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Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer(ITS)region of peritrich ciliates in environmental samples

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Journal of Oceanology and Limnology 2018年第3期36卷 818-826页

作者： SU Lei ZHANG Qianqian GONG Jun Laboratory of Microbial Ecology and Matter Cycles Yantai Institute of Coastal Zone ResearchChinese Academy of SciencesYantai 264003China University of Chinese Academy of Sciences Beijing 100049China

Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifi cations of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specifi c PCR primers were newly designed to amplify a fragment including the internal transcribed spacer(ITS) region of ribosomal rDNA from environmental samples. The primers showed high specifi city in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28 S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18 S rDNA in peritrichs. The newly designed specifi c primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in dif ferent systems.

关键词： Ciliophora Peritrichia clone library internal transcribed spacer(ITS) rDNA specific PCR primers

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

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Journal of Integrative Agriculture 2019年第5期18卷 1042-1049页

作者： WANG Yu-sheng TIAN Hu WAN Fang-hao ZHANG Gui-fen State Key Laboratory for Biology of Plant Diseases and Insect Pests/Key Laboratory of Integrated Pest Management of Crop Ministry of Agriculture/Institute of Plant Protection Chinese Academy of Agricultural Sciences Caofeidian Sub-Center of Hebei Entry-Exit Inspection and Quarantine Technical Center

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

关键词： Phenacoccus manihoti cassava mealybug invasive pest molecular identification species-specific COI primers

Specific primers design based on the superoxide dismutase b gene for Trypanosoma cruzi as a screening tool:Validation method using strains from Colombia classified according to their discrete typing unit

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Asian Pacific Journal of Tropical Medicine 2014年第11期7卷 854-859页

作者： Francisco Olmo Javier Escobedo-Ortegón Patricia Palma Manuel Sánchez-Moreno Ana Mejía-Jaramillo Omar Triana Clotilde Marín Grupo de Parasitologia Molecular Departmento de Parasitologia Universidad de Granada Laboratorio de Zoonosis y ETV's C.I.R.Dr.Hideyo NoguchiUniversidad Autonoma de YucatanAve.Itzaes #490 x 5997000 MeridaYucatan(Mexico) Grupo Biologiay Control de Enfermedades Infecciosas BCEI-SIU Instituto de BiologiaUniversidad de Antioquia

Objective:To classify 21 new isolates of Trypanosoma cruai(***) according to the Discrete Typing Unit(DTU) which they belong to,as well as tune up a new pair of primers designed to detect the parasite in biological ***:Strains were isolated,DNA extracted,and classified by using three Polymerase Chain Reactions(PCR).Subsequently this DNA was used along with other isolates of various biological samples,for a new PCR using primers ***,the amplified fragments were ***:It was observed the predominance of DTU i in Colombia,as well as the specificity of our primers for detection of ***,while no band was obtained when other species were ***:This work reveals the genetic variability of 21 new isolates of *** in *** primers confirmed their specificity for detecting the presence of ***.

关键词： Trypanosoma cruzi Polymerase chain reaction Colombia Superoxide dismutase gene b-based primers Discrete typing unit

Comparison of Extraction Methods for Genomic DNA of Fall Webworm [Hyphantria cunea( Drury) ] and Amplification Effect of Different primers on Sequences of CO I and CO II and Cytb

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Plant Diseases and Pests 2013年第4期4卷 36-38页

作者： Zhuang Mingjuan Chen Jingyun Zhan Guohui Cai Ping College of Architecture and Urban Environment Soochow University Suzhou Entry-Exit Inspection and Quarantine Bureau

[ Objective ] The paper was to compare different extraction methods for genomie DNA of fall webworm [ Hyphantria cunea （Drury） ] and study the am- plification effect of different primers on sequences of CO I and CO II and Cytb. [Method] With winter pupae of fall webworm as the test materials, genomic DNA of fall webworm were extracted by five different methods including CTAB method, SDS method, improved SDS method, kit method and different processing time. Six groups of different primers were used to amplify sequences of CO I and CO II and Cytb. [ Result~ Full DNA could be extracted by CTAB method, SDS method and improved SDS method, and the effort of low concentration SDS method was the best. Six groups of primers could amplify the corresponding fragment, and two groups of primers had the best effort for amplification of CO I . [ Conclusion] The study provides better methods for DNA extraction.

关键词： Fall webworm Extraction GenomJe DNA primers CO I CO II Cytb

Diversity shifts of rumen bacteria induced by dietary forages in dairy cows and quantification of the changed bacteria using a new primer design strategy

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Journal of Integrative Agriculture 2016年第11期15卷 2596-2603页

作者： JIN Di ZHAO Sheng-guo ZHANG Yang-dong SUN Peng BU Deng-pan Yves Beckers WANG Jia-qi State Key Laboratory of Animal Nutrition Institute of Animal ScienceChinese Academy of Agricultural Sciences Animal Science Unit Gembloux Agro-Bio TechUniversity of Liège

The partial 16 S r RNA gene sequences（100 to 500 bp） were widely used to reveal rumen bacterial composition influenced by diets, while quantification of the changed uncultured bacteria was inconvenient due to difficult designing of specific primers based on short sequences. This study evaluated the effect of forage resources on rumen bacterial diversity and developed new strategy for primer design based on short sequences to quantify the changed uncultured bacteria. Denaturing gradient gel electrophoresis（DGGE） analysis and subsequent band sequencing were used to reveal the distinct rumen bacteria composition in cows fed with two forage sources（single corn stover vs. mixed forages including alfalfa hay and corn silage）. The bacterial diversity in the rumen of dairy cows fed with corn stover was lower than that with mixed forages（P0.05）. The bacterium named R-UB affiliating to uncultured Succinivibrionaceae was identified, and it was abundant in the rumen of cows fed with mixed forages compared to corn stover. The full length 16 S r RNA gene sequences with identity of 97% to the R-UB 16 S r RNA gene sequence were obtained from Gen Bank and used to design specific primers to quantify uncultured bacterium R-UB. All sequences of amplicon from the new primers were of 100% identity to R-UB sequences indicating the high specificity of new primers. Quantitative PCR confirmed that abundance of R-UB in the rumen of cows fed with corn stover was lower than those fed with mixed forages（P0.01）. New strategy for designing primers based on partial 16 S r RNA genes to quantify targeted uncultured bacteria was successfully developed. The rumen bacteria descending significantly in the cows fed corn stover compared to those fed mixed forages was identified as uncultured R-UB from Succinivibrionaceae.

关键词： rumen bacterial diversity forage source primers qPCR

Genetic diversity and conservation of two threatened dipterocarps(Dipterocarpaceae) in southeast Vietnam

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Journal of Forestry Research 2019年第5期30卷 1823-1831页

作者： Dinh-Duy Vu Thi Tuyet-Xuan Bui Minh-Duc Nguyen Syed Noor Muhammad Shah Dinh-Giap Vu Yi Zhang Minh-Tam Nguyen Xiao-Hua Huang College of Forestry Northwest A&F UniversityYanglingShaanxi 712100People’s Republic of China Vietnam National Museum of Nature Vietnam Academy of Science and Technology(VAST)18 Hoang Quoc VietCau GiayHanoiVietnam Institute of Ecology and Biological Resource Vietnam Academy of Science and Technology(VAST)18 Hoang Quoc VietCau GiayHanoiVietnam Department of Horticulture Faculty of AgricultureGomal UniversityDera Ismail KhanPakistan Institute of Natural Products Chemistry Vietnam Academy of Science and Technology(VAST)18 Hoang Quoc VietCau GiayHanoiVietnam

Two threatened dipterocarp species,Dipterocarpus costatus and Dipterocarpus alatus are well-known endangered species in lowland forests of southeastern Vietnam,primarily from habitat loss and over-exploitation of their *** develop conservation strategies for these species,we analyzed 242 samples using nine microsatellite markers to determine the genetic variability within and among five populations of *** and three of ***,representing the natural range of dipterocarps in Southeast *** indicated low levels of genetic variability within populations with an average gene diversity of 0.223 for *** and 0.152 for *** of bottleneck tests indicated a reduction in population size of both species(P>0.05).Genetic differentiation among populations was high(FST=0.347 for *** and 0.274 for ***),indicating limited gene flow(Nm=0.662 for *** and 0.47 for ***)and isolated populations related to geographical *** of molecular variance showed high genetic variation within populations(72.92%for *** and 60.81%for ***)compared to among *** analysis and UPGMA tree also indicated the two optimal genetic clusters related to geographical *** results will provide a platform for the conservation,management and restoration of these species.

关键词： Dipterocarps Genetic conservation Gene diversity SSR primers

BanSatDB, a whole-genome-based database of putative and experimentally validated microsatellite markers of three Musa species

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The Crop Journal 2018年第6期6卷 642-650页

作者： Vasu Arora Neera Kapoor Samar Fatma Sarika Jaiswal Mir Asif Iquebal Anil Rai Dinesh Kumar Center for Agricultural Bioinformatics ICAR-Indian Agricultural Statistics Research Institute School of Sciences Indira Gandhi National Open University

The genus Musa is one of three genera in the family Musaceae, which includes bananas and plantains, which are monocotyledonous plants. Bananas have valuable nutritional content of vitamin C, B6, minerals, and dietary fiber and are a rich food energy source, given that carbohydrates account for 22%–32% of fruit weight. Molecular markers are valuable for crop improvement and population genetics studies. The availability of whole-genome sequence and in silico approaches has revolutionized bulk marker discovery. We describe an online web genomic resource, BanSatDB(http://***/bansatdb/) having the highest number(>341,000) of putative STR markers from Musa genera so far, represented by three species: M. acuminata(110,000), M. balbisiana(107,000), and M. itinerans(124,000)from 11 chromosomes of each species. BanSatDB has also been populated with 580 validated STR markers from the published literature. It is based on a three-tier architecture using MySQL, PHP and Apache. The markers can be retrieved by use of multiple search parameters including chromosome number(s), microsatellite types(simple or compound),repeat nucleotides(1–6), copy number, microsatellite length, pattern of repeat motif, and chromosome location. These markers can be used for Distinctness, Uniformity and Stability(DUS) tests of variety identification and for marker assisted selection(MAS) in variety improvement and management. These STRs have also proved to be helpful in classification of Musa germplasm to distinguish individual accessions and in the development of a standardized procedure for genotyping. These markers can also be used in gene discovery and QTL mapping. The database represents a source of markers for developing and implementing new approaches for molecular breeding, which are required to enhance banana productivity.

关键词： Musaceae Musa Banana Microsatellite Short tandem repeat(STR) primers

Isolation of microsatellite loci in the pollinating fig wasp of Ficus hispida, Ceratosolen solmsi

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Insect Science 2008年第4期15卷 303-306页

作者： Hao Yu Tong-Xin Zhang Hao-Yuan Hu Li-Ming Niu Hui Xiao Yan-Zhou Zhang Da-Wei Huang Institute of Zoology Chinese Academy of Sciences Beijing Graduate School of the Chinese Academy of Sciences Beijing Plant Protection College Shandong Agricultural University Tai' an Shandong China

Microsatellite loci were isolated for Ceratosolen solmsi, pollinator of the dioecious Ficus hispida. We developed nine polymorphic microsatellite loci based on the method of polymerase chain reaction isolation of microsatellite arrays （PIMA）. Enrichment of genomic libraries was performed by random amplified polymorphic DNA （RAPD）. A subset of 38 positive clones was sequenced; 15 clones showed microsatellite loci. We tested 15 designed primer pairs and nine of them produced polymorphic amplification in 48 individual wasps collected from different fruits of the dioecious host fig Ficus hispida in China. Among the 48 individuals, 49 alleles were obtained at the nine loci. The observed heterozygosity ranged between 0.357 and 0.634.

关键词： Ficus fig wasp markers microsatellite loci polymorphism primers

Development of Walnut EST-SSR Markers and Primer Design

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Agricultural Science & Technology 2011年第12期12卷 1810-1813页

作者：冯怡张智俊张舍龙罗淑萍浙江农林大学亚热带森林培育国家重点实验室培育基地浙江临安311300 新疆农业大学农学院新疆乌鲁木齐830052

[Objective] This research aimed to develop walnut EST-SSR markers and design corresponding primers.[Method] 5213 EST sequences of walnut（Juglans regia Linn.） public online in NCBI were used for character analysis with bioinformatics methods,and primers were designed for the selected EST sequences by using Primer 3.0 software.[Result] 207 SSRs were obtained from the EST sequences,including 188 non-redundant sequences,the detection rate was 3.97% with an average distribution distance of 21.12 *** 92 types of repeat motifs were involved,which were mainly composed of dinucleotide and trinucleotide,accounting for 31.40% and 35.27% of the total number of repeat motifs,respectively.30 pairs of primers were initially selected from the 50 randomly-selected SSR primers by PCR amplification.[Conclusion] This research would lay foundations for the development of EST-SSR molecular markers in walnut and design of the targeted EST-SSR primers by mining and analyzing the SSR sites in walnut EST sequences.

关键词： Walnut EST-SSR Microsatellite primers