玉米ae基因的分子标记及辅助选择研究
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摘要
玉米(Zea mays L.)是世界上重要的粮食、饲料与工业原料作物。玉米作为工业原料最主要的成份是淀粉,其中所含的直链淀粉在工业上用途很广。但普通玉米直链淀粉所占比重很低,限制了玉米中直链淀粉的应用。因此,开展高直链淀粉玉米育种,对发展高直链淀粉的生产和加工具有重要的意义。由于控制玉米直链淀粉含量的ae基因为单隐性基因,因此运用分子标记辅助选择的方法对后代进行快速选择,可以提高育种效率,加快育种进程。本文根据发表在GeneBank上的玉米淀粉分支酶基因(ae)序列,筛选分子标记,建立ae基因分子标记技术,并应用于高直链淀粉玉米回交后代的辅助选择,结果表明:
1.根据NCBI上公布的玉米淀粉分支酶基因(ae)序列设计引物,分别以高直链淀粉玉米ae-1和普通玉米鲁原92基因组为模板,扩增了ae基因的大部分序列,经测序并通过序列比对分析表明,高直链淀粉玉米和普通玉米在所测序列部分具有较高的同源性,除在3处有3-4bp连续碱基的缺失外,其余均为SNP和单碱基的插入/缺失。
2.在上述差异处分别设计引物,经过筛选,选择在基因第9和第10外显子之间一个4bp indel处设计的引物作为标记引物。在含有ae基因的玉米材料中能够扩增出一条496bp的特异性条带,称之为ae496标记。利用该标记对14种高直链淀粉玉米自交系和24种普通玉米自交系材料进行检测,高直链淀粉玉米自交系基因组中均能扩增出ae496条带,而普通玉米自交系中则全部扩增不出该条带。因此该标记可作为ae基因的显性分子标记。
3.用ae496标记对高直链淀粉玉米自交系和普通玉米自交系三个组合的F_1代、F_2代、BC_1F_1代进行检测。结果表明该显性分子标记符合孟德尔遗传规律。
4.通过高直链淀粉自交系与普通玉米自交系杂交组合F_2代籽粒直链淀粉含量和标记的分析,发现直链淀粉含量在32.1%以上的玉米籽粒均能扩增出ae496条带,而扩增不出ae496条带的玉米籽粒的直链淀粉含量均在25.8%以下。可以看出ae显性分子标记与籽粒直链淀粉含量之间存在相关性,通过分子标ae496可辅助选择ae基因获得高直链淀粉性状。
5.通过对BC_1F_1、BC_2F_1代分子标记辅助选择,分析其自交和回交后代的选择效果,结果显示通过标记辅助选择其后代可获得ae基因,其回交一代和回交二代分子标记辅助选择ae基因的效率较非标记选择分别高53.3%和73.3%,表明利用分子标记在回交后代中选择ae基因可显著提高选择效率。
Maize is one of the most important crops in the world.In maize,starch is the most important element as industrial raw material.In starch,amylose has a wide range of uses in industry.Amylose has a low proportion in dry weight of maize weight,which restricts application of amylase in maize.So to develop high amylose maize breeding has a great significance on the development of amylase production and procession.As the ae gene which controls amylose content in maize is a recessive one,the use of molecular marker-assisted selection methods for quick choices to future generations can improve breeding efficiency and speed up the breeding process.According to maize starch branching enzyme(ae) gene sequences published in Genebank,we screen the molecular marker,establish the technique of ae gene molecular marker,and apply the system to the selection of backcross generations in high amylose content maize.The result is as follows:
1.According to maize starch branching enzyme(ae) gene sequences published in NCBI,we choose a kind of high amylose content maize and a common maize genome as templates and design specific primers to amplificate most sequences of ae gene. Through compare and analysis with the PCR amplification fragment,we find that there is a high homology between the high amylose content maize and the common ones.Except 3 consecutive 3-4bp insertion or deletion,the major differences is SNP and insertion / deletion of single-base.
2.We design primers in the different place.With screening,we select a pair of primer between 9 and 10 exon with 4bp deletion as marker primer.We can amplificate a specific fragment of 496bp in maize which has ae gene.With the marker,we detect 14 kinds of high amylose content maize inbred lines and 24 kinds of common maize ones, a ae496 band can be amplificated in the high amylose maize inbred lines genomes, while the bands can't be amplificated in common maize inbred lines.So the marker can be used as ae gene dominant molecular marker.
3.We detect the F_1,F_2 and BC_1F_1 generations in 3 portfolios.The results show that the dominant molecular marker is in line with mendel law.
4.With analysis of the marker and the amylose content in F2 generation grain,we find that the amylose content of grain which is above 32.1%can be amplificated ae496 band,While the amylose content of maize grain which is below 25.8%can't be amplificated the ae496 band.We cann see the correlation between the ae dominant molecular marker and amylose content in grain.Through the ae496 marker we can select ae gene to obtain high amylose trait.
5.Through the molecular marker assisted selection in BC_1F_1,BC_2F_1 generations,we analyse the selective effect in self-hybrid and backcorssing generations.The results show that we can obtain ae gene in future generations by marker-assisted selection.The efficiency of selecting ae gene in backcross generation and its second-generation is 53.3%and 73.3%higher than random selection.It indicates that the use of molecular markers in future generations to select ae gene can enhance selective efficiency significantly.
1.根据NCBI上公布的玉米淀粉分支酶基因(ae)序列设计引物,分别以高直链淀粉玉米ae-1和普通玉米鲁原92基因组为模板,扩增了ae基因的大部分序列,经测序并通过序列比对分析表明,高直链淀粉玉米和普通玉米在所测序列部分具有较高的同源性,除在3处有3-4bp连续碱基的缺失外,其余均为SNP和单碱基的插入/缺失。
2.在上述差异处分别设计引物,经过筛选,选择在基因第9和第10外显子之间一个4bp indel处设计的引物作为标记引物。在含有ae基因的玉米材料中能够扩增出一条496bp的特异性条带,称之为ae496标记。利用该标记对14种高直链淀粉玉米自交系和24种普通玉米自交系材料进行检测,高直链淀粉玉米自交系基因组中均能扩增出ae496条带,而普通玉米自交系中则全部扩增不出该条带。因此该标记可作为ae基因的显性分子标记。
3.用ae496标记对高直链淀粉玉米自交系和普通玉米自交系三个组合的F_1代、F_2代、BC_1F_1代进行检测。结果表明该显性分子标记符合孟德尔遗传规律。
4.通过高直链淀粉自交系与普通玉米自交系杂交组合F_2代籽粒直链淀粉含量和标记的分析,发现直链淀粉含量在32.1%以上的玉米籽粒均能扩增出ae496条带,而扩增不出ae496条带的玉米籽粒的直链淀粉含量均在25.8%以下。可以看出ae显性分子标记与籽粒直链淀粉含量之间存在相关性,通过分子标ae496可辅助选择ae基因获得高直链淀粉性状。
5.通过对BC_1F_1、BC_2F_1代分子标记辅助选择,分析其自交和回交后代的选择效果,结果显示通过标记辅助选择其后代可获得ae基因,其回交一代和回交二代分子标记辅助选择ae基因的效率较非标记选择分别高53.3%和73.3%,表明利用分子标记在回交后代中选择ae基因可显著提高选择效率。
Maize is one of the most important crops in the world.In maize,starch is the most important element as industrial raw material.In starch,amylose has a wide range of uses in industry.Amylose has a low proportion in dry weight of maize weight,which restricts application of amylase in maize.So to develop high amylose maize breeding has a great significance on the development of amylase production and procession.As the ae gene which controls amylose content in maize is a recessive one,the use of molecular marker-assisted selection methods for quick choices to future generations can improve breeding efficiency and speed up the breeding process.According to maize starch branching enzyme(ae) gene sequences published in Genebank,we screen the molecular marker,establish the technique of ae gene molecular marker,and apply the system to the selection of backcross generations in high amylose content maize.The result is as follows:
1.According to maize starch branching enzyme(ae) gene sequences published in NCBI,we choose a kind of high amylose content maize and a common maize genome as templates and design specific primers to amplificate most sequences of ae gene. Through compare and analysis with the PCR amplification fragment,we find that there is a high homology between the high amylose content maize and the common ones.Except 3 consecutive 3-4bp insertion or deletion,the major differences is SNP and insertion / deletion of single-base.
2.We design primers in the different place.With screening,we select a pair of primer between 9 and 10 exon with 4bp deletion as marker primer.We can amplificate a specific fragment of 496bp in maize which has ae gene.With the marker,we detect 14 kinds of high amylose content maize inbred lines and 24 kinds of common maize ones, a ae496 band can be amplificated in the high amylose maize inbred lines genomes, while the bands can't be amplificated in common maize inbred lines.So the marker can be used as ae gene dominant molecular marker.
3.We detect the F_1,F_2 and BC_1F_1 generations in 3 portfolios.The results show that the dominant molecular marker is in line with mendel law.
4.With analysis of the marker and the amylose content in F2 generation grain,we find that the amylose content of grain which is above 32.1%can be amplificated ae496 band,While the amylose content of maize grain which is below 25.8%can't be amplificated the ae496 band.We cann see the correlation between the ae dominant molecular marker and amylose content in grain.Through the ae496 marker we can select ae gene to obtain high amylose trait.
5.Through the molecular marker assisted selection in BC_1F_1,BC_2F_1 generations,we analyse the selective effect in self-hybrid and backcorssing generations.The results show that we can obtain ae gene in future generations by marker-assisted selection.The efficiency of selecting ae gene in backcross generation and its second-generation is 53.3%and 73.3%higher than random selection.It indicates that the use of molecular markers in future generations to select ae gene can enhance selective efficiency significantly.
引文
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