[Objective] Grb-AST7 concatemer including 17 copies of Grb-AST7 was constructed by ＂self template-primer＂ PCR. [Method] Two primers of which were synthesized based on the amino acid sequence of Gryllus bimaculatus’...[Objective] Grb-AST7 concatemer including 17 copies of Grb-AST7 was constructed by ＂self template-primer＂ PCR. [Method] Two primers of which were synthesized based on the amino acid sequence of Gryllus bimaculatus’ Grb-AST7. [Result] Through splicing, a length of 570 bp, containing 17 copies of the Grb-AST7 gene repeats were obtained. Appropriate primer splicing conditions were as follows：splice time 20 min, 25 μl PCR system, containing 2 μl template. [Conclusion] The results laid foundation for the future studies on Grb-AST7 gene expression and bio-activity analysis.展开更多
The molecular phylogenetics of the Lepidoptera （butterflies and moths） is well studied, but that of Trichoptera （caddisflies）, the sister clade of Lepidoptera, is less studied. The PCR primer libraries developed f...The molecular phylogenetics of the Lepidoptera （butterflies and moths） is well studied, but that of Trichoptera （caddisflies）, the sister clade of Lepidoptera, is less studied. The PCR primer libraries developed for lepidopteran phylogenetics might work in Trichoptera. DNA from 8 caddisfly species （Asynarchus nigriculus （Banks, 1908）, Grammotaulius lorettae Denning, 1941, Hesperophylax occidentalis （Banks, 1908）, Limnephilus externus Hagen, 1861, Limnephilus picturatus McLachlan, 1875, Limnephilus secludens Banks, 1914, Limnephilus sublunatus Provancher, 1877 and Agrypnia deflata （Milne, 1931）） was used to screen for amplification. 107 primer pairs for 45 nuclear and 3 mitochondrial genes were tested. Primers for 1 new gene （40S ribosomalprotein $2 （RPS2）） and 8 genes previously used in Trichopteran phylogenetics were recovered （16S rRNA, 18S rRNA, carbamoyl-phosphate synthetase （CAD）, cytoehrome oxidase I （CO1）, cytochrome oxidase 11 （COIl）, elongation factor-1 alpha （EF-1 alpha）, isoeitrate dehydrogenase （IDH）, and RNA polymerase-II （POL-I1））. New primer pairs extended the genomic region sampled for many genes. Evolution rates among loci varied by 2 orders of magnitude. Differences among evolution rates and modes of inheritance offer flexible tools for resolving phylogenetic questions and examining genome evolution in the Trichoptera. Screening libraries of PCR primers is a useful approach for identifying PCR primers in related taxa with limited molecular genetic resources.展开更多
The information of single nucleotide polymorphisms （SNPs） is quite unknown in sweetpotato. In this study, two sweetpotato varieties （Xushu 18 and Xu 781） were sequenced by Illumina technology, as well as de novo t...The information of single nucleotide polymorphisms （SNPs） is quite unknown in sweetpotato. In this study, two sweetpotato varieties （Xushu 18 and Xu 781） were sequenced by Illumina technology, as well as de novo transcriptome assembly, functional annotation, and in silico discovery of potential SNP molecular markers. Tetra-primer Amplification Refractory Mutation System PCR （ARMS-PCR） is a simple and sufficient method for detecting different alleles in SNP locus. Total 153 sets of ARMS-PCR primers were designed to validate the putative SNPs from sequences. PCR products from 103 sets of primers were different between Xu 781 and Xushu 18 via agarose gel electrophoresis, and the detection rate was 67.32%. We obtained the expected results from 32 sets of primers between the two genotypes. Furthermore, we ascertained the optimal annealing temperature of 32 sets of primers. These SNPs might be used in genotyping, QTL mapping, or marker-assisted trait selection further in sweetpotato. To our knowledge, this work was the first study to develop SNP markers in sweetpotato by using tetra-primer ARMS-PCR technique. This method was a simple, rapid, and useful techn!que to develop SNP markers, and will provide a potential and preliminary application in discriminating cultivars in sweetpotato.展开更多
There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical ...There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.展开更多
基金Supported by National Science and Technology Support Projects(No.2007BAD48B02)Agriculture Science and Technology Achievement Transformation Project(No.2007GB23260410)Basic Research Operating Expenses in Central Public Research Institutes(2007HZS1J007)~~
文摘[Objective] Grb-AST7 concatemer including 17 copies of Grb-AST7 was constructed by ＂self template-primer＂ PCR. [Method] Two primers of which were synthesized based on the amino acid sequence of Gryllus bimaculatus’ Grb-AST7. [Result] Through splicing, a length of 570 bp, containing 17 copies of the Grb-AST7 gene repeats were obtained. Appropriate primer splicing conditions were as follows：splice time 20 min, 25 μl PCR system, containing 2 μl template. [Conclusion] The results laid foundation for the future studies on Grb-AST7 gene expression and bio-activity analysis.
基金provided by a University of Manitoba Graduate Fellowshipthe University of Manitoba Research Grants Program+3 种基金the Field Work Support Program of the Faculty of Science at the University of Manitobaa Rocky Mountain Biological Laboratory Research Fellowshipan NSERC Discovery Grant RGPIN386337-2011a Canada Foundation for Innovation Award
文摘The molecular phylogenetics of the Lepidoptera （butterflies and moths） is well studied, but that of Trichoptera （caddisflies）, the sister clade of Lepidoptera, is less studied. The PCR primer libraries developed for lepidopteran phylogenetics might work in Trichoptera. DNA from 8 caddisfly species （Asynarchus nigriculus （Banks, 1908）, Grammotaulius lorettae Denning, 1941, Hesperophylax occidentalis （Banks, 1908）, Limnephilus externus Hagen, 1861, Limnephilus picturatus McLachlan, 1875, Limnephilus secludens Banks, 1914, Limnephilus sublunatus Provancher, 1877 and Agrypnia deflata （Milne, 1931）） was used to screen for amplification. 107 primer pairs for 45 nuclear and 3 mitochondrial genes were tested. Primers for 1 new gene （40S ribosomalprotein $2 （RPS2）） and 8 genes previously used in Trichopteran phylogenetics were recovered （16S rRNA, 18S rRNA, carbamoyl-phosphate synthetase （CAD）, cytoehrome oxidase I （CO1）, cytochrome oxidase 11 （COIl）, elongation factor-1 alpha （EF-1 alpha）, isoeitrate dehydrogenase （IDH）, and RNA polymerase-II （POL-I1））. New primer pairs extended the genomic region sampled for many genes. Evolution rates among loci varied by 2 orders of magnitude. Differences among evolution rates and modes of inheritance offer flexible tools for resolving phylogenetic questions and examining genome evolution in the Trichoptera. Screening libraries of PCR primers is a useful approach for identifying PCR primers in related taxa with limited molecular genetic resources.
基金supported by the China Agriculture Research System (CARS-11)the National High-Tech R&D Program of China (2012AA101204)the Jiangsu Independent Inno vation Funds of Agriculture,China (CX(13)2032)
文摘The information of single nucleotide polymorphisms （SNPs） is quite unknown in sweetpotato. In this study, two sweetpotato varieties （Xushu 18 and Xu 781） were sequenced by Illumina technology, as well as de novo transcriptome assembly, functional annotation, and in silico discovery of potential SNP molecular markers. Tetra-primer Amplification Refractory Mutation System PCR （ARMS-PCR） is a simple and sufficient method for detecting different alleles in SNP locus. Total 153 sets of ARMS-PCR primers were designed to validate the putative SNPs from sequences. PCR products from 103 sets of primers were different between Xu 781 and Xushu 18 via agarose gel electrophoresis, and the detection rate was 67.32%. We obtained the expected results from 32 sets of primers between the two genotypes. Furthermore, we ascertained the optimal annealing temperature of 32 sets of primers. These SNPs might be used in genotyping, QTL mapping, or marker-assisted trait selection further in sweetpotato. To our knowledge, this work was the first study to develop SNP markers in sweetpotato by using tetra-primer ARMS-PCR technique. This method was a simple, rapid, and useful techn!que to develop SNP markers, and will provide a potential and preliminary application in discriminating cultivars in sweetpotato.
基金supported by the Shandong Seed Projectthe National Natural Science Foundation of China(No.31372524)Science and Technology Development Plan of Shandong Province,China(No.2014GHY 115002)
文摘There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.
