While STAT3 has been validated as a target for treatment of many cancers, including head and neck squamous cell carcinoma (HNSCC), a STAT3 inhibitor is yet to enter the clinic. We used the scaffold of C188, a small-molecule STAT3 inhibitor previously identified by us, in a hit-to-lead program to identify C188-9. C188-9 binds to STAT3 with high affinity and represents a substantial improvement over C188 in its ability to inhibit STAT3 binding to its pY-peptide ligand, to inhibit cytokine-stimulated pSTAT3, to reduce constitutive pSTAT3 activity in multiple HNSCC cell lines, and to inhibit anchorage dependent and independent growth of these cells. In addition, treatment of nude mice bearing xenografts of UM-SCC-17B, a radioresistant HNSCC line, with C188-9, but not C188, prevented tumor xenograft growth. C188-9 treatment modulated many STAT3-regulated genes involved in oncogenesis and radioresistance, as well as radioresistance genes regulated by STAT1, due to its potent activity against STAT1, in addition to STAT3. C188-9 was well tolerated in mice, showed good oral bioavailability, and was concentrated in tumors. Thus, C188-9, either alone or in combination with radiotherapy, has potential for use in treating HNSCC tumors that demonstrate increased STAT3 and/or STAT1 activation. Signal transducer and activator of transcription 3 (STAT3) is a member of a family of seven closely related proteins responsible for transmission of peptide hormone signals from the extracellular surface of cells to the nucleus . STAT3 iHNSCC was the first human cancer demonstrated to depend on constitutively activated STAT3 for growth . InhibitSTAT3 has been targeted in HNSCC xenograft models using the small-molecule, STAT3 inhibitor, Stattic, which was shown to enhance IR-sensitivity , 24. How705 followed by STAT3 tail-to-tail homodimerization (SH2 domain of each monomer binds the pY705 peptide domain of its partner). STAT3 homodimers accumulate in the nucleus, where they bind to specific STAT3 response elements in the promoter of target genes and regulate their transcription. Several small molecule drug-development programs have emerged directed at identifying drug-like compounds that target STAT3 at one or more stages of its activation [Ligand-engagement of receptors for growth factors or cytokines , 26 caustivation -29.Using virtual ligand screening, we docked 920,000 small molecules from 8 chemical libraries into the pY-binding pocket of the STAT3 SH2 domain and identified 3 hits–C3, C30, and C188–as direct STAT3 inhibitors. C188 demonstrated the greatest activity of the three , 30. Usiin vitro (EC50= 0.7 μM). To identify more potent STAT3 probes, we used the scaffold of C188 as a reference structure . However, the compounds in this group are, on an average, ~5x less active than the Group I compounds, which suggests the H atom of the 4-hydroxy group  with decreasing activity Figure . Additio50, or -log10IC50) of these compounds with the Phase field data calculated based on their aligned 3-D structures  = 0.58 (4), F-test = 133.0, and a pIC50 error of 0.19. To further validate the model, five leave-5-out training test sets were performed with good results yielding an r2 ≥ 0.90, q2 ≥ 0.57, F-test ≥ 95.8, as well as pIC50 errors of ≤ 0.24. 3-D QSAR was used to visualize the 4 major factors contributing to activity: H-bond donor  study, using the program Phase in Schrödinger (version 2010). These 40 compounds were built and their energy and geometry minimized using the OPLS-2005 force field in Maestro . The aligned ligands Figure  were imps Figure . As show4 Figure , q2 (no.50 = 3.3 – 10.5 μM) than C188  compared to C188 . C188-9 also was found to bind to STAT3 with high affinity (KD=4.7±0.4 nM), as determined by microscale thermophoresis , as well as in the primary human esophageal epithelial cell line (HEEpiC). Seven of 10 cell lines had increased basal pSTAT3 levels . We examWe next examined the effect of C188 and C188-9 on growth of tumor xenografts, which revealed that the greater growth inhibitory activity of C188-9 vs. C188 extended to UM-SCC-17B cell line xenografts. While established UM-SCC-17B xenograft tumors continued to grow in nude mice that received C188 , STA-21-peptide ), S3I-20-peptide ) and XZH-peptide ) were hid pSTAT3 ), a mark50 ~5 μM , 36). Ev50 ~5 μM , 37. Secof 86 μM ), such a50=35 μM ), and BP0=6.8 μM ) show a 0=6.8 μM .Several of small-molecule STAT3 inhibitors, as well as other classes of agents, have been shown to be effective in targeting STAT3 and inhibiting tumor growth in preclinical models, including HNSCC, , 40. In in vitro assays, as well as in tumor xenografts, demonstrated that C188-9 was as effective at targeting STAT1 as it was in targeting STAT3. This is not entirely surprising, given the high degree of similarity between the SH2 domains of STAT1 and STAT3 [In addition to promoting many hallmarks and enablers of cancer, STAT3 activation has been linked to resistance to radiation therapy in cells obtained from several normal tissues and cancers, including HNSCC , throughnd STAT3 .et al developed an IR-resistant, HNSCC cell line, Nu-61, from a xenograft in nude mice that grew out after multiple rounds of implantation and irradiation of xenografts starting with the IR-sensitive HNSCC cell line, SCC-61 [The role of increased STAT1 activity in tumors resistant to IR , 48, as , SCC-61 . Compari, SCC-61 ), was al, SCC-61 . Further, SCC-61 , 23. Our, SCC-61 . These f, SCC-61 . Our stu2 chambers at 37°C in DMEMF12 with 10% FBS and UM-SCC17B, HN30 and HN31 in DMEM with 10% FBS plus other common additives. SCC-61, SQ-20B and SCC-35 were maintained in 7% CO2 chambers in DMEMF12 with 20% FBS and other additives. The primary esophageal line, Human Esophageal Epithelial Cells (HEEpiC), were obtained (http://www.sciencellonline.com/OLDSITE/site/productInformation.php?keyword=2720) from the company ScienCell and maintained in its specific medium the Epithelial Cell Medium-2 (EpiCM-2) and was used within one month of first culturing it.HNSCC cell lines SCC-9, SCC-15, HN5, UM-SCC-1, HN30 and HN31 , SCC-61, SQ-20B, SCC-35  and UM-SCC-17B (obtained from Dr. Thomas E Carey from University of Michigan) were genotyped at 14 loci for authentication by the cell-typing core at MDACC. SCC-9, SCC-15, HN5, UM-SCC-1 were maintained in 5% COUsing C188 scaffold as a reference structure, 2-D fingerprint screening was performed using the Life Chemicals database, which contained over 490,000 compounds. We used 100 percent similarity as cutoff and employed Tanimoto coefficient/Unity/Sybyl/Tripos as the similarity comparing method.The 207 compounds resulted from 2-D fingerprint screening were converted into 3-D structures for analysis as described in 3-D quantitative structure activity relationship (QSAR) study was performed as described in Binding of STAT3  pre-incubated without or with C188/C188-9 to phosphorylated and control non-phosphorylated biotinylated EGFR derived dodecapeptides based on sequence surrounding Y1068  was meas50 values were calculated using the GraphPad Prism.Phosphoflow analysis was done as described in Xu et al . BrieflyLuminex bead-based assays were used to determine levels of pSTAT1, pSTAT3, and GAPDH, as described  as detainorm=Fhot/Fcold where Fcold is the homogeneous fluorescence distribution observed inside the capillary before the IR-Laser is switched on and Fhot is the steady low fluorescence state after the IR-Laser is switched on for 30s. Fnorm was calculated and plotted against the logarithm of the different concentrations of the peptide or C188-9 dilution series, to obtain a sigmoidal binding curve. This binding curve was fitted with the nonlinear solution of the law of mass action, and the dissociation constant KD was calculated.Binding of C188-9 or EGFR Tyr(P)-1068 phosphopeptide to WT STAT3β (127-722) was measured by microscale thermophoresis (MST). C188-9 was titrated between 0.305 and 10,000 nM and EGFR Tyr(P)-1068 was titrated between 0.025 and 800 nM to a constant amount (~80 nM) of fluorescently labeled STAT3 (127-722). Movement of STAT3 under a temperature gradient was measured by recording the change of fluorescence signal as the heated molecules moved away from the point of application of the IR-laser used for heating. This movement was traced in the fluorescence time trace. The change in fluorescence FCells were cultured in triplicates in in complete DMEM ± drug, in ultra-low attachment 96 well plates for 72 hrs or cell-culture treated plates for 48 hrs and viable cells were quantitated using MTT. Optical density (OD) was measured at 590 nm using a 96-well multi-scanner . Relative % viability (viability after any treatment ÷ viability of untreated cells × 100) was plotted along Y-axis. At least 2 replicates experiments were performed and were used for IC50 calculation using GraphPad software.6) were injected into the tongues of athymic, 8–10 week old, male, nude mice . Once tumors were established, mice  were randomized (average tumor vol ~ 15 – 20 mm3) to receive 5 times a week, intraperitoneal injections of either DMSO or C188 (50 mg/Kg) or C188-9 (100 mg/Kg). Tumor volumes were measured twice weekly. Average tumor volumes 6/π × (long dimension) × (short dimension)2) were calculated and normalized to the volume at first day of treatment and plotted Comparison was done by t test (* p<0.05).UM-SCC-17B cells (1.5 × 10Tissue was cryosliced (5μ) with Cryotome and total RNA was extracted using RNeasy Microarray tissue kit  followed by DNase digestion and Qiagen RNeasy column purification . The RNA integrity was verified using an Agilent Bioanalyzer 2100 . High-quality RNA (RNA Integrity number or RIN >9.0) was processed using an Illumina TruSeq RNA sample prep kit following the manufacturer's instruction . Detailed method is provided in Pharmacokinetic (PK) studies to assess plasma levels of C188-9 following IP and oral routes were performed in C57BL/6 mice. PK studies to assess tumor vs. plasma levels of C188-9 were performed in nude mice bearing xenograft tumors. Details regarding these studies are in the Student's t-test or a paired t-test was used to compare control- and C188-9 treated groups as indicated.


Induced mutagenesis was employed to create genetic variation in the lentil cultivars for yield improvement. The assessments were made on genetic variability, character association, and genetic divergence among the twelve mutagenized populations and one parent population of each of the two lentil cultivars, developed by single and combination treatments with gamma rays and hydrazine hydrates. Analysis of variance revealed significant inter-population differences for the observed quantitative phenotypic traits. The sample mean of six treatment populations in each of the cultivar exhibited highly superior quantitative phenotypic traits compared to their parent cultivars. The higher values of heritability and genetic advance with a high genotypic coefficient of variation for most of the yield attributing traits confirmed the possibilities of lentil yield improvement through phenotypic selection. The number of pods and seeds per plant appeared to be priority traits in selection for higher yield due to their strong direct association with yield. The cluster analysis divided the total populations into three divergent groups in each lentil cultivar with parent genotypes in an independent group showing the high efficacy of the mutagens. Considering the highest contribution of yield trait to the genetic divergence among the clustered population, it was confirmed that the mutagenic treatments created a wide heritable variation for the trait in the mutant populations. The selection of high yielding mutants from the mutant populations of DPL 62 (100 Gy) and Pant L 406 (100Gy + 0.1% HZ) in the subsequent generation is expected to give elite lentil cultivars. Also, hybridization between members of the divergent group would produce diverse segregants for crop improvement. Apart from this, the induced mutations at loci controlling economically important traits in the selected high yielding mutants have successfully contributed in diversifying the accessible lentil genetic base and will definitely be of immense value to the future lentil breeding programmes in India. Lens culinaris Medikus subsp. culinaris) is a self-pollinated, annual and diploid (2n = 2x = 14) grain legume crop with a relatively large genome of 41063 Mbp . T. T13]. Tσ2G (an estimate of genotypic variance) = (MSG −MSE)/r; .GCV (genotypic coefficient of variation) = 2bs (an estimate of broad sense heritability) = σ2G/σ2P, where σ2G is the genotypic component of variance and σ2P is the phenotypic component of variance.hP.h2bs/x) X 100, where, h2bs = Broad-sense heritability; σP = Phenotypic standard deviation of the mean performance of treated population; K = 2.64, constant for 1% selection intensity.GA (as % of the mean) =  in all the quantitative phenotypic traits including grain yield per plant, except days to flowering and seeds per pod, for which the treatments effect was comparatively lower  whereas, in the cv. Pant L 406, the highest GCV was obtained with G2 treatment. The estimated heritability shows considerable variation for days to maturity and was the highest at G1 (cv. DPL 62) and at H1+G1 (cv. Pant L 406) treatment. The highest genetic advance recorded with G1 treatment in the cv. DPL 62 and with G2 in the cv. Pant L 406.In the M3 generation. The population of treatments H2+G2 in cv. DPL 62 and H2 in cv. Pant 406, showed the highest gain in mean values of the fertile branches among all. The genotypic coefficient of variation increased in the mutagenized population. The H2 populations in both the cultivars showed maximum heritability for the trait fertile branches.There was a significant increase in mean values of the fertile branches in all the mutagen treatments in both the cultivars in the M2 generation, the mean shifted mostly in a positive direction with negative deviations in extreme treatments. Significant increases in a mean number of pods per plant were observed in lower and moderate doses of mutagens. The mean values were recorded to be increased more in the cv. DPL 62 with highest in H3+G3 than in the cv. Pant L 406 in H2 compared to respective control mean. The GCV was recorded to be higher with all the treatments of mutagens in both the cultivars. The highest GCV was recorded with H1 (cv. DPL 62) and G2 (cv. Pant L 406) treatments. The calculation on heritability and genetic advance showed considerable variation for the number of pods per plant in both the cultivars. The highest estimated heritability and genetic advance were found in H1+G1 and H1 respectively for cv. DPL 62. The cultivar Pant L 406 showed the maximum heritability and genetic advance withG3 rays and G2 treatments respectively.In the M3 generation. The mean values for the treated population differed significantly from the control mean. The genetic parameters were increased in all the treatments of gamma rays and HZ employed alone or in combination. The highest GCV and genetic advance at H1 and heritability at G2 were observed in the cv. DPL 62. The maximum values of genetic parameters for cv. Pant 406 were in G3.The mean values for the number of pods per plant increased over the controls in both the cultivars for all mutagenic treatments selected in the M2 generation, the significant shifts in mean values compared to control were observed in both the cultivars. The mean 100 seed weight (g) of cv. DPL 62 and Pant L 406 increased from controls to the maximum in H1 and H2, respectively. The highest GCV was recorded in H1+G1 in the cv. DPL 62 while the highest estimated GCV was obtained at H3+G3 in the cv. Pant L 406. The highest estimated heritability at H1 and genetic advance at H1+G1 were observed in cv. DPL 62, while the cv. Pant L 406 gave the highest estimated heritability and genetic advances with G3.In the M3 generation. The most effective treatments for the increase were H2 and G3 compared to controls in cultivars DPL 62 and Pant L 406, respectively. The genetic parameters were higher in the treated population as compared to controls. The highest GCV and GA % was recorded in combination treatment of H1+G1 and heritability in H1 in the cv. DPL 62, while the highest estimated GCV at H1+G1, heritability % at G3 and GA % at H1+G1 was obtained in the cultivar Pant L 406.Mean shift for 100 seed weight (g) were observed to be significantly positive in all the treated populations in the M2 population from mutagen treatments for rising M3 generation exhibited an increase in the mean values for grain yield per plant in both cv. DPL 62 and cv. Pant L 406. The mean values in these treated population significantly differed from that in the control. The mean seed yield of the control populations of cv. DPL 62 and cv. Pant L 406 were increased maximum at H1 and H2, respectively in the treated populations. The genetic parameters for grain yield per plant were also increased in the selected treated population as compared to control. The highest GCV was observed at H1, heritability at G2 and genetic advance at H1 in cv. DPL 62, whereas in the cv. Pant L 406, maximum GCV was at H2, heritability at G3 and genetic advance at H2 were observed.The selected M3 generation, mutagen treated population, selected for M3 generation, exhibited an increase in the mean values for grain yield per plant in both cv. DPL 62 and cv. Pant L 406. The mean values in the treated population significantly increased from that in the control. The grain yield per plant per plant increased from control to maximum at G1 in the cv. DPL 62 while at G3 in cv. Pant L 406. The genetic parameters for grain yield per plant increased in the treated population as compared to control. The highest GCV and GA were reported at H1 and heritability at G2 in the cv. DPL 62. In cv. Pant L 406, the highest GCV and GA at H2 and heritability at G3 were reported.In M2 generation, the mean shifted in both positive as well as in negative directions in the treated population. In general, the lower and moderate doses of gamma rays and HZ and the lower doses of gamma rays + HZ combined treatments showed a significant increase in mean yield per plant over the controls in both cv. DPL 62 and cv. Pant L 406. The mean values increased more in the cv. DPL 62 than in the cv. Pant L 406. The genotypic coefficient of variation, heritability, and genetic advance increased over the controls with all the treatments in both the cultivars. In the cv. DPL 62, the highest GCV at G1, heritability at H2+G2 and genetic advance at G1 were recorded highest, whereas GCV and heritability at H2+G2, genetic advance at G2 were highest in cv. Pant L 406. The high heritability coupled with high genetic advance was recorded for a number of the treatments with gamma rays and HZ alone or in combination, indicating that significant gains could be expected from the selection.In M3 generation showed that the mean values increased significantly with each treatment in both cv. DPL 62 and cv. Pant L 406. The genetic parameters increased in the treated population as compared to the controls. The values of GCV and heritability were highest at G2and GA at G1 treated populations of cv. DPL 62, while in cv. Pant L 406, GCV and GA at G2 and heritability at G3 was highest as compared to their respective control means.Data recorded for harvest index per plant in MThe recorded data on days to flowering, days to maturity, nodules per plant and seeds per pod resulted in non-significant deviation in most of the mutagen doses from the respective controls in both the cultivars DPL 62 and Pant L 406. The mean flowering time was reduced in all the treatments while the mean days to maturity were reduced only in few treated populations. The observation on nodules per plant showed positive deviations mostly in lower and moderate doses of mutagens while number decreases at the highest doses of the mutagens used alone or in combination. The seeds per pod in the both the lentil cultivars revealed that most of the single and combination treatments of gamma rays and HZ were not effective in inducing significant deviations in the mean number of seeds per pod.2 generations showed pods per plant , seeds per pod  and 100 seed weight  had strong correlation with grain yield for the cultivars DPL 62 and Pant L 406, respectively. This suggested that the selection based on these traits in the treated populations can be done for screening high yielding mutants.The results of the character association are given in The 12 treated populations and 1 control population from each cv. DPL 62 and Pant L 406 were clustered based on the ten quantitative phenotypic traits recorded. The number of clusters was determined to be three in the population of both the cultivars by the agglomerative coefficient graph using elbow method . The phy2 treated population relative to remaining PC. The second PC explained the most of the variability viz. 22.87% (DPL 62) and 27.80% (Pant L 406) that was uncorrelated and not summarized by the first PC. Therefore, the total variation in the original data set is broken down into independent components and their cumulative variation % was observed to be 84.24% and 79.27 in cv. DPL 62 and cv. Pant L 406, respectively. The PCA analysis revealed that the yield contributed maximum towards divergence in the populations, therefore, confirmed that the yield trait responded desirably to the mutagenic treatments for possible selection of the high yielding mutants. The loading plot of the biometrical traits in first two PCs clearly showed that the yield related traits had contributed considerably towards the overall genetic variation. The distribution pattern also indicated the existence of the significant amount of variability among the mutagenized population for the observed quantitative traits.The traits which contributed in separating the different treated population are presented in The wide genetic variability is prerequisite for initiate a breeding programme. The extent of genetic variation present in the crop genepool determines the success of breeding. Induce mutation technology have been proved to be best among the available techniques for creating a novel genetic blend in crop genome. It facilitates the plant breeders to screen and select the desired combination of expressed economic traits. The study of induced micromutations is an important and tedious work to be accomplished and needs extensive analysis of the yield related quantitative traits by statistical tools for selecting the best improved mutant genotype. It is evident from the several reported works in various crops , 17], , ,19],,,22] tha tha22] t2) and genetic advance (GA %) are needed. The observation on different traits revealed a wide extent of inter-traits variability is induced by the different treatments of gamma rays and hydrazine hydrates alone or in combination. Study on the shift in mean values of different quantitative traits showed mostly desirable shifts in different yield contributing traits with few insignificant exceptions where mutagenic treatments reduce the mean values. Since the large numbers of genes are involved in the expression of quantitative traits and considering the randomness with which mutagen interact with the targeted genome, the variations in both directions from the control means are very much expected. In the study, the analysis of variance using genetic parameters indicated significant variation for all six quantitative traits among the different mutagenized population compared to their control population, confirming the possibility for improvement through selection in the subsequent generations. The higher values of heritability resulted from narrow differences between phenotypic and genotypic coefficients of variation for most of the selected treatment populations in M3 generation showed minimum environmental variances at the expression level of the polygenic traits in both the lentil cultivars. The values for heritability and genetic advances showed an independent trend for most of the treatment populations in most of the quantitative traits which revealed the fact that high heritability and large genetic advance could not be expected always . R. R2) ands[et al.  and manyThe degree of the linear relationship between two traits considered to be the function of selection, gene linkage, and pleiotropy . The chaGenetic distance is defined as “any quantitative measure of genetic difference, be it at the sequence level or the allele frequency level that is calculated between individuals, populations or species” . Phenotyet al., [The inter-cluster distance is useful in determining the parental line in hybridization program and the higher distance between the clusters will give a broad spectrum of variability in the segregating generation. Rohman et al.,  suggeste2 populations was done to categorize the representative traits for phenotypic characterization of the mutated lentil populations, and thereby to identify superior high yielding plants for propagation into next M3 generation. The reduced numbers of the uncorrelated variable (PCs) are resulted by linear transformation of the original variables in the data set. The major contributors to the genetic divergence were found to be yield per plant and harvest index per plant; therefore, further selection on these traits in the subsequent generation may possibly lead to the isolation of more diverse yield trait mutants. Afuape et al. [In the present work, Principal component analysis of the yield related traits of Me et al.  also sug3 generation. Therefore, the high yielding mutant selection from these populations should be considered in future breeding program for developing elite mutant cultivars. Also, the divergent populations could be used for hybridization program for obtaining desirable segregants in the subsequent generations.The study suggests that quantitative phenotypic markers are the useful tool for preliminary assessment of genetic diversity. The high GCV together with high heritability and genetic advance resulted from the mutagenic treatments in the selected mutagenized populations compared to respective parents showed the wide possibility of selection for improving yield trait in the lentil cultivars. The character association studies clearly showed that the significant and positive correlation of pods per plant, seeds per pod and 100 seed weight (g) with yield. The hierarchical cluster analysis grouped the population of treatments and controls into three primary clusters in the lentil cultivars DPL 62 and pant L 406. Hence, selection of mutants must be based on the wider inter-cluster distance with the controls and superior mean performance for yield and yield components. The results of the assessment on induced micromutations for the yield trait indicated significant inter-population divergence have been created by the different mutagenic treatments. Based on the mean quantitative trait data and genetic parameters, six mutagenized population, namely, 0.2% HZ (H2); 100 Gy γ rays (G1); 200 Gy γ rays (G2); 200 Gy γ rays + 0.2% HZ (H2+G2) in cv. DPL 62 and 0.2% HZ (H2); 300 Gy (G3) in cv. Pant L 406, was found to be superior for grain yield in the MS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableDf = Degree of freedom, ** = at p < 0.05 level significance, ns = not significant, based on the One-way ANOVA analysis.(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(i) Experimental field. (ii) Seeds of lentil microsperma cv. Pant L 406. (iii) Seeds of lentil macrosperma cv. DPL 62. (iv) Fertile “dwarf” mutant (v) Fertile “tall” mutant (vi) Fertile normal plant. (vii) Fertile “semi-dwarf “mutant. (viii) Fertile “high yielded bushy” mutant. (ix) Fertile “high yielded tall” mutant. (x) Multiple pods per peduncle.(TIF)Click here for additional data file.


Despite the global value of barley, compared to its wild progenitor, genetic variation in this crop has been drastically reduced due to the process of domestication, selection and improvement. In the medium term, this will negatively impact both the vulnerability and yield stability of barley against biotic and abiotic stresses under climate change. Returning to the crop wild relatives (CWR) as sources of new and beneficial alleles is a clear option for enhancing the resilience of diversity and adaptation to climate change. Southeastern Anatolia constitutes an important part of the natural distribution of wild barley in the Fertile Crescent where important crops were initially domesticated. In this study, we investigated genetic diversity in a comprehensive collection of 281 geo-referenced wild barley individuals from 92 collection sites with sample sizes ranging from 1 to 9 individuals per site, collected from southeastern Anatolia and 131 domesticated genotypes from 49 different countries using 40 EST-SSR markers. A total of 375 alleles were detected across entire collection, of which 283 were carried by domesticated genotypes and 316 alleles were present in the wild gene pool. The number of unique alleles in the wild and in the domesticated gene pool was 92 and 59, respectively. The population structure at K = 3 suggested two groups of wild barley namely G1-W consisting wild barley genotypes from the western part and G1-E comprising those mostly from the eastern part of the study area, with a sharp separation from the domesticated gene pool. The geographic and climatic factors jointly showed significant effects on the distribution of wild barley. Using a Latent Factor Mixed Model, we identified four candidate loci potentially involved in adaptation of wild barley to three environmental factors: temperature seasonality, mean temperature of driest quarter, and precipitation of coldest quarter. These loci are probably the targets of genomic regions, with potential roles against abiotic stresses. Hordeum vulgare L. ssp. vulgare) was domesticated more than 10,000 years ago in the Fertile Crescent [H. vulgare L. ssp. spontaneum. Both taxa are diploid (2n = 14), predominantly self-pollinated and fully interfertile [Archeological evidence suggests that barley  have been identified as responsible for non-brittle rachis in domesticating barley. The btr1-type barley emerged in the Southern Levant prior to the appearance of btr2-types in the Northern Levant [Btr1 gene called btr1b has been reported to be and has been referred to wild accessions from Gaziantep region in Turkey [In the process of IBD, geographical distances confine the gene flow. It is expected that genetic differentiation will increase with increased physical distances between populations . On the n Levant . More ren Turkey . This caIn this study we utilized EST-SSR markers to investigate the patterns of genetic diversity of collected the largest wild barley sample collected to date from southeastern Anatolia. In addition, we incorporated a worldwide collection of diverse landraces and cultivars mainly maintained in gene banks to compare diversity between the two different gene pools. The major objectives of the study were: a) to investigate the genetic structure and diversity of wild barley in Turkey and compare it with the domesticated varieties worldwide, b) to examine the relative importance of geographical and environmental factors on the patterns of genetic variation in wild barley; and c) to identify the potentially most important eco-geographical variables associated with forming the genetic makeup and variation of barley in southeastern part of Turkey.ex situ repositories to increase the sample size and to fill collection gaps in the study area. This lead to a total number of 92 collection sites with sample sizes ranging from 1 to 9 individuals per site. The collection was further enriched with a diverse set of domesticated genotypes. A total of 281 wild and 131 domesticated genotypes (landraces and cultivars) genotypes were selected for the study. Geographic data including longitude, latitude, and altitude were recorded using global positioning system (GPS) to obtain the sampling location of wild barley . Four seeds from individual spikes per genotype were sown under greenhouse conditions. After three weeks, two fresh leaves were used for DNA extraction. Forty-five fluorescence-labeled EST-SSR markers covering all seven chromosomes of the barley genome, were selected for the study ,34 S2 T. PCR reaGenetic diversity parameters including the number of alleles per each locus, observed heterozygosity, gene diversity, polymorphism information content (PIC), and major allele frequency (MAF) were calculated using software Power Marker software version 3.25 . PIC valThe population structure was investigated at three levels (L): across the entire population of domesticated and wild barley (L1), wild and domesticated barley from Turkey (L2), and wild barley only (L3). To infer genetic structure of the population, a Bayesian clustering approach was used using STRUCTURE software version 2.3.4, which assigns individuals to pre-defined clusters (K) based on their membership of a cluster ,39. The www.worldclime.org). The current environmental layers (1950–2000) were downloaded with resolution of 30 arc/sec for 19 bioclimatic variables. For each collection site, the values of 19 bioclimatic variables were extracted and the geographical distribution of wild barley was mapped using ArcGIS 10.3 [vegan package in R v 3.3.2 [http://www.spss.com). We also performed a redundancy analysis (RDA), an alternative method to the Mantel test to examine the relative contribution of environmental and geographic data, and the combination of both in driving genetic structure [vegan package in R [For wild barley, the relationships between genetic diversity and geographical and environmental factors were investigated. Climate data for each collecting site were obtained from the Worldclim database 1.4  implemented in the LEA package in R  to inveshttp://webblast.ipk-gatersleben.de/barley_ibsc) and the Barleymap database (http://floresta.eead.csic.es/barleymap) to determine the candidate genes and annotation of loci surrounding the mapping positions of the SSR markers [Arabidopsis thaliana protein sequences by BLASTP to find the probable orthologues using EnsemblPlants (http://plants.ensembl.org).For this process, we used the latest barley reference genome available in the IPK database  and 363 bp (GBM1002) were obtained across the entire collection . The meaRegarding wild barley a total of 316 alleles with 92 unique alleles were detected with a mean value of 7.9 alleles per locus . MarkersAt the first level (L1) we performed a population structure analysis across the entire collection (n = 412). To determine the most probable value of K, the LnP(D) values from the STRUCTURE output were plotted against K, and the results showed that these values increased gradually after K = 3. This data suggests three major groups in this collection . HoweverAt the second level (L2) we confined our analysis to genotypes originating only from Turkey (n = 323); i.e., all wild  and 42 domesticated barley varieties. The results were consistent with what we obtained for the entire collection, where at K = 2, the wild barley genotypes were separated from the domesticated ones, and at K = 3, the former were separated into two groups . MoreoveAt the third level (L3), the population structure analysis was confined to wild barley only (n = 281). Similar to the previous results, accessions were clustered in the same groups: i.e., G1-E, and G1-W, with 19 being identified as admixed. This result was also confirmed by the population structure analysis of wild barley using TESS software . The samWe used the Mantel and partial Mantel tests to investigate the correlation between eco-geographical variables and genotypic data .The highest correlation value was correlated with environmental and geographical distances (r = 0.6399). Both environmental and geographical distances were significantly but not strongly correlated with the genetic distance . Since environmental and geographical distances were correlated, we performed partial Mantel tests to investigate the pure relationships IBE and IBD. The results showed that genetic distance has a reduced but significant correlation with both geographical and environmental distances . However, the correlation between geography and environment did not show a remarkable reduction (r = 0.5857). Based on the information provided by the Mantel test, it can be speculated that geographical and environmental factors have jointly affected the genetic structure of wild barley in the study area, under two groups (G1-E and G1-W), confirming the existence of a spatial structure in the wild gene pool.We used RDA to further reveal the amount of the genetic variation explained by environmental variables, geographic coordinates and the combination of both .The results indicated that the variation contributed by environmental variables have played a more important role than the geographic variables. By simple redundancy analysis, the impure proportions of climate and geography on genetic variation were found to be significant . However, the partial RDA was computed in order to separately determine the pure variation contributed by the environmental and geographic variables. Similar to the data obtained above, both factors significantly contributed to the genetic variation of wild barley but the pure environmental portion of genetic variation was higher than the geographic variation .According to the results presented in We detected four SSR markers (10%), which possessed at least one allele associated with one bioclimatic variable: GBM1256, GBM1008, GBM1405, and GBM1464 . One allin silico analysis of the associated markers to detect underlying candidate genes (SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1 (SARD1) in Arabidopsis thaliana, an important member of the calmodulin binding protein-like gene family. Of two markers associated with BIO-9, one (GBM1008) was found in the region corresponding to the gene encoding chaperone protein DnaJ (HSP40) and the other associated marker (GBM1405) appeared to underlie the gene encoding a protein belonging to the adenine nucleotide alpha hydrolases-like superfamily, located 306 Kb downstream of another interesting gene similar to serine hydroxymethyltransferase 7 (SHMT7) in A. thaliana. Finally, the marker associated with BIO-19 (GBM1464) was found in a gene region similar to SLK2 which encodes transcriptional corepressor SEUSS in A. thaliana, and was located 179Kb downstream of the gene similar to CML23 encoding calmodulin-like proteins in A. thaliana.Using the IPK database we performed an te genes . The firIn the present study, the genetic diversity analysis revealed a high number of alleles  across the entire collection of wild and domesticated barley. This is comparable to the results previously reported using the same marker set: a worldwide collection of 1,485 barley landraces genotyped with 42 markers with 372 alleles , a worldThe analysis of the population structure across the entire collection resulted in two major clusters of wild barley exist in southeastern Anatolia, indicating a selection pressure during evolution . The fewH. spontaneum in Turkey was not only common, but also at least partly adaptive [We performed Mantel tests and redundancy analysis as a complementary approach to detect the complex multivariate relationships. As stated above, both geographic and environmental  factors were important in explaining different aspects of the population structure of wild barley in Turkey. The significant correlations between geographical and genetic distances as well as between environmental and genetic distances indicate a spatial distribution, influenced by natural selection. Using the Mantel test, a strong significant correlation (r = 0.6399) was observed between geographical and environmental distances. The partial Mantel test showed that genetic distance still has a significant, but reduced correlation with environment or geography . Furtheradaptive . Thus, sadaptive . Similaradaptive –24.Abebe et al.  found thThe associations between EST-SSR alleles and environmental factors identified eight associated alleles from four different loci associated with both temperature and precipitation variables. Of the three environmental variables, BIO-9 (mean temperature of driest quarter) had the maximum number of associated alleles, followed by BIO-19 (precipitation of coldest quarter) and BIO-4 , with two and one correlated alleles, respectively. The distribution of wild barley is shown on the map of Turkey for the selected bioclimatic variables . The wilHordeum chilense from Chile, Castillo, et al. [In this study, GBM1008, GBM1256 (on chromosome 6H), and GBM1405 (on chromosome 3H) were associated with temperature factors while GBM1464 (on chromosome 7H) was associated with the precipitation variable. Among the eight alleles associated with climatic variables, one allele (GBM1464-126) was not present in the domesticated gene pool. In their investigation of 94 samples of , et al.  detected, et al.  reported2+ -dependent peroxygenase activity and have been shown to play an important role in response to drought and salinity stresses [A. thaliana, a positive regulator of plant immunity that promotes the production of salycilic acid (SA) and induces a wide range of SA-dependent and SA-independent genes [A. thaliana. In switchgrass, adenine nucleotide alpha hydrolases have been reported to be involved in flooding tolerance and that are implicated in leaf senescence [SLK2. A recent study reported that SLK1 and SLK2 interact with LEUNIG_HOMOLOG (LUH) forming SLK1-LUH and SLK2-LUH co-repressor [2+ sensors that play critical roles in interpreting crypted Ca2+ signals and regulate various types of proteins, most of them directly or indirectly regulating plant responses to environmental stresses [The loci associated with environmental variables were further searched in the database for putative candidate genes. We found that these associated markers were located in the regions containing candidates involved in various abiotic stresses . The assstresses ,63. Anotnt genes . The marnt genes . Five mant genes ,66. Thesnescence . SHMTs anescence . The marepressor . These cepressor . For exaepressor . Anotherstresses . In concS1 Fig(A), the mean logarithm of probability values, LnP(D) against the number of predefined clusters (K); (B), Magnitude of delta K vs. K values obtained from STRUCTURE HARVESTER.(PDF)Click here for additional data file.S2 FigAt K = 2, blue colour represents wild barley and green colour represents the domesticated group in L2. At K = 3, the wild barley were subdivided into two groups G1-E (light blue) and G1-W (dark blue). Assignment of individuals to each group was based on their membership coefficient (Q).(PDF)Click here for additional data file.S3 FigAt K = 3, wild barley from the western part (G1-W) was separated from those from the eastern part (G1-E), shown in dark blue and light blue, respectively in L3.(PDF)Click here for additional data file.S4 FigAll temperature variables are based on degrees in Celsius except for Bio3, which defined as the percentage and quantifies how large the day-to-night temperatures oscillate relative to the summer-to-winter  oscillations. All precipitation variables are based on millimeter, except for Bio15 and is expressed as a percentage which is a measure of the variation in monthly precipitation totals over the course of the year. Bio15 is the ratio of the standard deviation of the monthly total precipitation to the mean monthly total precipitation .(PDF)Click here for additional data file.S5 Fig®) available at http://www.worldclim.org/bioclim which multiplies the result by 100 (SD * 100), which was designed to preserve significant digits. This variable groups genotypes based on standard variation so that those which have the maximum standard deviation highlighted in red color and lower amounts are in other colors. For instance, red group shows genotypes which their standard variation is between 9200 to 9800. In fact, this group have experienced much thermal variation during years. BIO9: mean temperature of driest quarter . Note that according to values from WorldClim database, temperature data are in °C * 10. BIO19: precipitation of coldest quarter .BIO4: temperature seasonality (The amount of temperature variation over a given year (or averaged years) based on the standard deviation (variation) of monthly temperature averages. The values are based on original program Arc Map Language (AML(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


The Scots pine is often used in the biomonitoring of forests. Studies on the chemical composition plus variability of its needles morphological structure allow for an assessment of the state of environmental pollution. However, in their natural populations, the response of individual trees to stress differs. This study reports on the influence of long-term soil contamination with trace elements on the morphology of the needles, its possible relation to the differentiation of the genetic pool, and their implications for biomonitoring. In the natural and self-renewable pine stand growing near the point polluter , two categories of trees are observed with respect to their health status: pollution-tolerant (T) and pollution-sensitive (S). A detailed analysis of the trace element content of the needles reveals that the concentration of Cd, Zn, Pb, and Cu in the needles is significantly higher in S as compared to T individuals. The metal accumulation pattern decidedly follows the sequence Pb > Cd > Cu > Zn. An analysis of the fluctuating asymmetry (FA) of the needles reveals that sensitive trees showed an FA index ten times higher in comparison to tolerant ones. Moreover, the high differences between these S and T tree groups are also observed in the basic genetic diversity parameters investigated by an analysis of DNA simple sequence repeats (SSR). The concentration of trace elements in pine needles, distinct in sensitive and tolerant trees and in connection with their morphological and genetic characteristics, may reflect an adaptation process. The level of Mg and Fe content in the needles could be a physiological-toxicological index for evaluating trace element “lethality” expressed as Mg and Fe mineral-survival strategies. The example of differences described in this Scots pine population should be taken into consideration in ecotoxicological research to better interpret the obtained results. Pinus sylvestris L.), the main component of the coniferous forests of Europe, is a species commonly used for the passive biomonitoring of environmental pollution , the evaluation of the environmental risks offers researchers the ability to predict what might happen to the local population in the near future. However, the well-known individual variability of each specimen’s response must be taken into consideration when interpreting the results. The Scots pine . As stated by definition, FA represents small, non-directional deviations from perfect symmetry in morphological characters. This is the result of stress acting on the specimens under development  due to their very substantial variability and often non-neutral character, is increasingly used to assess the genetic variation of natural populations growing under strong stress conditions  an investigation of the developmental instability (expressed through FA index) of trees growing at an area chronically exposed to trace metals pollution; (2) a determination of the basic population-genetic parameters using microsatellite DNA markers in order to characterize the genetic pool of the population; (3) the verification of the hypothesis, that the concentrations of Mg and Fe in Scots pine needles could be a physiological-toxicological index of trace metals “lethality”; and (4) an application of data from sensitive and tolerant pine trees for biomonitoring purposes.P. sylvestris were used as bioindicators in determining the soil pollution around the point polluter at Miasteczko Śląskie, South Poland. Trees were growing in a strongly impacted area located 500–1000 m north–northeast of the point polluter. Needle pairs were collected in late summer at intervals of 4 years (2006–2010–2014) in a natural population according to the recommendations of the ICP Manual Forest  of ars 2006––2014 in 3 dm−3 (Gupta and Aten 3 (MARS5 CEM Corporation). The digests were then filtered, and the trace elements were determined by the AAS method. Analyses were performed in duplicate , indicating high repeatability.From each of the 198 specimens , 10 shoots were collected and 3960 needles were measured. Measurements (made twice by two independent persons) were taken under a stereomicroscope to the nearest 0.025 mm, next to the directional asymmetry. Anti-symmetry and trait size were checked according to the protocol described by Kozlov and Niemelä  and KozlThe genomic DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle ©). Amplifications were carried out separately for each marker in a 2720 Thermal Cycler (Applied Biosystems©) according to the protocol described by Celiński et al. (©) capillary electrophoresis system with GeneScan™600LIZ™ as an internal size standard. Individuals were analyzed and genotyped with the GeneMapper version 3.7 software (Applied Biosystems©).For microsatellite genotyping, a total of six loci were initially tested for the presence of amplification products. Four of these loci—SPAG 7.14, SPAC 11.4 . The arithmetic mean and standard error of mean were calculated. The Shapiro-Wilk test did not show the normal distribution of the FA index. Then, the non-parametric analysis of variance  and Wilcoxon test were conducted in order to determine whether statistically significant differences, in terms of the fluctuating asymmetry factor and trace element concentration in needles, occur between groups of trees (Ferguson and Takane NA), the effective number of alleles (NE), the number of private alleles (NAP), the heterozygosity observed (HO) and expected (HE), the heterozygosity individual for particular samples (Hind), the fixation index (F), the Hardy-Weinberg equilibrium test (HWE), and the genetic differentiation (ϕPT). In order to partition the total level of genotypic variance within and among the sensitive (S) and tolerant (T) groups of Scots pine trees, the hierarchical analysis of molecular variance (AMOVA) was calculated using GenAIEx v.6.5 : Pb (302.6) > Zn (95.3) > Cd (6.6) > Cu (5.6). The magnitude of metal pollution was evaluated by a comparison with the reference values (RefV) representing 10, 50, 1.0, and 0.05 mg kg−1 for Cu, Zn, Pb, and Cd, respectively. Only the RefV for Cu fluctuates within mean concentrations in tolerant (14.5 mg kg−1) and sensitive (8.9 mg kg−1) pine needles. This implies that Cu was not a mineral “stressor” impacting the normal growth of the investigated trees. Most striking were the concentrations of Mg as well as Fe, both slightly higher than the lowest values of their ranges: 300–1400 and 100–850 mg kg−1, respectively. Such levels reveal the response of these pines to the negative impact of metals; in particular, to Pb, Zn, and Cd, with visibly higher concentrations of Mg and Fe in the S trees as compared to the T trees.For the content of metals in the soil at a depth of 0–20 cm, the mean values were 322.7, 386.0, 4.79, and 39.6 mg kga et al. . The assa et al.  revealedR2 values particularly for Cd and Zn, and (ii) Fe with the highest R2 values particularly for Pb and Cd. Both groups, i.e., tolerant as well as sensitive, have exhibited responses to Cd that are indicative of the acute toxicity of this metal.The relationships reported in Fig. t test) were significant , suggesting the occurrence of asymmetry. An absence of anti-symmetry was revealed by the normality of the distribution of left minus right needle values (p = 0.19), while the zero mean value (p = 0.17) confirmed the absence of directional asymmetry. Thus, the observed asymmetry was classified as fluctuating asymmetry (FA) . Within groups between particular years, the Friedman ANOVA for S  = 2.46; p = 0.293) as well as for T  = 2.82; p = 0.244) did not show any significant differences (Table r = −0.91) and a medium negative correlation between FA and Fe (r = −0.44). There also was a medium positive correlation between FA and Mg (r = 0.46). The remaining correlations, between FA and Zn, Pb, and Cd, are negative and very weak. In both groups S and T, there are very strong negative correlations between FA and Mg (r = −0.90) and FA and Fe (r = −0.89) and strong positive correlations between FA and Pb (r = 0.77), FA and Cd (r = 0.72), and FA and Zn (r = 0.62). Between FA and Cu, there is a medium positive correlation (r = 0.40).The length differences between left and right needles in the fascicle (Student st Table . In everHind), FA, and concentration of trace metals in needles did not show any significant correlation in group S. In group T, there was a significant, negative, and medium correlation between Hind and the concentration of zinc .The principal coordinates analysis (PCoA) of the FA index, concentration of trace metals, and observed heterozygosity Fig.  showed tNA), i.e., 25 alleles were found in locus SPAG 7.14 in group S while only 18 alleles were noticed in group T. In the three other microsatellite loci, a higher number of alleles were detected in the T group than in the S group (Table NAP) was highly variable among particular SSR loci and the groups (Table HO) varied between loci and the two analyzed groups of trees (Table F), which amounted to 0.057 for the S group and 0.179 for the T group. The Hardy-Weinberg test (HWE) indicated that in the S group, all SSR loci were in equilibrium, while in the T group three of the four SSR loci were not. The Mann-Whitney U test revealed significant differences in the individual heterozygosity (Hind) values . An analysis of molecular variance (AMOVA) revealed that 2 % of total genetic variance was present among the sensitive and tolerant tree groups, while the remaining 98 % was observed within the Scots pine groups  and 52 alleles in the group of tolerant trees (T). There were no significant differences among the sensitive (S) and tolerant (T) groups of trees in the total number of alleles, the average number of alleles per locus, or in the average number of effective alleles Table . SignifiThe analyses performed at regular intervals of time should reflect the relationships between changes in the trees’ condition and changes to their nutritional status. The process of trace elements accumulation in plants and the resulting impacts on mineral nutrient concentrations depend on two basic factors: (i) genetic and biological characteristics, and (ii) mineral levels of the growth medium and abiotic pressure. Both factors interact strongly and shape what we express as the environmental bio-mineral adaptability. The case of the Scots pine, as reported in Table The first two metals are chemically toxic as compared to Cu and Zn, which exhibit phytotoxicity when their concentrations exceed a given threshold. The reference values, as suggested by Markert  and PaisTolerant pines: Pb (258) > Cd (196) > Zn (7) > Cu (1)Sensitive pines: Pb (560) > Cd (328) > Zn (9) > Cu (1.5)Lead and cadmium are the metals that were much more preferably accumulated by the C + 1 old pine needles. Next, their mobility in plants is strongly contrasting with Pb being less mobile (slow translocation), whereas Cd (similarly to Zn) experiences the opposite. The oldest needles may have developed a physiological translocation barriers of assimilates, making the phytotoxicity of the reported metals seem unavoidable. This process shapes and regulates survival reactions, which in turn strengthen the accumulation of vital elements—particularly Mg and Fe within the Scots pine.P. sylvestris, exhibit a specific morphology; for example, reduced leaf area, which in turn is highly efficient in light interception for active photosynthesis. Therefore, we have formulated the hypothesis that the concentrations of both Mg and Fe in Scots pine needles should serve as a physiological-toxicological index for evaluating trace metals “lethality.”It has been evident for a long time that magnesium, as well as iron, plays a key role in the processes of plant photosynthesis; they serve a vital plant mechanism, irrespective of growth conditions  Fe with the highest R2 values particularly for Pb and Cd. The key findings of these patterns confirm the process of mineral mobility with plant assimilates. Magnesium is very mobile, as are Cd and Zn, in contrast to Fe and Pb (Pais and Benton Jones Two R-R patterns are evident: (i) Mg with the highest Lythrum salicaria increased in a lead-polluted environment versus a non-polluted one. Kozlov et al.  was higher in group S (0.765), and the test for Hardy-Weinberg proportions showed highly significant (p < 0.001) heterozygote deficiency in tolerant trees (T) in respect to three loci. This can be interpreted as neutrality of SSR markers, but similar results were obtained in studying enzyme variation. A higher HO value in roughly half of the analyzed loci in P. sylvestris was higher in the sensitive tree group . Therefore, the 2 % genetic differentiation among the two groups of trees  found in our study may indicate the strong selective pressure that leads to the differentiation of the local population. Pollution-sensitive tress appeared to be in Hardy-Weinberg equilibrium, while the pollution-tolerant subpopulation, exhibiting a highly adapted genotype to this specific polluted habitat, had a significant excess of homozygotes in three out of four analyzed loci.According to Dickinson et al. , the lonFST values by 30–50 %. In our studies, the excess homozygosity at these loci looks to be fitness related . Tolerant trees demonstrated a lower degree of genetic variation than pollution-sensitive trees with respect to some nuclear microsatellite loci. This is in agreement with previous studies conducted on the same polluted site using isoenzyme markers , FA, and concentration of trace metals remained unchanged.Since SSRs have been widely applied to the process of elucidating genetic diversity and population structure in plants, there is a discussion over their suitability for this type of research  is a good indicator of environmental stress, and needles of pollution-tolerant (T) and pollution-sensitive (S) individuals exhibited different levels of metal accumulation. The genetic structure of the observed populations exposed to trace elements may be altered by a reduction of their genetic variation, e.g., through selective sweeps which affect the frequency of alleles. If so, changes in allele frequencies may ultimately lead to an adaptation of the populations to the environmental pollution. In the case of the Scotch pine, a high level of pollen migration from the neighboring population, normal in the case of wind-pollinated species, can restore the initial level of variation in the populations exposed to pollution.In the case of In biomonitoring, the selection of examined individuals must be based on the rigid criteria of spatial representativeness. During specimen observation, correct data have to take into account not only the underlying measuring or sampling methodology but also an individual response reaction to stress conditions. Currently, incorporating measurements of genetic diversity for diagnosing the state of environmental pollution is not frequent, but this could change, especially when new data confirm with greater precision the observed interdependencies. These studies show one of the aspects of the correlations between high trace metals contamination and the genetic differentiation of Scots pine populations growing in the polluted environment.


Two main demographic phenomena have substantially changed the condition of elderly: the growth of the urban population and the increase in longevity.The aim of the present review is to investigate how the elderly living in the cities perceive the sense of insecurity compared to those who reside in rural areas, and their Quality of Life (QoL).Studies published from January 2011 to August 2017 were identified on Google and PubMed combining the following terms: “elderly urban/rural QoL” or “old age urban/rural QoL”.We found 18 different papers published. However, there was only one study on how the elderly perceive the violence in the city. Studies on quality of life were not univocal. Studies on depressive disorders in old age were most homogeneous showing a condition worsening in the cities. A study on the perception of violence in US showed in residents of cities and neighborhoods with the entertainment arena and casinos an increase of criminality perception. In contrast, the crime decreased in both above-mentioned neighborhoods.The condition of elderly in the cities is changed considerably in the recent years. It is estimated that this trend will increase in the coming years. We do not know how older people are experiencing these changes and how they perceive the persistence of violence in the cities. Future researches must satisfy this need by addressing the issue with appropriate methodological tools. This is a public health priority. First one is the growth of the urban population. This trend has been so impetuous that was impossible to adapt the architecture and the urban designs, at least in most cities, to the new demographic emergency: in 1950 30% of the world population lived in urban areas, it has increased to 54% in 2014 and it is expected to reach 66% in 2050 [Although most of the new arrivals are made up of young people , the eldThe second phenomenon is the increase in longevity. In the world people aged over 60 were 607 million in 2000, then they became 901 million in 2015, and they will be around 1.4 billion in 2030. In 2050, the population of older persons will become double than of 2015, reaching around 2.1 billion. The people aged over 80, is growing even faster [In a city that seems to grow too fast, why you can't plan the necessary infrastructures? How can you provide the welfare and support to the weakest?In a field where the simplest solutions, as systematic institutionalization of elderly people with low autonomy, are certainly not the best solutions for their quality life (QoL), it isn't certain that they are even the least costly solutions.The above mentioned phenomena are of such an epochal relevance that their study and the implementation of any preventative action on their possible health and mental health consequences should be a priority in the fields of public health and global mental health. The aim of present review is to investigate how the elderly living in the cities perceive the sense of insecurity compared to those who reside in rural areas, and to study relation between this perception and QoL.2We carried out a systematic research on studies that compared “elderly urban/rural quality of life” or “old age urban/rural quality of life” (as key words used in Google and PubMed for search) from January 2011 to August 2017.31). Results were found in Zhejiang Province, China [We found 14 different studies with 18 papers published  who did not participate in the “community” household studies; by the cultural factors as determinants of adherence; by different adherence rates, usually lower in cities and often lower in the more disadvantaged neighborhoods; by the difference of accessibility to a home fixed telephone when searches were conducted on samples selected on telephone directories, or even conducted by telephone.Prospective studies are at the beginning. A recent cohort survey conducted in New Zealand highlighted how the QoL and depressive symptoms were related to: house ownership, economic living standard, urban versus rural residence, length of residence, ethnicity, age, and household composition .Studies on depressive and mood disorders in old age are most homogeneous concerning a condition worsening of elderly in the cities, at least in western societies as found by a German survey , by the Conversely, studies in China -32 and VThe results of such study therefore suggest that is likely a shift of people with high frequency of depression from a brief migratory experience in cities towards the return to villages. A phenomenon that has already been hypothesized in a study repeated over time in Sardinia , 35, 36.In general, all these researches may not be able to answer to questions but may suggest hypothesis on risk and protecting factors to be tested in well-equipped studies and with longitudinal designs . For exaUltimately, this step is necessary to reflect on what aspects need to be investigate and on which factors is possible to create a protective role.3.2About one third of the population of cities, and ever more in the megacities, lives in disadvantaged circumstances that are described as “slums”. Due to the impetuous flux of people to the cities this fraction is predicted to grow tremendously in the next years .A stimulating environment could offer many opportunities to change their lives for people becoming to conditions of extreme poverty or war. The theory of goal striving stress  has beenBut in a changing world, the adaptation has difficulties ever for the newcomers youngest, even more for older people who come from a rural world that has certainly slower pace, and also for who, on the contrary, already lived in suburban neighborhoods that are changing totally their own physiognomy.On the other hand, it is incontestable that some, not all, cities have become the most dangerous places in the world. For example the U.S. homicide rate declined by nearly half (49%), from 9.3 homicides per 100,000 U.S. residents in 1992 to 4.7 in 2011, falling to the lowest level since 1963. However, some cities maintained rates of homicide over 50 per 100,000  or over 40 (Detroit and New Orleans). The same trend was found in Europe that is known as “the safest continent in the world”. However, also some European cities, such as Rotterdam in Holland or Lodz in Poland’s, show rate of homicides really over the average of the continent or the respective countries [However, there is a total impressive lack of studies of how elderly live violence in the city and what consequences it has on their own life and autonomy.It is paradoxical that some indications come from a study not specifically conducted on the elderly and not conducted in particularly deprived areas . This stThis strange result is only understandable if one takes into account that a sample of predominantly old people had difficulty adjusting to a change. Although, this change meant an increase in the number of jobs and economic benefits, the respondents themselves found it.If this happens in suburbs rich and safe enough, it can only hypothesize what is perceived on security of elderly in depressed suburbs, with high rates of crime and an impressive increase in population from new arrivals with different cultures. This is a lack of knowledge that needs to be overcome.The condition of elderly in the cities is changed considerably in the recent years. This trend is estimated not to diminish but will increase in the coming years.However, we do not know how older people are experiencing these changes, how they perceive the persistence of violence in cities and how these phenomena impact their daily lives.Future researches must necessarily satisfy this need by addressing the issue with appropriate methodological tools.Given the proportions of the phenomenon of migration to the cities and the increase in life expectancy, this is a public health priority.


ST values. Haplogroups identified in Brazilian breeds are in agreement with previous findings in South American samples. The low variability observed mainly in locally adapted breeds, indicates that, to ensure conservation of these breeds, careful reproductive management is needed. Additional genetic characterization studies are required to support accurate decision-making.The first horses were brought to Brazil by the colonizers after 1534. Over the centuries, these animals evolved and adapted to local environmental conditions usually unsuitable for exotic breeds, thereby originating locally adapted Brazilian breeds. The present work represents the first description of maternal genetic diversity in these horse breeds based on D-loop sequences. A D-Loop HSV-I fragment of 252 bp, from 141 horses belonging to ten Brazilian breeds / genetic groups  were analysed. Thirty-five different haplotypes belonging to 18 haplogroups were identified with 33 polymorphic sites. Haplotype diversity (varying from 0.20 to 0.96) and nucleotide diversity (varying from 0.0039 to 0.0239) was lower for locally adapted than for specialized breeds, with the same pattern observed for F Horses have played an important role in shaping human civilization and their domestication occurred from several wild populations 4.000 to 6.000 years ago . Over thGenetic characterization of cryopreserved and live animals is an important approach for orientating conservation strategies , China . These s2, 1U Taq polymerase. Amplifications started with an initial denaturation step of 94 °C for 5 min, followed by 35 cycles of 1 min at 94 °C, 59 °C for 1 min, 72 °C for 1 min, and a final extension of 5 min at 72 °C. PCR products were purified with Exo-SAP enzyme and sequenced using a BigDye v3.1 sequencing kit  and a DNA sequencer ABI 3130 (Applied Biosystems) according to the manufacturer's manual.To investigate mtDNA diversity in specialized  and locally adapted breeds (creoles) of horses in Brazil, 141 animals belonging to 10 locally adapted and commercial Brazilian breeds/genetic groups were analysed . AnalyseFst distances between pairs of breeds were carried out using the Arlequin V. 3.0 (http://www.fluxus-engineering.com) was used for calculation of the median joining network  using SeqScape v2.6, as well as other sequences deposited in GenBank. After edition, the sequences  were analyzed in MEGA V.3.1  were expectedly low, varying between breeds , suggesting a low population structure level , or long term breeding strategies, such as with the Crioula, for which a herd book has been maintained for almost 100 years (Genetic diversity is essential to maintain current production needs in several environments, allowing sustained genetic improvement and facilitating faster adaptation to changing breeding objectives . Careful


Eptesicus fuscus) and their cimicid ectoparasite  in the lower Great Lakes region of the United States, in an area of about 160,000 km2. We genotyped 142 big brown bat and 55 C. adjunctus samples at eight and seven microsatellite loci, respectively, and inferred effects of various types of land cover on the genetic structure of each species. We found significant associations between several land cover types and genetic distance in both species, although different land cover types were influential in each. Our results suggest that even in a parasite that is almost entirely reliant on its hosts for dispersal, land cover can affect gene flow differently than in the hosts, depending on key ecological aspects of both species.Identification of landscape features that correlate with genetic structure permits understanding of factors that may influence gene flow in a species. Comparing effects of the landscape on a parasite and host provides potential insights into parasite‐host ecology. We compared fine‐scale spatial genetic structure between big brown bats ( The ave3.1C. adjunctus (one population at Clec104 and Cle015 and another population at Clec104). These incidences of deviation from Hardy–Weinberg equilibrium were not systematic across loci, which would have suggested presence of null alleles, or across populations. Therefore, we retained these two markers and two populations for our analyses. Genetic diversity indices were overall higher in big brown bats than in C. adjunctus across microsatellite markers  across the whole dataset were, on average, lower for big brown bats than for C. adjunctus .We found no significant evidence, after Bonferroni correction, of Hardy–Weinberg disequilibrium in big brown bats, nor linkage disequilibrium in either species. We found three significant cases of deviation from Hardy–Weinberg equilibrium in rs Table , and val3.2R2 = 0.04; Table p < .01) and proportion of open land cover (p < .01), but a negative relationship with developed land cover (p = .034). These results suggest that geographic distance and open land cover may act to limit gene flow in big brown bats, while developed lands may facilitate gene flow.In big brown bats, geographic distance, proportion of open land cover, and proportion of developed land cover had significant relationships Table  with genC. adjunctus, proportion of forested land cover and proportion of wetlands both had a marginally significant relationship with genetic distance , while the effect of wetlands was negative (p = .04). These results suggest that forests may act to limit gene flow in C. adjunctus while wetlands may facilitate gene flow.In 44.1First, our results support an earlier finding by Vonhof et al.  of a sigC. adjunctus. These were different than the types of land cover found to affect big brown bat genetic distance, even though C. adjunctus almost entirely depends on its hosts to move outside of roosts  compared to its host, the steelhead trout (Oncorhynchus mykiss), led to parasite genotypes providing more accurate population assignments in the host than could be obtained by examining genotypes of the host itself (Criscione, Cooper, & Blouin, Although there are many examples in which host and parasite movement or gene flow are correlated (Bruyndonckx, Henry, Christe, & Kerth, C. adjunctus than in the big brown bat. Two other studies on C. adjunctus conducted at two different spatial scales also found a much higher degree of genetic differentiation in the parasite (Talbot et al., C. adjunctus is a weak generalist ectoparasite of highly mobile hosts, with a generation time that is likely much shorter than that of its hosts. Results from our landscape analyses suggest that there may be additional differences between C. adjunctus and its bat hosts in the location and timing of gene flow that contribute to their different genetic structure.Our results support the pattern of higher differentiation in the parasite, with higher pairwise genetic distances in C. adjunctus can use several different bat species as hosts. In a range‐wide study of the genetic structure of C. adjunctus, Talbot et al. (C. adjunctus may switch host species somewhat regularly, a situation expected for a generalist ectoparasite. It is possible that the different responses of big brown bats and C. adjunctus to landscape composition partly reflect the fact that other bat species, such as M. lucifugus or M. septentrionalis, are also contributing to C. adjunctus gene flow. However, several key aspects of the ecology of big brown bats, including the use of wetlands for foraging, the use of temporary roosts while foraging, and seasonal patterns of gene flow, are shared with other bat species that are potential hosts of C. adjunctus (Adam & Hayes, C. adjunctus genetic differentiation arise not just from the behavior of big brown bats, but from the behavior of multiple potential host species. Furthermore, because big brown bats are among the more widely dispersing of C. adjunctus' potential hosts, this bat species is likely to determine the upper limit of gene flow, and hence patterns of genetic differentiation, in the parasite.Although all parasite samples used in this study came from the body of big brown bats or in a roost inhabited by big brown bats, t et al.  noted moWhile the effects of the landscape on gene flow and genetic structure of many animal species have been described (Manel & Holderegger, None declared.BT's work involved conception of the study, collection of some samples, execution of genetic and statistical analyses, and writing the first draft. NK coordinated the study, supervised the collection and interpretation of genetic data, and revised the writing. BF contributed to the collection of data, coordinated the study, supervised the interpretation of data, and revised the writing. MJV and HGB contributed with most of the sample collection and helped in the interpretation of data and revision of the writing. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Editorial on the Research TopicMental Imagery in Clinical DisordersMental imagery refers to the simulation or recreation of perceptual experience across different sensory modalities , 2. The van Veen et al. report two studies that examine how the speed of eye movement (EM) interacts with the emotionality and vividness of negative memories. Although a high rate of EM was associated with significant reduction in emotionality and vividness, contrary to expectation, the rate of EM did not interact with the extent of reported image vividness. The study reported by van Schie et al. examines the effect of EM on the retrieval of cued negative images acquired through word–image paired association. Dual-task EMs were found to have no effect on both self-rated vividness and emotionality of the images or latency responses. These studies highlight the importance of achieving greater understanding of the complex relationship between increased working memory load and selective interference effects on trauma-related imagery.One central theme across the topic is articles that have focused on the role of mental imagery in memories for negative events. Over the last decade, there has been a considerable impact in this area of, for example, working memory theory and dual task methodology  on pNelis et al. takes a different but equally important approach by examining the recall of positive rather than negative life events. Two studies are reported in which participants are asked to retrieve positive autobiographical memories followed by induction of either image- or verbal-based processing styles. A concrete/imagery-based processing style was associated with a larger increase in positive effect in comparison to verbal processing, which is consistent with the perspective that mental imagery can serve as an “emotional amplifier” of personal memories .A second theme represented in the topic explores the contribution of mental imagery across more diverse clinical disorders than those associated with trauma memory. Klein and Moritz, they argue that intrusive mental imagery lies on a continuum where the boundary between experiences associated with clinical disorders and those experienced by the wider population is not clearly defined. They discuss the findings from recent online studies investigating major depressive disorder (MDD) and obsessive–compulsive disorder (OCD), which find 1 in 5 patients with OCD and 1 in 10 patients with mild-to-moderate MDD report strong to extreme mental imagery associated with their pathological cognitions. The opinion article by May et al. focuses on the Elaborated Intrusion Theory (EI), originally developed as a theoretical framework to account for substance-related craving, but here considered in relation to understanding clinical disorders such as PTSD and suicidal imagery. They argue that EI theory can provide a “roadmap” to help treat mental imagery of maladaptive goals across a range of different clinical disorders. This approach includes identifying those situations associated with greater risk for temptation and loss of control and methods for reducing attention to intrusive imagery such as mindfulness or acceptance training.In the opinion article by Cili and Stopa who apply the self-memory system (SMS) theory of autobiographical memory  appears less important than its valence or content. The opinion article by Mace summarizes his work on involuntary autobiographical memory chains, in which the involuntary retrieval of one memory triggers other additional memories to quickly spring to mind. Mace’s theoretical account has been very useful in understanding how contextual priming of involuntary trauma memory might occur .Missbach et al. and Kemps and Tiggemann both discuss the function of mental imagery in the context of food consumption and food cravings. Missbach et al. argue that research on mental imagery can help better understand the eating behavior and also aid the design of more effective individual-level interventions. It can also help explain why some individuals are more successful self-regulators in their consumption of food than others. In a similar vein, Kemps and Tiggemann discuss the growing evidence that mental imagery plays a key role during the subjective experience of food craving, including an evaluation of imagery-based craving reduction techniques. These techniques use modality-specific cognitive tasks to interfere with mental imagery and help suppress associated craving. This field of research is valuable in relation to understanding mental imagery in clinical disorders as it helps suggest new avenues to explore for effective clinical interventions for treating involuntary imagery-based cognitions.The opinion articles by In conclusion, we believe that the range of submissions presented in this research topic make a strong contribution to help in establishing a theoretical and methodological foundation that can enable the effective comparison of imagery across different disorders and across different domains. There are informative parallels that can be drawn between the literature on clinical disorders and current theoretical models that assign a functional role for intrusive imagery during craving and addiction. Mental imagery processes may also underlie the effectiveness of clinical interventions such as imagery rescripting, imaginal exposure in Cognitive Behavioral Therapy, schema-focused therapy, and cognitive bias modification training. Nonetheless, there still remains an ongoing need for further systematic comparison of the role of imagery across different disorders, with the goal of establishing those common elements, which are most relevant to investigate mental imagery within the context of clinical psychology.All authors drafted the manuscript and provided critical revisions. All authors approved the final version of the manuscript for submission.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.


Thuja koraiensis Nakai is an endangered conifer of high economic and ecological value in Jilin Province, China. However, studies on its population structure and conservation genetics have been limited by the lack of genomic data. Here, 37,761 microsatellites  were detected based on 875,792 de novo-assembled contigs using a restriction-associated DNA (RAD) approach. Among these SSRs, 300 were randomly selected to test for polymorphisms and 96 obtained loci were able to amplify a fragment of expected size. Twelve polymorphic SSR markers were developed to analyze the genetic diversity and population structure of three natural populations. High genetic diversity  and moderate population differentiation  were found in this species. Molecular variance analysis suggested that most of the variation (83%) existed within populations. Combining the results of STRUCTURE, principal coordinate, and neighbor-joining analysis, the 232 individuals were divided into three genetic clusters that generally correlated with their geographical distributions. Finally, appropriate conservation strategies were proposed to protect this species. This study provides genetic information for the natural resource conservation and utilization of T. koraiensis and will facilitate further studies of the evolution and phylogeography of the species. Thuja koraiensis Nakai, an evergreen coniferous member of Cupressaceae, is mainly present in the Korean Peninsula and Changbai Mountain area of China [T. koraiensis populations, and their natural regeneration is usually poor [T. koraiensis has high adsorption ability of fluorine and therefore plays an important role in environmental protection [of China . A high lly poor . In 1992lly poor  and was lly poor . T. koraotection . Its woootection .T. koraiensis have focused mainly on its growth [Tapiscia sinensis [Pulsatilla patens [Antirrhinum charidemi [Origanum compactum [T. koraiensis and related species, making it difficult to implement feasible strategies for the conservation of this valuable endangered conifer tree.Previous studies of s growth , morphols growth ,7,8, ands growth , as wells growth ,10,11. Ts growth ,13. Moles growth ,15. Amons growth ,17,18. Tsinensis , Pulsatia patens , Antirrhharidemi , and Oriompactum , have beT. koraiensis has been hindered by a lack of genomic information, which are usually not available for rare and endangered species. The rapid developments of next-generation sequencing (NGS) technologies offer the possibility to generate large amounts of DNA sequences and develop molecular marker from nonmodel plants such as Ziziphus jujube [Hevea brasiliensis [Pistacia vera [Solanum melongena [Sisymbrium austriacum [Gossypium hirsutum [Arachis hypogaea [Lancea tibetica [The development of SSR markers for s jujube , Hevea biliensis , and Piscia vera . Restriccia vera ,27. Untielongena , Sisymbrstriacum , Gossypihirsutum , Arachishypogaea , and Lantibetica . Moreovetibetica ,34. Thustibetica .T. koraiensis populations. The genetic resources and genic SSR markers collected will substantially contribute to further evolutionary and population genetic studies in Thuja and other related species.The main aim of this study was to develop SSR markers based on RAD sequencing data, analyze the genetic diversity and population structure, and design conservation strategies for three natural populations including 232 individuals in northern China. This represents the first comprehensive description of the genetic diversity and population structure of existing natural T. koraiensis populations from May to June 2016. Five extant natural forestry centers were investigated in Jilin Province, but T. koraiensis samples were found and collected in only three populations. In Lenggouzi , more than 1000 trees were identified, and 155 samples were collected. Fifty individuals were found in Sandaogou , and 26 samples were collected. In Dajinggou , approximately 600 trees were identified, and 51 samples were collected  and Qubit 3.0 Fluorometer .A field survey was conducted on the extant ollected , Table 1EcoRI; then, the samples were heat-inactivated at 65 °C for 25 min. The P1 adapter was ligated to the products of the restriction reaction after digestion. One microliter of 10-µM P1 adapter was added to the sample, along with 1.5 µL T4 DNA Ligase and 2.5 µL 10× T4 DNA Ligase Buffer, and the reaction was incubated for 60 min at 4 °C, 15 min at 20 °C, and then held at 4 °C. Immediately after, DNA fragmentation was conducted; the VAHTS Fragmentase  was mixed thoroughly by gently vortexing for 3 s, and then the tube was transferred to ice. The fragmentation mix contained 25 µL DNA, 3 µL 10× VAHTS Fragmentase Reaction Buffer, and 2 µL VAHTS Fragmentase; and was incubated for 25 min at 37 °C and held at 4 °C. Samples were then separated by electrophoresis through a 2% agarose gel. The DNA fragments from 350 to 450 bp were isolated using a QIAquick Gel Extraction Kit  and plused poly(A) tail. The following components were added to 40 µL of reaction liquid obtained from the previous step: 2 µL nuclease-free water, 5 µL End Repair Reaction Buffer (10×), and 3 µL End Prep Enzyme Mix, followed by incubation at 25 °C for 20 min and 72 °C for 20 min, and was then held at 4 °C. One microliter of 10 µM P2 adapter was ligated, as described above for P1. The ligated product was purified using 1.0× Agencourt AMPure XP Beads .The DNA samples were processed into RAD libraries in a manner similar to that reported by Baird et al. . BrieflyPolymerase chain reaction (PCR) enrichment of the library was performed in PCR reactions of 50-µL volume that contained 25 µL 2× Kapa HiFi PCR Mix , 1.25 µL Universal PCR Primer/i5 Prime, 1.25 µL Index (X) Primer, and 2.5 µL nuclease-free water. The PCR conditions were: initial denaturation at 98 °C for 45 s, then 15 cycles of 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 7 min. PCR products were purified using 1.0× Agencourt AMPure XP Beads following the manufacturer’s protocol, and then pooled together using the Qubit 3.0 Fluorometer  and Qsep100 Capillary electrophoresis apparatus . Sequencing was performed by Illumina Hiseq 2500 platform  that generated paired-end reads of 150 nucleotides. The preparation of DNA sequencing libraries and deep sequencing and analysis were performed by Beijing Ori-Gene Science and Technology Corp., Ltd. . The raw data have been deposited in the NCBI Sequence Read Archive under the accession number SRR5338071.http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/). Adapter sequences, primers, Ns (unknown bases), short segments (<60 bp), and reads with quality scores below 30 were trimmed. The contigs were obtained using Trinity (http://trinityrnaseq.github.io/) and FLASH (http://www.cbcb.umd.edu/software/flash/) to splice high-quality sequences. Then, CD-HIT (https://github.com/weizhongli/cdhit) was used to cluster the sequences and remove the redundancy.For potential sequencing issues and contaminants, raw sequencing reads were filtered using FastQC (-5) was performed against the public databases, including TrEMBL (http://www.ebi.ac.uk/uniprot), Swiss Institute of Bioinformatics databases (Swiss-Prot) (http://www.expasy.ch/sprot), protein families (Pfam) database (http://pfam.xfam.org/), NCBI nonredundant protein (Nr) database (http://www.ncbi.nlm.nih.gov), Cluster of Orthologous Groups for eukaryotic complete genomes (KOG) database (http://genome.jgi-psf.org/help/kogbrowser.jsf), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg). Based on the results of the Swiss-Prot and TrEMBL databases annotation, the gene ontology (GO) annotation of the contigs was obtained by Blast2GO [For functional annotation of the de novo-assembled contigs, BLASTX alignment . The parameters were set as follows: the minimum repeat unit was defined as 10 repeats for mononucleotide motifs; six repeats for dinucleotide motifs; and five repeats for tri-, tetra-, penta-, and hexanucleotide motifs. SSR primers were designed using Primer3 (https://sourceforge.net/projects/primer3/). Primers were designed according to the following criteria: amplified regions that ranged from 100–350 bp, primer annealing temperatures that ranged from 55–60 °C, optimal primer length that ranged from 18–24 bp, and GC content that ranged from 40–60%.Sequences that contained SSR motifs were identified using the MISA search tool . These primers pairs were validated by PCR using the M13-tail technique, where an M13-tagged sequence (5′-TGTAAAACGACGGCCAGT-3′) was added at the 5′ end of all forward primers to allow fluorescent labeling . PCR amplifications, product separation, and scoring were performed as described .AN), effective number of alleles (EN), observed heterozygosity (OH), expected heterozygosity (EH), Shannon’s information index (I), and fixation index (F), were assessed at each locus and population levels using GeneAlEx version 6.5 [stF) and gene flow (mN) were also calculated using GeneAlEx version 6.5 [The genetic diversity indices, including the number of alleles  that contained 37,761 SSR markers were identified. Mononucleotide repeats were the most abundant, and accounted for 16,802 sequences (44.50%), followed by dinucleotide  and trinucleotide  motifs  motifs . The fre) motifs . The mosraiensis .EH values, respectively, and 0.798 and 0.124 for PIC values, respectively. Ten of the 12 polymorphic markers showed HWE, while the others showed significant deviations from HWE after a Bonferroni correction, which was probably because of small sample size and heterozygote deficiency  to 7.667 (LGZ), with a mean of 5.481, and the EN varied from 2.235 (DJG) to 2.750 (SDG), with a mean of 2.514. The average OH and EH values were 0.644 and 0.548, respectively, and ranged from 0.537 (DJG) to 0.766 (LGZ) and from 0.491 (DJG) to 0.582 (SDG), respectively. The I values, indicative of genetic diversity, had an average of 1.008. The average value of F was −0.128 and ranged from −0.036 in the DJG population to −0.279 in the LGZ population , Yushania brevipaniculata  [Epimedium sagittatum (average contig length = 224.9 bp) [T. koraiensis assembly, the total number, N50 value and average length of contigs are all superior to other RAD-sequenced species, which indicates better quality contig assembly [T. koraiensis assembled contigs was 41.38%, which is consistent with results from RAD sequencing studies in A. faberi (44%) [S. austriacum (41.5%) [Arabidopsis thaliana , V. vinifera [C. cardunculus (37.4%) [H. annuus (36.2%) [EcoRI (GAATTC) with rich AT may bias the dataset towards low GC content. These genome sequences will provide important information for developing molecular markers and conducting genome research on T. koraiensis.In this study, the sequence assembly generated 875,792 contigs with an average length of 262 bp (N50 = 274 bp). The number of contigs for dunculus , Heliants annuus , Arundina faberi , and L. tibetica , suggest contigs . The N50 240 bp) , and Epi24.9 bp) . In the assembly . The GC ri (44%)  and S. a (41.5%) ; somewhavinifera , C. card (37.4%) , and H.  (36.2%) , althougT. koraiensis contigs was searched against seven public databases. In the GO analysis, biological process was the most significantly enriched GO term, comprising 50,298 annotations, and metabolic process  was prominent, indicating that these are involved in important metabolic activities. The conifer species, including T. koraiensis, are adversity resistant, especially in cold resistance, but the mechanisms underlying this adaptation are unknown. In the KEGG pathway annotation, 1133 contigs were identified in signal transduction, such as ATP-binding cassette transporters (ABC transporters) and cyclic adenosine monophosphate (cAMP) and adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathways, which are relevant to stress responses. Among the environmental adaptation pathways, the circadian clock plays important roles in activating the adaptation of a species to the local environment by regulating physiological activities [Sabina chinensis [Platycladus orientalis [T. koraiensis.The functional annotation of tivities . In totahinensis  and Platcess 30,7 was promT. koraiensis is higher than those observed in S. austriacum [G. hirsutum [A. hypogaea (2.9%) [T. koraiensis and closely related species. In previous studies, di- or trinucleotide motifs were identified as most prevalent for most organisms [T. koraiensis revealed that mononucleotide motifs were the most abundant (44.50%), followed by dinucleotide (39.18%) and trinucleotide (13.56%) motifs. These results agree with other observations in Prunus armeniaca [A. thaliana, and Populus trichocarpa [G. hirsutum and S. melongena (AT/AT), but the most abundant trinucleotide motif is different from these species  [T. koraiensis, which strongly supports previous studies which indicated that CG/GC and CCG/CGG repeats are very infrequent in a large number of dicotyledonous plants, but the most predominant in monocots [Based on these contigs, we successfully developed 37,761 SSR loci. The frequency of RAD-derived SSR occurrence in striacum , G. hirshirsutum , and A. a (2.9%) , which irganisms , but therganisms ,61,62. Trmeniaca , A. thalchocarpa . The mosirsutum) ,30). It monocots ,65,66. Amonocots ,67.T. koraiensis individuals from three natural populations, with mean AN and EH values of 5.481 and 0.548, respectively. The genetic diversity of T. koraiensis was slightly lower than that of conifer species that have widespread geographic ranges, including Thuja occidentalis  [Pinus densiflora  [P. orientalis  [Paeonia jishanensis  [Taxus wallichiana  [Pinus dabeshanensis  [OH (0.644) was slightly greater than that of EH (0.548), and the value of F was negative at the population level, indicating that excess heterozygosity was existed within the entire natural distribution range of the species.A relatively high genetic diversity was found using the SSR marker analysis in 232 lations) , Pinus dlations) , and P. lations) . Comparelations) ,72. Howelations) , and Pinlations) . The avestF = 0.048–0.078) and weak population structure were found among the three natural populations, which may be subject to local habitat conditions. The AMOVA analysis revealed that most (83%) of the total molecular variance was within-population, which are similar to results obtained for other Cupressaceae species including Taxodium distichum (81.47%) [T. occidentalis (88%) [Juniperus thurifera (90.50%) [T. koraiensis is much greater than that within P. orientalis, in which the genetic variation was only 1.25% among populations, with greatest variation among individuals within populations and within individuals (98.75%) [P. orientalis compared with those of T. koraiensis. The genetic differentiation into distinct populations is strongly influenced by genetic drift, gene flow, long-term evolution, mating systems, selection, and mutations [mN value among T. koraiensis populations ranged from 2.940 to 4.958, indicating the frequent flow of genes and continuous distribution of populations. The population structure analysis divided the three populations into three clusters that were significantly related to their geographical origins, and some trees from the populations had a mixed ancestry, especially in the LGZ and SDG populations. According to botanical characteristics, T. koraiensis is wind-pollinated and its pollen can spread over long distances, which may explain the high level of genetic diversity. The studied trees of T. koraiensis were sampled within a 220-km radius of Jilin Province. The relatively small geographic range and anemophilous pollination could produce more frequent gene exchange events and result in little differentiation between populations.Moderate population differentiation (pairwise (81.47%) , T. occiis (88%) , and Jun(90.50%) , based o(98.75%) . This vautations . In thisT. koraiensis of LGZ, SDG, and DJG should serve as a valuable baseline for future monitoring of the effectiveness of a conservation strategy to maintain and restore genetic diversity in this conifer.The main goal of conservation is to develop a suitable strategy for maintaining current genetic diversity and ensuring the long-term evolution of an endangered species ,81. In t