Symmetries have not been completely determined and explained from the discovery of the DNA structure in 1953 and the genetic code in 1961. We show, during 10 years of investigation and research, our discovery of the Supersymmetry Genetic Code table in the form of 2 × 8 codon boxes, quadruplet DNA symmetries, and the classification of trinucleotides/codons, all built with the same physiochemical double mirror symmetry and Watson–Crick pairing. We also show that single-stranded RNA had the complete code of life in the form of the Supersymmetry Genetic Code table simultaneously with instructions of codons’ relationship as to how to develop the DNA molecule on the principle of Watson–Crick pairing. We show that the same symmetries between the genetic code and DNA quadruplet are highly conserved during the whole evolution even between phylogenetically distant organisms. In this way, decreasing disorder and entropy enabled the evolution of living beings up to sophisticated species with cognitive features. Our hypothesis that all twenty amino acids are necessary for the origin of life on the Earth, which entirely changes our view on evolution, confirms the evidence of organic natural amino acids from the extra-terrestrial asteroid Ryugu, which is nearly as old as our solar system. Ten years ago, our main challenge was to find a solution as to how, in an autonomous system such as the DNA molecule, Chargaff’s second parity rule (CSPR) , also caThe fundamental role of symmetry in a biological system is to decrease disorder (entropy) and to preserve the integrity of the system. Many scientists have investigated the importance of symmetries in biological processes, especially after the discovery of the DNA molecule and the genetic code as basic structures related to life on Earth. The general question is whether symmetries reflect some fundamental “laws” of genome evolution or whether they are a type of statistical pattern . The ideJacques Monod attached great significance to symmetry in biology, which must not be understood in purely geometrical connotations, but rather in a much wider sense, identical to that of order within a structure . In 1943, Schrödinger proposed in his lecture at Trinity College in Dublin that hereditary material must take the form of an “aperiodic crystal”, implying the presence of symmetries in the structure of DNA. Ever since Nirenberg’s discovery in 1961 , scientists searched for symmetries within genetic code. Up to the present discovery of the SSyGC, complete symmetry in the genetic code has not been found, leaving a doubt as to whether the symmetrical nature as the “protector” of order even exists. In a framework of symmetry as one of the guiding principles, we discuss a symmetry investigation of the DNA molecule and of genetic code based on programmable biomolecular mediated processes and physicochemical laws, with Einstein’s symmetry paradigm extended to the life sciences. More specifically, here, we discuss how the laws of DNA and genetic code can be considered as being related to physicochemical and mirror symmetries. Ariadne’s thread on the path of our discovery of DNA and genetic code symmetries was our trinucleotide classification ,9,10. TrAt first sight, a simple distribution of trinucleotides on A + T rich and C + G rich showed an important basic structural organization of our DNA classification in the form of 20 quadruplets: 10 A + T rich and 10 C + G rich . Each trFrom the classification of trinucleotides/codons, a symmetrical relationship between purines and pyrimidines in each quadruplet is the same between direct (D) and reverse (R), as well as between complement (C) and reverse complement (RC). Purines marked as “0” and pyrimidines as “1” for trinucleotides and codons give eight possible combinations from which the first two are in a D↔C relationship, and the other two are in a D↔R relationship: , . The point is that all four members of each quadruplet are specific, and it is free choice which is considered as direct (D). The others are adjusted according to Watson–Crick pairing for complement, reverse, and reverse complement functions . In the quadruplet classification of trinucleotides/codons, there arises a dominant role of double mirror symmetry: within each quadruplet as well as between the whole A + T rich and C + G rich groups of quadruplets . This isIn 1951, Chargaff’s first parity rule on nucleotide pairing in the DNA molecule was published , where tIn 1968, Chargaff´s unexpected second parity rule (CSPR) showed a marked similarity of frequencies also of nucleotides A and T, as well as C and G, within each of the two strands of DNA. It was published as an empirical global rule for long enough DNA segments and not being derived from a compelling principle like Watson–Crick base pairing underlying the first rule . This ruVarious other names have also been used for CSPR in the literature, such as “strand symmetry”, “intra-strand symmetry”, “word symmetry”, and “inversion symmetry”. According to its meaning, this rule could also be called Chargaff’s nonlocal pairing. For more than 50 years, a conclusive explanation of CSPR was still rather controversial. Namely, up until our discovery, CSPR revealed general species-independent properties and had remarkable implications for some unknown mechanism that seems to be present ,26.f(D) and f(RC(D)) increases, and for smaller lengths, any tendency of f(D) and f(RC(D)) frequency identity disappears. To solve this problem, we used the empirically estimated minimal length of a genomic sequence (~100 kb) as the reference value for trinucleotides (n = 3). We showed that for the estimate in this minimal sequence length, each trinucleotide must be present ~1500 times. Using this estimate as a gauge, we determined estimates for minimal lengths of oligonucleotides of other orders . Pe0,000 bp After the discovery of DNA quadruplet symmetry, a more complex problem led us to investigate the genome itself, i.e., how is it possible that each DNA species, despite mutations during evolution, preserves genome symmetries in the form of CSPR and quadruplet symmetry? This question was a large challenge for scientists from the very beginning of CSPR discovery . Sueoka We discovered the concept of the natural law of DNA creation and conservation owing to our fundamental DNA quadruplet mirror symmetry, which automatically leads to CSPR. The natural law is activated, according to which the same mono- or oligonucleotide insertion must be inserted simultaneously into both strands of DNA. However, regardless of the localization in the second strand, the new DNA segment with stable quadruplet symmetries in a bidirectional 5′3′↔3′5′ manner is created  9,10]. ,10. 9,10circulus vitae. From Nirenberg’s discovery in 1961 [Ariadne’s thread of our investigation leads us to the fundamental discovery of physicochemical symmetries of the genetic code and, in this way, closing  in 1961  in which in 1961  and all  in 1961 ,34. The 84 possible codon combinations of the genetic code [The stereochemical theory postulates that the structure of the code is determined by a physicochemical affinity between amino acids and codons or anticodons . Unfortutic code . The genetic code is degenerate because more than one type of codon  may encode a single amino acid. The intriguing algebraic approaches to the genetic code evolution through the progressive symmetry breaking theory explained the observed degeneracy of the genetic code with a mathematical technique for organizing the group theoretical structure ,53,54,55The evolution of the genetic code and life on Earth was a scientific challenge for many scientists, with interesting results, but without discovering complete physicochemical genetic code symmetries ,61,62,63A completely new approach is our discovery of the fascinating supersymmetry genetic code (SSyGC) table with five physicochemical symmetries between bases, codons, and amino acids: (1) purine–pyrimidine symmetry on the principle of Watson–Crick pairing , (2) direct–complement symmetry between codons, (3) double mirror symmetry between bases and codons, (4) A + T rich and C + G rich symmetry between codons, and (5) symmetry between the position of amino acids . During our discovery of the SSyGC table, we conformed to the traditional concept with 4 × 4 boxes as in the SGC table . In thisNamely, our path to the ordering of codons started from Serine because we noticed the unique regularity: its six codons are in two neighbouring boxes that are in a direct–complement relation. In this way, we discovered the logic of the third base in codons and the link between Serine and Arginine in the same column of boxes and between Serine and Leucine in the neighbouring column of boxes. After identifying this core structure of the SSyGC table, the further ordering of other codons according to a similar logic is straightforward.It is fascinating that the SSyGC table starts with a very important AUG start signal. This is an automatic result of the SSyGC symmetries. Namely, the position of each codon is unique and strictly localized. At the same time, all three stop signals  are positioned according to the mirror symmetry in both halves of the code in two boxes with the same 1-0-0 purine–pyrimidine relationship .Our SSyGC table also consists of 16 codon boxes, as in SGC, but in the form of two columns, each with eight boxes, and with the same codons in each box like the SGC but in a different arranging. The main difference in the SSyGC table is the position of the third base between direct and complement codon boxes on the principle of Watson–Crick pairing: GACU ↔ CUGA, AGUC ↔ UCAG. This leads to the physicochemical code symmetry with the double mirror symmetry .We postulate “the symmetry theory of genetic code”, which is based on the unique physicochemical purine–pyrimidine symmetry net between bases and codons of our SSyGC table. Because of the unique symmetry net, the position of each codon in the SSyGC table is strictly definite. The common purine–pyrimidine symmetry net as “the golden rule” of the SSyGC table, which is identical and universal for all RNA and DNA species on Earth, has remained unchanged during all of evolution. It is also identical for more than 30 known slightly alternative nuclear and mitochondrial genetic codes, including those that will be discovered in the future. We show that variations of the number of codons for individual amino acids inserted in the SSyGC table arise most often with a capture from the neighbouring codons from the split box according to the metabolic requirements ,34. ThisThe symmetry net structure of the SSyGC table enables an automatic transformation with the direct alignment of all codons from direct boxes (top strand) and all codons from complement boxes (bottom strand) of the genetic code into a DNA-type sequence with Watson–Crick pairing, as well as its form analogous to the 5′3′ codon and 3′5′ anticodon: Codons from the first column of the SSyGC table:Codons from the second column of the SSyGC table:The perfect symmetry of each individual codon is clearly visible. For example, all symmetric codons such as UUU-AAA, CCC-GGG, CAC-GUG, UGU-ACA, etc., strictly aligned one below the other in all four rows because of mirror symmetry. The horizontal third base alignment from direct boxes is GACU, GACU, AGUC, AGUC, AGUC, AGUC, GACU, and GACU with their complement in complement boxes. Therefore, it follows that the SSyGC table and DNA molecule are like a coin with two sides.It seems that codon–anticodon symmetry as a complete structural physicochemical form of the unique SSyGC table made possible, in the early Earth, the synthesis of an immature protein with anticodon stem-loop tRNA, later becoming double-stranded DNA; this is also the theoretical approach from earlier investigations ,63.RNA viruses have an identical SSyGC table with the unique common purine–pyrimidine symmetry net, like DNA species. This means that the symmetry net had a direction for RNA to DNA transformation on the principle of Watson–Crick pairing, which enabled the fingerpost for evolution from an RNA to DNA molecule. Translation is not limited to twenty amino acids. The additional Selenocysteine as the 21st amino acid takes possession of the UGA stop signal and Pyrolysine as the 22nd amino acid  takes thOne could assume that one new non-natural amino acid, because of a special metabolic need of species , adopts the codon of some other natural amino acid or stop signal of code without disturbing the physicochemical symmetry net of the SSyGC table. However, there is a question of the consequences of such artificial translation, with possible harmful or even lethal consequences.Escherichia coli with the entire synthetic DNA genome that utilized just 61 codons for protein synthesis compared to 64 in natural living organisms. Performing “synonymous codon compression”, they recompiled the E. coli genome with two  out of six codons encoding Serine and the TAG stop signal. Accordingly, this synthetic organism with “laboratory different genetic code” uses 59 codons and TAA and TGA to encode 20 amino acids and two stop signals. E. coli with “synthetic DNA” displayed only minor changes, with a slower growth rate, slightly elongated cells, and enabled deletion of previously essential tRNA. If the two codons and one stop signal are omitted from the SSyGC table, the remaining codons and stop signals stay in the same positions within the common purine–pyrimidine symmetry net, keeping their symmetrical mutual relationship. The positions of omitted codons and stop signals remain empty, because none of the remaining codons and stop signals can replace them without violating the symmetries, as each codon and stop signal has a strictly defined position within the SSyGC table. In this way, it is not a different genetic code but unfortunately an abnormal, mutilated, and artificial SSyGC table [E. coli could survive, but with mutation marks regarding the shape and growth rate. E. coli cannot survive without all codons of any amino acids. Fredens and coworkers (2019)  construcGC table . ConsequXerella Beyerinck, a single-cell green microalgae and one of the oldest living species with an estimated age of about 2.5 billion years. It also has the same SSyGC table, which consists of 50% proteins and possesses all twenty natural amino acids [An important example is the protist no acids ,66. The In 1961, Nirenberg and collaborators deciphered the genetic code, determining experimentally which codons correspond to each of the 20 natural amino acids, but without considering the codon’s regularity in the form of a genetic code table. This challenge was addressed by Nobel laureate F. H. Crick, and in 1968, a solution was published  under thIn the meantime, more than thirty different codes have been discovered for genomes of some bacteria and archaea as well as for some organellar mitochondrial and eukaryotic nuclear genomes. Because of this, the name of the Universal Genetic Code was changed to the Standard Genetic Code.The fact that the Standard Genetic Code is degenerate, i.e., that more than one codon can code for the same amino acid, the search for symmetries cannot be completely successful with amino acid arrangement. We realized that the key to the genetic code symmetries must be between codon purines and pyrimidines as the starting point. This approach led us to the discovery of the supersymmetry genetic code (SSyGC) table, characterized by codon physicochemical symmetries, also including the double mirror symmetry. The symmetry core of the SSyGC table is the purine–pyrimidine symmetry net as a “golden rule”, which is common for all RNA and DNA species and unchangeable during evolution.The Standard Genetic Code has an ordering of codons according to pyrimidines  and purines . This ordering is strictly established both in horizontal and vertical directions. Thus, the resulting genetic code has only alphabetic symmetry, with a UCAG ordering of bases. Therefore, the third base in codons does not differentiate among sixteen codon boxes in the Standard Genetic Code table and cannot fully contribute to the discovery of physicochemical symmetries of genetic code. This has, to this day, contributed to the problem of many studies based on the Standard Genetic Code table with a UCAG ordering of the third base ,101,102.On the contrary, with our SSyGC table, we have proved that the third base is a crucial point for the discovery of physicochemical symmetries on the principle of Watson–Crick base pairing (A↔U and C↔G like codon/anticodon) and for the discovery of double mirror symmetry. We have also proved that the DNA quadruplets have the same symmetries, as well as our classification of trinucleotides/codons . It is vBecause of the complete physicochemical symmetries of the SSyGC table, it is not necessary to involve “the frozen accident”. All more than thirty alternative genetic codes with a slight departure from the standard code can be incorporated in the SSyGC table . Thus, tOur symmetry-based theory of genetic code broadens the horizon for understanding evolution as a fundamental process in creation and the richness of life on Earth. This approach points out that besides mutation and natural selection, other factors also may have been responsible for evolution, as is the basic and unchangeable physicochemical purine–pyrimidine symmetry net as “the golden rule” of the genetic code structure.We point out that the fundamental role of symmetry in the genetic code is to decrease disorder (information entropy) and to preserve the integrity of a biological system ,11,27,34Regarding the results of our discovery of the multifaceted physicochemical symmetry of the SSyGC table remaining unchanged during all of evolution, we hypothesize that life on Earth was developed when all natural amino acids were already present . We concAn especially important discovery on the asteroid is also the presence of L-2-aminobutyric acid as a chiral precursor for the synthesis of non-natural L-amino acids in the later stage of origin ,63,105. Therefore, the first manifestation of life in the form of a single-stranded RNA molecule already had genomes with the complete code of life in the form of the SSyGC table for proteinogenesis, as well as instructions as to how to develop the DNA molecule on the principle of Watson–Crick pairing. Similarly, we discovered that DNA and genetic code with unchangeable symmetries during evolution decrease the disorder (entropy) of vital processes and preserve the genome’s integrity, which changes our view on all of evolution. After the discovery of natural amino acids on the asteroid Ryugu, the connecting of the genetic code degeneracy with symmetries seems to lose sense. Namely, if the prebiotic organic material was originating during the time of the creation of the solar system, then the natural amino acids were delivered to the early Earth at the time of the origin of life. Then, one would expect that each amino acid was coded by a different number of codons in accordance with the metabolic need of species; this is reflected in the structure of the genetic code.At the end of this journey investigating the symmetry of the genetic code and DNA molecule, we can ask: what would be the answer of biology, molecular biology, genetics, or medicine students to the question of what could be said about the Standard Genetic Code table? The answer could be that it is a table that consists of 4 × 4 boxes containing 61 codons and three stop signals ordered alphabetically as U-C-A-G (two pyrimidines and two purines), both horizontally and vertically. Simultaneously, besides the Standard Genetic Code table, there are also more than 30 alternative nuclear and mitochondrial genetic codes. There are many aspects of SGC in the recent literature, but without complete physicochemical symmetries between codons ,102,107.The SSyGC table starts with an AUG start signal.All 61 codons and three stop signals are arranged in 2 × 8 boxes, which alternate in each row on the principle of A + T rich and C + G rich codons but with the same purine and pyrimidine ordering.The ordering of purines and pyrimidines in both columns is identical.Vertically, between direct and complement boxes, purines, and pyrimidines as well as codons are regularly ordered on the principle of Watson–Crick pairing.Horizontally in the same row, purine in the first column transforms in the purine of the second column, and analogously pyrimidine transforms in pyrimidine, creating alternate A + T rich and C + G rich codons.In the SSyGC table, the codons of all amino acids are not scattered, including three sextets for Serine, Arginine, and Leucine.The SSyGC table has a central vertical and horizontal double mirror symmetry according to the mirror symmetry axis as well as a double mirror symmetry of DNA quadruplets and a classification of trinucleotides/codons.In this way, the physicochemical unique symmetry net of the whole SSyGC table is structured, creating full symmetries between bases, codons, and amino acids.The symmetry net is unique and common for all RNA and DNA living species on Earth.The symmetry net is also common for more than 30 nuclear and mitochondrial genetic codes, which differ from the Standard Genetic Code table.Due to the symmetry net, codons of the SSyGC table directly transform in the DNA molecule with Watson–Crick pairing .The unique symmetry net has remained unchanged during evolution and has the power of the natural law for the origin of life.The protection of the genetic code and DNA molecule symmetries during all of evolution reveals their role in decreasing entropy (disorder) and the preservation of species integrity.On the other hand, answers to the question regarding the characteristics of the SSyGC table would be completely different:Now, 60 years after Nirenberg’s empirical discovery, physicochemical symmetries of the SSyGC table for all RNA and DNA species on Earth are discovered, shedding, together with DNA symmetry, a new light on the evolutionary development of species.The recent discovery of extra-terrestrial abiotic amino acids with identical physicochemical symmetries to the genetic code and DNA molecule, and at the same time, the discovery of unchangeable genetic code during evolution for all RNA and DNA species are a challenge for scientists of genetics, biology, chemistry, physics, medicine, and philosophy to investigate and reveal the riddle of the origin of life.


Scutellarin promoted the up-regulation of various markers and AG490 neutralised the effect of scutellarin. In vivo, up-regulation of p-JAK2 and p-STAT3 after ischaemia is known. These results are consistent with previous reports. Scutellarin further enhanced this upregulation at 1, 3, and 7 d after MCAO. Scutellarin exerts its therapeutic effects on cerebral ischaemia by activating the astrocyte JAK2/STAT3 signalling, which provides a firm experimental basis for its clinical application.Scutellarin is used to treat brain ischaemia. However, its underlying mechanism of action remains unclear. This study aimed to elucidate the potential mechanism of action of scutellarin in brain ischaemia through network pharmacology and experimental verification. The JAK2/STAT3 signalling pathway was identified and experimentally verified. Expression of JAK2/STAT3 signalling related proteins in TNC-1 astrocytes with BV-2 microglia-conditioned medium (CM), CM + lipopolysaccharide (LPS) (CM + L), and CM pretreated with scutellarin + LPS (CM + SL) was analysed by Western Blot and immunofluorescence staining. Expression levels of JAK2, p-JAK2, STAT3, and p-STAT3 were evaluated in astrocytes pre-treated with AG490. Middle cerebral artery occlusion (MCAO) in rats was performed in different experimental groups to detect expression of the above biomarkers. Network pharmacology suggested that the JAK2/STAT3 signalling pathway is one of the mechanisms by which scutellarin mitigates cerebral ischaemic damage. In TNC-1 astrocytes Ischaemic stroke is mainly caused by occlusion of the middle cerebral artery (MCA), which triggers a cascade of neuroinflammatory and immune responses2. Currently, the treatment options for patients with ischaemic stroke are limited. Recombinant tissue plasminogen activator (rt-PA) is an effective treatment for ischaemic stroke, although its neurotoxicity must be considered3. However, the applications of thrombolytic therapy are limited due to its short therapeutic time window and many contraindications4. Brain injury caused by ischaemic stroke results from a complex set of neuropathophysiological and neuropathological events, including oxidative stress, amyloid production, and tau protein dysfunction7. Excessive reactive oxygen species (ROS) produced by oxidative stress are the main cause of neuronal necrosis and apoptosis8, and scavenging excessive ROS effectively mitigates the progression of neuronal death and improves prognosis9.Stroke, which often leads to long-term disability and death, is a life-threatening condition, and approximately 85% of strokes are classified as ischaemicErigeron breviscapus (Vant.) Hand.-Mazz. Scutellarin  is one of the representative flavone. Numerous studies have shown that many flavonoids have antioxidant activity and can improve patient survival12. Previous studies have shown that compared to edaravone, high-dose scutellarin (100 mg/kg) significantly reduces infarct volume after cerebral ischaemia in rats13. Fang et al.14 found that scutellarin significantly improved neurological function and reduced cerebral infarct volume in rats with experimentally induced cerebral ischaemia. Chen et al.15 demonstrated that scutellarin attenuates microglia-mediated neuroinflammation by downregulating inflammatory and pro-inflammatory mediators. However, the targets and mechanisms of action of scutellarin in the reduction of ischaemic brain injury remain unclear.Breviscapine is a crude extract of flavonoids of 14. Thus, the close temporal and spatial relationship between microglia and astrocytes at the lesion site of cerebral ischaemia may not be fortuitous. Indeed, cross-talk between microglia and astrocytes plays an important role in the pathophysiological process following brain ischaemia16.Recent studies have shown that microglia are the first cells to be activated following brain ischaemia. Activated microglia then swiftly migrate and accumulate in the infarct core and penumbral regions to phagocytose cellular debris. Activated microglia can also induce the activation of astrocytes, which are closely associated with microglia. The so-called “cross-talk” between activated microglia and astrocytes would consequently amplify the inflammatory cascade response19. Hou et al.20 suggested that part of the mechanism underlying the neuroprotective effect of resveratrol on cerebral ischaemia/reperfusion injury is mediated by activation of the JAK2/STAT3 pathway. The JAK family and its associated receptors, the STAT family, are the three major components of the JAK2/STAT3 signalling pathway. Phosphorylated JAK2 (p-JAK2) specifically activates STAT3, and phosphorylated STAT3 (p-STAT3) forms a dimer that translocates from the cytoplasm to the nucleus, regulating the transcription of target genes and inducing corresponding gene expression21.The Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) signalling pathway is involved in ischaemia, metabolism, hypoxia, and oxidative stress in various tissues and organs. Many studies have reported that activation of the JAK2/STAT3 signalling pathway enhances cerebral angiogenesis and promotes neurological recovery after ischaemic strokeTo identify the new signalling pathways by which scutellarin attenuates brain ischaemic injury, we combined a network pharmacology approach and experimental validation. As intercellular cross-talk plays a crucial role in the treatment of cerebral ischaemia, we used BV-2 microglia-conditioned medium to incubate astrocyte in an in vitro experiment, which will help us better understand the process by which scutellarin treat brain ischaemia Fig. .Figure 1According to the PharmMapper and SwissTargetPrediction prediction results, 189 targets were predicted for scutellarin, and duplicated targets were removed. The prediction results of the OMIM, GeneCards, and DisGeNET databases were intersected to obtain a total of 721 targets for brain ischaemia. A predicted target interaction network between scutellarin and brain ischaemia was constructed using the Cytoscape, including 4372 nodes and 107,457 edges. , molecular functions (MF), and top 15 cellular components (CC) were selected by functional annotation of 54 targets, among which several BP results were mainly concentrated in cellular responses to nitrogen compounds, DNA repair, regulation of cellular response to stress, and mitotic cell cycle processes Fig. A. The ma22, among which, the JAK2/STAT3 signalling pathway had played an important role in regulating the inflammatory response and was therefore, selected for further experimental validation.To explore the interactions of the 54 common target proteins, a PPI network of the common target proteins was obtained from the STRING database and further visualised using Cytoscape. The resulting network incorporated 54 nodes representing 54 protein targets and 438 edges representing 438 pairs of protein interactions. Compared to the cold colour and small nodes (proteins) in the network, the warm colour and large nodes (proteins) had more edges (interactions), representing a higher degree, and are more important for further research . Remarkably, p-JAK2 and p-STAT3 expression levels further increased in the CM + SL group (p < 0.05). There were no significant differences in the expression levels of JAK2 and STAT3 between the groups (p > 0.05)  Fig. A,B. Immu05) Fig. C. Therefp < 0.05). As expected, the expression levels of p-JAK2 and p-STAT3 were significantly lower in the CM + SL + I group than in the CM + SL group (p < 0.01). There were no significant differences in the expression of JAK2 and STAT3 between the groups (p > 0.05)  was added to the astrocytes for 1 h, and the medium was replaced with different BV-2 microglia-conditioned medium for 24 h. Western Blotting was used to detect the expression of p-JAK2, JAK2, p-STAT3, and STAT3 in astrocytes. The expression of p-JAK2 and p-STAT3 was significantly increased in the CM + SL group (05) Fig. A,B. Immu05) Fig. C. These p < 0.01). In the 1 d MCAO + S group, p-JAK2 and p-STAT3 protein expression was further augmented compared with the MCAO group (p < 0.05). JAK2 and STAT3 expression levels were not significantly different between the groups (p > 0.05) . In the MCAO + S group, p-JAK2 and p-STAT3 expression was markedly increased, which was significantly different from that observed in the MCAO group (p < 0.05). There were no significant differences in the expression levels of JAK2 or STAT3 among all groups (p > 0.05) . In rats administered scutellarin, the expression of p-JAK2 and p-STAT3 was further elevated and was significantly higher than in the MCAO group (p < 0.01). The expression levels of JAK2 and STAT3 were not significantly different among all groups (p > 0.05)  Fig. A,B. The 05) Fig. C,D,E,F. 23. To explore the mechanisms underlying the effects of scutellarin on cerebral ischaemia, a network pharmacology approach was used in the present study to systematically interpret the potential targets and related mechanisms of scutellarin in ischaemic stroke. Based on network analysis and literature reports, the JAK2/STAT3 signalling pathway was selected for experimental verification. In vitro experiments showed that the LPS-activated BV-2 microglia-conditioned medium activated the astrocytic JAK2/STAT3 signalling pathway. Scutellarin pretreatment further enhanced the protein expression of p-JAK2 and p-STAT3. In vivo experiments showed that the expression of p-JAK2 and p-STAT3 in rats at 1, 3, and 7 d in the MCAO group was higher than that in the sham-operated group. This increase was further enhanced by scutellarin, suggesting that the therapeutic effect of scutellarin on cerebral ischaemia is at least partially mediated by activation of the JAK2/STAT3 signalling pathway in astrocytes.Ischaemic stroke has become a major public health concern in recent years25 and that inhibition of the JAK2/STAT3 signalling pathway has a neuroprotective function in ischaemic stroke26. Nevertheless, pharmacological treatment of ischaemic stroke is a comprehensive and complex process. It has been shown that activated STAT3 leads to oxidative stress and mitochondrial dysfunction27. However, flavonoids also exhibit antioxidant activity. We hypothesised that scutellarin activates the JAK2/STAT3 signalling pathway, which, in turn, leads to a series of downstream cascades; however, scutellarin blocks downstream adverse factors of the JAK2/STAT3 signalling pathway, such as oxidative stress, and promotes favourable factors for the JAK2/STAT3 signalling pathway.However, several studies have suggested that aberrant activation of the JAK2/STAT3 signalling pathway contributes to the inflammatory response in ischaemic stroke28. In another study, we found that scutellarin significantly reduced STAT3 expression when acting alone on astrocytes or microglia (data not yet published). In this study, LPS-activated microglia were pretreated with scutellarin and astrocytes were incubated with microglia-conditioned medium. In in vitro experiments, there was cross-talk between microglia and astrocytes, and in vivo, there was cross-talk between multiple cells types , all of which may have multiple cytokine effects. We hypothesised that these factors may be responsible for the elevated STAT3 expression levels.Curcumin, a natural polyphenol, inhibits abnormal activation of STAT3 signalling29. However, multiple studies have shown that the activation of the JAK2/STAT3 signalling pathway plays a key role in ischaemic brain injury32. Clearly, our study supports the former view; however, further research is needed to interpret this. Third, it has been shown that oxidative stress, inflammation, and apoptosis interact with each other and form a complex signalling network that leads to neuronal cell necrosis, senescence and apoptosis35. However, previous studies have shown that scutellarin has antioxidant effects. Specific experimental validation has not been performed, and the downstream mechanisms of scutellarin after the completion of antioxidation are not known. However, further studies are required to confirm this hypothesis.Despite the encouraging results of our study, there were some limitations. First, our results do not prove that scutellarin plays a neuroprotective role by activating the astrocytic JAK2/STAT3 signalling pathway. This is because we did not demonstrate a reduction in stroke volume after scutellarin treatment or a neuroprotective effect in animals treated with scutellarin in parallel with AG490. Second, our study showed that scutellarin promoted the activation of the JAK2/STAT3 signalling pathway, which has been shown to activate the JAK2/STAT3 signalling pathway and promotes neurological recovery after ischaemic strokeIn conclusion, this study systematically analysed the possible targets and mechanisms of action of scutellarin in cerebral ischaemia. We experimentally verified the therapeutic effect of scutellarin on cerebral ischaemia by activating the astrocytic JAK2/STAT3 signalling pathway. We are confident that our study provides a reliable scientific basis for the clinical application of scutellarin in the prevention and treatment of brain ischaemia, and perhaps also in other ischaemic cardiovascular diseases. However, downstream mechanisms still need to be explored.http://www.lilab-ecust.cn/pharmmapper/)36 and SwissTargetPrediction (http://www.swisstargetprediction.ch/)37 databases were searched to predict related protein targets. These protein names were then entered into the UniProt database (http://www.uniprot.org/)38 to obtain their official gene names. A hundred scutellarin targets were obtained from SwissTargetPrediction. In total, 109 scutellarin targets were selected from PharmMapper (z-score > 0.5). Finally, 189 scutellarin targets were identified by combining the two databases. Disease targets for brain ischaemia were obtained from three databases: OMIM (https://omim.org/)39, GeneCards (https://www.genecards.org/)40 and DisGeNET (http://www.disgenet.org)41. In total, 401 brain ischaemia targets were obtained from the OMIM database, 200 from the GeneCards database (Relevance score > 13.34), and 202 from the DisGeNET database (Score > 0.02). Finally, a total of 721 targets for brain ischaemia were obtained by taking the ensemble from the three databases.To identify the corresponding targets of scutellarin, the PharmMapper 42. First, brain ischaemia and scutellarin networks were constructed based on the obtained targets. Second, the intersection of the two networks was used to construct a merged network, and the values of degree, Betweenness, ClosenessCentrality, LAC, and NeighborhoodConnectivity were limited to filter a new network. The merged network of scutellarin and brain ischaemia comprised 54 nodes and 675 edges.An intersection network of scutellarin and brain ischaemia was constructed using Cytoscape (https://string-db.org/)43, which is a database of known and predicted protein–protein interactions that stem from computational prediction, including both direct and indirect interactions among proteins. Ultimately, the PPI data with species limited to “Homo sapiens” and confidence scores of 0.9 were reserved for further research. Visualization via Cytoscape 3.70.Protein–protein interaction (PPI) data were extracted from the STRING database (http://metascape.org)44 online database was used to perform functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. GO analysis included biological processes (BP), cellular components (CC), and molecular functions (MF). Statistical significance was set at p < 0.01.The Metascape  and the Guide for the Care and Use of Laboratory Animals at Kunming Medical University. All experimental protocols and animal use were approved by the Laboratory Animal Ethics Committee of Kunming Medical University . Efforts were made to minimise the number of rats used and their suffering.All animal experiments were performed in accordance with the ARRIVE guidelines  were group-housed with a 12-h light‑dark cycle and provided chow and water ad libitum. They were randomly divided into sham-operated + saline (sham), middle cerebral artery occlusion (MCAO) + saline (MCAO), and MCAO + scutellarin (MCAO + S) groups. In the MCAO group, anaesthesia was induced by an intraperitoneal injection of sodium pentobarbital (50 mg/kg). A dental drill was used to create a circular aperture in the right parietal bone to expose the underlying main trunk of the MCA. The right MCA was occluded by electrocoagulation. The rats in the MCAO + S groups were administered an intraperitoneal injection of scutellarin  dissolved in saline 2 h before and 12, 24, 48, and 60 h after MCAO; the other two groups were injected with saline. The rats were sacrificed 1, 3, and 7 d after MCAO. Laser speckle imaging system  to measure cerebral blood perfusion.Adult male Sprague–Dawley rats , CM + L, CM + L + I, CM + SL and CM + SL + I group by incubating them with the above conditioned medium for 24 h. After various treatments, astrocytes were collected for protein extraction or immunofluorescence analysis.Both TNC-1 astrocytes and BV-2 microglia were purchased from the American Type Culture Collection . TNC-1 astrocytes and BV-2 microglia were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) at 37 °C in a humidified incubator under 5% CORat cerebral cortices in each group were lysed on ice with precooled lysis buffer  to prepare tissue homogenates, and cultured TNC-1 astrocytes were lysed similarly. Cells were harvested 15 min after cleavage using a cell scraper. The tissue homogenate and cell protein were centrifuged at 14,000 rpm at 4 °C for 20 min, and the supernatant was collected. The protein concentration was determined using a BCA protein assay kit . Protein samples were loaded and heated at 95 °C for 10 min and separated by SDS-PAGE electrophoresis. Protein bands were electro-blotted onto polyvinylidene difluoride (PVDF) membrane and blocked with 5% skim milk powder for 2 h. Membranes were cut prior to hybridisation with antibodies: p-JAK2 (1:1000), JAK2 (1:1000), p-STAT3 (1:2000), STAT3 (1:1000)  and β-actin . After 16 h of incubation with the respective primary antibodies, the membrane was incubated with the following secondary antibodies: goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP secondary antibody . Specific binding was revealed using an ECL chemiluminescence kit (Biosharp Life Sciences) and a gel imaging system (Bio-Rad). All experiments were repeated three times.Rats were deeply anaesthetised with 3% sodium pentobarbital and then sacrificed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed and embedded in an OCT-embedding agent. Coronal sections of the brain were cut at 10 μm thickness on a microtome. The sections were then rinsed with PBS. To block non-specific binding proteins, tissue sections were incubated in 5% goat serum diluted in PBS for 1 h at room temperature. Tissue sections were incubated with the following primary antibodies: GFAP , p-JAK2 , JAK2 , p-STAT3 (1:100) and STAT3 (1:1000) . After 16 h, the brain sections were washed with PBS and incubated with fluorescent secondary antibodies: Cy3-conjugated secondary antibody (1:200) and CoraLite488-conjugated secondary antibody (1:200)  for 1 h at room temperature. After washing with PBS, fluorescent sealing tablets containing DAPI were used to seal the sections. Images were captured using a Zeiss microscope (Axio observer Z1). The flowchart of the experiment is illustrated in Fig. p < 0.05. GraphPad Prism 6 software was used to analyse the data.Data are presented as mean ± standard deviation (m ± SD). After normality and homoscedasticity of variance tests, one-way ANOVA and Tukey post hoc test were used. Statistical significance was set at Supplementary Information.


Every week, 1–2 breeds of farm animals, including local cattle, disappear in the world. As the keepers of rare allelic variants, native breeds potentially expand the range of genetic solutions to possible problems of the future, which means that the study of the genetic structure of these breeds is an urgent task. Providing nomadic herders with valuable resources necessary for life, domestic yaks have also become an important object of study. In order to determine the population genetic characteristics, and clarify the phylogenetic relationships of modern representatives of 155 cattle populations from different regions of the world, we collected a large set of STR data , including unique native cattle, 12 yak populations from Russia, Mongolia and Kyrgyzstan, as well as zebu breeds. Estimation of main population genetic parameters, phylogenetic analysis, principal component analysis and Bayesian cluster analysis allowed us to refine genetic structure and provided insights in relationships of native populations, transboundary breeds and populations of domestic yak. Our results can find practical application in conservation programs of endangered breeds, as well as become the basis for future fundamental research. Bos indicus breeds has been discussed, for example, in the work of Yurchenko et al. [The erosion and extinction of the genetic resources of domesticated animal species is a global problem FAO 2021) [ [1]. Acco et al. . The auto et al.  have indBos grunniens) is a valuable resource for nomadic herders and an important object to study. Recent studies of Bos grunniens diversity demonstrated that STR (short tandem repeat) markers used to explore the genetic diversity of cattle [The domestic yak  and STR markers; mitochondrial DNA and the Y-chromosome variation. Microsatellites have long established themselves as reliable markers. They are evenly distributed throughout the eukaryotic genome  and are In order to determine the population genetic characteristics, and clarify the phylogenetic relationships for 155 cattle populations from different regions of the world, we analysed genetic variability of 12 highly variable microsatellite loci included in the panel recommended by ISAG-FAO . The stuSubchapters in Results and Discussion are subdivided into breed groups for convenience of narration and perception of the text.For the 12 microsatellites analysed, 215 alleles were detected, with 12 (Bm1824) to 26 (Tgla122) per locus. The mean number of alleles per locus across all samples was 17.9. The average percentage of the total number of alleles observed in the locus varied from 19.55% (Altai-Rus) to 63.76%  . We calcHigh AF values (AF > 0.8) were obtained for five loci: locus Eth10 with alleles 217 (Charolais and Romagnola), 219  and 221 ; locus Bm1824 with alleles 180 (Scottish Highlander and Muturu) and 182 (Heck Cattle); locus Csrm60 with allele 102 (Scottish Highlander); locus Eth225 with allele 140 ; locus Sps115 with alleles 248  and 256 (Yakut); and locus Eth3 with allele 117 .For each locus and each sample, the PIC values were estimated on the basis of the number and frequency of alleles at the locus. The mean PIC value appeared to be 0.795. The highest polymorphism levels were obtained for Tgla122 (PIC = 0.897), Csrm60 (PIC = 0.888), Bm1824 (PIC = 0.856) and ilsts006 (PIC = 0.842).The genetic individuality of a breed may be defined by private  alleles. Out of 215 alleles in 10,250 animals genotyped, 23 alleles were private. All the described private alleles were discovered in the genomes of local cattle breeds  and various populations of domestic yak. The remaining private alleles were revealed in the genomes of various populations of domestic yak. The highest numbers of private alleles equal to 3 were found in representatives of the Gyr breed from India, Siboney breed from Cuba, and in the domestic yak population. Within these particular breeds the largest number of private alleles per locus equal to 4 was detected for Tgla227 . The larp-value for the chi-square test of the Hardy–Weinberg equilibrium . The observed and expected heterozygosity values were compared using the Bartlett test. The results obtained showed that there was a difference between the mean Ho and He values, and the pooled sample deviates from the Hardy–Weinberg equilibrium . High Ho values (0.81) were found for Criollo Baja California and Criollo Pilcomayo and the lowest (0.57) for Aryg-Khem-Rus.Genetic variability in each animal sample was studied in terms of the number of alleles (A), allelic richness (Ar), observed (Ho) and expected (He) heterozygosity, inbreeding coefficient (Fis) and ilibrium . Ar variWe calculated the inbreeding coefficient (Fis) for each sample as Fis = (He − Ho)/He. High Fis values point to a decreased heterozygosity of the microsatellites due to inbreeding. A very severe deficiency of heterozygotes was observed in two samples: Pineywoods (Fis = 0.166) and Criollo Yacumeño (Fis = 0.162). The Fis values were below zero, and the prevalence of heterozygotes were found in 60 breeds; the lowest ones are Sindi, Altai-Rus and Blanco Orejinegro. This implies that mating within the breeding farms is random and nonassortative, and no inbreeding occurs. For the other breeds, we observed neither strong excess nor strong deficiency of heterozygotes. The mean Fis value in the pooled sample is 0.003.Bos grunniens) separated away from the rest of the breeds, which is consistent with the modern idea of the cattle phylogeny.Both on the phylogenetic tree  and on tp-values = 0.05; 0.01 and 0.005) were examined for genetic differentiation. Fst analysis demonstrates that studied samples are significantly distanced from each other. The greatest divergence was noted between the Japanese Wagyu and the Italian Marchigiana . Representatives of the Kyrgyz native and the Alatau breeds were most closely related to each other , which is consistent with the results of the previous study [p-value = 0.1239), the Podolica  and the Kyrgyz native breeds . At the same time, nonsignificant or weak genetic differentiation of Buryat cattle with most of the studied breeds should be noted. The greatest differentiation of Kalmyk cattle was observed with the Yakut , Chianina  and Wagyu  breeds. Thus, the results of the Fst analysis testify in favor of the presence of common ancestors of Kalmyk cattle, both with the representatives of the Grey root and with the representatives of the Turano-Mongolian root. The Istrian breed from the Istrian peninsula also entered the cluster of Italian breeds, which is probably due to the territorial proximity and possible hybridization.Most European breeds are grouped together in a distinct cluster on a phylogenetic tree, with only a few breeds from other breed groups included. Among them, there was also a Kalmyk breed from the Asian breed group, united with Podolica. PCA b showed us study . AnalysiNext to Italian breeds there is a cluster of the United Kingdom cattle: Dexter, Jersey, Aberdeen Angus, Shorthorn and British White Cattle. The French meat breed Charolais joined this group. Its clustering near the Aberdeen Angus breed can be explained by known cases of crossing representatives of these two breeds. Another large cluster is formed by Dutch breeds, including Brandrood Cattle, Maas Rijn IJssel, Verbeterd Roodbont, Dutch Friesian, Dutch Belted and Groningen Whiteheaded. It is noteworthy that this group is joined by a breed from Russia—Kholmogory. The history of its breeding dates back to the 17th century and is noted for crossing with “Dutch cattle” . The RedThe majority of Asian breeds were included in the European breeds sector. Breeds from the Turano-Mongolian root Buryat, Gobi, Khogorogo and Yakut united in one cluster, and Wagyu proved to be closer to European breeds. It is known that Wagyu is experiencing European influence, including a British one . Aulie-AThree more breeds of the Turano-Mongolian root , together with some European breeds, were localized in the Iberian cluster. In close proximity to Alatau were Kyrgyz native, Kostroma and Brown Swiss. All three breeds were used in the breeding of the Alatau breed .Iberian breeds form separate cluster near the European breed group. The territory of the Iberian Peninsula could act as a contact zone between the African and European continents through the Strait of Gibraltar . The VacBos indicus in the genomes of African breeds. On the PCA plot, zebu breeds also formed a separate group. The farthest of all was the breed of Indian zebu Gyr.All five breeds of this group formed a common cluster on the phylogenetic tree near the African breed group, which is explained by the high proportion of Bos taurus, humped Bos indicus, sanga (African humpless Bos taurus × humped Bos indicus hybrid) and zenga (sanga×zebu hybrid). The Muturu, Gabu and Bafata breeds living on the west coast of Africa belong to the African phylum Bos taurus [According to the classifications available in the literature ,47,48, ts taurus . On the s taurus . Baladi s taurus . On the s taurus ,53. A sis taurus . The Kurs taurus . At the s taurus .Bos taurus genes into the genomes of domestic yaks and zebu was recorded. The subsequent differentiation of breeds (K = 6) led to the isolation of the Yakut breed. A similar divergence of Yakut cattle is noted in these works: Li et al. [i et al. , Iso-Toui et al. , Yurcheni et al.  and Li ai et al. . The DexBos indicus. Among the animals of the Asian breed group we revealed another trace of probable introgression from the yak, in particular in the Buryat and Aulie-Ata breeds. Referring to According to the results of cluster analysis at K = 12, the domestic yak populations from Mongolia contain a Turano-Mongolian component. At the same time, we noted the influence of European breeds in the Aikol yak breed from Kyrgyzstan. Earlier, it was found that 1.3% of the genes of Mongolian yaks were inherited from bovine ancestors . The ideAs K values increased, European breeds were separated into their own clusters. In particular, at K = 12, the following breeds formed their own clusters: Holstein, Dexter, Hereford, Limousin and Scottish Highlander. At K = 10 , all stuBos taurus was previously described by Chen et al. [At K = 12, the predominance of bright orange colour in the cluster of Asian breeds is clearly visible. We assume that this colour indicates the Asian, probably Turano-Mongolian, origin . The larn et al. .The results of our structural analysis also indicate the presence of an Asian component in the genomes of some Italian breeds. In particular, at K = 12, among the Italian breeds, the Podolian Podolica, the Sicilian Modicana and Cinisara demonstrated the greatest contribution of the Asian ancestry. At K = 20, the putative Asian component in Podolica showed commonality with the ancestors of Buryat (Russia) and Khogorogo (Mongolia). At the same time, on the PCA plot, the Podolica grouped with other Italian breeds, away from the Asian breed group. Structural analysis of the Asian breed group at K = 20 shows that most of the populations have a clearly visible genetic component of European breeds.Structural analysis demonstrated that at K = 20, the breed Marismeña  joined tBos indicus is more widespread on the African and Creole breed group, which is consistent with the previously described history of their origin. The exception was made by several Creole breeds, where zebu influence was barely perceptible. Such breeds include Criollo Argentina (Argentina), Criollo Patagónico (Argentina), Criollo Patagónico Chileno (Chile) and Romosinuano (Colombia). A previous study showed the absence of male-mediated introgression of Bos indicus into the genomes of Argentine breeds [Bos indicus in the African breed cluster. They were previously separated from the African breed group and formed a separate cluster on both the phylogenetic tree and the PCA plot. These breeds belong to the African type Bos taurus. Ankole-Watusi and Eastern Shorthorn Zebu showed the highest percentage of indicine ancestry. At K = 4, all representatives of the Asian breed group demonstrate in their structure a trace of Bos indicus, best discernible in the Khogorogo breed from Mongolia. Earlier, the contribution of indicine ancestors to the genome of the Turano-Mongolian breeds [Bos indicus genome is most prominent in the following European breeds: Italian Cinisara, Modicana, Chianina, Romagnola and Podolica, Croatian Istrian, Swiss Simmental and Brown Swiss, English Jersey and Russian Tagil. Among the Iberian breeds, a noticeable trace of zebu is observed in the Spanish Betizu, Rubia Gallega, Retinta and the Portuguese Brava de Lide. However, already at K = 6, the zebu trail becomes barely discernible in the Tagil, Brava de Lide and Retinta breeds. Analysing the structure of the Bos indicus breeds, we noted that the greatest influence of taurine cattle at K = 4 is expressed in the Sindi and Brahman breeds. In the work of Koufariotis et al. [Bos taurus.Zebu breeds formed a separate cluster at K = 4 . The infe breeds . The Wesn breeds  was descn breeds ,58. At Ks et al.  devoted Bos indicus and Bos grunniens. Among other breeds of the European cluster, a possible trace of African breeds is also demonstrated by breeds from Russia, including Kholmogory and Kostroma. In the Asian breed group, the greatest contribution of the African component is observed in two breeds from Russia , Gobi from Mongolia and Alatau from Kyrgyzstan. The presence of an African component in the genomes of the Russian [Bos taurus is noticeable.The influence of the African component on other breed groups becomes clear at values K = 10. In alignment with previous studies ,34,40, I Russian  and Tura Russian . HoweverBos grunniens). Domestic yaks provide nomadic herders with valuable resources necessary for life. A large habitat of domestic yaks is in Russia. At the same time, to the best of our knowledge, large-scale studies involving large samples of yaks from Russia in the context of a wide list of Bos taurus breeds from different regions of the world and Bos indicus breeds have not yet been conducted. In order to determine the population genetic characteristics and to clarify the phylogenetic relationships of modern representatives of 155 cattle populations, a large body of data  was collected and analysed, including unique native cattle, 12 yak populations from Russia, Mongolia and Kyrgyzstan, as well as zebu breeds. Based on the results obtained, we provide a number of important clarifications regarding the genetic structure of the populations of native cattle and domestic yak.As the methods of genotyping became more and more accessible, the number of studies devoted to the assessment of genetic diversity and the establishment of phylogenetic relationships of indigenous cattle breeds grew ,4,5,6. HMITF) from cattle to yaks [MITF is a growth factor, which participates in the regulation of melanocytes by controlling the synthesis of pigment [Bos indicus. Among the animals of the Asian breed group, a trace of possible introgression from the yak was also revealed, in particular in the Buryat and Aulie-Ata breeds. Wu et al. reported about the yak haplotypes introgression in genes involved in the response-to-hypoxia pathway  from yak to Tibetan cattle, which contributed to the adaptation of the latter to high altitude [EPAS1 haplotypes were transferred from Denisovans to Tibetans [AQP5 gene in response to adaptation to high altitudes characterized by low temperatures have been observed in both yaks and cold-resistant Kholmogory cattle [Bos taurus and Bos indicus cattle have larger body sizes, increased milk yield and are better adapted to a warm climate than yaks. At the same time, hybrid males (F1) are sterile, while females retain fertility [The populations of domestic yak from Mongolia have a component of Turano-Mongolian cattle breeds while components of European cattle breeds found in the Aikol yak from Kyrgyzstan . By usin to yaks . MITF is pigment . As is k pigment . Medugor pigment . The gen pigment . At K = altitude . Cases oTibetans  and fromTibetans . In studTibetans . In addiTibetans . It is wy cattle . This may cattle . Yak hybertility . Males rertility .Based on archaeological data, the authors of a number of studies suggest two main ways of agricultural cattle breeding in Europe: (1) the Danube way, along which Neolithic farmers moved north through the Central European plains, and (2) the Mediterranean way running along the Mediterranean coast ,70. CattAmong the Italian breeds considered, the Sicilian root (Cinisara and Modicana breeds) and Podolian  can be distinguished . CinisarBos primigenius and were domesticated on the territory of the Pannonian Basin during the reign of the Arpad dynasty. One of the first mentions of the long-horned Hungarian cattle can be found in a document of the 16th century [The Podolian breed Hungarian Grey  appeared century . It has  century . However century . By 1962 century . Measure century ,86.The beginning and spreading of pastoralism in Russia dates back to the 6th century AD. It was associated with the migrations of ancient Slavic tribes . Over a Bos taurus), a later Eurasian ancestor (Bos taurus), and a new Chinese ancestor (Bos indicus) diverging from the Indian Bos indicus. Turano-Mongolian cattle belong to Bos taurus, but have morphological and genetic differences from European taurine cattle [Cattle domesticated on the Indian subcontinent or imported from the Fertile Crescent and Europe are considered by Decker et al. to be the ancestors of Asian cattle breeds . These ae cattle ,104. Thee cattle  include e cattle . As is ke cattle , belongie cattle . A book e cattle  describie cattle .Bos taurus asiaticus can be considered some of the progenitors of Podolian breeds [The presence of the Asian component in the genomes of some breeds at low K values may indicate ancient hybridization events between the Asian breed group and other breeds due to human migration. One of these routes could run through the Mediterranean and connect Asia with Central Italy . A numben breeds .The results of our structural analysis indicate the presence of the Asian component in the genomes of some Italian breeds. In particular, at K = 12 among Italian breeds, the greatest contribution of Asian ancestry was demonstrated by Podolian Podolica, Sicilian Modicana and Sicilian Cinisara. As noted above, at K = 20, the assumed Asian component in the Podolica breed reveals affinity with the ancestor Buryat (Russia) and Khogorogo (Mongolia). At the same time, on the PCA plot, Podolica formed a cluster with other Italian breeds, away from the Asian breed group. The exchange of cattle between Asia and Europe can also be traced in the genomes of Asian breeds. At K = 20, structural analysis of the Asian breed group shows that the genetic component of European breeds is clearly visible in most populations. The presence of the genetic component mentioned may be due to the export of European cattle to Asia, followed by hybridization of imported and local cattle. The results of cluster analysis at K = 15 and K = 20 by Decker et al. indicate the presence of an admixture of European taurine in the Mongolian breed and the Wagyu breed .Domesticated cattle were brought by Christopher Columbus to the Caribbean island of Hispaniola from the Canary Islands during his second voyage across the Atlantic in 1493 . Until 1Bos indicus is more widespread on the African and Creole breed group. In their work, MacHugh et al. noted that African cattle are characterized by a geographical gradient of indicine ancestry [Bos indicus genes increases on average to 56.9% (from 22.7% to 74.1%) [Bos indicus (mainly bulls) to Brazil in the 19th and 20th centuries [Bos indicus, best discernible in the Khogorogo breed from Mongolia. In a work by Yurchenko et al., the results of structural analysis (K = 3) obtained from genotyping data using SNP markers also demonstrate the contribution of indicine ancestors to the genome of Turano-Mongolian breeds [Bos indicus in the Mongolian and Kazakh breeds presumably occurred in the 2nd–7th centuries AD during the Silk Road period [Bos taurus [Bos taurus with African zebus may have occurred [The influence of ancestry . It was o 74.1%) . Speakino 74.1%) . At leaso 74.1%) , referrio 74.1%) , in a stenturies . The resenturies , as wellenturies ,117 and enturies  of Creolysis K =  obtainedn breeds , and in n breeds . Accordid period ,119. In s taurus . There ioccurred . Accordioccurred .Bos indicus genome to the European breed group can be noted, among others, in the Podolian cattle . The introgression of Bos indicus genes into the Podolian cattle genome has been repeatedly noted in previous studies [At K = 4  impact o studies ,121,122. studies . The tra studies . The Jer studies  reported studies  suggeste studies . Analysi studies .Bos taurus to almost pure Bos indicus [Bos indicus [Bos taurus males and Bos indicus males [When studying the origin of African breeds, the genetic diversity of populations from pure  indicus  was reve indicus ,126. A s indicus . Accordi indicus ,73. This indicus . At the us males ,129. Accus males ,130. Theus males . The preus males ,131. Basus males  suggesteus males ,40. Therus males  In suppous males  cite theTo sum up, we note that most of the results obtained are in line with the history of breeds and similar studies, including those performed using SNP markers. The low bootstrap support observed for large breed-clusters on the phylogenetic tree could be expected. This pattern has already been noted earlier in the analysis of many populations , especiaBos taurus showed that the genomic signature of early populations may be hidden in modern individuals by a later admixture.At the same time, we consider it necessary to describe the possible limitations of the approaches used. These include a different number of samples in breeds, a small number of individuals of some breeds, and a reduction in the number of STR markers used. In addition, samples of the genetic material of some breeds involved in the analysis were obtained far from the places of the historical origin of the breed. And, depending on the location, they could undergo mating with other breeds in order to improve certain economically useful or adaptive qualities. Yurchenko et al.  showed tBM1824, BM2113, CSRM60, CSSM66, ETH3, ETH10, ETH225, ILSTS006, SPS115, TGLA53, TGLA122 and TGLA227), which are included in the panel recommended by ISAG–FAO for studying the genetic diversity of cattle [Original STR analysis data for 15 cattle populations and 12 yak populations provided by our laboratory team in the course of previous studies ,17. The http://doi.org/10.5061/dryad.d4500); Ginja et al. [https://doi.org/10.5061/dryad.5dv41ns43.Open-access STR data of other cattle populations from different regions of the world were presented in the papers: Van de Goor et al. , data ava et al.  data avaThe object of our study is the statistical analysis of an STR dataset of 155 native and transboundary cattle populations, including domestic yak and zebu populations. Genotyping was carried out using 12 highly stable and polymorphic microsatellites (f cattle . The desBos indicus) and 833 animals from 12 populations of the Domestic yak breed group (Bos grunniens)  . Breed nBefore combining the data into one dataset, a data generalization procedure was performed. This procedure determines a nucleotide bias using the heat maps of the PopGenReport R package  and corrp-value were calculated by the pairwise.fst function from the hierfstat R package [The calculations were performed in the R environment version 4.1.2. Mean number of alleles (Na) and effective number of alleles (Ne) were calculated using the Genalex package . Allelic package  and visu package .The genetic Da distance matrix  was calcPCA was performed to investigate genetic relationships among studied breeds. The scripts used the ade4 and adegenet R-libraries. Calculations were carried out with the dudi.pca function, input data were scaled out using the scaleGen function with the “mean” method. Additional visualizations were performed on the adegraphics platform in R.The population structure of 155 breed populations  was studied using the STRUCTURE 2.3.4 software . We testrepeats) . For cla