As a major activator of transforming growth factor β (TGF-β), the RGD receptor αvβ8-integrin is involved in pathogenic processes related to TGF-β dysregulation, such as tumor growth, invasion, and radiochemoresistance, metastasis and tumor cell stemness, as well as epithelial-mesenchymal transition. The novel positron emission tomography (PET) radiopharmaceutical Ga-68-Triveoctin for in vivo mapping of αvβ8-integrin expression might enhance the prognosis of certain tumor entities, as well as support and augment TGF-β-targeted therapeutic approaches.NMe)K(pent-4-ynoic amide)) were synthesized by click chemistry (CuAAC), labeled with Ga-68, and evaluated in MeWo (human melanoma) xenografted SCID mice by means of PET and ex-vivo biodistribution. αvβ8-integrin expression in murine tissues was determined by β8-IHC. A human subject received a single injection of 173 MBq of Ga-68-Triveoctin and underwent 3 subsequent PET/CT scans at 25, 45, and 90 min p.i..Monomeric and trimeric conjugates of cyclo. Accordingly, biodistribution showed a higher tumor uptake  but a similar baseline upon blockade (0.25%IA/g for both). IHC showed an intermediate β8-expression in the tumor while most organs and tissues were found β8-negative. Low non-target tissue uptakes (< 0.4%IA/g) confirmed a low degree of unspecific binding. Due to its hydrophilicity (log D = − 3.1), Ga-68-Triveoctin is excreted renally and shows favorable tumor/tissue ratios in mice . A high kidney uptake in mice  is not reflected by human PET , which furthermore showed notable uptakes in coeliac and choroid plexus .The trimer Ga-68-Triveoctin exhibits a 6.7-fold higher αvβ8-integrin affinity than the monomer (ICGa-68-Triveoctin enables sensitive in-vivo imaging αvβ8-integrin expression in murine tumor xenografts. PET in a human subject confirmed a favorable biodistribution, underscoring the potential of Ga-68-Triveoctin for mapping of αvβ8-integrin expression in a clinical setting. Hence, Mounting experimental evidence suggested that explaining the biological role and significance of αvβ8-integrin requires, in essence, a closer view on a family of mammalian cytokines called transforming growth factor beta  , 5. AgaiGenerally, TGF-β acts as a growth suppressor onto normal cells and may function as a tumor suppressor . For ins68Ga-Triveoctin, a 68Ga-labeled trimer of the αvβ8 selective octapeptide c(GLRGDLp(NMe)K) [In view of its multifaceted role in human pathology and oncogenesis, we anticipate a substantial scientific and clinical value for in-vivo mapping of physiological and pathological αvβ8-integrin expression patterns. For this purpose, we developed p(NMe)K)  suitable68Ga radiolabeling [n-octanol/phosphate-buffered saline (PBS) distribution coefficients (log D) and ex-vivo biodistribution studies [Synthesis and characterization of Triveoctin is described in the Additional file labeling , cultivalabeling , determi studies , and µPE studies  were don68Ga labeling, non-processed eluate of a 68Ge/68Ga-generator with a SnO2 matrix  was adjusted to pH 2 by adding HEPES buffer (400 µL of a 2.7 M solution), used to label 2 nmol of Triveoctin or TRAP-AvB8 for 3 min at 95 °C, which was purified by solid-phase extraction using a SepPak® C8 light cartridge (Waters). Radiochemical yields were 95.8 ± 1.3% (n = 10), referring to the final product after purification. Quality control was performed by radio-TLC with citrate buffer, confirming > 99% purity . Distribution coefficients (log D) were determined by shake-flask method using n-octanol and PBS.For fully automated 2 atmosphere in DMEM/HAM  with 10% fetal calf serum (Thermo Fisher). Approx. 107 cells were subcutaneously injected with Matrigel®  into the right shoulder of 6–8-week-old female CB17 severe combined immunodeficiency (SCID) mice, which were used about 2–3 weeks later for PET and biodistribution.MeWo cells  were grown at 37 °C under 5% CO68Ga-Triveoctin. The eyes were excised, rinsed with PBS and embedded in Sakura Tissue-Tek®. After equilibrium had been reached at − 20 °C, lateral 50-μm cross sections were cut on a cryostat microtome (Leica CM1950) and thaw-mounted on SuperFrost Plus microscope slides. The slides were air-dried on a heating plate and exposed to an imaging plate from 45 min after sacrifice onwards overnight. The imaging plate was read out by a CR-35 Bio Scanner (Raytest). Gaussian smoothing was applied to the final images. The same sections were subsequently HE-stained in a standard automated process.For ocular autoradiography and histology, a mouse was sacrificed 50 min after injection of 12 MBq 68Ge/68Ga generator  was directly eluted into the reaction vial containing Triveoctin (35 µg) and adjusted to pH 4.5 with sodium acetate. After heating for 4 min at 95 °C, the mixture was passed over a solid-phase extraction cartridge (Waters Sep-Pak®light tC-18), which was purged with water (10 mL). Thereafter, 68Ga-Triveoctin was eluted with ethanol/water mixture , followed by isotonic saline (9 mL). The formulation containing approx. 5% ethanol was passed through a 0.22-µm filter into a sterile injection vial and dispensed for injection. Quality control was done by radio-HPLC using a Shimadzu system equipped with a column Chromolith® Performance RP18e , gradient 0–100% acetonitrile in water within 15 min, flow rate 1.4 mL/min, tR = 8.53 min, and met the in-house specifications for 68Ga-labeled compounds (> 95%).Using a fully automated synthesis module, the eluate of a 68Ga-Triveoctin was done according to §13/2b of the German Drug Act (Arzneimittelgesetz). A human subject received a single intravenous injection of 68Ga-Triveoctin . A standard low-dose CT was acquired from the whole body  and used for absolute scatter correction of the subsequent PET scan. The emission PET scan was acquired over 19 min using continuous bed motion with a speed of 2.2 mm s–1 for the legs and 1.4 mm s–1 for the remaining body. The PET scan was repeated 45 min p.i. without another CT scan. Another PET/CT imaging sequence was acquired 90 min p.i. as the subject had to leave the scanner for voiding. All scans were obtained during normal breathing. PET images were reconstructed using the TRueX algorithm with 4 iterations, 5 subsets, time-of-flight (TOF) application and without filtering. Resulting PET images had an image matrix size of 440 × 440 with a voxel size of 1.65 × 1.65 × 3.0 mm. The dosimetry values were calculated using OLINDA V1.1 [Application of NDA V1.1  on the b68Ga-peptides was done adopting a synthetically robust scheme [68Ga labeling afforded the novel trimer 68Ga-Triveoctin . Trimerization, however, resulted in a threefold and 6.7-fold higher target affinity for Ga-Triveoctin as compared to the neat peptide and the chelator monomer, respectively  validation of target expression confirms that there is a non-negligible αvβ8 expression density on epithelial cells of the lung Fig. b, which 68Ga-Triveoctin enables imaging αvβ8-integrin in tumor regions with low expression, which 68Ga-TRAP-AvB8 is not capable of. Figure 68Ga-TRAP-AvB8 allows only for delineation of the central part of the tumor with high β8 expression , which is not reduced completely upon blockade but only diminished by slightly more than half (16%IA/g remaining). A substantial fraction of the remaining uptake could be caused by the larger molecular size, i.e., to less advanced renal clearance observed at the same time point (60 min p.i.) because of slower excretion kinetics. The main reason for the elevated kidney uptake might however be the expression of αvβ8-integrin on mural mesangial cells [68Ga-Triveoctin can therefore be interpreted as another sign of its target sensitivity, i.e., its superior capability of visualizing mesangial αvβ8-integrin expression in mice.The µPET furthermore visualizes that compared to the kidney uptake of al cells , 33. The68Ga-Triveoctin showed only a low soft tissue uptake in a human subject, notable tracer accumulations with descending intensity were observed in kidneys, urinary bladder, choroid plexus, infraadrenal (likely a ganglia), spleen, gastric mucosa, retina, liver, larger vessels, salivary glands and intestine K) itself must therefore be regarded as the main reason why 68Ga-Triveoctin possesses a suitable hydrophilicity (log DOW = − 3.1), and its higher αvβ8-integrin affinity and subtype selectivity can actually translate into favorable imaging characteristics. The 68Ga chelator TRAP [68Ga labeling solutions, such as FeIII [II, and CuII [Several studies have shown quite consistently that combining more than a single copy of a given targeting molecule (frequently referred to as multimerization) reliably increases the overall avidity of such constructs ,tor TRAP  was chostor TRAP , 30, butas FeIII , ZnII, aand CuII , and theand CuII .Comparison of human PET data with murine ex-vivo distribution reveals several discrepancies, most strikingly, regarding eye uptake, which is high in mice but insignificant in human. Whether this is related to species or subject age  cannot be answered on the basis of the existing data, but eye uptake seems to be an artifact of the preclinical experiments. Likewise, the kidney-to-blood and -to-muscle ratios at 90 min p.i. in human  are about 8 and 16 times, respectively, lower than in mice . The relatively high kidney uptake in mice thus also appears to be a mouse artifact, which might in part be related to the expression of αvβ8-integrin on mural mesangial cells , 33, butThe existing insights into the expression and functions of αvβ8-integrin, which were gained mainly from cell studies and histological data, do not allow for a valid conclusion whether or not this receptor might be suited for a "if you can see it, you can treat it" radionuclide imaging and -therapy approach similar to the highly popular and successful sst2- and PSMA-targeted theranostics . While t68Ga-Triveoctin PET (up to 6 scans below the 20 mSv limit) might even allow for in-depth studies of target expression as a response to therapy.Beyond the classic theranostic paradigm, the involvement of αvβ8 in major oncogenic pathways, such as TGF-β1 signaling and EMT, might ultimately render αvβ8-PET useful for clinical reasoning and personalized medicine, such as for cancer prognosis and stratification of patients for certain chemotherapies. In view of a high relevance of PET imaging of the TGF-β signaling pathway adressing downstream targets , αvβ8-PE68Ga-Triveoctin was obtained by trimerization of the cyclic peptide cyclo(GLRGDLp(NMe)K) on the TRAP chelator scaffold. Because of its high αvβ8-integrin affinity, the tracer enabled sensitive in-vivo imaging in murine tumor xenografts. PET in human confirmed a favorable biodistribution with low background, underscoring the potential of 68Ga-Triveoctin for future clinical investigation of αvβ8-integrin expression, e.g., for conditions associated with TGF-β dysregulation.The αvβ8-integrin PET diagnostic Additional file 1: Synthesis and analytical data for Triveoctin, radio-TLC for 68Ga-Triveoctin, and small-animal biodistribution data for 68Ga-Triveoctin in numerical form (table).


Cannabis sativa is excluded from regulation by the United States Department of Agriculture (USDA). Distinctive Cannabis varieties are ostracized from registration and therefore nearly impossible to verify. As Cannabis has become legal for medical and recreational consumption in many states, consumers have been exposed to a wave of novel Cannabis products with many distinctive names. Despite more than 2000 named strains being available to consumers, questions about the consistency of commercially available strains have not been investigated through scientific methodologies. As Cannabis legalization and consumption increases, the need to provide consumers with consistent products becomes more pressing. In this research, we examined commercially available, drug-type Cannabis strains using genetic methods to determine if the commonly referenced distinctions are supported and if samples with the same strain name are consistent when obtained from different facilities.Unlike other plants, de-novo microsatellite markers using the “Purple Kush” genome to investigate potential genetic variation within 30 strains obtained from dispensaries in three states. Samples were examined to determine if there is any genetic distinction separating the commonly referenced Sativa, Indica and Hybrid types and if there is consistent genetic identity found within strain accessions obtained from different facilities.We developed ten Although there was strong statistical support dividing the samples into two genetic groups, the groups did not correspond to commonly reported Sativa/Hybrid/Indica types. The analyses revealed genetic inconsistencies within strains, with most strains containing at least one genetic outlier. However, after the removal of obvious outliers, many strains showed considerable genetic stability.We failed to find clear genetic support for commonly referenced Sativa, Indica and Hybrid types as described in online databases. Significant genetic differences within samples of the same strain were observed indicating that consumers could be provided inconsistent products. These differences have the potential to lead to phenotypic differences and unexpected effects, which could be surprising for the recreational user, but have more serious implications for patients relying on strains that alleviate specific medical symptoms.The online version of this article (10.1186/s42238-019-0001-1) contains supplementary material, which is available to authorized users. Cannabis sativa L. dates back thousands of years : glaucoma  Leafly  and variCannabis usage groups, which are well supported by genetic analyses  in the U.S., and marijuana or drug-types with moderate to high THC concentrations . Within the two major groups Cannabis has been further divided into strains  in the commercial marketplace, and particularly for the drug types, strains are assigned to one of three categories: Sativa which reportedly has uplifting and more psychotropic effects, Indica which reportedly has more relaxing and sedative effects, and Hybrid which is the result of breeding Sativa and Indica types resulting in intermediate effects. The colloquial terms Sativa, Hybrid, and Indica are used throughout this document even though these terms do not align with the current formal botanical taxonomy for Cannabis sativa and proposed Cannabis indica  and are produced through cloning and, in some cases, self-fertilization to produce seeds  related to flowers, cannabinoid profile, and terpene production. Phenotype is a product of genotype and environment. Cannabis is considerably variable and extraordinarily plastic in response to varying environmental conditions , olives (Olea europea L.) and apples . With the rapid development of new varieties in these types of crops, morphological characters have become increasingly difficult to distinguish. Currently, quantitative and/or molecular characters are often used to demonstrate uniqueness among varieties. Microsatellite genotyping enables growers and breeders of new cultivars to demonstrate uniqueness through variable genetic profiles  and The Plant Variety Protection Act of 1970  any genetic distinction separates the common perception of Sativa, Indica and Hybrid types; (2) consistent genetic identity is found within a variety of different strain accessions obtained from different facilities; (3) there is evidence of misidentification or mislabeling.We used a well-established genetic technique to compare commercially available Cannabis samples for 30 strains were acquired from 20 dispensaries or donors in three states (Table es Table . All sames Table  with 0.0es Table  were usees Table . The 30 es Table . ResultsCannabis draft genome from “Purple Kush” (GenBank accession AGQN00000000.1) was scanned for microsatellite repeat regions using MSATCOMMANDER-1.0.8-beta  to examine variation in the dataset. Lynch & Ritland (r) statistics were calculated between all 122 samples resulting in 7381 pairwise r-values showing degrees of relatedness. For all strains the r-mean and standard deviation (SD) was calculated averaging among all samples. Obvious outliers were determined by calculating the lowest r-mean and iteratively removing those samples to determine the relatedness among the remaining samples in the subset. A graph was generated for 12 popular strains  for two genetic groups, 46.56 forA Principal Coordinate Analyses (PCoA) was conducted using GENALEX Fig. . Principr) between all 122 samples was calculated in GENALEX. The resulting 7381 pairwise r-values were converted to a heat map using purple to indicate the lowest pairwise relatedness value (− 1.09) and green to indicate the highest pairwise relatedness value , second order relatives  share 25% genetic identity , and unrelated individuals are expected to have an r-value of 0.00 or lower. Negative values arise when individuals are less related than expected under normal panmictic conditions  to 0.68 (“Island Sweet Skunk”)  was removed and the overall r-means and SD values within strains were recalculated , first or higher order relatives , second order relatives , low levels of relatedness , and not related . Overall r-means are displayed for all 30 strains (Table r-means indicate only three strains are first or higher order relatives (Table r-means 0.26–0.45), ten strains show low levels of relatedness . The impact of outliers can be clearly seen in the heat map for “Durban Poison” which shows the relatedness for 36 comparisons , while 13 are not related . However, removal of two outliers, Denver 1 and Garden City 2, reduces the number of comparisons ranked as not related from 13 to zero.Individual pairwise ”) Table . Standared Table . In all Cannabis is becoming an ever-increasing topic of discussion, so it is important that scientists and the public can discuss Cannabis in a similar manner. Currently, not only are Sativa and Indica types disputed  when samples with high genetic divergence were removed from the data set  was generated to visualize genetic relatedness throughout the data set . For example, “Sour Diesel” , and no comparisons in “Blue Dream” have negative values. While “Blue Dream” has an initial overall r-mean indicating first order relatedness within the samples  are higher than within samples of “Sour Diesel”. Interestingly, “Golden Goat” is reported to be a hybrid descendant of “Island Sweet Skunk”  grew the variations  from seeds he found in a single bag of rt Danko . Howeverrt Danko . The STRrt Danko , and sixCannabis clones and stable seed, because currently they can only assume the strains they possess are true to name. There is a chain of events from seed to sale that relies heavily on the supplier, grower, and dispensary to provide the correct product, but there is currently no reliable way to verify Cannabis strains. The possibility exists for errors in plant labeling, misplacement, misspelling (e.g. “Chem Dog” vs. “Chemdawg”), and/or relabeling along the entire chain of production. Although the expectation is that plants are labeled carefully and not re-labeled with a more desirable name for a quick sale, these misgivings must be considered. Identification by genetic markers has largely eliminated these types of mistakes in other widely cultivated crops such as grapes, olives and apples. Modern genetic applications can accurately identify varieties and can clarify ambiguity in closely related and hybrid species , and the vernacular usage will likely continue to be used, despite a lack of evidence of genetic differentiation.This study shows that in neutral genetic markers, there is no consistent genetic differentiation between the widely held perceptions of Sativa and Indica Cannabis plants are propagated. Although it is impossible to determine the source of these inconsistencies as they can arise at multiple points throughout the chain of events from seed to sale, we theorize misidentification, mislabeling, misplacement, misspelling, and/or relabeling are all possible. Especially where names are similar, there is the possibility for mislabeling, as was shown here. In many cases genetic inconsistencies within strains were limited to one or two samples. We feel that there is a reasonable amount of genetic similarity within many strains, but currently there is no way to verify the “true” genotype of any strain. Although the sampling here includes merely a fragment of the available Cannabis strains, our results give scientific merit to previously anecdotal claims that strains can be unpredictable.Instances we found where samples within strains are not genetically similar, which is unexpected given the manner in which Additional file 1:Table S1. Twelve popular strains and their described assignment of Sativa and Indica according to six online data bases of Cannabis strain information. Table S2. Primer information includes the multiplex assignment, primer name, microsatellite repeat and number of units repeated in the “Purple Kush” draft genome , forward and reverse sequences (asterisk denotes the sequence to which the tag is attached), the universal tag , dye , optimized annealing temperature, MgCl uL volume,amplified fragment size range, and the number of alleles in the data set. (XLSX 52 kb)Additional file 2:Figure S1. STRUCTURE HARVESTER graph indicating K = 2 is highly supported. (ΔK = 146.56) as the number of genetic groups for this data. (PDF 55 kb)Additional file 3:Figure S2. Bar plot graphs generated from STRUCTURE analysis for individuals from twelve popular strains  values for 122 samples where purple indicates no genetic relatedness  and green indicates a high degree of relatedness . Sample strain names and location of origin are indicated along the top and down the left side of the chart. Pairwise genetic relatedness (r) values are given in each cell and cell color reflects the degree to which two individuals are related. (PDF 239 kb)


Solanum in the Canarian archipelago, where three endemic taxa are currently recognized. Inter-taxa hybridizations were performed, and morphological expression was assessed with a common-garden approach. Using the eggplant Single Primer Enrichment Technology (SPET) platform with 5,093 probes, 74 individuals of three endemic taxa  were sampled for SNPs. While morphological and breeding studies showed clear distinctions and some continuous variation, inter-taxon hybrids were fertile and heterotic for vigor traits. SPET genotyping revealed 1,421 high-quality SNPs and supported four, not three, distinct taxonomic entities associated with post-emergence geological, ecological and geographic factors of the islands. Given the lack of barriers to hybridization among all the taxa and their molecular differences, great care must be taken in population management. Conservation strategies must take account of the sexual and breeding systems and genotypic distribution among populations to successfully conserve and restore threatened/endangered island taxa, as exemplified by Solanum on the Canary Islands.Islands provide unique opportunities to integrated research approaches to study evolution and conservation because boundaries are circumscribed, geological ages are often precise, and many taxa are greatly imperiled. We combined morphological and hybridization studies with high-throughput genotyping platforms to streamline relationships in the endangered monophyletic and highly diverse lineage of  Islands have long been recognized as biological laboratories because they provide particularly insightful understanding into the evolution, ecology, and conservation of plant species . Their gNotably, a number of molecular-based studies have revealed sharp genetic discontinuities within and between islands in both narrowly distributed and widespread Canarian endemic plant lineages . These rSolanum lidii Sunding and S. vespertilio Aiton are endangered endemic Solanaceae from the Canary Islands that are notable for their suite of reproductive features [ is endemic to Gran Canaria, where it grows in ruderal shrublands on disturbed sites and rocky areas as inland cliffs  and S. vespertilio subsp. doramae (hereafter SVD) Marrero Rodr. & Gonzalez-Martin. SVV is restricted to Tenerife, the largest of the seven main islands in the Canarian archipelago, where the greatest concentration of individuals occurs in two areas: the palaeo-islands of Teno  and Anaga  (habitat) . While ohabitat) . ProposeS. vespertilio subsp. doramae  and ISSR markers. That study, conducted on limited population sampling , aimed to quantify the genetic differentiation among the three taxa to assess the potential for reintroduction of the threatened SVD in its natural habitats on Gran Canaria. Despite the relatively high levels of polymorphism detected with both molecular techniques, the barcode sequences (matK and rbcL) did not reveal distinction between the two subspecies of S. vespertilio, and only one position in trnL-F discriminated S. lidii from S. vespertilio. These results reinforced the close phylogenetic relationship among the taxa highlighted in the AFLP analysis by In the most recent molecular study of the relationships among these three taxa, Caujapé-Castells et al. (unpublished data) assessed five cpDNA sequences (S. melongena and one S. incanum accessions (Single nucleotide polymorphisms (SNPs) are among the most abundant and easy to automate molecular markers . As a recessions . The eggcessions .S. lidii and S. vespertilio s.l., and their crossability, (ii) provide new data to assess their genetic relationships and (iii) suggest effective conservation strategies for these endangered taxa, that include the extinct-in-the wild S. vespertilio ssp. doramae.Traditional and new molecular tools have been revealed as powerful and indispensable aids to conservation efforts, particularly with endangered species . Herein,Solanum taxa were sampled  in “common gardens” (research greenhouses at the University of Connecticut), and in hand-generated hybrids between all possible pairwise combinations of SVV, SVD, and SL. Measurements were taken on the topmost mature leaves on mature plants , and included length of the blade from tip to the lowest point on the basal lobes of the blades ; the width was taken at the widest point. The length/width ratio was then calculated.The plants used for the scoring or measurement of characters noted as key differences among the species  dependedt-tests were used to test for significant differences between various pairs in Artificial hybrids were generated between all three possible pairwise combinations: SVV with SVD, SVV with SL, and SVD with SL . Samples were diluted and sent to IGA Technology Services  for genotyping using the eggplant SPET platform, where genomic libraries were constructed and sequenced using an Illumina NextSeq 500 sequencer in single-end mode (150 bp). The eggplant SPET platform was designed using the SNP dataset (over 10.9 million SNPs) generated from the whole-genome resequencing of seven S. melongena and one S. incanum accessions   dendrogram based on ∗ program  . To clar program . The SVD program , 2015 is program . To conv program  was used program , and rep program .PRJNA556343. Raw reads of each accession are deposited under the accession numbers from SRX6649376 to SRX6649471. VCF files with the corresponding variants identified are available upon request to the corresponding author.The sequence data have been deposited into the NCBI Short Read Archive under the Bioproject identifier S. vespertilio are distinct from S. lidii in terms of leaf length and width, but less distinct in terms of the length/width ratio , in the average within-population gene diversity per locus  and in the average total population gene diversity per locus , respectively. As well, the average inbreeding coefficient (Fis) per locus was 0.085. However, the average fixation index per locus (Fst) was 0.331, indicating moderate to a high level of differentiation due to the members of some populations carrying unique alleles compared to the rest. On the other hand, the average gene diversity (Dst) per locus among subpopulations was low (stD = 0.088), and ranged from 0 to 0.403, similar to the average measure of population differentiation (Dest) per locus (estD = 0.116), which ranged from 0 to 0.510. Population SL_TE showed the highest average observed heterozygosity (Ho) per locus with a value of 0.230, followed by populations SVV_BR (0.134), SVV_AR (0.128), and SVV_ME (0.121); the lowest value was observed for SVD_O (0.039) , while population SVD_O presented the most negative average values with −0.798. Individual values for all the accessions for Ho, He, and Fis are reported in Fst values between pairwise populations displayed low values among SVV populations, ranging from 0.106 between SVV_ME and SVV_BR to 0.296 between SVV_AR and SVV_CO . Not surprisingly, the SL_TE pairwise values were generally uniformly higher among all the comparisons, interestingly the highest for the comparison with SVV_CO (0.431).Most of the SNPs identified were biallelic and only 13 presented more than two alleles. The average of the reference allele frequency  was 0.782 with a wide range from 0.014 to 0.993 . Overall (0.039) . A simild SVV_CO . The valS. vespertilio , and different homozygous alleles for the rest of the accessions of the two subspecies of S. vespertilio , were selected to create a subset called “Core private SNPs.” This subset presented 36 SNPs with five SNPs on chromosomes 3, 7, 10, and 12, four SNPs on chromosomes 1 and 6, three SNPs on chromosomes 4, two SNPs in 2 and 8 and just one SNP on chromosome 11, while chromosomes 5 and 9 did not bear any SNPs. Some of the private SNPs in the S. vespertilio, only one private SNP was found for SVV and none for SVD. However, for SVD, when the accession (SVD_I_1) is removed from the analyses, seven private SNPs were identified between SVV and SVD, although none of them was specific and universal and therefore did not meet the requirement for being included in the “Core private SNPs” subset.One of the main aims of this study, apart from establishing genetic relationships among the accessions assessed, was to provide genetic tools to easily discriminate the three taxa. As expected, given the larger morphological distinction, the largest set of private SNPs  was found for SL with 55 SNPs that discriminate it from the two subspecies of pertilio . PrivateThe UPGMA-based dendrogram obtained for 74 accessions revealed a sister basal clustering of all SL individuals, although the relationships among them were not always well supported by bootstrap values . InteresK = 5. Only two clusters (red and yellow) consisted mainly of the individuals from a single population (or species)  accessions from the SVD_I_1 (Moya) accession. Furthermore, this second coordinate separated almost all the SVV accessions from Lomo de Las Bodegas SVV_BO from the rest of the Anaga SVV populations SVV_BR, SVV_AR and SVV_ME. The latter showed a strong admixture pattern.Although there were some minor rearrangements in branch disposition, the two different methods for estimating species trees (UPGMA and SVDquartets), gave the same topology of accessions, confirming the results of the UPGMA analysis that we show herein, and suggesting that there are no significant artifactual groupings due to deep coalescence . The fasspecies) . In factspecies) . The firspecies) . The firS. lidii and S. vespertilio, two rare Canary Island endemics much more distantly related to the eggplant. Therefore, this study provides additional proof of the reliability of this technology as well as of its wide cross-species transferability. This new genotyping solution combines the benefits of (a) complexity reduction , (b) high-density DNA arrays , and (c) discovery of novel polymorphisms in addition to the target SNPs in regions of interest, thus allowing high-transferability across species , it would mean that these taxa are recent endemics on Gran Canaria and Tenerife, in spite of the fact that these two large islands are among the older in the archipelago  to Tenerife, where it proliferated in the more common moist habitats. In this scenario, S. vespertilio subsp. doramae  can be construed as one remnant of that split and further radiation into what are today increasingly uncommon undisturbed moist habitats on Gran Canaria. The position in the tree .the tree  of SVD_ICanarina canariensis , or Navaea phoenicea , a palaeo-endemic with a similar distribution to SVV, Solanum do not provide support for current gene flow between these widely separated areas, so that the separation between the distribution areas in Teno and Anaga might well be more ancient than the reported upheavals.Not all the lineages with an Anaga-Teno distribution are old. Northern Tenerife has been subjected to a number of geologic events such as volcanic landslides and avalanches in different periods of time from 2.2 Ma (Tigaiga landslide) to 170 ka   that liktropogon , and somcromeria  or Canarariensis , presentS. vespertilio on Tenerife may constitute separate subspecies, as genetically differentiated as S. vespertilio on Gran Canaria (SVD) is from SVV on Tenerife. The fact that the individuals from Teno show perfect intrapopulation uniformity with no indications of hybridization or migration from other groups in the fastSTRUCTURE analysis  the area of Anaga has been ecologically  stable historically, and (b) it reportedly acted as a source of genetic diversity for other regions in the archipelago , and the nesting of other accessions of SVD fully interspersed within the collections of SVV , a more arid region in the northwest of Tenerife. The Teno samples of SVV are notably different from all other Tenerife accessions. Thus, these data perhaps reflect the geological and geographical distinction of the Teno area as a paleo-island .As noted above, the SNP genotypic data highlight a very distinct genotypic population of o-island . A thoroS. lidii), has the lowest fixation index (Fis), implying low self-breeding, in spite of starkly declining population sizes.The rareness and vulnerability of all the taxa considered, especially the two from Gran Canaria, make this study extremely important for designing effective conservation strategies and restoration programs that are based on the full understanding of the relationships, genotypic diversity, ecological requirements and reproductive biology (including pollination biology) of the most threatened taxa. For instance, there is an interesting paradox in the context of mating systems  as appliFis values (range between 0.301 and 0.587) for the larger and more widespread S. vespertilio (SVV) populations. These values make sense in terms of the reproductive biology and population biology of SVV as we understand it. However, they do not for S. lidii (SL), where Fis is negative (-0.204), indicating an excess of heterozygotes. This is uncommon and could be caused by either: (a) previous hybridization between genetically very different SL populations that have not yet reached Hardy-Weinberg equilibrium, or (b) natural selection for highly heterozygous individuals. However, regardless of the underlying cause, these perspectives are important for effective conservation design. Hybridization of SVV, SVD, or SL with other Solanum species occurring in the Canary islands, including the cultivated S. melongena L., that might endanger the genetic integrity of these Canarian endemics, is unlikely given the different habitats and phylogenetic distance with other Solanum species occurring in the Canary islands  is likely to be more stable. In this way, for the distinctive Gran Canarian genotypes (SVD) to be maintained, conservation plans must limit the growth of introduced SVV near SVD. Indeed, the nesting of the presumably “pure” SVD individuals sampled at Finca de Osorio (Gran Canaria) within the SVV samples from Las Bodegas (Tenerife) , we suggest that the SVD_I source, which is the most differentiated from SVV, might be considered for conservation-restoration efforts. Similarly, independently of whether the two areas of distribution of S. vespertilio subsp. vespertilio on the palaeo-islands of Tenerife (Anaga and Teno) represent distinct subspecies or not, their clear genetic differentiation indicates that in situ conservation efforts should avoid genetic mixing between them because the populations could be the result of two different evolutionary histories. At the minimum, the ravine in Teno (“Los Cochinos”) harbors a genotypically and ecologically distinct population that makes it a biodiversity micro-reserve, a refugium perhaps, of Solanum diversity  see  indicateS. vespertilio in particular (Solanum kept at the seed bank of the JBCVCSIC should be reinforced to ensure success in future restoration efforts.Finally, as with most considerations of biodiversity, it is difficult to assess how future climate scenarios may affect the endemic populations we studied. However the current cloud forests of the Canaries generated by trade winds, crucial for the water balance in many of the natural habitats for rticular , have berticular . Given tSolanum of the Canary Islands indicates that complex morphological and molecular variation is distributed among these taxa. Although they are still capable of sexual reproduction, suitable habitats are diminishing and populations are seriously declining. To successfully protect and restore native populations of these Solanum and other oceanic island endemics (A broad combination of approaches employed in this study of the endemic endemics , the bioex situ management and in situ restoration of the Canarian Solanum and other island taxa. In this context, despite the small population sizes, we have found low levels for the inbreeding coefficient and a low genetic differentiation between SVV and SVD, suggesting that genetic flow that may compromise the genetic integrity of SVD may have taken place. Our results on relationships and genetic differentiation also suggest that an additional taxonomic entity, in addition to SVV and SVD, may exist within S. vespertilio.Our results showed that the three taxa display clear morphological differences, but they are fully cross-compatible, which may endanger their genetic integrity. The novel SNPs and SPET technology we utilized provides a powerful tool that, together with other multidisciplinary data generated, build the comprehensive foundation necessary for the successful S. vespertilio and S. lidii. We consider that dialogue among scientists and various stakeholders is necessary to implement recovery plans that reduce direct threats such as land development, feral herbivores, invasive species, and genetic contamination on these endangered Canarian endemisms.Importantly, the different complementary approaches that we applied provide solid foundations for the conservation strategies of PRJNA556343. Raw reads of each accession are deposited under the accession numbers from SRX6649376 to SRX6649471.The datasets generated for this study can be found in the sequence data have been deposited into the NCBI Short Read Archive under the Bioproject identifier PG and RJ-M: data curation, project administration, and writing – original draft preparation. PG, RJ-M, SV, and GA: formal analysis. PG, RJ-M, SV, JP, ÁM, JC-C, and GA: methodology, writing – review and editing, and data visualization. JP and JC-C: resources. JP, JC-C, and GA: supervision and research design and funding management.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genetic improvement of pearl millet is lagging behind most of the major crops. Development of genomic resources is expected to expedite breeding for improved agronomic traits, stress tolerance, yield, and nutritional quality. Genotyping a breeding population with high throughput markers enables exploration of genetic diversity, population structure, and linkage disequilibrium (LD) which are important preludes for marker-trait association studies and application of genomic-assisted breeding.Genotyping-by-sequencing (GBS) libraries of 309 inbred lines derived from landraces and improved varieties from Africa and India generated 54,770 high quality single nucleotide polymorphism (SNP) markers. On average one SNP per 29 Kb was mapped in the reference genome, with the telomeric regions more densely mapped than the pericentromeric regions of the chromosomes. Population structure analysis using 30,208 SNPs evenly distributed in the genome divided 309 accessions into five subpopulations with different levels of admixture. Pairwise genetic distance (GD) between accessions varied from 0.09 to 0.33 with the average distance of 0.28. Rapid LD decay implied low tendency of markers inherited together. Genetic differentiation estimates were the highest between subgroups 4 and 5, and the lowest between subgroups 1 and 2.Population genomic analysis of pearl millet inbred lines derived from diverse geographic and agroecological features identified five subgroups mostly following pedigree differences with different levels of admixture. It also revealed the prevalence of high genetic diversity in pearl millet, which is very useful in defining heterotic groups for hybrid breeding, trait mapping, and holds promise for improving pearl millet for yield and nutritional quality. The short LD decay observed suggests an absence of persistent haplotype blocks in pearl millet. The diverse genetic background of these lines and their low LD make this set of germplasm useful for traits mapping. Pennisetum glaucum (L.) R. Br. syn. Cenchrus americanus (L.) Morrone) is a climate-resilient crop grown in the arid and semi-arid areas of the world. Pearl millet is the most widely grown millet species, accounting for approximately half of the total worldwide production of millets [Pearl millet , maize (Zea mays L.), sorghum (Sorghum bicolor Moench), and rice (Oryza sativa L.) in its nutritional value [Pearl millet is also gaining a reputation as a health-promoting nutritious grain. The grain contains vital nutrients and is considered to be equal or superior to that of wheat  mapping that lays the foundation for marker-assisted breeding to improve the selection efficiency for faster development of cultivars , 27. MolCharacterization of the genetic diversity and population structure of pearl millet germplasm and breeding populations is needed in order to accelerate its genetic improvement for agronomic and nutritional traits. Toward this goal, a panel of 309 inbred lines derived from landraces and improved open pollinated varieties collected from different parts of Africa and India were evaluated for population genomics using genotyping-by-sequencing (GBS) markers. The present investigation employed pertinent population genomic approaches to understand the extent of genetic diversity of a pearl millet inbred lines population comprising both potential restorers and seed parents for hybrid variety development.A total of ~ 750 million total unfiltered reads were generated from 309 inbred lines arranged in four GBS plates run twice independently on an Ion Proton Next-Generation Sequencer . All the raw sequence reads for all the accessions have been submitted to the National Center for Biotechnology Information (NCBI) sequence read archive and deposited under the accession ID “BioProject ID”: PRJNA598172. Mapping the GBS reads to the pearl millet reference genome sequence initially detected 150, 977 unfiltered and non-imputed SNPs for the panel. Among these, 123,995 SNPs were distributed on chromosomes 1 to 7. A total of 26,982 SNPs were mostly from the unanchored genome sequences or from the part of the genome not covered in the reference genome sequence. The raw number of SNPs mapped on each chromosome ranged from 13,432 (chr 7) to 21,665 (chr 1).A high-density of high quality SNPs across the seven pearl millet chromosomes with most of the SNPs distributed in the telomeric regions than the pericentromeric regions of the chromosomes Fig.  were visT. minor allele frequency varied from 0.05 to 0.50 with a mean of 0.20  were much higher than transversion mutations  and the transition/transversion ratio was 1.63  of pairwise comparisons of the inbred lines varied from 0.09 to 0.33 with the average distance of 0.28 Fig. . The higAnalysis of the pearl millet inbred lines population using 30,893 LD pruned SNPs and the genetic distance matrix of the lines in ADMIXTURE  identifiTo confirm the ADMIXTURE results, principal component analysis (PCA) using the snpgdsPCA function of the R package SNPRelate  and geneAmong the five subgroups, subgroup 1 contained accessions such as ICTP 8203, GB8735, MC94, ICMV96490 and ICMR312, mainly known for their high levels of Fe and Zn content. Subgroup 2 was composed of landraces/improved cultivars mainly from WCA countries. Subgroup 3 was formed with inbred lines derived from landraces/improved cultivars collected from various sources, and showed more dispersion than the other subgroups. Results showed that the subgroup 4 contained inbred lines derived from the source populations IP-3110, IP-6745, IP-21142, PBPMPOP-1 and PBPMPOP-2, whereas group 5 comprised about 50% of inbred lines extracted from PBPMPOP-3.In accordance with the neutral allele distribution expected for inbred populations, the nucleotide diversity (π) and Tajima’s D statistics among the five subpopulations were presented in Table st-based analysis of the SNP data . The Fst coefficients among the groups varied from 0.044 to 0.110, indicating moderate differentiation. The highest level of differentiation was observed between subgroups 4 and 5. In contrast, the lowest Fst coefficient was observed between subgroups 2 and 3.Genetic differentiation of subgroups was calculated using Fta Table . The FstThe mean nucleotide diversity observed in subgroup 2 (9.77E-06) was similar to the whole panel (9.92E-06) Table . The nuc2) across the genome declined rapidly with increasing physical distance and most of the r2 values were below 0.05  mapping and association analysis . GBS is In this study, 309 pearl millet inbred lines were genotyped using GBS. Using the pearl millet genome  as a refGenome-wide marker density analysis across the chromosome arms identified an average of 35 SNPs per Mb (1 SNP per 29 Kb) of genome size. This SNP density is slightly lower than the previously reported 48 SNPs per Mb of genome . The repGermplasm resources and the genetic diversity of a crop species have paramount importance in the genetic improvement of a crop for desirable traits and conservation of genetic resources. Nucleotide polymorphism is a measure of genetic diversity and a key to understanding the effect of past selective forces on the gene pool. Average nucleotide diversity in the whole panel was 0.28 in this study, which was higher than reported for a global collection , but lowPopulation structure is a very important part of evolutionary genetics and depicts the diversity of a metapopulation that might have evolved independently. Knowledge about the genetic diversity and the population structure of a crop has important implications for a genome-wide association study. In the present study, population structure analysis using 30,893 SNPs detected five subgroups in a panel of 309 inbred lines and the grouping basically matches pedigree relationships or the parental source of the inbred lines. This number of subpopulations was validated by graphing kinship against the cross-validation error. Some genetic diversity studies reported six subpopulations in different panels of pearl millet , 32. A pA wide range of genotypic variations are prevalent in pearl millet for various agronomic traits and stress tolerance as a result of its cultivation in diverse agro-climatic conditions and soil types . HoweverFst values between the subgroups showed moderate differentiation. A previous study conducted in Niger revealed also a moderate level of differenciation on cultivated pearl millet accessions compared to the wild populations [Genetic patterns in the natural plant populations shows their biological importance in the ecology and economic prominence in agriculture . In pearulations . This vaFst revealed moderate differentiation among the five subpopulations. The nucleotide diversity and Tajima’s D statistics showed low diversity in subgroup 5, but high diversity in the other four subgroups. The genomic resources developed in this study can significantly contribute to the application of genomic-assisted breeding in pearl millet, especially in heterotic grouping and hybrid breeding.A better understanding of genetic diversity in pearl millet enhances the use of available germplasm for breeding and systematic conservation. Screening 309 pearl millet lines with 54,770 high quality genome-wide SNPs revealed that genetic diversity is preserved in pearl millet inbred lines from Africa and the Indian subcontinent. Population structure analysis detected five subpopulations that are mostly a reflection pedigree relationship. Population diversity analysis using A total of 309 inbred lines obtained from International Crop Research in Semi-Arid Tropics (ICRISAT-Niger) were used in this study  and MspI (5′-C/CGG-3′) restriction enzymes . Then the GBS [For library construction, 200 ng of DNA was double digested with  the GBS  librarie the GBS . The DNAhttps://cegresources.icrisat.org/data_public/PearlMillet_Genome/v1.1) [www.maizegenetics.net) [The sequencing runs generated a total of 750.2 million reads. The pearl millet reference genome (me/v1.1)  was usedics.net) . The mapics.net) . An 80 bGenotype and taxa summaries  were calculated using TASSEL v.5 software . Populat2) [ls function in R. Genetic difference among the identified subgroups were determined using Fst across the SNPs as a measure of population differentiation due to genetic structure. DNA sequence variation within and between populations was estimated by analyzing the genetic diversity (pi) and Tadjima’s D [LD analysis was performed using TASSEL as a standardized disequilibrium coefficient (D’)  and squa2)  among pajima’s D  in a sliAdditional file 1. List of materials with their pedigree and origin.Additional file 2. SNP markers  developed for 309 pearl millet inbred lines.Additional file 3. Relative kinship among pairs of inbred lines. (CSV 1417 kb)Additional file 4. Genetic distance among pairs of inbred lines. (CSV 1611 kb)


Lemur catta) are experiencing rapid population declines due to ongoing habitat loss and fragmentation, as well as increasing exploitation for bushmeat and the illegal pet trade. Despite being the focus of extensive and ongoing behavioral studies, there is comparatively little known about the genetic population structuring of the species. Here, we present the most comprehensive population genetic analysis of ring‐tailed lemurs to date from across their likely remaining geographic range. We assessed levels of genetic diversity and population genetic structure using multilocus genotypes for 106 adult individuals from nine geographically representative localities. Population structure and FST analyses revealed moderate genetic differentiation with localities being geographically partitioned into northern, southern, western and also potentially central clusters. Overall genetic diversity, in terms of allelic richness and observed heterozygosity, was high in the species . In fact, it is the highest among all published lemur estimates to date. While these results are encouraging, ring‐tailed lemurs are currently affected by ongoing habitat fragmentation and occur at lower densities in poorer quality habitats. The effects of continued isolation and fragmentation, coupled with climate‐driven environmental instability, will therefore likely impede the long‐term viability of the species.Madagascar's ring‐tailed lemurs ( Here, we present the most comprehensive population genetic analysis of ring‐tailed lemurs to date from across their likely remaining geographic range. Our study uses multilocus genotypes for 106 adult individuals from nine geographically representative localities to evaluate population genetic diversity and structure on a range‐wide scale. The results described herein demonstrate that, while Madagascar's remaining ring‐tailed lemur populations are geographically isolated, they have retained high levels of genetic diversity, with moderate genetic differentiation among populations despite a relatively large geographic distribution. Sample collection and export/import protocols adhered to Malagasy and International laws and were approved by Malagasy wildlife authorities and the US Fish and Wildlife Service.Our sample included 106 adult ring‐tailed lemurs from nine geographically representative localities from across their existing range Table . This da2.2n = 31) using QIAamp DNA Stool Mini Kits (QIAGEN) following Clarke et al.  was extracted from new samples , that is, the probability that two randomly drawn individuals from a population will show identical multilocus genotypes.Extraction products were amplified via PCR in a 13 μl reaction volume using 6.25 μl HotStarTaq DNA polymerase Master Mix, 20 mg/ml BSA, 10 μM primer pairs, and 3 μl (0.25–1 ng) gDNA using annealing temperatures outlined in the Appendix 2.42.4.1icro‐Checker , and expected (HE) heterozygosities, and Wright's FIS for each sampling location were calculated using GenoDive .To assess the genetic distances between sampling localities, Wright's enAlEx v.6.5 .The presence of isolation‐by‐distance (IBD) was evaluated using the program Gtructure v2.3.4  with no a priori information regarding the individuals' geographic sampling locations provided, so clusters were formed solely on genetic information. We evaluated the hypothesis K = 1–12, three more than the number of wild populations  was assessed using the highest value of ∆K , which indicates the most pronounced subdivision within the data and the optimal number of genetic clusters. We implemented a two‐step approach to evaluate further substructure in the dataset. We first identified the most likely number of clusters within the overall sample (n = 106) and then ran subsequent analyses within each of the K clusters  was performed in R, using the adegenet package  was performed with a standard genetic distance matrix  ranged from 4.33 to 8.67 . Pairwise distances (FST) between BER and BLK (0.053), BER and AMB (0.071), and between BEZA and TNP (0.034) indicate minimal to moderate genetic differentiation between these localities and are consistent with the clustering seen in the subsequent Structure analyses. Larger values of FST (>0.15), suggesting great differentiation, were observed between ISALO and sampling locales in the north (SAKA and TSARA) and south (AMB and BLK). Despite great geographic distance, there was only moderate FST between BEZA and BER (0.077), BEZA and BLK (0.083), and TNP and BER (0.091). The results of Mantel's test  with geographical distance explaining over 17% of the variation in genetic distance. However, there was a considerable amount of unexplained variation in the IBD data. For example, in the eastern cluster there is relatively little geographic distance between the three populations comprising the northern (ANJA‐SAKA‐TSARA) and southern groups (AMB‐BER‐BLK), yet some sampling sites within each of these triads reflect moderate genetic differentiation  could not confidently exclude K = 1. Mean L(K) and ΔK plots of all Structure  runs are provided in supplementary information  and an eastern cluster , as indicated by the highest value of ΔK Figure . Clustero et al.  and founn Figure .K‐means clustering estimated four genetic clusters. The majority of individuals clustered geographically with southern localities  showing a higher membership probability to Cluster 1, northern localities  to Cluster 2, and the central locality (ISALO) to Cluster 3. Individuals in the western localities (BEZA and TNP) showed higher membership probabilities to Cluster 4. From our initial analyses, we could not confidently group ISALO, as our Structure results indicated that ISALO clustered with southern localities (BER‐AMB‐BLK) despite the distance (301 km) and our DAPC results grouped the site into its own central cluster. Upon further investigation, we did find support in our Structure analysis for higher K‐values . In fact, the genetic diversity within ISALO was most similar to the smallest fragment included in our study, SAKA  at the time of this analysis.Although most of Madagascar's ring‐tailed lemur populations are geographically isolated, evidence described herein demonstrate that they have retained high levels of genetic diversity, with moderate genetic differentiation among populations despite being separated by a relatively large geographic distance. We describe levels of genetic diversity , meaning there was a positive correlation between genetic and geographic distances among populations. There are currently no records of subfossil ring‐tailed lemurs outside of their current distribution . Moreover, this dataset forms the basis for future landscape genetics analyses which will be used to infer migration and gene flow across the species' remaining range. Because ring‐tailed lemurs are highly terrestrial and are suspected to utilize river basins as dispersal corridors ; Formal analysis (lead); Investigation ; Visualization (lead); Writing‐original draft (lead); Writing‐review & editing . Jessica A. Knierim: Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Writing‐review & editing . Sohail Khan: Formal analysis (supporting); Investigation (supporting); Writing‐review & editing . Dominique L. Raboin: Formal analysis (supporting); Investigation (supporting); Writing‐review & editing . Sateesh Venkatesh: Formal analysis (supporting); Investigation (supporting); Writing‐review & editing . Tara A. Clarke: Data curation ; Formal analysis (supporting); Funding acquisition ; Investigation ; Resources ; Writing‐review & editing . Frank P. Cuozzo: Formal analysis (supporting); Funding acquisition ; Investigation ; Writing‐review & editing . Marni LaFleur: Data curation ; Funding acquisition ; Investigation ; Resources ; Writing‐review & editing . Richard R. Lawler: Formal analysis ; Visualization ; Writing‐original draft ; Writing‐review & editing . Joyce A. Parga: Data curation ; Formal analysis (supporting); Funding acquisition ; Investigation ; Resources ; Writing‐review & editing . Hantanirina R. Rasamimanana: Formal analysis (supporting); Investigation ; Writing‐review & editing . Kim E. Reuter: Formal analysis (supporting); Writing‐review & editing . Michelle L. Sauther: Formal analysis (supporting); Funding acquisition ; Investigation ; Writing‐review & editing . Andrea L. Baden: Conceptualization (lead); Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Resources ; Supervision (lead); Visualization ; Writing‐original draft (lead); Writing‐review & editing .Fig S1‐S2Click here for additional data file.Appendix S1Click here for additional data file.


Amphistegina lobifera to reveal the evolutionary processes responsible for its biogeographic distribution. We collected specimens from 16 sites encompassing the entire range of the species and analyzed hypervariable fragments of the 18S SSU rDNA marker. We identified six hierarchically organized genotypes with mutually exclusive distribution organized along a longitudinal gradient. The distribution is consistent with diversification occurring in the Indo‐West Pacific (IWP) followed by dispersal toward the periphery. This pattern can be explained by: (a) high dispersal capacity of the species, (b) habitat heterogeneity driving more recent differentiation in the IWP, and (c) ecological‐scale processes such as niche incumbency reinforcing patterns of genotype mutual exclusion. The dispersal potential of this species drives the ongoing range expansion into the Mediterranean Sea, indicating that A. lobifera is able to expand its distribution by tracking increases in temperature. The genetic structure reveals recent diversification and high rate of extinction in the evolutionary history of the clade suggesting a high turnover rate of the diversity at the cryptic level. This diversification dynamic combined with high dispersal potential, allowed the species to maintain a widespread distribution over periods of geological and climatic upheaval. These characteristics are likely to allow the species to modify its geographic range in response to ongoing global warming without requiring genetic differentiation.Understanding the role of dispersal and adaptation in the evolutionary history of marine species is essential for predicting their response to changing conditions. We analyzed patterns of genetic differentiation in the key tropical calcifying species of large benthic foraminifera  Genetic differentiation and distribution of populations can provide clues about processes shaping species evolutionary histories. We examined the genetic signature of globally distributed species of large benthic foraminifera in order to identify mechanisms acting at geological and ecological time scales. We found that while dispersal capacity allows populations to extend their range, geographic distribution was strongly shaped by biogeographic breaks and niche incumbency. Following the cleaning process, individual specimens were placed in 1.5‐ml tubes containing 200 µl of lysis buffer with added Proteinase K. DNA extractions of each specimen were then conducted according to manufacturer's instructions, and DNA concentration was quantified using the DropSense96 . After DNA extraction, between three and eight individual specimens per site generated enough DNA for downstream analysis. Total DNA concentration was standardized to 1 ng/µl of DNA across all samples. For amplification, we used a polymerase chain reaction (PCR) using the PHUSION® Hot‐start II polymerase . We selected specific primers that targeted hypervariable regions in the 18S SSU rDNA. In all cases, DNA templates were amplified utilizing a seminested PCR approach, as the extracted DNA is likely to be dominated by genetic material from the symbionts, and the rDNA template within a single foraminiferal cell is low  following manufacturer's instructions.Total DNA of each individual foraminifera was extracted with the QIAamp® TA Cloning Kit . Amplicons were ligated to a pCR 2.1‐TOPO® vector, transformed into One Shot™ TOP10 chemically competent Escherichia coli cells, and grown overnight on LB‐agar plates containing ampicillin (50 mg/ml). Eight to 16 clones per specimen were selected and placed in 0.5‐ml tubes containing 18.2 Ω MilliQ water. Bacterial cells were lysed through one freezing–thawing cycle, and a final PCR was performed using the primer set [S14f1‐1528R]. Positive PCR products were sequenced in both directions using an ABI 3730xl DNA Analyzer  at BaseClear . The obtained chromatograms were manually checked, complementary fragments of the same sequence were de novo assembled, primer sequences removed from both ends, and consensus sequences were deposited on NCBI under the accession number TBA.The purified PCR product was cloned using the TOPOA. lobifera from NCBI GenBank . Default options were selected for both trees, and in one of them, A. lessonii was indicated as an out‐group. We retained the delimitation returned by the maximum‐likelihood solution. The resulting tree was visualized with iTOL v4  using the Haversine formula in MATLAB v. R2017b. However, we calculated distances between O’ahu and Sicily, and O’ahu and Crete manually using the tracking tool in Google Earth going through the Equator. The Haversine formula calculates the shortest distance over the Earth's surface between two geographic points, and so it calculated these distances over the poles, which is not a relevant ecological representation of the distances between the sites in the Mediterranean and the Pacific Ocean. We estimated the relationship between the patristic genetic distance and geographic distance between all pairs of genotypes averaged per locality using a linear regression model .Based on the LTT plot, lineage I displays shallower diversification than lineage II Figure . Pybus γ3.4R2 = .48, p‐value < .01; Figure Genotypes IIb, IIIa, and IVa, which are restricted to oceanic islands, possess low genetic variability Figures  and 4. I4A. lobifera demonstrates that the species underwent repeated episodes of diversification, which remained morphologically cryptic. Lineage I has an extensive geographic range and demonstrates the high dispersal capacity of A. lobifera, while lineages III and IV are limited to isolated oceanic islands. The most remarkable pattern of the diversification is the large degree of mutual exclusion among genotypes. A similar pattern has also been observed in planktonic foraminifera , and Pohnpei. Similarly, the genotype IIc occurring in Ningaloo reef, Okinawa, and Indonesia is connected through the Leeuwin Current that allows genetic mixing of populations occurring in the Pacific and Indian Oceans , which showed significant divergence from each other in the central Pacific region Figures  and 2. T4.4A. lobifera is a successful Lessepsian migrant, colonizing the eastern Mediterranean Sea , much earlier than the opening of the Suez Canal in 1869 through a different natural water way connecting Indo‐Pacific to the Eastern Mediterranean ; Data curation (lead); Formal analysis (lead); Funding acquisition (lead); Investigation (lead); Methodology (lead); Project administration (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Raphaël Morard: Conceptualization (supporting); Data curation (supporting); Formal analysis ; Methodology ; Writing‐review & editing (supporting). Thomas Edward Roberts: Conceptualization (supporting); Data curation (supporting); Formal analysis (supporting); Writing‐review & editing (supporting). Steve S. Doo: Data curation (supporting); Writing‐review & editing (supporting). Jamaluddin Jompa: Resources (supporting); Writing‐review & editing (supporting). Christiane Schmidt: Data curation (supporting); Writing‐review & editing (supporting). Marleen Stuhr: Data curation (supporting); Writing‐review & editing (supporting). Willem Renema: Conceptualization (supporting); Data curation (supporting); Writing‐review & editing (supporting). Michal Kucera: Conceptualization (supporting); Supervision (lead); Validation (supporting); Writing‐review & editing (supporting).Figure S1Click here for additional data file.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.


Many of the world's major cities are located in coastal zones, resulting in urban and industrial impacts on adjacent marine ecosystems. These pressures, which include pollutants, sewage, runoff and debris, temperature increases, hardened shorelines/structures, and light and acoustic pollution, have resulted in new evolutionary landscapes for coastal marine organisms. Marine environmental changes influenced by urbanization may create new selective regimes or may influence neutral evolution via impacts on gene flow or partitioning of genetic diversity across seascapes. While some urban selective pressures, such as hardened surfaces, are similar to those experienced by terrestrial species, others, such as oxidative stress, are specific to aquatic environments. Moreover, spatial and temporal scales of evolutionary responses may differ in the ocean due to the spatial extent of selective pressures and greater capacity for dispersal/gene flow. Here, we present a conceptual framework and synthesis of current research on evolutionary responses of marine organisms to urban pressures. We review urban impacts on genetic diversity and gene flow and examine evidence that marine species are adapting, or are predicted to adapt, to urbanization over rapid evolutionary time frames. Our findings indicate that in the majority of studies, urban stressors are correlated with reduced genetic diversity. Genetic structure is often increased in urbanized settings, but artificial structures can also act as stepping stones for some hard‐surface specialists, promoting range expansion. Most evidence for rapid adaptation to urban stressors comes from studies of heritable tolerance to pollutants in a relatively small number of species; however, the majority of marine ecotoxicology studies do not test directly for heritability. Finally, we highlight current gaps in our understanding of evolutionary processes in marine urban environments and present a framework for future research to address these gaps. More than 40% of the world's population lives within a narrow coastal band, and urban density near the sea is predicted to continue to rise in future decades , protective structures , and residential or commercial waterfront construction . In addition, ocean sprawl includes structures associated with marine extraction  that may be located farther offshore. While there is no total estimate of the global extent of ocean sprawl, several trends indicate that most of these categories are likely to increase rather than decrease. For example, rising sea levels and the increasing frequency of coastal storms due to climate change ; moreover, discrepancies between modeling and genetic results suggested that patterns of differentiation were shaped both by average migration patterns and by rare events such as storms. While oil and gas platforms are not themselves structures that are typically associated with urbanization, they bear similarities to many urban structures in that they represent hardened, artificial habitat replacing soft‐bottomed habitat, and therefore, studies of these habitats may yield useful insights for predicting evolutionary patterns in urban environments.Ocean sprawl can affect marine evolution via several mechanisms depending on the nature of the artificial environments created, but perhaps the best‐studied effects are on genetic diversity and connectivity. Bishop et al.  review tSiphonaria denticulata (pulmonate limpets) are abundant on sea walls in Sydney harbor, individuals are small in size and the reproductive output of the population is lower, suggesting this artificial habitat may be selecting for these features over evolutionary time scales. A study of hydroid assemblages in harbor sea walls versus natural rocky cliffs demonstrated significant differences between the two habitats, showing that harbor species were more likely to be small, short‐lived, and with a free‐swimming medusa phase—in other words, the harbor environment appears to select for r‐strategists , pollutants acting as mutagens  and heavy metals. Toxic chemical pollutants can come from runoff, industrial dumping, as well as shipping, and include metals, tributyltin (an anti‐fouling compound), PBDEs, PCBs, PAHs, plastics, and microplastics , in which storm water and human sewage are merged and discharged into waterways, is a particularly acute issue that can result in significant ecosystem impacts. These effects include long‐term eutrophication, oxygen depletion and ammonia peaks  can result from terrestrial activities such as deforestation, or marine activities such as dredging and construction, and results in increased turbidity and smothering of benthic organisms , whereas both parameters decreased significantly in another species (U. pertusa). Nielsen, Brownlee, Coelho, and Brown  from the Hudson River. The very large effective population size of this species promotes relatively high standing genetic variation, and genomewide data suggest this standing variation is likely the source for adaptive alleles. Pollution tolerance was also observed in the congener of F. heteroclitus, the Gulf killifish F. grandis, with evidence that introgressed alleles from F. heteroclitus may have played a role in the evolution of tolerance to PAHS, PCBs and  and also by generating new opportunities for transport via ballast water and rafting on marine debris or tar balls. Sessile calcareous organisms such as serpulid polychaetes, bryozoans, barnacles, and gastropods are often found encrusted on floating plastic debris, and this mode of transportation has allowed, in some cases, long‐range dispersal away from native habitat  Do urban pollutants appreciably increase mutation rate in marine species? Previous studies have shown an increase in mutations upon exposure to mutagens such as PCBs and PAHs  were carried out using very few or anonymous markers Table . Newer mAnother valuable direction for future research would be use of historic specimens  to analyze genomic changes over many generations and to assess hypotheses about the origin of adaptive alleles. Here again, new methodologies such as Hy‐RAD and targeted capture have improved the chances of sequencing DNA from old specimens. Of particular interest would be urban marine populations near older cities (>400 years), where evolution has had more time to occur, such as Lagos, Osaka, Jakarta, Panama City, Singapore, New York, Boston, and Sydney. However, a challenge with older cities is that, due to greater urban sprawl, it is often more difficult to find nearby and relevant reference (nonurbanized) sites for comparison.With sea level rise, higher temperatures and increases in extreme weather events, urban dwellers, and policymakers are increasingly aware of the importance of ensuring the resilience of coastal marine ecosystems to protect coastlines and restore biodiversity (Dafforn et al., None declared.


Hepatoblastoma (HB) is a common liver malignancy in children. Our previous study has disclosed the crucial role of STAT3  in HB.Present study was designed to study the circular RNA (circRNA) STAT3 in HB.Gel electrophoresis revealed the circular characteristics of circ-STAT3. Function assays like EdU, transwell and sphere formation assay disclosed the function of circ-STAT3 in HB cells. Mechanism assays including ChIP, RIP, RNA pull down assay demonstrated the macular mechanism underlying circ-STAT3.Circ_0043800, which was originated from STAT3, was up-regulated in HB tissues and cells. More importantly, silencing of circ-STAT3 led to the inhibition on HB cell growth, migration and stem-cell characteristics. Circ_0043800 was predominantly located in the cytoplasm of HB cells. Then, circ_0043800 was found to up-regulate STAT3 via sponging miR-29a/b/c-3p. Besides, we identified that STAT3 overexpression partially rescued silenced circ_0043800, while miR-29a/b/c-3p inhibition completely rescued silenced circ_0043800 on HB cellular biological behaviors. Subsequently, Gli2 (GLI family zinc finger 2) was identified as another target of miR-29a/b/c-3p. Circ_0043800 served as a competing endogenous RNA (ceRNA) to up-regulate both Gli2 and STAT3 via sponging miR-29a/b/c-3p. Moreover, we figured out that Gli2 overexpression completely rescued silenced circ_0043800 on HB cell malignant behaviors. After that, we discovered that Gli2 transcriptionally activated circ_0043800. The in-vivo assays further revealed that circ_0043800 promoted HB tumor growth by up-regulation of Gli2 and STAT3.Gli2-induced circ_0043800 served as the ceRNA to promote HB by up-regulation of STAT3 and Gli2 at a miR-29a/b/c-3p dependent manner. Hepatoblastoma (HB) is a highly invasive malignancy in children and takes up around 50% in pediatric liver cancers . ApproxiWe have previously published a study that lncRNA LUCAT1 promotes cell proliferation, migration, and invasion in HB via regulation on the miR-301b/STAT3 axis . Thus, pEmerging endogenous circular RNAs (circRNAs) were identified in human cancers . CharactIt was also reported that circRNAs participate in the competitive endogenous RNA (ceRNA) pattern. The ceRNA pattern means that circRNAs served as the endogenous sponge of miRNAs to block the inhibition of miRNAs on mRNAs. CircRNAs served as the ceRNA to play their tumor facilitator or suppressor roles in multiple cancers. CircPUM1 sponges miR-615-5p and miR-6753-5p to up-regulate NF-κB and MMP2 and to facilitate ovarian cancer tumorigenesis and progression . CircMLLFifty paired hepatoblastoma tissue samples and adjacent non-cancerous tissue samples were collected between January 2014 and March 2019, with the ethical approval from the Ethics Committee of Qilu Hospital of Shandong University and PLA general hospital. Patients did not receive chemotherapy or radiotherapy before surgery. All samples were snap-frozen in liquid nitrogen and stored at -80 °C.2. RNase R (3 U/μg) was acquired from Epicentre Technologies .Human HB cell lines  and human normal liver cell line (THLE-3) used for thus study were available from the Chinese Academy of Sciences . DMEM supplying with the 10% FBS and 100 U/ml penicillin/streptomycin was procured from Invitrogen  for cell culture purposes at 37 °C in 5% COTotal RNAs were extracted from the collected tissues and cultured cells employing Trizol reagent (Invitrogen), PrimeScript™ RT reagent kit  was then used for generating cDNA. Quantitative analysis was performed with SYBR Premix Ex Taq II (Takara), and the comparative Ct method was applied for data analysis. U6 or GAPDH acted as the normalized gene.Cells of HepG2 and HuH-6 were fixed by 4% formaldehyde and dehydrated, then air-dried for incubation with the circ-STAT3-FISH probe . Following hybridization, cells were rinsed and cultured with Hoechst solution, then analyzed by fluorescence microscope .6 HB cells were rinsed in PBS, then centrifuged for separating the nuclear RNA and cytoplasmic RNA using the PARIS™ kit (Invitrogen). Expression level of circ-STAT3 was examined by qRT-PCR, with GAPDH and U6 detected as control of cell cytoplasm and cell nuclei, respectively1 x 10The shRNAs and NC-shRNAs were synthesized at Genepharma Company  to silence circ-STAT3 and Gli2 in HepG2 and HuH-6 cells utilizing Lipofectamine2000 (Invitrogen). Besides, the miR-29a/b/c-3p mimics/inhibitor and NC mimics/inhibitor, as well as pcDNA3.1/Gli2, pcDNA3.1/STAT3 and NC-pcDNA3.1 were procured from RiboBio. Forty eight h later, transfected cells were reaped for analysis.Transfected HB cells were incubated for 3 h with 100 μL of EdU medium diluent in 96-well plate. Cell proliferation was estimated by using EdU staining kit (Ribobio) as per guidebook. Cells were then fixed and stained in DAPI solution, finally observed under Olympus fluorescence microscope.After transfection, HB cells in 6-well plate were subjected to 14-day cell culture. Then, all cells were first fixed, stained by 0.1% crystal violet, then photographed and analyzed.4 transfected HB cells in serum-free medium, and the lower chamber was supplied with 100% culture medium. Cell invasive ability was examined after 24 h by counting cells on the bottom under microscope.Cell invasion assay was implemented employing the transwell chamber coating with matrigel . The upper chamber was seeded with 5 × 106 HepG2 and HuH-6 cells were plated in the 6-well plate, and then the wounds were created by use of 200-μL sterile tip after reaching about 90% cell density. The cell migration ability was assessed after 24 h under microscope, then photographed.5 x 10The 96-well ultralow attachment plate was commercially acquired from Corning Inc.  for seeding 10 HB cells per well with sphere medium. After 7-day of cell incubation, number of sphere cells was calculated. Sphere efficiency was confirmed by using “the number of cell spheres with diameter greater than 75um in each well” to divide “the total number of original inoculated cells in each well”. Besides, number of cell spheres with diameter greater than 75um in each well was manually calculated under the microscope.After lysing in RIPA lysis buffer, protein extracts were separated on 12% SDS-PAGE, shifted to PVDF membranes and cultured with 5% nonfat milk. The primary antibodies  against Tubulin, GAPDH and SOX2, Nanog, OCT4, STAT3, Gli2 were used for probing membranes. After washing in TBST, membranes were incubated with the HRP-tagged secondary antibodies (Abcam). ECL detection method  was applied for detecting protein signals.Thermo Fisher RIP kit was acquired for performing RIP assay as guided by supplier . In line with the user guide, cell lysates were conjugated with normal control IgG antibody (Millipore) or human Ago2 antibody  in magnetic beads. The recovered RNAs were examined using qRT-PCR.The STAT3, circ-STAT3 or Gli2 fragments covering the wild-type and mutated target sites of miR-29a/b/c-3p were synthesized and inserted into the pmirGLO luciferase vector . The recombinant vectors were named as STAT3-Wt/Mut, circ-STAT3-Wt/Mut and Gli2-Wt/Mut. The primer sequences for STAT3-Mut were shown as follows: STAT3-Mut (forward): 5-aagtacttagtctaccacgatacaaccttgactccctttctcc-3; STAT3-Mut (reverse): 5-atcgtggtagactaagtacttctcactaaaaggccaatacattac-3. The primer sequences for circ-STAT3-Mut were shown as follows: circ-STAT3-Mut (reverse) 5-gcttaatcgtggtaggaggctgttaactgaag-3; circ-STAT3-Mut (forward) 5-cctcctaccacgattaagcattcagcttccttc-3. T4 ligase was used to link the mutant sequences to the pmirGLO vectors. They were co-transfected into HB cells with miR-29a/b/c-3p mimics or NC mimics for 48 h. For gene promoter analysis, HB cells were co-transfected with the pGL3 luciferase vector (Promega) containing circ-STAT3 promoter, and pcDNA3.1/Gli2 or NC pcDNA3.1. Luciferase activity was examined using Dual-Luciferase Reporter Assay System (Promega).For RNA pull down, cell protein extracts were collected for mixing with the circ-STAT3 biotin probe or circ-STAT3 no-biotin probe as control and magnetic beads using the Pierce Magnetic RNA-Protein Pull-Down Kit as instructed (Thermo Fisher Scientific), following qRT-PCR analysis. For DNA pull down, the protein samples were cultured with Biotin circ-STAT3 promoter or No-biotin circ-STAT3 promoter as control and magnetic beads, following western blot analysis.The fixed HB cells were subjected to 15 min’s crosslink, then treated with ultrasonic for shearing chromatin into 200–1000 base-pair. The Gli2 or control IgG antibody was added into chromatin samples for ChIP assay. After adding beads, the immunoprecipitates were assayed by qRT-PCR.Xenograft tumor assay was approved by the Animal Health Committee of Qilu Hospital of Shandong University. The male BALB/c nude mice (6 weeks) were commercially and randomly acquired from Beijing Vital River Laboratory . The transfected HB cells with sh-circ-STAT3#2 or sh-NC were collected for subcutaneous injection into nude mice. For each group, 5 mice were used and a total of 10 mice were used for in-vivo assay. Tumor growth was monitored every 4 days. Nude mice were killed through cervical decapitation after 28-days, tumors were dissected carefully for weigh assessment.The tumor tissues collected from the xenograft tumor assay were initially fixed, then dehydrated, embedded in paraffin. Subsequently, the sections (4-μm-thick) were immunostained for IHC assay using specific antibodies to Gli2, STAT3, Nanog, OCT4, PCNA and Ki-67.p-value below 0.05.Data were all presented as the mean ± standard deviation (SD) from three independent bio-repeats. SPSS  was carried out for statistical analysis. Difference of groups was analyzed by Student’s t-test or ANOVA, with significant level specified as . Then, expression of circ_0043800 and circ_0043804 was detected in HB tissues and adjacent non-tumor tissues. The result revealed that only circ_0043800 was up-regulated in HB tissues while circ_0043804 expression showed no significance in tumor tissues and adjacent non-tumor tissues . Besides, expression of other 12 circRNAs exhibited no significant difference between HB tumor tissues and adjacent non-tumor tissues . We named circ_0043800 as circ-STAT3 for subsequent experiments. As was illustrated in Fig. FigureB revealed the Sanger sequencing as well as the backspliced junction of circ-STAT3. Then, convergent primers were used to amplify STAT3 while divergent primers were used for circ-STAT3 with cDNA and gDNA as the templates. It was revealed in qRT-PCR that circ-STAT3 was amplified by divergent primers only in cDNA while STAT3 was amplified by convergent primers in both cDNA and gDNA . Next, we detected expression of circ-STAT3 and STAT3 by treatment of RNaseR and it was disclosed that RNaseR treatment caused a significant decrease in STAT3 expression while circ-STAT3 expression was not influenced . Moreover, the subcellular location of circ-STAT3 was identified. It was demonstrated in FISH assay that circ-STAT3 was mainly distributed in the cytoplasm of HB cells , indicating that circ-STAT3 played a role at post transcription level. Also, subcellular fraction assay revealed that around 80% of circ-STAT3 was distributed in the cytoplasm of HepG2 and HuH-6 cells . Thus, we determined that circ-STAT3 was upregulated in HB tissues and cells and predominantly located in the cytoplasm.We firstly detected relative expression of 14 circRNAs which are derived from STAT3 in HepG2 and HuH-6 cells normalized to control cells. The 14 circRNAs were identified by the circBase tool . It was . Circ-STAT3 depletion reduced number of EdU positive cells and colonies in EdU staining and colony formation assay . Then, transwell and wound healing assay revealed that number of invaded cells and wound closure were reduced by circ-STAT3 depletion . Further, sphere formation assay revealed lessened sphere formation efficiency in circ-STAT3 silenced cells . Expression of stemness markers  was decreased by circ-STAT3 silence at the mRNA and protein level . Taken together, knockdown of circ-STAT3 played a suppressive role in HB cell proliferation, invasion, migration and stemness.Next, function of circ-STAT3 in HB cells was evaluated. The knockdown efficiency of circ-STAT3 was verified for the next loss-of-function assays Fig. a. Circ-Ssay Fig. b-c. Thenion Fig. d-e. Furtlls Fig. f. Expres, indicating that circ-STAT3 and STAT3 co-existed in RISC. Based on starBase database [. We detected expression of these 121 miRNAs in HepG2 cells and selected the first 10 abnormally expressed miRNAs (5 for up-regulated miRNAs and 5 for down-regulated miRNAs). As was revealed, miR-29a/b/c-3p exhibited the most significant down-regulation in HepG2 cells . Then, we enhanced expression of miR-29a/b/c-3p in HB cells and the overexpression efficiency of miR-29a/b/c-3p was verified through qRT-PCR analysis . Further, we identified that miR-29a/b/c-3p overexpression significantly reduced expression of STAT3 at the mRNA and protein level . The putative binding sites of STAT3/circ-STAT3 and miR-29a/b/c-3p were predicted from starBase database and were mutated for the following assays . The luciferase reporter assay disclosed that relative luciferase activity of wild STAT3 and circ-STAT3 was reduced by miR-29a/b/c-3p mimics compared with NC-mimics. In the meanwhile, mutation abrogated the effects of miR-29a/b/c-3p mimics on luciferase activity of STAT3/circ-STAT3 . Besides, the biotin labeled circ-STAT3 was applied to pull down miR-29a/b/c-3p and it was revealed that miR-29a/b/c-3p was significantly pulled down by biotin labeled circ-STAT3 while no productions were observed in non-biotin labeled circ-STAT3 . Moreover, the RIP assay revealed that circ-STAT3, miR-29a/b/c-3p and STAT3 were abundantly enriched in Anti-Ago2 precipitated RNA-induced silence complexes (RISCs), while no productions were seen in Anti-IgG . Besides that, we identified that miR-29a/b/c-3p promoted cell proliferation, invasion, migration and stemness . After that, we sought to examine the biological functions of cells expressing circ-STAT3 harboring different mutations for miR-29a/b/c-3p (termed as circ_0043800-Mut1/2/3). . We identified that overexpression of circ_0043800-Mut1/2/3 alone had positive impacts on HB cell proliferation, migration, invasion and stem-like characteristic . Whereas, overexpressed circ_0043800 without binding sites with miR-29a/b/c/−3p had no significant effect on above cell functions. More importantly, inhibition of miR-29a/b/c-3p completely reversed the effect of circ_0043800 silencing on cell proliferation . Based on these data, we concluded that circ-STAT3 served as a ceRNA by sponging miR-29a/b/c-3p.Since circular RNAs are widely reported to regulate their host genes, we wondered if circ-STAT3 had the regulatory effects on STAT3 expression. As presented in Fig. database , 121 miR. The next EdU and colony formation assay revealed that STAT3 up-regulation partially counteracted the suppressive effects of circ-STAT3 depletion on HB cell proliferation ability . Transwell and wound healing assay disclosed that cell invasion and migration ability were reduced by circ-STAT3 silence but was partially rescued by STAT3 up-regulation . Moreover, circ-STAT3 depletion-mediated suppressive influence on HB cell stemness was partially restored by overexpression of STAT3 . In this section, we draw the conclusion that STAT3 partially rescued effects of circ-STAT3 depletion on cell proliferation, invasion, migration and stemness.After we have verified the ceRNA mechanism of circ-STAT3, miR-29a/b/c-3p and STAT3, the functional rescue assays were in need. Overexpression efficiency of STAT3 was firstly verified in qRT-PCR analysis Fig. a. The neity Fig. b-c. Tranion Fig. d-e. MoreAT3 Fig. f-h. In t. Expression of these 673 mRNAs in HepG2 cells was evaluated and the top 10 abnormally expressed mRNAs (5 for up-regulated miRNAs and 5 for down-regulated miRNAs) were selected. It was revealed that Gli2 was the most significant up-regulated mRNA with the highest fold change . Importantly, depletion of circ-STAT3 remarkably reduced expression of Gli2 . Moreover, up-regulation of miR-29a/b/c-3p caused a noticeable reduce in Gli2 expression at both mRNA and protein levels . Intriguingly, the suppressive effects of miR-29a/b/c-3p on Gli2 were not as significant as that on STAT3. Next, potential binding sequences of miR-29a/b/c-3p and STAT3 were revealed . We mutated these binding sequences for the follow-up luciferase reporter assay. MiR-29a/b/c-3p overexpression led to the reduced luciferase activity of wild Gli2. When the binding sites were mutated, luciferase activity of Gli2 was not impacted by miR-29a/b/c-3p mimics . Besides, circ-STAT3, miR-29a/b/c-3p and Gli2 were significantly pulled down by anti-Ago2 but not anti-IgG . Thus, we concluded that Gli2 was another target of miR-29a/b/c-3p and was positively regulated by circ-STAT3.Considering the partial effects of STAT3 on circ-STAT3-mediated cellular function, we explored other targets of miR-29a/b/c-3p. Based on the search results of starBase database. The respective number of target genes for miR-29a-3p (777 targets), miR-29b-3p 756 targets) and miR-29c-3p (757 targets) were illustrated in Fig. 6 targets. Up-regulation of Gli2 completely rescued the suppressive effects of silenced circ-STAT3 on cell proliferation, invasion and migration ability . Also, silenced circ-STAT3 impaired cell stemness characteristics, but this reduction was completely restored by Gli2 overexpression . In a word, Gli2 took the complete rescue effects in circ-STAT3 on HB cell proliferation, invasion, migration and stemness.After we have identified Gli2 as another target of miR-29a/b/c-3p, whether Gli2 was required in circ-STAT3-mediated HB cell functions need to be further explored. At first, we verified the up-regulation efficiency of Gli2 Fig. a. Up-regity Fig. b-e. Alsoion Fig. f-h. In a. Meanwhile, silenced Gli2 significantly reduced the expression of circ-STAT3 and STAT3 . The following luciferase reporter assay and DNA pull down followed by western blot analysis revealed that Gli2 could bind to the promoter of circ-STAT3 . DNA motif of Gli2 and the putative binding sites of Gli2 on circ-STAT3 promoter were predicted from JASPAR (http://jaspar.genereg.net/) . We mutated the binding sites and found out that luciferase activity of wild circ-STAT3 promoter was enhanced by the overexpression of Gli2 while mutation led to abolished the increased tendency of luciferase activity . Accordingly, a ChIP assay revealed that compared with IgG, Gli2 antibody contained the significant enrichment of circ-STAT3 promoter . According to these data, we concluded that Gli2 was the transcription factor for circ-STAT3 to up-regulate circ-STAT3.The more significant suppressive effects of miR-29a/b/c-3p on STAT3 than on Gli2 as well as the complete rescue effects of Gli2 on circ-STAT3-mediated cell functions aroused our interest. It has been verified that circRNAs and their host genes could be regulated by the common transcription factor , and GliFig. a. MeanwhAT3 Fig. b. The foAT3 Fig. c-d. DNA t/) Fig. e. We mutity Fig. f. Accordter Fig. g. Accord. Further, we detected mRNA and protein level of stemness biomarkers and the results disclosed that expression of stemness biomarkers was attenuated by silenced circ-STAT3 . In all, circ-STAT3 promoted HB tumor growth in vivo.To further address the effects of circ-STAT3 on HB, the xenograft mouse model was established. As was shown in Fig. Fig. c. FurtheAT3 Fig. d-e. In a.Based on all findings, Gli2-induced circ-STAT3 served as a ceRNA against miR-29a/b/c-3p to elevate STAT3 and Gli2 expression, thus facilitating cell proliferation, invasion, migration, stemness and tumor growth in HB HB is a common malignancy in childhood cancer and has caused a great threat to children health. With the improvement of high throughput sequence analysis, circRNAs have been verified as the crucial contributors in cancer occurrence and progression. However, the role of circRNAs in HB was scarcely reported. STAT3 was a common oncogene in multiple cancers, HB included. Our previous study has disclosed the tumor facilitator role of STAT3 in HB via the ceRNA pattern. Also, STAT3 expression is enhanced by overexpression of NOS3 in Nitric Oxide treated HB cells . PresentCircRNAs were widely reported to regulate their host genes via the ceRNA pattern. For instance, circGFRA1 functions as the ceRNA of GFRA1 by regulating miR-34a in triple negative breast cancer . CircFBLHowever, it turned out that STAT3 partially rescued circ-STAT3 silence-mediated effects while miR-29a/b/c-3p completely rescued that in HB cells, which indicated that circ-STAT3 might sponge miR-29a/b/c-3p to regulate another mRNA. Subsequently, we identified Gli2 as the down-stream target of miR-29a/b/c-3p. The report of function of Gli2 in HB is limited. However, Gli2 was widely reported in hepatoma. Up-regulation of JCAD inhibits the ability of LATS2 to phosphorylate YAP, thus elevating CCND1 and Gli2 expression to promote hepatoma cell proliferation . Down-reThe circRNA-involved ceRNA mechanism is scarcely seen in HB but is commonly revealed in liver cancer. As Su Y et al. revealed, circRNA Cdr1as serves as a ceRNA to promote hepatocellular carcinoma progression through sponging miR-1270 to up-regulate AFP . NUDT21 On the whole, current study revealed that circ-STAT3 served as a sponge of miR-29a/b/c-3p to elevate STAT3 and Gli2 expression with Gli2 as the transcription factor for circ-STAT3. Circ-STAT3 facilitates cell proliferation, invasion, migration, stemness and tumor growth in HB via up-regulation of STAT3 and Gli2, indicating circ-STAT3 as a putative biomarker for HB.Additional file 1: Figure S1. A. Relative expression of 12 circRNAs in HB tissues and adjacent non-tumor tissues was assessed via qRT-PCR. Student’s t-test. B. Sanger sequencing and backsplice junction of circ-STAT3. The symbol “n.s.” indicates no significance.Additional file 2: Figure S2. A. Overexpression efficiency of miR-29a/b/c-3p was assessed via qRT-PCR. One-way ANOVA. B-E. EdU, transwell, wound healing and sphere formation assay revealed the function of miR-29a/b/c-3p upregulation in HB cells. One-way ANOVA. **P < 0.01.Additional file 3: Figure S3. A-D. EdU, colony formation, transwell, wound healing assay exhibited influence of overexpressed circ-STAT3-Mut1, circ-STAT3-Mut2, circ-STAT3-Mut3 or circ-STAT3-Mut1/2/3 on HB cell proliferation, invasion and migration. One-way ANOVA. E-G. Sphere formation assay, qRT-PCR and western blot analyses revealed influence of overexpression of circ-STAT3-Mut1/2/3 on HB cell stemness characteristics. One-way ANOVA. H. Knockdown efficiency of miR-29a/b/c-3p was verified in qRT-PCR. One-way ANOVA. I. EdU assay revealed the rescue effects of miR-29a/b/c-3p inhibitor in circ-STAT3 on cell proliferation. One-way ANOVA. **P < 0.01. The symbol “n.s.” indicates no significance.Additional file 4: Figure S4. Concept map of how circ-STAT3 mediated STAT3 and Gli2 in HB.