小麦三雌蕊基因Pis1连锁的SRAP分子标记开发
发布时间：2018-12-07 15:39
【摘要】：采用SRAP分子标记技术筛选与小麦三雌蕊基因Pis1连锁的分子标记。以小麦三雌蕊近等基因系CM28TP及其轮回亲本CM28(川麦28)为亲本,杂交获得F1,F1自交得到F2群体,提取基因组DNA,采用分离群体分组分析法(Bulked Segregant Analysis,BSA)构建三雌蕊性状池和正常雌蕊池,利用44条SRAP正向引物和44条SRAP反向引物组成1936个SRAP引物组合,逐步进行筛选,结果如下:1、为研究小麦三雌蕊性状的遗传规律,以CM28与CM28TP为亲本,杂交获得F1代312株,F1代自交得到1046株F2代群体。观察F1代单株,结果显示F1代全为三雌蕊性状;F2代统计结果为793株表现三雌蕊性状,253株为正常雌蕊。χ2检验结果表明,F2代三雌蕊和正常雌蕊分离比符合3:1比例,验证了三雌蕊性状由显性单基因控制。2、本实验采用传统的CTAB法和试剂盒法提取基因组DNA,试剂盒法包括植物基因组DNA提取试剂盒和多糖多酚植物基因组DNA提取试剂盒两种,并进行了比较,提取的结果表明:采用多糖多酚植物基因组DNA提取试剂盒提取的基因组DNA,纯度、浓度、OD260/OD280和OD260/OD230值等均符合要求。琼脂糖凝胶电泳检测的基因组DNA电泳条带,条带清晰且较亮,没有拖尾现象。3、选用44条SRAP正向引物和44条SRAP反向引物组成1936个SRAP引物组合,对两个亲本CM28和CM28TP进行SRAP分析,筛选出在亲本间具有差异的特异性引物组合共224对。用在亲本间表现出多态性的224对引物组合分别对三雌蕊池、正常雌蕊池进行扩增,有32对引物组合的PCR扩增产物在基因池间表现出差异性。用在三雌蕊池和正常雌蕊池间表现差异的32对引物组合,分别用构建基因池的11株三雌蕊单株和11株正常雌蕊单株进行PCR扩增,其中me2-em36引物组合在8个三雌蕊上扩增出一条约100bp的条带,而6个正常雌蕊上并没有扩增出。me3-em23引物组合在9个正常雌蕊上扩增出一条约200bp的条带,而7个三雌蕊上并没有扩增出。me16-em21引物组合在8个正常雌蕊上扩增出一条约50bp的条带,而7个三雌蕊上并没有扩增出。me16-em25引物组合在8个三雌蕊上扩增出一条约50bp的条带,而7个正常雌蕊上并没有扩增出。me16-em26引物组合在10个正常雌蕊扩增出一条约80bp的条带,而9个三雌蕊上并没有扩增出。me43-em27引物组合在8个三雌蕊上扩增出一条约80bp的条带,而7个正常雌蕊上并没有扩增出。me44-em39引物组合在8个三雌蕊上扩增出一条约80bp的条带,而8个正常雌蕊上并没有扩增出。将这7对引物组合在218株F2群体中进行验证,其中有三雌蕊159株,正常雌蕊59株。利用Mapmaker 3.0计算遗传距离,结果只有me2-em36和me16-em26这两对引物组合扩增的条带与Pis1基因连锁,将这两个标记命名为m2e36,m16e26。m2e36与Pis1的遗传距离为18.22cM,标记m16e26与Pis1的连锁距离为22.63cM。这两个分子标记分别位于Pis1基因的两侧。
[Abstract]:SRAP molecular marker technique was used to screen the molecular markers linked to the Pis1 gene of Tripistil gene in wheat. F _ 2 population was obtained by crossing with CM28TP and its recurrent parent CM28 (Chuanmai 28). Genomic DNA, was extracted from F _ 2 population by separating population grouping analysis (Bulked Segregant Analysis,). BSA) was used to construct a three-pistil trait pool and a normal pistil pool. 1936 SRAP primer combinations were composed of 44 SRAP forward primers and 44 SRAP reverse primers. The results were as follows: 1. Using CM28 and CM28TP as parents, the F _ 1 generation was crossed with 312 plants, and 1046 F _ 2 populations were obtained from F _ 1 generation. The single plant of F1 generation was observed, and the results showed that all F1 generations were three pistil characters. In F2 generation, 793 plants showed three pistil traits and 253 were normal pistil. 蠂 2 test showed that the segregation ratio of three pistils and normal pistil of F2 was 3:1, which confirmed that the trait of three pistil was controlled by dominant single gene. In this experiment, the traditional CTAB method and kit method were used to extract genomic DNA, from plants, including plant genomic DNA extraction kit and polysaccharide polyphenol plant genomic DNA extraction kit, and compared with each other. The results showed that the purity, concentration, OD260/OD280 and OD260/OD230 value of genomic DNA, extracted from polysaccharides polyphenol plant genomic DNA extraction kit met the requirements. The genomic DNA bands detected by agarose gel electrophoresis were clear and bright, and there was no trailing phenomenon. 3. 44 SRAP forward primers and 44 SRAP reverse primers were selected to form 1936 SRAP primer combinations. SRAP analysis was performed on two parent CM28 and CM28TP. A total of 224 pairs of specific primer combinations with different parents were screened. Two hundred and twenty-four pairs of primer combinations were used to amplify the three pistil cisterns and the normal pistil cisterns, and 32 pairs of primer combinations were used to amplify the PCR products among the gene pools. Using 32 pairs of primer combinations which showed the difference between the three pistil cisterns and the normal pistil cisterns, the PCR amplification was carried out by using 11 single plants of three pistil and 11 single plants of normal pistil, respectively. The me2-em36 primer combinations amplified a treaty 100bp band on 8 pistil, but not on 6 normal pistil. Me3-em23 primer combination amplified a treaty 200bp band on 9 normal pistil. Me16-em21 primer combinations amplified a treaty 50bp band on 8 normal pistil. Me16-em25 primer combinations amplified a treaty 50bp band on 8 triecium. Me16-em26 primer combinations amplified a treaty 80bp band in 10 normal pistil. The me43-em27 primer combination amplified a treaty 80bp band on 8 triecium. Me44-em39 primer combinations amplified a treaty 80bp band on 8 triecium, but not on 8 normal pistil. The 7 pairs of primer combinations were tested in 218 F2 populations. There were 159 gynoecium and 59 normal pistil. The genetic distance was calculated by Mapmaker 3.0. Only the bands amplified by the combination of me2-em36 and me16-em26 were linked to the Pis1 gene. The genetic distance between m2e36 m16e26.m2e36 and Pis1 was 18.22 cm. The linkage distance between marker m16e26 and Pis1 was 22.63 cm. These two molecular markers are located on both sides of the Pis1 gene, respectively.
【学位授予单位】：西华师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
本文编号：2367409
[Abstract]:SRAP molecular marker technique was used to screen the molecular markers linked to the Pis1 gene of Tripistil gene in wheat. F _ 2 population was obtained by crossing with CM28TP and its recurrent parent CM28 (Chuanmai 28). Genomic DNA, was extracted from F _ 2 population by separating population grouping analysis (Bulked Segregant Analysis,). BSA) was used to construct a three-pistil trait pool and a normal pistil pool. 1936 SRAP primer combinations were composed of 44 SRAP forward primers and 44 SRAP reverse primers. The results were as follows: 1. Using CM28 and CM28TP as parents, the F _ 1 generation was crossed with 312 plants, and 1046 F _ 2 populations were obtained from F _ 1 generation. The single plant of F1 generation was observed, and the results showed that all F1 generations were three pistil characters. In F2 generation, 793 plants showed three pistil traits and 253 were normal pistil. 蠂 2 test showed that the segregation ratio of three pistils and normal pistil of F2 was 3:1, which confirmed that the trait of three pistil was controlled by dominant single gene. In this experiment, the traditional CTAB method and kit method were used to extract genomic DNA, from plants, including plant genomic DNA extraction kit and polysaccharide polyphenol plant genomic DNA extraction kit, and compared with each other. The results showed that the purity, concentration, OD260/OD280 and OD260/OD230 value of genomic DNA, extracted from polysaccharides polyphenol plant genomic DNA extraction kit met the requirements. The genomic DNA bands detected by agarose gel electrophoresis were clear and bright, and there was no trailing phenomenon. 3. 44 SRAP forward primers and 44 SRAP reverse primers were selected to form 1936 SRAP primer combinations. SRAP analysis was performed on two parent CM28 and CM28TP. A total of 224 pairs of specific primer combinations with different parents were screened. Two hundred and twenty-four pairs of primer combinations were used to amplify the three pistil cisterns and the normal pistil cisterns, and 32 pairs of primer combinations were used to amplify the PCR products among the gene pools. Using 32 pairs of primer combinations which showed the difference between the three pistil cisterns and the normal pistil cisterns, the PCR amplification was carried out by using 11 single plants of three pistil and 11 single plants of normal pistil, respectively. The me2-em36 primer combinations amplified a treaty 100bp band on 8 pistil, but not on 6 normal pistil. Me3-em23 primer combination amplified a treaty 200bp band on 9 normal pistil. Me16-em21 primer combinations amplified a treaty 50bp band on 8 normal pistil. Me16-em25 primer combinations amplified a treaty 50bp band on 8 triecium. Me16-em26 primer combinations amplified a treaty 80bp band in 10 normal pistil. The me43-em27 primer combination amplified a treaty 80bp band on 8 triecium. Me44-em39 primer combinations amplified a treaty 80bp band on 8 triecium, but not on 8 normal pistil. The 7 pairs of primer combinations were tested in 218 F2 populations. There were 159 gynoecium and 59 normal pistil. The genetic distance was calculated by Mapmaker 3.0. Only the bands amplified by the combination of me2-em36 and me16-em26 were linked to the Pis1 gene. The genetic distance between m2e36 m16e26.m2e36 and Pis1 was 18.22 cm. The linkage distance between marker m16e26 and Pis1 was 22.63 cm. These two molecular markers are located on both sides of the Pis1 gene, respectively.
【学位授予单位】：西华师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
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