[Objective] This study aimed to explore an effective method for rapid salt- extraction of high-quality genomic DNA from dried seeds of plants. [Method] Seeds of seven varieties of crops were ground into powder. A hund...[Objective] This study aimed to explore an effective method for rapid salt- extraction of high-quality genomic DNA from dried seeds of plants. [Method] Seeds of seven varieties of crops were ground into powder. A hundred milligrams of seed powder was added to extracting solution for high salt-extraction of genomic DNA. The yield and quality of extracted DNA were determined by using ultramicro UV/Vis spectrophotometer detection method, PCR and restriction enzyme digestion. [Result] About 619.67-1 811.21 ng of genomic DNA was extracted from per 100 mg of dried seed powder of seven varieties of conventional crops. A260/A280 ratios of the obtained DNA solution all ranged from 1.87 to 2.07, the purity and quality of PCR were suitable for PCR and restriction enzyme digestion. Clear target bands of specific endogenous gene fragments of seven varieties of crops were amplified by PCR, and the obtained DNA could be fully digested with EcoRV and Hindlll.[Conclusion] This method could be used for rapid extraction of high-quality genomic DNA from dried seeds.展开更多
利用磁珠法对甘蓝、白菜、芥菜、洋葱、黄瓜、番茄、辣椒等7种形状、大小不同的蔬菜单粒种子和幼苗进行DNA提取,采用内参基因Actin进行实时荧光定量PCR评价DNA质量,利用KASP(kompetitive allele specific PCR)标记检验DNA质量是否达到K...利用磁珠法对甘蓝、白菜、芥菜、洋葱、黄瓜、番茄、辣椒等7种形状、大小不同的蔬菜单粒种子和幼苗进行DNA提取,采用内参基因Actin进行实时荧光定量PCR评价DNA质量,利用KASP(kompetitive allele specific PCR)标记检验DNA质量是否达到KASP检测要求。结果显示,单粒种子磁珠法提取的各蔬菜DNA平均Ct值均处于19~24范围内。KASP检测进一步证实种子提取DNA的质量达到KASP标记分型的要求。综上所述,本研究建立了一种简单、经济、实用性强的单粒种子DNA提取方法,该方法对不同大小的蔬菜种子具有较好的稳定性,且大幅缩短了获得基因型数据的时间。展开更多
Heterosis is an important biological phenomenon and widely applied in agriculture.Although many studies have been performed by using vegetative organs of F_(1) hybrid plants,how heterosis (or hybrid vigor) is initiate...Heterosis is an important biological phenomenon and widely applied in agriculture.Although many studies have been performed by using vegetative organs of F_(1) hybrid plants,how heterosis (or hybrid vigor) is initiated and formed,particularly the underlying molecular mechanism,remain elusive.Hybrid contemporary seeds of rice indica varieties 9311 and PA64 were innovatively used and analysis of DNA methylome of embryo and endosperm at early developing stages revealed the globally decreased DNA methylation.Genes,especially those relate to hormones function and transcriptional regulation present non-additive methylation.Previously identified heterosis-related superior genes are non-additively methylated in early developing hybrid contemporary seeds,suggesting that key genes/loci responsible for heterosis are epigenetically modified even in early developing hybrid seeds and hypomethylation of hybrid seeds after cross-pollination finally result in the long-term transcriptional change of F_(1) hybrid vegetative tissues after germination,demonstrating that altered DNA methylation in hybrid seeds is essential for initiation regulation and maintenance of heterosis exhibiting in F_(1) hybrid plants.Notably,a large number of genes show non-additive methylation in the endosperm of reciprocal hybrids,suggesting that endosperm might also contribute to heterosis.展开更多
In order to overcome the problems that DNA extraction from wheat leaves for PCR is restricted by seasons and chloroform is difficult to be acquired, a method of DNA extraction from wheat seeds without using chloroform...In order to overcome the problems that DNA extraction from wheat leaves for PCR is restricted by seasons and chloroform is difficult to be acquired, a method of DNA extraction from wheat seeds without using chloroform was estab- lished in this study. The DNA extracted from wheat leaves and seeds using chloro- form was used as control, the quality of DNA extracted from wheat seeds without using chloroform was examined by muiti-PCR. The results showed that the concen- tration of DNA extracted from wheat seeds by chloroform-free method was lower than those extracted from wheat leaves and seeds using chloroform. The amplifica- tion effect of DNA extracted from wheat seeds by chloroform-free method was also a little worse than those extracted from wheat leaves and seeds using chloroform, but this problem can be addressed though loading higher quantity of DNA sample or adopting polyacrylamide gel electrophoresis method. Therefore, the chloroform-free DNA extraction method can be used when there are no special requirements for DNA quality or chloroform cannot be acquired timely.展开更多
We developed a computerized seed identification system. Fifteen rice varietiesthat were widely used in China were analyzed by AFLP fingerprinting. 12 primerpairs were screened, In order to simplify the procedure and c...We developed a computerized seed identification system. Fifteen rice varietiesthat were widely used in China were analyzed by AFLP fingerprinting. 12 primerpairs were screened, In order to simplify the procedure and cut down the cost inseed identification. the least number of primer pairs for practical seed identifi-cation should be seleeled. In this study. 3 primer pairs were selected. They展开更多
Use of a single seed is very useful for genetic studies on Vitis vinifera. However, molecular markers require a fair amount of high purity DNA. Grapevine contains high concentrations of polysaccharides, polyphenols, t...Use of a single seed is very useful for genetic studies on Vitis vinifera. However, molecular markers require a fair amount of high purity DNA. Grapevine contains high concentrations of polysaccharides, polyphenols, tannins and other secondary metabolites. These compounds may hamper the DNA isolation processes and subsequent analysis. In this study we have compared two DNA isolation methods: the NucleoSpin Plant II method and a modified protocol from Doyle and Doyle. The average value of 260/280 nm absorption ratio, which is used to assess the purity of DNA and RNA was 1.8 (accepted as “pure” DNA) and 0.9 (presence of protein or other contaminants) for the first and second method, respectively. Using the NucleoSpin protocol, from a single seed (20 - 35 mg) we obtained an average yield of extracted DNA of 24.8 ± 5.2 to 38.4 ± 11.5 ng·mg-1 dry weight. Among the two protocols examined, the NucleoSpin method was more efficient and gave better quality of DNA values compared to those from the modified Doyle and Doyle procedures.展开更多
基金Supported by Project of Common Safety Assessment Technology for Genetically Modified Organisms of the Ministry of Agriculture of PRC (2011ZX08011-006)~~
文摘[Objective] This study aimed to explore an effective method for rapid salt- extraction of high-quality genomic DNA from dried seeds of plants. [Method] Seeds of seven varieties of crops were ground into powder. A hundred milligrams of seed powder was added to extracting solution for high salt-extraction of genomic DNA. The yield and quality of extracted DNA were determined by using ultramicro UV/Vis spectrophotometer detection method, PCR and restriction enzyme digestion. [Result] About 619.67-1 811.21 ng of genomic DNA was extracted from per 100 mg of dried seed powder of seven varieties of conventional crops. A260/A280 ratios of the obtained DNA solution all ranged from 1.87 to 2.07, the purity and quality of PCR were suitable for PCR and restriction enzyme digestion. Clear target bands of specific endogenous gene fragments of seven varieties of crops were amplified by PCR, and the obtained DNA could be fully digested with EcoRV and Hindlll.[Conclusion] This method could be used for rapid extraction of high-quality genomic DNA from dried seeds.
文摘利用磁珠法对甘蓝、白菜、芥菜、洋葱、黄瓜、番茄、辣椒等7种形状、大小不同的蔬菜单粒种子和幼苗进行DNA提取,采用内参基因Actin进行实时荧光定量PCR评价DNA质量,利用KASP(kompetitive allele specific PCR)标记检验DNA质量是否达到KASP检测要求。结果显示,单粒种子磁珠法提取的各蔬菜DNA平均Ct值均处于19~24范围内。KASP检测进一步证实种子提取DNA的质量达到KASP标记分型的要求。综上所述,本研究建立了一种简单、经济、实用性强的单粒种子DNA提取方法,该方法对不同大小的蔬菜种子具有较好的稳定性,且大幅缩短了获得基因型数据的时间。
基金supported by the Ministry of Science and Technology of China (2012CB944804)the National Transformation Science and Technology Program (2016ZX08001006-009)the National Key Research and Development Program of China (2016YFD0100501, 2016YFD0100902)。
文摘Heterosis is an important biological phenomenon and widely applied in agriculture.Although many studies have been performed by using vegetative organs of F_(1) hybrid plants,how heterosis (or hybrid vigor) is initiated and formed,particularly the underlying molecular mechanism,remain elusive.Hybrid contemporary seeds of rice indica varieties 9311 and PA64 were innovatively used and analysis of DNA methylome of embryo and endosperm at early developing stages revealed the globally decreased DNA methylation.Genes,especially those relate to hormones function and transcriptional regulation present non-additive methylation.Previously identified heterosis-related superior genes are non-additively methylated in early developing hybrid contemporary seeds,suggesting that key genes/loci responsible for heterosis are epigenetically modified even in early developing hybrid seeds and hypomethylation of hybrid seeds after cross-pollination finally result in the long-term transcriptional change of F_(1) hybrid vegetative tissues after germination,demonstrating that altered DNA methylation in hybrid seeds is essential for initiation regulation and maintenance of heterosis exhibiting in F_(1) hybrid plants.Notably,a large number of genes show non-additive methylation in the endosperm of reciprocal hybrids,suggesting that endosperm might also contribute to heterosis.
基金Supported by Science and Technology Innovation Team Project for Dryland Grain and Oil Crops of Zhejiang Province(2011R50026-13)Major Program of Science and Technology of Zhejiang Province(2012C12902-2-3)Special Science and Technology Innovation Project for Seeds and Seedlings of Wenzhou City(N20120023)~~
文摘In order to overcome the problems that DNA extraction from wheat leaves for PCR is restricted by seasons and chloroform is difficult to be acquired, a method of DNA extraction from wheat seeds without using chloroform was estab- lished in this study. The DNA extracted from wheat leaves and seeds using chloro- form was used as control, the quality of DNA extracted from wheat seeds without using chloroform was examined by muiti-PCR. The results showed that the concen- tration of DNA extracted from wheat seeds by chloroform-free method was lower than those extracted from wheat leaves and seeds using chloroform. The amplifica- tion effect of DNA extracted from wheat seeds by chloroform-free method was also a little worse than those extracted from wheat leaves and seeds using chloroform, but this problem can be addressed though loading higher quantity of DNA sample or adopting polyacrylamide gel electrophoresis method. Therefore, the chloroform-free DNA extraction method can be used when there are no special requirements for DNA quality or chloroform cannot be acquired timely.
文摘We developed a computerized seed identification system. Fifteen rice varietiesthat were widely used in China were analyzed by AFLP fingerprinting. 12 primerpairs were screened, In order to simplify the procedure and cut down the cost inseed identification. the least number of primer pairs for practical seed identifi-cation should be seleeled. In this study. 3 primer pairs were selected. They
文摘Use of a single seed is very useful for genetic studies on Vitis vinifera. However, molecular markers require a fair amount of high purity DNA. Grapevine contains high concentrations of polysaccharides, polyphenols, tannins and other secondary metabolites. These compounds may hamper the DNA isolation processes and subsequent analysis. In this study we have compared two DNA isolation methods: the NucleoSpin Plant II method and a modified protocol from Doyle and Doyle. The average value of 260/280 nm absorption ratio, which is used to assess the purity of DNA and RNA was 1.8 (accepted as “pure” DNA) and 0.9 (presence of protein or other contaminants) for the first and second method, respectively. Using the NucleoSpin protocol, from a single seed (20 - 35 mg) we obtained an average yield of extracted DNA of 24.8 ± 5.2 to 38.4 ± 11.5 ng·mg-1 dry weight. Among the two protocols examined, the NucleoSpin method was more efficient and gave better quality of DNA values compared to those from the modified Doyle and Doyle procedures.
