Except for soil zinc (Zn) concentration, all the nutrient concentrations were significantly higher in the topsoil (0–15 cm), compared with two lower soil depths indicating that Zn was intricate by changes in soil environmental conditions, root acquisition, and/or leaching to lower soil depth. Leaf N concentration significantly increased over time following seasonal environmental fluctuations, tree growth, and development. Thus, leaf N concentration remained above the optimum nutrient range implying lower N requirement under irrigation scheduling with SmartIrrigation, an App used to determine the daily irrigation duration to meet tree water requirement and split fertigation techniques. Root Manganese (Mn) and Zn concentrations were significantly higher in the root tissues of the treated than the control trees and translocated to the leaves accordingly. Meanwhile, a significantly higher LAI for trees budded on Swingle (Swc) rootstock however, larger TCV for trees budded on Volkameriana (Volk) rootstocks. The trees had significantly larger TCV when the trees received a moderate N rate during early study years and under foliar 9 kg ha−1 coupled with the ground 9 kg ha−1 Mn and Zn treatments during the late study years. Therefore, split ground application of 224 kg ha−1 of N, foliar applied 9 kg ha−1 coupled with ground-applied 9 kg ha−1 Mn and Zn were the suggested rates to sustain the essential leaf nutrient concentration within the optimum ranges and improve the deterioration of vegetative growth associated with HLB-induced problems of citrus trees.The fate of foliar and ground-applied essential nutrients is the least studied topic under citrus greening or Huanglongbing (HLB)-affected citrus, which is inherently suffering from severe root decline because of HLB-associated problems. The objective of this study was to evaluate if ground-applied coupled with foliar spray of essential nutrients can reverse the decline in tree growth and understand the fate of the nutrients in the soil-root-tree interfaces. The treatments were arranged in a split-split plot design in which nitrogen (N) was ground-applied in 20 splits biweekly and Mn, Zn, and B were foliar and /or ground-applied in three splits following the spring, summer, and late summer flush seasons. Soil nutrients in three depths , root, and leaf nutrient concentrations of the essential nutrients, leaf area index (LAI), and tree canopy volume (TCV) data were studied twice (spring and summer) for two years. A significantly higher soil NH Candidatus Liberibacter asiaticus are Gram-negative, phloem-limited, the primary cause of Huanglongbing disease , fast-proliferating, and the most destructive disease of citrus trees since its detection in Florida in 2005 [Diaphorina citri Kuwayama), the disease’s vector, is a widespread pest throughout Florida [The bacteria  in 2005 ,2,3,4. T Florida ,7. Once  Florida ,9. HLB a Florida ,11. Seve Florida ,13,14,15Foliar nutrition can complement ground-applied fertilizers to satisfy crop nutrient demand, particularly when citrus root systems are restricted, in soil having a severe nutrient deficiency, nutrients immobilized because of undesirable soil conditions ,16,17,18Phytolacca acinosa, Mn accumulation in tissues  increased with time with an increase in the supply of the amount of Mn [Nitrogen is one of the essential nutrients for citrus production that play a vital role in canopy growth and fruit yield ,24. Flornt of Mn ,34. Simint of Mn ,34. Mn dnt of Mn ,35. Thount of Mn ,36. On tnt of Mn ,19. The physiological impact of Zn on the leaves and fruit have not been identified ,18,30,32Citrus × sinensis L. Osbeck cv. “Valencia late” budded onto Volkameriana (Volk) lemon  or Swingle (Swc) citrumelo (Citrus × paradisi Macf. × Citrus trifoliata L. Raf.) rootstocks were planted in 2006. The test was conducted at the University of Florida, Southwest Florida Research and Education Center near Immokalee, FL from 2017 to 2019. The soil at the test site was classified as Immokalee fine sand . A randomly selected mature five leaves per tree were collected and processed for quantitative polymerase chain reaction (qPCR) analysis in HLB diagnostic laboratory located at Southwest Florida Research and Education Center  in February 2017. Leaf samples with cycle threshold (Ct) values of 30 or less were deemed HLB-positive, with Ct values of more than 32 or more were HLB-negative. Hence, foliar laboratory results of the HLB-affected trees under the study showed a cycle threshold value of 24.7 ± 0.19, indicating an active infection or more copies of the CLas bacteria were amplified during each cycle. Mature sweet orange trees (−1) were split within the large block of the rootstocks, the sub subplots which received either 0× (control), 1× (foliar only), 2× (1× foliar and 1× ground-applied), or 3× (1× foliar and 2× ground-applied) bentonite clay encapsulated micronutrient product containing elemental sulfur and Mn and Zn oxide, as previously suggested by the University of Florida, Institute of Food and Agricultural Sciences for citrus nutrition [−1 of S coated metallic Mn and Zn oxides and 2.3 kg ha−1 of B nutrients (2O) at 168 kg ha−1. The test comprised of three main effects as the main block of two rootstocks, intermediary subplots of three N rates, and sub subplots of three micronutrient rates in which each sub subplot consisted of 0.036 ha. The treatments were assigned in random in each experimental units where the three N rates  calculated with Penman-Monteith and crop coefficient related to the meteorological data of the study site [The N and K were fertigated as split applications on a biweekly basis between February and November in each study year. Trees were fertigated with a micro-sprinkler Max−14 , Fill-in blue emitters were placed 15 cm from the tree trunk. The irrigation was assisted with a 172.4 kpa irrigation pump at a flow rate of 45 L h−1 . Citrus udy site ,30.4-N and NO3−1-N concentrations were extracted using the 2M KCl by pipetting a 40 mL of 2M KCl solution into an extraction tube containing approximately 5.0 g (±0.05) of wet soil, capped, and shaken for 30 min on a reciprocating shaker [4-N and NO3-N concentrations were processed using a Microplate Reader spectrophotometric  at 660 and 520 nm, respectively, and calculate on a dry weight basis [Soil samples were collected within a 0.5–1.0 m radius under the tree canopy. The samples were collected at three different locations along with three soil profiles: 0–15, 15–30, and 30–45 cm per tree in spring (Feb. or Mar.) and summer (Aug. or Sept.) of 2017 and 2018 growing seasons. The collected samples were stored in a freezer until soil extraction ,30. Soilg shaker . Subsequht basis ,46.3COOH + 0.015 M NH4F + 0.013 M HNO3 + 0.001 M EDTA + 0.25 M H4NO3) was pipetted into an extraction tube containing dry soil samples of 2.5 g (±0.05) [A 25 mL of Mehlich III extractant solution (0.2 M CH (±0.05) ,47 to ex (±0.05) ,14,48. M (±0.05) ,46.Leaf and root samples were collected in the spring (February/March) and summer (August/September) seasons. Twenty leaves (4–6 month-old) were collected from non-fruiting branches . The lean = 48).Canopy density or leaf area index (LAI) is an indicator of above-ground vegetation growth. The measurement of LAI was determined by the amount of radiation transmission through the tree canopy using the radiative transfer principle  with a STCV (m3) = Tree canopy volume; r = mean canopy radius (m); h = canopy height (m).The average canopy diameter along the east-west and north-south and canopy height was measured to calculate the canopy volume using Equation (1). The TCV was measured at the beginning (Feb. or Mar.) of the experiment and every six months thereafter (Feb. or Mar. and Aug. or Sept.) for three years.p-value of ≤ 0.05 was performed using the Tukey–Kramer honestly significant difference grouping range test. All graphs were formed using the Sigma Plot 14 .Three-way interaction effect among the main factors: rootstock, N rates, and micronutrient rate treatment effects was tested on soil, root, and leaf nutrient concentration, LAI, and TCV. Field data on rootstocks, N rates, micronutrients were deemed as fixed effects, while replication in sub and sub-sub blocks were assigned as the random effect to the PROC General Linear Model (GLM) procedures SAS 14.1 . This allowed us to see all the main effects and interaction effects between rootstocks, N rates, and micronutrient rates. The data were tested for linearity, normality, homogeneity of variance, and independent errors before the derivable statistics analyses were performed. Mean separation among the means with a statistical difference at the 3-N was higher on the topmost depth (0–15 cm) as compared with the two lower soil depths (15–30 and 30–45 cm) regardless of the N rates (3-N significantly increased at the lowest soil depth (30–45 cm) with the moderate N rate and was lowest with the lowest N rate (168 kg ha−1)  in summer 2017. The relative accumulation and leaching in soil NO3-N concentration during 2017 was not conspicuous along with the soil profile and within the N rates. During spring 2018, the soil NO3-N showed an increase of 7%, 30%, and 23% of soil NO3-N concentration in the topmost soil depth as compared with its immediate soil depth pertained to 168 kg, 224 kg, and 280 kg ha−1, respectively. In summer 2018, 36% more soil NO3-N was accumulated in the topmost soil depth for trees that received 200 kg ha−1 than 168 kg ha−1.During the entire study seasons, the amount of soil NO N rates A–C. The kg ha−1) B. Meanwh3-N showed an increase of 9%, 42%, and 65% of soil NO3-N concentration in the topmost soil depth as compared with its immediate soil depth ascribed to 168 kg, 224 kg, and 280 kg ha−1, respectively. The overall range of the soil NO3-N concentration varies between 0.3–4.0 mg kg−1 throughout the study season. These findings are similar to those results documented under similar fertigation programs on HLB-affected Hamlin citrus trees with optimum irrigation scheduling [−1 N the soil NO3-N concentration was 8–15 mg kg−1, which was a value of 200 to 400% more than the current study [During summer 2018, the soil NOheduling ,17,55. Int study ,17,22,56nt study ,57.3-N, the soil of NH4-N concentration was significantly higher in the topsoil depth as compared with the two lower depths regardless of the N rates during the 2018 seasons , foliar (1×) couple with the ground (1×) and foliar (1×) couple with the ground (2×), respectively. Soil Cu and Fe concentrations were not significantly affected on the topmost depth soil in reaction to the ground-applied essential nutrient treatments in neither of the study seasons.A significant effect on soil Ca, Mg, and NH seasons . Soil Ca−1.The root Mn concentration remained significantly higher for trees that received foliar (1×) coupled with the ground (1×) and foliar (1×) coupled with the ground (2×) treatments as compared with control trees in the spring season A,B. Soil−1): Mn = 250; and Zn = 409 supporting the aforementioned statements [Foliar (1×) coupled with the ground (1×) treated trees showed a significantly higher root Zn concentration compared with the control trees A,B. No satements .−1) and highest (280 kg ha−1) N rates  coupled with the ground (1×) or foliar (1×) coupled with the ground (2×) treatments constantly showed above the optimum nutrient ranges , foliar (1×) coupled with the ground (1×), and foliar (1×) coupled with the ground (2×), respectively (−1) on Hamlin, Parson Brown, Valencia, and Sunburst Citrus varieties under the control trees [ectively B. Similaol trees . The shaol trees ,34. Howeol trees ,18.In summer 2017, a significantly higher LAI for tree budded on Swc than Volk rootstocks was detected attributed to nutrient effect . The LAI4-NO3) application [A value of 6.4–8.1 of LAI has been reported in response to N-application on 22-year-old Shamouti orange trees, in which the trees produce a higher leaf density to excess N  rates. These results indicated that trees budded on both rootstocks responded to increasing N rates. However, increasing N rates beyond the highest N rate (224 kg ha−1) eliminated variation in TCV among the two rootstocks. A linear relationship between N application and tree biomass was observed implying a persistent tree above-ground biomass, which led to the increasing in tree size rather than tree age [−1, respectively. Consequently, the higher wind resistance of the citrus trees budded on Volk rootstocks was eliminated from the grove because of the severe damage inflicted by the Hurricane. It was only during the third year, the trees responded to the micronutrient for TCV. The TCV was significantly affected by the interaction of rootstock × N rate during the early growing seasons . Results N rate 2 kg ha−1 −1 coupled with foliar (1×) coupled with ground (1×) treatments. Similar results were observed, in which TCV increment was reported when trees received the suggested N rate of 224 kg N ha−1 and foliar Zn nutrition as compared with other N rates and application methods [A significantly higher TCV was recorded for trees that received foliar (1×) coupled with the ground (1×) as compared with the rest of the treatments. Thus, as compared with the control trees, TCV showed an increase of 9.6%, 17.6%, 8.6% in spring 2019 and 8.0%, 20.8%, 4.3% in summer 2019 in response to foliar (1×), foliar (1×) coupled with the ground (1×) or foliar (1×) coupled with the ground (2×) treatments, respectively. TCV with high light intensity for peak net photosynthesis can be utilized to evaluate crop growth, which eventually produces fruit in proportion to the TCV . An incr methods .−1 increases the leaf N concentration, LAI, and the TCV. Yet, increasing the N beyond the moderate N rate did not significantly impact TCV inferring a lower N requirement. Adding beyond 9 kg ha−1 of each foliar and ground applications of Mn and Zn treatments did not significantly increase soil, root, and leaf Mn and Zn concentrations, LAI, and TCV. Hence, 224 kg ha−1 of N and foliar 9 kg ha−1 coupled with ground-applied 9 kg ha−1 Mn and Zn were the suggested doses of essential nutrients to fulfill nutrient requirement need and reverse the decline in HLB-induced problems, while conforming to the BMPs modalities.Irrigation scheduling using SmartIrrigation coupled with the split application of essential nutrients showed a significant amount of the ground-applied nutrients to retain in the topmost soil depth. The acquisition of Mn and Zn by the roots and their concentration in the root and leaf tissue was in line with the dose of the application. Increasing the rate of N to 224 kg ha


GWAS on a bread wheat panel with high D genome diversity identified novel alleles and QTLs associated with resilience to combined heat and drought stress under natural field conditions.Aegilops tauschii introgressions was evaluated under H and combined heat–drought (HD) stresses to identify quantitative trait loci (QTLs) associated with resilience to the stresses, and to assess the practicability of harnessing Ae. tauschii diversity for breeding for combined stress resilience. Using genome-wide analysis, we identified alleles and QTLs on chromosomes 3D, 5D, and 7A controlling grain yield (GY), kernel number per spike, and thousand-kernel weight, and on 3D (521–549 Mbp) controlling GY alone. A strong marker–trait association (MTA) for GY stability on chromosome 3D (508.3 Mbp) explained 20.3% of the variation. Leaf traits—canopy temperature, vegetation index, and carbon isotope composition—were controlled by five QTLs on 2D , 3D (155–171 Mbp), and 5D (407–413 Mbp); some of them were pleiotropic for GY and yield-related traits. Further analysis revealed candidate genes, including GA20ox, regulating GY stability, and CaaX prenyl protease 2, regulating canopy temperature at the flowering stage, under H and HD stresses. As genome-wide association studies under HD in field conditions are scarce, our results provide genomic landmarks for wheat breeding to improve adaptation to H and HD conditions under climate change.As heat (H) and drought stresses occur concurrently under field conditions, studying them separately offers limited opportunities for wheat improvement. Here, a wheat diversity panel containing The online version contains supplementary material available at 10.1007/s00122-021-03969-x. In many wheat-growing regions, heatwaves and drought episodes occur concurrently and are considered the most damaging climatic stressors for wheat  under H and HD in Wad Medani, Sudan, in 2019 and 2020, and identified novel alleles and QTLs for several traits, including GY and related traits. The loci for most leaf traits, including canopy temperature and normalized difference vegetation index (NDVI), were pleiotropic for GY and related traits. The identified candidate genes suggest the role of gibberellin homeostasis in maintaining GY stability and of CaaX prenylation in regulating canopy temperature under the combined stress. Our study provides new genetic materials and QTLs for breeding wheat with improved resilience to H and HD conditions.The objective of this study was to identify QTLs associated with H and HD stress resilience in bread wheat under field conditions and to assess the practicability of harnessing Ae. tauschii (DD genome) and the durum wheat cultivar ‘Langdon’ (AABB genome)  were crossed with the Japanese bread wheat cultivar ‘Norin 61’ . To reduce linkage drag, the F1 hybrids were backcrossed to N61 . The lines in the diversity panel were selected based on similar days to 50% heading (DH) and were evaluated under H and HD stress in Sudan during the 2018–19 (BC1F6 generation) and 2019–20 (BC1F7 generation) growing seasons .A diversity panel of 145 MSD lines and 5 check cultivars was used (Table S1). The study was originally designed with 155 MSD lines and 5 check cultivars, but 10 MSD lines did not flower due to vernalization requirement and were excluded. The 145 lines used are a subset of 400 MSD lines characterized for H tolerance in Sudan . The Gezira Research Farm is a dry, hot irrigated field categorized in mega-environment 5 for wheat cultivation  buried 20 cm in the soil. To create the HD condition, drought was imposed by withholding irrigation when 50% of all genotypes had reached flowering, while regular irrigation continued under the H condition. To avoid permanent wilting, plots under the HD condition were re-watered when the soil water potential approached − 900 kPa  at 0.75 g kgi et al. . Before kPa Fig. . We obtaMorphophysiological traits were measured according to Pask et al. . Chlorop13C composition of flag leaves was analyzed using an elemental analyzer connected to a continuous-flow isotope ratio mass spectrometer  as described in Itam et al.  − 1] × 1000, where R is the 13C/12C isotope ratio.The m et al. . The ana–1) were sent to Diversity Arrays Technology (DArT) Pty. Ltd., Australia (http://www.diversityarrays.com) for whole-genome scanning using DArT-seq markers. Complexity was reduced using a combination of restriction enzymes to obtain a subset of restriction fragments for each sample . Broad-sense heritability was estimated for each trait in Plant Breeding Tools v. 1.3 (http://bbi.irri.org). To assess genotype stability across different conditions, GY stability index was calculated using the Finlay–Wilkinson regression model: yij = μ + Gi + βiEj + εij, where the regressand yij is the mean GY of the genotype, the intercept μ + Gi corresponds to the genetic main effect, the slope βi corresponds to genotype variability in GY across environments , the regressor Ej is the population-wide variability in GY across environments , and εij is the error term (Finlay and Wilkinson Best linear unbiased estimates (BLUEs) were obtained for each trait under H and HD using the residual maximum likelihood method implemented in META-R  = 3, and the quantile–quantile and Manhattan plots were generated in the R package “qqman”   were explored. These values were square root transformed, and their 95th percentile was taken as the critical point. Polynomial regression-based curves were fitted on scatter plots of r2 against marker distance, and the critical r2 was then used to estimate the LD decay (Breseghello and Sorrells The linkage disequilibrium (LD) of the markers was calculated from observed and expected allele frequencies using TASSEL. To determine a critical point beyond which LD is due to true genetic linkage, rhttps://urgi.versailles.inra.fr/blast/). The genes within 1 Mbp of each SNP flanking region were screened based on the literature, and eight candidate genes were selected. Candidate gene annotations were obtained from Ensembl Plants release 103 .To identify candidate genes, we conducted a BLASTN search of the stable and pleiotropic MTAs that passed the FDR 0.05 threshold against the IWGSC RefSeq v. 1.0  in 2020.The genotype-by-environment (G × E) interaction effects were significant for DH, GFD, GY, KPS, SPAD, and TKW in 2019, and for DM, GY, HI, and KPS in 2020 (Table S2). DH, HI, and PH had consistently higher heritability values (ranging from 0.70 to 0.89) than other traits, whereas BIO, CT1, CT2, CT3, SN, and SPAD had relatively low heritability (Table p < 0.05) with most of the evaluated traits in both conditions in both years (Table S3). The GY–BIO and GY–HI correlations were consistently high, with correlation coefficients (r) ranging from 0.564 to 0.742 (p < 0.01) in both conditions in both years. In contrast, all CT values tended to correlate negatively with most traits, including GY and BIO, under H in 2019 and under both conditions in 2020 (Table S3). Similarly, δ13C was negatively correlated with GY in both conditions, and with BIO, HI, and SPAD under HD in 2019. DH and GFD were negatively correlated in both conditions in both years (Table S3). We found low positive correlations between the two conditions in most traits except DH and PH, which had high correlations , likely because the combined stress was imposed after heading.GY correlated significantly  and chromosomal positions is shown in Fig. Ae. tauschii, ‘Langdon’, or N61) and the possible effect of the alleles on individual traits . A sample of results of the GLM and MLM models for both GWAS under HD in 2020 are shown in Table S6. Many significant MTAs were identified for various traits, with 12% and 27% of the MTAs passing the FDR 0.05 and 0.2 thresholds, respectively. In 2019, 75 MTAs for H, 4 for HD, and 25 for DR passed the FDR tests; in 2020, 23 MTAs for H, 62 for HD, and 123 for DR passed . In total, 100 highly significant MTAs (FDR < 0.2) were associated with H, 68 with HD, and 150 with DR in both years . A summary of the MTAs . Two MTAs for GY , located between 508 and 522 Mbp on Chr. 3D, explained 10.6% and 11.8%, respectively, of the phenotypic variation  were collocated between 577.5 and 577.7 Mbp on Chr. 4A and explained 7.2%–9.7% of the variation under H and HD conditions. Four MTAs controlling PH were found between 25 and 37 Mbp on Chr. 4D. Two of them were stable across H and HD: 1,079,306|F|0–62 was stable in both years and explained 9.5–25.7% of the variation, whereas 4,005,784|F|0–33 was stable in 2020 and explained 7.2–9.7% of the variation in PH (Table S5). Eleven MTAs were identified for GFD and explained, on average, 10.3% of the variation. An MTA on Chr. 3D  was pleiotropic for DM in the DR in 2020, and an MTA on Chr. 6D  was pleiotropic for GY under H in 2020  harbored MTAs for CT1, NDVI, KPS, and GY, which explained 7.6–17.7%; and the third locus (606–614 Mbp) harbored MTAs for NDVI, TKW, and GY, which explained 7.1%–16.2%. The locus on Chr. 3D (155–171 Mbp) harbored MTAs for CT1, CT3, and NDVI, which explained on average 9.9% of the variation. The locus on Chr. 5D (407–413 Mbp) contained MTAs for CT1, CT2, CT3, NDVI, and BIO, which explained 7.7%–32.8% of the variation. As all these loci on Chrs. 2D, 3D, and 5D also harbored MTAs for GY and related traits under H, HD, and DR , we investigated the loci controlling CT, NDVI, and δ, we inveWe identified a locus on Chr. 3D (521–549 Mbp) controlling GY and explaining 9.6%–21.5% of the variation under H and DR, and another locus on Chr. 3D (79–90 Mbp) common to KPS and TKW explaining 9.3–22.4% of the variation under H and DR (Table S5). We identified another locus common to KPS and TKW on Chr. 5D (240.6 Mbp) explaining on average 8.5% of the variation in DR, and a locus controlling GY and KPS on Chr. 7A (517–556 Mbp) explaining a relatively high (21.0%–40.3%) proportion of the variation under DR.GA20ox TraesCS3D02G393900) on Chr. 3D . A candidate gene for CT3 was identified as a gene involved in CaaX prenylation  on Chr. 1D . Their expression patterns show highest expression in the endosperm  and in the roots, shoots and inflorescences . These relative expressions were sourced from many independent studies involving different genotypes, growth stages, and conditions implemented in Genevestigator. A list of the candidate genes is shown in Table S7.The markers in the AB genome are in high LD with LD decay extending up to 131 Mbp, whereas those in the D genome have an LD decay at 1.1 Mbp. A candidate gene for GY, GY stability, and NDVI was identified as a gibberellin-regulating gene  on most traits confirmed the high genetic diversity of the panel. Under HD, GY was drastically reduced (up to 58% of that under H), highlighting the detrimental effect of HD under natural field conditions. Similar observations have been reported under field . Similar results have been reported under field conditions , 3D (155–171 Mbp), and 5D (407–413 Mbp) were identified for most leaf traits, including CT1–CT3 and NDVI. The Chr. 2D locus at 606–614 Mbp (130.8 cM) was previously reported for GY under HD in a synthetic-derived parent/elite line RIL population  . CaaX prenyl proteases are involved in the prenylation of CaaX proteins, a step essential for protein–membrane interactions, plant development, and stress signaling, especially in abscisic acid signaling in Arabidopsis  was Ae. tauschii diversity for climate resilience breeding using the synthetic derivative approach  controlled GY alone, a locus on Chr. 7A (517–556 Mbp) controlled GY and KPS, and two loci on Chrs. 3D (79–90 Mbp) and 5D (240.6 Mbp) each controlled KPS and TKW under H and HD stresses. The Chr. 7A loci  was previously reported for GY in elite European varieties under moderate water deficit  in Zea mays L. (Zm00001d007894). GA20ox3 functions in gibberellin biosynthesis, and gibberellins play a central role in plant responses to abiotic stresses by integrating multiple hormone signaling pathways  have been reported to confer tolerance to heat and drought stress in wheat  was linked to the gene TraesCS3D02G393900, which is orthologous to Ae. tauschii accessions, and the study provides insights into the utilization of high-diversity breeding panels for wheat improvement. Since GWAS studies under HD in field conditions are scarce, the identified candidate genes, alleles, and QTLs will potentially serve as genomic landmarks for breeding to improve wheat adaptation to H and HD stresses under climate change.The wheat MSD panel used in this study represents the diversity of 37 Summary of marker–trait associations (MTAs). (a) Number of MTAs in each field condition in 2019 and 2020; (b) number of MTAs identified on each chromosome; (c) number of MTAs identified for each trait; and (d) the range of allelic effects of MTAs in each trait. BIO, biomass; CT1, canopy temperature at 7 days before flowering; CT2, canopy temperature at flowering; CT3, canopy temperature at grain filling; DH, days to 50% heading; DM, days to maturity; GC, ground cover; GFD, grain-filling duration; GY, grain yield; HI, harvest index; KPS, kernel number per spike; NDVI, normalized difference vegetation index; PH, plant height; SI, GY stability index; SN, number of spikes per plot; SPAD, chlorophyll content; TKW, thousand-kernel weight; δ13C, delta carbon-13 value (TIF 2273 kb)Representative Manhattan plots of plant height (PH) and grain yield (GY) showing marker–trait associations in all three subgenomes of bread wheat lines under combined heat and drought stress (for PH) and in the drought response (for GY). The quantile–quantile plots of the genome-wide analysis are shown on the right (TIF 3239 kb)Expression pattern of GA20ox (TraesCS3D02G393900), the candidate gene for grain yield stability in wheat. The expression in 55 anatomical parts is shown on a log2 scale and is relatively high in endosperm tissues. Data were retrieved from the wheat transcriptome database  implemented in Genevestigator. The data contains independent studies involving different genotypes, growth stages, and conditions (PDF 30 kb)Expression pattern of CaaX prenyl protease 2 (TraesCS1D02G228400), the candidate gene for canopy temperature at grain filling in wheat. The expression in 55 anatomical parts is shown on a log2 scale and the is relatively high in roots, shoots, and inflorescences. Data were retrieved from the wheat transcriptome database  implemented in Genevestigator. The data contains independent studies involving different genotypes, growth stages, and conditions (PDF 37 kb)Ae. tauschii accessions (XLSX 16 kb)List of plant materials. The pedigrees of the MSD lines include the origins and lineages of their donor Mean sums of squares for traits evaluated under heat and combined heat–drought stress in 2019 and 2020 (XLSX 12 kb)Correlation coefficients of the evaluated traits in 2019 and 2020 (XLSX 20 kb)MSD lines selected on the basis of grain yield (GY) and its stability under heat (H) and combined heat–drought (HD) stress in comparison with their backcross parent (Norin 61) and a Sudanese check cultivar (Imam) (XLSX 12 kb)Marker–trait associations detected under heat (H) and combined heat–drought (HD) conditions, and in the drought response (DR) in 2019 and 2020 (XLSX 31 kb)A sample of results of the GLM and MLM models for general and D-genome GWAS under the combined heat–drought (HD) conditions for 2020 (XLSX 43 kb)Selected candidate genes for marker–trait associations and their locations and functions (XLSX 12 kb)Below is the link to the electronic supplementary material.


C) is a cell surface GPI-anchored protein, usually known for its role in the pathogenesis of human and animal prionopathies. However, increasing knowledge about the participation of PrPC in prion pathogenesis contrasts with puzzling data regarding its natural physiological role. PrPC is expressed in a number of tissues, including at high levels in the nervous system, especially in neurons and glial cells, and while previous studies have established a neuroprotective role, conflicting evidence for a synaptic function has revealed both reduced and enhanced long-term potentiation, and variable observations on memory, learning, and behavior. Such evidence has been confounded by the absence of an appropriate knock-out mouse model to dissect the biological relevance of PrPC, with some functions recently shown to be misattributed to PrPC due to the presence of genetic artifacts in mouse models. Here we elucidate the role of PrPC in the hippocampal circuitry and its related functions, such as learning and memory, using a recently available strictly co-isogenic Prnp0/0 mouse model (PrnpZH3/ZH3).Cellular prion protein  was not enhanced in the PrnpZH3/ZH3 hippocampus. In addition, we observed a delay in neuronal maturation and network formation in PrnpZH3/ZH3 cultures.We performed behavioral and operant conditioning tests to evaluate memory and learning capabilities, with results showing decreased motility, impaired operant conditioning learning, and anxiety-related behavior in C promotes neuronal network formation and connectivity. PrPC mediates synaptic function and protects the synapse from excitotoxic insults. Its deletion may underlie an epileptogenic-susceptible brain that fails to perform highly cognitive-demanding tasks such as associative learning and anxiety-like behaviors.Our results demonstrate that PrPThe online version contains supplementary material available at 10.1186/s12915-021-01203-0. C) is a cell surface glycosylphosphatidylinositol (GPI) anchored protein expressed in several tissues with high levels in the nervous system. It is expressed ubiquitously in the brain, especially in neurons and glial cells [C is known for its crucial role in the pathogenesis of human and animal prionopathies [C is transformed into a misfolded β-sheet-rich isoform, the infectious prion protein (PrPSc) [C in prion pathogenesis contrasts with puzzling data regarding its natural physiological role/s [Prnp0/0 mouse model without PrPC protein, with high breeding capability to dissect biological relevance in specific processes [Cellular prion protein  . Increasl role/s –10. Inderocesses , 11–13.C has been previously described as neuroprotective, mainly by using loss-of-function approaches [C overexpression was associated with increased susceptibility to neurotoxicity and cell death [Prnp levels should be constrained to a certain level to develop their natural functions [PrPproaches –16, whilll death , 17–19. unctions , 15. Thiunctions , 21, humunctions ), and muunctions .C in vivo using Prnp0/0 mice. However, it was later demonstrated that some physiological functions were unfortunately misattributed to PrPC due to genetic artifacts generated during the production of the most commonly used knock-out model, still in use in some laboratories: the Zürich I (PrnpZH1/ZH1) mouse [PrnpZH1/ZH1 model [Prnp-linked loci polymorphic region containing numerous 129/Sv-associated “flanking genes” (FG) close to Prnp deletion [PrnpZH1/ZH1 mice with C57BL/6 J for more than ten generations to reduce FG, a remnant of ≈ 2–5% of the 129/Sv genome markers still persisted in B6.129 mice [Prnp is localized is 62, related to different functions  administration in vivo [Prnp background is not available, one of these FGs is the signal regulatory protein alpha (Sirpα), an important regulator of several innate immune functions [Sirpα0/0 mice [C [C increased when a recent study described a substrain-related dependence of Cu(I)-ATPase activity among Prnp0/0 mice related to the 129/Sv FGs and not PrPC [As indicated, numerous studies have explored the physiological roles of PrP1) mouse . This mo1) mouse , 25, anddeletion , 27. In 129 mice , 28, 29.g others ). Thus,  in vivo , 30–32. unctions . Althoug0/0 mice , it has  mice [C , 27. Thenot PrPC .C is transported along axons [C in neurotransmission is still elusive. For example, different studies reported reduced [Prnp0/0 mice compared to wild-type mice. Following these descriptions, the consequences of Prnp absence in memory, learning, and behavior lead to variable results in studies using mice carrying FGs [In neurons, PrPng axons  and enring axons –39. Howe reduced –43, norm reduced , or enha reduced , 44 longying FGs , 45–48 oying FGs , 49.C functions associated with neurotransmission, learning, and behavior, taking advantage of a recently generated Prnp0/0 mouse model: the Zürich 3 (PrnpZH3/ZH3) [PrnpZH3/ZH3 mouse activity, learning, and memory capabilities. In addition, basic synaptic functions, KA-mediated excitability, and LTP induction were evaluated electrophysiologically in alert behaving mice. Finally, PrPC roles during neuronal differentiation and activity were also evaluated in primary cortical cultures. Results indicate that adult PrnpZH3/ZH3 mice display reduced activity and anxiety-like behavior. They also fail to acquire different instrumental learning tasks. In addition, our experiments show that hippocampal CA3-CA1 PrnpZH3/ZH3 synapse fails to induce LTP, most likely due to an exacerbated endogenous excitability, further corroborated in vivo after KA administration. Lastly, our results are sustained by the observed alteration in the expression patterns of several genes associated with neuronal system function and synaptic protein-protein interactions in the PrnpZH3/ZH3 hippocampus by an RNAseq analysis and its RT-qPCR validation.In the present study, we focused our attention on reexamining some PrPZH3/ZH3) . This coZH3/ZH3)  and it iZH3/ZH3) . Here weC has been related to deficiencies in behavior, learning, and memory in several mouse models with different results [C in systemic behavioral tasks, we took advantage of the new knock-out model, the PrnpZH3/ZH3 mouse. First, we analyzed the nest building capacity between PrnpZH3/ZH3vs. Prnp+/+ mice as an indicator of mouse welfare. In contrast to Schmitz et al., where they used PrnpZH1/ZH1 mice  , resultPrnp+/+ and PrnpZH3/ZH3 mice (n = 49 for each genotype) were individually placed  in the open field arena for 15 min, and their activity was evaluated on the X-Y-Z axes   vs. the periphery (outer region of the field) for each mouse   and more immobility episodes  confirming an anxiety-like behavior   at the end of the training session. In contrast, all Prnp+/+ mice (100%) meet the selected criterion from the 6th session , 50% of PrnpZH1/ZH1 mice failed to reach the criterion, but 80% of wild-type mice reached it at the end of the sessions  present evident instrumental learning deficiencies. Also, Prnp+/+ mice pressed the lever significantly more times from session 3 onwards than PrnpZH3/ZH3 animals   were subjected to a more complex operant conditioning paradigm, the light ON/light OFF task . As expected, the total number of lever pulses during the OFF period was higher in the Prnp+/+ mice and drastically reduced along with sessions (Session 1 = 134.8 ± 13.3 vs. Session 10 = 39.7 ± 5.2)   and an incipient increase in the ON period (Session 1 = 23.4 ± 1.6 vs. Session 10 = 31.1 ± 2.9)  /) was evaluated. At the end of the experiment (sessions 7 and 8), the PrnpZH1/ZH1 mice showed lower values than wild-type mice , reinforcing the notion that the absence of PrPC decreases the instrumental learning goals in mutant mice  Fig. . CollectPrnp+/+ and PrnpZH3/ZH3 mice in the accelerating rotarod test. After a training session, the mice latency to fall from the rods was tested for five sessions during two consecutive days. In the first day, the PrnpZH3/ZH3 mice displayed a significantly lower latency just in the first two session compared to the control mice . In the second day, no significant difference was observed     to explore the second object less time ((second − first) / total) compared to the wild-type mice   and PrnpZH3/ZH3 (n = 24) mice at different inter-stimulus intervals (from 10 to 500 ms). As already reported for CA3-CA1 synapses [Prnp+/+ and PrnpZH3/ZH3 facilitation .PrPpocampus , 37. As  subunit , see als subunit  for revisynapses , this apC deficiency in hippocampal synaptic excitability at a large range of stimulus intensities , fEPSP1 and fEPSP2 increased steadily more or less in parallel after 0.18 mA stimulation, reaching asymptotic values at 0.32 mA . From 0.26 mA stimulation, fEPSP1 and fEPSP2 were statistically equal in Prnp+/+ mice; thus, there was no synaptic facilitation at high intensities , fEPSP1 and fEPSP2 did not increase in parallel, showing an increased facilitation to paired-pulse presentations, and therefore suggesting the absence of this protective mechanism . Prnp+/+ fEPSP1 increased steadily to greater asymptotic values than PrnpZH3/ZH3 fEPSP1 (from 0.3 mA stimulation ≈ 50% increased), but fEPSP2 were almost equal . Consequently, the increase in fEPSP1 related to fEPSP2 was ≈ 50% in Prnp+/+ individuals, but > 140% in their PrnpZH3/ZH3 counterparts.Next, we analyzed the consequences of PrPies Fig. d–g. The PrnpZH3/ZH3 connections   ratio (fEPSP2/fEPSP1 × 100). At high intensities, the PP ratio was larger in Prnp0/0 mice (see “Background”). So, to analyze LTP in PrnpZH3/ZH3 mice, we performed an LTP induction protocol based on high-frequency stimulation (HFS) in 40 mice (Prnp+/+ = 20 and PrnpZH3/ZH3 = 20)   from the first day     than those suffered by Prnp+/+ mice   that enables activation of the molecular mechanisms needed for LTP induction. Therefore, as published with chemoconvulsants models [C might exert protection against electrically induced seizures.In addition, s models , 31, PrPPrnpZH3/ZH3 mice, RNA-seq was performed from the hippocampus region of 8 Prnp+/+ and 8 PrnpZH3/ZH3 animals. The sequencing data have been deposited at the Gene Expression Omnibus (GEO) with accession code: GSE189691. . Around 700 genes showed alterations in their expression profile (323 upregulated and 390 downregulated in PrnpZH3/ZH3 compared to Prnp+/+) , the “neuronal system” , and “protein-protein interaction at synapses”  , the Gamma-Aminobutyric Acid Type A Receptor Subunit Rho2 (Gabrr2), the Potassium Voltage-Gated Channels: Subfamily J Member 2 (Kcnj2) and 6 (Kcnj6), Subfamily A Member 1 (Kcna1), and Subfamily Q member 3 (Kcnq3)  injections, as reported in PrnpZH1/ZH1 mice (B6129 and B6.129 backgrounds) [Prnp+/+ mice did not suffer any severe epileptic episodes (stages I–IV). Only 22% and 11% of wild-type mice reached stages V and VI, respectively. In contrast, 55% of PrnpZH3/ZH3 mice suffered severe epileptic episodes, 20% reaching stage V and 35% at stage VI    Fig. 5)C in the PrnpZH3/ZH3 mice showed relevant numbers of labeled cells in the pyramidal layer of the CA1 and CA3  from Prnp+/+ and PrnpZH3/ZH3 mouse embryos (E16.5–E17.5) expressing the GECI indicator GCaMP6f under the neuronal syntaxin promoter [PrPivo  , we think that these discrepancies reinforce the relevance of the homogenous genetic background in our study vs. the others. In fact, the differences we observed also extend to the rotarod test. Our results showed no motor alternations in the PrnpZH3/ZH3 mice, but they need more sessions to learn the task compared to controls. Previous publications also showed any motor alterations in two different knock-out strains [Our results also reveal that ice . StrikiPrnp0/0 mouse models [PrnpZH1/ZH1 behavior [PrnpZH1/ZH1 model [Prnp0/0 mouse behavior, where Prnp was conditionally deleted at 12 or 16 months, and the results included no alterations in the Morris water maze or object recognition test [Prnp0/0 mouse models with some discrepancies with the previous works, most likely related to age-related sampling and the mice’s background.Following our results, alterations in locomotor activity and increased latency to initiate exploration were previously reported in other e models , 47. Anxe models , depresse models , and alte models , 45. In behavior . This diH1 model . Anotherion test . We repoC has been described as a regulator of glutamate synapses [Prnp0/0 mice [Glutamate neurotransmission is, in large part, responsible for cortical signaling, and its impairment has been related to behavioral deficits . PrPC hasynapses . Even gl0/0 mice . Thus, o0/0 mice .PrnpZH3/ZH3 and Prnp+/+ animals. Therefore, PrPC deletion did not alter synaptic facilitation, at least in the living behaving mouse model, at the least at relatively low stimulation intensities  which carried the FG [PrnpZH3/ZH3 animals in KA susceptibility were similar to those previously obtained with the other available Prnp0/0 co-isogenic mouse model, the Edinburgh 129/Ola [Prnp0/0 synapse are likely associated with the mouse backgrounds, the FG effect, and the experimental approach. However, the ZH3 mice results demonstrate that PrPC indeed protects against KA-induced epilepsy.However, contradictory results were published by other groups, who described an elevated epileptic threshold in  and PTZ , and als and PTZ . Both std the FG . Differed the FG , 76. Our 129/Ola . TherefoPrnpZH3/ZH3 mice but fails to generate significant LTP at the hippocampus. Some controversial results have been published about the implication of PrPC in LTP generation. Different experimental approaches  and a mixture of mouse models with distinct backgrounds were used, generating non-comparable data , 42PrnpZs to LTP , 78. Thieceptors , 79, 80,eceptors  for reviPrnp0/0 hippocampal synapse is highly excitable and epileptogenic. Alterations in brain connectivity due to developmental alterations, traumas, or infections contribute to this neuromodulation imbalance [PrnpZH3/ZH3 animals came from neuronal connectivity alterations, we studied bursting and network formation in PrnpZH3/ZH3-derived primary neuronal cultures. Relevant PrPC expression in vitro is observed from 4 to 5 DIV [PrnpZH3/ZH3 cultures did not mature or connect properly; they displayed asynchronous and very low bursting compared to wild-type cultures. These results suggest that the absence of PrPC causes a delay in neuronal maturation, but more relevantly in neural network formation and function. In fact, the role of PrPC-mediated signals in neuritogenesis has been demonstrated [C. However, and as indicated by Benvegnu and coworkers [Prnp0/0 and wild-type hippocampus. Hence, changes in ion conductance or channel receptor expression might be involved in this delay. In this line, electrophysiological experiments with biochemical characterization might confirm the basis of the delay in maturation in vitro.The presented data show that mbalance . In ordeto 5 DIV . Our resnstrated –84. Howeoworkers , there aPrnpZH3/ZH3 and Prnp+/+ mouse hippocampus showed downregulation of genes associated with the neuronal system and protein-protein interaction at synapses, fitting the phenotype we observed in the knock-out mice. The significant downregulation of Gabrr2 and Grin2b in PrnpZH3/ZH3 mice would produce dysregulation in the excitatory/inhibitory balance, increasing the excitability of the system, as we describe with the KA and the HFS analyses. The alteration of the inhibitory neurotransmission was already shown in Prnp0/0 models since they were susceptible to suffering from epileptic crises (see references above). PrPC has been widely described as a regulator of glutamatergic neurotransmission and its receptors, as we show here with the Grin2b. Moreover, mutations at Grin2b have recently been related to a rare brain disease, the GRIN2B-related neurodevelopmental disorder that causes intellectual disability, autism-spectrum-like behavior, epilepsy, and, sometimes, locomotor deficiencies as well [Grin2b might contribute to the behavior and learning deficits observed in the PrnpZH3/ZH3 mice.Finally, the gene ontology analysis of the RNAseq of  as well , 87. TheC impairs neuronal network formation and connectivity, producing enhanced susceptibility to excitotoxicity insults such as HFS and KA exposure. This epileptogenic circuitry seems to impair highly cognitive-demanding functions such as associative learning, and it produces anxiety-like behavior.In conclusion, our study points that the absence of PrPPrnp+/+) were purchased from Charles River Laboratories . PrnpZH3/ZH3 mice line was provided by A. Aguzzi (Switzerland)  were used in the present study. In ZH1 mouse experiments, null PrnpZH1/ZH1 and control mice (Prnp+/+) were obtained by crossing heterozygous Prnp+/ZH1 mice to obtain a mixed background (B6.129). It is well described that behavior and neural physiology are different between male and female rodent models due to several hormone and non-hormone-derived reasons [Adult C57BL/6 J mice (nd) . Total lysates were obtained by 30 s of centrifugation at 4 °C. The protein concentration of the lysates was quantified using Pierce BCA Protein Assay Kit (Thermo Scientific). Then, 10–50 μg of proteins were loaded to SDS-PAGE gels and transferred and transferred to nitrocellulose membranes for 1 h. Membranes were blocked with Tris-buffered solution with 0.1% tween, 5% skimmed milk, and 2% of FBS for 1 h at room temperature (RT) and incubated with PSD95 , PrP  or Actin  antibodies at 4 °C O/N. Following HRP-linked secondary antibody (Dako) incubation for 1 h at RT, membranes were developed with ECL substrate (Thermo).Prnp+/+ = 63 and PrnpZH3/ZH3 = 64; ZH1: Prnp+/+ = 10 and PrnpZH1/ ZH1 = 10). Mice were housed alone in boxes on a 12/12 h light/dark cycle with constant ambient temperature (21 ± 1 °C). Water and food were provided ad libitum except for the instrumental learning tests (see below).A total of 147 animals were used in these sets of experiments (ZH3: PrnpZH3/ZH3 = 7 and Prnp+/+ = 7). On the first day of testing, one piece of tissue paper (36 × 12 cm) was placed in the cage to facilitate nest building  were used  were placed in a square open field altimeter box . The field had a grid (16 × 16 cm) of infrared lasers on the XY-axis and one on the Z-axis. Locomotor activity was measured for 15 min in mice with the MUX-XYZ16L software. Mice were placed in the box’s periphery for 15 min for two consecutive days, and their behavior was recorded. The first day was considered a training session to reduce mouse anxiety associated with manual handling, and the data analyzed and displayed in the manuscript corresponded to the second session. The system inferred mouse activity by counting laser intersections. For anxiety-related behavior measurement, the center (inner) square of the field (10 × 10 cm) was considered as the central zone and the rest of the square as the peripheral (outer) zone [vs. periphery) if it remained in the region at least 3 s. Rearing episodes were considered when the animal stood up for at least 3 s, and immobility episodes if immobile for an additional 3 s. Obtained data were analyzed, and the sum of the crossed X- and the Y-axes are presented together to show total mouse mobility in the experiments. The time spent in the maze periphery zones measures thigmotaxis or wall-hugging behavior and indicates anxiety-related behavior [T-test or Mann-Whitney U non-parametric test (GraphPad Prism 8 software). The asterisks indicate significant differences: **p < 0.01 and ***p < 0.001. The arena and the walls were cleaned with soap and ethanol between trials to remove olfactory cues between experiments.In this test, is model , 54. In er) zone   were trained to press the lever to receive food pellets in a fixed-ratio (1:1) schedule. Seven daily sessions (20 min/each) were held. The boxes were cleaned with soap and ethanol (30%) between trials. Obtaining ≥ 20 pellets for two consecutive sessions was defined as the criterion to assume the learning criteria achievement. Following this first operant conditioning test, we increased the paradigm complexity to test the mice in a more demanding cognitive task for an additional 10 days. Only animals that met the learning criterion were tested . The paradigm consisted of light (ON period) and dark periods (OFF period) randomly distributed during the session. The light was provided by a small light bulb located over the lever. During the ON period (20 s), lever presses were reinforced with food pellets at a ratio of 1:1. During the OFF period, lever presses were not rewarded and were penalized by adding ten additional seconds (20 ± 10 s) to the next ON period. The number of lever presses during the different conditioning paradigms was monitored and recorded with the MED-PC program . Statistical analysis was carried out using two-way ANOVA with repeated measures and Bonferroni’s multiple comparisons test (GraphPad Prism 8 software). Asterisks indicate significant differences: **p < 0.01; and ***p < 0.001. Data are presented as the mean ± S.E.M. or as a percentage (as indicated in each figure).The instrumental learning tests were performed as described in previous studies of our group . Six SkiPrnpZH3/ZH3 = 8 and Prnp+/+ = 7). Motor performance was tested using an accelerating rotarod. Mice were pre-trained to the task to reach a minimum of 30 s performance at 5 rpms on the 1st day of testing. In each training run, animals were placed on the rods at an initial speed of 5 rpm for 30 s. After that, the testing consisted of 5 consecutive trials with 15-min inter-trial intervals. Each trial consisted of 30 s at 5 rpm followed by 5 rpm increases every 15 s with a cut-off of 5 min. Results are expressed as the mean latency of animals to fall from the rod ± S.E.M. The statistical analysis was performed with the two-way ANOVA + Bonferroni’s multiple comparisons test (GraphPad Prism 8 software).For this test, a total of 15 mice of 4 months were used . One hour later, two identical plastic objects were placed in the center of the arena for the training session. A short-term memory test was performed 2–3 h later by changing one of the objects . Data are presented as the mean ± S.E.M. or as a percentage (indicated in each figure).The object recognition test was performed in a homemade arena (30 × 25 × 20 cm), as described . A totalfecation . StatistPrnp+/+ = 49 and PrnpZH3/ZH3 = 49). Four of them died during surgery, and 33 mice were excluded because of the inability to obtain reliable and clean recordings. Thus, the experiments were performed with 61 mice (Prnp+/+ = 31 and PrnpZH3/ZH3 = 30). Surgery was performed as described in [stratum radiatum of the CA1 area , and two stimulating electrodes were implanted in the Schaffer collateral pathway of the CA3 region . Electrodes were made of 50 μm Teflon-coated tungsten wires . Electrode localizations were checked according to the field excitatory postsynaptic potential (fEPSP) profile evoked by a single stimulation. A silver wire was fixed to the skull as ground. All the wires were soldered to a six-pin socket  fixed to the skull with dental cement. Recordings were started at a minimum of 1 week after the surgery.A total of 98 adult male (3-5 months) mice were implanted with stimulating and recording electrodes  using differential amplification at a bandwidth of 0.1 Hz–10 kHz . For each experiment, artefactual recordings were discarded. The stimulation intensity threshold of each animal was set with paired-pulse stimulations at 40 ms of inter-stimulus interval. The stimulus intensity was set to 40–60% of the amount necessary to evoke a suturing response. These intensity values were used for all the experiments.Animals were consecutively recorded in groups of six individuals since they reach the total number of animals used in each experiment. Each animal was placed in a small plastic cubicle (5 × 5 × 10 cm) inside a large Faraday box (30 × 30 × 20 cm). fEPSPs were recorded with a high impedance probe (2 × 10Prnp+/+ = 27 and PrnpZH3/ZH3 = 24) were stimulated at Schaffer collaterals with a pair of pulses at different inter-stimulus intervals  at 2 × threshold intensities (≈ 0.2 mA). Threshold values were previously defined for each mouse. As classically defined, threshold values were determined as the intensity evoking fEPSP responses in 50% of the cases. For all the inter-pulse intervals, the stimulations were repeated ten times. Data are represented as the mean percentage increases of fEPSP2 from fEPSP1 recordings (fEPSP2 / fEPSP1 × 100) ± S.E.M.For synaptic facilitation experiments, 51 mice (Prnp+/+ = 14 and PrnpZH3/ZH3 = 15) were stimulated with paired pulses at 20 increasing intensities  at 40 ms of inter-stimulus interval. For all the selected intensities, the stimulations were repeated ten times. Data are represented as the mean of fEPSP slopes (V/s) ± S.E.M. The same data are presented as the mean of paired-pulsed ratio (PP ratio) ± S.E.M. PP ratio is the percentage of the increase of the fEPSP2 from fEPSP1 recordings (fEPSP2 / fEPSP1 × 100). The area under the curve (AUC) was calculated from the PP ratio of all the animals using GraphPad Prism 8 software. Statistical analysis was performed using the Mann-Whitney-Wilcoxon non-parametric test or two-way ANOVA + Bonferroni’s multiple comparisons test (GraphPad Prism 8 software). The asterisks indicate significant differences: *p < 0.05; **p < 0.01; and ***p < 0.001 in the figure.Schaffer collaterals of 29 mice (Prnp+/+ = 20 and PrnpZH3/ZH3 = 20) were stimulated at Schaffer collaterals. In a first experimental step, fEPSP baseline values were evoked and recorded for 15 min, with paired-pulse stimulus presented every 20 s (40 ms inter-stimulus). Stimulus intensities were selected to evoke fEPSPs of about 0.2–0.3 mV of amplitude  essentially as described in [U non-parametric test (GraphPad Prism 8 software). The asterisk indicates significant differences: *p < 0.05 in the figure.Adult (3–4 months old) male mice were used for these sets of experiments . Libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit v2 according to the manufacturer’s protocol. Briefly, 500 ng of total RNA was used for poly(A)-mRNA selection using Oligo (dT) magnetic beads and subsequently fragmented to approximately 300 bp. cDNA was synthesized using reverse transcriptase  and random primers. The second strand of the cDNA incorporated dUTP in place of dTTP. Double-stranded DNA was further used for library preparation. dsDNA was subjected to A-tailing and ligation of the barcoded Truseq adapters. All purification steps were performed using AMPure XP Beads. Library amplification was performed with PCR using the primer cocktail supplied in the kit. Final libraries were analyzed using Agilent DNA 1000 chip to estimate the quantity, check the size distribution, and then quantified by qPCR using the KAPA Library Quantification Kit  before amplification Illumina’s cBot. Libraries were sequenced 1 × 50 bp on Illumina’s HiSeq 2500.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). An estimation of ribosomal RNA in the raw data was obtained with riboPicker [https://www.gencodegenes.org/mouse/release_M14.html). A raw count of reads per gene was also obtained with STAR (-quantMode TranscriptomeSAM GeneCounts option). The R/Bioconductor package DESeq2 [padj < 0.05 were used to disclose relevant pathway alterations in the REACTOME v77 pathway database gene expression. The analysis has been done just with the protein-coding genes. The gene difference was considered biologically relevant if they are upregulated or downregulated with a fold change of > 1.2 or < 0.85, respectively. A pathway was considered relevant if it was related to neuronal and/or cerebral functions, showed significance (padj < 0.05) and contained more than 10 deregulated genes. The sequencing data have been deposited at the Gene Expression Omnibus (GEO) with accession code: GSE189691 .The quality of the fast files was checked using the FastQC software . Actb Mm02619580_g1 and Aars Mm00507627_m1 were used as endogenous controls. The assay was performed using technical duplicates per sample in 384-well optical plates with ABI Prism 7900 Sequence Detection system  following the supplier’s parameters: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The Sequence Detection Software  was used for data processing. It was further analyzed with the ∆∆Ct method, which consists of obtaining ∆Ct by normalizing each target gene to its endogenous control, followed by subtracting the mean-∆Ct of the control group samples to each ∆Ct to obtain ∆∆Ct values, and finally using these ∆∆Ct values as the negative exponent with base 2, thereby obtaining fold change per sample.For RT-qPCR validations, cDNA was obtained with a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) following the supplier’s instructions. RT-qPCR reactions contained 4.5 μL cDNA and mixed with 0.5 μL 20X TaqMan Gene Expression Assays and 5 μL of 2X TaqMan Universal PCR Master Mix (Applied Biosystems) for a final volume of 10 μL. TaqMan probes used were as follows: 4 for 15 min. Following three rinses (2 min/each) in deionized water, they were incubated in a solution of 0.001% Fluoro-Jade B  containing 0.05% of DAPI in 0.1% acetic acid for 30 min. Finally, sections were rinsed in deionized water (3 min), dehydrated with ethanol, cleared with xylene, and coverslipped with EukittTM . Considering that wild-type mice did not displayed Fluoro-Jade B labeled cells in the CA1-CA3 regions after KA treatments and only a very few background could be seen at high magnification and long exposures times (> 500 ms), the Fluoro-Jade B fluorescence in the pyramidal layer of the dorsal hippocampal region  was photo-documented using an Olympus BX61 epifluorescence microscope equipped with a cooled DP12L camera . Photomicrographs were obtained using a × 40 objective with identical time exposure between preparations from each wild-type and respective knock-out mouse. No modifications were applied to the obtained pictures. Changes in Fluoro-Jade B labeling were determined by analyzing the corrected total cell fluorescence (CTCF) values  dissolved in 0.1 M phosphate buffer (pH = 7.3–7.4). Brains were dissected and postfixed overnight with the same fixative solution. The following day, they were cryoprotected in 0.1 M phosphate buffer containing 30% sucrose (w/v). After freezing in dry ice, 50-μm-thick coronal sections were obtained with a freezing microtome . Sections containing the dorsal hippocampus were selected and rinsed in 0.1 M phosphate buffer and mounted on gelatin-coated slides. The slides containing sections were dried at 37 °C overnight. The following day, they were heated at 50 °C for 45 min before staining to improve adhesion. The staining started with pretreatment for 3 min in absolute alcohol, followed by 1 min in 70% ethanol and 1 min in deionized water. After that, they were oxidized in a solution of 0.06% KMnOs et al. , for detPrnp+/+ and PrnpZH3/ZH3 mouse embryos, as explained elsewhere [d-lysine (Sigma-Aldrich). NeurobasalTM medium supplemented with 6.5 mg/ml glucose, 2 mM glutamine, penicillin/streptomycin, 5% of horse serum, and B27 was used as a culture medium . As Prnp0/0-derived cells are sensitive to serum removal [Primary cortical cultures were fashioned from E16.5–E17.5 lsewhere . Brains  removal , after 22 and 37 °C . The same region of the culture was recorded throughout the days following the culture dish grid references. Images (1024 × 1024 pixels) were captured using a × 20 objective and 470 nm wavelength  every 100 ms for 8–10 min using the CellSensTM software (Olympus) or the Micro-Manager Open Source Microscopy Software (https://micro-manager.org). Exposure levels and frequency were maintained between cultures and evaluation days. GCaMP6f activity was measured in four different identified squares of each culture dish during these 4 days.Primary cortical neurons were infected 24 h after seeding with AAV9-Synapsin-GCaMP6f   [Fi(t) in each ROI i along the recording was then extracted, corrected from global drifts and artifacts, and finally normalized as (Fi(t) − F) / F= fi(t), where F0,i is the background fluorescence of the ROI. The time series of fi(t) was analyzed with NETCAL to infer neuronal activation timing using the Schmitt trigger method. Our analysis used + 2 S.E.M. of the baseline noise as the high threshold, + 1.5 S.E.M. as the low threshold, and 200 ms as the minimum event length. Calcium traces were calculated, and raster plots of network activity were then constructed by representing the trains of detected neuronal spikes over time. Next, network bursts were analyzed to quantify the ability of the neuronal networks to exhibit collective dynamics, i.e., the collective activation of a group of neurons in a short time window. Bursts were investigated using two approaches. In the first approach, raster plots of spike events were scanned to detect collective occurrences in which at least 5% of the neurons in the network fired synchronously within a 500-ms window. This threshold of 5% was set to disregard random activations that coincided in time. In the second approach, the fluorescence time series of all neurons in the network were averaged. The resulting trace was analyzed with the Schmitt trigger method to detect sufficiently strong fluorescence peaks associated with bursting episodes. Both approaches produced consistent results. Although the detected bursts contained a different number of participating neurons, this information was disregarded in the present analysis and treated later. The total number of detected network bursts divided by the recording duration reflected the culture’s activity and was indicated as bursts/min.The recordings were analyzed offline using two MATLABcal.com) , 101 andcal.com) . In NeurNA was set as a proxy of the healthy population in the neuronal network. The average fraction of active neurons in each condition was then determined as f = NA/NT, where NT is the total number of detected ROIs.The fraction of active neurons in the network was calculated as follows. All detected ROIs were assigned as neurons. After inferring the spike trains, those neurons exhibiting at least two spikes along the recording were considered active, and their number Prnp+/+ and PrnpZH3/ZH3 cultures at a given DIV: *p < 0.05 and ***p < 0.001. The # symbols indicate significant differences between a given DIV with the initial value at 8 DIV: ###p < 0.001.At least ten videos of each genotype/day from different culture plates were consecutively analyzed. Data are presented as the mean of network burst/min ± S.E.M. Statistical analysis was performed using two-way ANOVA + Bonferroni’s multiple comparisons test (GraphPad Prism 8 software). Asterisks indicate significant differences between F) applied were indicated. All data analysis was performed blind to the experimental group by two independent experimenters. Unless otherwise stated, sample size was chosen in order to ensure a power of at least 0.8, with a type I error threshold of 0.05, in view of the minimum effect size that was expected.All statistical analysis was performed with GraphPad PRISM 8 . Unless otherwise stated, data is plotted as the mean ± SEM. All experiments were performed three times unless specified. Normality of the distributions was checked via Shapiro-Wilk test. All tests performed were two-sided, and adjustments for multiple comparisons and/or significantly different variances  and PrnpZH3/ZH3 mice (right). b, Quantification illustrating the mean of the nest score . Data are presented as mean ± S.E.M. Source data and individual data values are available in Additional file Additional file 2: Figure S2. PrnpZH1/ZH1 mice failed to acquire an instrumental learning test in the Skinner box test using a fixed ratio (1:1) schedule. a, Percentage of Prnp+/+ and PrnpZH1/ZH1 mice reached the selected criterion during the training sessions. b, Task accuracy ((lever presses during light ON – lever presses during the light OFF) / Total number of lever presses) during the light ON / light OFF conditioning paradigm. Data are presented as a percentage in a and as mean ± S.E.M. in b. Source data and individual data values are available in Additional file Additional file 3: Figure S3. PrnpZH3/ZH3 mice have deficits in acquiring instrumental learning but not motor impairment in the Rotarod test. After a training session in the Rotarod, mice were tested two consecutive days with 5 sessions in each. Latency of mice to fall (sec.) from the Rotarod in the first day (a) or in the second (b). Data are presented as mean ± S.E.M.. *p < 0.05 and **p < 0.01, two-way ANOVA + Bonferroni’s multiple comparisons test. Source data and individual data values are available in Additional file Additional file 4: Figure S4. Stressed-like behavior in PrnpZH3/ZH3 mice impairing object recognition test performance. PrnpZH1/ZH1 mice also showed stressed-like behavior and failed to acquire short-term memory. a, Scheme of object recognition protocol that consisted of 3 sessions (10 min each). First, mice were habituated in the empty arena. One hour later, they were placed again in the arena with two identical objects for the training session. Finally, 2-3 h later, they were once more placed in the arena, replacing one object with a novel one for the short-term memory test. Fecal bodies (PrnpZH3/ZH3) and rearing episodes (PrnpZH1/ZH1) were counted in the habituation session as an indicator of animal stress. b, Number of fecal bodies generated per animal (Prnp+/+ and PrnpZH3/ZH3) during the habituation session. Data are presented as mean ± S.E.M. c-d Time that Prnp+/+ (c), and PrnpZH3/ZH3 (d) mice interacted with the objects in the short-term test. Data are presented as the percentage of mice interacting with the object in each time interval (0 to 35 s). e, Number of rearing episodes in the habituation session (Prnp+/+ and PrnpZH1/ZH1). f-g, Relative time that the two exposed objects are explored to the total time invested. Object 1 (O1) and object 1’ (O1’) in the training session (f) and number, and object 1 (O1) and object 2 (O2) in the short-term memory test (g). Data are presented as percentages in C-D and as mean ± S.E.M. in b, e, f, g. *p < 0.05 and ***p < 0.001, Mann-Whitney U non-parametric test. Source data and individual data values are available in Additional file sessions  min eachAdditional file 5: Table S1. List of the protein-coding significantly downregulated genes in PrnpZH3/ZH3 hippocampus compared to Prnp+/+.Additional file 6: Table S2. List of the protein-coding significantly upregulated genes in PrnpZH3/ZH3 hippocampus compared to Prnp+/+.Additional file 7: Figure S5. Gene ontology of the genes significantly downregulated and upregulated in PrnpZH3/ZH3 hippocampus compared to Prnp+/+. a, Volcano plot of the protein-coding genes analyzed in the RNAseq (~ 16.000). In the X-axis is plotted the Fold change (log2) and in the Y-axis the padj (-log10). The red line separates the significantly expressed genes . The blue lines indicate the 0,85 and 1,2 fold changes. b, Gene ontology analysis of the downregulated and upregulated genes with the Reactome software . Validation of the main genes altered by RT-qPCR: Grin2b (c), Gabrr2 (d), Kcnj6 (e), Kcna1 (f), Kcnj2 (g), and Kcnq3 (h). Data are presented as mean ± S.E.M. *p < 0.05 and ***p < 0.001. Source data and individual data values are available in Additional file padj -log. The redAdditional file 8: Movie S1. Representative movie of Prnp+/+ (bottom and up-left) and PrnpZH3/ZH3 (middle and up-right) mouse behavior after KA (middle and bottom) and PBS (top) administration. Note the blinking and seizure episodes suffered by the PrnpZH3/ZH3 mice treated with KA (labeled with asterisks at the beginning of the video). Source data and individual data values are available in Additional file Additional file 9: Figure S6. Images of the original, uncropped blots for Figs. Additional file 10. Excel file 1. File providing individual data values for all figures from the manuscript.


In order to understand the genetic basis of starch pasting viscosity characteristics of Chinese spring wheat, we assessed the genetic variation of RVA parameters determined by the Rapid Visco Analyser in a panel of 192 Chinese spring wheat accessions grown in Er’shi, Shihezi and Zhaosu during 2012 and 2013 cropping seasons. A genome-wide association study with 47,362 single nucleotide polymorphism (SNP) markers was conducted to detect marker-trait associations using mixed linear model. Phenotypic variations of RVA parameters ranged from 1.6 to 30.7% and broad-sense heritabilities ranged from 0.62 to 0.91. Forty-one SNP markers at 25 loci were significantly associated with seven RVA traits in at least two environments; among these, 20 SNPs were located in coding sequences (CDS) of 18 annotation genes, which can lead to discovering novel genes underpinning starch gelatinization in spring wheat. Haplotype analysis revealed one block for breakdown (BD) on chromosome 3B and two blocks for pasting temperature (T) on chromosome 7B. Cultivars with superior haplotypes at these loci showed better starch pasting viscosity than the average of all cultivars surveyed. The identified loci and associated markers provide valuable sources for future functional characterization and genetic improvement of starch quality in wheat. Triticum aestivum L.) is one of the most important staple food crops worldwide. With the improvement of living standards, people pay more attention to the quality of end-use products of wheat. Improvement of quality traits has become a major objective in wheat breeding . Each genotype was sown in ten rows, with a row length of 3 m, a row-to-row distance of 25 cm and plant-to-plant distance of 10 cm. There were differences in climate and soil conditions among Er’shi, Shihezi and Zhaosu, and different temperatures between the years 2012 and 2013. Planting and harvest dates and trial management varied according to the recommendations of each location. The accessions were harvested at maturity and cleaned prior to quality test.Flour milling was performed in a mill  to flour extraction rates of around 65%. Prior to milling, the hard, medium hard (mixtures of hard and soft wheat) and soft wheats were tempered overnight to moisture contents of around 16, 15, and 14%, respectively.Pasting properties of flour were determined with a Rapid Visco Analyser . The 3 g flour was suspended in 25 ml of distilled water before the solution was placed inside RVA instrument. The programs of temperature in the following order: held at 50°C for 60 s, heated from 50 to 95°C at a rate of 1°C/5 s and held at 95°C for 150 s, then cooled to 50°C at a rate of 1°C/5 s and held at 50°C for 120 s. RVA parameters including peak viscosity (PV), trough viscosity (TV), breakdown (BD), final viscosity (FV), setback (SB), peak time (PT) and pasting temperature (T) were determined.Fresh leaf samples were collected from 10-day old seedlings and sent to the CapitalBio Technology company4 cycles were used, followed by a simulation runs of 105 cycles with an admixture model. The K values of 1–10 and 3 independent runs were selected to attain reliable results. Web-based analysis “Structure Harvester v0.6.93”The Bayesian clustering technique was used with 3400 SNP markers to classify groups of genotypically same individuals using the statistical software STRUCTURE v.2.3.4 . Burn-inA principal component analysis (PCA) using filtered SNPs was performed with the TASSEL v.5.2.43 and the first three PCA values were plotted in three dimensions.p < 4.18E-4 . Linkage disequilibrium (LD) was calculated by TASSEL 5.0 with the markers whose positions were known. The LD decay plot was generated by intra-chromosomal r2 and base pair distance using R package ggplot2.Phenotypic data in different environments and best linear unbiased prediction (BLUP) values were analyzed, respectively, for association analysis to identify the marker-trait associations (MTAs) employing mixed linear model (MLM) in TASSEL v.5.2.43 . The MLMThe Chinese spring reference genome (IWGSC RefSeq v1.0) and gene annotations in GFF3 format were retrieved from the Ensemble database release 44p) above the threshold, and the phenotypic values of accessions with different alleles reached significant level (p < 0.05) in multi-environment, haplotype analysis was carried out by Haploview version 4.2 software  of RVA traits were calculated as h2 = σ2g/(σ2g + σ2gl/l +σ2gs/s + σ2gls/ls), where σ2g, σ2gl, σ2gs, σ2gls variances for genotypic, genotype by location interaction, genotype by season interaction and genotype by location by season interaction, respectively, whereas l and s were the numbers of locations and seasons, respectively. SNP density plots, Manhattan and quantile-quantile (Q-Q) plots were generated in R package CMplot while histograms were performed in Origin 8.0.ANOVA and correlation analysis were carried out using SPSS version 22.0. The coefficient of variation (CV) was calculated by dividing the standard deviation by the average of trait values. The BLUP values of all traits over 2 years across environments were calculated by the R package Lme4 . Broad-sh2 estimates were 0.91, 0.77, 0.86, 0.89, 0.82, 0.62 and 0.85, respectively , BD (r = 0.81**) and FV (r = 0.63**); TV was significantly and positively correlated with FV (r = 0.94**), SB (r = 0.72**) and PT (r = 0.63**); FV was significantly and positively correlated with SB (r = 0.89**) and PT (r = 0.48**); BD was significantly and negatively correlated with PT  on chromosomes 7A (1), 7B (1) and 7D (2) at 534, 489 and 463 Mb, respectively, were significantly associated with both PV and TV, while one SNP  on chromosome 7D at 13 Mb was significantly associated with both PV and BD. Thirty-four SNP markers associated with RVA parameters were mapped in the annotated genes; 20 SNPs among these were located in the CDS of 18 annotation genes, which were considered as candidate genes  revealed significant differences (p < 0.05) of the traits between two alleles in at least four environments  associated with BD showed that the 776–782 Mb interval on chromosome 3B had an 862-kb block, and six SNPs including BS00023017_51 were clustered in the block (BS00024883_51/BS00023017_51/IAAV8892/RFL_Contig2578_862/wsnp_Ex_c33879_42293206/wsnp_CAP11_c59_99263) haplotypes showed significantly higher BD (p < 0.05) than the average of all cultivars surveyed in four environments  associated with T, and two blocks in 442–454 Mb interval on chromosome 7B were detected. Two cultivars with CC/AA/CC , 17 cultivars with AA/TT/GG/GG (wsnp_Ex_c64815_63464750/RAC875_c4438_419/IAAV3414/TA001679-0356) haplotypes showed higher T than the average of all cultivars surveyed, exhibiting significant differences in some locations  encodes granule-bound starch synthase, generating a higher PV and BD  around there.Significant and stable MTAs for PV were mainly clustered to chromosomes 2B, 3A, 3B, 4A, 4B, 5A, 7A, 7B and 7D ; severalV and BD . Some stosome 4A . In the radation . In the GENE-4993_69 on chromosome 7B, Excalibur_c9183_1397 and Tdurum_contig69003_459 on chromosome 7D, BS00067650_51, Wx-B1 on chromosome 4A, where no locus associated with FV was detected in this research. Nine SNPs in seven loci on chromosomes 2B (2), 5B (1), 6B (1), 7B (2) and 6D (1) were significantly associated with T  significantly associated with T was very close to TRAESCS6B02G418100  which was the homolog of phosphorylase  SNPs were mapped in intergenic regions, and 34 (83%) were mapped in genic regions . Among tndosperm . Other cBS00023017_51, Excalibur_c10124_361 and Kukri_c4560_110 had significant (p < 0.05) effects on traits in at least four environments, and could be used as optimal loci in marker-assisted breeding and quality improvement. The haplotype analysis was conducted on these loci and three blocks were detected  revealed one block for BD on chromosome 3B and two blocks for T on chromosome 7B, cultivars with superior haplotypes at these loci showed better starch pasting viscosity than the average of all cultivars surveyed. Validation studies for SNPs in the candidate genes and detected loci will be conducted in the future by designing KASP assays, which can be further used for marker-assisted breeding for improvement of grain starch quality in wheat.It is not feasible that phenotypic selection for improving wheat starch quality at the early stages in wheat breeding. Even at later stages in the breeding process, starch quality was also impacted greatly by environments. Therefore, genetic selection through MAS is a desirable way to improve wheat starch quality in wheat breeding programs. Based on dense SNPs across the whole genome, the GWAS has become a common approach to uncover genetic components of agronomic traits, which provides us with insightful information into genetic architecture of complex traits. In this study, GWAS analysis were performed for RVA parameters with 47,362 SNPs in 192 Chinese spring wheat accessions among six environments. Forty-one SNPs at 25 loci were stably detected in at least two environments, of which 20 SNPs were located in the CDS of 18 annotation genes. Haplotype analysis for regions harboring the SNPs (


Escherichia coli (ETEC) strains are an important cause of bacterial diarrheal illness in humans and animals. Infections arising from ETEC could potentially be treated through the use of bacteriophage (phage) therapy, as phages encode for enzymes capable of bacterial cell lysis. vB_EcoP_SU7 was isolated from the Käppala wastewater treatment plant in Stockholm, Sweden, and propagated on an ETEC strain exhibiting the O:139 serovar. Transmission electron microscopy confirmed that vB_EcoP_SU7 belongs to the Podoviridae family and has the rare C3 morphotype of an elongated head. Bioinformatic analyses showed that the genome was 76,626 base pairs long and contained 35 genes with predicted functions. A total of 81 open reading frames encoding proteins with hypothetical function and two encoding proteins of no significant similarity were also found. A putative tRNA gene, which may aid in vB_EcoP_SU7’s translation, was also identified. Phylogenetic analyses showed that compared to other Podoviridae, vB_EcoP_SU7 is a rare Kuravirus and is closely related to E. coli phages with the uncommon C3 morphotype, such as ECBP2, EK010, vB_EcoP_EcoN5, and vB_EcoP_SU10. Phage vB_EcoP_SU7 has a narrow host range, infecting 11 out of the 137 E. coli strains tested, a latency period of 30 min, a burst size of 12 PFU/cell, and an adsorption rate of 8.78 × 10−9 mL/min five minutes post infection. With a limited host range and poor infection kinetics, it is unlikely that SU7 can be a standalone phage used for therapeutic purposes; rather, it must be used in combination with other phages for broad-spectrum therapeutic success. Enterotoxigenic  Escherichia coli is a common Gram-negative bacterium that is often found in the gastrointestinal tract of mammals and humans [E. coli that live as a normal part of the commensal microflora  [E. coli (ETEC) strains are among the major causative agents behind diarrheal diseases in low- and middle-income countries (often referred to as traveler’s diarrhea) and in animal farming [d humans . While t humans) ,3, there humans) . Enterot farming ,6. ETEC  farming .E. coli, as well as those caused by multiple bacteria have the potential to be treated with a cocktail comprised of different phage combinations [E. coli species, as well as on their infection pharmacology and the development of phage-bacteria resistance. In this study, we present the isolation and genome annotation of vB_EcoP_SU7 (SU7), a Podoviridae coliphage with the C3 morphotype.ETEC infections are commonly treated with different antibiotics, such as fluoroquinolones (most used) and ciprofloxacin ,8. Howevinations , the effinations . For phaE. coli ECOR standard reference collection [The bacterial strains used for host range analyses were provided as follows: the llection  was kind600) was 0.6. SU7 was enumerated using the agar overlay (OA) method with 65% w/v (22.75 g/L) TYA as previously described [g for 10 min and the supernatant passed through a sterile 0.45 µm syringe filter . Phages were precipitated by adding solid NaCl and PEG8000  to the partially purified suspension to final concentrations of 1 M and 10% w/v, respectively, and then stored at 4 °C for two weeks. Following refrigeration, phages were centrifuged at 11,000× g for one hour at 4 °C, the pellet re-suspended in 50 mL phosphate-buffered saline (PBS), pH 7.4, and the phage content determined using the OA method.SU7 was previously isolated from the Käppala wastewater treatment plant located 15 km East of Stockholm, Sweden, in November 2016. For phage enrichments, fresh LB media was inoculated with 100 µL of overnight bacterial cultures and allowed to grow to the mid-log phase at 37 °C with shaking or until the optical density at 600 nm  and 100 µL of 109 serially diluted SU7. The OA was poured over the surface of a pre-prepared TYA plate and incubated for approximately 18 h at 37 °C. Plaques were imaged using a Samsung SM-G950U camera. Twenty diameters were calculated using ImageJ software [SU7 plaque morphology was determined using ETEC7 as the host bacterium and the OA method . In shorsoftware .5–106 plaque-forming units (PFU)/mL and the phage content was determined by the OA method. Prepared bacterial plates were then inoculated with approximately 10 µL of the diluted SU7 phage suspension using a stamper. The spots were allowed to dry at room temperature, plates incubated at 37 °C overnight, and then assessed for its ability to produce a plaque. Host-range experiments were replicated in triplicate unless otherwise stated in the text.The host range of SU7 was determined using the spot test assay as previously described . In brie11 PFU/mL) was negatively stained with 1% w/v uranyl acetate [PEG purified SU7 (1.1 × 10 acetate  and visu acetate .600 0.6). Once OD600 0.6 was reached, the bacterial suspension was removed; the final volume of the bacterial suspension used for the experiment was 44.982 mL. A total of 18 µL of the SU7 phage stock (1.50 × 1010 CFU/mL) was added to the mid-log-phase bacteria (approximately 5.03 × 107 CFU/mL) at a multiplicity of infection (MOI) of 0.12 and mixed by swirling (T = 0). Aliquots of 1 mL, withdrawn every five minutes for an experimental duration of 65 min, were centrifuged at 6000× g for one minute. Using the supernatant, 1:10 serial dilutions in PBS were done to determine the phage content during each time point. All experiments were performed in triplicates. The adsorption rate constant was determined using the following formula shown below, where N is the bacterial density, oP and P are the starting and ending phage titers, k is the adsorption rate constant, and t is the time in minutes over which adsorption occurs:The latency period, burst size, and adsorption rate constant for SU7 were determined using the modified one-step growth curve protocol used by Koonjan et al. (2020) . In brieIt should be noted that the adsorption rate constant was determined using only two time points (T = 0 and T = 5). The burst size was calculated by dividing the phage titer after the first burst (approximately at 35 min) with the number of adsorbed phages .8 PFU/mL using the Norgen Biotek phage DNA isolation kit  according to the manufacturing instructions but with an additional DNAse I treatment. Before carrying out the sequencing, the phage DNA concentration was quantified by fluorometry on a Qubit 2.0  and the purity assessed by gel electrophoresis.SU7 DNA was extracted from suspensions containing a minimum of 1 × 10Caudovirales using the Basic Local Alignment Search Tool (BLAST) BLASTx software [70 and phage promoter regions were found using BPROM [SU7 genome library preparation and sequencing on the Illumina Miseq platform, with paired-end 300 base-pair (bp) reads using a V3 600-cycle kit, were carried out by Eurofins . The 718,152 reads in the phage index were quality controlled using FastQC version 0.11.8 . Reads wsoftware . Hypotheng BPROM  and Phagng BPROM ,32,33, rng BPROM ,35. Ribong BPROM ,37. Genong BPROM ,39 and tng BPROM ,41. Phagng BPROM ,43.Caudovirales phages. Alignments were made in ClustalW with the default setting within the Mega-X software and in MAUVE version 20150226 with default settings [Phylogenetic analyses of the SU7 genome were conducted using complete nucleotide sequence alignments. Sequences for whole genomes were obtained from the NCBI genome databases . The gensettings ,45,46. APodoviridae family within the Caudovirales order. It has an elongated head, with a length of 134 nm (134 ± 6 nm) and width of 44 nm (44 ± 2 nm), and a very short non-contractile tail, which could not be measured  and include the E. coli phages vB_EcoP_SU10, Paul, and phiEco32 [−9 mL/min. C3 phages with larger elongated heads theoretically would have smaller plaque morphology  as well as lower virulence and inferior infection kinetics [SU7 forms transparent, circular plaques approximately 1 mm in diameter (1.4 ± 0.3 mm) upon a lawn of its host bacteria ETEC7 A. TEM comeasured B. OccurrphiEco32 ,48,49,50kinetics ,52. Morpkinetics ,53,54.E. coli strains, phage SU7 showed a narrow host range, infecting 11 out of 137 (8%) test strains . It is possible that the DTR region of SU7 could be longer if more mismatches were allowed during the PhageTerm searches.PhageTerm predicted the SU7 genome termini ends in short direct terminal repeats (DTR). Like phage PhiEco32, it is possible that SU7 has a replication strategy whereby the formation of either circular or linear concatemeric DNA during infection results in the duplication of its genome ends; thus, the beginning and ending have the same sequence repetition . Upon inE. coli phages ECBP2 (94%), EK010 (93%), vB_EcoP_EcoN5 (91%), and vB_EcoP_SU10 (90%)  .Kuravirus genus and are morphologically defined by an elongated head (the rare C3 morphotype). Phages belonging to Kuravirus share more than 61% DNA sequence and more than 69% protein homology [Kuravirus. References to the Kuravirus phages in the phylogenetic analyses and their accession numbers can be found in All phages that bear genomic similarity to SU7 belong to the homology . This caPodoviridae phage with the rare C3 morphotype belonging to the Kuravirus genus. Successful phage therapy entails the phage(s) given must be virulent and rapidly reproduce, ideally having a short latency period and large burst size, be small in size to allow for better diffusion and site-directed dosage, and have a broad host range [Kuravirus genomic diversity.Due to their therapeutic potential, phages are ideal candidates to treat antibiotic-resistant infections, such as those arising from ETEC. However, many phages need to be isolated and their genomes characterized before they can be used therapeutically. We have isolated and characterized the genome of vB_EcoP_SU7, a st range . Given t


The prognosis of patients with relapsed Ewing sarcoma is poor. In this study, we aimed to pooled-analyze the efficacy and safety of the combination of irinotecan and temozolomide in treating patients with relapsed Ewing sarcoma.PubMed, Cochrane CENTRAL, Web of Science, and EMBASE were systematically searched on September 27, 2021. The primary outcomes were rates of objective response and disease control, and the secondary outcomes were toxicities.Six retrospective studies with 184 patients were enrolled in the analysis. The median age ranged from 14 to 21. The integrated rates were 44%  for objective response and 66% (55–77) for disease control. Grade 3–4 neutropenia, thrombocytopenia, and diarrhea occurred in 8% (3–16), 7% (3–11), and 8% (5–10) of chemotherapeutic cycles, respectively. 18% (7–32) and 6% (2–11) of patients suffered grade 3–4 neutropenia and thrombocytopenia after irinotecan plus temozolomide treatment.Irinotecan plus temozolomide combination chemotherapy showed antitumor activity and an acceptable safety profile in patients with relapsed Ewing sarcoma. More future prospective studies are needed to confirm the retrospective results. 1. Nearly 44% of relapsed Ewing sarcoma patients achieved an ORR after IT treatment.2. Relapsed Ewing sarcoma treated with IT had an over 66% DCR.3. IT chemotherapy caused low incidences of neutropenia, thrombocytopenia, and diarrhea in treating relapsed Ewing sarcoma. Ewing sarcoma is a very rare tumor and usually occurs in childhood and young adults. Undifferentiated small round blue-cells are the main pathologic characteristics . HistogeIn order to increase the survival outcomes in advanced patients, a recently published prospective study had evaluated the activity and tolerability of irinotecan plus temozolomide as front-line chemotherapy in primary disseminated Ewing sarcoma. In the study, although grade 3–4 adverse events were observed in 3% of enrolled patients, the objective response rate (ORR) was 59%, with a 3-year OS of 36% and a 3-year EFS of 21% . HoweverIn the second- or later-line setting for patients with recurrent and primary refractory Ewing sarcoma, therapeutic chemotherapies recommended by National Comprehensive Cancer Network (NCCN) guideline include cyclophosphamide + topotecan, irinotecan + temozolomide ± vincristine, cabozantinib, docetaxel + gemcitabine, and ifosfamide + carboplatin + etoposide . Among tThe rEECur trial is the first randomized controlled study to compare the chemotherapeutic regimens in recurrent and primary refractory Ewing sarcoma. In this ongoing clinical trial, irinotecan plus temozolomide has a 20% response rate, a 4.7 months (95% CI: 3.4 to 5.7) progression-free survival (PFS), and a 13.9 months (95% CI: 10.6 to 18.1) OS, but the interim results find that the combination of irinotecan and temozolomide is less effective than topotecan plus cyclophosphamide, gemcitabine plus docetaxel, and high-dose ifosfamide [However, after reviewing the published retrospective studies, we noticed that the effects of irinotecan plus temozolomide combination therapy were much better in relapsed Ewing sarcoma –14. For Therefore, in this study, we synthesized the irinotecan and temozolomide chemotherapy data in treating patients with relapsed Ewing sarcoma in retrospective studies for comprehensively understanding the benefits and risks and future application.We conducted this pooled analysis according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guideline .A systematic search was performed in online databases  on September 27, 2021. The search terms included: (1) Ewing sarcoma, (2) irinotecan, and (3) temozolomide. References of relevant records in reviews were manually checked for more eligible studies.All evaluable studies were assessed to meet the following inclusion and exclusion criteria.Patients were diagnosed as relapsed Ewing sarcoma, including recurrent disease (defined as the patients who received up-front chemotherapy) and primary refractory disease (defined as the patients who progressed under upfront chemotherapy),Patients were treated with irinotecan plus temozolomide chemotherapy,Data of responses, survival outcomes, and/or toxicities were available.Inclusion criteria:Meeting abstractsCase reports,Basic or animal studiesIrinotecan and temozolomide concurrent with or followed by additional agents,Data of Ewing sarcoma could not be separated from other types of tumors,Non-English studies.Exclusion criteria:ORR and DCR were the primary outcomes. ORR should be defined as the percentage of patients who achieved composite complete or partial responses, and DCR is defined as the overall rate of complete response, partial response, and stable disease. Treatment-related adverse events were the secondary outcomes. Bi-Cheng Wang and Guo-He Lin independently extracted detailed data from each article, including the name of the first author, year of publication, study design, number of patients, median age, doses, cycles, survival outcomes, responses, and toxicities. Any discrepancies were resolved by consensus.The risk of bias was evaluated by sensitivity analysis and Egger's test. Rates of objective response and disease control and incidences of toxicities were pooled-analyzed in a random-effects model owing to the single-arm data syntheses. All above analyses were conducted through R (version 4.1) software and the “meta” package , 17.n = 17), conference abstracts (n = 13), registered trials (n = 2), case reports (n = 2), animal studies (n = 2), and non-English studies (n = 1). Finally, six retrospective studies were enrolled in the pooled analysis , and 40 mg/m2/day on day 1–5. While the therapeutic strategy of temozolomide was 100 mg/m2/day on days 1–5. The median number of cycles of the combination therapy ranged from 4 to 14.Table Response data for 172 of the 184 included patients were available and were utilized to analyze ORR and DCR. The pooled rate of objective response was 44%  , the ORR was only 20%, with a median PFS of 4.7 months and a median OS of 13.9 months [Besides the pooled results, the comparisons between irinotecan plus temozolomide and other second- or later-line chemotherapies deserve our attention.2/day on day 1–5) and temozolomide (125 mg/m2/day on day 1–5) plus vincristine (1.5 mg/m2/day on day 1). Median cycles were 4.1 per patient. Even the ORR was 54.5% and the DCR was 68.2%, the median time to disease progression was only three months (range 1.1 to 37.1) [In Raciborska’s study, 22 relapsed Ewing sarcoma patients received irinotecan (50 mg/mto 37.1) . CompareResearchers have tried to combine PARP inhibitors  with irinotecan and/or temozolomide in Ewing sarcoma to elevate the response rates and survival outcomes , 20. Whe2 (on day 1–5) and topotecan 0.75 mg/m2 (on day 1–5). The median number of chemotherapeutic cycles was four (range 1–10). The results showed that Ewing sarcoma patients had a 23% (3/13 patients) ORR and a 77% DCR (7/13 patients) with manageable toxicities [The study reported by Hunold detected the combination of cyclophosphamide (250 mg/m2/day on day 1–5) and topotecan (0.75 mg/m2/day on day 1–5) in 54 patients with relapsed Ewing sarcoma. With a median of 3 cycles of chemotherapy, the ORR and DCR were 32.6% and 59.1%, and the 1-year OS rate was 61% (95% CI 47–74%) . Additioxicities . Accordi2 ifosfamide. The median age was 17 years (6–45 years). 44% and 76% of patients achieved an ORR and a DCR, respectively. Nevertheless, extremely high incidences of grade 4 neutropenia (97% cycles) and thrombocytopenia (54% cycles) were observed [High-dose ifosfamide could be another treatment option for recurrent or advanced Ewing sarcoma patients. In Ferrari’s study, 37 Ewing sarcoma patients (including 33 patients with metastatic recurrent disease and four patients with progression during neoadjuvant chemotherapy) were administrated with 15 g/mobserved .Cabozantinib is a MET and VEGFR2 inhibitor. Italiano et al. conducted a multicenter phase 2 clinical trial to investigate the activity of cabozantinib in advanced Ewing sarcoma . 26% of 2/day on day 8) + gemcitabine (675 mg/m2/day on days 1 and 8) and ifosfamide (1800 mg/m2/day on days 1–4) + carboplatin (400 mg/m2/day on day 1) + etoposide (100 mg/m2/day on days 1–4) chemotherapies as the second-line strategies [The NCCN guideline has recommended docetaxel (75–100 mg/mrategies , 26. In rategies . It seemSeveral limitations existed in this analysis. First, even this study showed the comparison between data in prospective clinical trials and our pooled analysis of retrospective studies, we could not demonstrate that the differences were statistically significant. Second, only retrospective studies were eligible. The enrolled articles mainly described retrospective assessments from single centers in which toxicity and response monitoring might not be necessarily standard. Moreover, retrospective studies might exert higher selection bias and lack strict evaluation. Third, the doses and administration schedules varied widely and more specific information. Fourth, outside of a clinical trial, it is easy to imagine that disease control could be overestimated if patients are not undergoing imaging at standard time points, and toxicity might be underappreciated if there are no routine toxicity assessments.Irinotecan combined with temozolomide is an effective and safe chemotherapeutic strategy for patients with relapsed Ewing sarcoma. Based on the results in this integrated analysis, we provided informative data for both clinicians and patients. In future prospective clinical trials, irinotecan plus temozolomide chemotherapy could be a preferred control strategy in searching for more effective therapies for Ewing sarcoma patients.