DNA methylation, an epigenetic mechanism used by cells to control gene expression, has an important biological role in plant development and environmental fitness. Since plant DNA methylation is closely related to env...DNA methylation, an epigenetic mechanism used by cells to control gene expression, has an important biological role in plant development and environmental fitness. Since plant DNA methylation is closely related to environmental conditions, variation during the day is expected. Here, in genetically identical plants of Populus nigra clone N46, DNA methylation changes in leaves over a 24 h period were detected using the methylation-sensitive amplification polymorphism method. The results showed different DNA methylation patterns in mature poplar leaves: not only in individuals at the same time, but also in samples at each of the six time during the day. In addition, night samples had a higher percentage of methylation than in morning samples. However, no statistically significant differences were found among the samples gathered at different times. Similar results were obtained for three other P. nigra clones with different genetic backgrounds. Real time qPCR showed that the DNA methyltransferase genes Pt-MET1 and Pt-SOM1 involved in CG DNA methylation in poplar were stable over a 24 h period in leaves of P. nigra N46 compared with circadian-controlled genes. That could be part of the reason that methylation of CCGG sites is stable in those leaves. That DNA methylation differed even in genetically identical plants indicates the specificity of DNA methylation changes in their genomes. No statistically significant differences in methylation changes were found between day and night, suggesting that DNA methylation is more stable than expected and is unlikely to be involved in circadian regulation in plants.展开更多
In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for ...In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for 30 days to illustrate the epigenetic mechanism of osmoregulation.Thirty primers were used to analyze the difference of methylation level of different tissues.The results showed that the baseline methylation level of muscle,hepatopancreas and gill was 47.31%,22.94%and 17.69%,respectively.After exposed to low salinity stress,the methylation epiloci changed in the three tissues.Both demethylation and methylation processes occurred under low salinity stress.The methylation ratio decreased in muscle and gill but increased in hepatopancreas.These results indicated that DNA methylation is tissue-specific when P.trituberculatus responds to low salinity.展开更多
During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we perfo...During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH (genome in situ hybridization) and AFLP (amplified fragment length po-lymorphism) on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary struc-ture variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism (MSAP) analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translo-cation lines (CN12, 20.15%; CN17, 20.91%; CN18, 22.42%), but the ratios of hemimethylated sites were significantly lowered (CN12, 21.41%; CN17, 23.43%; CN18, 22.42%), whereas 16.37% were fully-me-thylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of me-thylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns (33.74%), 9 demethylation patterns (22.76%) and 7 uncertain patterns (4.07%). In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.展开更多
Current methods for single nucleotide polymorphism （SNP） analysis are timeconsuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification （MASA） method fo...Current methods for single nucleotide polymorphism （SNP） analysis are timeconsuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification （MASA） method for rapid SNP analysis. The method is a marriage of two technologies： MASA primers for target DNA and a double-stranded DNA-selective fluorescent dye, SYBR Green I. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experimental results showed that the different genotypes can be clearly discriminated by the assay. The real-time fluorescence MASA method will have an enormous potential for fast and reliable SNP analysis due to its simplicity and low cost.展开更多
A new approach combined the specificity of allele-specific amplification （ASA） with the sensitivity of electrochemiluminescence （ECL） assay for single nucleotide polymorphism （SNP） analysis was proposed. Briefly...A new approach combined the specificity of allele-specific amplification （ASA） with the sensitivity of electrochemiluminescence （ECL） assay for single nucleotide polymorphism （SNP） analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru（bpy）3 ^2＋（TBR）-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA-ECL assay. The method is useful in SNP analysis due to its sensitivity,safety, and simplicity.展开更多
An effective PCR protocol for detecting the sequence related amplified polymorphism （SRAP） in rice was developed. One hundred and ten pairs of SRAP primers were used for segregation analysis in an F2 population deri...An effective PCR protocol for detecting the sequence related amplified polymorphism （SRAP） in rice was developed. One hundred and ten pairs of SRAP primers were used for segregation analysis in an F2 population derived from a cross between Shennong 606 and Lijiangxintuanheigu. Among the 110 primer pairs, 35 pairs generated 143 polymorphic bands with an average of 4.09 polymorphic bands per primer pair, and 24 pairs （16.78%） showed the genetic distortion （P〈0.05）. Of the 24 primer pairs, 12 pairs deviated toward the male parent Shennong 606 and 11 pairs toward the female parent Lijiangxintuanheigu, only one toward heterozygote. It was found that the segregation distortion might be caused by the joint gametic and zygotic effects.展开更多
Investigation of the relationships of phenotypic and epigenetic variations might he a good way to dissect the genetic or molecular basis of phenotypic variation and plasticity in plants, Castor bean （Ricinus cornraun...Investigation of the relationships of phenotypic and epigenetic variations might he a good way to dissect the genetic or molecular basis of phenotypic variation and plasticity in plants, Castor bean （Ricinus cornraunis L）, an important non-edible oilseed crop, is a mono-species genus plant in the family Euphorbiaceae. Since it displays rich phenotypic variations with low genetic diversity, castor bean is a good model to investigate the molecular basis of phenotypic and epigenetic variations. Cytosine DNA methylation represents a major molecular mechanism of epigenetic occurrence. In this study, epigenetic diversity of sixty landrace accessions collected worldwide was investigated using the methylation- sensitive amplification polymorphism （MSAP） technique, Results showed that the epigenetic diversity （based on the polymorphism of DNA methylated loci） exhibited a medium variation （Ne = 1.395, He = 0.242, I = 0.366） at the population level though the variation was great, ranging from 3,80% to 3431% among accessions. Both population structure analysis and the phylogenetic construction （using the neighbor-joining criteria） revealed that the two main clades were identified, but they did not display a distinct geographic structure, After inspecting the location of polymorphic methylated loci on genome we identified that the polymorphic methylated loci occur widely in nuclear and organelle genomes. This study provides new data to understand phenotypic and epigenetic variations in castor bean,展开更多
DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth,development,and polyploidization.However,there is still no distinct evidence in tobacco regarding the distrib...DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth,development,and polyploidization.However,there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics.We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco,Nicotiana tabacum,using a methylation-sensitive amplified polymorphism(MSAP) technique.The results showed that methylation existed at a high level among tobacco accessions,among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic.A cluster analysis revealed distinct patterns of geography-specific groups.In addition,three polymorphic sites significantly related to tobacco mosaic virus(TMV) resistance were explored.This suggests that tobacco breeders should pay more attention to epigenetic traits.展开更多
We examined 50 Escherichia coli (E. coli) strains isolated from broiler chickens between January 2013 to March 2014 in order to evaluate the epidemiological prevalence of avian pathogenic E. coli (APEC) in Jordan by m...We examined 50 Escherichia coli (E. coli) strains isolated from broiler chickens between January 2013 to March 2014 in order to evaluate the epidemiological prevalence of avian pathogenic E. coli (APEC) in Jordan by multiplex PCR and random amplification of polymorphic DNA (RAPD) tests. The multiplex polymerase chain reaction (PCR) which was used as tentative criteria of APEC targets 8 virulence associated genes;enteroaggregative toxin (astA), Type 1 fimbria adhesion (fimH), iron-repressible protein (irp2), P fimbriae (papC), aerobactin (iucD), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), and colicin V plasmid operon (cva/cvi) genes. The number of detected genes could be used as a reliable index of their virulence. E. coli strains already typed as an APEC always harbor 5 to 8 genes, but non-APEC strains harbor less than 4 genes. Assuming the criteria of an APEC is possession of 5 or more virulence associated genes;we found that all 50 E. coli strains were classified as APEC strains. The RAPD analysis showed that the E. coli strains could be grouped into 35 of RAPD types by using these two different RAPD primer sets, RAPD analysis primer 4 5'AAGAGCCCGT5', and RAPD analysis primer 6 5'CCCGTCAGCA3'. The current study confirmed the endemic nature of APEC in broiler flocks in Jordan. It is essential that the biosecurity on poultry farms should be improved to prevent the introduction and dissemination of APEC and other agents. Furthermore, farmers need to be educated about the signs, lesions, and the importance of this agent.展开更多
Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins,The association of Single Nucleotide Polymorphism(SNP)in the calpastatin gene with meat tenderness is a...Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins,The association of Single Nucleotide Polymorphism(SNP)in the calpastatin gene with meat tenderness is an important topic in meat production.Therefore efficient procedure to investigate the SNP is necessary.The objectives of this study were to detect the SNP of calpastatin gene at domain L marker(G/C transversion)of the Kamphaengsaen beef breed(KPS cattle;n=26)by the Amplification Refractory Mutation System(ARMS)compared with the Restriction Fragment Length Polymorphism(RFLP)methods and to determine the genotypes of the KPS cattle at that marker.Genomic DNA of calpastatin gene extracted from blood of the KPS cattle was detected with ARMS and RFLP methods.The ARMS system has utilized two primer pairs to amplify the two different alleles of a polymorphism in single PCR reaction to detected single base mutation.In this method,the alleles-specific primers had a mismatch at 3'terminal base and a second deliberate mismatch at position-2 from 3'terminus.While the RFLP method detected a polymorphism using PCR-base technique follow by RsaI restriction enzyme.Amplification of the ARMS method revealed that the results were not different from the conventional method of RFLP.Analysis of genotypes revealed that the KPS cattle inherited the CC,CG and GG genotypes at domain L marker.These were reliable when verified by nucleotide sequence analysis of PCR products.The animals were genotyped and determined for tenderness phenotype with this marker that predicted variation of an intronic polymorphism at domain L of the calpastatin gene.Therefore,the ARMS method was simple,efficient technique,and suitable for detecting SNP at domain L marker of the calpastatin gene.展开更多
A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and te...A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.展开更多
【目的】蔗茅(Erianthus fulvus)是甘蔗重要的野生资源,可用于改良品种的抗逆性和产量。系统鉴定和开发蔗茅基因组中的简单重复序列(simple sequence repeat,SSR)位点,筛选可利用的多态性SSR标记,解析蔗茅资源的遗传多样性特征,开发重...【目的】蔗茅(Erianthus fulvus)是甘蔗重要的野生资源,可用于改良品种的抗逆性和产量。系统鉴定和开发蔗茅基因组中的简单重复序列(simple sequence repeat,SSR)位点,筛选可利用的多态性SSR标记,解析蔗茅资源的遗传多样性特征,开发重要性状关联分子标记,对于利用蔗茅基因资源改良品种性状具有重要意义。【方法】采用生物信息学软件TBtools中的SSRminer模块,对二倍体蔗茅全基因组序列进行系统性SSR位点挖掘,并对所得数据进行统计分析,揭示其在基因组中的分布模式及规律。利用Batch Target Region Primer Design功能批量设计SSR引物,并通过Primer check工具评估引物的特异性。在6个蔗茅种质中,通过对随机合成的50对SSR引物和14对来自甘蔗的SSR引物进行扩增效率和多态性比较分析,验证引物多态性。【结果】共鉴定出152707个蔗茅SSR位点,平均检测频率为5.64 kb/个,大部分分布在基因间区。SSR类型分布以单碱基、二碱基、三碱基为主。二核苷酸SSR类型中的基序重复次数变化最大,而五核苷酸的基序重复次数变异最低。共检测出883种不同的SSR基序重复类型,其中,A/T和AT/TA是最为丰富。共设计144692对SSR引物,其中85025对显示高特异性。这些特异性引物在基因组上呈现出两端密集、中间稀疏的分布特点。扩增试验显示,50对随机合成的SSR引物中,有42对在蔗茅中扩增出稳定清晰的条带,其中32对显示出多态性,多态率为64.0%。与甘蔗SSR引物相比,蔗茅SSR引物表现出更高的扩增效率和更好的多态性水平。经过筛选,从32对有效的蔗茅SSR引物中确定了16对多态性好且扩增条带清晰的引物。这16对引物共扩增出72条条带,多态性信息量(polymorphism information content,PIC)范围为0.63—0.83,平均PIC值为0.74,表明它们在蔗茅种质资源的多态性分析和分子标记研究中具有有效性和实用性。【结论】揭示了蔗茅基因组中高丰度和多样性的SSR分布特征。获得16对扩增效率高、多态性良好的SSR引物。展开更多
基金supported by National Nonprofit Institute Research Grant of Chinese Academy of Forestry(TGB2013010)
文摘DNA methylation, an epigenetic mechanism used by cells to control gene expression, has an important biological role in plant development and environmental fitness. Since plant DNA methylation is closely related to environmental conditions, variation during the day is expected. Here, in genetically identical plants of Populus nigra clone N46, DNA methylation changes in leaves over a 24 h period were detected using the methylation-sensitive amplification polymorphism method. The results showed different DNA methylation patterns in mature poplar leaves: not only in individuals at the same time, but also in samples at each of the six time during the day. In addition, night samples had a higher percentage of methylation than in morning samples. However, no statistically significant differences were found among the samples gathered at different times. Similar results were obtained for three other P. nigra clones with different genetic backgrounds. Real time qPCR showed that the DNA methyltransferase genes Pt-MET1 and Pt-SOM1 involved in CG DNA methylation in poplar were stable over a 24 h period in leaves of P. nigra N46 compared with circadian-controlled genes. That could be part of the reason that methylation of CCGG sites is stable in those leaves. That DNA methylation differed even in genetically identical plants indicates the specificity of DNA methylation changes in their genomes. No statistically significant differences in methylation changes were found between day and night, suggesting that DNA methylation is more stable than expected and is unlikely to be involved in circadian regulation in plants.
基金supported by the grants from the National Natural Science Foundation of China (No. 4147 6124)the Natural Science Foundation of Zhejiang Province (No. LY17C190005)+3 种基金the Major Agriculture Program of Ningbo (No. 2017C110007)the Ningbo Science and Technology Project (No. 2016C10037)the Open Fund of Ningbo University (No. xkzsc1505)K C Wong Magana Fund in Ningbo University
文摘In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for 30 days to illustrate the epigenetic mechanism of osmoregulation.Thirty primers were used to analyze the difference of methylation level of different tissues.The results showed that the baseline methylation level of muscle,hepatopancreas and gill was 47.31%,22.94%and 17.69%,respectively.After exposed to low salinity stress,the methylation epiloci changed in the three tissues.Both demethylation and methylation processes occurred under low salinity stress.The methylation ratio decreased in muscle and gill but increased in hepatopancreas.These results indicated that DNA methylation is tissue-specific when P.trituberculatus responds to low salinity.
基金the National Natural Science Foundation of China (Grants No. 30671136 and 30730065)the China Postdoctoral Science Foundation (Grant No. 20070411158)+1 种基金the Program for New Century Excellent Talents in University from Ministry of Education,China (Grant No. NCET-06-0810)the Youth Foundation of University of Electronic Science and Technology of China (Grant No. L08010901JX0677)
文摘During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH (genome in situ hybridization) and AFLP (amplified fragment length po-lymorphism) on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary struc-ture variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism (MSAP) analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translo-cation lines (CN12, 20.15%; CN17, 20.91%; CN18, 22.42%), but the ratios of hemimethylated sites were significantly lowered (CN12, 21.41%; CN17, 23.43%; CN18, 22.42%), whereas 16.37% were fully-me-thylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of me-thylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns (33.74%), 9 demethylation patterns (22.76%) and 7 uncertain patterns (4.07%). In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.
基金This research is supported by the National Natural Science Foundation of China(60378043,30470494)the Natural Science Foundation of Guangdong Province(015012,04010394).
文摘Current methods for single nucleotide polymorphism （SNP） analysis are timeconsuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification （MASA） method for rapid SNP analysis. The method is a marriage of two technologies： MASA primers for target DNA and a double-stranded DNA-selective fluorescent dye, SYBR Green I. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experimental results showed that the different genotypes can be clearly discriminated by the assay. The real-time fluorescence MASA method will have an enormous potential for fast and reliable SNP analysis due to its simplicity and low cost.
基金the National Natural Science Foundation of China （Nos. 30600128, 30670507,30470494） the Natural Science Foundation of Guangdong Province （No. 015012）.
文摘A new approach combined the specificity of allele-specific amplification （ASA） with the sensitivity of electrochemiluminescence （ECL） assay for single nucleotide polymorphism （SNP） analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru（bpy）3 ^2＋（TBR）-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA-ECL assay. The method is useful in SNP analysis due to its sensitivity,safety, and simplicity.
文摘An effective PCR protocol for detecting the sequence related amplified polymorphism （SRAP） in rice was developed. One hundred and ten pairs of SRAP primers were used for segregation analysis in an F2 population derived from a cross between Shennong 606 and Lijiangxintuanheigu. Among the 110 primer pairs, 35 pairs generated 143 polymorphic bands with an average of 4.09 polymorphic bands per primer pair, and 24 pairs （16.78%） showed the genetic distortion （P〈0.05）. Of the 24 primer pairs, 12 pairs deviated toward the male parent Shennong 606 and 11 pairs toward the female parent Lijiangxintuanheigu, only one toward heterozygote. It was found that the segregation distortion might be caused by the joint gametic and zygotic effects.
基金jointly supported by Chinese National Key Technology R & D Program (2015BAD15B02)National Natural Science Foundation of China (31661143002 and 31501034)
文摘Investigation of the relationships of phenotypic and epigenetic variations might he a good way to dissect the genetic or molecular basis of phenotypic variation and plasticity in plants, Castor bean （Ricinus cornraunis L）, an important non-edible oilseed crop, is a mono-species genus plant in the family Euphorbiaceae. Since it displays rich phenotypic variations with low genetic diversity, castor bean is a good model to investigate the molecular basis of phenotypic and epigenetic variations. Cytosine DNA methylation represents a major molecular mechanism of epigenetic occurrence. In this study, epigenetic diversity of sixty landrace accessions collected worldwide was investigated using the methylation- sensitive amplification polymorphism （MSAP） technique, Results showed that the epigenetic diversity （based on the polymorphism of DNA methylated loci） exhibited a medium variation （Ne = 1.395, He = 0.242, I = 0.366） at the population level though the variation was great, ranging from 3,80% to 3431% among accessions. Both population structure analysis and the phylogenetic construction （using the neighbor-joining criteria） revealed that the two main clades were identified, but they did not display a distinct geographic structure, After inspecting the location of polymorphic methylated loci on genome we identified that the polymorphic methylated loci occur widely in nuclear and organelle genomes. This study provides new data to understand phenotypic and epigenetic variations in castor bean,
基金supported by the Program for High-Quality Tobacco Development of China (No. [2010]221)the Foundation of Science and Technology of Guizhou Province (No. J[2010]2251)the Program for Guizhou Tobacco Science of China (No. 200910)
文摘DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth,development,and polyploidization.However,there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics.We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco,Nicotiana tabacum,using a methylation-sensitive amplified polymorphism(MSAP) technique.The results showed that methylation existed at a high level among tobacco accessions,among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic.A cluster analysis revealed distinct patterns of geography-specific groups.In addition,three polymorphic sites significantly related to tobacco mosaic virus(TMV) resistance were explored.This suggests that tobacco breeders should pay more attention to epigenetic traits.
文摘We examined 50 Escherichia coli (E. coli) strains isolated from broiler chickens between January 2013 to March 2014 in order to evaluate the epidemiological prevalence of avian pathogenic E. coli (APEC) in Jordan by multiplex PCR and random amplification of polymorphic DNA (RAPD) tests. The multiplex polymerase chain reaction (PCR) which was used as tentative criteria of APEC targets 8 virulence associated genes;enteroaggregative toxin (astA), Type 1 fimbria adhesion (fimH), iron-repressible protein (irp2), P fimbriae (papC), aerobactin (iucD), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), and colicin V plasmid operon (cva/cvi) genes. The number of detected genes could be used as a reliable index of their virulence. E. coli strains already typed as an APEC always harbor 5 to 8 genes, but non-APEC strains harbor less than 4 genes. Assuming the criteria of an APEC is possession of 5 or more virulence associated genes;we found that all 50 E. coli strains were classified as APEC strains. The RAPD analysis showed that the E. coli strains could be grouped into 35 of RAPD types by using these two different RAPD primer sets, RAPD analysis primer 4 5'AAGAGCCCGT5', and RAPD analysis primer 6 5'CCCGTCAGCA3'. The current study confirmed the endemic nature of APEC in broiler flocks in Jordan. It is essential that the biosecurity on poultry farms should be improved to prevent the introduction and dissemination of APEC and other agents. Furthermore, farmers need to be educated about the signs, lesions, and the importance of this agent.
文摘Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins,The association of Single Nucleotide Polymorphism(SNP)in the calpastatin gene with meat tenderness is an important topic in meat production.Therefore efficient procedure to investigate the SNP is necessary.The objectives of this study were to detect the SNP of calpastatin gene at domain L marker(G/C transversion)of the Kamphaengsaen beef breed(KPS cattle;n=26)by the Amplification Refractory Mutation System(ARMS)compared with the Restriction Fragment Length Polymorphism(RFLP)methods and to determine the genotypes of the KPS cattle at that marker.Genomic DNA of calpastatin gene extracted from blood of the KPS cattle was detected with ARMS and RFLP methods.The ARMS system has utilized two primer pairs to amplify the two different alleles of a polymorphism in single PCR reaction to detected single base mutation.In this method,the alleles-specific primers had a mismatch at 3'terminal base and a second deliberate mismatch at position-2 from 3'terminus.While the RFLP method detected a polymorphism using PCR-base technique follow by RsaI restriction enzyme.Amplification of the ARMS method revealed that the results were not different from the conventional method of RFLP.Analysis of genotypes revealed that the KPS cattle inherited the CC,CG and GG genotypes at domain L marker.These were reliable when verified by nucleotide sequence analysis of PCR products.The animals were genotyped and determined for tenderness phenotype with this marker that predicted variation of an intronic polymorphism at domain L of the calpastatin gene.Therefore,the ARMS method was simple,efficient technique,and suitable for detecting SNP at domain L marker of the calpastatin gene.
基金supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (20070075003, 20091301120003)the Natural Science Foundation of Hebei Province (B2009000170)
文摘A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.
文摘【目的】蔗茅(Erianthus fulvus)是甘蔗重要的野生资源,可用于改良品种的抗逆性和产量。系统鉴定和开发蔗茅基因组中的简单重复序列(simple sequence repeat,SSR)位点,筛选可利用的多态性SSR标记,解析蔗茅资源的遗传多样性特征,开发重要性状关联分子标记,对于利用蔗茅基因资源改良品种性状具有重要意义。【方法】采用生物信息学软件TBtools中的SSRminer模块,对二倍体蔗茅全基因组序列进行系统性SSR位点挖掘,并对所得数据进行统计分析,揭示其在基因组中的分布模式及规律。利用Batch Target Region Primer Design功能批量设计SSR引物,并通过Primer check工具评估引物的特异性。在6个蔗茅种质中,通过对随机合成的50对SSR引物和14对来自甘蔗的SSR引物进行扩增效率和多态性比较分析,验证引物多态性。【结果】共鉴定出152707个蔗茅SSR位点,平均检测频率为5.64 kb/个,大部分分布在基因间区。SSR类型分布以单碱基、二碱基、三碱基为主。二核苷酸SSR类型中的基序重复次数变化最大,而五核苷酸的基序重复次数变异最低。共检测出883种不同的SSR基序重复类型,其中,A/T和AT/TA是最为丰富。共设计144692对SSR引物,其中85025对显示高特异性。这些特异性引物在基因组上呈现出两端密集、中间稀疏的分布特点。扩增试验显示,50对随机合成的SSR引物中,有42对在蔗茅中扩增出稳定清晰的条带,其中32对显示出多态性,多态率为64.0%。与甘蔗SSR引物相比,蔗茅SSR引物表现出更高的扩增效率和更好的多态性水平。经过筛选,从32对有效的蔗茅SSR引物中确定了16对多态性好且扩增条带清晰的引物。这16对引物共扩增出72条条带,多态性信息量(polymorphism information content,PIC)范围为0.63—0.83,平均PIC值为0.74,表明它们在蔗茅种质资源的多态性分析和分子标记研究中具有有效性和实用性。【结论】揭示了蔗茅基因组中高丰度和多样性的SSR分布特征。获得16对扩增效率高、多态性良好的SSR引物。
