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In summary, we report phenotypic, genetic and genomic information regarding the interaction between the pathogen D. ampelina and grapevines, including two novel resistance loci, Rda1 and Rda2 located on chromosome 15 of V. cinerea B9 and chromosome 7 of ‘Horizon’, respectively. In the case of Rda1, our results suggest that the D. ampelina—V. cinerea B9 interaction is mediated by the action of one or more NB-LRR genes with homology to VIT_15s0046g02730 and VIT_15s0046g02800, providing the first hints about the molecular basis of the Phomopsis resistance phenotype.
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study
| 100.0 |
BIR and LCD: conceived of and designed the experiments, provided populations and resources and reviewed the manuscript. JL: conducted process and statistical analysis of RNA-Seq data. RSL: provided first field assessment of symptoms and designed the evaluation scale, MO: performed statistical analysis of RNA-Seq. PB: conceived of and designed the experiments, collected phenotypic data, performed molecular biology and microbiology laboratory work, genetic analysis and statistical analysis, and wrote the paper. KB and RT: conceived and designed greenhouse inoculation assays, collected phenotypic data, performed statistical analyses and contributed to and reviewed the manuscript. WFW: performed field identification and isolation of D. ampelina and reviewed the manuscript.
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| 99.9 |
Below is the link to the electronic supplementary material. Supplementary Figure S1: Distribution of number of quality reads per sample for differential expression (DE) and eQTL studies. (PDF 22 kb) Supplementary material 2 (PDF 23 kb) Supplementary material 3 (PDF 13 kb)
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other
| 99.9 |
Chemotherapy is one of the treatment modalities that arrests growth of malignancy either by killing transformed cells or by arresting their division. Chemotherapy is often used either as adjuvant or neoadjuvant or in conjunction with radiotherapy and surgery. However, the major drawback of using such cancer therapeutics is that it may lead to transient and/or permanent damage to normal tissues due to their direct toxicity to healthy cells or toxicity mediated by signals generated by target cancer cells [1, 2]. Nausea, vomiting, diarrhoea, inflammation of the intestinal and oral mucosa, loss of appetite, fatigue, anaemia, leukopenia, pain, and initiation of secondary neoplastic changes are some of the common symptoms of toxicities during cancer chemotherapy [3–6]. Generation of free radicals and initiation of uncoordinated inflammatory response are some of the principle mechanisms of toxicities derived from cancer chemotherapy or radiotherapy [7–10]. Bleomycin (BLM), a commonly used anticancer drug, has been reported to exert free radical mediated toxic effects in similar manner to that of ionized radiation (IR) . BLM induces DNA strand breaks, resulting in production of DNA adducts and excess reactive oxygen species (ROS) leading to oxidative stress, mitochondrial leakage, and apoptosis . Bleomycin at a concentration of 40 μg/mL can induce radiomimetic damage equal to 2 Gy of gamma radiation which is the standard fraction applied daily to cancer patients in fractionation regimes used in radiotherapy .
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review
| 99.8 |
Natural antioxidants have been considered to be promising protective agents against free radical mediated damage induced during cancer therapy. A number of effective compounds such as hoechst , cysteine , caffeine , calcium channel blockers , flavonoids , and 2-deoxy-D-glucose have been identified. Natural antioxidants have also demonstrated effective protection against ROS mediated cellular damage during cancer chemotherapy and radiotherapy [20–22]. However, there are comparatively few investigations that investigate the use of natural protectors during chemotherapy and/or radiotherapy. The importance of such protectors is emerging due to frequent use of chemotherapeutics in combinational therapy with radiation, causing higher magnitude of toxicity than either of the above two modes of therapy.
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review
| 99.9 |
Polyherbal decoction comprised of Nigella sativa seeds, Hemidesmus indicus roots, and Smilax glabra rhizome in equal proportions has traditionally been used by a particular family of Ayurvedic physician in Sri Lanka to treat cancer patients . Recent studies have demonstrated that the above decoction can provide significant protection against chemically induced hepatocarcinogenic changes in rats without producing any significant toxic effects and exert cytotoxicity in human hepatoma (HepG2) cells . Protection against oxidative damage , favourable immune modulation , and expression and suppression of proapoptotic and antiapoptotic genes are some of the mechanisms which mediate anticancer activity of the decoction. It was hypothesized that the claimed antioxidant activity of the decoction may play a dual role: (a) protecting healthy cells against the damage mediated by either chemotherapy or radiotherapy and (b) restricting the tumour progression by resisting mutation in cancer management.
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| 99.0 |
Therefore, the present study was carried out with the primary objective of evaluating the effectiveness of the decoction comprised of N. sativa seeds, H. indicus roots, and S. glabra rhizome, in protecting against bleomycin induced cytogenetic damage in peripheral blood lymphocytes (PBLs).
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| 99.94 |
Seeds of N. sativa (Ranunculacaea), roots of H. indicus (Asclepiadaceae), and rhizome of S. glabra (Smilacaceae) were purchased and authenticated by the botanist at the Bandaranayaks Memorial Ayurvedic Research Institute (BMARI), Nawinna, Maharagama, Sri Lanka. Voucher specimens (UKFM/B/2006/01, UKFM/B/2006/02, and UKFM/B/2006/03) have been deposited at the Department of Biochemistry and Clinical Chemistry, Faculty of Medicine, University of Kelaniya, Sri Lanka.
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other
| 99.94 |
Standardized decoction was prepared as previously described by Samarakoon et al. . Equal portions (20 g each) of N. sativa (seeds), H. indicus (roots), and S. glabra (rhizome) were mixed and boiled gently in 1.6 L of distilled water until a final volume of 200 mL was obtained. The extract was then filtered, freeze dried, and stored in a vacuum desiccator at −4°C. The percentage yield of the freeze dried plant material was 13.6%. For experimental purposes, the required weight of the freeze dried powder was reconstituted in 1% dimethyl sulfoxide (DMSO) and filter sterilized using 0.22 μm disposable filters.
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| 100.0 |
Study was approved and carried out according to the guidelines of Ethical Review Committee, Sri Ramachandra University, Chennai, India. Heparinized blood samples (~10 mL) were obtained with informed consent and according to institutional procedures from six healthy donors aged 24–34 years, without any history of smoking, tobacco chewing, or alcohol consumption, and they were not taking any drugs for medical or other reasons.
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| 99.94 |
Out of 10 mL of blood collected from each individual, equal volume of 4 mL was used for chromosome aberration (CA) studies and cytokinesis-blocked micronuclei (CBMN) assay while the remaining blood was used for γH2AX assay. For each CA and CBMN assays, 4 mL of blood was reconstituted with 36 mL of RPMI-1640 culture medium supplemented with 7.5% NaHCO3, 20% fetal calf serum, 200 mM L-glutamine, penicillin (100 units/mL), and streptomycin (100 μg/mL) and equally aliquoted into eight sterile vials. Two sets of vials (each set consisting of three vials) were added with the decoction at concentrations of 100, 300, and 600 μg/mL. Each set of vials served as decoction control and the test, respectively. All vials were then incubated at 37°C in 5% CO2 for 3 hrs. At the end of the 3rd hour, bleomycin at a concentration of 40 μg/mL was added to (a) a one set of vials containing three different doses of the decoction and (b) a vial without the decoction (bleomycin control) and all were further incubated for 2 hrs. A vial with addition of neither decoction nor bleomycin served as negative control. After 5 hrs of incubation, cells in each vial were washed with Hanks Balanced Salt Solution (HBSS) three times. Each sample was then reconstituted in 4.5 mL of culture medium (RPMI-1640) and used for CA and CBMN analysis as described below.
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| 100.0 |
For γH2AX assay, remaining 2 mL of blood was reconstituted with 18 mL of RPMI-1640 culture medium and equally aliquoted into four sterile vials. Decoction at a concentration of 100 μg/mL was then added to two vials (decoction control and test) and all vials were incubated at 37°C in 5% CO2 for 3 hrs. At the end of the 3rd hour, bleomycin at a concentration of 40 μg/mL was added to (a) a vial containing the decoction (test) and (b) a vial without the decoction (positive control). Subsequently, all vials were incubated for further 2 hrs and lymphocytes were isolated by layering 4 mL aliquot from each vial on to 2 mL of Histopaque–1077 and centrifuged at 1000g for 5 min at room temperature.
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study
| 100.0 |
Lymphocyte cultures for chromosomal aberration assay were processed as described earlier . Briefly, cultures were initiated by adding 0.2 mL of phytohemagglutinin into each vial containing 0.5 mL of blood and 4.5 mL of RPMI-1640 supplemented with 7.5% NaHCO3, 20% fetal calf serum, 200 mM L-Glutamine, penicillin (100 units/mL), and streptomycin (100 μg/mL) and incubated at 37°C in 5% CO2. At 46th hour of incubation, the cells were blocked at metaphase by adding colcemid at a final concentration of 0.1 μg/mL and the cultures were further incubated until 48 hrs. The samples were then harvested after hypotonic treatment (20 minutes with 0.45% KCl at 37°C), washed three times with Carnoy's fixative, and cast on clean precooled slides. All slides were stained with 10% Giemsa, air-dried, and mounted with cover-slip using DPX mounting medium to analyze the chromosomal aberrations.
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| 99.94 |
Lymphocyte cultures for micronuclei assay were set up according to method described by Fenech and Morley . Briefly, cultures were initiated as described in chromosomal aberration assay and at the end of 44th hour of culture Cytochalasin-B at a final concentration of 3 μg/mL culture was added to each vial. The cells were further incubated for 28 hrs at 37°C. The cells were harvested with brief hypotonic treatment and slides were prepared by fixing the cells with Carnoy's fixative. The cell suspensions were dropped onto a clean cooled slide and stained with Giemsa.
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study
| 99.94 |
Isolated lymphocytes were spotted onto microscope slides (six slides/each individual) at an optimized cell density for immunofluorescence staining. Cells were fixed in 3.7% paraformaldehyde in PBS (15 min at room temperature) and permeabilised using 0.5% Triton X 100 in PBS (5 min at 4°C). Samples were blocked in phosphate buffered saline (PBS) with 2% bovine serum albumin (BSA) for 3 × 5 min at room temperature. Samples were then incubated with anti-γH2AX antibody (clone JBW301) overnight at 4°C, and washed in PBS, 2% BSA, for 2 × 5 min. Finally slides were incubated with FITC-conjugated goat anti-mouse secondary antibody (Invitrogen, Paisley, UK) for 1 h at room temperature. Slides were washed in PBS for 2 × 2 min, stained with 4,6-diamidino-2-phenylindole (DAPI) for 5 min, washed for 2 min in PBS, and mounted using Mowiol Mounting Medium.
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| 99.75 |
For cytogenetic analysis, preparations were coded and scored blind by two examiners. Chromosomal aberrations were analyzed in 100 metaphase spreads for each individual. Cells were analyzed for chromosome and chromatid types of aberrations as described earlier .
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study
| 99.9 |
Scoring of MN was limited to binucleate (BL) lymphocytes only with preserved cytoplasm according to the criteria proposed by Fenech et al. . Identification of binucleate cells in cell groups required careful visual examination of the individual cell boundaries. The results are expressed as number of micronucleated cells per 1000 binucleate cells on the two different slides from the same culture. Proliferation kinetics data was calculated by considering the frequencies of mono-, di-, tri-, and tetranucleate cells per each treatment group. A nuclear division index (NDI) was calculated according to the formula proposed by Takeshita et al. as follows: NDI = (1M1 + 2M2 + 3M3 + 4M4)/N, where M1 to M4 represent the number of cells with one to four nuclei and N is the total number of cells scored.
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study
| 100.0 |
For the viewing of γ-H2AX immunofluorescence foci, an epifluorescence microscope (ProvisAX70, Olympus) was used. Fields were initially selected at 10x on the basis of DAPI counterstained nuclei. Acquired DAPI image was used to define the focusing area and the detection of foci was performed using 40x objectives at optimized detection parameters. Scoring of foci was carried out manually in double blinded manner.
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| 84.5 |
From the various types of aberration recorded the aberration frequency was calculated as follows:(1)Aberration Frequency=Number of aberrations observed per sampleNumber of metaphases scored per sample.The standard error for the aberration frequency was calculated as follows:(2)Standard error=Number of aberrations observed per sampleNumber of metaphases scored per sample.Multiple comparisons between different experimental groups were done using multifactor ANOVA.
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study
| 99.94 |
Dicentric chromosomes (DC), acentric fragments (AF), and chromatid breaks (ChB) were the indices used to evaluate the chromosomal aberrations during the present study. The results of unstable chromosomal aberrations in the four experimental groups are summarized in Table 1. The baseline chromosomal damage of the negative control was negligible (DC = 0.037, AF = 0.043, and ChB = 0.063) and was comparable to that in published literature. Further, it was observed that there was no significant change in chromosomal aberrations due to introduction of the decoction at the dose range of 100–600 μg/mL.
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study
| 100.0 |
Chromosomal aberrations in bleomycin control were significantly high when compared with the negative control. On exposure to bleomycin, there was a 7-fold increase in the frequency of cells with dicentrics (0.27 per 100 cells) when compared to the index in negative control group (0.037 per 100 cells). The increases observed in AF (8-fold increase) and ChB (7-fold increase) were similar to that observed with respect to DC. In the test cells (lymphocytes exposed to bleomycin plus the decoction) there was a significant reduction (p < 0.05) in chromosomal damage when compared with the bleomycin control. The reductions in DC, AF, and ChB at the decoction dose of 300 μg/mL were nearly 2-fold that of the bleomycin control. The maximum percentage of inhibitions for DC (48%), AF (47%), and ChB (37%) was observed at the decoction dose of 300 μg/mL. However, a slight deterioration in the protection was observed at a decoction dose of 600 μg/mL (Table 1).
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study
| 100.0 |
Prevalence of MN in four experimental groups is illustrated in Table 2. The mean MN frequency in control PBLs was 10 MN per 1000 binucleated cells. Similar to effect in chromosomal aberration study, the decoction by itself did not induce any excess MN. A slight statistically insignificant elevation in MN count was observed in PBLs exposed to the decoction (100–600 μg/mL), with respect to the control samples. These results suggest that the decoction at a concentration range of 100–600 μg/mL does not induce any significant damage to lymphocyte genome in vitro.
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study
| 100.0 |
As expected nearly 9-fold increase in MN (89 MN per 1000 binucleated cells) was observed in lymphocytes exposed to bleomycin for 2 hrs at a dose of 40 μg/mL (equal to that of 2 Gy of gamma radiation). This increase was consistent in all three subjects tested.
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study
| 100.0 |
Bleomycin induced damage was significantly reduced (p < 0.05) in lymphocytes preexposed to the decoction at the tested dose range. Overall reduction was nearly 72% (25 MN per 1000 binucleated cells) when compared with the bleomycin control (88 MN per 1000 binucleated cells). This result suggests a significant protective effect of the decoction against genomic damage induced by bleomycin.
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study
| 100.0 |
Nuclear division index (NDI) was calculated to assess the effects of the decoction on the proliferative capacity of lymphocytes. NDI in lymphocytes treated with the decoction is same as that of control, suggesting no interference of the decoction in human lymphocyte proliferation. However, a significant delay in lymphocyte proliferation in the presence of bleomycin at a concentration of 40 μg/mL was observed. This was manifested by a change in relative numbers of M1 to M4 cells. However, restoration of NDI was observed in lymphocytes pretreated with the decoction prior to bleomycin induction. This reflects a successful overcome of cytotoxic effects caused by bleomycin, by modulating the lymphocyte proliferation.
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study
| 100.0 |
The effect of the decoction on bleomycin induced γ-H2AX foci in peripheral lymphocytes is illustrated in Figure 1 and Table 3. Baseline DNA damage in the present study was at 0.11 ± 0.04 foci per cell, represented by 674 cells without a single foci and 218 and 108 cells with single and double foci, respectively. None of the control cells indicated more than two (2) foci within a cell. Decoction by itself (at a concentration of 100 μg/mL) did not induce any significant DNA damage, as it indicated 0.14 ± 0.06 foci per cells with a distribution at 0–2 foci per cell. This observation is compatible with the previous results of MN and CA assays. Bleomycin (40 μg/mL), an equal dose to 2 Gy irradiation, significantly induced γ-H2AX foci (3.16 ± 1.13), with a distribution range of 0–10 foci per cell. Out of 1000 cells analyzed, approximately 700 cells were bearing either 2, 3, or 4 foci per cell. This was compatible with previous reports on lymphocytes exposed to 2 Gy irradiation.
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study
| 100.0 |
It was interesting to observe a significant reduction in γ-H2AX yield in lymphocytes exposed to bleomycin (40 μg/mL) pretreated with the decoction at a dose of 100 μg/mL. The number of foci per cell stood at 2.42 ± 1.01 foci within the range of 0–9 foci per cell. However, majority of cells (approx. 600 cells) indicated either 0, 1, or 2 foci per cell while 340 cells were reported to contain 3 foci per cell. The present results further confirm the ability of the decoction to protect against cytogenetic damage mediated by irradiation.
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study
| 100.0 |
A wide variety of chemotherapeutic agents are used routinely in clinical oncology. These therapies can also lead to cytotoxic and genotoxic effects in normal cells of the body, in addition to their effects on tumour cells [1, 3]. Any agent that could modulate such toxicity to normal cells would therefore be of much value in reducing the very unpleasant side effects experienced by cancer patients receiving chemotherapy.
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review
| 99.5 |
It is well documented that bleomycin, like most other chemotherapeutic drugs, can mediate DNA changes in both cancer cells and normal cells. As with ionizing radiation, Bleomycin mediates its genotoxic effects by inducing DNA double strand breaks (dsbs) which is one of the critical lesions for cellular death . This “radiomimetic” agent induces such double strand breaks by highly specific, concerted free radical attack on deoxyribose moieties in both DNA strands [33, 34]. Although the lesion induced by bleomycin is supposed to be only a small subset of the many lesions induced by ionizing radiation, effects of this radiomimetic agent in cells are remarkably similar to those of ionizing radiation .
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study
| 99.94 |
The present investigation has shown that the decoction comprised of N. sativa seeds, H. indicus roots, and S. glabra rhizomes can significantly protect against bleomycin induced (a) chromosomal aberrations, (b) formation of micronuclei, and (c) DNA dsbs in normal human peripheral blood lymphocytes. Decoction itself induces no significant chromosomal aberrations, no micronuclei formation, nor DNA dsbs in lymphocytes.
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study
| 100.0 |
Nuclear Division Index (NDI) is calculated to assess the effects of decoction on the proliferative capacity of human lymphocytes . From NDI values calculated in the present investigation, it was apparent that bleomycin caused a delay in cell proliferation, manifested by a change in relative numbers of cells with one nucleus to cells with four nuclei (M1 to M4 cells). The NDI values in lymphocytes exposed to bleomycin alone are similar to those reported by other investigators . In general, different types of ionizing radiation delay lymphocyte cell cycle and the extent of such delay depends on the dose delivered. An average mitotic delay of about 1 h per 1 Gy of gamma radiation has been observed once lymphocytes are exposed to ionizing radiation . These delays in cell cycle allow the cell to repair DNA and try to reduce the adverse effects of the irradiation . Normalization of NDI in bleomycin exposed lymphocytes that are previously treated with the decoction supports the protection mediated by the decoction against radiation induced damage.
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study
| 100.0 |
Antioxidant activity is considered to be one of the major mechanisms by which many plants and phytochemicals are reported to offer radioprotection . The potent antioxidant compounds present in the decoction may therefore be mainly responsible for the protective effect demonstrated by this herbal drug against bleomycin induced DNA damage in normal peripheral lymphocytes. The antioxidant potency of this polyherbal preparation is probably due to the collective contribution of antioxidant compounds present in the individual plant components used in its preparation. Thus, previous in vivo studies demonstrate that N. sativa seed oil can significantly reduce blood oxidative stress markers of rats exposed to a single dose of 6 Gy radiation . Similarly, extracts of H. indicus that can protect against radiation induced strand breaks in plasmid DNA have also been shown to protect against oxidative stress induced lipid peroxidation . Although these studies demonstrate the ability of the individual plants in the decoction to reduce oxidative stress, none of them have evaluated the ability of these plants to protect against cytogenetic damage induced by chemotherapy alone or in combination with radiotherapy.
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study
| 99.9 |
Since the polyherbal decoction under current investigation consists of a number of active ingredients, previously reported multimechanistic approach of the decoction in counteracting tumour progression can reasonably be justified. Counteract to oxidative stress is one of such mechanisms that may render a resistance to chemotherapy or radiotherapy in malignant cells, but effective in protecting healthy cells during the said treatment modalities. However, such compromised anticancer activity of the decoction will be well compensated by the existence of the remaining anticancer mechanisms of the decoction.
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study
| 99.94 |
In conclusion, the present investigation indicates that the decoction comprised of N. sativa seeds, H. indicus roots, and S. glabra rhizomes has the potential to protect against cytogenetic damage inflicted by bleomycin in human peripheral lymphocytes. Since the decoction under current investigation has previously been reported to mediate anticancer effect against certain malignancies, the observed radioprotective potential has a definite clinical advantage during its use among cancer patients. Thus, it could protect the patient against radiation induced cell damage while restricting tumour progression.
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study
| 99.94 |
In the present investigation, the effect of the decoction was evaluated at a single preincubation period of 3 hrs. However, the in-cooperation of different preincubation periods may have a significant impact on the claimed radioprotective potential of the decoction under current investigation. Despite these limitations, the findings of the present study clearly indicate the broad functional spectrum of the decoction comprised of N. sativa seeds, H. indicus roots, and S. glabra rhizomes in cancer management.
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study
| 99.94 |
Alzheimer’s disease (AD) is the most common type of dementia, and is characterized by progressive memory impairment and cognitive decline (Roberson & Mucke, 2006). Mounting evidence has indicated that dementia attributed to synaptic dysfunction and neuronal loss in the hippocampus and its associated cortex (Youssef et al., 2008; Selkoe, 2002; Niikura et al., 2002; Scheff et al., 2007), which are caused by the accumulation of soluble Aβ oligomers (Rowan et al., 2007; Lacor et al., 2004; Haass & Selkoe, 2007). Aβ is the major constituent in senile plaques and cerebral amyloid angiopathy, which are two most distinctive histopathologies in AD. The accumulation of Aβ in the brain initiates a cascade of events, such as activating astrocytes and microglia, initiating inflammatory responses, which lead to oxidative injury, altering neuronal ionic homeostasis, kinases/phosphatase activities, and so on (Klafki et al., 2006). These cascades result in a wide range of neuronal/synaptic dysfunction and loss, as well as loss of neurotrophin retrograde transport, therefore causing patients to present with the symptoms of dementia. Adjustment of the pathological progress of amyloid peptide is a key strategy to slow down the AD progression. In addition to reduction of the Aβ levels in the brain (Klafki et al., 2006), the concomitant application of neuroprotective agents may be the alternative therapeutics for AD, and a strategy to prevent progressive synaptic and cognitive degeneration (Klafki et al., 2006). Neurotrophins (NT) are synthesized and secreted by the target tissue, and after binding to its receptors and transported in a retrograde manner to the cell body. They exert a wide range of actions, including neuronal survival and differentiation, modulation of neuronal excitability, development and maintenance of synapses and modification of synaptic structure and function (Poo, 2001). The application of neurotrophic factors such as BDNF and CNTF could enable the modulation of neuronal survival and synaptic connectivity (Lu, Christian & Lu, 2008; Garcia et al., 2010)
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review
| 99.9 |
Dendrobium nobile is a traditional Chinese herbal medicine. In our previous studies, alkaloids extract from Dendrobium nobile Lindl. (DNLA) showed neuro-protective activity. For example, DNLA can prevent neuronal damages induced by LPS (Li et al., 2011; Zhang et al., 2011), and oxygen-glucose deprivation and reperfusion (Wang et al., 2010), decrease neuronal apoptosis, hyperphosphorylation of tau protein (Yang et al., 2014) and Aβ deposition in rat brain (Chen et al., 2008). The present study aimed to explore the effects of DNLA in protecting neurons from Aβ25–35-induced neurotoxicity in mice, and analyzed the mechanism from the aspects of promoting the secretion of neurotrophic factors.
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study
| 99.94 |
Dendrobium was collected from Dendrobium planting regions of Xintian Traditional Chinese Medicine Industry Development co., LTD of Guizhou Province in 2014. The dried stems of the herb (10 kg) were extracted by 95% ethanol solution. DNLA was isolated from the extracts, and analyzed by LC-MS/MS. Alkaloids accounted for 79.8% of DNLA, and mainly contained Dendrobine (C16H25O2N, 92.6%), Dendrobine-N-oxide (C16H25O3N, 3.3%), Nobilonine (C17H27O3N, 2.0%), Dendroxine (C17H25O3N, 0.9%), 6-Hydroxy-nobilonine (C17H27O4N, 0.32%), and 13-Hydroxy-14-oxodendrobine (C16H23O4N, 0.07%). The chemical structures of these ingredients were shown in the Fig. 1A, and the chromatograms of the sample solutions were shown in the Fig. 1B.
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study
| 100.0 |
Chemical structures and chromatograms of Dendrobium alkaloids (A) Dendrobine, (B) Dendrobine-N-oxide, (C) Nobilonine, (D) Dendroxine, (E) 6-Hydroxy-nobilonine, and (F) 13-Hydroxy-14-oxodendrobine. (G) Chromatogams of Dendrobium alkaloids. Differences between the accurately tested and calculated m/z values were <2 ppm for [M+H]+ions: (1) Dendrobine, (2) Dendrobine-N-oxide, (3) Nobilonine, (4) Dendroxine, (5) 6-Hydroxy-nobilonine, and (6) 13-Hydroxy-14-oxodendrobine.
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study
| 99.9 |
Aβ25–35 (Sigma, St Louis, MO, USA) was dissolved in physiological saline to a final concentration of 2.5 g/L, and incubated for one week at 37 °C to reach a state of aggregation. CNTF antibodies (ab190985) and GDNF antibodies (ab18956) were obtained from Abcam (Cambridge, England). BDNF (sc-546) was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). TUNEL kits (In Situ Cell Death Detection Kit and POD) were purchased from Roche (Switzerland).
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other
| 99.6 |
Male Kunming mice (20–25 g) were purchased from the Laboratory Animal Center, Chongqing, China (Grade: specific pathogen-free [SPF], Certificate no.: SCXK 2012-0005). Mice were housed in SPF-grade animal facilities (Certificate no.: SYXK 2014-003), with 22–23 °C, and a 12-hour light/dark cycle. Mice were given food and water freely. All animal procedures were approved by the animal experimental ethical committee of Zunyi Medical College.
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other
| 99.9 |
The animals were randomly divided into three groups (n = 6): sham surgery, model, and DNLA (40 mg/kg) treatment group. Mice were anesthetized with 7% chloral hydrate (35–45 mg/kg, i.p.) and fixed on a stereotaxic instrument (RWD Life Science, Guangdong, China). Then Aβ25–35 (2.5 µg/µL, 2 µL) was injected into both lateral cerebral ventricle in the model and DNLA groups via a 5-µl microinjector. The injection site was posterior from the bregma (AP) = −0.4, mediolateral from the midline (MR) = 1.2, and dorsoventral from the skull (DV) = 2.7. Mice in the sham group were treated the same as the model group except for the injection of Aβ25–35 instead of sterile normal saline (Nie et al., 2010). Mice in the DNLA group were administered intragastrically with DNLA (40 mg/kg) on the first day after the injection for 19 consecutive days, while mice in the sham and model groups were administered with distilled water.
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study
| 100.0 |
The ability of spatial learning and memory of the mice was evaluated through the Morris Water Maze (MWM) test (Edwards et al., 2014; Milner et al., 2014). The test was performed on the 14th day after drug administration. The pool of MWM was 120 cm in diameter, filled with water (22–25 °C) and divided into four quadrants. A hidden platform was placed in the center of the target quadrant 1.5–2.0 cm under the surface of the water. MWM included the place navigation test and the spatial probe test. In the place navigation test, all mice were trained three times per day for four days. Mice were initially placed in the three quadrants which did not have a platform. Each trail lasted for 60 s or ended as soon as the mouse climbed on the platform. The time was recorded as the escape latency, if the mouse climbed on the platform within 60 s. If the mouse failed to find the platform within 60 s, its escape latency was recorded as 60 s. The spatial probe test was performed following the end of the place navigation test. In this test, the platform was removed, and each mouse was allowed to swim for 60 s. The searching distance in the target area and total area were measured. Data was recorded and analyzed by Top View Animal Behavior Analyzing System (Version 3.00).
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study
| 100.0 |
After the MWM test, three mice were selected randomly from each group and anesthetized with 7% chloral hydrate. Then these mice were perfused with phosphate-buffered saline (0.1 M, 4 °C) via the ascending aorta, followed by 4% paraformaldehyde until the tail and limbs were rigid. Thereafter, the brains were removed and cut in half. Half of the brain was fixed with 4% paraformaldehyde for seven days and cut into coronal sections (4-µm thick) for hematoxylin-eosin (H&E), Nissl and TUNEL staining (Yang et al., 2014; Liu et al., 2015). The other half was fixed with 2.5% glutaraldehyde for electron microscope detection.
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study
| 100.0 |
Western blot analysis was performed as previously reported (Liu et al., 2015; Li et al., 2015). Three mice from each group were sacrificed after WMW test, and the hippocampal and cortical tissues were collected and homogenized in RIPA lysis buffer (1:5, w/v). Protein concentrations were determined by BCA protein assay kit. An aliquot of 45 µg of protein was applied for electrophoresis followed by the transferring of protein to the polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat dry milk in TBST buffer for one hour at room temperature, and incubated with the primary antibody: anti-CNTF (1:1,000), BDNF (1:1,000), and GDNF (1:1,000) at 4 °C overnight. Next, HRP-labeled goat anti-rabbit IgG (Beyotime Biotechnology, Jiangsu, China; A0208, 1:2,000) was incubated with the membrane at room temperature for one hour. The blots were visualized using the enhanced ECL Western blot detection kit (7Sea Biotech, Shanghai, China) and scanned to Gel Imaging. The band intensity was quantified using Quantity One 1-D analysis software v4.52 (BioRad, Hercules, California, USA).
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study
| 100.0 |
Compared with the sham group, the escape latency of the model group was significantly prolonged in the navigation test, and the explored distance in the target area was decreased in the spatial probe test. After treatment for 14 days, the escape latency of the DNLA group was decreased and the explored distance in the target area was increased as compared with the model group. The results indicated that DNLA could protect the mice against learning and memory deficits which were induced by Aβ25–35 injection (Fig. 2).
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study
| 100.0 |
Effect of DNLA on Aβ25−35-induced learning and memory impairment. (A) The graph represented the escape latency of the different groups. (B) Graphs described the adjusted searching distance in the space probe test. Data were expressed as mean ± SEM (n = 6). ∗P < 0.05 vs. the sham group; #P < 0.05 vs. model.
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study
| 100.0 |
H&E staining was performed to observe the morphological changes of cortex and hippocampus tissues in mice brain. The results showed that the morphology of brain tissues in the sham group was normal, and the cell density was higher. However, loose structures in hippocampal and cortical neurons with disordered hippocampal pyramidal cell layers occurred in the model group. Furthermore, the images showed that not only the number of neurons was reduced, but also that neurons staining were abnormal with nuclear condensation in the model group. DNLA treatment significantly decreased the number of abnormally stained neurons. Also, the morphology of brain tissues was generally normal in the DNLA group (Fig. 3).
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| 100.0 |
Effects of DNLA on morphological alterations in the hippocampus and cortex induced by Aβ25−35. Sections of the hippocampus CA3 region and cortex were obtained and stained with H&E (magnification, 200×). (A–C) showed the CA3 hippocampal cyto-architecture of mice in sham, model and DNLA groups, respectively; (D–F) showed the cortical cyto-architecture of mice in the sham group, model group and DNLA groups, respectively.
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| 100.0 |
The effect of DNLA on neuronal apoptosis induced by Aβ25–35 was observed using the TUNEL method. The results revealed that the number of apoptotic cells in the hippocampus and cortex were not affected in the sham group, but were increased in the model group. DNLA treatment decreased the number of apoptotic cells (Fig. 4).
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| 100.0 |
Effects of DNLA on Aβ25-35-induced neuronal apoptosis in hippocampus and cortex. Sections of the hippocampus CA3 region and cortex were obtained and stained with TUNEL 184 staining (magnification, 200×). (A–C) showed the CA3 hippocampal cyto-architecture of mice in sham, model and DNLA groups, respectively; (D–F) showed the cortical cyto-architecture of mice in the sham group, model group and DNLA groups, respectively. The cells dyed brown are apoptotic cells. (G) The number of the apoptotic neurons in hippocampus and cortex (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\bar {x}\pm ~SEM,n=3$\end{document}x ¯±SEM,n=3). **P < 0.01 vs. the sham group; ##P < 0.01 vs. the model group.
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| 100.0 |
As a neural characteristic structure, the number of Nissl bodies reflects the state of neurons. In physiological conditions, the Nissl bodies were big and abundant, showing that the function of neuronal protein synthesis was strong; on the other hand, when nerve cells were damaged, the number of Nissl bodies will be reduced or even disappear. Intraneural Nissl bodies of the cortex and hippocampus were lightly stained and appeared to be sparsely arranged in the model group. However, deeper stained Nissl bodies with higher density in cortex and hippocampus neurons were found in the sham and DNLA groups (Fig. 5).
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study
| 100.0 |
Effects of DNLA on Aβ25−35-induced neuronal Nissl bodies in the hippocampus and cortex. Sections of the hippocampus CA3 region and cortex were obtained and stained with Nissl staining (400×). (A–C) showed the CA3 hippocampal cyto-architecture of mice in sham, model and DNLA groups, respectively; (D–F) showed the cortical cyto-architecture of mice in the sham group, model group and DNLA groups, respectively.
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| 100.0 |
The ultrastructures of neurons were observed under electron microscope. The results revealed that the nuclear morphology of mice in the sham group was normal, and the chromatin was normally distributed. In the cytoplasm, mitochondrial morphology was normal with clear and regularly-arranged cristae. Furthermore, a number of ribosomes could be observed in these regularly-arranged rough endoplasmic reticulum. However, in the model group, the ultrastructures of neurons were abnormal. It could be found that nuclear membrane had folded or had obscure boundaries, mitochondria were swelling even without the cristae, and the endoplasmic reticulum was swelling. DNLA treatment could reduce the injury. Compared with the model group, nuclear membrane was more smooth, swelling of mitochondrial was reduced, and partial rupture of cristae were observed in the DNLA group (Fig. 6). The neuronal synapses were also observed under electron microscope. Injection of Aβ25–35 would reduce the number of synapses and synaptic vesicles, and cause the shape and size of synaptic vesicles uneven. The structure of synapses was obscured, and the structure of the post-synaptic lattice became thin. DNLA inhibited the loss of synapses significantly (P < 0.05). Also, in the DNLA group, the generally normal synaptic structures were maintained (Fig. 7).
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study
| 100.0 |
Effects of DNLA on the ultrastructures of neurons in Aβ25−35-injected mice. The ultrastructures were observed under electron microscope (12kx ×). (A–C) showed the ultrastructures of hippocampal neurons in sham, model and DNLA groups, respectively; (D–F) showed the ultrastructures of cortical neurons in sham, model and DNLA groups, respectively.
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| 100.0 |
The neuronal synapses were observed under electron microscope (50kx ×). (A–C) showed the synapses of hippocampal neurons in sham, model and DNLA groups, respectively; (D–F) showed the synapses of cortical neurons in the sham, model and DNLA groups, respectively. (G) showed the effects of DNLA on the number of neuronal synapses in Aβ25−35-induced mice. Data were expressed as mean ± SEM (n = 3). ∗P < 0.05 vs. the sham group; #P < 0.05 vs. the model group.
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study
| 100.0 |
The protein expressions were analyzed by Western blot. Compared with the sham group, the expressions of BDNF, CNTF and GDNF significantly decreased in the model group. DNLA group had significantly increased expressions of BDNF, CNTF and GDNF in the cortex and hippocamous (Fig. 8).
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study
| 100.0 |
Effects of DNLA on the protein expressions of BDNF, CNTF and GDNF in brain tissues. (A) Protein contents of hippocampal tissues were plotted for sham, model and DNLA groups, respectively. The corresponding quantitation of BDNF, CNTF, GDNF protein were showed in (B–D) respectively. (E) Protein contents of cortex tissues were plotted for sham, model and DNLA groups, respectively. The corresponding quantitation of BDNF, CNTF, GDNF protein were showed in (F–H) respectively. The relative optical density was normalized to β-actin. Data were expressed as mean ± SEM (n = 3). Significance ∗P < 0.05, ∗∗P < 0.01 compared to the sham group, #P < 0.05, ##P < 0.01 compared to the model group.
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study
| 100.0 |
Numerous literatures have reported that intra-hippocampal or intra-cerebroventricular injections of Aβ1–42 or Aβ25–35 fragments into rats or mice could induce neuronal death, and alter spatial learning and memory performance. Aβ could induce a variety of injuries, such as oxidative injury and disturbed neuronal ionic homeostasis, which could eventually result in neuronal dysfunction and selective neuronal loss (Lue et al., 1999; Niikura et al., 2002). Excessive neuronal loss can damage brain functions, and the cognitive ability is destroyed firstly. In this study, we established the mice model which was induced by the intra-cerebroventricular injection of Aβ25–35, and found that there were a lot of apoptotic neurons in the hippocampus CA3 region and cortex. The results of MWM test indicated that neuronal apoptosis was accompanied with an obvious failure in spatial learning and memory performance. On the other hand, DNLA treatment could significantly improve the ability of spatial learning and memory, and decreased apoptosis in TUNEL and H&E staining. This result demonstrated that DNLA could improve learning and memory deficits in AD model mice, and the protection may be due to the decreased apoptosis induced by Aβ25–35.
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study
| 99.94 |
As it is known, when neurons are damaged, Nissl bodies are most sensitive. The main changes of Nissl bodies associated with neuronal injury include the dissolution and disappearance of Nissl bodies. In the model group, the number of Nissl bodies was decreased in cells. After the administration of DNLA, the number was increased. The results indicated DNLA could alleviate neuronal damage.
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study
| 100.0 |
It has reported that memory disorders in AD patients begin with subtle changes in hippocampal synaptic efficacy earlier than extensive neuronal degeneration. Davies et al. (1987) performed a quantitative morphometric analysis to measure the densities of neurons and synapses in cerebral cortical biopsy tissues collected from AD patients. They found that the numerical density of synapses decreased 25%–35%, and the number of synapses per cortical neuron decreased 15%–35% in AD patients (Davies et al., 1987). Evidence also suggested that synaptic dysfunction is caused by oligomeric assemblies of Aβ (Lacor et al., 2004; Rowan et al., 2007). Even in very mildly impaired patients, the degree of synaptic loss in the cortex showed a significant association with soluble Aβ levels. Our results revealed that Aβ25–35 induced synapse loss in the hippocampus and cortex, and DLNA could dramatically inhibit the loss of synapses and improve synaptic structures, thereby improving the learning and memory abilities of mice.
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study
| 99.94 |
The application of neurotrophic factors could enable the modulation of neuronal survival and synaptic connectivity (Poo, 2001). Neurotrophic factors are synthesized and secreted by the target tissue. After binding with receptors (tyrosine receptor kinase [Trk] and pan-neurotrophin receptot [p75]), these factors are internalized and transported to the cell body in a retrograde manner, where they would affect neuronal survival and differentiation (Poo, 2001; Barker & Shooter, 1994). The loss of neurons in the AD process is at least partly due to the lack of one or more neurotrophic factors. It has been found that the mRNA expression level of BDNF is low in the hippocampus and cortex of AD patients. Christensen et al. (2008) reported that the injection of Aβ1–42 in the hippocampus of rat would lead to learning and memory impairment and reduce cortical BDNF levels. These results indicate that memory deficit induced by Aβ1–42 is associated with the disorder of the expression of BDNF, which is reflected in lower cortical BDNF levels. Another study has shown that secretory vesicles containing BDNF exist within axon terminals, dendrites of pyramidal, and granule cells. BDNF can be secreted into the synaptic gap and binds with TrkB. After binding, BDNF serves as a key regulator in synaptic plasticity and memory, and plays a role in long-term and short-term memory (Lu, Christian & Lu, 2008). The present study proved that intracerebroventricular injection of Aβ25–35 could decrease the expression of BDNF in the hippocampus and cortex. However, DNLA have significantly increased BDNF expression, suggesting that the abilities of DNLA to reduce neuronal apoptosis and synaptic loss in mice may be partly associated with increased BDNF protein level.
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study
| 99.94 |
The maintenance of the neuronal survival and function needs simultaneous support from a variety of neural survival factors, which form an information network through signal transduction pathways in nerve cells, and generate an amplified effect via intracellular generalization (Poo, 2001). Therefore, we examined two other neurotrophic factors, CNTF and GDNF. CNTF is a member of a cytokine family (Duff & Baile, 2003), and is known for its neuroprotective effects, being as a survival factor for sympathetic, sensory and hippocampal neurons. Garcia et al. reported that CNTF reduced the impairments of synaptic and cognitive functions in the Tg2576 AD mouse model, and rescues neurons from degeneration induced by Aβ in vitro and in vivo (Pasquin, Sharma & Gauchat, 2015; Garcia et al., 2010). GDNF family and its different receptor systems are now recognized as one of the major neurotrophic networks in the nervous system, which are important for the development, maintenance and function of neurons and glial cells. GDNF signaling through NCAM can result in the activation of kinases MAP and Src-like kinases, and contributes to the regulation of several different processes, including synapse formation and neurite outgrowth (Allen et al., 2013). In hippocampal neuronal cultures, GDNF has been found to increase the number of synapses. Although most studies on the function of GDNF and its receptors has been concentrated on midbrain dopaminergic neurons, the receptors of GDNF are observed in many other brain regions. A study analyzed 250 blood plasma samples of AD patients, and identified GDNF as one of 18 signaling proteins. GDNF level was lower in AD patients than the controls. It is not difficult to envision how a signaling molecule such as GDNF, which has potent effects on neuronal maturation, cell survival and synapse formation, can effect cognitive functions; and if aberrantly or insufficiently expressed or secreted, contribute to cognitive decline (Ibáñez & Andressoo, 2016; Pertusa et al., 2008). Our study also found DNLA can significantly increase the expression of CNTF and GDNF. The results indicated that the ability of DNLA to prevent neuronal and synaptic loss may be attributed to increase the expression of BDNF, CNTF and GDNF.
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study
| 99.94 |
In summary, the present study demonstrates that DNLA could improve Aβ-induced learning and memory impairment in mice. This may be due to the prevention of neuronal apoptosis and synaptic loss via the increased expression of BDNF, GDNF and CNTF in the hippocampus and cortex (Fig. 9).
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study
| 100.0 |
The file shows the raw data of the place navigation test in day 1. Every mouse have trailed three times, each trail lasted for 60 s or ended as soon as the mouse climbed on the platform. The time was recorded as the escape latency, if the mouse climbed on the platform within 60 s. If the mouse failed to find the platform within 60 s, its escape latency was recorded as 60 s.
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other
| 99.7 |
The file shows the raw data of the place navigation test in day 2. Every mouse have trailed three times, each trail lasted for 60 s or ended as soon as the mouse climbed on the platform. The time was recorded as the escape latency, if the mouse climbed on the platform within 60 s. If the mouse failed to find the platform within 60 s, its escape latency was recorded as 60 s.
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other
| 99.7 |
The file shows the raw data of the place navigation test in day 3. Every mouse have trailed three times, each trail lasted for 60 s or ended as soon as the mouse climbed on the platform. The time was recorded as the escape latency, if the mouse climbed on the platform within 60 s. If the mouse failed to find the platform within 60 s, its escape latency was recorded as 60 s.
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other
| 99.7 |
The file shows the raw data of the place navigation test in day 4. Every mouse have trailed three times, each trail lasted for 60 s or ended as soon as the mouse climbed on the platform. The time was recorded as the escape latency, if the mouse climbed on the platform within 60 s. If the mouse failed to find the platform within 60 s, its escape latency was recorded as 60 s.
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other
| 99.7 |
The file shows the raw data of the spatical probe test. In this test, the platform was removed, and each mouse was allowed to swim for 60 s, the searching distance in the target area and total area were measured. The target area is ’center in 3’ of the file.
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other
| 99.9 |
The file shows the raw data of TUNEL staining. 3 animals are picked up randomly from each group and 3 pieces of each brain are required to be stained with TUNEL staining. we have taken photos of them from 3-4 perspectives randomly magnified 400 times.Then, calculated the numbers of total cells and apoptotic cells.
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other
| 99.6 |
American beech (Fagus grandifolia Ehrl.) is native to the eastern North American deciduous forests and is the only species of this genus in North America . The slow-growing, deciduous tree usually reaches about 37 m height (120 ft) and may attain ages of 300 to 400 years . The native range of American beech is within an area from Nova Scotia in southeastern Canada, west to Wisconsin and south to eastern Texas and northern Florida in the United States. Beech wood is easily workable, excellent for turning and steam bending and used for flooring, furniture, veneer and containers .
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other
| 99.94 |
Genetic research has been centered mostly on three genera (Fagus, Castanea and Quercus) of the family Fagaceae. The American beech (Fagus grandifolia Ehrh.) genome is estimated at 610 Mbp and has yet to be sequenced. The number of chromosomes is generally stable within the Fagaceae family (2n = 24), with occasional changes (2n = 24 + 1, 2, 3) resulting from irregular segregation at mitosis . Both Fagus grandifolia and F. sylvatica have the most rudimentary genomes within the family, making their genomes attractive for comparative genomics studies .
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study
| 99.75 |
The thinness of beech bark makes it vulnerable to a range of scale insects. Beech bark disease (BBD) is a scale-fungal complex disease, initiated when a specific scale insect, Cryptococcus fagisuga Lind., attacks the bark of beech trees and renders it susceptible to bark canker fungi of the genus Neonectria . Neonectria (Neonectria faginata or Neonectria ditissima) is the most common genus of ascomycete fungi associated with beech bark disease [6–8]. The disease develops through feeding activity of the beech scale insect that creates points of entryway for the fungal pathogen. Once established on the tree, the fungus is no longer influenced by fluctuations in beech scale density caused by environmental factors or habitat quality . The cankering response of beech to fungal infection produces bark microstructure fissures and when it progresses the cankers may coalesce girdling or partially girdling the tree leading to wilting foliage and even mortality of trees. Individuals that survive infection are more susceptible to abiotic and biotic stress .
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study
| 99.6 |
Beech bark disease first occurred when the scale insect was accidently introduced on plant material imported to Nova Scotia from Europe around 1890 . It is found throughout northeastern U.S. states and southeastern Canadian provenances and is still expanding. The disease distribution is generally attributed to the initial phase of the insect life cycle (“crawlers”) just after hatching, which is the only mobile phase of the life cycle. The “crawlers” can move to other areas on the same tree and can be dispersed by wind, birds, animals or humans. The mortality rate in beech has been significant in areas throughout the eastern United States. The loss of beech trees in some areas, where other hardwood species are rare, is causing even greater impact on wildlife, especially for black bear (Ursus americanus) [6, 12]. Pesticide control proved to not be fully effective in reducing the number of scale insects due to their protective waxy covering . Neither pesticide application nor removal of infested trees is practical in large natural areas, because of labor, financial and environmental constraints . Attempts to control C. faggisuga using a bio-agent such as a predatory mite [Allothrombium mitchelli Davis (Acari: Trombidiidae)] is currently under investigation .
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study
| 99.06 |
Several field trials showed some beech trees remain after infestations that appear to be naturally resistant to BBD, usually clustered in small groups [16, 17]. About 1% of American beech trees remain disease free in forests long-affected by BBD . There have been a small number of studies to understand the genetics of resistance of American beech to BBD [17–20]. Several studies have been conducted using molecular markers such as isozymes, RAPD (random amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSRs (simple sequence repeat) to differentiate between resistant and susceptible individuals and identify markers correlated with resistance, evaluate spatial and population genetic structure, and perform parentage analysis [19, 21–24]. Although genetic marker studies enhanced the efforts to clarify modes of inheritance, no markers have been discovered that correlate with resistance. To estimate heritability, resistance to the beech scale insect, the artificial infestation technique developed by Houston , was used to test parent trees and their full and half-sibling progeny for resistance . Individuals were classified as susceptible if five or more scale insects were present on the bark surface 1 year after scale insect eggs were affixed to the test tree. Low levels of resistance were found in families with only one resistant parent and a higher proportion of resistant progeny were only observed in families where both parent trees were resistant, confirming that resistance to beech scale insect is a heritable trait [19, 24]. Current screening for resistance aims to increase the proportion of resistant trees and remove the susceptible trees for breeding purposes. These results formed the basis of a regional breeding program for BBD-resistant American beech [5, 25]. Identification of genetic markers associated with the resistance phenotype could potentially accelerate breeding efforts and reduce costs through the implementation of indirect selection methods, reducing the need for the costly, time consuming and labor intensive methods currently used to test trees for resistance to the scale insect.
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study
| 99.7 |
Association mapping (AM) is an alternative approach that may, in theory, overcome limitations of pedigree-based quantitative trait loci (QTL) mapping . It has been used in model species with available genomic resources, however with recently available genome sequences for forest species, this approach has the potential to increase the chance of understanding the genetic architecture of complex traits. The candidate-gene-based approach has been used for forest species where genome sequences were not yet developed . In our study, we conducted a genome wide association study (GWAS) to discover loci associated with BBD resistance. A case-control design was used, which compared marker frequencies between a group of affected individuals (cases) and a group of unaffected individuals (controls). In this approach, we aimed to examine genetic architecture of disease resistance in American beech and identify candidate genes associated to BBD. Once identified, a marker breeding based approach could be used in breeding programs for BBD resistance.
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study
| 99.94 |
Trees sampled for RNA preparation were all part of the U.S. Forest Service Northern Research Station’s American beech breeding program in Delaware, OH. Tissues were sampled in the summer of 2009 from selected trees growing in naturally forested areas, grafted ramets of parent trees, and seedling progeny. The selected trees, or grafted ramets of the selected trees, were previously tested for beech scale resistance using an artificial infestation procedure . To maximize genetic diversity, five resistant and five susceptible trees originating from a diverse geographic range, that included New Brunswick (Canada), Maine (USA), the lower peninsula of Michigan (USA), the upper peninsula of Michigan (USA), Pennsylvania (USA), and both northern and southern Ohio (USA) were selected for RNA sequencing and SNP discovery. Outer bark tissues including periderm, vascular cambium, and phloem, were harvested using grafting knives sterilized with liquid ethanol and were immediately frozen in a dry ice ethanol bath before transfer to a − 80 °C freezer until shipping overnight to the Schatz Center at Pennsylvania State University in a dry nitrogen shipper to prevent thawing.
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study
| 100.0 |
RNA samples were sequenced using Roche 454 sequencing technology. The main aim was to establish a database for network analysis to determine tissue specific expression patterns. Individual total RNA samples were prepared from bark tissues using the method first described by and modified by . At least five grams of frozen bark tissues were weighed, ground to a fine powder under liquid nitrogen, and dispersed in CTAB buffer. Following two chloroform extractions, RNA was precipitated with LiCl2, extracted again with chloroform and precipitated with ethanol. The resulting RNA pellet was re-suspended in 40–100 μl of DEPC-treated water, and the quality was assessed with an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies). Poly(A) RNA was purified from total RNA using the Ovation RNA-Seq System kit (NuGen) following supplier’s instructions. Reverse-transcription was performed using the Just cDNA kit (Stratagene) and random hexamer primers.
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study
| 100.0 |
Individual sequencing libraries for each cDNA preparation were constructed and sequenced using a 454 sequencer as previously described . The cDNA preparations were sheared to approximately 500 bp fragment lengths. Adaptor sequences containing unique barcodes for each library were ligated to the fragmented cDNAs and immobilized on beads. The libraries for the five disease-resistant trees were pooled and the libraries for the five disease-susceptible trees were combined for separate multiplex sequencing, each on a different half of the same plate. Multiplex sequencing of the library pools was performed on an FLX model 454 DNA sequencer (Roche Diagnostics) at Penn State University. The DNA sequence files for each of the ten cDNA libraries were selected and compiled from the batch sequencing raw data files using a Newbler 454 software utility, based on the unique barcodes assigned to each of the libraries. Sequences generated in this study were submitted to the Short Read Archive at the National Center for Biotechnology Information, accessions numbers SRX1781388 to SRX1781397, for NCBI BioProject Accession PRJNA321730 (NCBI: http://www.ncbi.nlm.nih.gov).
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study
| 100.0 |
The SeqMan NGEN (DNASTAR) program for next generation transcriptome sequence data assembly was used to assemble contigs from the pooled 454 sequence data files for all five BBD-resistant tree libraries. Similarly, the pooled dataset consisting of 454 sequence data files for the five BBD-susceptible tree libraries was also assembled using the NGEN program. Finally, to obtain a reference transcriptome for F. grandifolia, all of the 454 sequencing data files for the ten libraries were pooled and assembled into a combined set of transcript contigs.
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study
| 99.94 |
Mapping of the reads from each library onto the assembled transcriptomes revealed a high level of several sequences of ribosomal origins. These transcripts accounted for 34% (180,292 out of 533,261), 27% (139,718 out of 521,505) and 18% (186,745 out of 1,026,995) of the resistant, susceptible and combined assemblies, respectively. To determine the amount of structural non-coding RNA (ncRNA) sequences present in the libraries, the RFAM structural RNA database was downloaded to serve as a local BLAST database. All of the sequence reads and contig sequences in the three assembled transcriptomes were aligned to the RFAM database, using a conservative BLAST e-value threshold of e-70, the ribosomal content was about 30% of the sequenced reads. ncRNAs accounted for 729, 1506 and 2110 contigs for the resistant, susceptible and combined assemblies, respectively. These contigs were removed from the assemblies, prior to SNP discovery.
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study
| 100.0 |
Due to the large amount of ncRNA reads, another assembly was conducted using the Newbler program (Roche) which incorporated filtering for structural RNAs, low quality reads, and abbreviated reads. The BLAST cut-off e-value used in the filtering ncRNAs was set at e-50, resulting in 349,613 out of 1,406,316 reads in total to be removed prior to assembly. Again, assemblies were conducted for the 5 BBD-resistant libraries, the 5 BBD-susceptible libraries, and all 10 libraries combined.
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study
| 99.94 |
Detection of putative polymorphic sites was performed by mapping reads filtered for structural RNAs to the beech reference transcript contigs, using the Newbler’s gsMapper program. Both DNASTAR and Newbler all-library combined contig sets were used, separately, as reference transcriptomes in the SNP discovery. A minimum depth of coverage of 15 reads from each library was required to call putative SNPs. High confidence SNP sites were identified as those with a minimum of 100 bases flanking the SNP site and where the reference nucleotide at the SNP site was non-ambiguous. The stress-response genes and EST-based DNA markers served as a resource (Fagus grandifolia Transcriptome, Hardwood Genomics Project, www.hardwoodgenomics.org) for the construction of linkage maps and a framework for a GWAS study.
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study
| 100.0 |
Resistant trees (R = 254) and susceptible trees (S = 260) were located and positions mapped in six US states and nine stands. Trees in Penobscot county (Maine), Berkshire county (MA), Randolph county (WV) and Pascataquis county (ME) and two Canadian provenances: Prince Edward Isl. Canada, Kings county, and Sissiboo Falls, Digby county (NS) were located and mapped as reported in previous studies [18, 22]. Additional trees were located and mapped in Ludington State Park, Mason county (MI), Luce county (MI), Mckean county (PA), Clearfield county (PA), Clinton county (PA) and Licking county (OH) (Fig. 1).Fig. 1Sampling locations of mapping population. Highlighted by red full circles are sampling locations of American beech full-sib individuals used for association mapping study across stands in six U.S. states and two Canadian provinces. The map graphic was reproduced in the package ‘ggmap’ (Spatial visualization in ggplot2) v.2.6.1 (Kahle and Wickham) in software R with Google Maps and Stamen Maps. For Fig. 1 a copyright permission was not required
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study
| 99.94 |
Sampling locations of mapping population. Highlighted by red full circles are sampling locations of American beech full-sib individuals used for association mapping study across stands in six U.S. states and two Canadian provinces. The map graphic was reproduced in the package ‘ggmap’ (Spatial visualization in ggplot2) v.2.6.1 (Kahle and Wickham) in software R with Google Maps and Stamen Maps. For Fig. 1 a copyright permission was not required
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other
| 99.94 |
Phenotypes were determined for 514 individuals through field assessment of all trees at the time of tissue collection. Trees exempt of beech scale insects and any apparent signs of fungal infection were classified as resistant. A subset of trees was artificially inoculated with scale eggs to confirm resistance either in the field or on grafted ramets of the original trees [16, 17, 25]. A complete description of sampled trees and their phenotypes is included in Additional file 1.
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study
| 100.0 |
Extraction of DNA from leaf and dormant bud tissues was carried out as described in . Dormant buds were collected as described in and stored at −70 °C until used for DNA extractions. For the 514 samples, DNA quality and quantity was tested before submitting for genotyping. Approximately 20 K SNPs, originating from EST transcriptome assemblies, were submitted for design scores and 16 K of these were selected for inclusion on an Affymetrix Axiom™ Genome-Wide 1.5 K - 50 K array (Santa Clara, CA). Genotypes were successfully determined for 506 of the 514 samples and were used in downstream analysis with an initial call rate of 97% or greater.
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study
| 100.0 |
Controlled cross-pollinations were carried out between two confirmed beech scale resistant American beech trees 1505 (R) and 1504 (R) in Ludington State Park, MI (USA) as previously described to create the mapping population . Additional cross-pollinations were carried out on grafted containerized ramets of the parents in 2010 to supplement the original family (N = 46), bringing the total number of progeny to 117.
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study
| 99.94 |
Linkage analysis was performed on 115 of the 117 progeny that were successfully genotyped at 5838 SNPs sites. A genetic map was constructed to order SNP markers used in association testing and served as graphical displays of the genome wide significant associated SNPs. The map construction was performed using the software Join-Map 2.0 . All SNPs that successfully “passed” a 1:2:1 or 1:1 segregation ratio test in the offspring, were used in the linkage analysis, assigned to linkage groups and ordered to determine map genetic distances in cM (centimorgans).
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study
| 100.0 |
Discriminant Analysis of Principal Components (DAPC) was used to cluster individuals based on genotypes. This well-known method aims to maximize group differences while minimizing within cluster variances . DAPC was applied on a matrix composed of 506 individuals and 5838 SNPs using an implementation available in the R package adegenet 2.0.0. To identify clusters, the principal component analysis (PCA) of the matrix data was first computed followed by Discriminant Analysis on the number of retained principal components as provided by DAPC method.
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study
| 100.0 |
A test for population stratification was also performed with qqman package in R software v.3.2.0, a common tool to visualize GWAS results and estimate the rate of genomic inflation. We measured genomic inflation also defined as λ (lambda) to provide evidence of population stratification or cryptic relatedness.
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study
| 99.94 |
To avoid statistical biases during population structure analysis and IBS score computations, SNPs with Pearson’s correlation coefficients (r2) higher than 0.8 were rejected. Due to the absence of a genetic scaffold, a chromosomal location-based SNP filtering, as implemented in common GWAS packages, such as PLINK v.1.9 , was replaced by iterative SNP filtering. Pairwise Pearson’s r2 across SNPs were computed and, at each step during the iteration, the SNP correlated (r2 > 0.8) with the most number of other SNPs was rejected. The iteration stopped when no remaining SNPs were correlated (all pairwise r2 < 0.8). This procedure left 3220 SNPs out of original 5838 SNPs. Filtered SNPs were then used to compute IBS scores, namely pairwise Pearson’s correlation coefficients (r2), between individuals. Here again iterative filtering was applied across trees to prune individuals with IBS > 0.1875. Out of 506 trees, 327 passed IBS-based filtering.
|
study
| 100.0 |
A logistic regression model was used to perform association tests between SNPs and disease scores. Population stratification was controlled using the first 20 ancestry principal components (PC’s) as covariates in the logistic model. GWAS was performed using the PLINK 1.9 package (, https://www.cog-genomics.org/plink2, ) and 3220 SNPs on 327 independent individuals (172 cases and 155 controls, all pairwise IBS < 0.185). Prior to association testing, individuals with missing genotype rate > 10% were deleted, leaving effectively 172 cases (susceptible or diseased trees) and 155 controls (resistant or symptom-free trees). SNPs were then filtered for Hardy-Weinberg equilibrium (p-values >10−5), minor allele frequencies (MAF > 0.05) and missing genotypes across individuals (< 10%). In total, 3155 SNPs were included in the association test after filtering. The genotyping rate was equal to 0.99. For every SNP included in the case-control test, the exact P value [P] and the estimated odds ratio [OD] for the association between the minor allele [A1] and the disease phenotype were calculated. Resulting p-values underwent genomic inflation control.
|
study
| 100.0 |
Haplotypes were identified using Haploview (v.4.2) , and with default parameters (exclusion of markers separated by >500 kb and individuals with >50% missing genotype). Pearson’s coefficient of determination (r2) was used to determine the pairwise correlation between genetic markers.
|
study
| 99.94 |
The sequencing of 10 cDNA libraries yielded 1,406,316 reads covering 508,764,432 bases. The libraries from BBD-resistant trees yielded between 87,964 and 210,340 reads, while 70,218 to 205,945 reads were obtained for libraries from BBD-susceptible trees (Table 1). While the quality and quantity of the sequence data was acceptable (Table 1; Additional file 2), the mRNA poly-A selection step left a large proportion of structural non-coding RNAs in the samples, including ribosomal RNA. Moreover, up to a third of the transcriptome sequence reads were mapped to ncRNAs. Subsequently, the quality of the mRNA preparations was re-assessed with an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies), revealing remnant rRNA peaks (Additional file 3). Because the ncRNA contamination was detected after the first transcript assemblies were conducted, a second assembly with additional filtering was conducted (see below).Table 1Result metrics for the sequencing of 10 beech cDNA librariesLibrary Name (based on tree number)Number of readsAverage read lengthTotal basesBBD-Resistant trees Beech_1228R210,34037578,912,594 Beech_2692R131,30635446,467,779 Beech_1504R123,19437045,498,709 Beech_1208R87,96435831,467,605 Beech_2276R147,78137455,192,118Resistant tree Sub-Totals700,585366.2257,538,805BBD-Susceptible trees Beech_1973S205,94535873,702,265 Beech_DN00726S159,66034955,759,976 Beech_3128S130,91735946,933,567 Beech_2143S138,99135749,586,742 Beech_Holden70,21836025,243,077Susceptible tree Sub-Totals705,731356.6251,225,627Totals for all libraries1,406,316361508,764,432
|
study
| 100.0 |
Assemblies of 454 sequence reads into contigs were conducted using both the SeqMan NGEN (DNASTAR) program and the 454 Newbler (Roche) assembler. Contigs were built using reads from, either five BB-resistant libraries, five BBD-susceptible libraries or combination of both libraries.
|
other
| 97.4 |
The NGEN assembly resulted in a total of 28,592, 27,544 and 44,065 contigs for pools of BBD-resistant, BBD-susceptible and the combination of both libraries, respectively. NGEN assemblies incorporated 76%, 73% and 73% of the sequence reads from the BBD-resistant, BBD-susceptible and the combined samples. NGEN contig lengths averaged approximately 360 bases, with a median varying from 475 to 542 bases. The longest transcripts obtained in the resistant, susceptible and combined assemblies were 11,704, 8168 and 10,800 bases, respectively. Table 2 summarizes the results of the three F. grandifolia transcriptome assemblies using the NGEN program.Table 2 F. grandifolia transcriptome NGEN assembly statisticsBBD-Resistance LibrariesBBD-Susceptible Libraries“Combined” Reference transcriptomeAssembled Reads533,261521,5051,026,995Unassembled Reads167,324184,226379,321Total Number of Reads700,585705,7311,406,316Assembled Reads (%)76.1273.9073.03Assembled Contigs28,59227,54444,065Contigs >2 K6222711115Av. Length of Contigs362354357
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study
| 100.0 |
The Newbler filtered assembly resulted in 10,690 contigs for the BBD-resistant data, 7630 contigs for the BBD-susceptible data, and 16,285 contigs for all libraries combined. The Newbler assemblies incorporated 84%, 81% and 86% of the sequence reads from the BBD-resistant, BBD-susceptible and the combined data, respectively. The longest transcripts obtained by Newbler in the resistant, susceptible and combined assemblies were 4651, 4336 and 10,681 bases respectively. The average length of the Newbler contigs across the three assemblies was 679 bases. The detailed Newbler assembly statistics are shown in Table 3. The “large contigs” from the Newbler assembly are those with 500 bases or longer, which overall averaged 949 bases in length.Table 3 F. grandifolia transcriptome sequence assembly summary obtained from NewblerAssembled Contig SequencesBeech “Resistant” LibraryBeech “Non-Resistant” LibraryBeech “Combined” LibraryAligned Bases (%)157,365,926(83.63%)143,162,558(80.91%)314,816,063(86.23%)Aligned Reads (%)438,153(82.04%)412,164(78.90%)893,216(84.54%)Number of Contigs, All10,690763016,285Total Contig Bases7,845,7004,478,07811,664,012Average Contig Length734587716Number of Large Contigs714740189943Average Large Contig Size9618751009N50 Large Contig Size10058931081Largest Contig Size4651433610,681
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study
| 100.0 |
SNP site discovery was performed using both the DNASTAR NGEN and Newbler combined assemblies as reference transcriptomes. The Newbler gsMapper SNP calling program generated two output files. One output file contained all possible SNPs. The other output file contained only the high confidence SNP calls, which was used as the starting point for selecting SNPs for the mapping study. A summary of the SNP discovery results is displayed in Table 4. As shown in the last two columns of Table 4, 15,542 and 12,119 candidate SNP sites were discovered using the Newbler and DNASTAR reference transcriptomes, respectively. For each candidate SNP with a minimum 100 bases of sequence flanking the SNP, 50 bases from each flanking side of the SNP site was extracted for DNA marker development. The values in parenthesis in the last two rows of Table 4 represent the number of SNP sites selected, based on sufficient flanking sequence. The sequences and statistics for the high quality and most informative SNPs used in the GWAS are presented in a table in Additional file 4.Table 4Summary of SNP discovery results for F. grandifolia using the reference transcriptomes generated by DNASTAR NGEN and NewblerStatisticsDNASTAR Reference TranscriptomeNewbler Reference TranscriptomeNumber of Contigs in Reference43,21214,977Number of Bases in Reference28,676,24211,580,835Number of Mapped Reads (%)1,357,629 (96.55%)938,418 (88.82%)Number of Mapped Bases (%)494,805,288 (97.45%)312,081,222 (85.48%)Fully Mapped Reads (%)444,838 (31.64%)396,609 (37.54%)Partially Mapped Reads (%)35,728 (2.54%)135,858 (12.86%)Non-Unique Mapped Reads (%)871,685 (61.99%)147,210 (13.93%)Chimeric Reads (%)5378 (0.38%)258,741 (24.49%)Unmapped Reads (%)32,025 (2.28%)101,623 (9.62%)Reads Too Short (%)16,470 (1.17%)16,470 (1.56%)High Confidence SNP Calls2119 (12,069)15,542 (14,574)HC SNP Calls (Not Ambiguous)10,971 (10,934)15,541 (14,573)
|
study
| 100.0 |
In total, 514 DNA samples were submitted to genotyping using the Affymetrix Axiom™ Genome-Wide 1.5 K - 50 K array and after quality filtering, resulted in genotypes for 506 samples (R = 249 and S = 257). Of the initial 16,709 SNPs, 5838 Poly High Resolution SNPs passed Affymetrix filtering metrics and only these were included in downstream analysis. A set of 5838 SNPs was visualized for the cluster pattern in SNPolisher R Package v.1.5.1 (Affymetrix Inc.).
|
study
| 99.94 |
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