Striga hermonthica is a serious biotic stress limiting maize production in sub-Saharan Africa. The limited information on the patterns of genetic diversity among maize inbred lines derived from source germplasm with mixed genetic backgrounds limits the development of inbred lines, hybrids, and synthetics with durable resistance to S. hermonthica. This study was conducted to assess the level of genetic diversity in a panel of 150 diverse maize inbred lines using agronomic and molecular data and also to infer the population structure among the inbred lines. Ten Striga-resistance-related traits were used for the phenotypic characterization, and 16,735 high-quality single-nucleotide polymorphisms (SNPs), identified by genotyping-by-sequencing (GBS), were used for molecular diversity. The phenotypic and molecular hierarchical cluster analyses grouped the inbred lines into five clusters, respectively. However, the grouping patterns between the phenotypic and molecular hierarchical cluster analyses were inconsistent due to non-overlapping information between the phenotypic and molecular data. The correlation between the phenotypic and molecular diversity matrices was very low (0.001), which is in agreement with the inconsistencies observed between the clusters formed by the phenotypic and molecular diversity analyses. The joint phenotypic and genotypic diversity matrices grouped the inbred lines into three groups based on their reaction patterns to S. hermonthica, and this was able to exploit a broad estimate of the actual diversity among the inbred lines. The joint analysis shows an invaluable insight for measuring genetic diversity in the evaluated materials. The result indicates that wide genetic variability exists among the inbred lines and that the joint diversity analysis can be utilized to reliably assign the inbred lines into heterotic groups and also to enhance the level of resistance to Striga in new maize varieties. Zea mays L.) is an important cereal crop playing a crucial role in global food security and as a source of income for smallholder farmers in sub-Saharan Africa (SSA). It is referred to as the queen of cereals because of its high yield potential, ease in processing, and low cost [Maize .Genetic diversity is the foundation for crop improvement, and it plays an important role in breeding programs . Informa7 to S9 stages of inbreeding having diverse reactions to S. hermonthica were randomly selected from a large number of lines with different genetic backgrounds and used in the present study. The inbred lines were derived from diverse source populations  and Mokwa  in Nigeria during the main rainy seasons of 2017 and 2018. The lines were arranged in a 15 × 10 alpha lattice design with two replications and planted in strips. Within each strip, an inbred line was planted in a 4 m long row, with 0.75 m inter-row spacing and 0.25 m intra-row spacing.S. hermonthica seeds from the soil through suicidal germination. S. hermonthica seeds used for artificial infestation of the infested blocks were collected from farmers’ sorghum fields in the previous planting year. Two maize seeds were planted in a 6 cm deep hole injected with 8.5 g of sand mixed with Striga seeds. The mixture contains approximately 3000 germinable Striga seeds. Two weeks after planting, all maize plants were thinned to one plant per hill to attain a population density of 53,333 plant ha−1. Fertilizer was applied at the rate of 30 kg/ha of Nitrogen, 60 kg/ha each of phosphorus and potassium at planting, and an additional 30 kg/ha nitrogen was applied four weeks later. Weeds other than Striga were removed from plots manually throughout the planting season.The field was treated with ethylene gas two weeks before planting to remove Striga-infested conditions include: plant stand, anthesis and silking days, plant height, ear aspect, and grain yield . DNA was extracted from young leaves of each inbred line using a modified cetyltrimethylammonium bromide (CTAB) protocol . PurifieiSeq2500 . Raw floiSeq2500 . Reads aStriga infested conditions based on mixed-model analysis with the restricted maximum likelihood procedure in SAS version 9.4 [Analyses of variance combined across the four year–location combinations were computed for all traits measured under sion 9.4 . In thission 9.4 . The optsion 9.4  while thsion 9.4 . Correlak) ranging from k = 2 to k = 10 [Over 560,000 SNPs were received from the Elshire group in New Zealand. To determine the quality of the data, quality control (Q.C.) was performed to retain only bi-allelic sites, SNPs with minor allele frequency (MAF) less than 5% and missing data more than 10% were removed from the data set. In addition, SNPs with high linkage disequilibrium (L.D) were pruned using the indep-pairwise function implemented in PLINK  . Based oo k = 10 ,49. The o k = 10 ,52.Striga infestation, genotypic and joint distance matrices were assessed using a Mantel test based on the Monte-Carlo method with 9999 permutations. Genetic groups were defined using a combination of the agronomic trait-based and genotypic dissimilarity matrices. The joint matrix was generated by the summing the genotypic and agronomic trait-based dissimilarity matrices. Similarities between the hierarchical clusters generated from the agronomic trait-based and genotypic data were accessed using the tanglegram function implemented in the dendextend R package . The relStriga infestation except for ear aspect  observed between an IWDS inbred line and a TZLC derived inbred line whereas the lowest genetic distance (0.03) was observed between an IWDS line and a line derived from a composite.In the combined analyses of variance, differences among environments and inbred lines were significant for all traits measured under r aspect . Heritabr aspect . Grain yS. hermonthica (Striga susceptible line (5057) and a line derived from IWDS that supports the largest number of emerged Striga plants. The two inbred lines in this cluster also had the highest Striga damage symptom rating at 8 and 10 weeks after planting. Cluster II consists of twenty-nine inbred lines that support few emerged Striga plants, with moderate Striga damage symptoms and they produced intermediate grain yield. Inbred lines in this cluster are characterized as tolerant to Striga infestation. Cluster III consists of one hundred and five inbred lines that support fewer emerged Striga plants with varying levels of Striga damage symptoms. These lines are characterized as moderately resistant to Striga infestation. Cluster IV consists of five maize inbred lines with high Striga emergence count and damage symptoms. These lines yielded more than the susceptible lines and they are characterized as moderately susceptible to Striga infestation. Cluster V had nine inbred lines with low Striga emergence and Striga damage symptoms. These lines are characterized as resistant to Striga infestation.Assessment of the phenotypic diversity using the agronomic traits grouped the inbred lines into five clusters based on their reaction pattern to monthica . ClusterThe results of principal component analysis (PCA) showed that the first three components together accounted for 81% of the total phenotypic variation . The firStriga damage rating at 8 and 10 weeks after planting. In contrast, there was a negative correlation between grain yield and ear aspect, Striga damage rating at 8 and 10 weeks after planting, and Striga count at 8 and 10 weeks after planting  had 1% heterozygosity rate, which is the expected amount of residual heterozygosity in inbred lines. The expected heterozygosity, also known as gene diversity, varied from 0.10 to 0.50, with an average of 0.32. Polymorphic information content values ranged from 0.10 to 0.47, with an average of 0.26 .k = 5, which grouped all the 150 inbred lines into five clusters  was observed between two inbred lines showing low emerged Striga plants.The population structure analysis performed through Admixture revealed a rapid elbow at clusters . In addiclusters , the optthe DAPC . EstimatStriga plants. Cluster II consists of 27 inbred lines derived from an intermediate maturing synthetic (IWDS). Most of the lines in this cluster are high yielding and support fewer emerged Striga plants. Cluster III consists of eight inbred lines, with seven of them originating from an early maturing composite (TZEC) and the remaining from a late-maturing synthetic (TZLC). Inbred lines in cluster II and III all had a white kernel color  maintained the same group across both hierarchical clusters  showed that the inbred lines clustered into three distinct groups  between the agronomic and the molecular dissimilarity matrices. However, a high correlation (r = 0.85) was observed between the joint matrix and the agronomic dissimilarity matrix, while a moderate correlation (r = 0.50) was observed between the joint matrix and the genotypic data-based matrix  platform were used to assess the genetic diversity and population structure of the inbred lines. The three complementary approaches used to define the optimal number of groups in this study  all grouped the inbred lines into five distinct groups. The assigning of group membership in DAPC is in agreement with the hierarchical cluster analysis; this finding is consistent with reports from other studies ,57,58. Testation  and thesS. hermonthica. Seven inbred lines maintained the same cluster positions across both hierarchical clusters when compared with each other. The six inbred lines from the ZDIP and the TZLC groups are high yielding and support low emergence of Striga plants; they also have low Striga damage rating. These lines can be used as desirable parents to broaden and diversify the genetic base in Striga resistance breeding programs. The last inbred line is a known susceptible inbred line.The combined dissimilarity matrix of the agronomic-trait-based and molecular marker data grouped the inbred lines into three distinct clusters based on their reaction pattern to The low correlation observed between the genotypic and agronomic-trait-based distance matrices in this study should not be regarded as a limitation to access the genetic diversity but as an indicator of the complementarity of these methods . The lowThe high and moderate correlation observed between the combined dissimilarity matrix and the phenotypic and genotypic dissimilarity matrices, respectively, is an indication that genetic diversity analysis based on the joint matrix is an invaluable tool to cumulatively and reliably allocate genotypic and phenotypic information. This result is consistent with the findings of other studies ,61,62. TS. hermonthica. The molecular diversity analysis was able to group the inbred lines based on their genealogy and historical background than the phenotypic diversity analysis. However, the grouping patterns between both clusters were inconsistent due to non-overlapping information between the data types. The joint diversity analysis explores the synergy of the two approaches (molecular and agronomic) by capturing the information to provide a comprehensive understanding of the entire diversity among the inbred lines.In conclusion, assessment of the genetic diversity using both agronomic traits and molecular data revealed the existence of considerable genetic variability among the evaluated materials. It also provides invaluable information on the reaction patterns of the inbred lines to


Disparities across racial and ethnic groups are present for a range of health outcomes. In this opinion piece, we consider the origin of racial and ethnic groupings, a history that highlights the sociopolitical nature of these terms. Indeed, the terms race and ethnicity exist purely as social constructs and must not be used interchangeably with genetic ancestry. There is no scientific evidence that the groups we traditionally call “races/ethnicities” have distinct, unifying biological or genetic basis. Such a focus runs the risk of compounding equity gaps and perpetuating erroneous conclusions. That said, we suggest that the terms race and ethnicity continue to have purpose as lenses through which to quantify and then close racial and ethnic disparities. Understanding the root cause of such health disparities—namely, longstanding racism and ethnocentrism—could promote interventions and policies poised to equitably improve population health. DNA studies and archeological findings tell us that present-day humans descend from hominids in Ethiopia and surrounding territories. Early humans migrated “out of Africa” some two million years ago Fig. . The finHomo sapiens. Linnaeus further divided Homo sapiens into Europaeus (white skin), Asiaticus (yellow skin), Americanus (red skin), and Afer (black skin) on the basis of geography and skin color [Today’s racial and ethnic categories arose long before the field of genetics. The term “race” was first formally used in the English language around 1580, from the old French “rasse” and the Italian “razza,” to categorize modern humans. In the sixteenth and seventeenth centuries, scientists classified humans according to geographic locations using skin color, stature, and other distinguishing physical characteristics. Later, “nations” and “types” were introduced to describe humans living on different continents. Carl Linnaeus (1758), the Swedish biologist who developed the technique for classification of plants and animals, gave modern humans the scientific name in color . From thModern DNA sequencing technologies revolutionized our understanding of the human genome in ways that could not have been imagined before. We can now read all 3.2 billion nucleotides in a matter of hours. Scientists have investigated the DNA of thousands of people from around the world trying to find a genetic underpinning for racial or ethnic differences. They have found that human DNA is 99.9% similar, whether we are from Europe, Asia, the Americas, or Africa. In fact, there is more genetic diversity within a single racial/ethnic group than between two or more groups . Two indPlasmodium falciparum infection, and altered Th1/Th2 cytokine responses compared to their geographic neighbors. Sickle cell anemia (caused by hemoglobin-Beta gene mutations) and East African sleeping sickness  gene represent two examples where environmental conditions selected some alleles for local adaptation as protection from indigenous diseases like malaria and Trypanosoma parasitism, respectively. However, these adaptations are not without physiologic consequences: sickle cell disease leads to painful crises and organ damage, and the APO1 variant is associated with increased susceptibility to chronic kidney disease (CKD). The APO1 variant, however, is missing among inhabitants of the Ethiopian highlands, where the Tsetse fly vector for the trypanosome parasite is absent, paralleling a very low incidence of CKD. Thus, small changes in our genetic makeup do occur, but they do so to adapt to environmental conditions. These genetic changes are not, however, race- or ethnicity-specific. Instead, changes can happen to any human population in the world in response to specific environmental conditions no matter their racial or ethnic categorization.Small existing genetic differences helped us adapt to local environmental conditions across the continents since the migration out of Africa . This isTrying to find a biological basis for differences across races or ethnicities is like looking for a needle in a haystack, akin to seeking a biological basis for why certain individuals and populations speak different languages. Our ability to speak a specific language, or more than one language, is not built upon our genes. Instead, language and culture are learned. They may be based on where we grow up or live; they can be altered and redefined . SimilarIn the United States (US), there are marked racial and ethnic disparities with respect to a range of socioeconomic and environmental factors like poverty, educational attainment, access to healthcare, and proximity to pollutants . LookingTo be clear, there is no evidence that genes drive racial and ethnic health disparities. Rather, racial disparities in health are better explained structural racism than by genetic differences. In the US, for every preventable disease, African Americans (AA) have the highest mortality rate and shortest expected years of life compared to non-Hispanic Whites –11. ThisIt is this context, and inequities therein, that drive between-group differences—disparities—in a range of health outcomes. We know that historically disadvantaged populations—including AA and Hispanic patients—have a higher burden of the comorbidities traditionally used by hospitals to stratify patients by risk. This is largely because of structural and socioeconomic factors. Studies and statistics suggest that, compared to their white counterparts, AA patients are 40% more likely to have high blood pressure, twice as likely to have heart failure, four times as likely to die from asthma-related complications, three times more likely to have chronic kidney disease, twice as likely to be diagnosed with colon and prostate cancer, and represent 44% of the HIV positive population. Across the US, we are seeing alarming statistics about the disproportionate toll of COVID-19 on AA and Latino people . There hThese alarms, and the continued reality of racial and ethnic disparities, are real and require attention; they follow closely behind differences in the sociopolitical context that different racial and ethnic groups confront. There is plenty of evidence linking marginalization, segregation, and discrimination to a laundry list of adverse social, economic, environmental, and, yes, health outcomes. Race and ethnicity have been used to justify horrific discrimination across generations. This justification has been both explicit and implicit; it is at the root of the horrors we are now seeing in our streets. Indeed, George Floyd, Breonna Taylor, Ahmaud Arbery, Daniel Prude, Jacob Blake, and many more have borne the brunt of a classification schema built to perpetuate hierarchical tiers of status and power. These different tiers drive differences in the cleanliness of the air we breathe, the homes in which we live, and the resources we enjoy. Differences in health outcomes follow.While there has been substantial effort in reconceptualizing the notion of racial disparities beyond genes/biology, the utility of the terms race and ethnicity remains problematic among scholars, the lay public, and other stakeholders. Genetic data can provide data on the nature of human biological diversity. How we incorporate this information into structural racism and race information will be critical in ensuring that modern understandings of human variation are viewed with anti-racist, anti-ethnocentric lenses. Yet, race and ethnicity must always be de-biologize and never be used interchangeably with genetics. Instead, they should be recognized as the sociopolitical  construcHomo sapiens evolved in Africa, and all humans indisputably share a common, unifying genetic origin. Genetic variation across Europe and Asia, and the Americas and Australia, is essentially a subset of the genetic variation in Africa. Regardless of differences in skin color, cultural background, religion, or language, we all are more similar than different! Genes do not cause disparities, rather systemic, structural inequities have long been the culprit. Just as one learns a new language through study and immersion, so too can we learn more about one another through social means, through interaction and advocacy for equity. Disparities did not emerge overnight nor are they amenable to simple, overnight solutions. Instead, we must recognize the context that underlies them, driven by sociopolitical and not genetic factors. Racial and ethnic disparities represent a multi-level, large scale problem in need of multi-level, large scale solutions .


The JAK/STAT3 signaling pathway plays an essential role in various types of cancers. Activation of this pathway leads to increased tumorigenic and metastatic ability, the transition of cancer stem cells (CSCs), and chemoresistance in cancer via enhancing the epithelial–mesenchymal transition (EMT). EMT acts as a critical regulator in the progression of cancer and is involved in regulating invasion, spread, and survival. Furthermore, accumulating evidence indicates the failure of conventional therapies due to the acquisition of CSC properties. In this review, we summarize the effects of JAK/STAT3 activation on EMT and the generation of CSCs. Moreover, we discuss cutting-edge data on the link between EMT and CSCs in the tumor microenvironment that involves a previously unknown function of miRNAs, and also discuss new regulators of the JAK/STAT3 signaling pathway. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway played a crucial role in many biological functions during the multistep development of human tumors, including proliferation, inflammation, and survival. The JAK/STAT signaling pathway comprises of the receptor and adaptor proteins of interleukin 6 (IL-6), interferon-alpha (IFN-α), and interferon-gamma (IFN-γ) that mediate pleiotropic functions upon binding to their respective ligands ,2.The IL-6 family of cytokine comprises IL-6, IL-11, IL-27, IL-31, oncostatin M (OSM), cardiotrophin 1 (CT-1), ciliary neurotrophic factor (CNTF), cardiotrophin-like cytokine factor 1 (CLCF1), and leukemia inhibitory factor (LIF). Elevated expression of the cytokines belonging to this family is implicated in the development of various human diseases . Upon biThere are four JAK family non-receptor tyrosine kinases, JAK1, JAK2, JAK3, and TYK2. JAK1, JAK2, and TYK2 are ubiquitously expressed, whereas JAK3 is predominantly expressed in hematopoietic cells . The JAKUntil now, seven members of the STAT family  have been identified. Each of the STAT proteins shares highly conserved domains, including amino-terminal, coiled-coil, DNA binding, SH2, and transactivation domains . The AspSTAT proteins are differentially implicated in cancer tumorigenesis. Although STAT1 is known to be involved in mediating the anti-tumor immunity and other STAT families are known to be involved in the promotion of cancer development, it is STAT3 that is most well studied as a significant intrinsic transcription factor in the induction of the EMT and in the pathogenesis of cancer  16]. IL. IL16]. STAT3 acts as a transcriptional activator through binding to the promoter of target genes in a tyrosine phosphorylation-dependent manner . For insThe activation of JAK2/STAT3 signaling is triggered by various hormones, cytokines (including the IL-6 family), and growth factors through a variety of molecular mechanisms. OSM, another IL-6 cytokine family member, is crucial for JAK/STAT activation. It interacts with the extracellular matrix (ECM) proteins, and OSM/OSM receptor (OSMR)-mediated JAK2/STAT3/FAK/Src activation promotes EMT and CSC production ,37,38,39Hormones, growth factors, and chemokines are associated with JAK/STAT3 activation. The gastrointestinal (GI) peptide hormone, gastrin, promotes the invasiveness of cancer cells by the induction of MMP-1, -2, and -9 secretion . GastrinThe heparin-binding growth factor, Midkine (MK), induces EMT via activating the nuclear accumulation of the Notch-2 receptor through the formation of the MK-Notch-22 complex . In addiNon-canonical Wnt signaling, which is independent of the β-catenin/TCF pathway, induces EMT through STAT3 activation. Upregulated Wnt5a/b and its receptor, Wnt receptor, Frizzled2 (Fzd2), induce cell migration by non-canonical pathways, and activation of STAT3 by the Fzd2/Fyn/STAT3 complex upregulates STAT3 target genes .G-protein coupled receptors (GPCRs) and human GH (hGH) have been reported to be involved in the regulation of JAK/STAT signaling in cancer. Activation of PAF/platelet-activating factor receptor (PAFR) promotes lung cancer progression via the activation of Src/STAT3 or JAK2/STAT3 . MoreoveThe contribution of intracellular signaling components to the induction of EMT through the activation of the JAK/STAT signaling in cancer progression has been extensively studied B. Potential mechanisms by which tyrosine or serine/threonine kinases activate the JAK/STAT3 signaling. Nuclear interactions, as well as cytoplasmic interactions between EGFR and STAT3, increase the expression of iNOS, cyclin D1, and c-fos via direct binding of EGFR/STAT3 complex to their promoters . Moreove2+ permeability, enhance metastasis by induction of EMT through increased STAT3 expression [Serine/threonine kinases as the components of the ion channel are involved in STAT3 activation. The production of calcium signal by EGF induces EMT via activation of STAT3 by increasing the levels of TRPM7, a serine/threonine kinase . Anotherpression . TRPC1-ipression . Increaspression  and PIM-pression . Furtherpression .Several proteins regulate STAT3 activation through direct interaction with STAT3. Pin1 is highly expressed in a majority of cancers ,93, and Diverse proteins are upregulated during cancer progression and facilitate constitutive JAK/STAT activation. The levels of G3BP1, which plays a critical role in stress granule (SG) formation, are increased in renal cell carcinoma (RCC) and promote IL6/STAT3 activation . FUZ is The activation of fundamental components of cellular metabolism and DNA methylation involves tumor progression by regulation of STAT3 activity. Upregulation of G6PD, an enzyme involved in the pentose phosphate pathway, and PKM2, the key enzyme catalyzing the final step of glucose metabolism is correlated with metastasis and poor survival of patients with HCC and ESCC and promotion of EMT via STAT3 and STAT5 ,108. MorThe Annexin (Anx) family contains calcium-dependent phospholipid-binding proteins and is associated with the invasiveness of cancer. AnxA1 induces invasiveness, drug-resistance, and generation of CSCs in prostate and pancreatic cancer by increasing the p-STAT3 level . In addiHypoxia-responsive proteins are involved in STAT3-mediated EMT induction. Non-canonical TGF-β and EGF signaling induce the accumulation and upregulation of hypoxia-inducible factor 1α (HIF-1α) in the nucleus. During this process, the degradation of HIF-1α is blocked by the formation of STAT3-HIF-1α complex, and this is followed by induction of Twist-1 expression by binding of both STAT3 and HIF-1α to the Twist-1 and VEGF promoters ,119,120.Direct activation of STAT3 by the NPM-ALK chimeric protein, which plays a central pathogenic role in anaplastic large cell lymphoma (ALCL), promotes invasiveness of ALK-positive ALCL through induction of Twist-1 . The kinJAK/STAT3 signaling also can be induced by oncoproteins, including RPL34 , HOXB8 , CD146 , and AkiRecently, it was reported that inhibition of the canonical TGF-β signaling due to loss of SMAD4 might be associated with cancer progression and drug resistance. Loss of SMAD4 induces constitutive activation of STAT3 in pancreatic cancer, which cooperates with non-canonical TGF-β signaling to promote cancer progression . ScreeniThere are multiple intrinsic regulators, which can prevent inappropriately sustained STAT3 activation A. NitricAMPK has been implicated in the inhibition of tumor proliferation via suppression of STAT3 activation. AMPK suppresses tumor survival through inhibition of mTOR by increasing the expression of p-TSC2, a tumor suppressor gene , and leaTyrosine phosphatase is involved in the negative modulation of STAT3 activity. SHP-1 promotes the inactivation of JAK/STAT3 ,146, andSome negative regulators inhibit IL-6/JAK/STAT signaling by downregulating the expression of these proteins. ESE3/EHF, ETS transcription factor, inactivates IL6/JAK/STAT3 signaling through the transcriptional repression of IL-6 by direct binding to the IL-6 promoter . FrequenAlongside the transcriptional repression, critical negative regulation occurs via inhibition of IL-6/JAK/STAT3 activation. Mucosal barrier disruption by loss of CLDN-3 promotes CRC malignancy through induction of the Wnt/β-catenin signaling by activation of IL-6/JAK/STAT3 signaling. In contrast, the upregulation of CLDN-17 activates Tyk2/STAT3 signaling to promote malignancy in HCC ,156. In Recent studies have identified that other negative regulators inhibit JAK/STAT3 activation by interfering with the interaction between STAT3 and downstream targets or non-receptor tyrosine kinase. DNA JB4 inhibits Src phosphorylation by direct binding to the SH3 domain of c-Src and suppresses invasion of cancer by disrupting the interaction between Src and Src-downstream targets such as EGFR, FAK, and STAT3 . RKIP inCytokines secreted by either cancer cells or surrounding stromal cells act as negative regulators. For instance, IL-32α and IL-32θ inhibit TGFβ-induced EMT, metastasis, and self-renewal in various cancer cells by inhibition of STAT3 activation ,168. AddCancer stem cells have been proposed to explain cancer progression and therapeutic resistance. Multiple extracellular stimuli and intracellular signaling pathways have been implicated in the activation of the JAK/STAT signaling pathway or in the induction of cross-talk between various JAK/STAT pathways to promote the generation of cancer stem cells and acquisition of drug resistance ,171. Of Osteopontin (OPN)/integrin complex upregulates the expression of CD44 variants. OPN binding to CD44 enhances metastasis and generation of CSC through activation of STAT3 ,182,183.JAK/STAT3 activation by positive regulators is involved in drug- or radiation-resistance. CXCR4-mediated STAT3 activation induces gefitinib-resistance and transition into stem-like cancer cells, which possess self-renewal capacity and radiation resistance . AdditioIn addition to JAK2/STAT3 signaling, which is mainly regulated by proteins (positive or negative modulators), the expression and functions of JAK/STAT are also controlled by other regulatory mechanisms. Notably, many studies have established a link between non-coding microRNAs (miRNAs) and JAK/STAT signaling. JAK/STAT3 signaling induces miRNAs to regulate the target genes. For instance, the loss of p53, IL-6, and LIF induces mTOR and STAT3 activation ,189,190,miRNAs are upregulated in response to STAT activation, and the JAK/STAT3, in turn, is often activated by miRNAs. miR-221/222 induces the EMT via activation of IL-6-dependent NF-κB and STAT3 through suppression of adiponectin receptor 1 (ADIPOR1) ,198. AddBesides, increasing the stabilization of STAT3 through SOCS-1 or SOCS-2—both of which are downregulated by miR-155-5p or miR-194—promotes the spread of prostate cancer  and is amiRNAs lead to the inactivation of the JAK/STAT signaling pathway through several different mechanisms. They act as negative regulators by suppressing the expression of IL-6/JAK/STAT3. miR-93 prevents the generation of CSCs and metastasis through the downregulation of multiple stem cell regulatory genes, including JAK1 and STAT3 . AdditioAdditionally, miRNAs act as negative regulators by direct or indirect suppression of IL-6/JAK/STAT3 activation. miR-876-3p inhibits glioma progression, including EMT, by inhibiting JAK2/STAT3 activation via suppression of KIF20A expression . GATA3-iTCF-4/KIFC1-mediated HMGA1 induces the expression of STAT3, MMP2, and Twist-1 by binding to their promoters . AdditioThe upregulation of thyroid hormone (T3) and its receptor (TR) in HCC suppresses miR-130b expression. However, increased miR-130b and miR-345 levels result in the upregulation of interferon regulatory factor 1 (IRF1) via T3/TR, which controls cell migration and invasion and represses the expression of p-mTOR, p-STAT3, p-AKT, and p-ERK1/2 ,224. TheAlong with its essential role in the progression of cancer, the IL-6/JAK/STAT3 signaling axis incorporates various unexpected components and miRNAs that contribute to JAK/STAT3 activation in cancer. Unpredicted functions of various components and miRNAs that have been identified as new positive or negative regulators of STAT3 may need further investigations, for us to be able to selectively target JAK/STAT3 signaling for cancer therapy. Thus, therapies that target these proteins or miRNAs, which are currently not thoroughly explored, may be implicated in suppressing invasion, dissemination, and transition into CSCs in the tumor microenvironment. Although small RNA-target based therapy, including miRNAs, is challenging due to the broad off-target effects, studies that investigate the use of small RNAs as medical interventions have expanded the preclinical and clinical applications. Recently, the FDA approved Onpattro (patisiran) for polyneuropathy in patients with hereditary amyloidosis; it is the first FDA approval for an siRNA drug. Thus, miRNAs or novel identified regulatory proteins may serve as potential targets in further clinical developments and represent significant conceptual advances in terms of repression of JAK/STAT3 activation in human diseases, including cancer, diabetes, inflammation, and neurodegenerative disorders. Thus, the development of therapeutic strategies based on the targeting of the JAK/STAT3 pathway is of utmost clinical relevance.


Asperula naufraga is a rare and threatened obligate chasmophyte, endemic to Zakynthos island . In this study, we provide a combined approach (including monitoring of demographic and reproductive parameters and study of genetic diversity) to assess the current conservation status of the species and to estimate its future extinction risk. The five subpopulations of A. naufraga were monitored for five years (2014–2018). Population size markedly fluctuated between 68–130 mature individuals during the monitoring period. The extent of occurrence (EOO) was estimated at 28.7 km2 and the area of occupancy (AOO) was 8 km2. Stage-structure recordings were similar for all subpopulations, characterized by high proportions of adult and senescent individuals, following a common pattern, which has been observed in other cliff-dwelling plants. Preliminary genetic analysis with SSRs markers revealed low heterozygosity within subpopulations and significant departure from H-W equilibrium, which combined with small population size suggest increased threat of genetic diversity loss. Our results indicate that the species should be placed in the Critically Endangered (CR) IUCN threat category, while according to Population Viability Analysis results its extinction risk increases to 47.8% in the next 50 years. The small population size combined with large fluctuations in its size, low recruitment and low genetic diversity, indicate the need of undertaking effective in situ and ex situ conservation measures. The Mediterranean Basin constitutes the second largest hotspot of plant diversity and endemism at a global scale . The divHabitat specialization to the harsh conditions of cliff surface often leads to low dispersal ability and rarity , 8. HoweThe advantages of multidimensional approaches regarding the biology and ecology of threatened plants towards the implementation of effective conservation management planning have been repeatedly highlighted e.g. , 17. HowUnderstanding genetic structure and variability of plants facilitates investigation of their biological characteristics, evolutionary history, and adaptive potential . EndemicAsperula naufraga Ehrend. & Guterm. is a perennial chasmophyte, forming loose cushions [Asperula sect. Cynanchicae (DC.) Boiss. and had been provisionally included in the informal A. ser. Palaeomediterraneae [A. sect. Cynanchicae revealed that this group does not represent a monophyletic unit and turns out to be highly artificial [A. sect. Cynanchicae are blurred because of poor species divergence and hybridization, combined with some cases of non-monophyletic species [Asperula naufraga, however, is a well-differentiated species in morphological terms, easily distinguished from all related species within A. sect. Cynanchicae [Asperula daphneola O. Schwarz [The west coasts of the Ionian Islands (Greece) have some of the largest and most impressive cliff systems in the Mediterranean, hosting a rich chasmophytic flora. cushions . It is acushions . It is aerraneae , 28. Thetificial . Phyloge species . Asperulanchicae . Informaanchicae –32. Auto Schwarz .A.naufraga and to estimate its future risk of extinction. More specifically, we aim to: (a) accurately define the geographical distribution of the species after exploring all potentially suitable habitats, (b) assess its population dynamics and reproductive biology, (c) estimate genetic diversity and potential gene flow within and among subpopulations, and (d) propose strategies and conservation measures for its management and maintenance.In this study, we combine demographic and genetic approaches to assess the current conservation status of The terms mature individual, population, subpopulation, population size, location, Extent of Occurrence (EOO) and Area of Occupancy (AOO) are used according to the definitions established by the International Union for the Conservation of Nature . EOO is A. naufraga  were surveyed for five consecutive years (2014–2018), in order to identify its distribution range. The exploration of the west coast cliffs of Zakynthos was aided by the use of binoculars. A GPS device was used for the capture of location data in the field. Detailed mapping, the polygon of the EOO for the total population, as well as the polygons of the local EOO for each subpopulation of the species were constructed using the ArcGIS 10.5.1 (ESRI) software package. AOO was estimated as the sum of the occupied grid cells 1 × 1 km.All known locations, as well as all suitable habitats for 2, which gives a rough estimation of plant density [For the estimation of population size , censuses of the mature individuals during the flowering/fruiting period in all subpopulations were carried out for five consecutive years. As an individual in this study we defined a cluster of shoots, in order to exclude the possibility of counting potential clones, although there is no evidence that the species can reproduce vegetatively. The number of plants per m density , was est density . Annual For the study of reproductive characteristics, i.e., fecundity   and RelaA. naufraga is a schizocarp, consisting of two mericarps, each one containing a single seed). In order to estimate the number of sound seeds per flower and per stem, 1–3 stems were collected from tagged individuals during each fruiting period and the seeds (seeds remain attached to the fruit during the first stages of its formation) were observed with a stereoscope (ZEISS Stemi 305). RRS was calculated by dividing the actual production of sound seeds to the potential maximum seed production. Seed rain was estimated according to Andreou et al. [A. naufraga reaches its first reproduction is 3 years.The number of flowering stems per individual, and the number of flowers per flowering stem and per individual were recorded during each flowering period. The number of mericarps per stem was recorded during each fruiting period  and the duration of flowering period was examined with stepwise multiple linear regression analysis, in order to investigate the impact of climatic variables on flowering period. Meteorological data were obtained from Hellenic National Meteorological Service. Statistical analyses were performed using Statistica 8.0 software (StatSoft Inc.). Moreover, to identify possible pollinators, all insect species that were associated with A. naufraga pollination were photographed and samples were collected for further identification to the lowest possible taxonomic level.The duration of flowering (and fruiting) period of Comparisons of data regarding reproductive biology were performed by One Way ANOVA. Differences among pairs of means were checked by Tukey’s Method using Statistica 8.0 software (StatSoft Inc.).t/Nt+1). The analysis was implemented with provision for the next 10 and 50 years, using RAMAS Ecolab v.2 software package [Population Viability Analysis (PVA) was carried out using the total number of mature individuals during the first year of monitoring (i.e. 2014) as initial abundance and the survival rate of mature individuals as survival rate, taking into consideration demographic stochasticity and without considering any density-dependent parameters. Population growth calculated as the interannual variation in the total number of mature individuals  in March 2018. All sampled individuals were located at least 5 m apart. The number of individuals sampled depended on the size and the accessibility of each subpopulation examined , 0.3 mM dNTP Mix (Kapa Biosystems), 0.3 pmol/μL forward primer, 0.3 pmol/μL reverse primer, 1 U HiFi HotStart DNA Polymerase (Kapa Biosystems). Reactions were carried out in a thermal cycler (Bio-Rad Laboratories Inc.) under the following conditions: an initial denaturation step at 95°C for 3 min., followed by 35cycles of 20 s denaturation at 98°C, annealing at 60–62°C  for 15 s, extension at 72°C for 30 s. The PCR program was completed by a final extension step at 72°C for 1 min. Negative controls without DNA were used to exclude (cross) contamination in the PCR reaction. The PCR products were separated by electrophoresis on a 3% agarose gel (BDH Laboratory Supplies) in 0.5 X TBE buffer, stained with Midori Green (Nippon Genetics) and visualized using Alpha Imager-Mini (Alpha Innotech). Molecular weights were estimated by comparison with a 20-bp DNA ladder (TaKaRa Bio Inc).After preliminary testing of five nuclear microsatellite loci already developed for the closely related folia L. , three wNa), number of effective alleles per locus (Ne), unbiased expected heterozygosity (uHe), observed heterozygosity (Ho), percentage of polymorphic loci (P) and Shannon’s index of diversity (I). GenAlEx was also used to perform Chi-Square Tests for Hardy-Weinberg Equilibrium for the subpopulations studied.The population genetic software GenAlEx ver. 6.502  was usedThe Polymorphism Information Content (PIC) for each SSR locus was calculated from allele frequencies , 45 withD) between subpopulations were estimated according to Nei [F) on a per locus basis and Wright’s F-statistics [FIS, FIT and FST were calculated using GenALEx. The estimation of gene flow (Nm), calculated as the number of migrants entering a subpopulation in each generation, was made according to Wright [FST estimation, we used the Excluding Null Alleles (ENA) method as implemented in the program FreeNA [Unbiased genetic distances , based on the hierarchical variance of gene frequencies. The test of significance for the AMOVA was carried out on 9,999 permutations of the data. Principal Coordinate Analysis (PCoA) based on Nei’s genetic distances and on the pairwise Ne)   by 0.4 and 0.1, representing a common range of Ne/Nc ratios as suggested by Garner et al. [Effective population size . Specifically, flowering period is significantly elongated by higher mean annual temperatures . Contrariwise, higher minimum temperatures , higher maximum temperatures  and increased precipitation (mm)  lead to shortened flowering period. In addition, the duration of the flowering period seems to have a positive effect on RRS .Flowering and fruiting periods of tuations . FloweriA. naufraga, during flowering period. Coptocephala rubicunda subsp. rubicunda  is considered to be polinophagous (feeding on pollen) [A. naufraga could be expected. The other species  belongs to a genus of blood-sucking insects, although it is possible that adults feeding on flower nectar may provide pollination services [Two different insect species were repeatedly recorded on the flowers of  pollen) , hence tservices .A. naufraga. Since the average number of amplified alleles per locus was four  ranged from 2.00 for An-S to 3.67 for An-Pl and An-PV, respectively. The number of effective alleles (Ne) per locus ranged from 1.71 (An-S) to 2.86 (An-PV). The Chi-Square Tests performed to test for Hardy-Weinberg Equilibrium, revealed that except for loci GA-50 and GA-1 in An-S and GA_30 in An-Pl, in all other cases the subpopulations deviated significantly from Hardy-Weinberg equilibrium.The number of alleles per locus and subpopulation ; subpopulation An-S, which is the smallest subpopulation of the species, showed the lowest value (uHe = 0.474) whereas the largest subpopulation (An-PV) exhibited the highest (uHe = 0.661).The mean observed heterozygosity within the subpopulations of A. naufraga was relatively low (Ho = 0.278). Subpopulation An-F exhibited the lowest value (Ho = 0.178), whereas subpopulation An-N the highest (Ho = 0.359)  of subpopulations was moderate (u= 0.359) .F) on a per locus basis, representing reduction in heterozygosity, ranged from 0.302 (An-Pl) to 0.706 (An-F) (average F = 0.449), indicating in most cases inbreeding or undetected null alleles. F-statistic also indicates high inbreeding level  as FIS measures the reduction in heterozygosity, due to non-random mating within each subpopulation, and high departure of genotype frequencies from Hardy–Weinberg expectations (shortage of heterozygotes) relative to the entire population  (I) varied from 0.615 to 1.148 for An-S and An-PV respectively (Nm) among the A. naufraga subpopulations was 1.282 individuals per generation.The Fixation Index ( 0.0001) . Shannonectively . MoreoveA. naufraga subpopulations. High pairwise genetic distances were observed between An-N and An-Pl (0.759), An-N and An-S (0.643), as well as between An-N and An-F (0.614). Low pairwise genetic distance was observed between An-Pl and An-F (0.124)  . Likewis alleles .FST value either calculated with (0.157) or without (0.164) the ENA correction for null alleles, indicates moderate genetic differentiation among subpopulations.The analysis of molecular variance (AMOVA) indicated higher within subpopulation variation which accounted for 79% of the total variation, while variation among subpopulations accounted for 21% of the total variation. The global A. naufraga. All An-N individuals with some from An-PV an An-F and one individual from An-Pl  are grouped into one basic cluster, while the remaining individuals are assigned to two different genetic clusters. The major mode  and loss of alleles [The estimated  alleles .A. naufraga using the total number of mature individuals was projected for the next 10 and 50 years  the development of recreational areas (threat code 1.3) and the recreational activities (threat code: 6.1) such as hiking, rock-climbing and the construction of secondary roads (threat code: 4.1) at location An-N; (b) gathering of plants (threat code: 5.2) mainly near the hiking trails at location An-N; (c) increase in frequency of human induced fires at locations An-N and An-PV (threat code: 7.1.1); (d) threats from catastrophic geological events, and more specifically from earthquakes (threat code: 10.2), which result in the destruction of the species and its habitat at locations An-Pl, An-F and An-PV; (e) ranching, i.e. domestic or semi-domesticated animals allowed to roam in the wild (threat code: 2.3.1) at location An-S and An-F.The direct threats recognized for A. naufraga is classified as Critically Endangered (CR) c refers to AOO, which is less than 10 km2, related to severely fragmented subpopulations, continuing decline observed, estimated, inferred or projected in: EOO, AOO, area-extent-quality of habitat, number of mature individuals, and extreme fluctuations in: EOO, AOO and number of mature individuals.Based on monitoring data, red (CR)  accordinA. naufraga, an emblematic endemic chasmophyte of Zakynthos island, were monitored for five years. The presence of five small and remote subpopulations along about 30 km in the western coastal cliff system of Zakynthos, indicates a long colonization and extinction history. Habitat traits, i.e. the rocky calcareous substrate and open sites with low vegetation cover, were similar in all subpopulations studied. The EOO and the AOO of A. naufraga were stable during the monitoring period. The local EOO, however, which expresses the true extent of the species in nature, was decreased in subpopulations An-N and An-F due to anthropogenic activities/pressures  and vegetation succession .Plant population monitoring programs are especially scarce because they are time- and resource- consuming, and are therefore restricted to a few threatened species . Thus, oA. naufraga markedly fluctuated over the monitoring period. These annual fluctuations were not only caused by local changes in the habitat or threats/pressures. This becomes clear if we consider that the fluctuation pattern was common to all subpopulations. According to our results, A. naufraga is a perennial species with a rather short life span. A strong correlation between life span of perennial herbs and population stability has been evidenced [A. naufraga. Interestingly, maximum fecundity coincided with minimum survival rate of mature individuals during the monitoring period. Moreover, small populations can exhibit strong annual fluctuations in their size due to stochastic forces  [A. naufraga may be related to climatic conditions (mean temperature and precipitation), assuming that similar environmental factors affect neighboring subpopulations, while the effect of precipitation and mean temperature on population dynamics of perennial herbs has been demonstrated in several studies [e.g. A. naufraga. Seedling establishment is especially difficult in vertical cliffs; survival of existing individuals is far more important than the establishment of new plants [The few existing studies on population dynamics of chasmophytes suggest unusually stable population sizes and high local population persistence , 61–63. videnced , while tvidenced , 65, whisticity) . Environsticity) . The fluies e.g. , 69. Cerw plants , while hw plants , 64.Highw plants .A. naufraga fluctuates in response to variation in climatic factors , while there is a strong positive correlation between the duration of flowering period and RRS. Extended flowering is likely to assure plant reproduction. Individual plants flowering for long periods have several advantages related to higher reproductive success, higher outcrossing rates and more time for seed maturation [A. naufraga, the elongated flowering period leads to the production of a larger mean number of seeds, which ultimately leads to higher reproductive success.Flowering phenology is a critical life-history trait that strongly influences reproductive success . The durturation . For insturation . In the Asperula naufraga according to IUCN categories and criteria [A. naufraga should move from the “Endangered” (EN) to the “Critically Endangered” (CR) category. PVA results indicate that three of the five subpopulations of the species, namely An-PV, An-F and An-S, might go extinct within the next 50 years, while the extinction risk for the whole population is 47.8%.Monitoring data collected during this study were used to re-evaluate the conservation status of criteria , using Rcriteria . AccordiA. naufraga. The number of alleles revealed for the SSR loci studied, was sufficient to acquire a first rough estimation of genetic variation, characterize the subpopulations, and describe population structure.In this study, three microsatellite loci were used in a preliminary attempt to gather genetic information about the extremely rare and threatened A. naufraga, the Shannon’s information index (I) was low, following the same trend as Ho and uHe. Generally, Ho values are lower than uHe values, which may indicate non-random mating [Ho computed on the SSR loci studied, was lower than expected for endemic plant populations. On the other hand, uHe values of A. naufraga are similar to uHe values of short lived perennial plants as reported by Nybom [Regarding the diversity parameters within m mating . Howeverby Nybom .Ho among all subpopulations was similar to that reported by Gargiulo et al. [Asperula crassifolia populations, although in our case uHe is higher. Genetic diversity maintained within a species is considered to be a result of both historical events and recent evolutionary processes. Low heterozygosity estimates may be caused by the effect of small population size, genetic drift, high selection pressure in closed population, inbreeding, and restricted gene flow, which eventually contribute to a reduction in genetic diversity [The mean o et al. , 32 for iversity , 76.F) for the loci studied indicate a high number of homozygotes. The same also stands for FIS within subpopulations of A. naufraga and FIT, which accounts for the entire population. Positive FIS values may also be recorded in outcrossing populations under certain circumstances. If we exclude the presence of clonal individuals, the causes of such values of F indices may be the occurrence of null alleles, non-random mating , allele dropout, selection and the restricted gene flow (Nm = 1.282), especially for the An-N subpopulation. Although we have not specifically tested our data for the occurrence of a recent bottleneck, the low genetic diversity and the relatively high fixation index may suggest that subpopulations of A. naufraga have suffered from a reduction in their size. In AMOVA, the mean FST value is significantly lower than the values reported for narrow/endemic plant populations by Nybom [FST calculated in our study indicates a moderate genetic differentiation, significantly lower than that observed (mean FST = 0.293) between the three populations of A. crassifolia [A. crassifolia populations compared to those of Α. naufraga.The positive values of fixation index  indicating extensive gene flow, which is also in accordance with the low value for Nei’s D (0.03) between these two subpopulations, as well as their geographical proximity. Interestingly, the individuals from these two subpopulations belong to the same genetic cluster as deduced by the STRUCTURE analysis and they are positioned next to each other in PCoA. There is also increased gene flow between subpopulations An-Pl and An-PV and An-F and An-PV . In all other cases, gene flow ranged between 0.893 and 1.686. According to Couvet [The mean value of o Couvet  one migro Couvet . NotablyNe, which is in line with the results of the Conservation Status Assessment and should be considered as additional information to assessments as suggested by Garner et al. [Census of the population size facilitate the estimation of r et al. .A. naufraga were unknown, the results of the present study provided relevant new knowledge, crucial for species conservation. Geographical distribution and population size of A. naufraga are constrained by the availability of suitable habitat and recruitment limitations. Our results indicate that stable population size is not a common feature shared by all perennial obligate chasmophytes. Cliff systems are usually stable habitats, but cliff plant populations may be subject to remarkable fluctuations even in short time scales, as indicated by our monitoring results. The population of A. naufraga proved to be dominated by adult and senescent individuals, suggesting low recruitment. Survival ability of mature individuals counterbalance recruitment limitations and enhances the survival of the species in harsh environments like cliffs [As (census) population size, reproductive aspects and genetic structure of e cliffs , 79, 80,e cliffs , which cNe. A pragmatic in situ conservation approach for A. naufraga should give priority to minimize/eliminate the impacts of the threats it faces. In particular, according to field observations and the results of PVA and genetic analyses, the subpopulations at the locations Faros Keriou, Sxiza and Navagio should be prioritized for conservation measures. The ownership status at the locations of subpopulations An-F, An-S and An-N constitutes a challenge for their conservation. Subpopulations An-F and An-S are partly located in areas that are privately owned, and are also used for animal keeping. Grazing control in order to reduce trampling pressure from herbivores in subpopulations An-F and An-S is required. In addition, informing the local authorities and the community is deemed necessary at the location Navagio, a famous recreational area where accidental trampling and gathering of plants have been observed. Moreover, ex situ conservation of the species in seed banks can offer insurance against extinction by providing a source population for future reintroduction or reinforcement of wild population. Seed banking is a necessary and cost-effective complement to in situ conservation of wild plants, and it provides a vital source of material to assist in ecological restoration of damaged and degraded habitats [Population data indicates an increased extinction risk due to the small population size combined with large fluctuations, low recruitment, low genetic diversity and inbreeding. The same is also evidenced by the estimated habitats . GeneticS1 Fig(TIF)Click here for additional data file.S2 FigA. naufraga are shown.A) Subpopulation An-PV, B) subpopulation An-S, C) subpopulation An-F. The average (line), ±1 standard deviation and minimum and maximum (dots) numbers of the subpopulations of (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Plasmodium vivax CSP (PvCSP) have demonstrated their effectiveness in preventing malaria, at least in part. However, genetic diversity of pvcsp in the natural population remains a major concern.Circumsporozoite surface protein (CSP) of malaria parasites has been recognized as one of the leading vaccine candidates. Clinical trials of vaccines for vivax malaria incorporating Plasmodium vivax in Myanmar were analysed in this study. The pvcsp was amplified by polymerase chain reaction, followed by cloning and sequencing. Polymorphic characteristics and natural selection of pvcsp population in Myanmar were analysed using DNASTAR, MEGA6 and DnaSP programs. The polymorphic pattern and natural selection of publicly accessible global pvcsp sequences were also comparatively analysed.A total of 171 blood samples collected from patients infected with pvcsp sequences were divided into two subtypes VK210 and VK247 comprising 143 and 28 sequences, respectively. The VK210 subtypes showed higher levels of genetic diversity and polymorphism than the VK247 subtypes. The N-terminal non-repeat region of pvcsp displayed limited genetic variations in the global population. Different patterns of octapeptide insertion (ANKKAEDA in VK210 and ANKKAGDA in VK247) and tetrapeptide repeat motif (GGNA) were identified in the C-terminal region of global pvcsp population. Meanwhile, the central repeat region (CRR) of Myanmar and global pvcsp, both in VK210 and VK247 variants, was highly polymorphic. The high level of genetic diversity in the CRR has been attributed to the different numbers, types and combinations of peptide repeat motifs (PRMs). Interestingly, 27 and 5 novel PRMs were found in Myanmar VK210 and VK247 variants, respectively.Myanmar pvcsp population suggests a complex genetic profile of pvcsp in the global population. These results widen understanding of the genetic make-up of pvcsp in the global P. vivax population and provide valuable information for the development of a vaccine based on PvCSP.Comparative analysis of the global  Plasmodium species has threatened human health since ancient time [Plasmodium vivax is the most prevalent species outside of Africa. More than 7.5 million cases of malaria were caused by the parasite, which accounted for 56% of total malaria cases in South East Asia [Plasmodium vivax has been linked to benign malarial infection due to its mild clinical manifestations compared with Plasmodium falciparum; however, concerns that P. vivax also causes serious clinical illnesses and even death have increased [P. vivax, known as hypnozoites, can remain dormant and survive for weeks to months in liver cells, and be reactivated [Malaria caused by ent time . The morent time . Among fast Asia . Plasmodncreased , 4. The P. vivax CSP (PvCSP) consists of peptide repeat motifs (PRMs). Based on the composition of one of the three major PRMs in the CRR, pvcsp is classified into three allelic variants: VK210, VK247 and P. vivax-like. The two most abundant alleles include VK210 and VK247, which carry nonapeptide repeat motifs GDRA(D/A)GQPA and ANGAG(N/D)QPG, respectively [P. vivax-like variant contains APGANQ(E/G)GAA motifs in the CRR [Circumsporozoite protein (CSP) is the most abundantly expressed protein on the surface of sporozoites. CSP plays multiple and crucial roles in the development, migration and invasion of sporozoites into hepatocytes . Thus, tectively . Meanwhipvcsp, including VK210 and VK247 [pvcsp population may influence the effectiveness of PvCSP-based vaccine, which may be a barrier for the development of a universal vaccine. Therefore, a comprehensive analysis of genetic diversity and structure of the global pvcsp population is necessary. In this study, genetic polymorphism and natural selection of Myanmar pvcsp has been examined. A comparative analysis of global pvcsp was also conducted to obtain a deeper insight into the genetic nature of pvcsp in the global P. vivax population.Although no licensed vaccine against vivax malaria is currently available, a few notable advances in the development of a potential vaccine have been reported. A vivax malaria subunit vaccine named VMP001 has been clinically tested. This vaccine uses a chimeric recombinant protein containing repeat sequences of two major alleles of nd VK247 . The phand VK247 . HoweverP. vivax-infected Myanmar patients were used in this study. The blood samples were collected from the patients in field surveys in towns and villages in Naung Cho, Pyin Oo Lwin, and Tha Beik Kyin in Upper Myanmar during 2013 to 2015  for confirmation by species-specific polymerase chain reaction (PCR) targeting 18S ribosomal RNA (rRNA) gene [One hundred seventy-one blood samples from NA) gene . Informepvcsp gene was performed with primers by using nested PCR. The primers for first round PCR were 5ʹ-ATGTAGATCTGTCCAAGGCCATAAA-3ʹ and 5ʹ-AATTGAATAATGCTAGGACTAACAATATG-3ʹ. The thermal cycling parameters for primary PCR were as follows: one cycle of initial denaturation at 95 °C for 5 min, 25 cycles of 94 °C for 1 min, annealing at 58 °C for 2 min and extension at 72 °C for 2 min, followed by a final extension at 72 °C for 5 min. The nested PCR was performed with primers, 5ʹ-GCAGAACCAAAAAATCCACGTGAAAATAAG-3ʹ and 5ʹ-CCAACGGTAGCTCTAACTTTATCTAGGTAT-3ʹ, and similar amplification condition except the annealing temperature was 68 °C. Ex Taq DNA polymerase  with proof-reading activity was used in all PCR steps to minimize the nucleotide mismatching during the amplification. Each PCR product was analysed by electrophoresis on 2% agarose gel. The resulting PCR product was extracted from the gel and was cloned into T&A cloning vector . Each ligation mixture was transformed into Escherichia coli DH5α competent cells. To identify positive clones with appropriate insert, colony PCR with nested PCR primers was performed. The nucleotide sequences of the cloned pvcsp were analysed by the Sanger method with M13 forward and reverse primers. Plasmids from at least two independent clones from each transformation mixture were analysed to confirm the sequence accuracy. These nucleotide sequences analysed in this study were deposited at GenBank under the accession numbers MN821829–MN821999.Genomic DNA was extracted from dried blood spots using QIAamp DNA Blood Kit  following the manufacturer’s protocol. Amplification of pvcsp were analysed using Editseq and Seqman in the DNASTAR package . Two major variants of pvcsp sequences, Salvador I  and Papua New Guinea , were used as reference sequences to analyse Myanmar pvcsp sequences. The values of number of segregating sites (S), haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π), and average number of pair-wise nucleotide differences within a population (K) were calculated with DnaSP ver. 5.10.00 [P < 0.05) with MEGA6 program [The nucleotide and deduced amino acid sequences of Myanmar  5.10.00 . The rat program  using Ne program  with the program . Tajima’ program  to evalu program , 20. A npvcsp among the global P. vivax population was also analysed. The pvcsp sequences deposited in public database were used in this study; Cambodia (n = 41), India (n = 79), Iran (n = 50), South Korea (n = 39), Brazil (n = 41), Mexico (n = 19), Colombia (n = 25), Sudan (n = 30), and Vanuatu (n = 21) .The genetic diversity of mbia n = 5, Sudan  5.10.00  and MEGA 5.10.00  as descrpvcsp genes were successfully amplified form the genomic DNA samples used in this study. The size of the amplified pvcsp genes ranged from 0.5 to 1.3 kb. Sequence analysis of the amplified pvcsp genes revealed only two variants of pvcsp, VK210 and VK247, in Myanmar pvcsp, but not the P. vivax-like variant. The VK210 variants were prevalent  and the frequency of VK247 variants was 16.4% (n = 28). No mixed infection with the two different variants was detected.A total of 171 Myanmar pvcsp showed a limited range of genetic diversity. Alignment of the deduced amino acid sequences of Myanmar pvcsp revealed 7 distinct haplotypes of VK210 variants and 7 haplotypes of VK247 variants . An alanine insertion at the end of the RI conserved motif (KLKQP) was identified in haplotypes 2, 3 and 4. The N86I was found in haplotype 3. Two dimorphic (K91R and P95S) and one trimorphic (K93E/N) amino acid changes were observed in the RI motif of haplotypes 4, 5, 6, and 7  sequence, was the most prevalent, accounting for 57.1% of 28 Myanmar VK247 variants. All amino acid changes identified in Myanmar VK247 variants were all dimorphic . Furthermore, eight amino acids (97DGAGNQPG104) were not detected in haplotypes 6 and 7 , Vanuatu (47.6%), Myanmar (21.7%), Brazil (9.8%), and India (7.6%). No alanine insertion was found in the VK210 sequences identified in Iran, South Korea, and Mexico. Indian VK210 variants showed the highest genetic diversity with amino acid substitutions at 10 positions including E83K/G, K85T/Q, N86K/Y/I, P87A, R88G, N90I, L92V, K93N, Q94H/P and P95G, even though their frequencies were low, less than 15%. Meanwhile, A82T, N86I, N86S, K91R, P95S, and K93E/N showed uneven geographic distribution in the global VK210 variants with very low frequencies. The N-terminal non-repeat region of global VK247 variants were also well-conserved, although low frequencies of uneven amino acid changes were identified  variations in length of polymorphisms in CRR; however, the CRR of Myanmar VK210 variants had 24 different length polymorphisms consistent with various types and different compositions of PRMs. The overall genetic diversity in CRR of Myanmar VK247 variants was much lower than that of Myanmar VK210 variants. A total of 23 VK247 haplotypes, each CRR comprising different numbers and combinations of 8 types of PRMs, were identified Fig. . Five ofpvcsp suggested a high level of genetic diversity. Among VK210 variants, the frequency of ANKKAEDA insertion differed by country , overall haplotype diversity (Hd), and nucleotide diversity (π) were 0.098, 0.096 ± 0.034, and 0.0024 ± 0.0009, respectively (Table K), the overall haplotype diversity (Hd), and the nucleotide diversity (π) for the C-terminal non-repeat region of Myanmar VK210 variants were 0.209, 0.186 ± 0.044, and 0.0035 ± 0.0009, respectively (Table P < 0.05) and –2.2251 (P < 0.01), respectively (Table K), overall haplotype diversity (Hd) and nucleotide diversity (π) of the N-terminal non-repeat region were 0.405, 0.405 ± 0.094 and 0.0090 ± 0.0021, respectively (Table P > 0.1) and –1.9719 (P < 0.05) for the N- and C-terminal non-repeat regions, respectively  and positive Tajima’s D (0.9518), Fu and Li’s D (1.1271), and Fu and Li’s F (1.2185). However, Myanmar and Cambodia VK247 variants revealed positive values for dN–dS and negative values for Tajima’s D, Fu and Li’s D, and Fu and Li’s F. The C-terminal non-repeat region of VK267 variants from Cambodia and Colombia also showed nucleotide diversity, which was lower than that of Myanmar VK247 variants. Similar to Myanmar VK247 variants, the C-terminal non-repeat region of Cambodia VK247 variants showed a negative values of dN–dS (–0.0172), Tajima’s D (–1.4009), Fu and Li’s D (–1.5866), and Fu and Li’s F (–1.7190). However, the dN–dS of Colombia variants was estimated to be positive (0.0017), although the values of Tajima’s D, Fu and Li’s D, and Fu and Li’s F were negative.The nucleotide diversity and natural selection in the global VK247 variants were analysed Table . AnalysiP. vivax [pvcsp population on vaccine efficacy remains unclear. In this study, genetic polymorphism and natural selection in Myanmar pvcsp and global pvcsp populations were comprehensively analysed. Among the three pvcsp allelic variants, only two allelic variants, VK210 and VK247, were identified in P. vivax isolates from Myanmar analysed in this study. The VK210 was the dominant allele occurring in 83.6% of Myanmar pvcsp population, which is consistent with previous studies reporting that VK210 is the most common allelic variant in the pvcsp populations from Iran, India, China, Brazil, Thailand, Bangladesh, and Azerbaijan [Anopheles mosquito species may affect the prevalence of VK210 and VK247 variants in endemic areas [Anopheles are distributed throughout the country, and at least 10 species including Anopheles dirus, Anopheles minimus, and Anopheles aconitus may transmit malaria [Anopheles species is not clear; however, the differences in adaptability of each allelic variant to mosquito vectors affects the prevalence of VK210 in Myanmar. The predominance of VK210 may also be associated with its genetic diversity. VK247 showed a lower level of genetic diversity than VK210 in the Myanmar pvcsp population, suggesting different polymorphisms may increase the adaptability of VK210 to different mosquito vectors.PvCSP is one of the leading candidates for vivax malaria vaccine. Several recent PvCSP-derived vaccines were designed as multivalent formulations or chimeric molecules in an attempt to induce protective immunity against P. vivax , 21, 22.erbaijan –31. Meanerbaijan . It has ic areas , 33. In  malaria . Differepvcsp was relatively well-conserved. Only a few amino acid changes were identified in the latter portion of the N-terminal non-repeat region of Myanmar pvcsp. The RI motif (KLKQP), a cell adhesive motif exposed by proteolytic cleavage after the interaction between sporozoite and hepatocyte [pvcsp, although the prevalence of the insertion differed by geographically. Amino acid changes were also identified in global VK210 variants, but they showed uneven geographic distribution with low frequencies. The N-terminal non-repeat region of global VK247 variants was also relatively well-conserved, although uneven and minor amino acid changes were also identified. It has been shown that the invasion of malaria parasite is inhibited by antibodies directed against RI motif [pvcsp suggest that this region including RI motif may represent an attractive candidate for formulation of a PvCSP-based vaccine.The N-terminal non-repeat region of Myanmar patocyte , was welRI motif . The higpvcsp. Both VK210 and VK247 variants showed great genetic diversity in CRR resulting in 118 and 23 different haplotypes for VK210 and VK247, respectively. Insertions and deletions of nonapeptide sequences probably resulted from either sexual recombination during meiosis or intrahelical strand slippage during mitotic DNA replication [pvcsp. Interestingly, 47 different types of PRMs including 27 novel types were identified in the CRR of Myanmar VK210 variants. In the case of VK247 variants, a total of 8 distinct PRMs were identified, in which 5 were novel ones. These findings also suggest point mutations as one of the major factors increasing the genetic complexity of Myanmar pvcsp. Similar patterns of high genetic polymorphisms were also identified in the CRR of global pvcsp population; however, the overall diversity due to different types and repeats of PRMs was greater in the pvcsp from Myanmar compared with other countries. Especially, the repeated numbers of PRMs varied widely in both Myanmar VK210 and VK247 variants compared with those from other countries.As expected, high level of genetic diversity was identified in the CRR of Myanmar pvcsp also showed genetic polymorphisms. Differences in ANKKAEDA octapeptide insertion and GGNA tetrapeptide repeat motifs and amino acid substitutions throughout the region contributed to genetic diversity in Myanmar and global VK210 variants. Despite the unclear biological functions of ANKKAEDA octapeptide and GGNA tetrapeptide in VK210 variants, they may also be generated via intrahelical recombination, and parasites carrying the ANKKAEDA octapeptide show a high degree of delayed infections [pvcsp, both in VK210 and VK247 variants, were differed by country, no clear geographic clustering was identified. Considering the limited genetic information of the C-terminal region in global pvcsp in current, a further study employing larger numbers of pvcsp sequences in diverse geographical origins is necessary to understand the genetic nature and evolutionary aspects of the C-terminal region in global pvcsp population.The C-terminal regions of Myanmar and global fections . Similarpvcsp suggests that these regions are likely to be under natural selection, which may maintain or generate genetic diversity in the global pvcsp population. The dN–dS values for Myanmar and Cambodia VK210 and VK247 variants were positive, implying that balancing selection might act in these regions. However, the values were 0 or negative for both VK210 and VK247 variants from other countries except the N-terminal region of South Korea VK210 and the C-terminal region of Colombia VK247. Tajima’s D and Fu and Li’s D and F values also revealed complex patterns of natural selection that were unique to pvcsp from each country. While pvcsp populations from a few countries showed no genetic diversity involving the N- and C-terminal regions, a few pvcsp populations showed negative Tajima’s D values, suggesting purifying natural selection. The overall trend indicated that the global pvcsp population decreased the genetic diversity in the two terminal non-repeat regions. Due to size polymorphisms induced by different numbers and arrangements of PRMs in CRR, a direct analysis of natural selection in CRR was not possible in this study. However, evidence supporting natural selection in the CRR have been reported [pvcsp.Analysis of the two non-repeat regions in global reported –38. Thespvcsp were greater than those reported from other countries analysed. Similar patterns of high genetic diversity in several polymorphic genetic markers including apical membrane antigen-1, merozoite surface protein-1 and -2, and CSP of Myanmar P. falciparum population have also been reported [P. vivax isolates analysed in this study were collected in restricted areas of Myanmar and, therefore, not fully representative of the nation-wide genetic diversity and population structure of Myanmar pvcsp. A further comprehensive study involving a larger number of P. vivax isolates collected from different regions in Myanmar is necessary.Despite the remarkable reduction in malaria transmission in Myanmar during the last decades , 38, thereported –42. Desppvcsp population on the efficacy of PvCSP-based vaccine remains unclear. This study highlights an additional level of complexity in the application of this antigen for the development of a vivax vaccine. Global pvcsp population revealed polymorphic characters, especially in the CRR. The size polymorphisms and the appearance of novel PRMs as well as new CRR arrays primarily contribute to the genetic diversity of global pvcsp. Natural selection may also be a major force affecting the genetic diversity of global pvcsp. The results of this study provide not only an insight into the genetic nature of global pvcsp but also valuable information for the development of a universal vaccine based on PvCSP. Continuous monitoring of genetic diversity of global pvcsp population is necessary to elucidate the polymorphic nature and evolutionary aspect of pvcsp in global P. vivax population.PvCSP is a leading candidate for vivax vaccine; however, the impact of natural genetic variations in the global Additional file 1: Figure S1. Schematic structure of pvcsp. The gene is separated into three regions; an N-terminal non-repeat region, a central repeat region (CRR), and a C-terminal non-repeat region. The CRR consists of two major repeat peptide motifs (PRMs), termed VK210 and VK247.Additional file 2: Table S1. Global pvcsp sequences analysed in this study.


Plasmodium species. Knowledge about their genetic diversity can help predict vaccine efficacy and the spread of novel parasite variants. Thus, we investigated pvcsp gene polymorphisms in 219 isolates . Forty-eight polymorphic sites were detected, 46 in the central repeat region (CR), and two in the C-terminal region. Also, the CR presents InDels and a variable number of repeats. All samples correspond to the VK210 variant, and 24 VK210 subtypes based on CR. Nucleotide diversity (π = 0.0135) generated a significant number of haplotypes (168) with low genetic differentiation between the Brazilian regions (Fst = 0.208). The haplotype network revealed similar distances among the BA and AF regions. The linkage disequilibrium indicates that recombination does not seem to be acting in diversity, reinforcing natural selection’s role in accelerating adaptive evolution. The high diversity (low Fst) and polymorphism frequencies could be indicators of balancing selection. Although malaria in BA and AF have distinct vector species and different host immune pressures, consistent genetic signature was found in two regions. The immunodominant B-cell epitope mapped in the CR varies from seven to 19 repeats. The CR T-cell epitope is conserved only in 39 samples. Concerning to C-terminal region, the Th2R epitope presented nonsynonymous SNP only in 6% of Brazilian samples, and the Th3R epitope remained conserved in all studied regions. We conclude that, although the uneven distribution of alleles may jeopardize the deployment of vaccines directed to a specific variable locus, a unique vaccine formulation could protect populations in all Brazilian regions.Circumsporozoite protein (CSP) is the primary pre-erythrocytic vaccine target in  Plasmodium falciparum is responsible for most cases and deaths from malaria, P. vivax is by far the most widespread, accounting for almost half of non-African malaria cases  in 201 (92%) samples. The motif 20 disclosure four nsSNPs [G(D/N)(R/G/S)A(A/G)GQ(A/P)A] in 177 (81%) samples (Considering now the three TCD4and P25) , 49, onl samples .PNEKSVKEYL(D/N)KVRATVG] [In the Th2R epitope [KVRATVG] , locatedpvcsp is a single copy gene located in chromosome 8 of P. vivax. It is highly expressed on sporozoites’ surface, playing an essential role in adhesion and invasion of hepatocytes [pvcsp genetic diversity in Brazil is limited since the majority of pvcsp studies had been focused on the immunological response triggered by CSP epitopes [pvcsp variants associated with the response to treatment, or even in the detection of such variants in Anopheles mosquitoes [pvcsp gene in two different epidemiological sites, the Brazilian Amazon rainforest (BA) and the Rio de Janeiro Atlantic Forest (AF).atocytes . Due to atocytes . The knoepitopes , 52 or tsquitoes , 34. Nowpvcsp’s gene variant VK210 was detected in both BA and AF; consequently, no association with the presence of VK210 and the geographic origin of the parasites could be made. Although other two variants—VK247 and vivax-like—have already been detected in P. vivax parasites from the Brazilian Amazon [Only n Amazon , 28, 53 n Amazon , the cosn Amazon , Nicaragn Amazon , Pakistan Amazon , Sri Lann Amazon , Myanmarn Amazon , Cambodin Amazon , Iran [5n Amazon , Vanuatun Amazon , and Chin Amazon .pvcsp variants differ according to transmission intensity [An. Darlingi and An. albitarsis, while in AF malaria transmission occurs almost exclusively by An. (Kerteszia) cruzii vector [P. vivax parasite variant from the immune system.It has been claimed that the presence and extension of ntensity . Howeverntensity , Hondurantensity , and Chintensity , do not i vector . AltogetAnopheles mosquito salivary gland and human hepatocytes during the invasion processes [pvdbp gene [Sequence analyses revealed high CR diversity, contrasting to none or low diversity in the N- and C- terminals (5’ and 3’ ends), respectively. Indeed, the pre-central (Region I) and the post-central (Region II) repeat sequences that contain both B- and T-cell epitopes  remainedrocesses . Conversdbp gene , 68.P. falciparum parasites [P. vivax sequences contain one or more copies of two nonapeptide motifs displaying three synonymous substitutions . The persistence of these two common allotypes suggests its maintenance by selective pressure. These silent polymorphisms could protect the parasites against stochastic reductions in variability, such as those resulting from bottlenecks. On the other hand, the allotype 3, considered a T-helper cell epitope, revealed an “excess” of nonsynonymous substitutions in the great majority of the samples. The low Sal-1 allotype 3 frequency has already been reported in China [P. vivax parasites’ survival, but they might help to evade the host immune response.A large number of VK210’s substitutions and length polymorphisms have been found worldwide , 69. Howarasites . In thisin China , Mexico in China , and alsin China , 28, 71.GNGAGGQAA), also present in Belem 1 strain (GenBank: EU401923), a Brazilian reference strain, was detected in high frequency (71%) mostly in BA isolates. Other investigations in Korea [GNRAAGQPA)–was already reported in Sri Lanka [GNSAGGQPA), only detected in French Guyana, suggested that this allotype is not circulating in Brazil. Unfortunately, the number of samples from Amapá state, which borders French Guiana, was too small to confirm the lack of allotype 13 in Brazil.The allotype 10 , which is a significant factor leading to global isolates’ genetic in China , Cambodiin China , and evein China . InteresRemarkably, samples with deletion showed the most conserved DNA sequence, and, inversely, those with insertion were more polymorphic. Therefore, most allotypes had been arising in the final CR portion (motifs 19 and 20), suggesting more significant selective pressure on these motifs, most likely mediated by the host immune system.P. vivax populations with great genomic diversity in several low endemic regions across the world [As the sexual recombination during meiosis in the mosquito, CR diversity could be raised by intrahelical strand-slippage events during mitotic DNA replication , 70, inche world , 36, 73,P. vivax could also affect the pvcsp population structure. Despite the considerable distance between the Brazilian Amazon and Atlantic Forest, the low mobility of infected people between these areas, and the smaller parasite population’s size in AF that generally could reduce gene flow between BA and AF, identical polymorphisms were found in parasite populations of these regions.The genetic diversity in South America has been attributed to different parasite lineages originating from geographically diverse regions , 36, 73,P. vivax cases, increasing the chances of recombination; and iii) BA parasites showed more variation in SNP numbers than AF.The low genetic differentiation between AF and BA populations was also verified with a haplotype network, in which sequences of both regions are interlinked and have similar distances between their haplotypes. AF isolates were formed more clusters than the BA ones. Thus, the isolates of the same geographic region may share similar evolutionary histories. The extensive haplotype distance in BA parasite populations could be a result of i) BA geographic area is much larger than the AF; ii) BA area concentrates approximately 99% of Brazilian Although AF and BA have distinct epidemiological profiles, including different vector species, transmission rates, and hosts with different immune pressure, migration rates, and regional-temporal fluctuations, we did not detect any fixed nucleotide differences in isolates from these geographic areas. Remarkably, AF autochthonous cases, indicative of zoonotic transmission to humans , 10, wheP. vivax populations harbored high genetic diversity and low level of genetic differentiation between AF and BA parasite populations. These findings were already observed in parasite populations from Colombian geographically distinct regions [Contrary to the expectations, all sampled  regions .pvcsp sequence suggest that recombination has not played a crucial role in shuffling the nucleotide variations. Alternatively, the central region of CSP may contain a mutational “hot spot” that could mask the presence of recombination in the pvcsp [The low values of linkage disequilibrium across the he pvcsp .On the other hand, insertion positions of a small block at the end of the central region with high LD in AF samples could suggest a recent bottleneck and/or clonal expansions, as expected in places with a reduction in transmission rates.pvcsp gene, Tajima’s D neutrality test was performed, and the results suggest that diversity was mediated by balancing selection, probably by the presence of multiple B- and T- cell immunodominant epitopes [To better understand if selection acts upon the epitopes , 48, 49.P. vivax peptides are located in the C-terminal region and they are orthologous to those of P. falciparum CSP [P. ovale [P. malariae [P. knowlesi [P. falciparum [It is well known that a single amino acid change or clustered replacements in B- or T- cell epitopes may potentially reduce the antibody response and the peptide-binding pocket of HLA, respectively. Here, we investigated eleven epitopes previously described to trigger the host immune response , 48, 49.arum CSP . The Th2P. ovale , but difmalariae , P. knowknowlesi  and P. flciparum  parasitepvcsp-gene in isolates from different Brazilian endemic areas, showing high CR diversity and low geographic pvcsp population’s structure, probably modulated by natural selection and host immunity. Although the uneven distribution of allelic genes may jeopardize vaccines’ deployment directed to a specific variable locus, a unique vaccine formulation could protect human populations in all Brazilian regions.To summarize, this study provides information on the genetic polymorphisms of S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Acipenser ruthenus to ensure that the two were distinguishable. We then investigated the fitness-related traits and genetic diversity of the purebred fish and their hybrid crosses. The hybrids grew faster and exhibited a higher genetic diversity than purebred fish, suggesting their superior fitness for commercial aquaculture. We did not observe adverse effects of the population cross, and an investigation of future generations may provide insight into the consequences of using non-native stock in conservation programs.Sturgeon are an ancient fish family widely distributed in the northern hemisphere, and many are listed as critically endangered, facing risk of extinction in the wild due to habitat destruction and overfishing to supply caviar. As well as breeding for commercial aquaculture, conservation programs use stock derived from other areas along with local native restocking projects. The fitness of introduced non-native sturgeon and their progeny from crossing with native stock is unknown. To assess the consequences of such interbreeding populations that have adapted to local conditions, we first analyzed the genetic makeup of hatchery-reared Danube and Volga populations of sterlet Hatchery-reared sterlet originating from the Danube and Volga river basins that showed population-discriminatory alleles on at least one microsatellite locus were used to produce purebred (within-population) and hybrid crosses to evaluate intraspecific hybridization with respect to the genetic polymorphism and physiological fitness of fish for commercial aquaculture and, conservation programs. Reciprocal crossing assessed the effect of parent position. The fish were reared in indoor and outdoor tanks and monitored over 504 days for growth traits. The highest final mean body weight (144.9 ± 59.5 g) was recorded in the Danube (♀) × Volga (♂) hybrid and the highest survival in the Volga (♀) × Danube (♂) hybrid. The Volga purebred exhibited the lowest mean body weight (124.8 ± 57.6 g). A set of six microsatellites was used to evaluate the heterozygosity. The mean number of alleles was highest in the Danube (♀) × Volga (♂) hybrid and lowest in the Volga purebred, suggesting an influence of the parent position in the hybridization matrix. The higher level of genetic polymorphism, as in the Danube (♀) × Volga (♂) hybrid, may confer greater fitness in a novel environment. Our analysis revealed that the intraspecific hybrids performed better than the purebred fish in the controlled and suboptimal rearing conditions. A. baerii, A. gueldenstaedtii, and the hybrid H. dauricus × A. schrenckii  × 100, in which F1 = the value of hybrid, MP = the mean value of the purebred crosses.The fish were measured to calculate the mean weight and cumulative survival at 77, 175, 229, 325, 386, and 504 dph. The specific growth rate  from fin clips for the population divergence study and from swim-up larvae for the assessment of heterozygosity in groups. Six microsatellite markers, AciG 35 [A), pairwise GST, DA matrix, gene diversity at each locus, fixation index, and expected (He) and observed (Ho) heterozygosity of the Danube and Volga populations were calculated using GeneAlex [A) and expected (He) and observed (Ho) heterozygosity used to assess the level of polymorphism in the analyzed progeny fish groups were calculated using GeneAlex [Whole genomic DNA was extracted using the Nucleo Spin AciG 35 , AfuG 13 AciG 35 , Aox 45  AciG 35  were use AciG 35 . A microGeneAlex . LikewisGeneAlex . The visp = 0.05, and Tukey’s post-hoc (parametric data), Kruskal–Wallis, and Dunn’s post-hoc tests were used for the non-parametric data fertilization rate, the hatching rate, the mean BW, the growth heterogeneity, and the specific growth rate. The differences in survival between the three tank replicates were evaluated using a Pearson’s Chi-square test at a significance of p = 0.05. The significance of the differences between the individual groups was tested by an ANOVA with a significance at p = 0.05. The significance of the differences in NA, Ho, and He between the Danube and Volga populations and between the hybrids and purebreds was tested with a one-way ANOVA at a significance of p = 0.05.A statistical analysis was conducted using Statistica 13 . The data were analyzed for normal distribution using the Kolmogorov–Smirnov test. Multiple comparisons were carried out by a one-way analysis of variance (ANOVA) at a significance of ST and DA matrix values of the Danube and Volga populations were 0.136 and 0.549, respectively. These values indicate a moderate genetic differentiation between the Danube and Volga stock.The highest allele diversity was found in the Danube population for locus Aox 45 (0.882) . The allThe fixation index values of the Danube and Volga populations are shown in The highest fertilization rate (81.73 ± 2.16) was recorded in the D × V hybrid, and the highest hatching rate was in the Danube purebred (75.85 ± 1.89) . The lowThe observed mean BW and cumulative survival at different sampling points are shown in The SGR was significantly higher in the D × V hybrid (3.10 ± 0.02) and significantly lower in the V × D hybrid (1.77 ± 0.1) from 58–175 dph, compared to other groups. There were no significant between-group differences in the SGR at later sampling points. Growth heterogeneity differed significantly among the groups from 58 to 175 dph, with the D × V hybrid exhibiting the highest GH and the V × D hybrid the lowest. There were no significant GH differences between the Danube and Volga purebreds at the later sampling points, whereas the reciprocal hybrids showed significant differences in the GH from 386–504 dph .The mean number of alleles was significantly higher in the D × V hybrids, and the Volga purebred displayed the lowest mean number of alleles . The obsAt first, the genetic structure of the two geographically distant populations—i.e., Volga sterlet and Danube starlet—was investigated in this study to ensure that the sample sets of the populations displayed genetic heterogeneity, since it is reported that 23% of wild Danube sterlet possess a partial or complete Volga genotype . The DanRegular monitoring of the genetic diversity of broodstock and progeny is crucial to maintaining the genetic integrity of a fish farm population . The selWe evaluated the relative fitness of the purebred and inter-population crosses, rearing hatchery-derived Danube and Volga sterlet under identical conditions. Significant between-group differences were detected in fitness-related traits, suggesting a variation in the potential for fish farming. Hybridization is commonly used in aquaculture to increase fitness, manifested as an improvement in relevant physiological traits compared to the parent species ,41. The Pangasianodon gigas, rohu Labeo rohita, and common carp Cyprinus carpio [The hybrids showed a higher mean BW compared to either purebred at most sampling points. Our results are consistent with those reporting an effect of the parent position in the hybridization matrix in the Mekong giant catfish s carpio ,45,46. Ts carpio .Our results are also in agreement with the results of studies exploring sturgeon hybridization to increase the growth rate and improve productivity through hybrid vigor ,48,49,50Hybridization may lead to an increase in genetic polymorphism and heterozygosity , resultiThe competitive risk to wild populations from intraspecific hybrids is generally less than that from interspecific hybrids, since the level of genetic polymorphism and the occurrence of rare alleles is comparatively lower. Also, intraspecific hybrids possess the same number of chromosomes, eliminating the production of sterile hybrids. Nevertheless, intraspecific hybridization can present risks to future generations associated with outbreeding depression that should be considered in the selection of non-native broodstock for increasing productivity . The escSalmo salar and assessed the correlation between genetics and reproductive quality in their broodstock [The sterlet hybrids produced in the present study can be exploited for commercial aquaculture to increase growth rate, improve productivity through hybrid vigor, and transfer desirable traits to future generations. They can be potential candidates for enclosed common-environment studies to determine their fate under target wild conditions. Researchers have studied the consequences of stocking multiple strains of Atlantic salmon oodstock . Althougoodstock .Our analysis showed the intraspecific hybrids to perform better than the purebreds, similar to our previous results on interspecific sturgeon hybrids . There wIn addition to genetic and phenotypic plasticity as a source of the observed differences in fitness-related traits, we cannot exclude likely environmental and sampling biases. The growth rates of the fish under study were lower than those commonly reported, which could be because of the suboptimal rearing environment. The fish with lost tags in the communal tanks were excluded from the analysis, which may have influenced the average measures of mean body weight and survival. The effect of cannibalism and tank-specific population density  on the depressed growth rates and cumulative survival cannot be neglected. However, as these fish were reared in pooled stock, the examined fitness-related traits were equally affected by unfavorable rearing conditions. Hence, we believe that the observed differences were most likely caused by genetic origin (purebred vs. hybrid). To the best of our knowledge, this study brings the first observation of the effect of intraspecific hybridization on fitness-related traits in hatchery-reared populations of sterlet. Therefore, it has an important implication for additional comparative studies focusing on the survival and fitness-related traits of hybrid crosses in the natural environment. Nevertheless, any generalization of our results to conventional sturgeon aquaculture should be undertaken with caution.The hatchery-reared Danube and Volga sterlet broodstock differed genetically. The D × V hybrid grew faster than the purebred sterlet, and its reciprocal hybrid (V × D) showed a higher survival at three of six sampling points compared to the purebred. The observed genetic diversity was significantly higher in the D × V. Our results indicate that the D × V hybrid shows potential for achieving a better growth rate and can be recommended over purebreds for commercial aquaculture. Future research focusing on the investigation of the fecundity, gamete quality, survival of F1 hybrids as well as the study of fitness-related traits in future generations should be undertaken in a more natural setting or, ideally, the native habitat. This can provide insight into the fate of intraspecific sturgeon hybrids, which could serve as potential information for conservation efforts.