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 experti...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.展开更多
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 ...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.展开更多
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 acc...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.展开更多
In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic spe...In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic specific PCR primers for 1B chromosome. Sequence analysis revealed that LMWXY22-2 was composed of 1 364 bp nucleotides, including a 317 bp promotion region and a 1 047 bp coding region which could be translated into a mature protein of 349 amino acids. In spite of a few minor mutations, the sequence of 5＇ untranslated region （UTR）, the coding region, the deduced N- and Cterminus comparisons indicated that LMWXY22-2 belonged to the reported subunits of LMW-m type and type lII group 5, respectively. Inner gene markers for 1D chromosome together with the phylogenetic analysis revealed that this gene was classified into Glu-D3, which was not in agreement with the I B locus-specific primers for LMW genes completely.展开更多
Objective:To establish a new detecting method for disease susceptibility loci R1628P and G2385R of Parkinson’s disease(PD)related gene LRRK2.Methods:Sequence specific primers were designed to make a genotyping of DNA...Objective:To establish a new detecting method for disease susceptibility loci R1628P and G2385R of Parkinson’s disease(PD)related gene LRRK2.Methods:Sequence specific primers were designed to make a genotyping of DNA markers with known genotypes by use of quantitative fluorescence real-time PCR(RT-PCR).100 cases of PD samples with unknown genotypes were tested,and verified by use of polymerase chain reaction linked restriction fragment length polymorphism(PCR-RLFP).Results:The genotyping results of DNA markers proved to be correct,and 100 cases of samples to be tested had a completely consistent genotyping result with PCR-RLFP genotyping result.Conclusions:Sequence specific primer and quantitative fluorescence RT-PCR can successfully make a genotyping for disease susceptibility loci R1628P and G2385R of LRRK2.展开更多
[Objective] This study aimed to establish molecular identification methods for Bacillus licheniformis. [Method] Based on clone sequencing and difference analysis for 16S and ITS sequences of B. licheniformis TS-01, sp...[Objective] This study aimed to establish molecular identification methods for Bacillus licheniformis. [Method] Based on clone sequencing and difference analysis for 16S and ITS sequences of B. licheniformis TS-01, specific primers were designed using region sequences as the targets used for amplifying all test strains. [Result] The specific primers of B. licheniformis were designed from the ITS and 16S rDNA regions. The optimal annealing temperature of the specific primers for PCR was 67.2 ℃ with 24 cycles. A 905 bp marker fragment was amplified for B. licheniformis TS-01, while all other test strains showed negative results. This indicated that a specific 16S-ITS marker was obtained, which accurately identified the strain at the species level. [Conclusion] This molecular identification method for B. licheniformis TS-01 has laid the foundation for molecular diagnosis of B. licheniformis.展开更多
Plants adapt to phosphorus （P） deficiency through a complex of biological processes and many genes are involved. Tomato （Solanum lycopersicum L. ＇Hezuo906＇） plants were selected to grown hydroponically to study ...Plants adapt to phosphorus （P） deficiency through a complex of biological processes and many genes are involved. Tomato （Solanum lycopersicum L. ＇Hezuo906＇） plants were selected to grown hydroponically to study the temporal and spatial gene expression patterns of the 14-3-3 gene family and their roles in response to P deficiency in tomato plants. Using real-time reverse-transcriptase polymerase chain reaction （RT-PCR）, we investigated the expression profiles in different tissues （root, stem and leaf） at short-term and long-term P-deficient stress phases. Results revealed that i） four members of 14-3-3 gene family （TFT1, TFT4, TFT6 and TFTT） were involved in the adaptation of tomato plants to P deficiency, ii） TFT7 responded quickly to P deficiency in the root, while TFT6 responded slowly to P deficiency in the leaf, iii） expression response of TFT4 to P-deficient stress was widely distributed in different tissues （root, stem and leaf） while TFT8 only displayed stem-specific expression, and iv） temporal and tissues-specific expression patterns to P deficiency suggested that isoform specificity existed in tomato 14-3-3 gene family. We propose that TFT7 （one member of e-like group in tomato 14-3-3 family） is the early responsive gene and may play a role in the adaptation of tomato plants to shortterm P deficiency, while TFT6 （one member of non-e group in tomato 14-3-3 family） is the later responsive gene and may play a role in the adaptation of tomato plants to long-term P deficiency.展开更多
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 Chin...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.展开更多
The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in ...The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbc L and mat K gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825,which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbc L gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68%interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the mat K gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbc L and mat K regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR,designed from rbc L sequences, that could differentiate quinoa from 64 genera including13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years(Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.展开更多
基金Supported by the National Natural Science Foundation of China(Nos.31572255,41522604,31301867)the Strategic Priority Research Program of CAS(No.XDA11020702)the Science and Technology Development Program of Yantai(No.2014ZH073)
文摘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.
基金supported by the National Natural Science Foundation of China (30700526)the Postdoctoral Science Foundation of China (55920)the Science Foundation of the Fujian Province,China (2009N0013)
文摘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.
基金supported by the National Key R&D Program of China(2017YFC1200600,2016YFC1201200 and 2015BAD08A16)the Science and Technology Innovation Program of CAAS(caascx-2013-2018-IAS)
文摘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.
基金supported by grants from the Significant Special Found of "13115" S&T Innovation Project of Shaanxi Province,China(2007 ZDKG-01)"13115" Technology Innovation Engineering and Engineering Technology Research Center of Shaanxi Province,China(2008 ZDGC-02)the Special Capital for the Construction of Modern Agriculture Technical System of Shaanxi Province,China (NYCYTX-001)
文摘In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic specific PCR primers for 1B chromosome. Sequence analysis revealed that LMWXY22-2 was composed of 1 364 bp nucleotides, including a 317 bp promotion region and a 1 047 bp coding region which could be translated into a mature protein of 349 amino acids. In spite of a few minor mutations, the sequence of 5＇ untranslated region （UTR）, the coding region, the deduced N- and Cterminus comparisons indicated that LMWXY22-2 belonged to the reported subunits of LMW-m type and type lII group 5, respectively. Inner gene markers for 1D chromosome together with the phylogenetic analysis revealed that this gene was classified into Glu-D3, which was not in agreement with the I B locus-specific primers for LMW genes completely.
文摘Objective:To establish a new detecting method for disease susceptibility loci R1628P and G2385R of Parkinson’s disease(PD)related gene LRRK2.Methods:Sequence specific primers were designed to make a genotyping of DNA markers with known genotypes by use of quantitative fluorescence real-time PCR(RT-PCR).100 cases of PD samples with unknown genotypes were tested,and verified by use of polymerase chain reaction linked restriction fragment length polymorphism(PCR-RLFP).Results:The genotyping results of DNA markers proved to be correct,and 100 cases of samples to be tested had a completely consistent genotyping result with PCR-RLFP genotyping result.Conclusions:Sequence specific primer and quantitative fluorescence RT-PCR can successfully make a genotyping for disease susceptibility loci R1628P and G2385R of LRRK2.
文摘[Objective] This study aimed to establish molecular identification methods for Bacillus licheniformis. [Method] Based on clone sequencing and difference analysis for 16S and ITS sequences of B. licheniformis TS-01, specific primers were designed using region sequences as the targets used for amplifying all test strains. [Result] The specific primers of B. licheniformis were designed from the ITS and 16S rDNA regions. The optimal annealing temperature of the specific primers for PCR was 67.2 ℃ with 24 cycles. A 905 bp marker fragment was amplified for B. licheniformis TS-01, while all other test strains showed negative results. This indicated that a specific 16S-ITS marker was obtained, which accurately identified the strain at the species level. [Conclusion] This molecular identification method for B. licheniformis TS-01 has laid the foundation for molecular diagnosis of B. licheniformis.
基金Supported by the National Natural Science Foundation of China (Nos. 31272229 and 41171234)the National Basic Research Program (973 Program) of China (No. 2013CB127402)
文摘Plants adapt to phosphorus （P） deficiency through a complex of biological processes and many genes are involved. Tomato （Solanum lycopersicum L. ＇Hezuo906＇） plants were selected to grown hydroponically to study the temporal and spatial gene expression patterns of the 14-3-3 gene family and their roles in response to P deficiency in tomato plants. Using real-time reverse-transcriptase polymerase chain reaction （RT-PCR）, we investigated the expression profiles in different tissues （root, stem and leaf） at short-term and long-term P-deficient stress phases. Results revealed that i） four members of 14-3-3 gene family （TFT1, TFT4, TFT6 and TFTT） were involved in the adaptation of tomato plants to P deficiency, ii） TFT7 responded quickly to P deficiency in the root, while TFT6 responded slowly to P deficiency in the leaf, iii） expression response of TFT4 to P-deficient stress was widely distributed in different tissues （root, stem and leaf） while TFT8 only displayed stem-specific expression, and iv） temporal and tissues-specific expression patterns to P deficiency suggested that isoform specificity existed in tomato 14-3-3 gene family. We propose that TFT7 （one member of e-like group in tomato 14-3-3 family） is the early responsive gene and may play a role in the adaptation of tomato plants to shortterm P deficiency, while TFT6 （one member of non-e group in tomato 14-3-3 family） is the later responsive gene and may play a role in the adaptation of tomato plants to long-term P deficiency.
基金supported financially by the National Natural Science Foundation of China (31301610)
文摘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.
基金Financial support received from Department of Biotechnology,Government of India vide grant No.BT/PR-8953/BCE/08/533/2007project sanctioned against grant No.BT/04/NE/2009financial support from Department of Science&Technology,Government of India in the form of a research fellowship under the INSPIRE program
文摘The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbc L and mat K gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825,which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbc L gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68%interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the mat K gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbc L and mat K regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR,designed from rbc L sequences, that could differentiate quinoa from 64 genera including13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years(Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.
