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Goose astrovirus genotype 1 (GAstV-1) has emerged in goose farms in some provinces of China in recent years and is considered to be one of the pathogens of gout in goslings in China. However, few studies have been conducted on the dynamic distribution, tissue tropism, and pathogenesis of GAstV-1 in goslings. In 2022, an epidemiological investigation of goose astrovirus (GAstV) in goslings was conducted in seven provinces of China. During the investigation, a GAstV-1 designated as GAstV-JSXZ was identified in the kidney of an 8-day-old gosling and was successfully isolated from a goose embryo. The full genome sequence of GAstV-JSXZ was determined using the next-generation sequencing technique. The complete genome of GAstV-JSXZ was 7299-nt-long. Interestingly, the phylogenetic analysis revealed that Chinese GAstV-1 has formed two distinct subgroups based on the ORF 2 genomes, designated GAstV-1 1a and GAstV-1 1b. The GAstV-JSXZ shared the highest identity with GAstV-1 1a strain FLX and TZ03 in nucleotides (ORF1a: 98.3–98.4%; ORF1b: 92.3–99.1%; ORF2: 95.8–98.8%) and amino acid sequences (ORF1a: 99.4–99.5%; ORF1b: 98.2–98.8%; ORF2: 97.0–99.4%). To evaluate the pathogenicity of GAstV-1, 1-day-old goslings were inoculated with the virus by oral and subcutaneous injection routes, respectively. The results revealed that the virus causes extensive pathological organ damage, especially in the kidney, liver, and thymus. Virus-specific genomic RNA could be detected in the cloacal swabs and tissues of infected goslings throughout the experiment. The viral copy numbers examined in the kidney and intestine were the highest, followed by the liver and spleen. These results are likely to provide a new understanding of the pathogenicity of GAstV-1 in geese. [ABSTRACT FROM AUTHOR]

Qingye ramie is a multi-purpose crop, used as a feed ingredient in southern China, that is susceptible to drought. Qingye ramie was studied to investigate the effects of high temperatures and drought on its growthh. The results show that, after drought, ramie leaves turn yellow and that the height of the plant, the number of tillers, and its antioxidant activity decreased. To elucidate the molecular mechanism of drought tolerance, we performed RNA sequencing (RNA-seq) on drought-stressed samples and found that 3893 differentially expressed genes showed significant changes; 1497 genes were upregulated, and 2796 genes were downregulated. These genes were categorized into four metabolic pathways and were mainly enriched in plant hormone signal transcription, phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, and starch and sucrose metabolism. Among these, we mapped the regulatory mechanism of Qingye ramie under drought and adversity. Of these, the expression of MAPK-related genes in the plant hormone pathway was the most significant. The expression of three MAPK serine/threonine protein kinase genes was upregulated by 2.62- to 3.45-fold and the expression of PP2C-related genes increased by 3.34- to 14.12-fold. The expression of PYR/PYL genes decreased significantly by 2.92–7.09-fold. Furthermore, in addition to NAC, ERF, MYB, bHLH, bZIP, C2H2, GeBP, and WRKY transcription factors that have been shown to regulate drought. Some other transcription factors, such as CCL, ASD, SAU, and SPS, were also up- or downregulated in Qingye ramie. Then, the samples were analyzed by qRT-PCR and the variations were consistent with the sequencing results. Consequently, we suggest that the changes after drought stress in green-leaf ramie may be regulated by these transcription factors. Further studies can be carried out in the future, which will provide valuable and important information on the plant's drought resistance mechanism and deepen our understanding of the mechanisms of drought resistance in Qingye ramie. [ABSTRACT FROM AUTHOR]

In order to reveal the mechanisms of photosynthetic regulation of Lavandula angustifolia Mill. under low temperature stress, photosynthesis-related genes were screened and the molecular mechanism were analyzed for this species growing in Harbin, northeast of China. RNA-seq technique and photosynthetic physiology measurement were performed under 20°C, 10°C, and 0°C in this study. The results showed that the observing modified rectangular hyperbola mode could accurately reflect the light-response processes under low temperature stress and the low temperature reduced the light energy utilization of L. angustifolia. The stomatal conductance decreased with the temperature dropping, which was associated with the up-regulation of LaBAM1s, LaMPK4-1 and LaMMK2. The up-regulation of LaMPK4-1 and LaMMK2 was beneficial for ROS scavenging. The improvement of cold resistance in L. angustifolia was related to the up-regulated expression of LaFBA and LaOMTs and down-regulated expression of LaGAPAs, LaGOX, and LaTKL1s with the temperature decreasing. The up-expression of LaPSY at 10°C than it at 20°C could protect the photosynthetic organs from oxidative damage. Moreover, the photosynthetic rates at 10°C and 0°C were close to the measured values, which was related to the interactions of RCA with SBPase and Rubisco with SBPase. These findings could provide a theoretical reference for further exploring the cold tolerance mechanism of L. angustifolia, as an important aromatic plant resource, and promoting its cultivation and distribution in the northeast of China. [ABSTRACT FROM AUTHOR]

Centipedegrass (Eremochloa ophiuroides (Munro.) Hack.) is a species originating in China and is an excellent warm-season turfgrass. As a native species in southern China, it is naturally distributed in the phosphorus-deficient and aluminum-toxic acid soil areas. It is important to research the molecular mechanism of centipedegrass responses to phosphorus-deficiency and/or aluminum-toxicity stress. Quantitative Real-Time PCR (qRT-PCR) is a common method for gene expression analysis, and the accuracy of qRT-PCR results depends heavily on the stability of internal reference genes. However, there are still no reported stable and effective reference genes for qRT-PCR analysis of target genes under the acid-soil-related stresses in different organs of centipedegrass. For scientific rigor, the gene used as a reference for any plant species and/or any stress conditions should be first systematically screened and evaluated. This study is the first to provide a group of reliable reference genes to quantify the expression levels of functional genes of Eremochloa ophiuroides under multiple stresses of P deficiency and/or aluminum toxicity. In this study, centipedegrass seedlings of the acid-soil-resistant strain 'E041' and acid-soil-sensitive strain 'E089' were used for qRT-PCR analysis. A total of 11 candidate reference genes (ACT, TUB, GAPDH, TIP41, CACS, HNR, EP, EF1α, EIF4α, PP2A and actin) were detected by qRT-PCR technology, and the stability of candidate genes was evaluated with the combination of four internal stability analysis software programs. The candidate reference genes exhibited differential stability of expression in roots, stems and leaves under phosphorus-deficiency and/or aluminum-toxicity stress. On the whole, the results showed that GAPDH, TIP41 and HNR were the most stable in the total of samples. In addition, for different tissues under various stresses, the selected reference genes were also different. CACS and PP2A were identified as two stable reference genes in roots through all three stress treatments (phosphate deficiency, aluminum toxicity, and the multiple stress treatment of aluminum toxicity and phosphate deficiency). Moreover, CACS was also stable as a reference gene in roots under each treatment (phosphate deficiency, aluminum toxicity, or multiple stresses of aluminum toxicity and phosphate deficiency). In stems under all three stress treatments, GAPDH and EIF4α were the most stable reference genes; for leaves, PP2A and TIP41 showed the two highest rankings in all three stress treatments. Finally, qRT-PCR analysis of the expression patterns of the target gene ALMT1 was performed to verify the selected reference genes. The application of the reference genes identified as internal controls for qRT-PCR analysis will enable accurate analysis of the target gene expression levels and expression patterns in centipedegrass under acid-soil-related stresses. [ABSTRACT FROM AUTHOR]

Rhododendron pulchrum Sweet is a traditional ornamental plant cultivated in China and presents a great variation in petal coloration. However, few studies have been performed to reveal the genes involved and the regulatory mechanism of flower color formation in this plant. In this study, to explore the underlying genetic basis of flower color formation, transcriptome analysis was performed by high-throughput sequencing techniques on four petal samples of different colors: purple, pink, light pink, and white. Results show that a total of 35.55 to 40.56 million high-quality clean reads were obtained, of which 28.56 to 32.65 million reads were mapped to the reference genome. For their annotation, 28,273, 18,054, 24,301, 19,099, and 11,507 genes were allocated to Nr, Swiss-Prot, Pfam, GO, and KEGG databases, correspondingly. There were differentially expressed genes among the four different petal samples, including signal-transduction-related genes, anthocyanin biosynthesis genes, and transcription factors. We found that the higher expressed levels of genes associated with flavonol synthase (FLS) might be the key to white formation, and the formation of red color may be related to the higher expression of flavanone 4-reductase (DFR) families. Overall, our study provides some valuable information for exploring and understanding the flower color intensity variation in R. pulchrum. [ABSTRACT FROM AUTHOR]

Davidia involucrata Baill. (D. involucrata), a rare and endangered wild plant, is native to China and is globally recognized as an ornamental tree species. However, D. involucrata exhibits inherent biological characteristics that contribute to its low reproductive efficiency. To address this challenge, somatic embryogenesis, a biotechnological method, offers numerous advantages, including enhanced reproductive efficiency, a large reproductive coefficient, and a complete structural composition. Consequently, somatic embryogenesis holds significant value in the propagation and genetic improvement of this particular tree species. In a previous study, we utilized immature zygotic embryos of D. involucrata as explants and induced somatic embryogenesis from embryogenic callus, thereby establishing a rapid propagation and plant regeneration scheme. In this study, we utilized Illumina RNA sequencing to compare the transcriptomes of the embryogenic callus (EC) and non-embryogenic callus (NEC) of D. involucrata. The analysis revealed 131,109 unigenes assembled from EC and NEC, and 12,806 differentially expressed genes (DEGs) were identified. To verify the authenticity of the transcriptome sequencing results, qRT-PCR was performed and 16 DEGs were screened, with the stable reference gene UBQ being selected. Our analysis focused on genes related to plant growth regulators and somatic embryogenesis, such as the Aux, IAA, ARF, GH3, AHP, ARR, CYCD, BBM, WUS, GRF, SERK, and WOX gene families. We found that certain genes in these families were significantly upregulated in EC induction compared to NEC, indicating that they play crucial roles in D. involucrata cell proliferation, differentiation, and cell totipotency. These results offer new insights into the role of these gene families in EC, and may guide efforts to improve the somatic embryo induction, culture conditions, and genetic transformation efficiency of D. involucrata. [ABSTRACT FROM AUTHOR]

The fall armyworm, Spodoptera frugiperda (Noctuidae: Lepidoptera), is a wide-reaching notorious insect pest of important cereal crops, which has developed resistance to multiple classes of insecticides. It invaded the Sichuan Province of China in 2019. In this study, we performed resistance monitoring of insecticides for 11 field-collected populations from Sichuan, and all the populations were susceptible to emamectin benzoate and chlorpyrifos. The variations in resistance level to indoxacarb (resistance ratio (RR), 9.23–45.53-fold), spinetoram (RR, 4.32–18.05-fold), and chlorantraniliprole (RR, 2.02–10.39-fold) were observed among these populations. To investigate the resistance mechanism of chlorantraniliprole, synergism tests were performed and showed that piperonyl butoxide had a slight synergistic effect on chlorantraniliprole for the QJ-20 population (1.43-fold) in moderate resistance (RR, 10.39-fold) compared with the treatment group without synergist. Furthermore, the expression scanning for resistance-related genes showed that five P450 genes (CYP6AE43, CYP321A8, CYP305A1, CYP49A1, and CYP306A1) and the ryanodine receptor gene (Ryr, chlorantraniliprole target) were overexpressed in the QJ-20 population. These results indicated that the fall armyworm in Sichuan has exhibited diverse susceptibilities to several classes of insecticides, and the overexpression of Ryr and several P450 genes may contribute to the development of resistance in S. frugiperda to chlorantraniliprole. [ABSTRACT FROM AUTHOR]

Tilianin is a flavonoid glycoside with potential applications in the prevention and treatment of myocardial infarction, but its specific targets and pharmacological mechanisms are unclear. Therefore, in this study, a reverse screening and molecular docking approach was used to predict the pharmacological targets and mechanisms of tilianin on myocardial infarction based on network pharmacology and experimental validation. Firstly, we screened various databases to obtain potential core targets of tilianin. Secondly, we used protein-protein interaction, gene ontology, Kyoto Encyclopedia of Genes and Genomes, and ClueGO to correlate the key targets before molecular docking of new target genes. Finally, the gene expression of tilianin in HL-1 cells was experimentally validated with quantitative real-time polymerase chain reaction and immunofluorescence. The result showed that tilianin may significantly modulate pro-inflammatory effects in the tumor necrosis factor-a signaling pathway, which activates and regulates various cellular activities. Molecular docking revealed that protein kinase B1, proto-oncogene cellular sarcoma, tumor necrosis factor-a, matrix metalloproteinase-9, nitric oxide synthetase 2, mitogen-activated protein kinase 14 and phosphatase and tensin homolog-induced kinase 1 may be the key targets for the systematic regulatory effect of tilianin against myocardial infarction. Finally, quantitative real-time polymerase chain reaction and immunofluorescence results showed that tilianin significantly influences the expression of the key genes in myocardial infarction. Using network bioinformatic analysis, molecular docking techniques, and experimental validation, we predicted and preliminarily validated potential target sites for tilianin. These findings provide a new perspective for further exploration of the mechanism of action of tilianin in myocardial infarction therapy. Meanwhile, this study provides an objective basis for further experimental studies in the future. [ABSTRACT FROM AUTHOR]

Chinese bayberry (Myrica rubra) is an important tree in South China, with its fruit being of nutritional and high economic value. In this study, early ripening (ZJ), medium ripening (BQ) and late ripening (DK) varieties were used as test materials. Young leaves of ZJ, BQ and DK in the floral bud morphological differentiation periods were selected for transcriptome sequencing to excavate earliness related genes. A total of 4,538 differentially expressed genes were detected. Based on clustering analysis and comparisons with genes reportedly related to flowering in Arabidopsis thaliana, 25 homologous genes were identified. Of these, one gene named MrSPL4 was determined, with its expression down-regulated in DK but up-regulated in ZJ and BQ. MrSPL4 contained SBP domain and the target site of miR156, and its total and CDS length were 1,664 bp and 555 bp respectively. The overexpression vector of MrSPL4 (35S::35S::MrSPL4-pCambia2301-KY) was further constructed and successfully transfected into tobacco to obtain MrSPL4-positive plants. Based on the results of qRT-PCR, the relative expression of MrSPL4 was up regulated by 3,862.0-5,938.4 times. Additionally, the height of MrSPL4-positive plants was also significantly higher than that of wild-type (WT), with the bud stage occurring 12 days earlier. Altogether, this study identified an important gene -MrSPL4 in Chinese bayberry, which enhanced growth and flowering, which provided important theoretical basis for early-mature breeding of Chinese bayberry. [ABSTRACT FROM AUTHOR]

Watermelon is an important fruit that is widely distributed around the world. In particular, the production and consumption of watermelon in China ranks first in the world. Watermelon production is severely affected by a variety of biotic and abiotic stresses during cultivation, and polyploidization can promote stress resistance and yield. However, the morphological and physiological characteristics of tetraploid watermelon and the underlying molecular mechanisms are still poorly understood. In this study, we revealed that the leaves, fruits, and seeds of tetraploid watermelon were significantly larger than those of the diploid genotype. Some physiological characteristics, including photosynthetic rate (Pn) and stomatal conductance (Gs), were greater, whereas the intercellular CO2 concentration (Ci) and transpiration rate (Tr) were lower in tetraploid than in diploid watermelon. Two-dimensional gel electrophoresis combined with tandem mass spectrometry (MALDI-TOF/TOF) was performed to compare proteomic changes between tetraploid and diploid watermelon. A total of 21 differentially expressed proteins were identified; excluding the identical proteins, 8 proteins remained. Among them, four proteins were upregulated and four were downregulated in tetraploid versus diploid genotypes. qRT-PCR results showed inconsistencies in gene expression and protein accumulation, indicating a low correlation between gene expression and protein abundance. Generally, this study extends our understanding of the traits and molecular mechanisms of tetraploid watermelon and provides a theoretical basis for watermelon polyploid breeding. [ABSTRACT FROM AUTHOR]

Oat is a food and forage crop species widely cultivated worldwide, and it is also an important forage grass in plateau regions of China, where there is a high level of ultraviolet radiation and sunlight. Screening suitable reference genes for oat under UV-B and high-light stresses is a prerequisite for ensuring the accuracy of real-time quantitative PCR (qRT–PCR) data used in plant adaptation research. In this study, eight candidate reference genes (sulfite oxidase, SUOX; victorin binding protein, VBP; actin-encoding, Actin1; protein PSK SIMULATOR 1-like, PSKS1; TATA-binding protein 2-like, TBP2; ubiquitin-conjugating enzyme E2, UBC2; elongation factor 1-alpha, EF1-α; glyceraldehyde-3-phosphate dehydrogenase 1, GAPDH1;) were selected based on previous studies and our oat transcriptome data. The expression stability of these reference genes in oat roots, stems, and leaves under UV-B and high-light stresses was first calculated using three frequently used statistical software (geNorm, NormFinder, and BestKeeper), and then the comprehensive stability of these genes was evaluated using RefFinder. The results showed that the most stably expressed reference genes in the roots, stems, and leaves of oat under UV-B stress were EF1-α, TBP2, and PSKS1, respectively; the most stably expressed reference genes in the roots, stems, and leaves under high-light stress were PSKS1, UBC2, and PSKS1, respectively. PSKS1 was the most stably expressed reference gene in all the samples. The reliability of the selected reference genes was further validated by analysis of the expression of the phenylalanine ammonia-lyase (PAL) gene. This study highlights reference genes for accurate quantitative analysis of gene expression in different tissues of oat under UV-B and high-light stresses. [ABSTRACT FROM AUTHOR]

Soybean mosaic virus (SMV) is a worldwide and hardly controlled virus disease in soybean. Kefeng-1 is an elite variety resistant to SMV in China. In order to discover resistance genes and regulation networks in Kefeng-1, we analyzed transcriptome data of resistant (Kefeng-1) and susceptible (NN1138-2) soybean varieties in response to infection of the SMV strain SC18 at 0, 6, and 48 hours post-inoculation (hpi) and 5 days post-inoculation (dpi). Many differentially expressed genes (DEGs) were identified with Kefeng-1 and NN 1138-2. Based on the enrichment analysis for gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, we found that 48 hpi was the best time point for the defense response of the two soybean varieties in response to the SMV infection. The expression of seven candidate genes was further verified by qRT-PCR and was relatively consistent with the results of RNA-Seq. The expression of genes for Glyma.11G239000 and Glyma.18G018400, members of the ethylene-insensitive 3/ethylene-insensitive3-like (EIN3/EIL) protein family involved in ETH, were downregulated in NN1138-2 but not in Kefeng-1 and the expression of Glyma.14G041500 was upregulated in Kefeng-1 at 5 dpi. The expression of jasmonic acid repressor genes (TIFY/JAZ) was downregulated in NN1138-2 but not in Kefeng-1. NPR1 involved in the salicylic acid signaling pathway was downregulated in NN1138-2 at 48 hpi but upregulated in Kefeng-1. It shows that ethylene, jasmonic acid, and salicylic acid signaling pathways may be involved in the disease resistance process to the SMV strain SC18. Our findings would help to understand the molecular mechanism of soybean resistance to SMV. [ABSTRACT FROM AUTHOR]

Herbaceous peony (Paeonia lactiflora Pall.) is a traditional flower, famous in China, which owns highly ornamental value due to its various colors, especially double-colored species. However, the researches on color formation of double colored are rare reported. In this study, the P. lactiflora variety 'Hebao Jinlian' with red-yellow double colored petals was used as materials. The anatomical structure and flavonoid components were observed and determined. It was found that colored cells were mainly distributed in the upper and lower epidermis, six kinds of flavonoid components were identified. The total amount of anthocyanins and anthoxanthins decreased gradually with the growth of flowers, and the content in the outer petals was higher than that in the inner petals. Furthermore, by transcriptome sequence, the differential expression genes (DEGs) of outer and inner petals were enriched in flavonoid biosynthesis, phenylpropane biosynthesis and so on at four developmental stages. The accuracy of transcriptome sequence was verified by the expression pattern of DEGs detected by quantitative real-time polymerase chain reaction (qRT-PCR). Combined the analyses of flavonoid assay with transcriptome sequence, the synergistic effect of chalcone synthase (CHS), flavonoid 3-hydroxylase (F3H) and anthocyanindin synthase (ANS) may be the main reason for the formation of red outer petals, while the expression of chalcone isomerase (CHI) and flavonol synthase (FLS) were conducive to the formation of yellow inner petals. The results will lay a foundation for clarifying the color mechanism of double colored petals of P. lactiflora, and also provide a reference for the study of flower color in other ornamental plants. [ABSTRACT FROM AUTHOR]

Anthracnose, a destructive disease in strawberry worldwide, is caused by the pathogenic fungus Colletotrichum ssp.. To date, numerous studies focusing on its identification, pathogenicity and biological characteristics have been published. However, research on quantifying the colonization of this pathogen is still lacking, which is crucial to the study of anthracnose resistance. In this study, we developed a sensitive primer pair targeting the cutinase gene that is capable of distinguishing Colletotrichum gloeosporioides (C. gloeosporioides) species complex, the main species causing strawberry anthracnose in China, including C. fructicola, C. gloeosporioides, C. aenigma and C. siamense, from other Colletotrichum species and typical fungal pathogens. On this basis, we established a quantitative real‐time PCR (qRT‐PCR) assay to accurately quantify pathogen amounts from both pure cultures and infected strawberries, with detection limits of 105–101 copies. Using this method, we characterized quantitative traits of C. fructicola colonization in strawberry leaves and discovered differences in pathogen populations at different positions. Moreover, monitoring of pathogen growth during infection showed a dynamic change in the amount of C. fructicola. Finally, a comparison of the absolute and relative quantification results further proved this method's validity. Therefore, we propose that this specific PCR method enables rapid detection and quantification of C. gloeosporioides species complex colonization in strawberry and will be a useful tool for anthracnose management and strawberry resistance studies. [ABSTRACT FROM AUTHOR]

The coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), is a major destructive pest of Coffea arabica L. (Gentianales: Rubiaceae), widely planted in many Asian countries, including China. Quantitative real-time polymerase chain reaction (qRT-PCR) is a common method for quantitative analysis of gene transcription levels. To obtain accurate and reliable qRT-PCR results, it is necessary to select suitable reference genes to different experimental conditions for normalizing the target gene expression. However, the stability of the expression of reference genes in X. quadripes has rarely been studied. In this study, the expression stability of nine candidate reference genes were investigated under biotic and abiotic conditions for use in qRT-PCR's normalization. By integrating the results of four algorithms of NormFinder, BestKeeper, geNorm, and RefFinder, the optimal reference gene combinations in different experimental conditions were performed as follows: RPL10a and EIF3D were the optimal reference genes for developmental stage samples, EIF4E, RPL10a, and RPS27a for tissue samples, V-ATP and EF1α for the sex samples, EIF3D and V-ATP for temperature treatment, RPS27a and RPL10a for insecticide stress, and RPL10a, RPS27a, and EF1α for all the samples. This study will help to obtain the stable internal reference genes under biotic and abiotic conditions and lay the foundation for in-depth functional research of target genes or genomics on olfactory molecular mechanisms, temperature adaptability, and insecticide resistance in X. quadripes. [ABSTRACT FROM AUTHOR]

Salsola ferganica is a natural desert herbaceous plant in the arid area of western and northwestern China. Because of its salt tolerance and drought resistance, it is of great significance in desert afforestation and sand-fixing capacity. There has been much research on the genes involved in plants under desert stresses in recent years. The application of the best internal reference genes for standardization was a critical procedure in analyzing the gene expression under different types. Even so, the reference gene has not been reported in the application of gene expression normalization of S. ferganica. In this study, nine reference genes (TUA-1726, TUA-1760, TUB, GAPDH, ACT, 50S, HSC70, APT, and U-box) in S. ferganica were adopted and analyzed under six different treatments (ABA, heat, cold, NaCl, methyl viologen (MV), and PEG). The applicability of candidate genes was evaluated by statistical software, including geNorm, NormFinder, BestKeeper, and RefFinder, based on their stability values in all the treatments. These results indicated that the simultaneous selection of two stable reference genes would fully standardize the optimization of the normalization research. To verify the feasibility of the above internal reference genes, the CT values of AP2/ERF transcription factor family genes were standardized using the most (ACT) and least (GAPDH) stable reference genes in S. ferganica seedlings under six abiotic stresses. The research showed that HSC70 and U-box were the most appropriate reference genes in ABA stressed samples, and ACT and U-box genes were the optimal references for heat-stressed samples. TUA-1726 and U-box showed the smallest value in gene expression levels of cold treatment. The internal reference groups of the best applicability for the other samples were U-box and ACT under NaCl treatment, ACT and TUA-1726 under MV stress, HSC70 and TUB under PEG treatment, and ACT in all samples. ACT and U-box showed higher stability than the other genes based on the comprehensive stability ranking of RefFinder, as determined by the geometric mean in this study. These results will contribute to later gene expression studies in other closely related species and provide an important foundation for gene expression analysis in S. ferganica. [ABSTRACT FROM AUTHOR]

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Blueberries are particularly rich in anthocyanins and are favored extensively for their health benefits. In this study, the anthocyanin profiles of fruits of four blueberry cultivars (Gardenblue, Legacy, Misty, and Brightwell) were investigated. Total anthocyanin content gradually increased in all four cultivars during development, but mature fruits of Brightwell and Gardenblue had a higher anthocyanin content than those of Legacy and Misty. Thirty-two kinds of anthocyanins were quantified using UPLC-ESI-MS/MS. Principal component analysis showed that the anthocyanin profiles of Gardenblue and Legacy were similar to those of Misty and Brightwell, respectively. Levels of malvidin 3-O-galactoside, malvidin 3-O-glucoside, malvidin 3-O-rutinoside, delphinidin 3-O-glucoside, delphinidin 3,5-O-diglucoside(delphin), petunidin 3-O-glucoside, and petunidin 3-O-rutinoside in mature fruits of Gardenblue were higher than those in the other three cultivars. The content of delphinidin 3-O-(6"-O-malonyl)-beta-D-glucoside, petunidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-arabinoside, delphinidin 3-O-arabinoside, and delphinidin 3-O-galactoside was the highest in mature fruits of Misty. Levels of petunidin 3,5-diglucoside and petunidin 3-O-galactoside in mature fruits of Brightwell were higher than those in the other three cultivars. Quantitative real-time reverse transcription-polymerase chain reaction analysis showed that ANS, F3ʹ5'H, and UFGT were preferentially expressed in fruits of Gardenblue, possibly attributing to higher levels of delphinidin 3-O-glucoside and delphinidin 3,5-O-diglucoside (delphin) in this cultivar. This study adds to our knowledge of anthocyanin content and profiles in the four important blueberry cultivars grown in China. [ABSTRACT FROM AUTHOR]

Red pitaya (Hylocereus polyrhizus) is widely cultivated in southern and southwestern China. To provide a basis for studying the molecular mechanisms of the ripening of this fruit, we carried out RNA sequencing (RNA‐seq) analysis to identify differentially and stably expressed unigenes. The latter may serve as a resource of potential reference genes for normalization of target gene expression determined using quantitative real‐time PCR (qRT‐PCR). We selected 11 candidate reference genes from our RNA‐seq analysis of red pitaya fruit ripening (ACT7, EF‐1α, IF‐4α, PTBP, PP2A, EF2, Hsp70, GAPDH, DNAJ, TUB and CYP), as well as β‐ACT, which has been used as a reference gene for pitayas in previous studies. We then comprehensively evaluated their expression stability during fruit ripening using four statistical methods (GeNorm, NormFinder, BestKeeper and DeltaCt) and merged the four outputs using the online tool RefFinder for the final ranking. We report that PTBP and DNAJ showed the most stable expression patterns, whereas CYP and ACT7 showed the least stable expression patterns. The relative gene expression of red pitaya sucrose synthase and 4, 5‐dihydroxyphenylalanine‐extradiol‐dioxygenase as determined by quantitative real‐time PCR and normalized to PTBP and DNAJ was consistent with the RNA‐seq results, suggesting that PTBP and DNAJ are suitable reference genes for studies of red pitaya fruit ripening. [ABSTRACT FROM AUTHOR]

Long winters led to a one‐crop‐a‐year cultivation system until the winter wheat Dongnongdongmai 1 (Dn1) was successfully cultivated in northeast China. This crop variety is resistant to extremely low temperatures (−35 °C).To better understand the adaptability of winter wheat Dn1 to low temperatures, gas chromatography time‐of‐flight mass spectrometry (GC‐TOF/MS) and metabolomics analysis was conducted on the tillering nodes of winter wheat during the overwintering period. Enzyme‐regulating genes of the metabolic products were also quantitatively analysed.The metabolomic results for the tillering nodes in the overwintering period showed that disaccharides had a strong protective effect on winter wheat Dn1. Amino acid metabolism (i.e. proline, alanine and GABA) changed significantly throughout the whole wintering process, whereas organic fatty acid metabolism changed significantly only in the late stage of overwintering. This result indicates that the metabolites used by winter wheat Dn1 differ in different overwintering stages.The relationship between field temperature and metabolite changes in winter wheat Dn1 during overwintering periods is discussed, and disaccharides were identified as the osmotic stress regulators for winter wheat Dn1 during the overwintering process, as well as maintenance of the carbon and nitrogen balance by monosaccharides, amino acids and lipids for cold resistance. [ABSTRACT FROM AUTHOR]

Prunus triloba, a well-known ornamental plant, is native to northern China. There are three kinds of petals of P. triloba: simple flower, semi-double flower and double flower. At present, the specific molecular mechanisms of floral development in P. triloba is not clear. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has been widely used to measure gene expression level to further explore gene function, while suitable reference genes must maintain stable expression under different conditions to obtain accurate data. In order to select appropriate reference genes for three cultivars of P. triloba with different petal types, 9 candidate reference genes were evaluated, namely Pt18sRNA, PtTEF2, PtACTIN, PtGAPDH, PtEF 1-α1, PtEF 1-α2, PtEF 1-α3, PtRPL13 and PtRPII. The geNorm and NormFinder algorithms were used to analyse the data from three kinds of flower buds and organs of different petal-type cultivars (P. triloba 'Dahuazi', P. triloba 'Fuban Tiaozhi' and P. triloba 'Hanyan'). Our results show that the suitable internal reference genes are different among cultivars: PtRPL13 and PtRPII present greater stability in P. triloba 'Dahuazi' and P. triloba 'Fuban Tiaozhi', whereas PtGAPDH is more stable in the study of gene expression of P. triloba 'Hanyan' organs by qRT-PCR. [ABSTRACT FROM AUTHOR]

Syringa pinnatifolia Hemsl. (Oleaceae) is a species of shrub with a limited distribution in China. Several chemical compounds with pharmacological effects have been isolated from S. pinnatifolia, including new lignans and sesquiterpenes. Studies of gene expression in this species require the identification of suitable reference genes that are stably expressed under different conditions and in different tissues. To identify candidate reference genes, here we used the geNorm, NormFinder, and BestKeeper algorithms to analyze the stability of 12 candidate genes. The geometric mean of the rankings generated with these algorithms was used to obtain a comprehensive ranking. TBP and PP2A were found to be appropriate reference genes for calli treated with different external stimuli, and TIP41 and TBP were found to be appropriate reference genes in differentiated tissues. When calli and differentiated tissues were considered together, TBP and TIP41 were found to be the most reliable reference genes. The selected genes were validated by analysis of HMGR expression in calli and differentiated tissues. This study is the first to screen candidate reference genes in the genus Syringa and could help guide future molecular studies in this genus. [ABSTRACT FROM AUTHOR]

Background: Paeonia lactiflora 'Hangshao' is widely cultivated in China as a traditional Chinese medicine 'Radix Paeoniae Alba'. Due to the abundant unsaturated fatty acids in its seed, it can also be regarded as a new oilseed plant. However, the process of the biosynthesis of unsaturated fatty acids in it has remained unknown. Therefore, transcriptome analysis is helpful to better understand the underlying molecular mechanisms. Results: Five main fatty acids were detected, including stearic acid, palmitic acid, oleic acid, linoleic acid and α-linolenic acid, and their absolute contents first increased and then decreased during seed development. A total of 150,156 unigenes were obtained by transcriptome sequencing. There were 15,005 unigenes annotated in the seven functional databases, including NR, NT, GO, KOG, KEGG, Swiss-Prot and InterPro. Based on the KEGG database, 1766 unigenes were annotated in the lipid metabolism. There were 4635, 12,304, and 18,291 DEGs in Group I (60 vs 30 DAF), Group II (90 vs 60 DAF) and Group III (90 vs 30 DAF), respectively. A total of 1480 DEGs were detected in the intersection of the three groups. In 14 KEGG pathways of lipid metabolism, 503 DEGs were found, belonging to 111 enzymes. We screened out 123 DEGs involved in fatty acid biosynthesis (39 DEGs), fatty acid elongation (33 DEGs), biosynthesis of unsaturated fatty acid (24 DEGs), TAG assembly (17 DEGs) and lipid storage (10 DEGs). Furthermore, qRT-PCR was used to analyze the expression patterns of 16 genes, including BBCP, BC, MCAT, KASIII, KASII, FATA, FATB, KCR, SAD, FAD2, FAD3, FAD7, GPAT, DGAT, OLE and CLO, most of which showed the highest expression at 45 DAF, except for DGAT, OLE and CLO, which showed the highest expression at 75 DAF. Conclusions: We predicted that MCAT, KASIII, FATA, SAD, FAD2, FAD3, DGAT and OLE were the key genes in the unsaturated fatty acid biosynthesis and oil accumulation in herbaceous peony seed. This study provides the first comprehensive genomic resources characterizing herbaceous peony seed gene expression at the transcriptional level. These data lay the foundation for elucidating the molecular mechanisms of fatty acid biosynthesis and oil accumulation for herbaceous peony. [ABSTRACT FROM AUTHOR]

Background. Quantitativereal-time reverse transcriptase polymerase chain reaction is the common method to quantify relative gene expression. Normalizating using reliable genes is critical in correctly interpreting expression data from qRT-PCR. Euscaphis konishii is a medicinal plant with a long history in China, which has various chemical compounds in fruit. However, there is no report describing the selection of reference genes in fruit development of Euscaphis konishii. Methods. We selected eight candidate reference genes based on RNA-seq database analysis, and ranked expression stability using statistical algorithms GeNorm, NormFinder, BestKeeper and ReFinder. Finally, The nine genes related to the anthocyanin synthesis pathway of Euscaphis konishii were used to verify the suitability of reference gene. Results. The results showed that the stability of EkUBC23, EkCYP38 and EkGAPDH2 was better, and the low expression reference genes (EkUBC23 and EkCYP38) were favourable for quantifying low expression target genes, while the high expression reference gene (EkGAPDH2) was beneficial for quantifying high expression genes. In this study, we present the suitable reference genes for fruit development of Euscaphis konishii based on transcriptome data, our study will contribute to further studies in molecular biology and gene function on Euscaphis konishii and other closely related species. [ABSTRACT FROM AUTHOR]

There has been an increasing incidence rate of rice false smut in global rice cultivation areas. However, there is a dearth of studies on the environmental concentrations and hazards of ustiloxin A (UA), which is the major mycotoxin produced by a pathogenic fungus of the rice false smut. Here, the concentrations of UA in the surface waters of two paddy fields located in Enshi city, Hubei province, China, were measured, and its toxicity in T. Thermophila was evaluated. This is the first study to detect UA in the surface waters of the two paddy fields, and the measured mean concentrations were 2.82 and 0.26 μg/L, respectively. Exposure to 2.19, 19.01 or 187.13 μg/L UA for 5 days significantly reduced the theoretical population and cell size of T. thermophila. Furthermore, treatment with 187.13 μg/L UA changed the percentages of T. thermophila cells in different cell-cycle stages, and with an increased malformation rate compared with the control, suggesting the disruption of the cell cycle. The expressions of 30 genes involved in the enriched proteasome pathway, 7 cyclin genes (cyc9 , cyc10 , cyc16 , cyc22 , cyc23, cyc26, cyc33) and 2 histone genes (mlh1 and hho1) were significantly down-regulated, which might be the modes of action responsible for the disruption of cell cycling due to UA exposure. Image 1 • For the first time, ustiloxin A was detected in the surface water of paddy fields. • Exposure to ustiloxin A reduced the theoretical population of T. Thermophila and caused cell malformation. • Cell cycle was disrupted by ustiloxin A. • Expressions of genes related to cell cycling were changed due to ustiloxin A exposure. For the first time, it was founded that ustiloxin A (UA) was detected in surface waters of paddy fields, and exposure to UA in T. Thermophila caused toxicity by disrupting cell cycle. [ABSTRACT FROM AUTHOR]

The ability to survive annual temperature minima could be a key determinant of distribution limits for insects under global climate change. Recent studies have suggested that insect aquaporins are indispensable for cellular water management under conditions that lead to dehydration and cold stress. Aquaporins are integral membrane water channel proteins in the major intrinsic protein superfamily and promote selected solutes and the movement of water across biological membranes. We cloned and characterized nine full-length aquaporins from Dendroctonus armandi (DaAqps), the most destructive forest pest in the Qinling Mountains of Shaanxi Province, China. Eight of the DaAqps belong to three classical aquaporin grades, including the Drosophila integral protein, the Pyrocoelia rufa integral protein, the entomoglyceroporins and one that belongs to the unorthodox grade of aquaporin 12-like channels. The DaAqps were increasingly expressed during different developmental stages and in different larval tissues, and expression peaked in mid-winter. They were tested under cold conditions for different lengths of time, and the expression of almost all DaAqps was down regulated with decreasing temperatures and long-term exposure to cold conditions. However, when the lowest temperatures were reached, the levels were immediately upregulated. These genes indicate that cold tolerance can improve through mortality responses at low temperatures after RNA interference of DaAqps. In our study, we analyzed the molecular response, expression patterns, and RNA interference of DaAqps and clarified the crucial role of protective compounds (aquaporins) underlying D. armandi cold tolerance and provide a new pest control method. [ABSTRACT FROM AUTHOR]

Grass carp shares the largest portion of the aquaculture production in China, but hemorrhagic disease caused by grass carp reovirus (GCRV) often results in tremendous loss of fingerlings and yearlings, causing significant economic damages. However, it is difficult to study antiviral mechanisms in grass carp in vivo due to its large size and long reproductive cycle. Therefore, a small cyprinid species named rare minnow with high sensitivity to GCRV, is regarded as a useful model to study the mechanisms of this disease. In this study, rare minnows were infected with the type-IIGCRV (GCRV-HZ08), and pathogenesis was investigated by BGISEQ-500 transcriptome sequencing of four cDNA libraries, hepatopancreas, gills, head-kidney and spleen, and real time quantitative PCR (qRT-PCR). We obtained 51.22 Gb bases in total, and de novo assembled 107,756 unigenes with an average length of 1,441 bp. GO analysis revealed that the differentially expressed genes (DEGs) involved in the defense mechanisms were the most enriched GO terms in all four tissues. KEGG analysis revealed that the most enriched pathways were "Influenza A", "Herpes simplex infection", "Transcriptional misregulation in cancer" and "Metabolic" pathways. We also performed a comparative transcriptomic study between GCRV-infected rare minnow and grass carp data. This revealed that "IL-17 signaling pathway", "NF-kappa B signaling pathway" and "Influenza A" pathways are conserved (important) in the regulation of anti-GCRV infection in both species, and need to be further investigated. Furthermore, a total of four immune-related DEGs were selected for qRT-PCR validation, and the results confirmed the RNA-seq data. These results enhance our understanding of the antiviral responses of cyprinid fish infected by GCRV. • We analyzed the transcriptome of rare minnow after viral challenge with type-II GCRV. • Influenza A pathway plays a critical role in defense mechanism against GCRV in fish. • Rare minnow is a suitable model to study the anti-GCRV immune responses in vivo. [ABSTRACT FROM AUTHOR]

Abstract Carex rigescens is an ornamental turfgrass in northern China which has a relatively low maintenance cost and robust tolerance to many adverse environmental conditions, so it could be considered a new material for researching into plant stress resistance. However, suitable reference genes are vacant for obtaining reliable results in quantitative real-time PCR (qRT-PCR) analysis of C. rigescens in adversity research. In this study, we tested the expression stability of nine potential reference genes in leaves and roots under five different abiotic stress conditions, including cold, salt, heat, osmotic and cadmium (Cd). We then selected the best reference genes according to the analysis results calculated by three algorithmic programs (geNorm, NormFinder and BestKeeper) and used the RankAggreg package to merge the outputted data. The results showed that combinations of at least two reference genes should be used for reliable normalization except in heat-treated root samples, which require three reference genes. eIF-4α , GADPH , SAND and PEPKR1 and their combination were found to be the most stably expressed reference genes, while SAM , TUA-α and UPL7 were the three least stable reference genes among most of experimental samples. In addition, five stress-induced genes (Cu-Zn SOD , P5CS , LEA , GST , and APX) were chosen to verify the stability of the selected reference genes in various tissues and under various stress conditions. The results of this study will provide an important fundamental basis both for gene expression verification for transcriptomic and proteomic analyses and for gene expression analysis for future gene function research in C. rigescens. Highlights • At least two reference genes should be used in qRT-PCR analysis in all experimental conditions. • eIF-4α , GADPH , SAND , PEPKR1 and their combination performed superiorly in this study. • SAM , TUA-α and UPL7 were the least stable reference genes in all experimental samples. • Cu-Zn SOD , P5CS , LEA , GST , and APX were chosen to verify reference genes stability. [ABSTRACT FROM AUTHOR]

Background: Stellera chamaejasme Linn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion of S. chamaejasme has greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs) as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization in S. chamaejasme has been reported. Method: In this study, 10 candidate RGs namely, 18S, 60S, CYP, GAPCP1, GAPDH2, EF1B, MDH, SAND, TUA1, and TUA6, were singled out from the transcriptome database of S. chamaejasme, and their expression stability under three abiotic stresses (drought, cold, and salt) and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH) were estimated with the programs geNorm, NormFinder, and BestKeeper. Result: Our results showed that GAPCP1 and EF1B were the best combination for the three abiotic stresses, whereas TUA6 and SAND, TUA1 and CYP, GAPDH2 and 60S were the best choices for ABA, GA, and ETH treatment, respectively. Moreover, GAPCP1 and 60S were assessed to be the best combination for all samples, and 18S was the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes (P5CS2 and GI) further verified that the RGs that we selected were suitable for gene expression normalization. Result: Our results showed that GAPCP1 and EF1B were the best combination for the three abiotic stresses, whereas TUA6 and SAND, TUA1 and CYP, GAPDH2 and 60S were the best choices for ABA, GA, and ETH treatment, respectively. Moreover, GAPCP1 and 60S were assessed to be the best combination for all samples, and 18S was the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes (P5CS2 and GI) further verified that the RGs that we selected were suitable for gene expression normalization. [ABSTRACT FROM AUTHOR]

Grass carp ( Ctenopharyngodon idellus ) is an economical aquaculture species in China, and the Grass Carp Reovirus (GCRV) that causes hemorrhagic disease seriously affects the grass carp cultivation industry. Substantial evidence indicates that there is an association between the membrane-associated RING-CH family of E3 ligase (MARCH) family and immune defense in mammals, while functional studies on non-mammalian MARCH proteins are limited. In order to know the characteristics of the MARCH genes in C. idellus , eight MARCH genes ( MARCH1, 2, 5, 6, 7, 8, 9 and 11 ) were cloned and the open reading frames (ORF) were identified in grass carp. All MARCH proteins in grass carp contained an RING-CH domain, which is characteristic of the MARCH protein. The phylogenetic analysis revealed that different MARCH proteins gathered into their separate clusters. All eight members of the MARCH gene family were detected in all tissues sampled, but the relative expression level differed. In addition, the mRNA expression of all the MARCHs was regulated at different levels in the immune organs after a GCRV challenge, and they responded robustly in both the intestine and liver. The mRNA expression of MARCH8, MHC II, TfR, IL1RAP, EGR1, and DUSP1 in the intestine after GCRV infection was analyzed, and the results showed that MARCH8 could negatively regulate TfR, IL1RAP, EGR1, and DUSP1, which signaled via the MAPK or NF-κB-activation pathways that play vital roles in immunity. Our findings identified a novel gene family in C. idellus and provided novel evidence that MARCH genes are inducible and involved in the immune response. Moreover, MARCH8 might function to negatively regulate immune receptors in C. idellus . Therefore, the MARCH might play a vital role in regulating the immune response of C. idellus . [ABSTRACT FROM AUTHOR]

Gentiana straminea is a popular Tibetan medicine that has been used for thousands of years in China to treat various diseases and conditions. Although it has multiple pharmaceutical purposes and important economic plant resource in China, transcriptome and molecular base still known limited. In flowering season, samples were collected from different tissues, using the NGS Illumina. Solexa platform, about 58.85 million sequencing reads were generated and assembled de novo, yielding 78,764 high quality unigenes with an average length of 1090 bp. Gene Ontology (GO), KEGG pathway mapping showed that 49,033 of these were identified as putative homologs of annotated sequences in the protein databases. Among them, candidate genes associated with iridoid, flavonoid and anthocyanin were identified. Further the key enzymes involved to iridoid and flavonoid synthesis pathway were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) on different tissues, the flower and root had the higher expression than leaves. In addition, 7591 SSR markers were identified from the unigenes of the G . straminea transcriptome. The foundation of G . straminea provided the important resource for facilitating to study molecular and functional genomics of it and related this species on the Qinghai–Tibet Plateau. [ABSTRACT FROM AUTHOR]

Aim: To explore the pattern of expression of circulating miRNAs in patients with polycystic ovary syndrome (PCOS). Materials and methods: Microarray and qRT-PCR were used to investigate circulating miRNAs in PCOS during clinical diagnosis. The targets of dys-regulated miRNAs were predicted using bioinformatics, followed by function and pathway analysis using the databases of Gene Ontology and the KEGG pathway. Results: BMI, triglyceride, HOMA-IR, Testosterone and CRP levels were significantly higher, while estradiol was significantly lower in PCOS than in control groups. After SAM analysis, 5 circulating miRNAs were significantly up-regulated (let-7i-3pm, miR-5706, miR-4463, miR-3665, miR-638) and 4 (miR-124-3p, miR-128, miR-29a-3p, let-7c) were down-regulated in PCOS patients. Hierarchical clustering showed a general distinction between PCOS and control samples in a heat map. After joint prediction by different statistical methods, 34 and 41 genes targeted were up-and down-regulated miRNAs, in PCOS and controls, respectively. Further, GO and KEGG analyses revealed the involvement of the immune system, ATP binding, MAPK signaling, apoptosis, angiogenesis, response to reactive oxygen species and p53 signaling pathways in PCOS. Conclusions: We report a novel non-invasive miRNA profile which distinguishes PCOS patients from healthy controls. The miRNA-target database may provide a novel understanding of PCOS and potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Powdery mildew (PM), caused by fungus Erysiphe necator, is one of the most devastating diseases of grapevine. To better understand grapevine-PM interaction and provide candidate resources for grapevine breeding, a suppression subtractive hybridization (SSH) cDNA library was constructed from E. necator-infected leaves of a resistant Chinese wild Vitis quinquangularis clone "Shang-24". A total of 492 high quality expressed sequence tags (ESTs) were obtained and assembled into 266 unigenes. Gene ontology (GO) analysis indicated that 188 unigenes could be assigned with at least one GO term in the biological process category, and 176 in the molecular function category. Sequence analysis showed that a large number of these genes were homologous to those involved in defense responses. Genes involved in metabolism, photosynthesis, transport and signal transduction were also enriched in the library. Expression analysis of 13 selected genes by qRT-PCR revealed that most were induced more quickly and intensely in the resistant material "Shang-24" than in the sensitive V. pseudoreticulata clone "Hunan-1" by E. necator infection. The ESTs reported here provide new clues to understand the disease-resistance mechanism in Chinese wild grapevine species and may enable us to investigate E. necator-responsive genes involved in PM resistance in grapevine germplasm. [ABSTRACT FROM AUTHOR]

Simple Summary: Reverse transcription-quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most accessible and widely-applied method for quantifying gene transcription. Stable reference genes need to be normalized to facilitate gene expression studies and obtain more accurate qRT-PCR data. In this research, eight reference genes from different functional categories and gene families were used to assess the stability level of gene expression under different conditions in H. cunea. Finally, the expression pattern of the target gene serine proteases (HcSP1) was evaluated under different conditions to verify the precision and reliability of the results. This study provides a comprehensive list of suitable reference genes for analyses and optional reference genes for other gene function research in H. cunea. The relative quantification of gene expression is mainly achieved through reverse transcription-quantitative PCR (qRT-PCR); however, its reliability and precision rely on proper data normalization using one or more optimal reference genes. Hyphantria cunea (Drury) has been an invasive pest of forest trees, ornamental plants, and fruit trees in China for many years. Currently, the molecular physiological role of reference genes in H. cunea is unclear, which hinders functional gene study. Therefore, eight common reference genes, RPS26, RPL13, UBI, AK, RPS15, EIF4A, β-actin, α-tub, were selected to evaluate levels of gene expression stability when subjected to varied experimental conditions, including developmental stage and gender, different tissues, larvae reared on different hosts and different larval density. The geNorm, BestKeeper, ΔCt method, and NormFinder statistical algorithms were used to normalize gene transcription data. Furthermore, the stability/suitability of these candidates was ranked overall by RefFinder. This study provides a comprehensive evaluation of reference genes in H. cunea and could help select reference genes for other Lepidoptera species. [ABSTRACT FROM AUTHOR]

Flavonoids play a key role as a secondary antioxidant defense system against different biotic and abiotic stresses, and also act as coloring compounds in various fruiting plants. In this study, fruit samples of purple (Passiflora edulis f. edulis) and yellow (Passiflora edulis f. flavicarpa) passion fruit were collected at five developmental stages (i.e., fruitlet, green, veraison, maturation, and ripening stage) from an orchard located at Nanping, Fujian, China. The contents of flavonoid, anthocyanin, proanthocyanin, and their metabolites were determined using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), activities of key enzymes involved in flavonoid metabolism were measured, and expression profiling of related genes was done using quantitative real-time PCR (qRT-PCR). The results revealed that total flavonoids, anthocyanins, and procyanidins were found to be increased in the fruit peel of both cultivars with fruit maturity. Total flavonoids, anthocyanins, procyanidins, flavonoid metabolites (i.e., rutin, luteolin, and quercetin), and anthocyanin metabolites (i.e., cyanidin-3-O-glucoside chloride, peonidin-3-O-glucoside, and pelargonidin-3-O-glucoside) were found abundant in the peel of purple passion fruit, as compared to yellow passion fruit. Principle component analysis showed that the enzymes, i.e., C4H, 4CL, UFGT, and GST were maybe involved in the regulation of flavonoids metabolism in the peel of passion fruit cultivars. Meanwhile, PePAL4, Pe4CL2,3, PeCHS2, and PeGST7 may play an important role in flavonoid metabolism in fruit peel of the passion fruit. This study provides new insights for future elucidation of key mechanisms regulating flavonoids biosynthesis in passion fruit. [ABSTRACT FROM AUTHOR]

• Long-term ENF decreased the abundance of soil diazotrophs. • Long-term ENF decreased species richness and diversity of soil diazotrophs. • Soil pH and EC values were the main factors affecting soil diazotrophs. • The maize rhizosphere could enrich soil diazotrophs. • The diazotrophic bacterial variations under ENF might inhibit early maize growth. Excessive nitrogen (N) fertilization (ENF) and low utilization efficiency of fertilizer-derived N in high-input, high-yield cropping systems are serious ecological and economic problems in China. To examine the effects of long-term ENF on soil diazotrophs in relation to maize growth, we performed a 5-year field trial whereby 0–300 kg N ha−1 were added to black soil under a maize monocropping system in Northeast China. At the end of the 5-year field trial, the abundance and community structure of diazotrophs in the bulk soil and rhizosphere were investigated at the maize jointing stage by real-time quantitative polymerase chain reaction (qRT-PCR) and high-throughput sequencing. The results showed that: 1) ENF (N300) limited maize growth in the early stages and did not increase grain yield compared with moderate N supplementation (N180); 2) ENF significantly reduced nifH copy number, species richness, and Shannon index values of soil diazotrophs by 53.7%–79.7%, 37.2%–47.6%, and 20.0%–31.6%, respectively, reduced the relative abundance of Burkholderia , and increased the relative abundance of Sphingobium ; 3) ENF decreased soil pH and increased electrical conductivity, which were the main factors affecting soil diazotrophic community structure as determined by Mantel test; and 4) diazotrophs were 1.3–3.0 times more abundant in the rhizosphere than in bulk soil, but no differences in α -diversity and community composition of diazotrophs were observed between rhizosphere and bulk soil. Thus, long-term ENF has negative impacts on the soil diazotrophic community, inhibiting maize growth in the early stage. Split N fertilization or slow/controlled-released N fertilizer should be used to avoid the negative effects of ENF on the soil diazotrophic community structure and early maize growth. [ABSTRACT FROM AUTHOR]

Organic acids are key components that determine the taste and flavor of fruits and play a vital role in maintaining fruit quality and nutritive value. In this study, the fruits of two cultivars of passion fruit Yellow (Passiflora edulis f. flavicarpa) and purple (Passiflora edulis f. edulis) were harvested at five different developmental stages (i.e., fruitlet, green, veraison, near-mature and mature stage) from an orchard located in subtropical region of Fujian Province, China. The contents of six organic acids were quantified using ultra-performance liquid chromatography (UPLC), activities of citric acid related enzymes were determined, and expression levels of genes involved in citric acid metabolism were measured by quantitative real-time PCR (qRT-PCR). The results revealed that citric acid was the predominant organic acid in both cultivars during fruit development. The highest citric acid contents were observed in both cultivars at green stage, which were reduced with fruit maturity. Correlation analysis showed that citrate synthase (CS), cytosolic aconitase (Cyt-ACO) and cytosolic isocitrate dehydrogenase (Cyt-IDH) may be involved in regulating citric acid biosynthesis. Meanwhile, the PeCS2, PeACO4, PeACO5 and PeIDH1 genes may play an important role in regulating the accumulation of citric acid. This study provides new insights for future elucidation of key mechanisms regulating organic acid biosynthesis in passion fruit. [ABSTRACT FROM AUTHOR]

Cryptomeria fortunei has become one of the main timber afforestation species in subtropical high-altitude areas of China due to its fast growth, good material quality, and strong adaptability, showing broad application prospects. Quantitative real-time PCR (qRT-PCR) is the most accurate and widely used gene expression evaluation technique, and selecting appropriate reference genes (RGs) is essential for normalizing qRT-PCR results. However, suitable RGs for gene expression normalization in C. fortunei have not been reported. Here, we tested the expression stability for 12 RGs in C. fortunei under various experimental conditions (simulated abiotic stresses (cold, heat, drought, and salinity) and hormone treatments (methyl jasmonate, abscisic acid, salicylic acid, and gibberellin) and in different tissues (stems, tender needles, needles, cones, and seeds) using four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper). Then, geometric mean rankings from these algorithms and the RefFinder program were used to comprehensively evaluate RG stability. The results indicated CYP, actin, UBC, and 18S as good choices for studying C. fortunei gene expression. qRT-PCR analysis of the expression patterns of three target genes (CAT and MAPK1/6) further verified that the selected RGs were suitable for gene expression normalization. This study provides an important basis for C. fortunei gene expression standardization and quantification. [ABSTRACT FROM AUTHOR]

Chilo suppressalis Walker (Lepidoptera: Crambidae) is a widely destructive pest occurring in rice, particularly in the rice-growing regions of Asia. In recent years, C. suppressalis has developed resistance to several insecticides because of the extensive use of insecticides. The resistance levels to four insecticides were determined among populations from different regions of Sichuan Province, China, using a drop-method bioassay. Based on LC 50 values of a laboratory susceptible strain, all field populations showed moderate level of resistance to triazophos (23.9- to 83.5-fold) and were either susceptible or had a low level of resistance to abamectin (2.1- to 5.8-fold). All field-collected populations had a low or moderate level of resistance to chlorpyrifos (1.7- to 47.1-fold) and monosultap (2.7- to 13.5-fold). The synergism experiment indicated that the resistance of the XW19 to triazophos may be associated with cytochrome P450 monooxygenases (P450s), with the highest synergistic ratio (SR) of 3.05-fold and increased ratio (IR) of 2.28-fold for piperonylbutoxide (PBO). The P450 activity of the TJ19 population was the greatest among the six field populations. Moreover, the relative expression levels of four resistance-related P450 genes were detected with qRT-PCR, and the results indicated that CYP324A12 , CYP321F3 and CYP9A68 were overexpressed in the resistant population, especially in the XW19 population (by 1.2-, 3.4 -, and 18.0-fold, respectively). In addition, the relative expression levels of CYP9A68 among the CZ19 and TJ19 populations were also enhanced 10.5- and 24.9-fold, respectively. These results suggested that CYP324A12 , CYP321F3 and CYP9A68 may be related to the resistance development of C. suppressalis to triazophos. Unlabelled Image • All population s of C. suppressalis were susceptible to moderate levels of resistance. • Increased P450s activity could contribute to the metabolic detoxification of C. suppressalis to triazophos. • The overexpression of three P450 genes could result in the development of resistance of C. suppressalis to triazophos. [ABSTRACT FROM AUTHOR]

Ginkgo biloba, a deciduous tree species in the Ginkgo family, has a long history of cultivation in China and is widely used in garden landscapes, medicine, food, and health products. However, few reports have focused on the systematic selection of optimal reference genes based on transcriptomic data in G. biloba. The purpose of our research was to select an internal reference gene suitable for different experimental conditions from thirteen candidate reference genes by the delta cycle threshold (ΔCt) method, geNorm, BestKeeper, NormFinder, and RefFinder programs. The reference genes were used for gene expression analyses of Ginkgo biloba. These results showed that elongation factor 1(EF1) and ubiquitin (UBI) were the best choices for samples of different ginkgo genotypes. The expression of UBI and HAS28 presented the most stable at different developmental stages of ginkgo, and EIF3I and RPII were considered as suitable reference genes in different tissues of ginkgo. For methyl jasmonate (MeJA) treatment, ACA and ACT were identified as the optimal reference genes. For cold stress treatment, RPII and EIF4E were chosen for the gene expression normalizations. HAS28 and GAPDH presented the most stable expression for the heat treatment. To validate the above results, a chalcone synthase gene (GbCHS) in ginkgo was amplified by quantitative real-time polymerase chain reaction (qRT-PCR). Our results provide different suitable reference genes for further gene expression studies in ginkgo. [ABSTRACT FROM AUTHOR]

The parasitic dinoflagellates in the genus Amoebophrya infect and kill a number of free-living dinoflagellates. The parasitoid has the potential to regulate harmful dinoflagellate blooms due to its high offspring production rates and a certain degree of host specificity. The planktonic dinoflagellate Prorocentrum donghaiense Lu is a commonly observed harmful algal species in coastal waters of China, and limited studies have been conducted to explore the ecological roles of parasitism in association with bloom dynamics. Thus, the in situ occurrence, prevalence, and dynamics of Amoebophrya during the recurrent blooms of P. donghaiense were systemically studied via morphological observations and high-throughput sequencing. The parasitic infections were identified microscopically in six dinoflagellate taxa with prevalence up to 5.13%, while Amoebophrya infection in the bloom-forming species P. donghaiense was rare (<0.01%), suggesting that P. donghaiense was free of the top-down control of Amoebophrya in the area. In addition, high-throughput sequencing of environmental DNA revealed that Amoebophrya co-dominated both the initiation and termination stages of the P. donghaiense bloom (up to 21.20% of OTU), but was less abundant during persistence of the bloom (3.70% of OTU). Further, the dynamics of Amoebophrya in the water column possessed similar patterns as revealed by qRT-PCR, in which high copy numbers of Amoebophrya (up to 3.60 × 109 copies L−1) were detected in the initiation and termination stages of the P. donghaiense bloom and low copy numbers (1.16 × 109 copies L−1) during persistence of the bloom. These findings manifested the paradoxical dynamics and complicated ecological strategies of Amoebophrya organisms during dinoflagellate blooms that need further study. Image 1 • Prorocentrum donghaiense bloomed in Sansha Bay, China in the early summer of 2018. • Amoebophrya infection in the bloom-forming species P. donghaiense was rare. • Molecular techniques revealed high abundance of Amoebophryidae organism in the water column. • Paradoxical dynamics of Amoebophrya was observed during the dinoflagellate bloom. [ABSTRACT FROM AUTHOR]

• We obtained the first complete open reading frame (ORF) of Foxl2 from H. cumingii. • Hc-Foxl2 expression was different between female and male gonads and juveniles. • In situ hybridization indicated that Hc-Foxl2 was expressed on the oocyte membrane. • Double-stranded RNA can effectively decrease the transcript levels of Hc-Foxl2. • Hc-Foxl2 is a female-related gene in H. cumingii and is involved in sex differentiation. The freshwater mussel Hyriopsis cumingii , is the most important species for pearl culture in China. At present, the mechanisms underlying sex differentiation and determination remain unclear in this species. Herein the open reading frame (ORF) of Foxl2 from H. cumingii (Hc-Foxl2) was cloned, and Hc-Foxl2 expression levels in six tissues (the gonad, gill, adductor muscle, foot, mantle, and kidney) were determined. Further, we performed quantitative real-time PCR to compare expressions levels between 1 and 8 months of age and 1-, 2-, and 3-year-old H. cumingii. The localization of Hc-Foxl2 expression in the ovary was analyzed by in situ hybridization, and its function was explored using RNA interference. We found that the ORF region of Hc-Foxl2 was 1215 bp in length, encoded 404 amino acids, and contained conserved FH domains. Hc-Foxl2 was expressed in the male and female tissues, with the expression levels being significantly higher in the ovary than in the testis. In 1–8-month-old H. cumingii , Hc-Foxl2 was expressed at the highest level at 5 months of age, and the gonads began to differentiate at the same time. Moreover, in 1-, 2-, and 3-year-old individuals, Hc-Foxl2 expression levels in the ovaries gradually decreased, but they were higher than those in the testis. Strong hybridization signals for Hc-Foxl2 were detected on the oocyte membrane in 3-year-old female mussels. We also performed double-stranded RNA (dsRNA) interference experiments using three dsRNA strands, which were injected into 5-month-old H. cumingii ; the interference effects were the best at 12 h and 48 h post-injection. After interference with Hc-Foxl2 , the expression levels of Wnt4 , which has an antagonistic relationship with Foxl2 during ovarian development, were slightly increased. Thus, we speculate that Hc-Foxl2 is a female-related gene in H. cumingii and that it is involved in sex differentiation and ovarian development. [ABSTRACT FROM AUTHOR]

Common varieties of genetically modified (GM) cotton increasingly display insect-resistant properties via expression of bacterial-derived toxins from Bacillus thuringiensis (Bt). This necessitates a deeper understanding of the possible effects of these crops on non-target insects. The mirid bug Apolygus lucorum is a major pest in cotton production in China, however, the effect of GM cotton on this non-target species is currently virtually unknown. This insect is exposed to these transgenic plants by consuming genetically modified (GM) leaves. In this study, laboratory experiments were conducted to assess the toxicity of CCRI41 and CCRI45, (genetically modified cotton varieties which express the toxins Cry1Ac and CpTI (Cowpea Trypsin Inhibitor)) on nymphs and adults of A. lucorum. There was no detectable increase in mortality after A. lucorum fed on GM cotton leaves for 20 days. While we detected trace amounts of Cry1Ac proteins in both A. lucorum nymphs and adults (<10 ng/g fresh weight), the expression of genes related to detoxification did not detectably differ from those feeding on non-GM cotton. Our binding assays did not show Cry1Ac binding to receptors on the midgut brush border membrane from either A. lucorum nymphs or adults. Our findings collectively indicate that feeding on leaves of the GM cotton varieties CCRI41 and CCRI45 have few toxic effects on A. lucorum. Image 1 • Bioassay results showed no effect of Cry1Ac -expressing cotton on Apolygus lucorum survival rate. • Cry1Ac -expressing cotton did not alter the detoxification genes expression of A. lucorum. • No cry1Ac binding protein was detected in the midgut of A. lucorum. • Planting transgenic Cry1Ac cotton will pose a negligible risk to A. lucorum. Capsule: Our study indicated that the GM cotton varieties CCRI41 and CCRI45 have few adverse effects on non-target insect pest A. lucorum. [ABSTRACT FROM AUTHOR]

• A TaqMan-based one-step real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay for the rapid detection of GoAstV was developed. • The qRT-PCR method we established was rapid, sensitive and specific. • This method has a higher positive coincidence rate for GoAstV detection than conventional RT-PCR. The outbreak of an infectious disease characterized by severe symptom of gout has set great threat to several major goose-producing regions in China since December 2016. The causative agent for the novel infection has been identified was a novel goose-origin astrovirus (GoAstV). Lack of effective detection methods indeed hinders further research, as well as prevention and control of GoAstV. Keep this in mind, a TaqMan-based one-step real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay for rapid detection of GoAstV was developed. Primers and probe were targeting the capsid protein gene sequence (ORF2). The method is capable of detecting quite low number of targeting nucleic acid as low as 10 copies/μL. What's more, it is also of great specificity and repeatability for GoAstV detection. No cross-activity was found with other goose-origin viruses. The assay had excellent intra-assay and inter-assay repeatability with the coefficient of variation (CV) value from 0.48% to 0.99%. A total of 340 GoAstV specimens from different regions of China were used in this study to verify the feasibility and effectiveness of this method in clinical diagnosis. The results indicated that qRT-PCR is a highly sensitive, specific and repeatable method for quantitative detection of GoAstV, which can be used to detect this virus, thereby facilitating epidemiological investigations of gout in goslings. [ABSTRACT FROM AUTHOR]

Recently, a novel goose astrovirus (N-GoAstV) was discovered in China, with the transmission route of N-GoAstV unclear. In this study, we developed a TaqMan-based real-time RT-PCR (qRT-PCR) assay for the detection of N-GoAstV infection. After the optimization of the qRT-PCR assay conditions, the results demonstrated that the lower limit of detection for N-GoAstV was 33.4 copies/μL. No cross-reactivity was observed with other goose-origin viruses. Intra-assay and inter-assay variability were ≤1.36% and 2.34%, respectively. N-GoAstV was detected in both field samples, embryos and newly hatched goslings by qRT-PCR assay, provided the view that N-GoAstV may be both horizontally and vertically transmitted. The established qRT-PCR method showed high specificity, sensitivity, and reproducibility, which can be used in future investigations on the pathogenesis and epidemiology of N-GoAstV. • We established a TaqMan real-time RT-PCR method for detection of novel goose astrovirus (N-GoAstV). • The method showed high sensitivity, specificity and reproducibility for detection of N-GoAstV. • This study provided the view that N-GoAstV may be possible vertically transmitted. [ABSTRACT FROM AUTHOR]

Background: Getah virus (GETV) is a neglected mosquito-borne Alphavirus that causes pyrexia, body rash, and leg oedema in horses and foetal death and reproductive disorders in pigs. Infected animals may play a critical role in the amplification and circulation of the virus. The present study aimed to investigate GETV infection in clinically infected cattle and vector mosquito species in northeastern China. Results: Serum samples were collected from beef cattle that presented sudden onset of fever in forest grazing areas, and metagenomic sequencing was conducted, revealing 29 contigs from ten serum samples matching the GETV genome. Quantitative RT-PCR (RT-qPCR) was performed with GETV RNA from 48 beef cattle serum samples, showing that the overall prevalence of GETV in the beef cattle samples was 6.25% (3/48). Serological investigation indicated that GETV neutralizing antibodies were detected in 83.3% (40/48, 95% CI 67–100) of samples from the study region. The GETV JL1808 strain was isolated from clinically infected cattle showing fever. Sequence comparisons showed high identity with the HuN1 strain, a highly pathogenic swine epidemic isolate obtained in Hunan province in 2017, at the nucleotide level (99.5%) and at the deduced amino acid level (99.7–99.9%). The phylogenetic analysis of JL1808 clustered in Group III, and also revealed a close genetic relationship with the HuN1 strain. Additionally, about 12,000 mosquitoes were trapped in this region. The presence of GETV infection was detected in mosquitoes, suggesting that the minimum infection rate (MIR) was 1.50‰, with MIRs of 1.67‰ in Culex pseudovishnui, 1.60‰ in Culex tritaeniorhynchus, and 1.21‰ in Anopheles sinensis. Conclusions: To the best of our knowledge, this is the first report of GETV infection in cattle. These results demonstrated that a highly pathogenic, mosquito-borne swine GETV can infect and circulate in cattle, implying that it is necessary to conduct surveillance of GETV infection in animals in northeastern China. [ABSTRACT FROM AUTHOR]

qRT-PCR is a powerful molecular research tool to study the regulation of gene expression. However, to accurately calculate gene expression levels, an experiment should include proper reference genes that show no changes in their expression level. Pinus massoniana, P. hwangshanensis, and their introgression hybrid in Mountain Lushan, China, are an ideal model for studying introgression and speciation. Although some research on reference gene selection for P. massoniana has been reported before, no studies on this subject have been performed where P. massoniana and its introgression hybrid were evaluated simultaneously. Here, we investigated ten genes (upLOC, SDH, ACT, EF, TOC75, DMWD, FBOX, PGK1, UBQ, and CL2417C7) identified from transcriptome data of these two taxa for reference gene potential. These ten genes were then screened across multiple tissues such as cone, young and mature stems, and young needles according to qRT-PCR thermal cycling and dissociation. Correlation coefficient, amplification efficiency, and cycle threshold value (Ct) range were applied to evaluate the reliability of each gene. The stability of candidate reference gene expression was calculated using three algorithms: geNorm, NormFinder, and BestKeeper. Base on the reliability and stability, we then offered a list of genes of recommended and not recommended for seven different tissue type and species. Our results demonstrated that different sample lines require different genes as reference to evaluate. [ABSTRACT FROM AUTHOR]