利用SSR分子标记技术对5个贵州柑橘地方品种的遗传背景进行分析．结果表明，24对SSR引物在5个地方品种和参与D N A指纹比对的14个栽培品种中共检测到92个等位基因，每个位点的等位基因数变幅为2～7个，平均为3.8个；多态性信息含量（PI...利用SSR分子标记技术对5个贵州柑橘地方品种的遗传背景进行分析．结果表明，24对SSR引物在5个地方品种和参与D N A指纹比对的14个栽培品种中共检测到92个等位基因，每个位点的等位基因数变幅为2～7个，平均为3.8个；多态性信息含量（PIC）变化范围在0.36～0.78之间，平均为0.49．经过DNA指纹比对，发现其中5对引物扩增的8条特征谱带可作为地方品种鉴别的参考性标记．利用NTSYS软件进行相似性系数计算，采用 UP‐GM A法聚类，结果显示，相似性系数为0.80，可将5个地方品种划分在不同的亚组中；地方品种米柑和牛肉红金橘均单独成组，其遗传背景独特；土柑与皱皮柑的亲缘关系较近；大果型冰糖橙与冰糖橙的遗传背景相同；红香柚与强德勒柚的遗传背景基本一致．展开更多
[Objective] This study aimed to establish a multiplex PCR system for de- tecting transgenic ingredients from Citrus. [Method] Based on the pBI121 plasmid sequences published in GenBank and actin gene sequence of Citru...[Objective] This study aimed to establish a multiplex PCR system for de- tecting transgenic ingredients from Citrus. [Method] Based on the pBI121 plasmid sequences published in GenBank and actin gene sequence of Citrus, the primers specific to CaMV35S promoter, NOS promoter, NOS terminator and actin gene were designed, to establish a multiple PCR system which could detect four types of sequences. In addition, orthogonal tests were performed to determine the optimal concentrations of all the components in PCR reaction system, as well as the optimal PCR cycle parameters. [Result] The optimal PCR reaction system should contain 2.5μl of 10xPCR buffer, 2.0μl of MgCI2 （25 mmol/L）, 2.0 μl of dNTP mixture （2.5 mmol/L of each dNTP）, 1.0 μl of actin gene primers （10μmol/L）, 1.0μl of 35S promoter primers （10 μmol/L）, 1.5 μl of NOS promoter primers （10 μmol/L） and 0.5 μl of NOS terminator primers （10μmol/L）, 0.1 μg of template DNA, 1.25 U of Taq DNA polymerase; ddH20 was added to the total reaction system of 25μl. The PCR reaction program consisted of pre-denaturing at 94℃ for 5 min; 31 cycles of denaturing at 94℃ for 30 s, annealing at 64.1℃ for 45 s and extension at 72℃ for 50 s; final extension at 72℃ for 10 min. The reaction system optimized with the orthogonal tests could detect as less as 0.1% transgenic component in the tested samples. [Conclusion] The MPCR detection system established in this study can meet the requirements in theory for detecting the genetically modified ingredients in Citrus or the deep-processed products.展开更多
文摘利用SSR分子标记技术对5个贵州柑橘地方品种的遗传背景进行分析．结果表明，24对SSR引物在5个地方品种和参与D N A指纹比对的14个栽培品种中共检测到92个等位基因，每个位点的等位基因数变幅为2～7个，平均为3.8个；多态性信息含量（PIC）变化范围在0.36～0.78之间，平均为0.49．经过DNA指纹比对，发现其中5对引物扩增的8条特征谱带可作为地方品种鉴别的参考性标记．利用NTSYS软件进行相似性系数计算，采用 UP‐GM A法聚类，结果显示，相似性系数为0.80，可将5个地方品种划分在不同的亚组中；地方品种米柑和牛肉红金橘均单独成组，其遗传背景独特；土柑与皱皮柑的亲缘关系较近；大果型冰糖橙与冰糖橙的遗传背景相同；红香柚与强德勒柚的遗传背景基本一致．
基金Supported by the Special Fund for Key Laboratories of Chongqing (CSTC)National Technology Research and Development Program of Ministry of Science and Technology for Countryside Field (863 Program,2011AA100205)+1 种基金Special Fund for Agro-scientific Research in the Public Interest of Ministry of Agriculture of China(201003067)Key Science and Technology Research Program of Ministry of Education of China (109131)~~
文摘[Objective] This study aimed to establish a multiplex PCR system for de- tecting transgenic ingredients from Citrus. [Method] Based on the pBI121 plasmid sequences published in GenBank and actin gene sequence of Citrus, the primers specific to CaMV35S promoter, NOS promoter, NOS terminator and actin gene were designed, to establish a multiple PCR system which could detect four types of sequences. In addition, orthogonal tests were performed to determine the optimal concentrations of all the components in PCR reaction system, as well as the optimal PCR cycle parameters. [Result] The optimal PCR reaction system should contain 2.5μl of 10xPCR buffer, 2.0μl of MgCI2 （25 mmol/L）, 2.0 μl of dNTP mixture （2.5 mmol/L of each dNTP）, 1.0 μl of actin gene primers （10μmol/L）, 1.0μl of 35S promoter primers （10 μmol/L）, 1.5 μl of NOS promoter primers （10 μmol/L） and 0.5 μl of NOS terminator primers （10μmol/L）, 0.1 μg of template DNA, 1.25 U of Taq DNA polymerase; ddH20 was added to the total reaction system of 25μl. The PCR reaction program consisted of pre-denaturing at 94℃ for 5 min; 31 cycles of denaturing at 94℃ for 30 s, annealing at 64.1℃ for 45 s and extension at 72℃ for 50 s; final extension at 72℃ for 10 min. The reaction system optimized with the orthogonal tests could detect as less as 0.1% transgenic component in the tested samples. [Conclusion] The MPCR detection system established in this study can meet the requirements in theory for detecting the genetically modified ingredients in Citrus or the deep-processed products.
