[Objective] This research aimed to search a best method for extracting the genomic DNA of Cymbidium ensifolium and establish the optimized ISSR-PCR reaction system.[Method] Genomic DNA was extracted from C.ensifolium ...[Objective] This research aimed to search a best method for extracting the genomic DNA of Cymbidium ensifolium and establish the optimized ISSR-PCR reaction system.[Method] Genomic DNA was extracted from C.ensifolium leaves by modified CTAB method.ISSR-PCR reaction system for C.ensifolium was optimized.[Result] High-quality genomic DNA was obtained from C.ensifolium.The 25 μl optimized ISSR-PCR reaction system for C.ensifolium contained 2.5 μl 10× PCR buffer,2.5 mmol/L MgCl2,240 ng template DNA,160 μmol/L dNTPs,1.25 U Taq DNA polymerase,0.4 μmol/L primer and 15.78 μl ddH2O.The optimal PCR procedures were：94 ℃ pre-denaturation for 5 min and then 40 cycles,94 ℃ denaturation for 30 s,50-60 ℃ annealing for 30 s （annealing temperature according to different primers）,72 ℃ extension for 50 s and a 72 ℃ extension for 7 min.[Conclusion] An optimized ISSR-PCR reaction system for C.ensifolium was established,which provides a basis for further study on genetic diversity of C.ensifolium by using ISSR molecular marker technique.展开更多
[Objective] The aim was to provide molecular basis for the identification of species in the moss family Bryaceae by the construction of inter-simple sequence repeats （ISSR） fingerprinting. [Method] In order to seek ...[Objective] The aim was to provide molecular basis for the identification of species in the moss family Bryaceae by the construction of inter-simple sequence repeats （ISSR） fingerprinting. [Method] In order to seek standardizing PCR reaction set-up, an orthogonal design was used to optimize ISSR-PCR amplification system of Bryaceae in five factors （Mg2＋, dNTPs, primer, DNA template, Taq DNA polymerase） at four levels respectively. [Result] A suitable ISSR reaction system was obtained, namely： 20 μl reaction system containing 5 ng of DNA template, 0.2 μmol/L primer, 2.25 mmol/L MgCl2, 0.6 U of Taq DNA polymerase, 0.4 mmol/L dNTPs. Proper annealing temperature was found at 48-50 ℃.The above system and six ISSR-PCR primers were used for the PCR amplification of 14 samples from Bryaceae and the related species in Mniaceae. A total of 86 bands were amplified, all showed polymorphism. NJ cluster analysis showed a star-shaped cladogram. [Conclusion] The results manifested that ISSR fingerprinting could provide the appropriate degree of polymorphism at low taxonomic level, so it would be a useful tool to provide additional evidence for resolving taxonomic relationships at the species level of Bryaceae.展开更多
[Objective] The experiment aimed to determine the optimum ISSR-PCR reaction system of Picea crassifolia kom. [Method] Picea crassifolia kom. was used as material to select and optimize influencing factors of ISSR-PCR ...[Objective] The experiment aimed to determine the optimum ISSR-PCR reaction system of Picea crassifolia kom. [Method] Picea crassifolia kom. was used as material to select and optimize influencing factors of ISSR-PCR such as Mg2+, dNTPs, Taq DNA polymerase, template DNA, primers, annealing temperature. [Result] The optimum ISSR-PCR reaction system in 20 μl reaction system was consisted of 1 μl 10×buffer, 1.5 mmol/L Mg2+, 0.2 mmol/L dNTPs, 1.0 U Taq DNA polymerase, 40 ng template DNA, 0.6 μmol/L primers. According to gradient test of annealing temperature in optimum ISSR-PCR reaction system of Picea crassifolia kom, it was found that the optimum annealing temperature of UBC 818 was 54.2 ℃ and the annealing temperature was different for different primers.[Conclusion]The construction of ISSR-PCR reaction system provided technical basis for classification of germplasm resources, construction of genetic map, gene mapping of Picea crassifolia kom. through using ISSR technology.展开更多
[Objective] By using the genomic DNA of Cymbidium faberi Rolfe as template,the factors that affect the result of ISSR-PCR reaction system were researched and the optimal system was established.[Method] The genomic DNA...[Objective] By using the genomic DNA of Cymbidium faberi Rolfe as template,the factors that affect the result of ISSR-PCR reaction system were researched and the optimal system was established.[Method] The genomic DNA was extracted from C.faberi Rolfe with method of modified CTAB.Different factors which affected ISSR amplification reaction were optimized.[Result] High-quality genomic DNA was obtained from C.faberi Rolfe.And the optimal reaction system was as follows：25 μl amplification reactions system contained 2.5 μl 10 × PCR buffer,2.0 mmol/L MgCl2,60 ng template DNA,160 μmol/L dNTPs,1.25 U Taq DNA polymerase,0.4 μmol/L ISSR primer and 15.85 μl ddH2O.The optimal amplification procedures were pre-denaturing for 5 min at 94 ℃,followed by 40 cycles of denaturing for 30 s at 94 ℃,annealing for 30 s at a temperature of 2-3 ℃ lower than melting temperature of each primer pair,extension for 50 s at 72 ℃.Then extension step of 7 min at 72 ℃ was performed.[Conclusion] The optimal system could provide a favorable basis for further study on genetic diversity of C.faberi Rolfe by using ISSR molecular marker technique.展开更多
Based on single-factor experiment and L9 （34 ） orthogonal experiment, a stable ISSR-PCR reaction system of Salvia przewalskii Maxim. was established and optimized. The resuhs indicated that the optimal concentration...Based on single-factor experiment and L9 （34 ） orthogonal experiment, a stable ISSR-PCR reaction system of Salvia przewalskii Maxim. was established and optimized. The resuhs indicated that the optimal concentrations of various components in a 20 ill ISSR-PCR reaction system of S. przewalskii Maxim. were ： 2 ul of 10 x buffer （ Mg2＋）, 0.4 mmol/L dNTPs, 1.0 U of Taq DNA polymerase, 0.3 umol/L ISSR primers and 30 ng of DNA template. This study laid the foun- dation for the utilization of S. przewalskii Maxim. germplasm resources.展开更多
[Objective] To investigate the impacts of ISSR-PCR amplification factors, for the establishment and optimization of ISSR-PCR reaction system for Ligusticum chuanxiong hort. [Method] Using genomic DNA of Chuanxiong lea...[Objective] To investigate the impacts of ISSR-PCR amplification factors, for the establishment and optimization of ISSR-PCR reaction system for Ligusticum chuanxiong hort. [Method] Using genomic DNA of Chuanxiong leaf extracted via an improved CTAB method as template, single factor analysis was performed to investigate the impacts of DNA template concentration, Mg2＋ concentration, dNTPs concentration, primer concentration, Taq DNA polymerase concentration on ISSR-PCR amplification and to optimize this system for Ligusticum chuanxiong hort. [Result] The ISSR-PCR amplification（25 μl） suitable for Ligusticum chuanxiong hort. was determined to be composed of 2.5 μl of 10×reaction buffer, 2.1 mmol/L MgCl2, 300 μmol/L dNTPs, 0.4 μmol/L primer, 1.0 U Taq DNA polymerase and 20-40 ng genomic DNA. [Conclusion] Our study laid basis for analyzing the genetic diversity of Ligusticum chuanxiong hort. resources distributed in 17 different areas of China.展开更多
Virgin Coconut Meal (VCM) was used for the development of instant wheat sooji (semolina) halwa mix with better nutritional attributes. Central composite rotatable design (CCRD) with 2 independent variables (sugar and ...Virgin Coconut Meal (VCM) was used for the development of instant wheat sooji (semolina) halwa mix with better nutritional attributes. Central composite rotatable design (CCRD) with 2 independent variables (sugar and VCM) and 4 responses (lightness, redness, taste and overall acceptability) was used for the optimisation. VCM incorporated instant halwa mix prepared using optimised levels of ingredients contained moisture 0.95%;fat 26.2%;protein 7.65%;total ash 0.86%;fibre 1.02% and received overall acceptability score of 8.5 on a 9 point hedonic scale providing 523.86 K·cal/ 100g. The changes in quality of stored VCM incorporated instant halwa mix packed in polypropylene (PP, 75 μ) and laminates of metallised polyester (MP, 90 μ) were monitored in order to assess the shelf-life. Instant halwa mix remained stable and acceptable for one year in both the packaging materials under ambient temperature conditions (15℃ - 34℃). However, the rate of lipid peroxidation was found to be slightly higher in PP packed samples as compared to MP packed ones. Fatty acid composition of VCM incorporated instant halwa mix remained practically unchanged during storage. Oleic acid was the major fatty acid present in fat extracted from halwa mix followed by palmitic and lauric acids.展开更多
An one factor test was used to optimize ISSR-PCR amplification system on macadamia in four levels of five factors(Taq DNA polymerase,template DNA,dNTPs,primer and Mg 2+,respectively) in this study.The results showed t...An one factor test was used to optimize ISSR-PCR amplification system on macadamia in four levels of five factors(Taq DNA polymerase,template DNA,dNTPs,primer and Mg 2+,respectively) in this study.The results showed that the 25 μL reaction system consisted of 1×PCR buffer,1 U Taq DNA polymerase,20 ng template DNA,0.15 mmol·L-1 dNTPs,0.25 μmol·L-1 primer and 2.5 mmol·L-1 Mg 2+.In addition,adding 0.4% formamide was able to reduce the background noise.The optimal PCR amplification process was as the following:1 cycle initial denaturalization at 94 ℃ for 5 min,followed by 35 cycles,which included denaturalization at 94 ℃ for 30 s,annealing for 1 min,and extension at 72 ℃ for 2 min,and then extension at 72 ℃ for 7 min,and finally holding the samples at 4 ℃.展开更多
文摘[Objective] This research aimed to search a best method for extracting the genomic DNA of Cymbidium ensifolium and establish the optimized ISSR-PCR reaction system.[Method] Genomic DNA was extracted from C.ensifolium leaves by modified CTAB method.ISSR-PCR reaction system for C.ensifolium was optimized.[Result] High-quality genomic DNA was obtained from C.ensifolium.The 25 μl optimized ISSR-PCR reaction system for C.ensifolium contained 2.5 μl 10× PCR buffer,2.5 mmol/L MgCl2,240 ng template DNA,160 μmol/L dNTPs,1.25 U Taq DNA polymerase,0.4 μmol/L primer and 15.78 μl ddH2O.The optimal PCR procedures were：94 ℃ pre-denaturation for 5 min and then 40 cycles,94 ℃ denaturation for 30 s,50-60 ℃ annealing for 30 s （annealing temperature according to different primers）,72 ℃ extension for 50 s and a 72 ℃ extension for 7 min.[Conclusion] An optimized ISSR-PCR reaction system for C.ensifolium was established,which provides a basis for further study on genetic diversity of C.ensifolium by using ISSR molecular marker technique.
基金Supported by Natural Science Foundation of Hebei Province(C2006000147)Zhengzhou Science and Technology Program(10PTGN449-6)~~
文摘[Objective] The aim was to provide molecular basis for the identification of species in the moss family Bryaceae by the construction of inter-simple sequence repeats （ISSR） fingerprinting. [Method] In order to seek standardizing PCR reaction set-up, an orthogonal design was used to optimize ISSR-PCR amplification system of Bryaceae in five factors （Mg2＋, dNTPs, primer, DNA template, Taq DNA polymerase） at four levels respectively. [Result] A suitable ISSR reaction system was obtained, namely： 20 μl reaction system containing 5 ng of DNA template, 0.2 μmol/L primer, 2.25 mmol/L MgCl2, 0.6 U of Taq DNA polymerase, 0.4 mmol/L dNTPs. Proper annealing temperature was found at 48-50 ℃.The above system and six ISSR-PCR primers were used for the PCR amplification of 14 samples from Bryaceae and the related species in Mniaceae. A total of 86 bands were amplified, all showed polymorphism. NJ cluster analysis showed a star-shaped cladogram. [Conclusion] The results manifested that ISSR fingerprinting could provide the appropriate degree of polymorphism at low taxonomic level, so it would be a useful tool to provide additional evidence for resolving taxonomic relationships at the species level of Bryaceae.
文摘[Objective] The experiment aimed to determine the optimum ISSR-PCR reaction system of Picea crassifolia kom. [Method] Picea crassifolia kom. was used as material to select and optimize influencing factors of ISSR-PCR such as Mg2+, dNTPs, Taq DNA polymerase, template DNA, primers, annealing temperature. [Result] The optimum ISSR-PCR reaction system in 20 μl reaction system was consisted of 1 μl 10×buffer, 1.5 mmol/L Mg2+, 0.2 mmol/L dNTPs, 1.0 U Taq DNA polymerase, 40 ng template DNA, 0.6 μmol/L primers. According to gradient test of annealing temperature in optimum ISSR-PCR reaction system of Picea crassifolia kom, it was found that the optimum annealing temperature of UBC 818 was 54.2 ℃ and the annealing temperature was different for different primers.[Conclusion]The construction of ISSR-PCR reaction system provided technical basis for classification of germplasm resources, construction of genetic map, gene mapping of Picea crassifolia kom. through using ISSR technology.
基金Supported by a Program from the State Environmental Protection Administration(20061A0013)~~
文摘[Objective] By using the genomic DNA of Cymbidium faberi Rolfe as template,the factors that affect the result of ISSR-PCR reaction system were researched and the optimal system was established.[Method] The genomic DNA was extracted from C.faberi Rolfe with method of modified CTAB.Different factors which affected ISSR amplification reaction were optimized.[Result] High-quality genomic DNA was obtained from C.faberi Rolfe.And the optimal reaction system was as follows：25 μl amplification reactions system contained 2.5 μl 10 × PCR buffer,2.0 mmol/L MgCl2,60 ng template DNA,160 μmol/L dNTPs,1.25 U Taq DNA polymerase,0.4 μmol/L ISSR primer and 15.85 μl ddH2O.The optimal amplification procedures were pre-denaturing for 5 min at 94 ℃,followed by 40 cycles of denaturing for 30 s at 94 ℃,annealing for 30 s at a temperature of 2-3 ℃ lower than melting temperature of each primer pair,extension for 50 s at 72 ℃.Then extension step of 7 min at 72 ℃ was performed.[Conclusion] The optimal system could provide a favorable basis for further study on genetic diversity of C.faberi Rolfe by using ISSR molecular marker technique.
基金Supported by Project of Qinghai Science and Technology Department(2011-NF19)Fund for Middle-aged and Young Scientists of Qinghai University(2012-QYT-1)
文摘Based on single-factor experiment and L9 （34 ） orthogonal experiment, a stable ISSR-PCR reaction system of Salvia przewalskii Maxim. was established and optimized. The resuhs indicated that the optimal concentrations of various components in a 20 ill ISSR-PCR reaction system of S. przewalskii Maxim. were ： 2 ul of 10 x buffer （ Mg2＋）, 0.4 mmol/L dNTPs, 1.0 U of Taq DNA polymerase, 0.3 umol/L ISSR primers and 30 ng of DNA template. This study laid the foun- dation for the utilization of S. przewalskii Maxim. germplasm resources.
基金Supported by the10th Five Years Key Programs for Science and Technology Development of China(2004BA721A31)~~
文摘[Objective] To investigate the impacts of ISSR-PCR amplification factors, for the establishment and optimization of ISSR-PCR reaction system for Ligusticum chuanxiong hort. [Method] Using genomic DNA of Chuanxiong leaf extracted via an improved CTAB method as template, single factor analysis was performed to investigate the impacts of DNA template concentration, Mg2＋ concentration, dNTPs concentration, primer concentration, Taq DNA polymerase concentration on ISSR-PCR amplification and to optimize this system for Ligusticum chuanxiong hort. [Result] The ISSR-PCR amplification（25 μl） suitable for Ligusticum chuanxiong hort. was determined to be composed of 2.5 μl of 10×reaction buffer, 2.1 mmol/L MgCl2, 300 μmol/L dNTPs, 0.4 μmol/L primer, 1.0 U Taq DNA polymerase and 20-40 ng genomic DNA. [Conclusion] Our study laid basis for analyzing the genetic diversity of Ligusticum chuanxiong hort. resources distributed in 17 different areas of China.
文摘Virgin Coconut Meal (VCM) was used for the development of instant wheat sooji (semolina) halwa mix with better nutritional attributes. Central composite rotatable design (CCRD) with 2 independent variables (sugar and VCM) and 4 responses (lightness, redness, taste and overall acceptability) was used for the optimisation. VCM incorporated instant halwa mix prepared using optimised levels of ingredients contained moisture 0.95%;fat 26.2%;protein 7.65%;total ash 0.86%;fibre 1.02% and received overall acceptability score of 8.5 on a 9 point hedonic scale providing 523.86 K·cal/ 100g. The changes in quality of stored VCM incorporated instant halwa mix packed in polypropylene (PP, 75 μ) and laminates of metallised polyester (MP, 90 μ) were monitored in order to assess the shelf-life. Instant halwa mix remained stable and acceptable for one year in both the packaging materials under ambient temperature conditions (15℃ - 34℃). However, the rate of lipid peroxidation was found to be slightly higher in PP packed samples as compared to MP packed ones. Fatty acid composition of VCM incorporated instant halwa mix remained practically unchanged during storage. Oleic acid was the major fatty acid present in fat extracted from halwa mix followed by palmitic and lauric acids.
文摘An one factor test was used to optimize ISSR-PCR amplification system on macadamia in four levels of five factors(Taq DNA polymerase,template DNA,dNTPs,primer and Mg 2+,respectively) in this study.The results showed that the 25 μL reaction system consisted of 1×PCR buffer,1 U Taq DNA polymerase,20 ng template DNA,0.15 mmol·L-1 dNTPs,0.25 μmol·L-1 primer and 2.5 mmol·L-1 Mg 2+.In addition,adding 0.4% formamide was able to reduce the background noise.The optimal PCR amplification process was as the following:1 cycle initial denaturalization at 94 ℃ for 5 min,followed by 35 cycles,which included denaturalization at 94 ℃ for 30 s,annealing for 1 min,and extension at 72 ℃ for 2 min,and then extension at 72 ℃ for 7 min,and finally holding the samples at 4 ℃.
