, especially in susceptible, imported or naïve cattle [T. annulata-infected cattle when compared to the lymphoid tissue of cattle infected with T. parva, where an extensive population of lymphoid cells were present [T. annulata did not cause the destructive disease, it was shown that schizont-infected macrophages possess a powerful mechanism of immune subversion. They inhibit the proliferation of schizont-infected lymphocytes and also suppress the non-specific activation of lymphocytes induced by the superantigen-like molecule  produced by both infected and non-infected macrophages, influence the outcome of infection, suppressing the growth of parasitized cells but inducing clinical signs and causing tissue damage [e cattle . The deae cattle . Signifie cattle . Previoue cattle ,96. The e cattle ,95,96. T present . The imm present . In caseiewed in ). The duiewed in ). Therefe damage .T. annulata-infected cattle showed a significant decrease in the T (CD4+ and CD8+) and B cell populations in case of high parasitaemia, while an increase in T and B cells was observed in cattle infected with <1% parasitaemia [T. annulata predominately infect macrophages, dendritic cells and B cells and are no longer regarded as protozoa that cause lymphoproliferation, while T. parva infects T cells, thereby causing lymphoproliferation. The different cell tropism was inferred by the type of cytokines produced by T. annulata schizont-infected cells, which are only characteristic of macrophages. In fact, the cytokine profile of schizont-infected monocytes/macrophages includes IFN-α, TNF-α, IL-1α, IL-1β, IL-6, and IL-10 [T. annulata schizonts predominately have an affinity for CD3− CD11b+ phagocytic cells [T. annulata-infected mononuclear cells are found throughout the lymphoid organs, and their metastasis is due to their expression of MMP [Flow cytometric analysis of samples from sitaemia . The decsitaemia ,98. Baldsitaemia  and Spoositaemia  confirmeic cells , which eic cells . In vivon of MMP  and of an of MMP . When scn of MMP .Theileria parva transforms lymphocytes and inhibits cell apoptotic pathways to secure its own survival within the host cells [Theileria-infected immune cells have been extensively reviewed by Tretina et al. [T. parva-infected lymphocytes possess a cancer-like phenotype. However, this is a reversible process as transformed T. parva-infected lymphocytes return to a resting phenotype upon clearance of the parasites by the theilericidal drug buparvaquone. Therefore, the transformation of T. parva-infected lymphocytes is not dependent on defined genomic changes in the host cell [T. annulata bovine lymphosarcoma cell lines and bovine leukaemia cells, which died from apoptosis within a couple of days [T. parva-infected cells to proliferate indefinitely, avoiding apoptosis, was attributed to the suppression of p53 activity, whose primary role is to mediate host cell apoptosis but which is quickly re-activated after treatment with theilericidal drugs [2-terminus and thereby impedes its transcriptional activity [T. parva-infected cell lines. This thereby suggested that an increase in MDM2b mRNA levels is associated with lymphocyte proliferation [T. parva-infected cells through p53-independent mechanisms. The p53 protein is mainly localized within the nucleus of infected cells, but conflicting reports have shown that it is associated with the schizont membrane [T. parva transformed cells, and they concluded that the active degradation of p53 by MDM2 and cytoplasmic sequestration contribute to the continuous immortalization of the host cells and the proliferation of the protozoa [T. parva-infected cell lines and promote apoptosis [T. parva-infected cells [st cells . This past cells . Therefost cells . The feaa et al. . Brieflyost cell . However of days ,106. Theal drugs . The p53al drugs ,109. In al drugs . The mouactivity  and can activity . Many spactivity . The oveactivity . MDM2b iactivity  and was feration . Hayashiferation  opined tmembrane . Howeverprotozoa . it is wprotozoa . Additiopoptosis . Among ted cells .+ macrophages  amongst other intralesional macrophages in the pulmonary, lymphoid, splenic and hepatic tissues of T. parva-challenged cattle [+ macrophages in tissues have been correlated with poor prognosis [Theileria parva schizont-infected lymphocytes produce a significant amount of IL-10 [+ expression. Protozoa induce alternatively activated macrophages and trigger the anergy, exhaustion and apoptosis of T cells as a means of host immune evasion [Toxoplasma gondii infection in mice [Trypanosoma congolense [T. brucei [+ haemoglobin/haptoglobin receptor protein on their cell surfaces and play a role as a haemoglobin or haptoglobin scavenger. The CD163+ macrophage response include an interference with CD8+ T cell responses and the inhibition of the proliferative activity of CD8+ T cells [T. parva-infected lymphocytes and erythrocytes leads to the release of free iron and proinflammatory conditions, resulting in necrosis and the subsequent activation of an enhanced CD163+ macrophage response in acute East Coast Fever [T. parva-infected erythrocytes and lymphocytes result in increased levels of proinflammatory cytokines that activate an enhanced CD163+ macrophage response. Interleukin-17 is a proinflammatory cytokine that plays a role in the pathogenesis of several protozoal diseases, such as ocular toxoplasmosis [T. parva-infected cattle and is a consequence of IL-17 secreting CD163+ macrophages.Immunohistochemistry demonstrated an increased number of infiltrating IL-17 secreting CD163d cattle . Increasrognosis . Theilerof IL-10 , which i evasion . Many so evasion . Alterna in mice  and in bngolense  and T. b. brucei  infectio T cells . The lysst Fever . It is plasmosis , avian clasmosis , cutaneolasmosis , and in lasmosis . Severe lasmosis . The binlasmosis ,127. Intlasmosis . The lymT. parva-challenged cattle [T. parva schizont-infected lymphocytes leads to a secondary systemic macrophage activation syndrome and a lethal vasculitis [An increased number of infiltrating macrophages is observed in the enlarged spleen and lymph nodes, the liver, and the lung of d cattle . Macrophd cattle , also knd cattle , lung cad cattle  and protd cattle . In summsculitis .Tentative diagnosis of theileriosis is made based on suggestive clinical signs, such as enlarged lymph nodes, pyrexia, anorexia, a loss of condition and pale mucous membranes. Confirmatory diagnosis is obtained with the microscopic examination of Giemsa-stained blood smears and lymph node fine needle aspirate smears, serological and molecular techniques. The use of the optical light microscopy method has been, in the past, the only available diagnostic tool that provided the morphological identification of blood parasites in ruminants. However, diagnosis solely based on the blood or lymph node smear method has low accuracy and is associated with technical problems . The micTheileria-specific antibodies by employing ELISA assays such as the T. annulata surface protein (TaSP)-ELISA [Theileria species [T. annulata infection in cattle under field conditions. A recombinant antigen ELISA based on MPSP has been developed for detection of T. orientalis [Serological methods measure P)-ELISA , and theP)-ELISA . The ind species . Mohamed species  demonstrientalis .® quantitative real-time PCR (qRT-PCR) assay [Theileria species and strains. Moreover, PCR molecular techniques can detect newly emerging and mutant strains [Theileria species include the loop-mediated isothermal amplification (LAMP) assay for the detection of T. annulata [Theileria [Molecular diagnostic techniques, such as PCR based on the 18S ribosomal RNA gene, MPSP gene, 28S ribosomal RNA genes and the sequencing of PCR amplicons ,78,138 aR) assay , are reg strains . Serolog strains ,140 and annulata , bead-baannulata  and randannulata ,144. A lheileria .Immunization is one of the most successful strategies for the prevention of infectious diseases and vaccines against bovine theileriosis are among the few vaccines available for protozoal diseases of animals .T. parva infection [T. parva parasites, alongside the treatment with expensive depot formulation of antibiotics. The ITM is not cost-effective and has a cumbersome production process as it requires large numbers of cattle for vaccine production. It is also difficult to standardize, store and distribute [The infection and treatment method (ITM) is currently the only immunization protocol available for nfection ,135. Thestribute . Live atstribute .Theileria subunit vaccine . Controlled release and needle-free  approaches are new delivery methods that are still in the research and development stage.The aims of an ideal vaccine is to produce the same immune protection that usually follows natural infection but without causing disease to generate long-lasting immunity, to prevent clinical disease and mortality after natural challenge and to interrupt the spread of infection to susceptible animals. Therefore, to achieve successful immunization, several factors have to be considered and they include the choice of appropriate antigen and adjuvant, dosing or immunization schedule and delivery platform. The choice of antigen is highly dependent on the ability of the antigen to express immunodominant epitopes and whether it possesses the ability to induce the production of fully neutralizing antibodies and activate cytotoxic T cell response. Adjuvants enhance the immunogenic properties of vaccines by prolonging antigen persistence, enhancing co-stimulatory signals, increasing local inflammation and stimulating lymphocytes via induced cytokines. Proinflammatory cytokines such as IL-12 and IL-2 stimulate both an innate and adaptive immune response and promote T-lymphocyte proliferation. These two cytokines could act as immunopotentiators if added to a iewed in ). The roT. parva infections. Challenges encountered facing the production of a global T. parva subunit vaccine include genetic complexities of T. parva strains [T. parva strains [T. parva subunit vaccination [Several vaccine trials utilizing various antigens and delivery routes have been performed against  strains , the hig strains , the bio strains , and thecination .T. parva vaccine antigens, Bastos et al. [T. parva-immune cattle develop both humoral and cellular immune response to this antigen and significant amounts of IFN-γ were produced by CD4+ T cells following ex vivo exposure to recombinant, mammalian-expressed Tp9. Therefore, recombinant Tp9 can be further evaluated as a component of a T. parva subunit vaccine.In order to produce new s et al.  investig+ T cell response—are stimulated by antigens such as T. parva schizont antigens (Tp1-Tp12) [T. parva sporozoite p67 antigen [T. annulata sporozoite antigen SPAG1 [+ T cells have been tested for their ability to induce immune responses and have been found to be vaccine candidates. These antigens also play a role in preventing or reducing the entry of sporozoites into host lymphocytes [T. parva [Theileria lestoquardi surface protein (TlSP) and Theileria annulata surface protein (TaSP)—are expressed in T. lestoquardi [T. annulata [T. parva and T. annulata and suggested that a T. annulata subunit vaccine is likely to protect against T. parva infections. This is because p67 and SPAG1 antigens can confer cross-species immunity [T. annulata sporozoites, and vice versa.Mucosal and/or systemic antibodies—and most especially the CD8p1-Tp12) ,152,153, antigen  and T. aen SPAG1 . Specifiphocytes . The polT. parva , and it T. parva . The antT. parva . Antigentoquardi  and T. aannulata , respectannulata . The devannulata  extensivimmunity , even thimmunity . The abiT. orientalis complex may not be feasible, although the development of a live vaccine based on one or two of the T. orientalis benign genotypes may be considered. Even though a certain immunological and genetic diversity exists among them, they are clustered together on one clade of the phylogenetic tree. It was proposed that, since they are grouped together, a cross-immunity between genotypes may exist [T. orientalis has had no success to date, and progress for this approach has been negligible over the years. Globally, there is no suitable vaccine against T. orientalis complex infection in cattle to date [T. orientalis and there is a low parasitaemia [buffeli/chitose genotypes are more closely related to each other than to the ikeda genotype; therefore, this genetic diversity may have implications for vaccine design [buffeli/chitose genotypes can stimulate heterologous immunity against the ikeda genotype. This lack of suitable vaccines further complicates T. orientalis management. Despite the development of a live vaccine being much more feasible than the subunit vaccine, Jenkins and Bogema [T. orientalis complex.The development of an effective subunit vaccine or live vaccine against ay exist . The use to date ,165. Thisitaemia . The bufe design . It is nd Bogema  opined tT. parva subunit vaccine is next-generation vaccine technology based on DNA vaccines. A DNA vaccine is particularly attractive for the prophylaxis of intracellular pathogens such as herpes simplex virus and mycobacteria, and since the Theileria parasites are intracellular pathogens, a DNA vaccine that expresses cytokines should be appropriate [+ T cells can aid in the response to chronic or latent infection. An increased IFN-γ level enhances the activities of cytotoxic T lymphocytes and NK cells. The use of DNA constructs encoding molecular adjuvants such as IL-6 and TNF-α has proven useful in several cattle diseases, such as foot and mouth disease. The addition of a molecular adjuvant enhanced antigen-specific cell mediated responses elicited by the DNA vaccine [Staphylococcus aureus in dairy cows [+ T cell responses [T. parva-challenged cattle induced CD4+ and CD8+ T cell IFN-y responses but not the antigen-specific cytotoxic T -ymphocyte (CTL) response [A possible solution to challenges facing the use of a ropriate ,169. In ropriate . For ins vaccine . DNA vaciry cows . Recombiiry cows . The adjiry cows . These cesponses . Howeverresponse .T. parva codon-optimized and native sequence PIM antigen was delivered through the intra-dermal route in Holstein steers. This method is also known as gene gun immunization, whereby DNA-encoded antigens are delivered directly into the nucleus of epidermal and dermal professional and non-professional antigen-presenting cells. These antigens are then expressed and processed to elicit an immune response. The gene gun has been used for the delivery of influenza vaccine in ferrets [T. parva infection elicited a robust protective immune response characterized by significant antibody and cell-mediated responses (Th1/IgG2 and INF-γ responses). Although the antibody response mounted was not enough to prevent East Coast fever in the T. parva-challenged calves, gene gun immunization may serve in the future as a suitable vaccine platform against T. parva and other bovine blood pathogens in cattle that sufficiently express the MHC class I molecules, with the role of binding and presenting PIM epitopes to cytotoxic T cells [By delivering DNA via different routes, DNA vaccines can generate a different type of immune response . For instance, intradermal injection of DNA vaccine elicits a predominate Th1 response, while the so called biological ballistic or biolistic DNA injections mainly stimulate a Th2 or a balanced Th1/Th2 response . Fry et  ferrets . The adv ferrets . The use T cells . Theileria species-infected cattle remain carriers for life and serve as a source of infection to naïve cattle thanks to the wide distribution of tick vectors and suitable tick habitats. Therefore, an effective vaccine able to prevent at least losses caused by acute clinical diseases from the most pathogenic Theileria species would have a significant economic impact. We propose a further investigation into DNA vaccine technology for the control and prevention of bovine theileriosis, since this technology is potent against latent/persistent or chronic infections and is characterized by sustained cellular and humoral immune responses. The epidermal injection of plasmid DNA encoding Theileria antigens generates both humoral and cellular immune response against invading protozoa. Moreover, DNA therapeutic vaccines can be delivered repeatedly without initiating an immune response against the DNA plasmid. Importantly, they are simple and cost-effective to produce in large quantities and are more heat stable than conventional vaccines. Their temperature stability is a great advantage for use, particularly in countries where cold chain maintenance is problematic.Co-immunization with immune-modulatory adjuvants such as plasmids encoding specific cytokines and chemokines as part of the vaccine formulation boosts the adaptive immunity. Chitosan—an easily absorbed, biodegradable nanoparticle—should be considered as a suitable antigen carrier as it permits slow release and sustains gene expression. The administration of plasmid DNA-encoding immunogenic proteins of a pathogen stands out as a novel approach for developing new-generation vaccines for the prevention of bovine theileriosis.Theileria vaccines. However, the complexity of immunization against theileriosis emerged and, in the future, an integrated approach for effective bovine theileriosis control and eradication programs could combine the administration of next-generation vaccines and theilericidal drugs and the genetic selection of Theileria-tolerant or resistant cattle.Currently, research is continuing into the exploration of the optimal route of DNA vaccination, the best antigen candidates, the dose and dosing/immunisation schedule for


Babesia species reported from domestic cats. Description of the four species  named based on morphology and/or host specificity is documented. Feline babesiosis is of major veterinary concern only in South Africa. Reports of the rare occurrence of feline babesiosis cases in Europe  and Asia  are documented. Molecular characterization has revealed that cats can harbor a variety of Babesia species. The previous practice of referring to all piroplasms, especially small ones, seen on feline blood smears as B. felis is therefore no longer tenable. The near-full-length 18S rRNA gene sequences entered into GenBank in 2001 (accession no. AF244912) are designated as definitive for B. felis sensu stricto. All published literature relating to molecular characterization of feline Babesia species that could be traced was critically assessed. Four Babesia species are now known to be involved in causing feline babesiosis in South Africa: the closely related B. felis s.s. and Babesia leo (clade I), Babesia lengau (clade II), and Babesia species cat Western Cape . Clade VI also includes Babesia canis presentii and Babesia hongkongensis reported from cats in Asia. Six other Babesia species have been reported from domestic cats: the dog-associated B. canis s.s., Babesia gibsoni, and B. vogeli, as well as Babesia lohae, Babesia microti, and Babesia vulpes. Phylogenetic relationships of all named species were assessed and are presented as trees. The relatively high prevalence of B. vogeli in clinically healthy cats  suggests that immunocompetent cats can harbor the infection with no discernible untoward effects. Reports of occurrence of B. felis and other Babesia species in domestic cats should be accepted only if they are supported by credible molecular provenance.This is the first comprehensive review of the literature pertaining to  In the first reference to feline babesiosis, published in 1904, it was claimed that “spontaneous pyroplasmosis” occurred in tame and wild cats in India . SporadiBabesia species was based on descriptions, detailed or otherwise, of morphology (including dimensions) of the organism. This led to the notion that Babesia species can be classified either as “large” or “small.” This approach had its pitfalls because piroplasms undergo marked morphological changes during their development in erythrocytes. In addition, there can be substantial overlap between measurements of “large” and “small” babesias. A second approach was to assume that Babesia species are more or less host specific. The logical outcome of such reasoning was that any Babesia seen on a blood smear of a wild felid should, by definition, be the same species as that found in domestic cats. Neither approach was satisfactory, and caution should be exercised when quoting names of Babesia species used in such publications.Before the advent of molecular characterization of species, one approach to identifying Babesia species from domestic cats. A similar situation exists in African great ape populations, for example, where molecular characterization is revealing an unexpected diversity of Plasmodium species that had not been detected previously , were regarded as definitive for Babesia felis sensu stricto  in Sudan. Trophozoites varied in diameter from less than 1 μm to 2.25 μm, the majority being about 1.25 μm; the four pear-shaped merozoites were arranged in a cruciform manner  shipped from San Francisco arrived at the Cairo Zoological Gardens, Egypt, where they were housed in close proximity to other large felids  Since Mangrulkar  was established artificially in spleen-intact domestic cats, which did not develop overt clinical signs (Babesia herpailuri (Babesia pantherae from leopards (Panthera pardus), a piroplasm smaller than B. herpailuri, was established in splenectomized domestic cats  may be a reservoir of infection. The disease was subsequently reported from Knysna, a coastal town approximately 430 km east of Cape Town   .Babesia, with mean dimensions 3.6 × 2.3 μm. In Poland, a 10-year-old cat showing weakness, anemia, fever, and hematuria recovered fully after administration of imidocarb  sequencing of a 559-bp fragment of the 18S rRNA gene showed only 95% homology with B. canis s.s.The only clinical case in Germany, reported in 1997 , was a 1midocarb . PiroplaB. canis s.s. was incriminated in one feline babesiosis case in Spain  of cats presented at a university clinic . Blood sBabesia canis subspecies presentii was described and named from two cats in Israel; one was sick, and the other a subclinical carrier  for classifying organisms to species level based on this variation. Greay et al.  are regarded as definitive for B. felis s.s.  (One (2.9%) of 34 healthy cats in Qatar was positive for Y452707) . BecauseBabesia species (11/45) than the conventional PCR (5/45). The authors claimed that the piroplasms involved were B. felis. These finding cannot be verified, however, because close inspection of the conventional and LAMP PCR primers used in the study indicated that they could amplify any Babesia species Furthermore, no sequencing was done to confirm the PCR and LAMP results.In a study done by Salim et al.  to develBabesia canis subspecies presentii was described from two cats in Israel (GenBank accession nos. AY272047 and AY272048) (AY15057) of the 18S rRNA gene sequenced when B. canis s.s. was identified in cats in Spain and Portugal .Y272048) . The relPortugal , specimeB. gibsoni in cats was from St Kitts in the Caribbean, where B. gibsoni was found in 5 (4%) of 119 apparently healthy cats (KY073362) were identical and showed 100% identity to 37 B. gibsoni sequences in GenBank .The only report of thy cats . The parBabesia species and tentatively named B. hongkongensis, was found in blood and kidney specimens of a free-ranging cat in Hong Kong (JQ867357) had 90.4% identity with those of B. gibsoni (AB499087.1). The near-full-length 18S rRNA gene sequence (1 612 bp) (GenBank accession no. JQ867356) had 96.7% nucleotide identity to various Babesia sequences found in feral raccoons (Procyon lotor) and dogs. Phylogenetic analysis of the 18S rRNA gene sequence data showed that B. hongkongensis falls into a distinct branch of the Babesiidae.An apparently new species, genetically and geographically distinct from other previously described ong Kong . Its mitBabesia lengau was described from cheetahs (Acinonyx jubatus) in South Africa   (Q411417) . SequencGQ411430 . BabesiaBabesia leo (GenBank accession no. AF244911) was described from lions (Panthera leo) in Kruger National Park, South Africa  was 99% similar to B. leo (h Africa . BabesiaBabesia lohae (GenBank MG593272 and MG593273) was identified in a female Ixodes holocyclus tick recovered from a cat, as well as in an Ixodes tasmani collected from a brushtail possum (Trichosurus vulpecula) in Queensland, Australia  to B. microti previously described in Ixodes ricinus ticks removed from dogs in Warsaw, Poland (GenBank accession no. EU882727). In Sicily, 6 of 23 cats were PCR positive for B. microti  of 260 cats tested positive for onal PCR . Both ca microti .B. felis and B. microti (B. microti (MK095343) had 100% sequence identity with B. microti Otsu strain (AB119446) from Japan, and 99% and 98% sequence identity with B. microti Gray (AY693840) and B. microti Munich (AB071177) strains, respectively.One clinically affected cat in Cape Town, South Africa, was infected with both  microti . This B.B. microti . Two partial 18S rRNA gene sequences of B. microti from the Pakistani cats are in GenBank (accession nos. MF401440 and MF401441). These sequences were marked as “UNVERIFIED” in GenBank . A BLASTn analysis showed that although MF401440 had 100% sequence identity to B. microti (KX758442), it had only 75% sequence coverage.In a survey in Pakistan, 21 (13.2%) of 159 cats presented at veterinary clinics were reported to be infected with  microti . PolymerB. vogeli infection in cats was from Bangkok, Thailand, where 21 (1.4%) of 1,490 stray cats tested positive (EU697608). The authors reported that a BLASTn search indicated 98% identity with a B. vogeli from dogs in Brazil (GenBank accession no. not given)  . BLASTn  Bangkok .B. vogeli; specimens from three of these cats that were sick showed 100% identity with B. vogeli accession no. JX871885 in GenBank (B. vogeli (GenBank accession nos. AB896788–AB896795)  of 320 cats were positive for  GenBank . In a seB896795) . In a su. vogeli .Babesia (KF970926–KF970929) (499–650 bp) showed 99% identity to B. vogeli (GenBank accession no. HM590440). Also in Brazil, specimens from 2 (4%) of 30 cats in Rio Grande do Sul State were positive, their sequences (GenBank accession nos. KT323932 and KT323933) (764–766 bp) showing 100% identity with B. vogeli (GenBank accession no. HM590440)  of 37 free-ranging cats in a zoo in Sao Paolo were positive for Babesia . Their sM590440) .B. vogeli was found in 15 (13%) of 119 apparently healthy cats (KY073363) (545 bp) and showed 100% identity to 19 B. vogeli sequences in GenBank . In Qatar, one (2.9%) of 34 healthy cats was positive for B. vogeli   .B. vulpes (then referred to as Theileria annae) (Babesia vulpes (referred to as B. annae) was also incriminated in a clinical case in a cat in France  , 56. Then France . Cats we. vulpes .Babesia s.s. clade was recently discovered in South Africa (Babesia species cat Western Cape (GenBank KR611133–KR611159). All previously reported feline babesiosis cases in South Africa had been caused by B. felis, B. lengau, or B. leo.A pathogenic piroplasm from the h Africa . Seven cBabesia 18S rRNA gene sequences have been deposited in the public DNA sequence databases. Apart from a limited number of 5S rRNA gene and internal transcribed spacer sequences, only 18S rRNA gene sequence data are available for the phylogenetic analysis of feline Babesia species of these, very few are full or near-full-length sequences; the majority are partial 18S rDNA sequence data.In recent years, many novel Babesia species reported from domestic cats, we used near-full-length 18S rRNA Babesia gene sequences available in GenBank to perform a multiple sequence alignment using ClustalX (version 1.81 for Windows)  substitution model (Hepatozoon felis (AY628681) and C. felis (AF399930) were included as outgroup. All positions containing gaps and missing data were eliminated. The final data set comprised a total of 1,351 positions. Evolutionary analyses were conducted in MEGA7.In determining the phylogenetic relationships of the Windows) . None ofon model , determion model , was useBabesia species from cats fell into three distinct clades . This clade also includes dog-infecting B. gibsoni, B. canis s.s., Babesia rossi, and B. vogeli, the human-infecting Babesia venatorum, as well as species infecting ungulates (such as B. divergens and Babesia odocoilei), and Babesia species infecting other carnivores such as bears, cougars, and raccoons, as well as field rodents.The t clades , in conct clades . Clade Iern Cape  fell intB. hongkongensis in clade VI [also referred to as the “carnivore/rodent clade” by Schnittger et al.  to include the ifferent . Note thPortugal  could noBabesia species known to cause disease in cats in South Africa fall in three distinct clades . In retrospect, differences in clinical manifestation, for example, presence or absence of pyrexia and response to specific treatment, reported in South African literature (1937 to 1980) suggest that more than one Babesia species was involved.The four t clades . The cloB. vogeli appears to be quite widespread in domestic cats, with reports from Brazil  of 320 cats in north and central Portugal (The dog-associated m Brazil , 55 and m Brazil  in the Am Brazil , 52 in Em Brazil , and Tham Brazil  in Asia.n Brazil , 13% on n Brazil , and 8.1n Brazil , suggest. vogeli . The EurPortugal .Babesia rossi, which causes severe babesiosis in dogs in sub-Saharan Africa, has not been reported from cats. Specimens from 27 cats tested specifically for B. rossi were negative (Haemaphysalis elliptica, the only known vector of B. rossi, is the most prevalent and abundant tick on cats in the Western Cape Province of South Africa (B. rossi may be recorded in local cats. Three separate attempts at artificial transmission of B. rossi from dogs to cats were unsuccessful, however (negative . Since Hh Africa , where c however –67.Babesia species and in identifying and describing new taxa. Molecular characterization has made it abundantly clear that referring to all piroplasms encountered in cats merely as B. felis is no longer acceptable. In view of the body of literature on the topic published since 2001, persisting in doing so is scientifically irresponsible. Reports of occurrence of B. felis s.s. and other Babesia species in domestic cats should only be accepted if they are supported by credible molecular provenance.In the absence of easily discernible morphological differences, molecular characterization is indispensable in distinguishing between BP performed the literature searches and drafted the basic manuscript. MO performed the phylogenetic analyses and wrote the relevant sections.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This special issue is a continuous effort of our successful 2018 special issue to discover novel biomarkers in the risk prediction, screening, diagnosis, progression, and prognosis cardiovascular diseases. We are happy to see real-time advances in the field with different study methods in different stages of diseases.C. Fu et al. reported that low expression of bradykinin B2 receptor on circulating progenitor cells indicated the poor outcomes of myocardial infarction, based on data from 174 myocardial infarction patients. V. A. M. Goulart et al. were able to identify alterations in the glycerophospholipids, alpha-linolenic acid, and sphingolipid metabolisms in ST-segment elevation myocardial infarction patients, using ultra-high-performance liquid chromatography-tandem mass spectrometry and MS-based flow injection analysis. J. Zhao et al. reported by meta-analysis that circulating microRNA-499 can be used as a diagnostic biomarker for acute myocardial infarction. Q. Ouyang et al. studied the effects of apelin on left ventricular-arterial coupling and mechanical efficiency in rats with ischemic heart failure and found that rats with ischemic HF were characterized by deteriorated left ventricular mechanoenergetics. Apelin improves mechanical efficiency by the inhibiting cardiac fibrosis and apoptosis in left ventricular myocardium, reducing collagen deposition in the aorta and dilating the resistant artery. K. Pieszko et al. employed machine learning techniques and hematological markers in a robust enrollment of 5053 patients and found that neutrophil count and red cell distribution width have a strong association with all-cause mortality after acute coronary syndrome. R. Rajtar-Salwa et al. showed the time-synchronized relationship between ischemia and left ventricular dysfunction assessed by high-sensitive troponin I and N-terminal pro B-type NT-pro natriuretic peptide. M. Mocan et al. reviewed biomarkers of inflammation in left ventricular diastolic dysfunction, which had been previously associated with a heart failure mechanism.P. Perge et al. found that vitamin D deficiency predicts poor clinical outcomes in heart failure patients undergoing cardiac resynchronization therapy. C.-H. Wang et al. showed the feasibility of amino acid-based metabolic profile in functional and prognostic assessment for heart failure outpatients. D. Simeunovic et al. showed the role of glutathione transferase P1 polymorphism in individual susceptibility to oxidative stress, inflammation, and endothelial dysfunction in coronary artery disease and idiopathic dilated cardiomyopathy. T.-L. Chuang et al. investigated the role of bone mineral density in the prediction of cardiovascular disease through atherogenic indexes in nonobese adults.T. Fang et al. found that the preoperative serum CEA, CA125, and CA19-9 levels can help predict the resectability of cholangiocarcinoma. X. Liu et al. showed the preoperative abnormal changes in respiratory rate, TPTEF/TE, VPEF/VE, and lung compliance are indicative of the risk of postoperative pulmonary complications in infants with congenital heart diseases. W. Su et al. reported the use of integrated microfluidic device for enrichment and identification of circulating tumor cells from the blood of patients with colorectal cancer. M. Walentowicz-Sadlecka et al. found that placental soluble fms-like tyrosine kinase-1 (sFlt-1) and sFlt-1/25(OH)D ratio can be used as a diagnostic tool in gestational hypertension, preeclampsia, and gestational diabetes mellitus. I. Mozos et al. presented the links between high-sensitivity C-reactive protein (hsCRP) and pulse wave analysis in middle-aged patients with hypertension and high normal blood pressure (HNBP). Furthermore, vitamin D level, hsCRP, and low-density lipoprotein cholesterol provide valuable information in middle-aged hypertensive and HNBP patients, related to arterial stiffness and early arterial aging.With so many novel and valuable biomarkers found and are being continuously found, we are closer to the essence of the phenomena and are more confident in the battle against cardiovascular diseases in the future.Shipeng WeiAgata M. Bielecka-DabrowaZhongjie Shi


In connection with the development of breeding and the creation of new plant varieties, the problem oftheir genotyping and identification is becoming increasingly important, therefore the use of molecular methods toidentify genetic originality and assess plant genetic diversity appears to be relevant. As part of the work performed,informative ISSR and IRAP DNA markers promising for the study of genetic diversity of the Rosa L. genus were soughtand applied to analysis of genetic relationships among 26 accessions of the genus Rosa L. from the gene pool collectionof Nikita Botanical Gardens. They included 18 cultivated varieties and 8 accessions of wild species. The speciessample included representatives of two subgenera, Rosa and Platyrhodon. The subgenus Platyrhodon was representedby one accession of the species R. roxburghii Tratt. Cultivated roses were represented by varieties of garden groupshybrid tea, floribunda, and grandiflora. The tested markers included 32 ISSRs and 13 IRAPs. Five ISSR markers  and three IRAPs  were chosen as the most promising.They were used for genotyping the studied sample of genotypes. In general, they appeared to be suitable for furtheruse in studying the genetic diversity of the genus Rosa L. The numbers of polymorphic fragments ranged from 12 to31, averaging 19.25 fragments per marker. For markers UBC 864 and UBC 843, unique fingerprints were identified ineach accession studied. The genetic relationships of the studied species and varieties of roses analyzed by the UPGMA,PCoA, and Bayesian methods performed on the basis of IRAP and ISSR genotyping are consistent with their taxonomicpositions. The genotype of the species R. roxburghii of the subgenus Platyrhodon was determined genetically as themost distant. According to clustering methods, the representative of the species R. bengalensis did not stand out fromthe group of cultivated varieties.When assessing the level of genetic similarity among the cultivated varieties of gardenroses, the most genetically isolated varieties were ‘Flamingo’, ‘Queen Elizabeth’, and ‘Kordes Sondermeldung’; formost of the other varieties, groups of the greatest genetic similarity were identified. This assessment reflects generaltrends in phylogenetic relationships, both among the studied species of the genus and among cultivated varieties. According to the Plant List database (www.theplantlist.org),the genus Rosa L. includes 373 recognized species. Sixteenof them occur in the natural flora of the Crimea .Currently, the world range of garden roses includes morethan 30 thousand varieties. The international classificationdivides all this diversity into 36 garden groups accordingto its decorative and biological characteristics . Rose breeding efforts have increased in recent yearsin Japan, China, India, Canada, and New Zealand . In Russia, breeding work with roses has been successfullycarried out in the Nikita Botanical Gardens (NBG)since 1824 . Roses from the garden groupsfloribunda, grandiflora, miniature, and hybrid tea have floweringperiods from 180 to 200 days a year, depending on thevariety, and are the most promising and popular in gardening.The mobilization and preservation of genetic resources ofthe entire diversity of rose cultivars and species that participatedin their creation is one of the main directions in the creationof new varieties that would meet the requirements of moderndecorative floriculture for specific regions of cultivation ofthis crop .The current development of DNA marking methods andtheir introduction into scientific practice contributes to theimprovement of the efficiency of research aimed at clarifyingthe genetic relationships of varieties at the intra- and interspecieslevel; study of the genetic structure of collections of genepools; creation of collections; certification and registration ofthe existing gene pool. In addition, DNA marking methodscan be effectively used to seek donors of genes for breedingvaluabletraits, identify duplicate accessions, and resolvedisputes when classifying newly received specimens. Theuse of data at the level of genetic similarity in combinationwith phenotypic characteristics of varieties in the formationof parent pairs in breeding programs can be promising.The earliest phylogenetic studies of the genus Rosa L.using molecular genetic markers include the work by Millanet al. (1996). The study used RAPD markers (RandomAmplified Polymorphic DNA) to assess polymorphism atthe intraspecific and interspecific levels in representatives ofvarious sections of Rosa. Three clusters were established bythe UPGMA method: the first cluster included accessions of sections Pimpinellifoliae and Synstylae, the second cluster wasformed by sections Chinenses (Indicae) and Gallicanae, andthe third was represented by species of sections Cassiorhodon(Cinnamomeae) and Caninae. However, a later work with asignificantly broader sample of 109 specimens belonging to39 species  failed to obtain an unambiguousdistribution of samples by taxa. These results were notcompletely consistent with a slightly earlier study by Americanauthors , who also used RAPD markers toanalyze another sample of 119 accessions of 36 Rosa species.Koopman et al. (2008) used AFLP DNA markers for phylogeneticanalysis. By analysis of a series of 92 samples belongingto 46 species of the genus Rosa L., the phylogeny ofspecies within the genus Rosa was reconstructed. MultilocusDNA-markers have been widely used to elucidate geneticrelationships, both in gene pool collections and in the study ofnatural populations of various species of the genus Rosa. Thus,using complex data obtained by ISSR and RAPD analysis, agroup of scientists from Turkey analyzed genetic relationshipsamong 27 Rosa species growing in Turkey . RAPD, ISSR, and SSR markers were used to study thegenetic relationship between Taif rose accessions and thosecollected in Syria and Egypt, including the Damascus rose. Theanalysis of the degree of genetic similarity based on the resultsof genotyping revealed the greatest degree of genetic affinityof Taif roses to Damascus roses of Gory variety growing inSyria . Molecular analysis methods,including ISSR markers, were also successfully used in anumber of works aimed at studying the genetic relationshipsin the genus Rosa and clarifying issues related to phylogenyperformed by Russian scientists .The objective of this work is to study the genetic relationshipsamong Rosa accessions of various origins from theNBG’s collection using IRAP and ISSR multilocus markers.As plant material for genotyping, 26 accessions of the genusRosa L. were selected in the study, 18 of which were cultivatedvarieties, and 8 belonged to wild species (Table 1). The sampleof species included representatives of two subgenera Rosa andPlatyrhodon. The subgenus Platyrhodon was represented by asingle specimen of the species R. roxburghii Tratt. Cultivatedroses were represented by hybrid tea, floribunda, and grandiflora groups of varieties. DNA was extracted from youngleaves by the CTAB method .ISSR and IRAP markers from various literature sourceswere chosen for DNA genotyping .A total of 32 ISSR markers and 13 IRAP markers were used.The markers were tested for the applicability to genotypingsamples of the genus Rosa. The PCR schedule was as follows:predenaturation at 95 °C for 3 min; 35 cycles: denaturation at95 °C for 35 s, annealing of primers at 50 °C (55 °C in caseof IRAP markers) for 1 min, elongation at 72 °C for 1.5 min;postextension at 72 °C for 5 min. Concentrations of reagents inthe PCR mixture: 2.5 μl of 10-fold buffer for Taq DNA polymerase, 0.5 or 2.5 μl of dNTP (2.5 mM),1 unit of Taq DNA polymerase, 2 μl of primer (3.75 mM) and40–50 ng of total DNA in the total volume of 25 μl. Electrophoresisof PCR products was performed at 100 V in 2.5 %agarose gel stained with ethidium bromide (2 % agarose gelwas used for testing markers at 120 V). DNA was visualizedunder ultraviolet illumination.On the base of the genotyping results, a binary matrix wasconstructed for further use of data in statistical processingprograms. For statistical processing of the results of ISSRand IRAP genotyping and analysis of genetic relationshipsof the studied gene pool, the program PAST version 2.17c was used. Structure 2.3.4 was used to evaluate the genetic structureof the series. Various values of hypothetical populations fromK = 2 to K = 7 were used in the calculation.The main criterion for choosing DNA markers was the qualityof fingerprints of the tested markers on the rose genotypes. The spectra of amplified fragmentsfrom the studied markers are shown in Fig. 1.Markers with the best fingerprints were selected for furtherwork. The quality criteria for fingerprints included the numberof DNA fragments, their clarity, and brightness in the electrophoreticimage. This is necessary for reliable evaluation ofgenotyping results. Five ISSR , and three IRAP  markers were chosen for genotyping.A series of 26 genotypes of representatives of the Rosa genuswas analyzed with 5 ISSR and 3 IRAP markers (Table 2).For various markers, the ranges of polymorphic alleles variedfrom 12 to 31 fragments, 19.25 fragments per marker onthe average. The UBC 864 marker had the largest numberof polymorphic fragments (31), and significant numbers ofpolymorphic fragments were also found with TDK 2R andASSR29. Two markers from the set (UBC 864 and UBC 843)gave unique fingerprints for each accession. The UBC 864marker also had the largest number of unique fragmentsidentified in a single instance in one of the accessions of thesample. Based on the results of genotyping, a binary matrixwas constructed for further use of data in statistical processingprograms.Eight markers used allowed us to obtain 153 polymorphicDNA fragments from a series of 26 accessions. This numberis sufficient for phylogenetic analysis. Application of themethod of principal coordinates (PCoA) to all accessionsidentified a separate group, including cultivated varieties ofroses . It should be noted that the species R. bengalensiswas considered varietal. Among the rose species, the farthestposition is occupied by the genotype of R. roxburghii. Thespecies R. hugonis and R. foetida are located at a distance fromthe bulk of genotypes in the series. The species most closelyrelated to domestic varieties are R. bracteata, R. multiflora,and R. indica. However, R. indica is located separately withregard to the two above-listed species, and its position is closerto domestic forms.The Bayesian analysis of the results of genotyping accessionswith K values ranging from 2 to 7 was performed usingStructure 2.3.4 program. At K = 2, accessions of R. multiflora,R. foetida, R. hugonis, R. indica, R. bracteata, and R. roxburghiiwere allocated to a separate group. The second group was formed by domestic varieties . The accessions ofR. bengalensis, ‘Queen Elizabeth’, and ‘Flamingo’ occupiedan intermediate position between these groups. At further increaseof K, the trend towards this distribution persists. Withinthese groups, differentiation by this method is poorly visible.Thus, the method allowed us to reliably divide the series intotwo major groups: wild rose species and domestic forms.The results of clustering by the UPGMA method revealedpatterns in the distribution of the studied genotypes, whichwere also noted when using the PCoA analysis . Ingeneral, the clades of the constructed dendrogram show lowbootstrap values. The most remote genotype is R. roxburghii.Two genotypes representing the species R. hugonis andR. foetida form a separate cluster. The next cluster is representedby R. bracteata and R. multiflora. The species R. indicaoccupies a separate position relative to other genotypes. Allspecies accessions except for R. bengalensis occupy an externalposition relative to the cluster that includes cultivatedforms of roses. Among the rose varieties, the most remotefrom the total mass of genotypes are ‘Queen Elizabeth’ and‘Flamingo’. Also, a remote position is occupied by the grouprepresented by varieties ‘Kordes Sondermeldung’ and ‘Alisa’.Other varieties can be divided into 4 clusters: (1) ‘Rouletii’,‘La France’, ‘Traviata’, and R. bengalensis; (2) ‘KorallovyiSyurpriz’, ‘Ayu-Dag’, ‘Gloria Dei’, ‘Kronenbourg’, and‘Miskhor’; (3) ‘Chrysler Imperial’, ‘Chatyr-Dag’, ‘Princede Monaco’, ‘Yves Piaget’, and ‘Mechta’; and (4) ‘Klimentina’and ‘Prekrasnaya Tavrida’.In this work, we evaluated the genetic relationship of varietiesfrom the gene pool collection of roses of the Nikita BotanicalGardens. The interpretation of the sample distribution inclustering using various methods is described below.The Bayesian analysis allows us to determine the geneticcontribution of ancestral forms for each genotype studied.Since most rose varieties have a hybrid origin, the analysis ofthe genetic relationship of samples found that diploid speciesare clearly separated from cultivated varieties, without makinga significant contribution to the gene pool of the studiedvarieties. However, two varieties have a minor contribution ofwild species ‘Flamingo’ and ‘Queen Elizabeth’, in its turn, thegenotype of R. bengalensis occupies an intermediate positionbetween the groups of wild species and cultivated varieties.This distribution stems from the origin of cultivated rosesfrom few wild rose species, with one of these species beingR. bengalensis.The isolation of domestic rose forms in clustering can beclearly shown by other methods, such as PCoA and UPGMA.However, in contrast to Bayesian analysis, the other twomethods provide a more detailed picture. The PCoA methoddifferentiates wild species by their distance from domesticforms. Similar data on the distribution of samples were obtainedby clustering using the UPGMA method. In turn, thelow confidence values of the dendrogram clade may be due tothe complex hybrid origin of both species and varietal accessions. Therefore, the UPGMA resultscan be further interpreted when compared with similar dataobtained by other methods.Summarizing the data on the distribution of species samplesand cultivated varieties of roses, we can make certain inferences.The phylogenetic data obtained from the results ofIRAP and ISSR genotyping are consistent with the informationabout the systematic position of the studied accessions. Thespecies most distant from the cultivated forms is R. roxburghii,representing the subgenus Platyrhodon of the genus Rosa,and the other species and domestic varieties belong to thesubgenus Rosa of the same genus. This distribution agreeswith taxonomy data. However, the results of a number ofmolecular studies on the phylogeny of the genus Rosa do notdistinguish the species R. roxburghii from the group of speciesof the subgenus Rosa . Within the Rosasubgenus, two species of the section Pimpinellifoliae occupya separate position. Studies conducted on chloroplast DNAmarkers confirm the proximity of these species . This section of the Rosa subgenus is probably theleast close to the cultivated varieties in the series. The types ofR. multiflora, R. bracteata, R. indica form the closest clusterswith varietal accessions, and their contribution to the formationof the domestic gene pool of roses is not ruled out. Rosabengalensis forms a common genetic group with domesticvarieties. The contribution of this species to the formationof cultivars is beyond question. It should also be noted thatthe genetic unity of cultivars indicates the generality of theirgene pool.The analysis of genetic relationships among rose varietiesfrom the results of IRAP and ISSR genotyping was based onPCoA and UPGMA clustering data. Comparing the data ofthese two methods, one can identify the most reliable groupsof varieties that are consistently detected by both methods. Thefirst group is ‘Rouletii’ and ‘La France’. Both varieties are ofWestern European origin, and their relationship is not obvious.The second group is ‘Prince De Monaco’, ‘Yves Piaget’,‘Chatyr-Dag’, and ‘Chrysler Imperial’. Two varieties fromthis group have ‘Chrysler Imperial’ among their ancestors:‘Yves Piaget’ and ‘Chatyr-Dag’. Whereas the relationshipof the three varieties ‘Chrysler Imperial’, ‘Yves Piaget’, and‘Chatyr-Dag’ can be explained by the origin of the last twofrom the first, their clustering into one group with ‘PrinceDe Monaco’ is difficult to explain. The third group includes‘Gloria Dei’, ‘Kronenbourg’, ‘Ayu-Dag’, and ‘KorallovyiSyurpriz’. There are two varieties in this group whose parentshave ‘Kordes Sondermeldung’, namely, one variety ‘GloriaDei’ and its sport  ‘Kronenbourg’.Varieties such as ‘Flamingo’, ‘Queen Elizabeth’, ‘KordesSondermeldung’ and ‘Alisa’ are the most genetically contrasting However, a number of varieties in the series arederived from these three, such as ‘Ayu-Dag’, ‘Klimentina’,‘Korallovyi Syurpriz’ (‘Kordes Sondermeldung’), ‘KorallovyiSyurpriz’ (‘Queen Elizabeth’) and ‘Alisa’ (‘Flamingo’).The distribution of species accessions of roses in clusters basedon their genetic relationships is consistent with the generallyrecognized phylogeny. It is worth to note that the accession ofthe species R. bengalensis is included in the cluster formed bycultivated varieties. This fact may point to a contribution ofwild roses of Indian origin to the formation of the gene pool ofmodern rose varieties. In turn, the analysis of the relationshipof cultivated varieties of garden roses reveals that the varieties‘Flamingo’, ‘Queen Elizabeth’, ‘Kordes Sondermeldung’, and‘Alisa’ stand out from the total mass of the studied varieties.The remaining rose varieties were divided into groups withthe greatest genetic similarity. Most of the results of cultivarclustering were explained based on information about thepedigree of varieties, but the position of some varieties wasdifficult to interpret. Further research is required to determinetheir relationships. Thus, the markers used in this work haveshown their effectiveness in the study of the genus Rosa.The authors declare no conflict of interest.Arzate-Fernandez A.M., Miwa M., Shimada T., Yonekura T., Ogawa K.Genetic diversity of Miyamasukashi-yuri (Lilium maculatum Thunb.var. bukosanense), an endemic and endangered species at MountBuko, Saitama, Japan. Plant Species Biol. 2005;20:57-65.Atienza S.G., Torres A.M., Millan Т., Cubero J.I. 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