A Rapid Identification Method of Bactrocera cilifera (Hendel) with Species-Specific Primers(SS-COI)

[Objective]The paper was to establish a rapid identification method of Bactrocera cilifera(Hendel)with species-specific primers(SS-COI).[Method]Using B.cilifera(Hendel)as the positive control,and 19 species of fruit flies such as B.diaphora(Coquillett)and B.dorsalis(Hendel)as the negative controls,a pair of species-specific primers,YF290 and YR511,were designed and screened for accurate identification of B.cilifera,based on mitochondrial DNA COI sequence.[Result]The PCR products were amplified and detected by electrophoresis.Only a clear and single band was observed at about 222 bp in the positive control,while no bands were found in the other negative controls.[Conclusion]The established rapid identification method with species-specific primers(SS-COI)is of great practical significance for rapid identification of fruit flies intercepted from import and export fruits and vegetables at ports,and for rapid clearance and early warning of import fruits and vegetables at ports.

Development of Species-Specific PCR Primers and Sensitive Detection of the Tylenchulus semipenetrans in China

Tylenchulus semipenetrans is the most economically important and widespread nematode pest of citrus in China.rDNA-ITS of 14 populations of T.semipenetrans which were collected from different citrus groves or Chinese fir（Cunninghamia lanceolata） plantations in China were amplified and sequenced.The species-specific primers were designed for the first time to diagnosis T.semipenetrans based on the sequences of rDNA-ITS regions of geographic population above.The primers were sensitive to amplify the expected band size（297 bp） from DNA template of a single second-stage juvenile（J2） or different life stages of T.semipenetrans.No specific band was amplified from 15 non-target nematode species which were commonly found in citrus groves.Specificity and reliability of the primers were validated by further PCR amplification of 16 extra populations of T.semipenetrans collected from 4 provinces of China.The primers successfully detected a single J2 of T.semipenetrans within a whole nematode community comprising a large numbers of non-target nematode.The developed diagnostic technique can be used for accurate identification of T.semipenetrans and also as a decision tool for nematode management for citrus or Chinese fir in China.

Species-specific COI primers for rapid identification of a globally significant invasive pest, the cassava mealybug Phenacoccus manihoti Matile-Ferrero

The globally invasive cassava mealybug Phenacoccus manihoti Matile-Ferrero is a pernicious pest of cassava,and its recent introduction into Asia has raised considerable alarm.To slow or prevent further invasion,an accurate,simple,and developmental-stage-independent detection method for P.manihoti is required.In the present study,a PCR method based on a species-specific mitochondrial DNA cytochrome oxidase I(SS-COI)marker was developed for rapid identification of P.manihoti.One pair of SS-COI primers(PMSSZW-1F and PMSSZW-1R)was designed based on sequence variations in the COI gene among P.manihoti and related mealybug species.Specificity of the primer pair was validated on 21 closely related species.Sensitivity tests were performed on four immature developmental stages and female adults.Efficacy tests demonstrated that at the relatively low concentration of(135.2±14.7)pgresuspended DNA,the specific fragment was detected in all replicates.Furthermore,the SS-COI primer pair was assayed on three populations of P.manihoti from major exporting countries of cassava.The PCR assay was proved to be a rapid,simple,and reliable molecular measure for the identification of P.manihoti.This tool will be useful for quarantine,monitoring,and management of this invasive pest.

Species-specific PCR-based assays for identification and detection of Botryosphaeriaceae species causing stem blight on blueberry in China

Botryosphaeriaceae species are important causal agents of blueberry stem blight worldwide. Blueberry stem blight has become an important disease, potentially affecting the quality and production of blueberries in China. It is difficult and time-consuming to identify at the species level using morphological methods. The aim of this study was to develop polymerase chain reaction（PCR） assays for the diagnosis and early detection of latent infections of blueberry stems by Botryosphaeria spp. Species-specific primers, based on the ribosomal DNA internal transcribed spacer region and β-tubulin gene, were designed and selected for use in PCR assays. Three primer pairs, Lt347-F/R for Lasiodiplodia theobromae, Np304-F/R for Neofusicoccum parvum and FaF/Bt2b for Botryosphaeria dothidea, successfully amplified specific PCR fragments of different sizes on pure cultures or from blueberry stems inoculated and naturally infected blueberry plants with three pathogens, respectively. These primers did not amplify any PCR fragments from other blueberry stem disease-associated pathogens, such as Phomopsis spp. and Pestalotiopsis spp. This PCR protocol could detect as low as 1 00 pg to 1 ng of purified fungal DNA. This PCR-based protocol could be used for the diagnosis and detection of these pathogens from pure cultures or from infected blueberry plants.

Construction of Agropyrum intermedium 2Ai-2 Chromosome DNA Library and Cloning of Species-Specific DNA Sequences

The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) wasidentified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes weremicroisolated and collected. After two rounds of PCR amplification, the PCR products wereranged from 150-3 000 bp,with predominant fragments at about 200-2 000 bp. Using Ag.intermedium genomic DNA as a probe, Southern blotting analysis confirmed the products originatedfrom Ag. intermedium genome. The products were purified, ligated to pUC18 and then transformedinto competence E.coli DH5αto produce a 2Ai-2 chromosome DNA library. The microcloningexperiments produced approximately 5 ×105 clones, the size range of the cloned inserts was 200-1 500 bp, with an average of 580 bp. Using Ag.intermedium genomic DNA as a probe, dot blottingresults showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences inthe library. Four Ag. intermedium clones were screened from the library by RFLP, and threeclones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitivesequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitiveDNA sequence.

Identification of various Biomphalaria alexandrina strains collected from five Egyptian governorates using RAPD and species-specific PCR techniques

The first generation of Biomphalaria snails collected from five Egyptian governorates (Giza, Fayoum, Kafr El-Sheikh, Ismailia and Damietta) were sub-jected to species-specific PCR assays and the results showed that snails collected from the field were B. alexandrina, and there was no evidence for the pres-ence of B. glabrata. The snails were subjected also to RAPD- PCR technique. The results showed that dif-ferent fingerprints with each B. alexandrina strain were produced with varying numbers of bands rang-ing in size from 123.6 to 796.6 bp depending on the snail strain and the primer used. Many specific bands were obtained with the four primers in each strain. Primer OPA-1 amplified the highest number of spe-cific bands (26 bands) and gave the highest poly-morphism among the primers used (100% polymor-phism). The estimated similarity coefficients among B. alexandrina strains based on the RAPD-PCR pro-files ranged from 0.56 to 0.72. The highest similarity coefficient (0.72) was recorded between the strains of Ismailia and Kafr El-Sheikh, while the lowest coeffi-cient (0.56) was reported between the strains of SPSC and Ismailia.

Collagen derived species-specific peptides for distinguishing donkey-hide gelatin(Asini Corii Colla)

Objective:As an important food therapy product with traditional Chinese medicine(TCM) applications,donkey-hide gelatin(Asini Corii Colla,ACC) has been used for thousands of years.However,till now few effective strategy had been proposed to distinguish ACC from other animal hide gelatins,especially closely related horse-and mule-hide gelatins,which was an embarrassment of ACC quality control.Methods:Combined mass spectrometry and bioinformatic methods have been applied to identify and verify two ACC-specific peptides(Pep-1 and Pep-2) capable of distinguishing ACC from other closely related animal gelatins with high selectivity.Results:It confirmed that these two peptides could be not only used for distinguishing ACC from highly homologous horse-hide and mule-hide gelatins as well as other animal hide gelatins.Conclusion:The present study provides a simple method for species-specific peptides discovery,which can be used for assessing the quality of animal gelatin products,and ensure they are authenticable and traceable.

Molecular evolution and species-specific expansion of the NAP members in plants

he NAP（NAC-Like, Activated by AP3/PI） subfamily is one of the important plant-specific transcription factors, and controls many vital biological processes in plants. In the current study, 197 NAP proteins were identified from 31 vascular plants,but no NAP members were found in eight non-vascular plants.All NAP proteins were phylogenetically classified into two groups（NAP I and NAP II）, and the origin time of the NAP I group might be relatively later than that of the NAP II group.Furthermore, species-specific gene duplications, caused by segmental duplication events, resulted in the expansion of the NAP subfamily after species-divergence. Different groups have different expansion rates, and the NAP group preference was found during the expansion in plants. Moreover, the expansion of NAP proteins may be related to the gain and loss of introns.Besides, functional divergence was limited after the gene duplication. Abscisic acid（ABA） might play an important role in leaf senescence, which is regulated by NAP subfamily. These results could lay an important foundation for expansion and evolutionary analysis of NAP subfamily in plants.

Factors determining the occupancy of nest-boxes by Great Tits(Parus major)in eucalypt plantations

Providing nest-boxes as surrogate tree cavities can be of great importance to increase the breeding populations of cavity-nesting birds in managed forests.However,the exact placement of nest-boxes should be taken into consideration to enhance their occupancy according to species-specific preferences.In this study,we investigated which factors can better predict nest-box occupancy by the Great Tit(Parus major)in eucalypt plantations.We used generalised linear mixed-effects models to analyse the influence of topography,nest-box positioning,vegetation cover and landscape variables on three-year occupancy records from 80 newly provided nest-boxes.Non-random patterns of nest-box occupancy were found with respect to all categories except topography.Results suggest that Great Tits prefer to occupy high-placed nest-boxes,close to areas that can provide them with supplementary resources either within or in the vicinity of the stand(i.e.,trees other than eucalypts,riparian vegetation,and large patches of adjacent habitats).Overall,this study provides important recommendations for nest-box placement and spatial distribution in managed forests and enhances the potential of nest-box interventions as a biodiversity offset and management tool.

Supplemental blue light increases growth and quality of greenhouse pak choi depending on cultivar and supplemental light intensity

To evaluate the supplementary blue light intensity on growth and health-promoting compounds in pak choi（Brassica campestris ssp.chinensis var.communis）,four blue light intensity treatments（T0,T50,T100 and T150 indicate 0,50,100,and 150μmol m^（-2） s^（-1）,respectively）were applied 10 days before harvest under greenhouse conditions.Both of cultivars（greenand red-leaf pak choi）under T50 had the highest yield,content of chlorophyll and sugars.With light intensity increasing,antioxidant compounds（vitamin C and carotenoids）significantly increased,while nitrate content showed an opposite trend.The health-promoting compounds（phenolics,flavonoids,anthocyanins,and glucosinolates）were significantly higher under supplementary light treatment than T0,so as the antioxidant capacity（2,2-diphenyl-1-picrylhydrazyl and ferric-reducing antioxidant power）.The species-specific differences in photosynthetic pigment and health-promoting compounds was found in green-and red-leaf pak choi.T50 treatment could be used for yield improvement,whereas T100 treatment could be applied for quality improvement.Results showed that blue light intensity can regulate the accumulation of biomass,morphology and health-promoting compounds in pak choi under greenhouse conditions.

Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA（rDNA） and internal transcribed spacer（ITS）.A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae.

Chaffinch (<i>Fringilla coelebs</i>L.) song in populations of the East Europe

At studying of song organizations of chaffinch (Fringilla coelebs L.) are found out unique parities of song types in different local populations of a species-specific area. Thus in different areas of the East Europe are forming original (different) song cultures of chaffinch—the certain set of song types including so-called “dialect song forms”. The complex interrelation of geographical variability of chaffinch song in many respects gives a support at an evolutionary view in the given aspect.

Degree of prevalence of different song types of chaffinch (<i>Fringilla coelebs</i>L.) in populations of the European Russia

When studying song organizations of chaffinch (Fringilla coelebs L.), we find the unique typological parities (ratio) of songs in different local populations of a species-specific area. The degree of prevalence of different chaffinch song types can’t correspond to percentage proceeding from their general (common) number in a population. Thus different areas of Russia are forming original (different) song cultures of chaffinch, consisting of the certain set and a parity (ratio) of song types, frequently including the so-called dialect song forms, that have been observed. The complex interrelation of geographical variability and structural variability of chaffinch song in many respects gives a support in an evolutionary view from the given aspect.

Molecular interaction of zp3 to zp3r reveals a cross-species fertilization mechanism

Objective: To evaluate the role of ZP3R in the species-specific fertilization mechanism. Methods:ZP3/ZP3R protein sequences ofMus musculus,Rattus norvegicus, andCavia porcellus were downloaded from UNIPROT. Percentage of amino acids that was calculated by using the SIAS program. Protein sequences modeled was established by using the Modeller 9.14 program and glycosylation of the ZP3 using GlyProt program. Docking simulation of the ZP3R-ZP3 was performed between the same species and different species with PatchDock program.Results:Comparison of the ZP3R and ZP3 structure between species showed that ZP3 in these three species was more similar than ZP3R. Docking simulations of protein showed that changes in the pattern of the ZP3-ZP3R domain for interaction on cross-species compared to the same species. Changes in the pattern of binding ZP3R-ZP3 made sperm-egg binding was not functional and could inhibit cross-fertilization.Conclusions: ZP3R-ZP3 interaction is species-specific, and the role of ZP3R is greater than ZP3 in determining the species-specific recognition stage and sperm-egg binding.

Vocal Variability of Chaffinch Song (<i>Fringilla coelebs</i>L.) as a Condition of Cultural Evolution in Local Populations

There are such characteristics of a matter of the nature, as variability and stability (tradition, norm). Probably, these opposites process as if other qualities of different forms of energy also create “movement” development. Thus the given properties of a matter of the nature can be considered at different levels of its organization. The singing of many passerine birds is incontrovertible feature of their life which determines and builds a reproductive cycle. By studying song repertoire of many sparrow species in details, it was revealed that the individual has not only one, but some types of songs. Thus spring singing represents the multifunctional phenomenon and can bear (carry) various values. The singing is not only a means of attracting females, but also a means of intimidation of the contender and delimitation of nested territory. Variants or types of species-specific song are individually various and distributed between individuals of a population. The complex interrelation of geographical variability of chaffinch song in many respects gives a support at an evolutionary view in the given aspect.

Differences in Amino Acid Composition between <i>α</i>and <i>β</i>Structural Classes of Proteins

The amino acid composition of α and β structural class of proteins from five species, Escherichia coli, Thermotoga maritima, Thermus thermophilus, yeast, and humans were investigated. Amino acid residues of proteins were classified into interior or surface residues based on the relative accessible surface area. The hydrophobic Leu, Ala, Val, and Ile residues were rich in interior residues, and hydrophilic Glu, Lys, Asp, and Arg were rich in surface residues both in α and β proteins. The amino acid composition of α proteins was different from that of β proteins in five species, and the difference was derived from the different contents of their interior residues between α and β proteins. α-helix content of α proteins was rich in interior residues than surface ones. Similarly, β-sheet content of β proteins was rich in interior residues than surface ones. The content of Leu residues was very high, approximately 20%, in interior residues of α proteins. This result suggested that the Leu residue plays an important role in the folding of α proteins.

Molecular Characterization and Prevalence of Trypanosoma Species in Cattle from a Northern Livestock Area in Cote d’Ivoire

Background: African animal trypanosomiasis (AAT) is caused mainly by Trypanosoma congolense, T. vivax, and T. brucei brucei and is the major constraint for livestock productivity in Sub-Saharan African countries. Information about animal trypanosomiasis status in Ivory Coast is missing, especially regarding molecular epidemiology. Therefore, this study intended to apply molecular tools to identify and characterize trypanosomes in Ivory Coast for sustainable control. Methods: 363 cattle blood samples were collected from Ferkessedougou Region in northern Ivory Coast in 2012. Buffy coat technique (BCT) and species-specific PCR assays were used to detect trypanosome species. Results: Out of 363 cattle examined with BCT, 33 were found positive with all trypanosomes species accounting for an average of 9.09% prevalence whereas polymerase chain reaction (PCR) using species-specific primers showed that 81 out of 363 cattle were infected with trypanosomes with an overall prevalence of 22.31%. Trypanosoma congolense savanah type, T. Vivax and T. brucei sl. accounted for 28.39%, 49.38% and 23.45% of the infection rate respectively. No infection with T. congo forest?type was detected. T. vivax infection was the most prevalence in the area investigated compared to the two other trypanosome species. Mixed infections with different trypanosomes species were detected accounting for 7.32% of prevalence. Regarding sexrelated prevalence, male cattles were slightly more infected than female but the difference was not significant. Conclusion: Our results showed that there was a high prevalence of AAT in livestock in Ferkessedougou Area. There is therefore a need to strengthen control policies and institute measures that help prevent the spread of the parasites for sustainable control of animal trypanosome in this area.

Comparative Genome Analysis of Scutellaria baicalensis and Scutellaria barbata Reveals the Evolution of Active Flavonoid Biosynthesis

Scutellaria baicalensis(S.baicalensis)and Scutellaria barbata(S.barbata)are common medicinal plants of the Lamiaceae family.Both produce specific flavonoid compounds,including baicalein,scutellarein,norwogonin,and wogonin,as well as their glycosides,which exhibit antioxidant and antitumor activities.Here,we report chromosome-level genome assemblies of S.baicalensis and S.barbata with quantitative chromosomal variation(2 n=18 and 2 n=26,respectively).The divergence of S.baicalensis and S.barbata occurred far earlier than previously reported,and a whole-genome duplication(WGD)event was identified.The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement.Comparative genome analysis of the congeneric species revealed the species-specific evolution of chrysin and apigenin biosynthetic genes,such as the S.baicalensis-specific tandem duplication of genes encoding phenylalanine ammonia lyase and chalcone synthase,and the S.barbata-specific duplication of genes encoding 4-Co A ligase.In addition,the paralogous duplication,colinearity,and expression diversity of CYP82 D subfamily members revealed the functional divergence of genes encoding flavone hydroxylase between S.baicalensis and S.barbata.Analyzing these Scutellaria genomes reveals the common and species-specific evolution of flavone biosynthetic genes.Thus,these findings would facilitate the development of molecular breeding and studies of biosynthesis and regulation of bioactive compounds.

Phyllosticta species associated with citrus diseases in China

Phyllosticta species associated with diseases of four commercial Citrus species grown in China are reported.Totally,496 Phyllosticta strains were isolated from mandarins(Citrus reticulata),pomeloes(C.maxima),oranges(C.sinensis)and lemons(C.limon)in the main citrus producing regions across China,and 74 strains were selected for phylogenetic analysis.Analyses inferred from the sequences of internal transcribed spacer region(ITS1,5.8S nrDNA and ITS2),partial translation elongation factor 1-alpha(TEF1)and partial actin gene(ACT),showed these representative Phyllosticta isolates clustered in four distinct clades corresponding to three known,and one undescribed species.The newly resolved taxon,Phyllosticta citrichinaensis was isolated from leaves and fruits of all four Citrus species and is introduced in this paper.This taxon caused minor damage,showing irregular spots or freckles.Phyllosticta citriasiana,associated with tan spot of pomeloes,was isolated only from pomeloes,and never from lemons,mandarins and oranges.Phyllosticta citricarpa,the citrus black spot pathogen,which is presently subjected to phytosanitary legislation in the EU and United States,was isolated from lemons,mandarins and oranges,but never from pomeloes.The isolates of P.citricarpa clustered in two subclades,one from mandarins,the other from oranges and lemons.P.capitalensis was isolated from all four Citrus species as an endophyte,causing false melanose,or together with P.citricarpa or P.citriasiana.Morphological,cultural and biochemical characters were consistent with the results of phylogenetic analysis.In addition,a specific primer pair Pca8/ITS4 was designed and selected,and its corresponding PCR procedure was developed for the detection of P.citriasiana in this study.

Identification of salivary proteins in the whitefly Bemisia tabaci by transcriptomic and LC-MS/MS analyses

The whitefly Bemisia tabaci is a notorious agricultural pest of many crops worldwide.Although it is thought that B.tabaci secretes saliva into the host plant to counter plant defenses,knowledge on the whitefly salivary proteome is limited.Here,we characterized the gene/protein repertoires of B.tabaci salivary glands and secreted saliva by transcriptomic and liquid chromatography tandem mass spectroscopy analyses.A total of 698 salivary gland-enriched unigenes and 171 salivary proteins were identified.Comparative analysis between the B.tabaci salivary proteins and those of different arthropod species revealed numerous similarities in proteins associated with binding,hydrolysis,and oxidation-reduction,which demonstrates a degree of conservation across herbivorous saliva.There were 74 proteins only identified in B.tabaci saliva,of which 34 were B.tabaci-specific.In addition,13 salivary proteins,of which I1 were B.tabaci-specific,were differentially regulated when B.tabaci fed on different hosts.Our results provide a good resource for future functional studies of whitefly salivary effectors,and might be useful in pest management.