Tag Library Research Articles

Flavonoids and caffeoylquinic acids in Chrysanthemum morifolium Ramat flowers: A potentially rich source of bioactive compounds

Varieties of chrysanthemums are among the world’s most valuable edible ornamental crops. However, the availability and relationship between the bio-chemicals of chrysanthemums and their morphological variations remain unclear. We developed liquid chromatography mass spectrometry to construct a spectral tag library to identify and quantify chemicals of 7 caffeoylquinic acids, 21 flavones and flavonols, 4 carotenoids, and 13 other compounds in 27 cultivars and representative tea of Chrysanthemum morifolium. A correlation analysis found that more acacetin 7-O-galactoside (23) resulted in lighter colored flowers and less acacetin (43) and kaempferol (44) was associated with yellow flowers. Hot-H2O extraction of C. morifolium tea showed that most flavonoids and caffeoylquinic acids dissolved out at 30 min, with 20.977 and 8.958 mg/g GW indicated that C. morifolium, which is used in food and tea, is rich in flavonoids and carotenoids. The results improve our understanding of flavonoid biosynthesis and the mechanisms responsible for flower color.

Development of signature-tagged mutagenesis in Riemerella anatipestifer to identify genes essential for survival and pathogenesis

Riemerella anatipestifer causes epizootic infectious disease in ducks, geese, turkeys and other birds, and serious economic losses especially to the duck industry. However, little is known about the molecular basis of its pathogenesis. In this study, signature-tagged transposon mutagenesis based on Tn4351 was developed in R. anatipestifer to identify genes essential for survival and pathogenesis. Seventeen tagged Tn4351 random mutation libraries of the R. anatipestifer strain WJ4 containing 5100 mutants were screened for survive using a duckling infection model. Twenty mutants that could not be recovered from the infected ducklings, were identified, and 17 mutated genes were identified by inverse PCR or genome-walking PCR. Of these genes, FIP52_03215, FIP52_04350 and FIP52_09345, were inserted into two mutant strains, and FIP52_03215 and FIP52_03175 were found exclusively on the chromosome of serotype 1 R. anatipestifer strains. Twelve out of 17 genes encoding for proteins were predicted to be involved in amino acid, nucleotide, coenzyme, or lipid transport and metabolism, one gene was predicted to be involved in signal transduction, one gene was predicted to be involved in DNA replication, recombination and repair, the other three genes had an unknown function. Animal experiments showed that the virulence of mutants 16-284, 7-295, 24-231, 9-232 and 19-214 were significantly attenuated compared to that of the wild-type WJ4. Moreover, the median lethal dose of mutant 16-284 was greater than 1010 CFU, and its virulence to ducklings was partially restored when it was complemented with the shuttle expression plasmid pRES-FIP52_09345. The results in this study will be helpful to further study the molecular mechanisms of the pathogenesis of R. anatipestifer infection.

An Expanded Surface-Enhanced Raman Scattering Tags Library by Combinatorial Encapsulation of Reporter Molecules in Metal Nanoshells.

Raman-encoded gold nanoparticles (NPs) have been widely employed as photostable multifunctional probes for sensing, bioimaging, multiplex diagnostics, and surface-enhanced Raman scattering (SERS)-guided tumor therapy. We report a strategy toward obtaining a particularly large library of Au nanocapsules encoded with Raman codes defined by the combination of different thiol-free Raman reporters, encapsulated at defined molar ratios. The fabrication of SERS tags with tailored size and predefined codes is based on the in situ incorporation of Raman reporter molecules inside Au nanocapsules during their formation via galvanic replacement coupled to seeded growth on Ag NPs. The hole-free closed-shell structure of the nanocapsules is confirmed by electron tomography. The unusually wide encoding possibilities of the obtained SERS tags are investigated by means of either wavenumber-based encoding or Raman frequency combined with signal intensity, leading to an outstanding performance as exemplified by 26 and 54 different codes, respectively. We additionally demonstrate that encoded nanocapsules can be readily bioconjugated with antibodies for applications such as SERS-based targeted cell imaging and phenotyping.

Abstract 4290: Multi-omic single cell sequencing for deep cell immune profiling and identification of potential biomarkers for cell therapy and immunotherapy

Abstract Introduction: Rapidly advancing single cell genomics technology enables uncovering cell heterogeneity missed by bulk sequencing/analysis. Multi-omic single cell sequencing has ability to assess up to a hundred cell surface proteins, hundreds of transcripts to whole transcriptome, or TCR/BCR gene rearrangement in thousands of cells. Here we report deep cell immune profiling of PBMC by assessing selected cell surface proteins by AbSeq and targeted immune response genes using two single cell sequencing platforms. Experimental procedures: For targeted RNASeq on 10xGenomics Chromium platform, droplets containing single PBMC cell/bead were generated. After cDNA synthesis, libraries with genes of interest were generated using BD Rhapsody Human Immune Response Targeted Panel. For BD Rhapsody multi-omic single cell analysis, PBMC were stained using tagged AbSeq antibodies and sample specific cell tag antibodies. Then viable cells after incubation with cell viability dyes were counted on BD Rhapsody scanner. Some samples were processed for removal of apoptotic cells using EasySep Dead Cell Removal (Annexin V) Kit (Stem Cell Tech). Labeled PBMC from multiple subjects were pooled and loaded to a BD Rhapsody chip. Followed by cell capture beads loading and lysis, mRNA and antibody tags were captured. cDNA was synthesized and libraries containing genes of interest were generated using Human immune response panel along with AbSeq and sample tag libraries. Sequencing was performed on NextSeq 500 (Illumina). Data analysis from 10xGenomics were conduct using its Cell Ranger software. Data analysis from BD Rhapsody workflow were conduct using Seven Bridges Genomics Pipeline and SeqGeq (FlowJo). For comparison, PBMC were also profiled for general major cell populations by flow cytometry. Summary and Conclusions: Targeted RNASeq using both single cell sequencing platforms enables deep cell profiling with benefits of lower cost and higher sensitivity compared with whole transcriptome RNASeq. The results for general cell populations generated by single cell sequencing are comparable to those generated by flow cytometry, however, single cell sequencing classifies sub-populations in greater details based on gene expression markers. Including tagged AbSeq antibodies to assess cell surface proteins in the workflow further helps classification in the case of post-transcriptional regulation, while identifying alternatively spliced isoforms (CD45RO and CD45RA) elusive by RNASeq. One of the challenges is the requirement for viable cells. Although some apoptotic cells could be removed, less viable cells leftover could interfere single cell sequencing and thus careful data QC bioinformatics is warranted. Overall, multi-omic single cell sequencing could enable deep cell profiling and assessing patient samples has great potential to provide biological insights and identify predictive biomarkers. Citation Format: Wenge Shi, Christian Laing, Jane Gao, Kerri Burns, Shyam Sarikonda, Reinhold Pollner, Hua Gong. Multi-omic single cell sequencing for deep cell immune profiling and identification of potential biomarkers for cell therapy and immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4290.

Amplification and Preparation of Cellular O-Glycomes for Functional Glycomics.

Mucin-type O-glycans play key roles in many cellular processes, and they are often altered in human diseases. A major challenge in studying the role of O-glycans through functional O-glycomics is the absence of a complete repertoire of the glycans that comprise the human O-glycome. Here we describe a cellular O-glycome preparation strategy, Preparative Cellular O-Glycome Reporter/Amplification (pCORA), that introduces 4-N3-Bn-GalNAc(Ac)3 as a novel precursor in large-scale cell cultures to generate usable amounts of O-glycans as a potential O-glycome factory. Cultured human non-small cell lung cancer (NSCLC) A549 cells take up the precursor, which is extended by cellular glycosyltransferases to produce 4-N3-Bn-α-O-glycans that are secreted into the culture medium. The O-glycan derivatives can be clicked with a fluorescent bifunctional tag that allows multidimensional HPLC purification and production of a tagged glycan library, representing the O-glycome of the corresponding cells. We obtained ∼5% conversion of precursor to O-glycans and purified a tagged O-glycan library of over 100 O-glycan derivatives, many of which were present in >100 nmol amounts and were sequenced by sequential MS fragmentation (MSn). These O-glycans were successfully printed onto epoxy glass slides as an O-glycome shotgun microarray. We used this novel array to explore binding activity of serum IgM in healthy persons and NSCLC patients at different cancer stages. This novel strategy provides access to complex O-glycans in significant quantities and may offer a new route to discovery of potential diagnostic disease biomarkers.

Cloning and Expression of a Perilla frutescens Cytochrome P450 Enzyme Catalyzing the Hydroxylation of Phenylpropenes.

Phenylpropanoid volatile components in plants are useful and valuable not only as flavorings, but also as medicines and food supplements. The pharmacological actions and toxicities of these compounds have been well studied but their synthetic pathways are generally unclear. In this study, we mined expressed sequence tag libraries of pure strains of perilla maintained for over 30 years for their oil type and conducted gas chromatography-mass spectrometry analyses of the perilla oils to confirm the presence of monohydrates speculated to be intermediates of the phenylpropene synthetics pathways. These putative monohydrate intermediates and their regioisomers were synthesized to identify the reaction products of assays of heterologously expressed enzymes. An enzyme involved in the synthesis of a phenylpropanoid volatile component was identified in perilla. Expression of this enzyme in Saccharomyces cerevisiae showed that it is a member of the cytochrome P450 family and catalyzes the introduction of a hydroxy group onto myristicin to form an intermediate of dillapiole. The enzyme had high sequence similarity to a CYP71D family enzyme, high regiospecificity, and low substrate specificity. This study may aid the elucidation of generally unexploited biosynthetic pathways of phenylpropanoid volatile components.

Viromes of Ten Alfalfa Plants in Australia Reveal Diverse Known Viruses and a Novel RNA Virus.

Alfalfa plants in the field can display a range of virus-like symptoms, especially when grown over many years for seed production. Most known alfalfa viruses have RNA genomes, some of which can be detected using diagnostic assays, but many viruses of alfalfa are not well characterized. This study aims to identify the RNA and DNA virus complexes associated with alfalfa plants in Australia. To maximize the detection of RNA viruses, we purified double-stranded RNA (dsRNA) for high throughput sequencing and characterized the viromes of ten alfalfa samples that showed diverse virus-like symptoms. Using Illumina sequencing of tagged cDNA libraries from immune-captured dsRNA, we identified sequences of the single-stranded RNA viruses, alfalfa mosaic virus (AMV), bean leafroll virus, a new emaravirus tentatively named alfalfa ringspot-associated virus, and persistent dsRNA viruses belonging to the families Amalgaviridae and Partitiviridae. Furthermore, rolling circle amplification and restriction enzyme digestion revealed the complete genome of chickpea chlorosis Australia virus, a mastrevirus (family Geminiviridae) previously reported only from chickpea and French bean that was 97% identical to the chickpea isolate. The sequence data also enabled the assembly of the first complete genome (RNAs 1–3) of an Australian AMV isolate from alfalfa.

The cytosolic protein GRP1 facilitates abscisic acid- and darkness-induced stomatal closure in Salvia miltiorrhiza

By screening an expressed sequence tag (EST) library of Salvia miltiorrhiza, we detected an acidic protein, SmGRP1, with no significant similarities to the other sequences in public databases. SmGRP1 encodes a peptide of 151 amino acids, 33.77 % of which are glutamic acid residues, and the peptide was positive according to “stains-all” staining. Expression analysis revealed that SmGRP1 was expressed in all examined tissues of S. miltiorrhiza but was most highly expressed in the leaves and stems. Without a signal peptide, SmGRP1 localized to the cytoplasm in protoplasts in subcellular localization experiments. SmGRP1 expression was prominently enhanced by ABA and darkness treatments; the protein could also be induced by high temperature, NaCl, and dehydration treatments, while low temperature suppressed its expression. Furthermore, although there were no obvious phenotypic differences in SmGRP1 overexpression and SmGRP1 knockdown mutants compared with control plants under normal culture conditions, the stomata of the knockdown lines remained open when treated with ABA, darkness, NO, and H2O2. In addition, the water loss rate of the knockdown mutants was faster than that of the control lines and overexpression mutants when exposed to air. These observations indicate that SmGRP1 is a novel acidic protein with potential calcium-binding capability and is involved in stomatal movement and stress resistance.

MMHub, a database for the mulberry metabolome.

Mulberry is an important economic crop plant and traditional medicine. It contains a huge array of bioactive metabolites such as flavonoids, amino acids, alkaloids and vitamins. Consequently, mulberry has received increasing attention in recent years. MMHub (version 1.0) is the first open public repository of mass spectra of small chemical compounds (<1000 Da) in mulberry leaves. The database contains 936 electrospray ionization tandem mass spectrometry (ESI-MS2) data and lists the specific distribution of compounds in 91 mulberry resources with two biological duplicates. ESI-MS2 data were obtained under non-standardized and independent experimental conditions. In total, 124 metabolites were identified or tentatively annotated and details of 90 metabolites with associated chemical structures have been deposited in the database. Supporting information such as PubChem compound information, molecular formula and metabolite classification are also provided in the MS2 spectral tag library. The MMHub provides important and comprehensive metabolome data for scientists working with mulberry. This information will be useful for the screening of quality resources and specific metabolites of mulberry. Database URL: https://biodb.swu.edu.cn/mmdb/

48P - Detection of low mutations in hepatocellular carcinoma by using circulating tumour DNA

BackgroundLiver cancer (LC) is presumed to be the sixth most common and newly diagnosed cancer and the fifth leading causing of cancer death around the world in 2018. Hepatocellular carcinoma (HCC) is the most frequent liver cancer and the chronic infection with hepatitis B/C virus is the major risk factor of HCC. Unlike tissue biopsy, liquid biopsy is based on collection of a sample in non-invasive and convenient way at multiple time points over the course of disease. For circulating cell free DNA, it can be used for Next Generation Sequencing (NGS) to detect genetic mutations. MethodsWhole Blood of patients are collected and plasma was separated. The circulating tumor DNA (ctDNA) is extracted from the plasma samples and quantified using Qubit High Sensitive assay and Agilent D1000 assay. The extracted ctDNA is used for molecular barcoded library construction and captured with probe for next generation sequencing. All samples were then analyzed to identify genetic mutations. ResultsCurrently, 20 HCC patients were collected and samples were extracted, sequenced and analyzed. Two thirds of the patients were hepatitis B-viral infected and the one third of the patients were Non-B, Non-C HCC. Most patients were staged 3 others were staged 1 or 2 in HCC. The total ctDNA in 2ml plasma of each patietns were ranged from 2.0ng to 200ng. As total amount of ctDNA were varied, input ctDNA to construct library was differed ranging from 2ng to 50ng. After molecular tagged libraries were constructed, were pooled and captured with probe for next generation sequencing on Nextseq 500/550 300cycles. Data analysis was performed: TP53 mutations were most frequent and the frequency of mutations are ranged from 3% to 40%. ConclusionsTo fully utilize advantages of the liquid biopsy and circulating tumor DNA, detection of low frequency mutations in the cancer is essential. Detection of low frequency mutation in circulating tumor DNA of hepatocellular carcinoma via optimization of circulating tumor DNA Isolation in plasma samples, optimization of library preparation and target capture condition by using molecular barcoding and customized somatic mutation probe, may improve prognosis, diagnosis, prediction and monitoring of therapeutic response and detection of residual tumor burden. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Text synthesis from keywords: a comparison of recurrent-neural-network-based architectures and hybrid approaches

This paper concerns an application of recurrent neural networks to text synthesis in the word level, with the help of keywords. First, a Parts Of Speech tagging library is employed to extract verbs and nouns from the texts used in our work, a part of which are then considered, after automatic eliminations, as the aforementioned keywords. Our ultimate aim is to train a recurrent neural network to map the keyword sequence of a text to the entire text. Successive reformulations of the keyword and full-text word sequences are performed, so that they can serve as the input and target of the network as efficiently as possible. The predicted texts are understandable enough, and the model performance depends on the problem difficulty, determined by the percentage of full-text words that are considered as keywords, that ranges from 1/3 to 1/2 approximately, the training memory cost, mainly affected by the network architecture, as well as the similarity between different texts, which determines the best architecture.

Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast

Clone collections of modified strains (“libraries”) are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks avenues for increasing throughput in functional genomics and cell biology research.

Design and Implementation of Power Market Operation Analysis Tag Library

Responding to the continuous development of power reform and power market, referred to research of the power market operation, this paper designs operational analysis tag library, which is carried out in four dimensions: market supply and demand analysis, market structure analysis, market behaviour analysis and market operation analysis, to display and track the trend of power market operations more intuitively, and describes the operation status of the power market more comprehensively. Besides, Power Market Operation Analysis Tag Library is designed to deal with power trading management from a macro perspective with decision support.

An efficient expression tag library based on self-assembling amphipathic peptides

BackgroundSelf-assembling amphipathic peptides (SAPs) may improve protein production or induce the formation of inclusion bodies by fusing them to the N-terminus of proteins. However, they do not function uniformly well with all target enzymes and systematic research on how the composition of SAPs influence the production of fusion protein is still limited.ResultsTo improve the efficiency of SAPs, we studied factors that might be involved in SAP-mediated protein production using S1 (AEAEAKAK)2 as the original SAP and green fluorescent protein (GFP) as the reporter. The results indicate that hydrophobicity and net charges of SAPs play a key role in protein expression. As hydrophobicity regulation tend to cause the formation of insoluble inclusion bodies of protein, an expression tag library composed of SAPs, which varied in net charge (from + 1 to + 20), was constructed based on the random amplification of S1nv1 (ANANARAR)10. The efficiency of the library was validated by polygalacturonate lyase (PGL), lipoxygenase (LOX), l-asparaginase (ASN) and transglutaminase (MTG). To accelerate preliminary screening, each enzyme was fused at the C-terminus with GFP. Among the four enzyme fusions, the SAPs with + 2 – + 6 net charges were optimal for protein expression. Finally, application of the library improved the expression of PGL, LOX, ASN, and MTG by 8.3, 3.5, 2.64, and 3.68-fold relative to that of the corresponding wild-type enzyme, respectively.ConclusionsThis is the first report to study key factors of SAPs as an expression tag to enhance recombinant enzyme production. The SAP library could be used as a novel plug-and-play protein-engineering method to screen for enzymes or proteins with enhanced production.

Root Hair Single Cell Type Specific Profiles of Gene Expression and Alternative Polyadenylation Under Cadmium Stress.

Transcriptional networks are tightly controlled in plant development and stress responses. Alternative polyadenylation (APA) has been found to regulate gene expression under abiotic stress by increasing the heterogeneity at mRNA 3′-ends. Heavy metals like cadmium pollute water and soil due to mining and industry applications. Understanding how plants cope with heavy metal stress remains an interesting question. The Arabidopsis root hair was chosen as a single cell model to investigate the functional role of APA in cadmium stress response. Primary root growth inhibition and defective root hair morphotypes were observed. Poly(A) tag (PAT) libraries from single cell types, i.e., root hair cells, non-hair epidermal cells, and whole root tip under cadmium stress were prepared and sequenced. Interestingly, a root hair cell type-specific gene expression under short term cadmium exposure, but not related to the prolonged treatment, was detected. Differentially expressed poly(A) sites were identified, which largely contributed to altered gene expression, and enriched in pentose and glucuronate interconversion pathways as well as phenylpropanoid biosynthesis pathways. Numerous genes with poly(A) site switching were found, particularly for functions in cell wall modification, root epidermal differentiation, and root hair tip growth. Our findings suggest that APA plays a functional role as a potential stress modulator in root hair cells under cadmium treatment.

Polystyrene Encapsulated SERS Tags as Promising Standard Tools: Simple and Universal in Synthesis; Highly Sensitive and Ultrastable for Bioimaging.

Surface coating determined the sensitivity and stability of surface-enhanced Raman scattering (SERS) tags in bioanalysis. The reported various coatings suffered from the drawbacks of a lack of rigidity, stability, or synthesis versatility. Herein, we demonstrated robust polystyrene (PS) coated SERS tags that could be prepared by an easy and universal approach. Taking advantages of biocompatible, transparent, compact properties of PS shell, the coated tags showed satisfactory sensitivity, biocompatibility, and superior structural stability in cell and in vivo imaging applications. More importantly, the PS coating strategy allowed for the encapsulation of SERS tags encoded with not only thiolated but also nonthiolated Raman reporters without loss of sensitivity, as exemplified in the synthesis of 9 different resonant dye-encoded tags. Moreover, the coating of SERS tags with various kinds of substrates was achieved via the same standard protocol. Comparing with widespread silica coated tags, the PS coated ones were more stable in harsh conditions and had an easily expanded ultrasensitive (resonant) tags library with much lower cost (no need of expensive sulfhydryl/isothiocyano reporters with limited types), illustrating great promise as standard analytical tools of commercialized value for bioanalysis, medical diagnostics, and environmental science studies.

Characterization of Der f 22 - a paralogue of the major allergen Der f 2

We previously identified an expressed sequence tag clone, Der f 22, showing 41% amino acid identity to published Der f 2, and show that both genes are possible paralogues. The objective of this study was to characterize the genomic, proteomic and immunological functions Der f 22 and Der f 2. The full-length sequence of Der f 2 and Der f 22 coded for mature proteins of 129 and 135 amino acids respectively, both containing 6 cysteine residues. Phylogenetic analysis of known group 2 allergens and their homologues from our expressed sequence tag library showed that Der f 22 is a paralogue of Der f 2. Both Der f 2 and Der f 22 were single gene products with one intron. Both allergens showed specific IgE-binding to over 40% of the atopic patients, with limited of cross-reactivity. Both allergens were detected at the gut region of D. farinae by immunostaining. Der f 22 is an important allergen with significant IgE reactivity among the atopic population, and should be considered in the diagnostic panel and evaluated as future hypoallergen vaccine therapeutic target.

Ovine keratome: identification, localisation and genomic organisation of keratin and keratin-associated proteins.

Wool is composed primarily of proteins belonging to the keratin family. These include the keratins and keratin-associated proteins (KAPs) that are responsible for the structural and mechanical properties of wool fibre. Although all human keratin and KAP genes have been annotated, many of their ovine counterparts remain unknown and even less is known about their genomic organisation. The aim of this study was to use a combinatory approach including comprehensive cDNA and de novo genomic sequencing to identify ovine keratin and KAP genes and their genomic organisation and to validate the keratins and KAPs involved in wool production using ovine expressed sequence tag (EST) libraries and proteomics. The number of genes and their genomic organisation are generally conserved between sheep, cattle and human, despite some unique features in the sheep. Validation by protein mass spectrometry identified multiple keratins (types I and II), epithelial keratins and KAPs. However, 15 EST-derived genes, including one type II keratin and 14 KAPs, were identified in the sheep genome that were not present in the NCBI gene set, providing a significant increase in the number of keratin genes mapped on the sheep genome.

DNA Sequence Dimorphisms in Populations of the Clubroot Pathogen Plasmodiophora brassicae.

To develop genetic markers for differentiation between pathotypes of the clubroot pathogen Plasmodiophora brassicae, DNA polymorphisms of 85 P. brassicae genes were investigated by comparing the sequences of these genes from published expressed sequence tag libraries to their sequences in the two released whole genomes. A significant portion of the identified sequence differences across all polymorphic genes are between an isolate from New Zealand and the two whole-genome sequenced isolates. Four genes with a high density of polymorphisms were selected and their partial sequences were amplified by polymerase chain reaction (PCR) from the old pathotypes 2, 3, 5, 6, and 8 (based on the Williams differential set) and the new virulent populations 3-like and 5-like. On the sequences of two of the four genes, the old pathotypes are all identical to the two whole-genome sequenced isolates and all of the new virulent populations are identical to the New Zealand isolate. Based on the dimorphism on the sequence of these two genes, an RNase H-dependent PCR protocol was developed. This protocol was demonstrated to be useful for virulent pathotype identification and may also be used to study the population dynamics of P. brassicae and the in planta interaction of different pathotypes.

Application of UAV Remote Sensing for a Population Census of Large Wild Herbivores—Taking the Headwater Region of the Yellow River as an Example

We used unmanned aerial vehicles (UAVs) to carry out a relatively complete population census of large wild herbivores in Maduo County on the Tibetan Plateau in the spring of 2017. The effective area covered by aerial surveys was 326.6 km2, and 23,784 images were acquired. Interpretation tag libraries for UAV images were created for wild animals, including Kiang (Equus kiang), Tibetan gazelle (Procapra picticaudata), and blue sheep (Pseudois nayaur), as well as livestock, including yaks and Tibetan sheep. Large wild herbivores in the survey transect were identified through manual imagery interpretation. Densities ranged from 1.15/km2 for Kiang, 0.61/km2 for Tibetan gazelle, 0.62/km2 for blue sheep, 4.12/km2 for domestic yak, and 7.34/km2 for domestic sheep. A method based on meadows in the cold and warm seasons was used for estimating the densities and numbers of large wild herbivores and livestock, and was verified against records of livestock numbers. Population estimates for Kiang, Tibetan gazelle, blue sheep, domestic yak, and domestic sheep were 17,109, 15,961, 9324, 70,846, and 102,194, respectively. Based on published consumption estimates, the results suggest that domestic stock consume 4.5 times the amount of vegetation of large wild herbivores. Compared with traditional ground survey methods, performance of UAV remote sensing surveys of large wild herbivore populations was fast, economical and reliable, providing an effective future method for surveying wild animals.