黄瓜节间性状的遗传分析与SRAP标记
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摘要
本研究从黄瓜矮化综合性状出发,重点研究了节间长度的遗传表达与基因标记,节间长度是决定黄瓜植株高矮最主要的性状,而合理的株型是关系黄瓜产量的重要因素,随着黄瓜大田栽培面积的增速渐缓和保护地栽培面积的不断扩大,选育适合保护地栽培的合理株型的黄瓜植株逐渐被提上品种选育日程。
本研究以矮杆短节材料黄瓜“矮生一号”为母本,选取节间较长、差异明显的“A38”黄瓜材料为父本进行杂交,并采用新型分子标记SRAP技术,结合群分法(Bulked SegregantAnalysis,BSA)策略,对黄瓜节间长度这一遗传基础相对比较复杂的性状进行分子标记,结果如下:
1.F_2代苗期下胚轴和节间长度调查统计表明,节间性状呈正态分布,属数量性状遗传。
2.以CTAB法和植物基因组快速提取试剂盒法分别提取DNA,均能获得满足本实验要求的DNA模板。
3.利用正交设计法,建立了黄瓜SRAP-PCR的最优扩增体系,20μL体系中各组分分别为:Primer1(0.1ug/uL)0.3μL;Primer2(0.1ug/uL)0.3μL;25mMMgCl_2 1.4μL;2.5mM dNTPs1.6μL;10×PCR buffers2μL;DNA约40ng,ddH_2O补足20μL。
4.实验中共使用引物1026对;其中982对引物扩增出多态性,292对引物扩增出差异带。引物多态性为95.7%,引物有效率(扩出差异带引物/总引物对)为28.5%。充分体现出SRAP标记的优越性。
5.通过引物对ME23/EM28扩增,获得一条稳定的与节间性状连锁的差异带。
6.经克隆测序,获得与节间相关的标记序列,总长度为369bp。
Research was made on the dewarf character of cucumber,the inheritance,expression and marking of the length of cucumber internode were mainly studied,for that it is the most important factors to effect the hight of cucumber plant and the pattern of the plant.Reasonable pattern is the key factor to decide the total cucumber yield.With the growing area of protected cultivation and the decreaseing area of the open field cucumber planting,breeders are demanded to put out the reasonable pattern cucumber varieties.
Short internode cucumber Aisheng No.1 was selected as female parent,long internode material was used as male parent,the length of internode was marked by SRAP combined with Bulked Segregant Analysis,the results are described below:
1.The Survey of F_2-generation seedlings hypocotyl and internode length show that the characters were normal distribution,which is the genetic of quantitative characters.
2.By CTAB or by plant genome quick extraction kit the extracted DNA has high quality to meet the requirement of molecular marking.
3.By orthogonal design cucumber SRAP-PCR system was set up.In 20μL volum Primerl (0.1ug/μL) is 0.3μL,Primer2(0.1ug/μL) is 0.3μL,25mM MgCl_2 is 1.4μL;2.5mM dNTP is 1.6μL;10×PCR buffer is2μL,DNA is about 40ng,ddH_2O is complicated to 20μL.
4.1026 pairs primers were tested,polymorphism bands can be amplified by 982 pairs primers,different bands can be amplified by 292 pairs primers and the primer polymorphism is 95.7%,the effective rate(primer with polymorphism/overall primer used) is 28.5%,showing the superiority of SRAP.
5.By ME23/EM28 a stable significant band was amplified to linked with the length of internode.
6.By clone and sequence,a DNA marker about 369bp was get.
本研究以矮杆短节材料黄瓜“矮生一号”为母本,选取节间较长、差异明显的“A38”黄瓜材料为父本进行杂交,并采用新型分子标记SRAP技术,结合群分法(Bulked SegregantAnalysis,BSA)策略,对黄瓜节间长度这一遗传基础相对比较复杂的性状进行分子标记,结果如下:
1.F_2代苗期下胚轴和节间长度调查统计表明,节间性状呈正态分布,属数量性状遗传。
2.以CTAB法和植物基因组快速提取试剂盒法分别提取DNA,均能获得满足本实验要求的DNA模板。
3.利用正交设计法,建立了黄瓜SRAP-PCR的最优扩增体系,20μL体系中各组分分别为:Primer1(0.1ug/uL)0.3μL;Primer2(0.1ug/uL)0.3μL;25mMMgCl_2 1.4μL;2.5mM dNTPs1.6μL;10×PCR buffers2μL;DNA约40ng,ddH_2O补足20μL。
4.实验中共使用引物1026对;其中982对引物扩增出多态性,292对引物扩增出差异带。引物多态性为95.7%,引物有效率(扩出差异带引物/总引物对)为28.5%。充分体现出SRAP标记的优越性。
5.通过引物对ME23/EM28扩增,获得一条稳定的与节间性状连锁的差异带。
6.经克隆测序,获得与节间相关的标记序列,总长度为369bp。
Research was made on the dewarf character of cucumber,the inheritance,expression and marking of the length of cucumber internode were mainly studied,for that it is the most important factors to effect the hight of cucumber plant and the pattern of the plant.Reasonable pattern is the key factor to decide the total cucumber yield.With the growing area of protected cultivation and the decreaseing area of the open field cucumber planting,breeders are demanded to put out the reasonable pattern cucumber varieties.
Short internode cucumber Aisheng No.1 was selected as female parent,long internode material was used as male parent,the length of internode was marked by SRAP combined with Bulked Segregant Analysis,the results are described below:
1.The Survey of F_2-generation seedlings hypocotyl and internode length show that the characters were normal distribution,which is the genetic of quantitative characters.
2.By CTAB or by plant genome quick extraction kit the extracted DNA has high quality to meet the requirement of molecular marking.
3.By orthogonal design cucumber SRAP-PCR system was set up.In 20μL volum Primerl (0.1ug/μL) is 0.3μL,Primer2(0.1ug/μL) is 0.3μL,25mM MgCl_2 is 1.4μL;2.5mM dNTP is 1.6μL;10×PCR buffer is2μL,DNA is about 40ng,ddH_2O is complicated to 20μL.
4.1026 pairs primers were tested,polymorphism bands can be amplified by 982 pairs primers,different bands can be amplified by 292 pairs primers and the primer polymorphism is 95.7%,the effective rate(primer with polymorphism/overall primer used) is 28.5%,showing the superiority of SRAP.
5.By ME23/EM28 a stable significant band was amplified to linked with the length of internode.
6.By clone and sequence,a DNA marker about 369bp was get.
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