【作者】 陈亮；

【导师】 胡银岗；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2010， 硕士

【摘要】 春化作用特性、光周期反应特性是小麦的重要农艺性状,直接影响小麦品种的利用、种植区划、引种及栽培技术,并且还间接影响产量、抗病、抗逆等许多重要的农艺性状,研究小麦的春化特性、光周期特性对引种、育种和栽培管理都具有重要意义。Ecotilling是检测生物自然群体相关基因等位变异的高通量反向遗传学研究方法,能够快速有效的识别出目的基因的差异位点。因此,将Ecotilling用于对春化基因和光周期基因的研究,有助于我们更加深入的了解小麦抽穗开花的分子调控机制及各基因存在的等位变异,为通过分子育种方法来改良小麦生育期性状提供参考。本研究优化了传统的Ecotilling技术,采用琼脂糖凝胶电泳检测Ecotilling的酶切片段,达到了较为满意的效果。并用琼脂糖凝胶Ecotilling技术对部分小麦材料中春化基因VRN-A1及光周期基因Ppd-D1相关区域存在的等位变异进行了检测,分析了目的片段中的SNP及其与性状的关系。主要结果如下:(1)初步建立了以琼脂糖凝胶检测为核心的Ecotilling技术体系。在验证CELⅠ具有较好活性的基础上,对以荧光检测技术为基础的Tilling/Ecotilling技术进行改进,用含有错配碱基的两种阳性质粒构建杂合双链,然后用琼脂糖凝胶电泳检测酶切产物,建立了琼脂糖凝胶电泳检测酶切片段的Ecotilling技术体系。(2)以小麦春化基因VRN-A1为参照优化了小麦Ecotilling技术体系的具体参数。结果表明,对于多倍体物种,酶切片段的量是检测的关键,当DNA池构建的供试材料与对照的比例为1:1、反应体系含DNA模板160ng、酶用量2.4μL /体系、切割时间45 min、2%琼脂糖凝胶电泳时,Ecotilling酶切片段检测结果较好,为应用琼脂糖凝胶Ecotilling技术检测小麦基因的SNP等位变异奠定了基础。利用建立的琼脂糖凝胶Ecotilling技术,对108份小麦品种的春化作用相关基因VRN-A1的SNP等位变异进行了检测。结果表明,根据酶切带型可将参试材料分为A、B、C三种单倍型,其中,A单倍型存在6处变异,B单倍型存在5处变异,C单倍型存在3处变异,测序表明这些变异包括单碱基的转换、颠换、缺失、以及小片段的插入。各单倍型材料的冬春性分析结果表明,A和B两种单倍型与品种的冬性表型有关,而C单倍型与品种的春性表型有关。并发现了3个调控小麦冬春性表型转变的关键位点的SNP等位变异。(3)采用建立的琼脂糖凝胶Ecotilling技术,对108份小麦品种的光周期基因Ppd-D1核心编码区的SNP等位变异进行了检测。结果发现,小麦农家种红芒麦、红芒麦2和宁春27可归为同一单倍型,都有三条相同的特异性酶切条带,其序列与对照相比,存在两处单碱基突变(C/T;G/A);坝农1号、定西35、QW6285等7份材料可归为另一种单倍型,呈现出两条特异的酶切条带,其序列与对照相比含有一个5bp的缺失;其余材料与对照相比则没有发生突变。初步分析表明,这三处SNP与小麦光周期敏感性没有明显关联,其与抽穗期的关系还需进一步分析。更多还原

【Abstract】 Vernalization and photoperiod response are important traits of wheat. They are direct influenced the wheat growing areas, seed introduction and cultivation techniques, and it’s also have great impact on yield, disease resistance, stress resistance, and many other important agronomic traits. So the study on vernalization and photoperiod are very important to seed introduction, breeding and management of cultivation. Ecotilling is a high throughput reverse genetic research method for detecting single nucleotide polymorphism (SNP) in the genes of a natural population; it can identify mutation sites rapidly in the target gene of natural population. Therefore, using Ecotilling to analyze vernalization gene and photoperiod gene can help us understanding the molecular regulation of flowering and the alleles of wheat, and it can also give us references on improving growth-period trait that based on molecular breeding.In our research, we optimized the traditional Ecotilling technique, using agarose gel electrophoresis to detect the digestion fragments, and achieved better results. We detect the partial coding region of vernalization gene VRN-A1and photoperiod gene Ppd-D1 in some wheat materials, and analyzed the relationship between the SNPs and the corresponding phenotype. The main results were summarized as follows:(1) Optimized the traditional Ecotilling technique, using positive plasmids which have base mismatch to create heteroduplex, then we detect the digestion fragment by agarose gel electrophoresis successfully. However we established Ecotilling that based on agarose gel electrophoresis basically.(2) We detect SNP in wheat vernalization gene VRN-A1 using agarose gel electrophoresis as an example to determine the optimal parameters in wheat Ecotilling. The result shows that the best amount of template used in PCR was 160 ng, the best ratio is 1 : 1 used for DNA pooling, enzyme amount used in digestion is 2.4μl, the digestion time is 45min, and 2% agarose gel is also the best choice. Therefore, an efficient method for detecting SNP in genes of polyploidy materials such as wheat was established by optimizing the Ecotilling procedure and the agarose gel electrophoresis detection.SNPs in the VRN-A1 gene associated with vernalization among 108 wheat accessions were detected using agarose gel Ecotilling. Based on the SNPs detected, these accessions were found to fall into different haplotype categories, namely, A, B, and C, each with five, six, and three variations, respectively. The sequencing analysis showed that the variations included conversion, transversion, and deletion of a single base, as well as the insertion of a small fragment. At the same time, the winter phenotype was found to be mainly determined by haplotype A and B, while the spring phenotype was mainly determined by haplotype C. Three SNPs are considered associate with wheat vernalization and another three mutations’function is unsure.(3) We used Ecotilling that based on agarose gels to screen SNPs of key coding regions of photoperiod gene Ppd-D1 in 108 wheat varieties. the results showed that there were three specific digestion bands for the landrace Hongmangmai,Hongmangmai2 and Ningchun27, the sequencing result indicated that there exited two SNPs (C/T;G/A) in the target region compared with the control. However, Banong1, Dingxi35, QW6285 and other four materials have two specific digestion bands, and their target region have a 5bp deletion compared with the control. Further analysis revealed there was no clear correlation between the SNPs in Ppd-D1 with the photoperiod of wheat, the influence of the SNPs on heading date were to be further clarified; the sequences of the remaining varieties are identical to the control.更多还原