CsSOD, CsPOD, CsCAT and CsAPX) expression of antioxidant enzyme biosynthesis. Taken together, our results confirmed that melatonin protects tea plants against abiotic stress-induced damages through detoxifying ROS and regulating antioxidant systems.Melatonin is a biological hormone that plays crucial roles in stress tolerance. In this study, we investigated the effect of exogenous melatonin on abiotic stress in the tea plant. Under cold, salt and drought stress, increasing malondialdehyde levels and decreasing maximum photochemical efficiency of PSII were observed in tea leaves. Meanwhile, the levels of reactive oxygen species (ROS) increased significantly under abiotic stress. Interestingly, pretreatment with melatonin on leaves alleviated ROS burst, decreased malondialdehyde levels and maintain high photosynthetic efficiency. Moreover, 100 μM melatonin-pretreated tea plants showed high levels of glutathione and ascorbic acid and increased the activities of superoxide dismutase, peroxidase, catalase and ascorbate peroxidase under abiotic stress. Notably, melatonin treatments can positively up-regulate the genes ( Camellia sinensis (L.) O. Kuntze) plant is an important economic crop that has been planted in China for nearly 2000 years [The tea  and superoxide anion (O2−) [Plants grown under environmental stress show the inhibition of growth and serious morphological, metabolic, and physiological anomalies ranging from chlorosis or leaf rolls to lipid peroxidation and protein degradation ,5,6. Malon (O2−) ,12. The on (O2−) ,14. Moreon (O2−) . In respon (O2−) . TherefoN-acetyl-5-methoxytryptamine) is an indole hormone involved in multiple biological processes [Melatonin , which has stronger antioxidant activity than melatonin, can also directly and efficiently scavenge ROS [Many researchers have demonstrated that melatonin can protect plants against ROS and consequent alleviation of oxidative stress ,27. Melaenge ROS ,28,29. Wenge ROS ,31. ROS-enge ROS ,33. Receenge ROS  and exogenge ROS ,35. As aenge ROS ,37. HoweAlthough remarkable progress has been made in investigating the role of melatonin in responses to abiotic stress, very less information about the effects of melatonin on abiotic stress tolerance in tea plants is available. In the present study, we explored the regulatory mechanisms controlling melatonin-mediated abiotic stress tolerance in tea plants and tried to understand the impact of melatonin on abiotic stress in tea plants. Hence, we analyzed the effects of melatonin on lipid peroxidation, ROS accumulation, antioxidant defense system, and photosynthetic capacity in tea seedling exposed to cold, high salt and drought stresses.The effect of exogenous melatonin on maximum photochemical efficiency of PSII (Fv/Fm) of tea plants under cold (4 °C), salt (NaCl) and drought stress are shown in A change in the contents of MDA with melatonin treatment is presented in 2O2 and O2− contents were assayed among control and melatonin-pre-treated plants  and 48 h of 4 °C treatment (1.70 μmol/g FW), respectively  cycle, the contents of GSH and ASA were examined in non-treated and melatonin-pretreated plants under control and abiotic stress conditions. As shown in CsSOD, CsPOD, CsCAT and CsAPX were analyzed, which are responsible for the biosynthesis of antioxidant enzymes. Similar to the changes in the activities of antioxidant enzymes, the transcript levels of CsSOD, CsPOD, CsCAT and CsAPX were all induced by 4 °C, NaCl and drought treatment   at a relatively low level.To cope with oxidative stress induced by adverse conditions, plants have evolved an effective antioxidant defense system, including enzymatic and non-enzymatic antioxidants. It was reported that under oxidative stress, ROS generation increases antioxidant enzymes activities in plants . Many std CsAPX) . Exogeno species ,49. Cons species ,50,51. I and CAT ,52. In t and CAT ,46. TakeIn conclusion, this study shows the ameliorative effects of exogenous application of melatonin on abiotic stress to tea plants. The present study provides the first evidence of the protective roles of exogenous melatonin response to multiple abiotic stresses in tea plants. To be specific, melatonin improves abiotic stress tolerance in tea plants, possibly through the enhancement of photosynthesis, the elimination of ROS and the upregulation of some key components of the antioxidant system. Our work also provides a case study that melatonin may have great potential for improving tea plant yield. However, further studies need to be conducted to provide more molecular and genetic evidence to support the mechanisms of melatonin-induced abiotic stress tolerance in tea plants.C. sinensis cv. ‘Longjing 43’) were cultivated in a chamber at the Tea Research Institute of Nanjing Agricultural University . Tea plants were grown in a growth chamber under 25 °C for 14 h with 200 μmol m−2 s−1 light intensity at daytime, at 18 °C for 10 h during night time and 70% of relative humidity. All plants were allowed to acclimatize for around 1 month before treatment, and every 60 tea cuttings seedlings were used for each treatment. Hoagland’s nutrient solution was used for fertilization with applications every 6 days, and proper moisture level was maintained by daily watering. Two-year-old vegetatively propagated cuttings of tea plants (−1 PEG) [The tea plants were randomized into eight groups: control condition (CK), control condition plus melatonin (CK + melatonin), cold condition (4 °C), cold condition plus melatonin (4 °C + melatonin), high salt condition (NaCl), high salt condition plus melatonin (NaCl + melatonin), drought condition (Drought) and drought condition plus melatonin (drought + melatonin). For the control, plants were treated with 25 °C and irrigated with deionized water until the experiment ended. For the melatonin treatment, tea plants were pretreated with 100 μM melatonin  solution for 7 days (once a day). This concentration was selected based on our preliminary tests and all treatments were sprayed as a volume that saturated the canopy (approximately 100 mL per plot). After pre-treatment, the plants were subjected to cold stress (4 °C), high salt (300 mM NaCl) and drought stress (200 g·L−1 PEG) ,54. Leafv/Fm. Minimum fluorescence (Fo) was measured during the weak measuring pulses, a 0.8-s pulsed light (4000 μmol·m−2·s−1) was applied to measure the maximum fluorescence (Fm). Fv/Fm was calculated as (Fm − F0)/Fm [The chlorophyll fluorescence parameters of tea leaves were determined with a portable photosynthesis system . The tea plants were pre-adapted in the dark for 30 min before measurement to ensure sufficient closure of all PSII reaction centers, the 2nd leaves from the top buds of tea plants were used to determine F− F0)/Fm .The level of lipid peroxidation in leaves was assessed by measuring the MDA content using 2-thiobarbituric acid as described by Hodges et al. .2O2 assay, 0.3 g of tea leaf samples were grounded with 3 mL of pre-cooled HClO4 (1 M) using a pre-chilled mortar and pestle, and the homogenate was centrifuged  at 6000× g for 5 min at 4 °C [g for 5 min at 4 °C. Afterward, the resulting supernatant was passed through an AG1x8 prepacked column  and H2O2 was eluted with 4 mL double-distilled H2O. The sample (800 μL) was mixed with 400 μL reaction buffer containing 4 mM 2,2′-azino-di (3-ethylbenzthiazoline-6-sulfonic acid) and 100 mM potassium acetate at pH 4.4, 400 μL deionized water and 0.25 U of horseradish peroxidase (HRP). H2O2 content was measured at 412 nm using UV spectrophotometer .For the H at 4 °C . The pH 2− production was estimated as described by Elstner and Heupel [g for 10 min. Then, 1 mL of the supernatant was mixed with 0.9 mL of 65 mM phosphate buffer (pH 7.8) and 0.1 mL of 10 mM hydroxylamine hydrochloride. The mixture was incubated at 25 °C for 20 min. Then, 0.375 mL of 17 mM sulfanilamide and 0.375 mL of 7 mM α-amino-phenylsulfonic were added to the mixture for another 20 min of incubation at 30 °C. Next, ethylether in the same volume was added and centrifuged at 2000× g for 5 min. The absorption of the reaction mixture was measured at 530 nm. Sodium nitrite was used as a standard solution to calculate the production rate of superoxide anion.Od Heupel  with sliw/v) polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 4 °C for 15 min at 12,000× g [Fresh leaves (0.5 g) were grounded on ice with 0.5 g quartz sands and 5.0 mL of 50 mM precooled phosphate buffer (pH 7.5) containing 0.1 mM ethylene diamine tetraacetic acid (EDTA) and 5.0%  (DTNB), and 700 μL of 50 mM phosphate buffer (pH 7.5) containing 2.5 mM EDTA. Then, 20 μL of GR (50 U/mL) was added to the supernatant and the increase in absorbance at 412 nm was used for calculating the content of GSH.The content of GSH was measured following the procedure described by Griffith . 0.1 g o4. The crude extract was centrifuged at 12,000× g for 10 min at 4 °C, the resulting supernatant was collected for further analysis. AsA was assayed by determining the absorbance difference of the supernatant at 265 nm in 200 mM sodium acetate buffer (pH 5.6) before and after 15-min incubation with 1.5 units of AsA oxidase.The content of AsA was assayed by the method of Logan et al. . The cruTM 2000 spectrophotometer . The cDNA of tea leaves was synthesized using a PrimeScriptTM RT reagent kit , following the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed on an IQ5 multicolor real-time PCR detection system . The reaction program was set as follows: 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s and 55 °C for 25 s. The reaction volume was 20 μL, which contained 2 μL diluted cDNA strand, 7.2 μL ddH2O, 10 μL SYBR® Premix Ex Taq , and 0.4 μL each primer. The C. sinensis β-actin gene was selected as an internal control gene. These qRT-PCR experiments were repeated three times, based on three separate RNA extracts from three samples. Relative gene expression levels were calculated using the 2−ΔΔCT method [Total RNA was isolated from tea plants using the Quick RNA Isolation Kit . The concentration and purity of RNA were measured using a NanoDropT method . DetectiP < 0.05.The experiment had a completely randomized design. Each value was expressed as the mean ± SE of the three independent experiments. All data were analyzed using SPSS 20.0 . Significance was determined by Duncan’s test and ANOVA. Different letters in figures indicate significant differences at


Escherichia coli (STEC), including E. coli O157:H7. Shiga toxin is encoded by resident prophages present within this bacterium, and both its production and release depend on the induction of Shiga toxin-encoding prophages. Consequently, treatment of STEC infections tend to be largely supportive rather than antibacterial, in part due to concerns about exacerbating such prophage induction. Here we explore STEC O157:H7 prophage induction in vitro as it pertains to phage therapy—the application of bacteriophages as antibacterial agents to treat bacterial infections—to curtail prophage induction events, while also reducing STEC O157:H7 presence. We observed that cultures treated with strictly lytic phages, despite being lysed, produce substantially fewer Shiga toxin-encoding temperate-phage virions than untreated STEC controls. We therefore suggest that phage therapy could have utility as a prophylactic treatment of individuals suspected of having been recently exposed to STEC, especially if prophage induction and by extension Shiga toxin production is not exacerbated.Hemolytic–uremic syndrome is a life-threating disease most often associated with Shiga toxin-producing microorganisms like  Prophages are bacteriophage (phage) genomes that replicate alongside their bacterial host’s genome until induced to produce viral particles. This carriage state, termed a lysogenic cycle, is characteristic of temperate phages , and the prophage-carrying bacterial host is termed a lysogen. Recent reviews provide information on the diverse and impactful biology and distribution of temperate phages, along with methods for temperate phage detection ,2,3. OneEscherichia coli (STEC) [Notable among prophage-encoded virulence factors are exotoxins, such as those associated with the O157:H7 serotype of Shiga-toxigenic i (STEC) ,9. Thesei (STEC) ,11,12. Oi (STEC) ,15,16,17i (STEC) ,19,20. Si (STEC) ,22, but i (STEC) .Certain antibiotics that induce the STEC SOS response also can induce Shiga-toxigenic prophages, resulting in new intracellular Shiga toxin production and subsequent phage lysis-associated toxin release ,24,25,26Based on the above assumptions, we reasoned that lysis of STEC O157:H7 by strictly lytic phages might eliminate STEC O157:H7 without further contributing to Shiga toxin production. If true, then such lytic phages might be employed as a means of anti-STEC treatment, and by extension as anti-Shiga-toxigenic phage agents—in effect an anti-temperate phage form of phage therapy. E. coli O157:H7.Here we test this hypothesis through in vitro experiments designed to explore the use of strictly lytic phages, unrelated to Shiga toxin-encoding prophages, as anti-STEC bactericidal agents, in order to assess the potential impact of phage therapy on the production of Shiga-toxigenic 933W phages by From the American Type Culture Collection (ATCC—identifier ATCC43895) we acquired the STEC serotype O157:H7 whose genome sequence is published under strain EDL933 ,12. In oWith a fully-sequenced working strain, we then assessed spontaneous prophage induction in STEC as follows. STEC cultures were grown in triplicate for 5 h, treated with chloroform for 2.5 h to lyse the cells and release encapsidated phage DNA, and 0.2 µm-filtered to remove cells and large cellular debris. Samples were then treated with DNase to minimize free DNA and enrich for encapsidated DNA. The DNA was then extracted and sequenced, and the resulting reads were mapped to the STEC genome, including prophage regions. Given that most free bacterial DNA was removed with DNase, elevated read recruitment across the entirety of any prophage region would indicate induction and subsequent encapsidation of the prophage region(s). This read recruitment methodology is especially useful for identifying which prophages are induced within polylysogens, as previously shown ,29,30.Mean read recruitment coverage values were calculated per host or prophage region and normalized by the sequencing depth and the sequence length of either the 933W genomic region  or the STEC genome without the 933W prophage . This revealed that prophage 933W, which encodes the Stx2 genes and is responsible for much of STEC’s pathogenesis ,31, had stx2a encoded by 933W. To this end, we grew and sampled STEC as done for the whole-genome induction screen above, and found that the prophage 933W-encoded stx2a copy number increased eight-fold from the start to the end of the aforementioned 7.5 h experiment (~105 to 8 × 105 per µL of filtrate)  approach targeting the Shiga toxin gene iltrate) . This coE. coli O157:H7 [8 and ~4.6 × 108 plaque-forming units per ml for phages p000v and p000y, respectively. Indeed, by the end of the experiment, addition of these phages had decreased the levels of 933W prophage induction, as quantified by qPCR. Namely, while the qPCR-measured ratio of stx2a copies per µL between 7.5 and 0 h was ~8 without phage (stx2a copies (a proxy for 933W prophage induction) and suggest that Shiga toxin production would also be reduced, due to both no further stimulation of prophage induction upon lytic phage infection, on the one hand, and reduction in the number of lysogens present on the other.Given that 933W induction is known to be associated with Shiga toxin production in  O157:H7 , we next O157:H7 , and whiut phage , with thut phage , Table 2E. coli O157:H7 in particular, Shiga toxin production and release is associated with prophage induction, mostly prophage 933W [The primary question regarding the potential for using phage therapy to treat pathogenic lysogens is whether such treatment might exacerbate patient exposure to toxins produced upon prophage induction. For Shiga-toxigenic age 933W ,33,34,35age 933W ,37. There are three related routes by which Shiga toxin exposure could occur . First, A second route of Shiga toxin release  used in this study was obtained from the American Type Culture Collection (ATCC) under identifier 43895, which is published as EDL933 under GenBank accession numbers CP008957 and CP008958 [https://www.doi.org/10.7946/P2HP89), and in GenBank under accession numbers MK047717 and MK047718, respectively.The Shiga-toxigenic CP008958 ,12. The CP008958 , and canCP008958  under DOBacteria were streaked onto TSA  plates from glycerol stocks, grown overnight at room temperature (RT), and held at 4 °C. Single colonies were then inoculated in TSB  and grown shaking at ~200 rpm at 37 °C overnight.8 CFUs/mL). Phages were added to 10–50 mL of the host culture at a low MOI (10-6–0.1), and incubated shaking at ~200 rpm at 37 °C for ~5 h. Chloroform was added to the infection at 1% (v/v) and incubated shaking at ~70 rpm at RT for 2 h. The chloroform was allowed to settle for 30 min, and the aqueous phase was 0.2 μm-filtered to remove any remaining cells. Some lysates were also concentrated via polyethylene glycol (PEG)-precipitation. For those, both NaCl (6.5 g) and PEG-8000 (10 g) were added per 100 mL lysate. This was incubated overnight at 4 °C, then centrifuged at 10,000 g in a Beckman J2-MC centrifuge  for 10 min. The supernatant was removed, and the pellet resuspended in phage buffer , and 1 mL 1 M MgSO4 per L).An overnight bacterial culture was diluted 1:50 in fresh TSB and grown shaking at ~200 rpm at 37 °C until the optical density (OD) reached ~0.3 (2.94 × 10The STEC strain obtained from ATCC (ATCC43895) was sequenced. For that, its genomic DNA was extracted using the ZymoBIOMICS DNA mini kit  following the manufacturer’s protocol. Similarly, sequenced phage infections and phage-free cultures from which prophage induction was assessed were also sequenced. The DNA of these samples was extracted using the Phage DNA Isolation Kit  following the manufacturer’s protocol. Any remaining host DNA was degraded by adding 10 µL (20 U) of DNase I from the RNase-free DNase I kit  prior to proteinase K treatment. Bacterial host DNA concentrations were quantified using the Qubit 3.0 Fluorometer and the Qubit dsDNA High Sensitivity Kit . Extracts from the previous step were prepared for sequencing on the Illumina MiSeq platform  using the Nextera XT Library Preparation Kit  according to the manufacturer’s protocol . The magnetic bead normalization step was replaced with a manual normalization step, based on library concentration and average size as measured by the Qubit 3.0 Fluorometer and Qubit dsDNA High Sensitivity Kit  and the Fragment Analyzer , respectively. Paired-end sequencing was performed using the MiSeq Reagent v3 (600 cycle) kit . Two sample types were obtained for sequencing whole bacterial and phage genomes, mapping reads to such genomes, and assessing prophage induction: phage-free and phage-infected bacterial cultures. The phage-infected samples were lysates grown as described in the “Lysates for Phage Amplification” section. The phage-free samples were mock lysates prepared as the infection samples, but without phages and in 30 mL containing 3.5 mL of phage buffer. After DNA extraction and library preparation procedures as described, samples were sequenced via the MiSeq technology described.11 to derive more comprehensible whole-genome coverage values. Coverage values per base were visualized by creating bedgraph files, using the bedtools [Reads from each of the lysates were mapped to the STEC and respective phage genome using the Burrows–Wheeler Aligner (BWA)  version bedtools  version bedtools  and Circbedtools  version 10 cells, which was then added to 100 mL of TSB. This was grown shaking at ~200 rpm at 37 °C; the OD was read after ~30 min and then every 10 min until the reading was 0.25–0.3. Phages were added to 2–5 mL of the host culture at MOIs lower than 0.1 or close to 6. A 0.3–1 mL sample was taken immediately and 0.2 μm-filtered to remove any cells, then stored at 4 °C. The infected culture was then incubated shaking at ~200 rpm at 37 °C for 5 h. Chloroform was added to the infection at 1% (v/v) and incubated shaking at ~70 rpm at RT for 2.5 h. The chloroform was allowed to settle for 20 min, and the aqueous phase was 0.2 μm-filtered to remove cells. Another 0.3–1 mL sample was taken and stored at 4 °C. DNA was extracted from the two filtered samples. First, the viral DNA was inactivated using DNase in a ratio of 1 µL of DNase to 9 µL of sample. Ethylenediamine tetraacetic acid (EDTA) and Ethylene glycol tetraacetic acid (EGTA) were added at 100 mM to inactivate the DNase. After the DNA was inactivated, extraction was continued using a Wizard DNA Clean-up Kit . Then, 1 mL of DNA clean-up resin was added to each sample, and they were mixed by inversion. The samples were put into a syringe and pushed through a Wizzard minicolumn , followed by the addition of 2 mL 80% isopropanol pushed through the column, 1 mL at a time. Samples were centrifuged for 2 min at 10,000 g to remove any excess isopropanol. Each sample was eluted in 50 µL of Tris EDTA (TE) that had been warmed to 80 °C. At the addition of TE, each sample was briefly vortexed and then centrifuged at 10,000 g for 30 s to elute the DNA. The samples were then analyzed for their prophage content via qPCR.The OD of an overnight bacterial culture was read to determine the volume containing 10stx2a forward primer: 5′-ATGTGGCCGGGTTCGTTAAT-3′; reverse primer: 5′-TGCTGTCCGTTGTCATGGAA-3′). The qPCR reaction was carried out with the following thermocycler conditions: initial denaturation and enzyme activation at 95 °C for 5 min, 40 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, and extension at 72 °C for 30 s, followed by one cycle of 95 °C for 15 s, 57 °C for 15 s, and 95 °C for 15 s for the dissociation curve. Fluorescence signal was collected at the end of the extension step and of the ramping period of dissociation curve. Serial dilution of the genomic DNA of strain STEC was used to generate the standard curve (R2 > 0.99). To run the qPCR, 2 µL of DNA extracted from a phage lysate was used as the template in the 15 µL qPCR reaction that contained 1× Perfecta SYBR Green FastMix  and 300 nM of each of the forward and reverse primers targeting prophage 933W (gene With the rise in antibiotic resistance in bacterial pathogens, phage therapy presents a promising alternative for treatment. Importantly, though, many of pathogens contain prophages that not only are commonly the source of pathogenesis ,6, but h


Transcriptome analysis of radial glia, newborn neurons and mature neurons using single cell sequencing identified distinct transcriptional profiles, including novel markers for each population. Specifically, we detected two separate newborn neuron types, which showed diversity of cell fate commitment and location. Further analyses showed that these cell types are homologous to neurogenic cells in the mammalian brain, identified neurogenic commitment in proliferating radial glia and indicated that glutamatergic projection neurons are generated in the adult zebrafish telencephalon. Thus, we prospectively isolated adult newborn neurons from the adult zebrafish forebrain, identified markers for newborn and mature neurons in the adult brain, and revealed intrinsic heterogeneity among adult newborn neurons and their homology with mammalian adult neurogenic cell types.Zebrafish display widespread and pronounced adult neurogenesis, which is fundamental for their regeneration capability after central nervous system injury. However, the cellular identity and the biological properties of adult newborn neurons are elusive for most brain areas. Here, we have used short-term lineage tracing of radial glia progeny to prospectively isolate newborn neurons from the her4.1 Summary: A combination of lineage tracing and single cell sequencing identifies specific markers and functional subpopulations of adult newborn neurons in the zebrafish forebrain. Adult neurogenesis, the generation and integration of additional neurons to the brain circuitry in adulthood, is widespread across vertebrates, although the extent greatly varies among species . It is tIn most mammals, adult neurogenesis is confined to the major neurogenic niches in the subgranular zone of the hippocampal dentate gyrus and the sub-ependymal zone of the lateral ventricles. Newborn neurons (NBNs) integrate only into the granular zone of the dentate gyrus, and into the olfactory bulb in rodents or into the striatum in humans . Outsidetbr1, neurod1 and bhlhe22  proliferation of radial glia, (2) neuron generation and (3) integration of newborn, differentiated neurons in the parenchyma . Within Recently, cellular differentiation trajectories were reconstructed using single cell sequencing – alone or in combination with cellular barcoding – in vertebrate embryos  or in thelavl3:gfp . Quantification showed that mCherryhigh/GFPneg RG represented 8.7%, whereas mCherrylow/GFPpos cells (designated as NBNs) accounted for 10.9% of all live forebrain cells. Mature neurons  were the dominating cell population in the adult forebrain with 72.1%, and lineage marker-negative cells accounted for 8.2% of forebrain cells  in the adult zebrafish telencephalon, we developed a short-term lineage-tracing protocol, based on retention of fluorescent proteins in cell type-specific, fluorescent reporter lines. To this end, we combined the neuronal reporter line avl3:gfp  with themarks RG . Althougrca 24 h , are inhrca 24 h . Using tgfp fish A. When aavl3:GFP C. These in cells D. Directin cells E.Fig. 1low/GFPpos cells could also represent mature neurons with low level activation of this pathway instead of being NBNs that inherited mCherry protein from their RG precursors. To exclude this possibility, we combined the elavl3:gfp neuronal reporter line with a newly generated radial glia specific reporter line: gfap:nls-mcherry, which expresses nuclear-localized mCherry in telencephalic radial glia cells  under the control of the radial glia-specific gfap promotor . Quantification confirms similar percentages of mCherryhigh/GFPneg and mCherrylow/GFPpos cells . To verify that the mCherrylow/GFPpos double-positive population is enriched with adult generated neurons, an EdU pulse-chase analysis was performed. Adult her4.1:mcherry; elavl3:gfp fish were injected three times with EdU in 12 h intervals to label sufficient numbers of proliferating cells in the adult brain. EdU incorporation was assessed at 2 h, 7 days or 4 weeks after the last injection to identify proliferating cells and their immediate progeny, early progeny and late progeny of the initially proliferating cells, respectively  and mCherryneg/GFPpos neurons (1.5±0.8%). After 4 weeks of chase, EdU incorporation in mCherrylow/GFPpos neurons was reduced to 0.7±0.1% compared with the 7 days chase, mCherryhigh/GFPneg RG showed a trend towards reduced EdU incorporation (3.9±1.6%), whereas mCherryneg/GFPpos neurons showed no significant change (0.6±0.1%)  B,C. Thesher4.1:mCherryhigh/elavl3:GFPneg bona fide RG.Together, these data provide substantial evidence that radial glia-derived NBNs in the adult zebrafish forebrain can be identified by retention of fluorescent proteins expressed in radial glia with simultaneous expression of a neuronal marker. Our analyses reveal a population of NBNs that is equal in number compared with the high/GFPneg), 169 NBNs (mCherrylow/GFPpos) and 30 mature neurons  from the adult telencephalon of six fish and sequenced them using SMARTSeq2   (sv2a), neurogranin a (nrgna) and calmodulin-dependent kinase 2a (camk2a) . The cells from the three FACS gates segregated into five transcriptome clusters: one cluster (RG) consisted almost exclusively of cells sorted as RG, three clusters  contained mostly cells sorted as NBN, and one mixed cluster (MN) contained cells from the NBN and MN gates . We assigned cell type identity to each cluster based on known marker genes , her4.1 and glula (encoding glutamine synthase), but also novel markers such as si:ch211-251b21.1 (encoding the glutamate receptor grik-1), fgfbp3, atp1a1b, selenop and mdka. The major pure NBN cluster (NBN.1) was characterized by specific expression of tubb5, cd99l2, ppp1r14ba, cnp and tuba2. The smaller pure NBN cluster (NBN.2) expressed the transcription factor ebf3a, kcnj19a, tbr1b, msi2b and podxl2. The mixed NBN and MN cluster (cluster MN) showed specific expression of sult4a1, sybu, ube2ka, gad1b and sprn2, while the OPC cluster predominantly expressed the marker genes aplnra, sema5a, si:ch211-286c4.6 (encoding Cd59), traf4a and cd82a, which were recently identified as genes expressed in zebrafish OPCs . Retention of mcherry, expressed from a RG-specific promotor in these OPCs, as well as shared marker expression ] revealed that 28.6±3.9% of all olig2:GFP cells were mCherrylow/GFPhigh double positive . We also observed numerous cells showing high levels of mCherry fluorescence and low levels of olig2-driven GFP fluorescence, possibly representing nascent RG progeny differentiating towards OPCs . In summary, these data provide robust evidence that her4.1-expressing RG generate heterogeneous and diverse progeny comprising different neuronal and oligodendroglial cell types in the normal adult telencephalon.Next, using our lineage tracing protocol, we sorted 171 RG (mCherryMARTSeq2 . On averd s100b)  Fig. 3A insm1a) , but sho(camk2a) , compareA insm1a of olig2  .To infer the differentiation trajectories of radial glia-derived NBNs, we performed pseudotime-based ordering of our single cell gene expression profiles based on diffusion maps A. In conFig. S5C). To infer the heterogeneity of cell identity within the zebrafish cell clusters, we also determined the cell type of the single corresponding, most similar murine cell for each zebrafish cell. We omitted cells that show only limited similarity to mammalian cells, identified by Fig. S5C and Materials and Methods). The results show that cells forming the RG cluster in the adult zebrafish brain mainly correspond to various glial cell types in the murine brain, i.e. astrocytes and radial glia-like neural stem cells (RGLs) of different maturation stages, neurogenic intermediate progenitor cells (nIPCs) with minor proportions corresponding to neuroblasts, OPCs and microglia. Zebrafish OPCs corresponded only to murine OPCs. Consistent with the pseudotime analysis, NBN.1 cells corresponded to neuroblasts and immature neurons of pyramidal, granule cell or GABAergic identity, while NBN.2 and MN cells mainly corresponded to mature neurons of GABAergic and pyramidal identity as well as juvenile granule cell neurons , we used ccnd1 as general proliferation marker to compare the transcriptome profile of +ccnd1, proliferating RG and ccnd1− quiescent RG. Apart from the expected enrichment of proliferation genes, such as snrpd1, actl6a and mcm7  and s100b, were enriched in quiescent RG . Strikingly, out of the four neuronal fate determinants/neuronal genes  that were most substantially expressed in RG  and that many divisions from proliferating RG are neurogenic. Taken together, we identify RG as the only proliferating cell type in the cell trajectory derived from her4.1-positive RG and provide strong evidence that most proliferating RG are committed to neurogenesis.As a substantial proportion of NBN.1 cells showed similarity to neuroblast, which contain proliferating cells in mammalian adult neurogenic niches, we asked whether different subtypes of proliferating cells, e.g. radial glia, progenitors or neuroblasts , dorsal telencephalon glutamatergic fate , ventral forebrain GABAergic fate  and glutamatergic or GABAergic neuron identity  along the differentiation trajectory. Pan-neurogenic markers and eomesa were induced in differentiated RG, were abundantly expressed in the NBN1 cluster, and continued to be expressed, although in fewer cells, in both the NBN.2 and the MN cluster  or ebf3a, msi2b and tbr1b   in the lateral anterior diencephalon  to NBNs that form a common cluster with mature neurons . These results suggest that, with our lineage-tracing approach, we detect various differentiation states of NBNs, present at steady state in the uninjured telencephalon  expressed under the control of the her4.1 promotor, and by the presence of mcm5-driven EGFP  and NBN.1 marker genes (tmsb and marcksb) is enriched in proliferating RG . OPCs with a very similar marker profile were also found in the juvenile zebrafish brain . Given that more than 25% of olig2:GFPpos cells are also positive for her4.1:mCherry, the double-positive cells are far too numerous to be accounted for by the small subpopulation of olig2:GFPpos precursors in the midline of the ventral telencephalon , we also identify two more mature populations of adult-generated neurons. One of these populations forms a common cluster with mature neurons that do not retain her4.1-driven mCherry fluorescence and expresses telencephalic identity markers such as neurod and dlx genes. The NBNs in this cluster are considered to be generated from adult RG within the last 4 weeks, based on the retention of her4.1:mCherry . Within the MN cluster, cells expressing glutamatergic or GABAergic markers are found. Likely, the small number of analyzed cells in this cluster precludes the separation of these cell types into separate clusters, as found recently . Wild-type experimental fish were in the AB background. Fish were raised at a density of 50-60 fish/12 l tank . Fish ofher4.1:mcherry, the elavl3:gfp and the olig2:gfp lines have been described previously (gfap reporter line Tg(gfap:nls-mcherry), the gfap promoter (her4.1 promoter of the T2her4.1:mCherry-T2A-CreER plasmid (gfap promotor. The mCherry reporter was PCR amplified and flanked by unique restriction sites using the primers Cherry-Fse-for (atatGGCCGGCCgccaccatggctccaaagaagaagcgtaaggtaatggccatcatcaaggagttcatc) and Cherry-Asc-rev (cgccGGCGCGCCgaattaaaaaacctcccacacc). The nuclear localization sequence (nls) was introduced into the sequence by a 5′ overhang of the PCR forward primer. The PCR product was subloned into the pCR2.1-TOPO vector. Next, the TOPO vector with the reporter and the pTol(gfap:mcherry-T2A-CreERT2) construct were digested using the enzymes AscI and FseI, and ligated to replace the mCherry-CreERT2 cassette with the nls-reporter. For germ-line transformation, linearized plasmid DNA with transposase mRNA was injected into fertilized eggs (F0), raised to adulthood and crossed to AB wild-type fish as previously described . For the identification of transgenic fish, F1 embryos were examined under a fluorescent microscope and positive embryos were raised.The eviously . For thepromoter  was PCR  plasmid . The lowher4.1:mcherry;elavl3:GFP transgenic fish were sacrificed by an overdose (0.024%) of MS-222 (Sigma) (Wild type or  (Sigma)  until cehigh/GFPneg), NBN (mCherrylow/GFPpos) and MN (mCherryneg/GFPpos) were sorted into single cavities of 96-well plates containing 2 µl lysis buffer using index sort mode and a 100 µm nozzle. The samples were immediately frozen until preparation of sequencing libraries.Cells expressing mCherry and GFP were detected on a LSR II cell sorter (BD Biosciences) after 488 nm excitation and a bandpass-filter 564/42 nm and 530/30 nm, respectively. Calcein Blue was detected after 405 nm excitation and a bandpass-filter 450/40 nm. Forward and side scatter were used to select all events that show a characteristic scatter profile of zebrafish brain cells. From this selection, all events that showed incorporation of the Calcein Blue were gated as living cells. Living cells were plotted for GFP and mCherry fluorescence to gate for the respective cell populations. Detection of all fluorescent samples was controlled against unstained wild-type cells. Single cells gated as RG  using a 100 µm nozzle. For the two mCherrypos populations, 8000-30,000 cells were sorted; for the mCherryneg/GFPpos population, sorting was stopped at 100,000 cells. Cells were incubated for 15 min in the dark and EdU was detected using the Click-IT Plus EdU AlexaFluor 647 Flow Cytometry Assay Kit (Thermo) according to the manufacturer's instructions. After EdU detection, the cell pellet was resuspended in 200 µl HBSS (Thermo), including 10 µg/ml (w/v) and incubated for 10 min at room temperature to stain the DNA in cell nuclei. Cells that are positive for AlexaFluor 647-labeled EdU and/or Hoechst were detected on a LSR II cell sorter (BD Biosciences) using 633 nm and 405 nm excitation, and a bandpass filter .For the analysis of EdU incorporation into the different cell types of forebrains from +/mCherry+) cells, mCherry only-positive cells (GFP−/mCherry+), GFP only-positive cells (GFP+/mCherry−) and negative cells (GFP−/mCherry−) were analyzed and quantified for the appearance of GFP and DsRED within the same cell, doublets and cells with attached fluorescent debris.For imaging flow cytometry, dissected zebrafish forebrains were dissociated and processed as for conventional flow cytometry (see above). The dissociated cells were analyzed for reporter fluorescence (GFP and mCherry) and Calcein Blue as viability marker using an ImageStream X Mark II imaging flow cytometer (Amnis), after gating forebrain cells for singlets (aspect ratio versus area). Data were processed offline using IDEAS software (Amnis). At least 20 individual images of double-positive  at a final 1× concentration and 0.1 µM UP-primer under the following cycling conditions: initial denaturation at 98°C for 3 min; 22 cycles of 98°C for 20 s, 67°C for 15 s and 72°C for 6 min; and final elongation at 72°C for 5 min. The amplified cDNA was purified using 1× volume of hydrophobic Sera-Mag SpeedBeads  and DNA is eluted in 12 µl nuclease free water. The concentration of the samples was measured with a Tecan plate reader Infinite 200 pro in 384 well black flat bottom low-volume plates (Corning) using AccuBlue Broad range chemistry (Biotium).Cells were sorted by FACS into a 96-well plate containing 2 µl of nuclease free water with 0.2% Triton-X 100 and 4 U murine RNase Inhibitor (NEB), spun down and frozen at −80°C. After thawing the samples, 2 µl of a primer mix was added . RNA was denatured for 3 min at 72°C and the reverse transcription was performed at 42°C for 90 min after filling up to 10 µl with reverse transcription buffer mix for a final concentration of 1× superscript II buffer (Invitrogen), 1 M betaine, 5 mM DTT, 6 mM MgClFor library preparation, 700 pg cDNA in 2 µl were mixed with 0.5 µl Tagment DNA Enzyme and 2.5 µl Tagment DNA Buffer  and tagmented at 55°C for 5 min. Subsequently, Illumina indices were added during PCR  with 1× concentrated KAPA Hifi HotStart Ready Mix and 0.7 µM dual indexing primers. After PCR, libraries were quantified with AccuBlue Broad range chemistry, equimolarly pooled and purified twice with 1× volume Sera-Mag SpeedBeads. This was followed by Illumina sequencing on a Nextseq500 aiming at an average sequencing depth of 0.5 million reads per cell. Primers used were as follows: dT-primer, C6-aminolinker-AAGCAGTGGTATCAACGCAGAGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN, where N represents a random base and V any base beside thymidine; TSO-primer, AAGCAGTGGTATCAACGCAGAGTACATrGrGrG, where rG stands for ribo-guanosine; UP-primer, AAGCAGTGGTATCAACGCAGAGT.Libraries were sequenced on an Illumina NextSeq 500 system, resulting in about 250,000 to 900,000 single end reads per cell. FastQC was used to examine quality of the reads after sequencing. Alignment of the reads to the GRCz10 reference, inclusive the 92 ERCC Spike-In transcripts, was carried out with GSNAP (v 2017-08-15), and Ensembl gene annotation version 87 was used to detect exon spanning reads. featureCounts (v1.5.3) was used with the same Ensembl annotation to count the uniquely aligned reads to the genes and to create a counts table.To identify low quality cells, quality control metrics were calculated from the raw count matrix in R using the package scater . Only ceFurther analysis was performed using the scanpy package (master branch commit 623f0d4) , 2019. UProliferative RG were defined as those that had one or more reads of ccnd1. All other RG were classified as non-proliferative. Differential expression analysis between the two groups was performed using a Wilcoxon rank-sum test. To examine the statistical significance of the over-representation of the top 100 marker genes for the NBN.1 cluster in the top 100 genes that are higher expressed in proliferative RG compared with quiescent RG a hypergeometric test was performed. From the 11 genes that were present in both, the top 100 marker genes for the NBN.1 cluster and the top 100 genes that are higher expressed in proliferative RG, four genes associated with neurogenesis were curated. For each cell, the number of expressed genes (>0 reads) among those four genes was calculated. To test the statistical significance of the increase in the number of cells that express genes associated with neurogenesis in proliferative RG, a one-sided Wilcoxon rank-sum test was performed.www.ensembl.org/biomart/martview/ ; filters, Orthologous Mouse Genes: Only; Attributes, Gene stable ID, Gene name, Mouse gene stable ID, Mouse gene name). It should be noted, that this is not a list of only one-to-one matches, i.e. a single species-specific gene can be contained in multiple pairs of orthologues genes. A cross-species count matrix was constructed in which each row corresponds to an orthologues gene pair and the columns correspond to the cells from both species. Let zja denote the normalized expression of zebrafish gene a in zebrafish cell j and mkb denote the normalized expression of mouse gene b in mouse cell k. Then, the elements of this cross-species count matrix were assigned asi, and all orthologues gene pairs a, b. To align mouse and zebrafish data, batch correction was performed using the combat function of scanpy with the species as the batch key. Highly variable orthologous gene pairs were selected using the scanpy function highly_variable_genes with parameter flavor ‘cell_ranger’. A principal component analysis was performed on these highly variable orthologues gene pairs. Hierarchical clustering in the space of the first 50 principal components using a Pearson correlation distance was performed using the dendrogram function in scanpy. For each zebrafish cell, the Pearson correlation distance to and the cell type of the nearest mouse cell in the space of the first 50 principal components were computed. To reduce the number of false positives, zebrafish cells with distances to mouse cells above the 66% quantile of the distance distribution were disregarded from the analysis.For the combined cell type homology analysis, scanpy version 1.4.4 was used. Read counts and metadata on cell type identities from the developing mouse dentate gyrus were acquired from GEO accession number GSE104323. Read counts from zebrafish and mouse were normalized using the scanpy function normalize_per_gene and log-transformed using the log1p function of scanpy. Next, the normalized count matrices for zebrafish and mouse were transformed into a count matrix for all orthologues gene pairs in the following way. A list of all pairs of orthologues genes between mouse (GRCm38.p6) and zebrafish (GRCz11) was downloaded from in situ and fixed at 4°C overnight in 2-4% paraformaldehyde/0.1 M phosphate buffer (pH 7.5). They were washed twice with phosphate-buffered saline (PBS) and transferred for decalcification and cryoprotection to 20% sucrose/20% EDTA in 0.1 M phosphate buffer (pH 7.5). Brains were frozen in 7.5% gelatine/20% sucrose and cut at 14 µm. Sections were stored at −20°C.Brains were exposed in situ hybridization on sections and on whole-mount brains and RNA probe generation was essentially performed as previously described (in situ riboprobe for tbr1b can be found elsewhere (in situ probe for tubb5 was obtained from the Zebrafish Gene Collection (cDNA clone MGC:85895). Probes for ebf3a and msi2b were cloned from zebrafish embryonic cDNA with the following primers: ebf3a-F, CAGCCAGTGGAGATCGAAAGGACAG; ebf3a-R, TGCCGTAGGGAGAGTTCGCAGAGGA; msi2b-F, GTTAGCCATGGAGGGAGACG; msi2b-R, GCGTCTTGGAAAGGCAACTT). The sections were washed at 60-65°C in washing solution  for 1×15 min and 2×30 min followed by 2×30 min MAB with 0.1% Tween-20 (MABT) washes. Sections were incubated for 1 h at room temperature in 2% DIG-blocking reagent (Roche) and incubated with anti-DIG antibody  diluted 1:4000 in 2% DIG-blocking reagent overnight at 4°C. Subsequently, sections were washed for 4×20 min in MABT, equilibrated with staining buffer and stained with the substrate NBT/BCIP. The staining was controlled using a stereomicroscope. Finally, sections were washed for 2×5 min in PBS, postfixed with 4% PFA for 20-30 min, washed again for 2×10 min in PBS and mounted with 70% glycerol in PBS. All washing steps were performed on a shaker, all incubation steps in a humid chamber.RNA escribed . Brieflylsewhere . The in www.thermofisher.com/antibody/product/HuC-HuD-Antibody-clone-16A11-Monoclonal/A-21271. Alexa Fluor 633-conjugated secondary antibodies were used . EdU was detected with the EdU Alexafluor 488 Plus Imaging Kit  according to the manufacturer's instructions.Immunohistochemistry was carried out as previously described . Brieflyher4.1:mcherry; elavl3:gfp-double transgenic fish) or for three times on consecutive days . After further survival for up to 1 month, fish were sacrificed by overdose with 0.024% MS-222 (Sigma) in fish water  or processed for cryosectioning .Adult (6-12 months old) fish were anaesthetized with 0.01% MS-222 (Sigma) in fish water  until sesh water  until cein situ hybridization or immunofluorescence were imaged with a Zeiss Axioimager Z1 microscope with Apotome structured illumination to achieve optical sections in fluorescent images, using a Zeiss Plan-Apochromat 5×/0.16 and Zeiss Plan-Apochromat 40×0.95 objectives. To minimize crosstalk between the channels in multicolored specimens, sequential image acquisition was performed. The images were processed using ImageJ v.1.44 (rsb.info.nih.gov/ij/) and Adobe Photoshop CC 2015. Figures were assembled using Adobe Illustrator CC 2015.Images of brain sections, stained using n indicates the number of forebrain cell pools, where cells from 7-10 telencephali were pooled (EdU incorporation) or the number of single quiescent od proliferating RG cells (neurogenic commitment in proliferating RG). Statistical significance was determined using one way ANOVA with Welch's correction for heteroskedatic data followed by Bonferroni's multiple comparison post hoc test (EdU incorporation) or using a one-sided Mann and Whitney U-test (neurogenic commitment in proliferating RG). Statistical tests were performed with Graph Pad Prism 8. No animals or data points were excluded from the analyses. Cells in the single cell analysis were excluded based on pre-determined technical quality control parameters. No methods were used to pre-determine sample size or randomize group assignment.For statistical analysis,


Improving N use efficiency of crops has been an effective strategy for promotion of the sustainable agriculture worldwide. The regulatory pathways associating with N uptake and the corresponding biochemical processes impact largely on plant N starvation tolerance. Thus, exploration of the molecular mechanism underlying nitrogen use efficiency (NUE) and the gene wealth will pave a way for molecular breeding of N starvation-tolerant crop cultivars.Nitrate , in mediating the plant N starvation response. TaNBP1 protein harbors a conserved W40 domain and the TaNBP1-GFP (green fluorescence protein) signals concentrate at positions of cytoplasm membrane and cytosol. TaNBP1 transcripts are induced in roots and leaves upon N starvation stress and that this upregulated expression is recovered by N recovery treatment. TaNBP1 overexpression confers improved phenotype, enlarged root system architecture (RSA), and increased biomass for plants upon N deprivation relative to the wild type, associating with its role in enhancing N accumulation and improving reactive oxygen species (ROS) homeostasis. Nitrate transporter (NRT) gene NtNRT2.2 and antioxidant enzyme genes NtSOD1, NtSOD2, and NtCAT1 are transcriptionally regulated under TaNBP1 and contribute to the improved N acquisition and the increased AE activities of plants.In the current study, we characterized the function of TaNBP1 is transcriptional response to N starvation stress. Overexpression of this gene enhances plant N starvation adaptation via improvement of N uptake and cellular ROS homeostasis by modifying transcription of NRT gene NtNRT2.2 and antioxidant enzyme genes NtSOD1, NtSOD2, and NtCAT1, respectively. Our research helps to understand the mechanism underlying plant N starvation response and benefits to genetically engineer crop cultivars with improved NUE under the N-saving cultivation conditions.Altogether, The online version of this article (10.1186/s12870-018-1374-6) contains supplementary material, which is available to authorized users. Nitrogen (N) is an essential macronutrient for the plant growth and development. For a long time, increased input of the N fertilizers has contributed greatly to the improvement of the crop productivity. However, overdose of N input during crop production has also caused serious environment problem in addition to the increased investment . ImproveANR1) and nodule inception-like protein 7 gene (NLP7) as well as maize DNA finger binding protein gene (ZmDOF1), act as crucial mediators in the regulation of N starvation tolerance via mediating external nitrate response and internal N assimilation [CHL1 and NRT2.1) play important roles in sensing nitrate signal, taking up external nitrate, and tanslocating internal N across tissues [Plants have evolved multifaceted strategies in response to external N availabilities , 3. Uponmilation –6. The A tissues . It is n tissues . HoweverP. syringae, a kind of bacterial pathogen, via regulating programmed cell death (PCD) and reactive oxygen species (ROS) homeostasis [P. syringae infection and injury through adjustment of stomata movement [GTP-binding proteins (G-proteins) are heterotrimeric consisting of three subunits, Gα, Gβ, and Gγ. In plant species as well as other eukaryotes, proteins in this functional class play essential roles in transducing the signals initiated by internal cues as well as environmental stressors , 10. Themovement . Severalmovement , 14.Subunit Gβ involves the constitution of the G-protein and exerts similar biological functions to Gα and Gγ, two other G-protein subunits. It is also involved in plant pathogen defensiveness . In addiT. aestivum L.) is one of the important cereal crops cultivated around the world. Thus far, a large set of investigations has been performed focusing on understanding of the physiological and biochemical mechanisms underlying plant N starvation response [T. aestivum species. Previously, based on microarray analyses, we identified a guanine nucleotide-binding protein (NBP) encoding TaNBP1 gene (GenBank accession No. AK332651), a beta subunit (Gβ) gene for G-protein of wheat, to be upregulated in expression under N starvation condition. In this study, we functionally characterized TaNBP1, a subunit Gβ encoding gene of wheat, in regulating the N starvation stress tolerance. Our results indicate that TaNBP1 is N-starvation inducible and acts as an essential modulator in plant N deficiency tolerance through regulating N acquisition and cellular ROS homeostasis.Wheat (response . Howeverhttps://www.mega.com). Conserved domain shared by the NBP proteins including TaNBP1 and its plant counterparts was specified as previously described [TaNBP1 and its homologous genes was established using the DNAStar software (https://www.dnastar.com).Sequence similarities of TaNBP1 and its homologous proteins in plant species were determined using MEGA7 software (escribed . Phylogehttps://swissmodel.expasy.org/interactive) that simulates protein three-dimensional structure was adopted to predict the 3-D structure of TaNBP1. To define the subcellular localization of TaNBP1 sorted from endoplasmic reticulum (ER), we generated an expression cassette harboring a TaNBP1-GFP (green florescence protein encoding gene) fusion gene as previously described [TaNBP1 was amplified by RT-PCR using specific primer pairs  [An online tool referred to as SWISS-MODEL algorithm  .TaNBP1 at two N supplies. With this aim, the seeds were germinated under 25 °C in darkness. After germination, the young seedlings were cultured hydroponically in a growth chamber. To this end, roots of the young seedlings were immersed into standard Murashige and Skoog (MS) solution (16 mM N) through holes of plastic foam that floated on the nutrient solution, which was renewed twice one week and air-circulated by a mini pump. Growth conditions for the seedlings were as follows: photoperiod of 16 h/8 h (light/ dark), temperature of 22 °C, light intensity of 230 μmol m− 2 s− 1 during light phase, and air humidity of 65 to 75%. At the third leaf-expansion stage, wheat seedling were subjected to N starvation treatment by transferring into a modified MS solution that contained reduced N (0.02 mM N), which was established by reducing NH4NO3 and KNO3 amounts and supplementing KCl in the meantime to sustain unaltered K content in the solution. Additionally, a followed N recovery treatment was initiated by retransferring the 27 h N-deprived seedlings again to a standard MS solution. At 0 h (before N starvation), 1, 3, 9, and 27 h after N starvation and N recovery treatments, root and leaf tissues were collected. Transcripts of TaNBP1 in both tissues examined were determined based on qRT-PCR. Briefly, total RNA in the tissues was extracted by TRIzol reagents . After treatment with RNase-free DNase  to avoid genomic DNA contamination, the total RNA (2 μg) was subjected to first-strand cDNA synthesis using RT-AMV transcriptase  in 20 μL reaction volume using oligo (dT)18 at 42 °C for 30 min, according to the manufacturer’s instructions. qRT-PCR analysis was performed in a total volume of 25 μL containing 12.5 μL of SYBR Premix ExTaqTM , 0.5 μL of forward and reverse primers, 1 μL cDNA and 10.5 μL nuclease-free water. TaNBP1 transcripts in tissues examined were calculated based on the 2-ΔΔCT method using wheat Tatubulin as an internal control. The gene-specific primers used for qRT-PCR analysis are shown in Additional file Shiyou 20, an elite high yielding wheat cultivar in North China used in our microarray analyses to identify genes differentially expressed upon N starvation stress, was selected to investigate the expression patterns of N. benthamian lines with TaNBP1 overexpression were generated to characterize the gene function in mediating N starvation tolerance. With this aim, an expression cassette harboring the TaNBP1 CDS was constructed using conventional approach. Briefly, the TaNBP1 CDS was amplified by RT-PCR with specific primers  gene expression patterns in the transgenic lines (Lines 2 and 3) and WT were evaluated after the N starvation treatment. Of which, the N concentrations were assessed as described previously  . Plant N qRT-PCR . TobaccoNtNRT2.2 was shown to be significantly upregulated in expression in the N-deprived TaNBP1 overexprresion lines (Lines 2 and 3) compared with wild type, suggesting its involvement in the TaNBP1-mediated N starvation response. To evaluate the function of this NRT gene in mediating plant N uptake, transgenic N. benthamian lines with NtNRT2.2 overexpression were generated using similar procedure for establishment of TaNBP1 overexpression lines. Primers for amplification of NtNRT2.2 CDS are shown in Additional file NtNRT2.2-mediated N acquisition characterization, NtNRT2.2–1 and NtNRT2.2–2, two lines with high expression of this NRT gene  and superoxide anion in the N-deprived TaNBP1 overexpression lines (Lines 2 and 3) and wild type. The parameters described above were determined as reported by Huang et al. 2010 [TaNBP1-mediated SOD, CAT, and POD activities, a suite of AE genes of tobacco, including five encoding SOD proteins , six coding for CAT proteins , and eleven encoding POD proteins , were subjected to expression evaluation using the N-deprived TaNBP1 overexpression lines based on RT-PCR or qRT-PCR. Accession numbers and primer pairs used for these AE genes are shown in Additional file Given the close relation between cellular reactive oxygen species (ROS) homeostasis and plant N starvation response, we assessed the activities of a set of antioxidant enzymes (AEs), including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), and the contents of malondialdehyde (MDA), hydrogen peroxide  or modified 1/2 MS salts with reduced N (0.06 mM N) which was established similarly to that for N-deprived wheat seedlings as aforementioned. In addition, same growth conditions were adopted for culturing these transgenic and WT seedlings. Three weeks after treatments, the transgenic and WT seedlings were subjected to assay of AE activities performed similarly to those for TaNBP1 overexpression lines.Two representative lines for each AE gene were selected to define the gene-mediated AE activities under both N normal and N starvation treatments. With this purpose, seeds of WT and transgenic lines at T3 generation with overexpression of the AE genes, including NtSOD1–1 and NtSOD1–2 for The mean values of the plant biomass, N concentration, N amount, activities of SOD, CAT, and POD, content of MDA, and qRT-PCR data in WT and transgenic lines under sufficient or deficient N conditions were derived from results of four replicates. Standard errors of the mean values and the significant differences among the mean values were analyzed using the Statistical Analysis System software (SAS Corporation).TaNBP1 cDNA is 1273 bp-long that encodes a 335 aa polypeptide , B. distachyon (XM_003567896), O. sativa (CT833917), and S. italica (XM_004961250)  prediction analysis, it was revealed that TaNBP1 harbors a typical β-propeller feature domain initiated by conserved WD40 repeats (repeats I to VII) Fig. . Based oTaNBP1 in tissues of roots and leaves were detected under the varied external N levels. Under N normal condition (16 mM N), the TaNBP1 transcripts were shown to be low in both roots and leaves. Upon N starvation stress (0.02 mM N), the TaNBP1 expression in both tissues was gradually upregulated over a 27 h treatment regime, reaching a peak at 27 h after the treatment  and WT were assessed after the N starvation treatment. No obvious variations in both N concentration and N accumulative amount were observed in transgenic and WT plants under N normal condition Fig. . In contNtNRT1.1-s, NtNRT1.1-t, NtNRT1.2-s, NtNRT1.2-t, NtNRT2.1, and NtNRT2.2, in the N-deprived transgenic lines (Lines 2 and 3) and WT plants. Among them, the NtNRT2.2 transcripts were drastically upregulated in lines 2 and 3 with respect to those in WT plants  among the transgenic and WT plants. These results suggest that the transcription of NtNRT2.2 is under control of TaNBP1 and the upregulated expression of this NRT gene possibly contributes to increased N accumulation in transgenic lines.The expression of a suite of tobacco NRT encoding genes, including nts Fig. , contrasNtNRT2.2 in improving N accumulation under N starvation stress was evaluated based on transgene analysis. NtNRT2.2–1 and NtNRT2.2–2, two NtNRT2.2 overexpression lines possessing more target transcripts than others  exhibited comparable SOD, CAT, and POD activities and MDA, H2O2 and superoxide anion contents with WT plants , six for CAT , and eleven for POD , was subjected to expression evaluation in the N-deprived TaNBP1 overexpression lines. Among them, two SOD genes  and one CAT gene  exhibited significantly upregulated in expression in Line 2 and 3, contrasting to other AE genes examined that showed unaltered transcripts abundance among the transgenic and WT plants  [AGB1) and a set of genes encoding subunit Gβ-like [AGG1, AGG2 and AGG3) that encode Gγ1, Gγ2 and Gγ3, respectively [The subunits Gα, Gβ and Gγ involved in assembly of G-proteins are conserved in all eukaryotes. The members in family constituting these subunits are generally limited in plant species. For example, only a subunit Gα encoding gene (GPA1) , a subun Gβ-like , and threctively , have beectively , 10.P. syringae defensiveness [+-channel activation [Subunit Gβ of G-proteins is involved in regulation of various physiological processes in plants, including to mediate plant pathogen siveness , modulatsiveness , regulattivation , and modtivation , 31. In TaNBP1 exhibited variation under modified N input conditions, showing that the TaNBP1 transcripts were gradually increased in roots and leaves over a 27 h regime under N starvation treatment. Moreover, the upregulated expression of this wheat gene was gradually downregulated once the N-deprived plants were resubjected to a N normal recovery treatment. These expression patterns suggest that TaNBP1 is involved in plant N starvation response. Previously, a cis-acting regulatory element referred to as NRE was shown to be critical in transcriptional regulation of a suite of N-responsive genes, including those enconding nitrite reductase (NIR) and NRT proteins in Arabidopsis, such as NRT2.1 and NRT2.2 [cis-regulatory elements underlying N starvation response of TaNBP1 can help understand its transcriptional mechanism upon low-N stress.Distinct N signaling is elicited upon N starvation stress, which initiates transcriptional alteration on a large set of the regulatory and the functional genes . A suited NRT2.2 . Further+-channel responses in the guard cells [TaNBP1 in regulating plant N starvation tolerance. Under N starvation treatment, the lines with TaNBP1 overexpression exhibited improved phenotypes, increased N accumulative amounts, and higher plant biomass with respect to the wild type  and high-affinity transport system (HATS) are the two major N uptake systems in plant species and other eukaryotes, which play crucial roles in the mediation of plant N acquisition and internal N translocation across tissues upon various N input conditions . Of whiccapacity –41. In tnts Fig. . These fNRT2.1 involves the physiological processes of initiation and extension of the primary and secondary roots, aside from its role in regulating NO3− uptake [TaNBP1 or NtNRT2.2 revealed that they all displayed enlarged RSA system under N starvation treatment compared with wild type , whose root growth is inhibited under high nitrate conditions and the inhibition intensity is correlated with the reduction on auxin concentration in root tips. Alleviation of transduction of the auxin signaling from shoot to root then involves modulation of the RSA behavior [TaNBP1 and NtNRT2.2 can help understanding of the root phenotype formation of plants.Root system architecture (RSA) impacts largely on the water acquisition and the inorganic nutrient uptake from growth media. In addition to the NRT protein mediated N uptake, the RSA modulated by low-N stress also drastically impacts on the plant N uptake, whose establishment has been reported to be associated with the involvement of distinct NRT genes. For example, the Arabidopsis − uptake . In thispe Figs.  and 5c. behavior . Moreovebehavior ; Arabidobehavior . These fO. sativa) are regulated by RGG1, a γ subunit of G-protein; overexpression of RGG1 confers plants enhanced CAT activities and improved salt tolerance [TaNBP1 overexpression lines, we revealed the function of TaNBP1 in mediating cellular ROS homeostasis under N deprivation. Our results indicated that the TaNBP1 overexpression lines exhibited increased SOD, CAT, and POD activities, lowered MDA contents, and reduced H2O2 and superoxide anion accumulative amounts  consisting of a sevenfold β-propeller in TaNBP1 are highlighted by blue background. Figure S2. Phylogenetic relations between TaNBP1 and its homologous genes from various plant species. Figure S3. Target gene transcripts in lines overexpressing TaNBP1 and NtNRT2.2a, TaNBP1 transcripts in transgenic lines; b, NtNRT2.2 transcripts in transgenic lines. WT, wild type. Line 1 to Line 7, independent transgenic lines with TaNBP1 overexpression. NtNRT2.2–1 to NtNRT2.2–6, independent lines with NtNRT2.2 overexpression. In a, TaNBP1expression levels in transgenic lines are normalized by the constitutive Tatubulin transcripts. In b, NtNRT2.2 expression levels in transgenic lines are normalized by the constitutive Nttubulin transcripts. Internal standard reference genes are set an expression level of 1. Figure S4. Target gene transcripts in lines overexpressing differential AE genes a, NtSOD1 transcripts in transgenic lines; b, NtSOD2 transcripts in transgenic lines; c, NtCAT1 transcripts in transgenic lines; WT, wild type. Expression levels of the AE genes in transgenic lines are normalized by the constitutive Nttubulin transcripts whose expression level is set as 1. (DOC 162 kb)


However, in terms of public health, higher DLM residues in cheese show that further specific studies should be performed on double dosage efficacy and pharmacokinetic and pharmacodynamics properties of ectoparasites in lactating goats.The distribution of pyrethroid insecticide deltamethrin (DLM) in goat milk and cheese (caciotta) following pour-on administration at the sheep dosage (DLMS-10 mL/60 kg body weight) and double dosage (DLMD-20 mL/60 kg body weight) was studied. DLM concentrations were measured in milk collected from study animals (No.14) before treatment and at 2, 4, 8, 12, 16, 24, 30, 36, 48, 56, until 168 h (7 days) post treatment and in caciotta cheese at 12 and 24 h post treatment. At both dosages, the maximum level of DLM residues in goat milk and cheese was below the maximum residue limit (MRL) of 20 μg kg Goat milk and its products play a significant role in human nutrition ; it is bThe most commonly used insecticides in ruminants are mainly topically used as pour-on formulations ; among tThe analytical method for the detection of DLM was fully validated prior in milk and cheese analysis. 4]+ and the most abundant transitions were at m/z 522.7 –––> 280.8 (quantification ion) and m/z 524.7 –––> 282.8 (confirmatory ion). The method was applied to fortified negative goat milk samples from different animals and the trueness, sensitivity and specificity were calculated.The limit of detection and limit of quantification (LOD and LOQ) are defined as the lowest concentration of the analyte with S/N = 3 and S/N = 10, respectively. S/N > 10 was obtained for all the measurements. Transitions and cone voltages for DLM and IS are reported in −1) [Chromatographic traces of DLM at specific retention time are reported in −1)   and cheese were spiked with DLM at seven levels: 1, 2, 4, 10, 20, 30 and 40 μg kg2 > 0.998). The limit of quantitation of the method, as determined from the lowest standard on the calibration curve was 1 μg kg−1. Intra-assay variation was determined by analyzing five samples within a single run; reproducibility (RSDs) were typically less than or equal to 6%. Mean trueness of the analytes was between 95 and 100%. Mean recovery of analytes was 95% or higher  and acceptable signal-to-noise ratio for applied transitions.Mean calibration curves in milk and cheese were linear (Rr higher . Inter-ar higher . CCα and−1) in milk and cheese samples, respectively.During the study, the DLM pour-on formulation  was well tolerated by all the treated animals with no adverse reactions. The dermal safety study on the DLM-groups showed the absence of adverse effects caused by the treatment (Draize score = 0) in all the goats. −1 after DLMS dose and from 0.17 to 1.74 μg kg−1 after DLMD dose. The highest levels were determined in samples collected at 30 h post treatment at DLMS dose; when DLMD dose was used, highest levels were detected at 36 h post treatment. The concentrations of DLM decreased progressively until 120 h after treatment, where they were less than the detection limit at both dose rates. There were no differences (P < 0.05) in the average of DLM milk residue for time sampling among the two dosage (DLMS and DLMD).DLM residues were not detected in milk until 8 h post treatment and levels in milk ranged from 0.16 to 1.67 μg kg−1 for DLMS dose and from 9.45 to 11.15 μg kg−1 for DLMD dose; highest levels were observed after 24 h for both treatments. In cheese, differently from milk, DLM residues following DLMD dose, at 12 h, were significantly higher (5.75 µg kg−1 vs. 9.45 µg kg−1) than DLMS dose whilst at 24 h values were similar at both treatments. Significant differences (P < 0.05) between different dosage and sampling time were found.DLM residues measured in cheese, obtained with pooled milk, were higher than those recovered in milk and ranged from 5.75 to 10.3 μg kg−1 for 0.1 g DLM and 53 μg L−1 for l g DLM). Results of present studies showed that, considering a similar administration dose , the time to reach the maximum level was slightly longer (36 h vs. 33 h) compared to the trials conducted in the cattle and concentrations were significantly different (1.3 μg Lβ1 vs. 9 μg L−1). There are few studies on the fate of DLM in lactating species. According to Venant et al. (1990) , after a−1 established for bovine milk [Other drugs belonging to the same pyrethroid insecticides group of DLM, such as α-cypermethrin, showed a different fate. Smaldone et al. 2013)  found α-ine milk  after poine milk  reportedine milk  showed t13  foundThe Committee for Medicinal Products for Veterinary Use defines goats as a minor species ; in thisThere are no studies on the fate of pyrethroids in dairy products. The finding of this research could be useful to characterize the lipid solubility property of DLM that may concentrate in curd of milk-producing animals during manufacturing process.4) and ammonium acetate (C2H7NO2) (purum p.a. grade). Preweighed mixture of 1.5 g anhydrous MgSO4 and 0.5 g of C18 bulk sorbent in 50 mL centrifuge tubes were obtained from UCT, Inc. . Sodium chloride (>99.5%) was obtained from AppliChem GmbH . Ultra-pure water (18.2 MΩ) was generated in-house using a Milli-Q Plus water purification system. PTFE syringe filters  were obtained from Agilent Technologies . An ultra-turrax probe blender from IKA , a Mistral 3000i centrifuge from MSE , a VWR International  during sample preparation multi-tube vortexer, and a Transonic 780 LH ultrasonic bath from Mason Technology . For a chromatographic separation was used an “Acquity UHPLC system” . The analyte was separated using the chromatographic conditions developed previously [−1 deltamethrin standard solution. The inter-channel delay and inter-scan delay were set to obtain maximum instrumental response.DLM analytical standard  was purchased from Sigma-Aldrich,  and deltamethrin-D5 internal standard (98%) was obtained from Toronto Research Chemicals . SpS-grade acetonitrile and methanol were obtained from Romil Chemicals . Sigma-Aldrich supplied anhydrous magnesium sulphate (MgSOeviously . Deltame4 (4 g) and NaCl (1 g) to induce phase separation. Samples were subsequently centrifuged  and the supernatant was transferred to dispersive solid phase extraction (d-SPE) tubes containing pre weighed C18 bulk sorbent and MgSO4. The samples were then vortexed for 1 min and centrifuged . The obtained extract was filtered through 0.2 μm PTFE syringe filters and 5 µL was injected onto the UHPLC-MS/MS system.A QuEChERS based approach was adapted to isolate deltamethrin residues from goat milk and cheese samples. Milk (10 ± 0.1 g) and cheese samples (1 ± 0.01 g) were weighed into 50 mL polypropylene centrifuge tubes . The samples were then fortified with internal standard (IS) solution (200 μL) and cheese samples were diluted with 9 mL of water followed by homogenisation using an ultra-turrax probe blender. The samples were extracted with acetonitrile MeCN (10 mL) by shaking vigorously for 1 min in the presence of MgSO−1 and 100 μg mL−1, respectively, in ethanol. DLM-D5 was used as internal standard (IS). Intermediate standards were prepared in acetonitrile at 100 μg mL−1 and 1 μg mL−1 for DLM and IS, respectively. Working standard fortification solutions at concentrations of 0.05 (std 1), 0.1 (std 2), 0.2 (std 3), 0.5 (std 4), 1 (std 5), 1.5 (std 6) and 2 (std 7) μg mL−1 were prepared in acetonitrile. Stock standard solution of DLM and DLM-D5 were prepared at concentrations of 2000 μg mL−1 in the matrix. Recovery was assessed by analyzing milk sample extracts spiked post-extraction with DLM at concentrations of 2 and 30 μg kg−1 in duplicate. Commercial goat milk purchased in a supermarket, found not to contain any detectable DLM residues, was used as the negative control. Matrix calibration curves were prepared by fortifying negative milk samples prior to extraction with 200 μL of each calibration standard solution (std 1–7) to give matrix concentrations of 1, 2, 4, 10, 20, 30 and 40 μg kg−1, which was listed by Commission Regulation (EU) No. 37/2010 for DLM residue in bovine milk [The following performance parameters were investigated: intra-assay variations, inter-assay variations, linearity, recovery, decision limit (CCα) and detection capability (CCβ). The accuracy and precision of the method were determined using negative goat milk samples fortified at 0.5, 1 and 1.5 times the MRL level of 20-μg kgine milk .2) to avoid physical contact and licking. On day 0, the research animals received DLM pour-on  at the manufacturers recommended sheep dose of 10 mL per animal for the the DLMS group, and at the double sheep dose of 20 mL per animal for the DLMD group. The pour-on formulation was applied topically with a syringe along the midline of the back from the withers to the trailhead. Milk samples were collected from each of the study animals (n = 14) before treatment (day 0) and at 2, 4, 8, 12, 16, 24, 30, 36, 48, 56, 96, 120, 144 and 168 h post treatment. The total daily production of the treated DMLS and DMLD goats was collected separately at the day 0, 12 and 24 h after treatment and kept at 4 °C. The milk was processed in the same time points to produce caciotta cheese with ripening period of 30 days. The processing techniques of caciotta cheese are described in Rubino et al., 2004 [The study was conducted in an experimental small ruminant farm  located in southern Italy, Potenza province (40°21′ N and 15°30′ 25″ E). For this study were selected fourteen Saanen lactating goats (Capra hircus), with a mean age of 3 ± 2 years and a mean bodyweight of 50 ± 10 kg. The animals were fed with concentrated food, with hay during the study period, and were milked twice a day; water was given ad libitum throughout the course of the study. Research animals were assigned at random to the following treatment groups of 7 animals each: DLM sheep dose group (DLMS-group), DLM sheep double dose group (DLMD-group). Throughout the experimental period each animal was isolated from the others in a single indoor box (6.25 mANOVA was used to compare concentrations of DLM between different dosage (DLMS and DLMD groups) in milk and cheese samples and different steps of time sampling. Statistical significance was considered to be at the 0.05 level. All of the statistical analysis was run using SPSS, Version 11.5, SPSS Inc., Chicago, IL, USA. −1, which has been set for bovine milk by EU Commission Regulation 37/2010. Other specific studies must be conducted to provide more information on double dosage efficacy and pharmacokinetic and pharmacodynamics properties of drugs against ectoparasites in goats since they are milk-producing animals. This could have important implications in terms of public health and trade of goat milk and byproducts.The findings of the present study show that the use of DLMD, with higher antiparasitic activity, pose no residues problems for goat milk and dairy products. In fact, highest levels of DLM in milk and cheeses after administration of 20 mL/60 kg body weight of DLM were always below the MRL of 20 μg kg


MSRB3) is instrumental in ossification and fat deposition, which regulate thegrowth and development of cattle directly. The purpose of this study wasaimed to explore insertions and deletions (indels) in MSRB3 gene and investigatetheir association with growth traits in four indigenous cattle breeds . Four indelswere identified by sequencing with DNA pool. Association analysis showedthat three of them were associated with growth traits  in cattle breeding.Methionine sulfoxide reductase B3 ( Chineseindigenous cattle breeds have the characteristics of slow growth, low yield,and low-grade beef . With the rapid development oflivestock husbandry, beef cattle improvement has become a heated and difficultproblem . However, because of the slow progress intraditional breeding, we need to seek a more efficient and accurate way ofbreeding urgently . A noteworthy method is molecular breeding technology,which emerged and developed into an indispensable breeding method, andindels is one of the genetic markers used for assisted selection .MSRB3 (methionine sulfoxide reductase B3) is an essential member of the MSRB gene family, whichcan reduce the catalysis effect of methionine-R-sulfoxide to methioninespecifically as an oxidoreductase . Previous researchhas shown that MSRB3 gene could influence ear shape and size, otitis media,heat resistance, oxidation resistance, auditory system, reproductive traits,and size of the hippocampus .In recent years, based on the analysis of GWASs (genome-wide associationstudies), gene silencing, and protein precipitation, MSRB3 was regarded as acandidate gene to affect bovine growth traits . At thesame time, it inhibited the proliferation of mouse embryonic fibroblasts andhuman dermal fibroblasts by acting on the p53, p21, and p27 pathways .The identification of candidate genes for growth traits has attracted moreand more attention. MSRB3 gene and its association with the body size of Chinese indigenouscattle through the indel screening and association analysis with growthtraits.Consequently, the purpose of this research is to obtain mutation informationon 2All experiments performed in this study were approved by the InternationalAnimal Care and Use Committee of the Northwest A&F University(IACUC-NWAFU). Furthermore, the care and use of animals was fully compliantwith local animal welfare laws, guidelines, and policies.2.1A total of 673 Chinese indigenous cattle were used in this study, includingLuxi cattle . DNA samples were diluted to a standard concentration  .Genomic DNA was isolated from leukocytes and the method refers toa whole-blood genomic DNA extraction kit . DNA quantity and purity (Table 1) . PCR amplification was performed in a 20 Based on the whole-genome sequencing of Chinese indigenous cattle in ourlab, seven possible indel loci were found in cattle 2.4http://www.msrcall.com/, last access: 25 August 2018), which wasbased on Nei's method . Distribution differences for genotypic and allelicfrequencies among different breeds were analyzed using the http://analysis.bio-x.cn, last access: 25 August 2018) . SPSS software (version 18.0) was used to calculate the associationsbetween different genotypes and growth traits; the structure of the model isThe sequences were contrasted and analyzed by Bioedit software (UK).Hardy–Weinberg equilibrium (HWE), homozygosity (Ho), heterozygosity (He),effective allele numbers (Ne), and polymorphism information content (PIC)were calculated and analyzed using the MSRcall website ; P2 was a 18 bp insertion(NC_007303.6 g.48816764-48816765 ins TTCTTTTGGCAACTGCAG); P6 was a 22 bpdeletion (NC_007303.6 g.48896074-48896095 delTTTTTCTTTGTCTGGTACACTT); P7 was a 37 bp insertion (NC_007303.6 g.48903091-48903092 ins AGCTGATGTATAACCTCCATAACTTGCTTTCCCCCCT); andno variation was found in P3, P4, or P5 . All the samples weregenotyped by PCR-AFLP (amplified fragment length polymorphism). As shown inthe Fig. 1a, wild type (II) showed a 161 bp band, mutant (DD) showed a131 bp band, and heterozygote (ID) showed 131 and 161 bp bands; as shown inthe Fig. 1b, wild type (DD) showed a 284 bp band, mutant (II) showed a302 bp band, and heterozygote (ID) showed 284 and 302 bp bands; as shown inthe Fig. 1c, wild type (II) showed a 221 bp band, mutant (DD) showed a199 bp band, and heterozygote (ID) showed 199 and 221 bp bands; as shown inthe Fig. 1d, wild type (DD) showed a 332 bp band, mutant (II) showed a369 bp band, and heterozygote (ID) showed 369 and 332 bp bands.In our previous study, four indel loci  were identified fromseven potential variations in bovine MSRB3 genewhich were detected by 10 % PAGE gel electrophoresis, including (a) theinsertion and insertion type (II genotype) consisting of a 161 bp band,deletion and deletion type (DD genotype) consisting of a 131 bp band, and theheterozygote type (ID genotype) that showed 161 and 131 bp bands inMSRB3-1 locus; (b) the insertion and insertion type (II genotype) consisting of a 302 bpband, deletion and deletion type (DD genotype) consisting of a 284 bp band, and theheterozygote type (ID genotype) that showed 302 and 284 bp bands inMSRB3-2 locus; (c) the insertion and insertion type (II genotype) consisting ofa 221 bp band, deletion and deletion type (DD genotype) consisting of a 199 bp band, and theheterozygote type (ID genotype) showed 221 and 199 bp bands in MSRB3-6 locus; and (d) the insertion and insertion type (II genotype) consisting of a 369 bp band,deletion and deletion types (DD genotype) consisting of a 332 bp band, and theheterozygote type (ID genotype) that showed 369 and 332 bp bands in MSRB3-7 locus.PCR products showed three genotypes in all of the indel loci in the cattle 3.2Genetic diversity is the prerequisite for species survival, adaptation, andevolution, and is also important for improving the performance of meat production.Genotype frequency, allele frequency, and genetic parameters were estimatedand shown in Table 2. For P1 locus, the breeds of QC, NY, and JX belonged toa low polymorphic locus . The results of GWASs have shown that MSRB3 is involved inregulation of ossification and adipose tissue development in cattle . Some studies have shown that the knockout of MSRB3 leads to adecrease in fibroblast proliferation, which could influence the function ofproteins, glycosaminoglycan and glycoproteins . Furtherstudy revealed that inhibition of MSRB3 could activate the expression of P53, andenhance the expression level of P21 and P27 subsequently .Above-mentioned studies strongly suggest that MSRB3 inhibited cellproliferation by acting on p53, p21, and p27 pathways, which suggests thatMSRB3 is an important candidate gene for animal growth and development . However, there was nostudy on marker-assisted selection in bovine MSRB3 gene. Therefore, we furtherverified the relationship between four indel loci in bovine MSRB3 gene and theirgrowth traits.In previous studies, the function of MSRB3 included functional and nonfunctional types. Functionalindels affect the gene product and growth traits, whereas nonfunctionalindels may be strongly linked with functional indels of MSRB3.In our study, P6 was published by NCBI and the remaining loci were newlydiscovered. The MSRB3 gene significantlyaffected growth traits, meaning that P1, P6, and P7 could be used as DNA markersfor eliminating or selecting excellent individuals in marker-assisted selection (MAS) breeding inrelation to growth traits, and MSRB3 gene could be used as a candidate gene forbreeding beef cattle.In a nutshell, the indels of P1, P6, and P7 within 5MSRB3 gene firstly, which wereassociated with notable growth traits in four Chinese indigenous cattlebreeds. Meanwhile, functional indels could be utilized as promisingmolecular markers in early marker-assisted selection for cattle breedingafter the further extensive research.This research identified four indels within


AbstractTrichogrammadanausicida (Nagaraja) and Trichogrammacacaeciae  (Hymenoptera: Trichogrammatidae), are reported for the first time in Israel. Moreover, our discovery of T.danausicida is the first report of this parasitoid species outside of India. The occurrence of those trichogrammatids was first discovered and documented in May 2016 during a survey of egg parasitoids of the False codling moth Thaumatotibialeucotreta (Lepidoptera: Tortricidae). The field survey was conducted on castor bean fruits (Ricinuscommunis) in the Israeli central coastal plain. The identity of the parasitoids was revealed by means of sequencing a portion of the cytochrome oxidase I gene (COI) of the studied parasitoids.The egg parasitpoids  Thaumatotibialeucotreta (Meyrick); i.e., FCM) (Lepidoptera: Tortricidae), native to African regions south of the Sahara, was first reported in Israel in 1984 on macadamia nuts (Ricinuscommunis) serves as a preferred host plant for the FCM, providing fruits nearly all year round for FCM development and survival (Trichogrammatoideacryptophlebiae (Nagaraja) . Other reported egg parasitoids that attack the FCM are Trichogrammatoideafulva (Nagaraja) and Trichogrammatoidealutea (Girault)  . Moreoveorchards . T.cryptGirault) . We wereRicinuscommunis) in the Israeli central coastal plain  gene using Polymerase Chain Reaction (PCR). PCR was performed in a total volume of 50 μL containing 25 μL of Ready Mix , 13.5 μL double distilled water, 0.75 μL of 20 pmole for each primer used, and 10 μL of DNA template . The primer sequences used for PCR are LCO_1490F 5’-GGTCAACAAATCATAAAGATATTGG-3’ and HCO_2198R 5’-TAAACTTCAGGGTGACCAAAAATCA-3’. PCR cycling conditions were 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 sec, 45 °C for 45 sec, and 72 °C for 1 min, with a final extension at 72 °C for 10 min.DNA was extracted from single parasitoids in 25 μL lysis buffer . This DNTrichogrammatoideacryptophlebiae parasitoids obtained from South Africa  were tested with the same pair of primers mentioned above, however, the obtained sequences did not match any sequences in GenBank (www.ncbi.nlm.nih.gov/Genbank), thus, an additional pair of primers that amplify a portion of the Internal Transcribed Spacer 2 sequences (ITS 2), located in the 5.8S and 28S region of the rDNA complex bordering the ITS 2 region, were used. Their sequences are: ITS2-F 5’-TGTGAACTGCAGGACACATG-3’ and ITS2-R 5’-GTCTTGCCTGCTCTGAG-3’. The PCR conditions were as follows: 94 °C for 3 min, followed by 33 cycles of 94 °C for 40 sec, 55 °C for 1 min and 72 °C for 1 min, with a final extension period at 72 °C for 5 min (or 5 min . Each PCK2P) genetic distances . In May 2016, in location number 5 (Table T.cryptophlebiae wasps from South Africa) and obtained their COI sequences. Those sequences were aligned with other Hymenoptera sequences and other outgroup sequences of species from other orders such as the Coleoptera, Diptera and Lepidoptera (obtained from GenBank). All species for which multiple specimens were sampled showed no interspecies variation. The maximum likelihood analysis of the COI gene resulted in a tree typology that showed the presence of two different species of trichogrammatids: Trichogrammadanausicida (Nagaraja)(3 specimens)  (one specimen)   and Tricpecimen)  Fig. 3)T.cryptopTrichogramma spp. are minute endoparasitoids of insect eggs. Currently, more than 230 species of Trichogramma are described worldwide, making them the largest genus in the Trichogrammatidae family. More than 200 insect species are being attacked by different Trichogramma species. Moreover, many species of Trichogramma are important biological control agents of numerous agricultural pests . Both egg parasitoids, T.danausicida and T.cacaeciae, apparently play only a minor role in keeping FCM population low in castor bean plants, and therefore are not potentially recommended biological control agents for FCM control. Similarly, T.cacaeciae collected from parasitised tortricid eggs found on pears and apples in North America, was extremely low (less than 1%). However, our findings may contribute to better knowledge of trichogrammatids fauna in Israel and the Middle East. Since information is lacking on those two parasitoids in scientific literature, biological and ecological studies are needed to determine their biology, host list, and their impact on their host biological control.al pests .Two speche world , the parin India . These t