单性花山葡萄遗传图谱的构建

发布时间：2018-01-29 19:16

本文关键词： 单性花山葡萄遗传图谱 SSR SRAP　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：山葡萄(Vitis amurensisRupr.)具有很强的抗寒性与抗病性,是培育抗寒、抗病新品种及砧木育种的宝贵植物材料。本研究选用雌能花山葡萄'左山一'和雄性花山葡萄'043'为亲本杂交创建了 F1代作图群体,采用SSR和SRAP两种分子标记技术,构建了山葡萄分子遗传图谱,为后期实现与性别相关基因位点的定位提供理论依据,也为抗寒抗病等育种工作打下良好基础,主要研究结果如下:1.应用SSR-PCR技术筛选出两对具有父母本特异性条带的SSR引物,并对148株杂交后代进行杂种真实性鉴定,共得到142株真杂种,真杂种率为95.9%。并创建了由142个真杂种组成的F1代作图群体。2.建立并优化了 SSR-PCR和SRAP-PCR反应体系。两者最佳反应体积都是16 μ 1,前者反应体系中,模板DNA含量为1.5ng,Mg2+浓度为1.0mmol/L,上下游引物浓度均为0.5mmol/L,dNTPs浓度为0.6mmol/L,TaqDNA聚合酶含量为0.5U。后者反应体系中,模板DNA浓度为0.9ng,Mg2+浓度为0.8mmol/L,上下游Primer浓度均为0.6 μ mol/L,dNTPs浓度为1.2mmol/L,Taq DNA聚合酶含量为0.6U,其余部分是灭菌后双蒸水。并验证了建立的SSR-PCR和SRAP-PCR反应体系的可靠性,表明俩个最佳体系可用于杂种鉴定及山葡萄遗传图谱构建等后续试验。3.得到153对至少在一个亲本上表现为杂合,在双亲间表现出多态性且在后代产生分离的SSR引物。其中在父本上得到96个分离位点,在母本上得到107个分离位点。用167对多态性良好的SRAP引物以作图群体扩增,一共得到258个分离位点。4.构建得到雌能花山葡萄'左山一'的分子遗传图谱,包括19个连锁群,覆盖总图距为1551.6cM,连锁群平均长度为81.7cM,位点之间平均距离为5.6cM,每个连锁群平均包含14.6个,位点位点间最短距离是0.1cM;雄性花山葡萄'043'的分子遗传图谱,包括19个连锁群,覆盖总图距为1540.9cM,连锁群平均长度为81.1cM,位点之间平均距离为5.3cM,每个连锁群平均包含15.3个位点,位点间最短距离是0.3cM。
[Abstract]:Vitis amurensis Rupr.) has strong cold resistance and disease resistance. In this study, the female grape 'Zuoshan' and the male 'Zaoshan' were used as parents to create the F1 generation mapping population for new resistant varieties and valuable plant materials for rootstock breeding. The molecular genetic map of Vitis vinifera was constructed by using SSR and SRAP molecular markers, which provided the theoretical basis for the localization of sex-related gene loci in the later stage. It also laid a good foundation for cold resistance and disease resistance breeding. The main results are as follows: 1. Two pairs of SSR primers with parental specific bands were screened by using SSR-PCR technique. A total of 142 true hybrids were obtained by identifying the hybrids of 148 hybrids. The true hybrid rate is 95.9.The F1 generation mapping population, which consists of 142 true hybrids, has been established and optimized. The optimum reaction volume of SSR-PCR and SRAP-PCR was 16 渭 1. In the former reaction system, the concentration of template DNA was 1.0 mmol / L and the concentration of upstream and downstream primer was 0.5 mmol / L. The concentration of dNTPs was 0.6 mmol / L, the concentration of Taq DNA polymerase was 0.5 U.In the latter reaction system, the concentration of template DNA was 0.9ng. The concentration of Mg2 was 0.8 mmol / L, and the concentration of upstream and downstream Primer was 0.6 渭 mol / L, the concentration of dNTPs was 1.2 mmol / L. The content of Taq DNA polymerase was 0.6U, and the rest was double-steamed water after sterilization. The reliability of the established reaction system of SSR-PCR and SRAP-PCR was verified. The results showed that the two optimal systems could be used in further experiments such as hybrid identification and genetic map construction of Vitis vinifera. 153 pairs were found to be heterozygotes on at least one parent. The SSR primer showed polymorphism between parents and produced segregation in offspring. 96 segregation loci were obtained on male parent. 107 segregation loci were obtained on the female parent, and 167 pairs of polymorphic SRAP primers were used to map the population amplification. A total of 258 segregation loci were obtained. The molecular genetic map of 'Zuoshan' was constructed, including 19 linkage groups, covering a total map distance of 1551.6 cm. The average length of linkage group was 81.7 cm, the average distance between loci was 5.6 cm, the average length of linkage group was 14.6, and the shortest distance between loci was 0.1 cm. The molecular genetic map of male Grape Grape, including 19 linkage groups, covers a total map of 1540.9 cm, and the average length of linkage group is 81.1 cm. The average distance between loci was 5.3 cm, and the average number of loci in each linkage group was 15.3, and the shortest distance between loci was 0.3 cm.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S663.1

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