【作者】 高兰英；

【导师】 马庆； 张增艳；

【作者基本信息】 内蒙古农业大学 ， 作物遗传育种， 2009， 硕士

【摘要】 小麦黄矮病(wheat yellow dwarf disease,WYD)是小麦的主要病害之一,是由蚜虫传播的大麦黄矮病毒(BYDV)引起,常造成巨大损失。小麦的近缘种一中间偃麦草对大麦黄矮病毒具有高度抗性,已至少包含三个抗黄矮病基因,分别定位于7St、7E、和2Ai-2染色体上。其中有一个基因被精确定位在中间偃麦草7X(St)染色体长臂端部,被命名为Bdv2。通过远缘杂交与生物技术已将中间偃麦草的黄矮病抗性基因(Bdv2)导入普通小麦基因组,选育出抗黄矮病的小麦-中间偃麦草易位系HW642等。为了检测追踪通过远缘杂交导入小麦背景的外源染色体,使用含外源染色体的附加/易位系及其亲本材料,基于小麦第七群已定位EST,开发外源染色体特异的EST-PCR标记,旨在快速有效地追踪和检测与小麦第七同源群抗黄矮病基因相连锁的标记,本试验共设计了75对基于定位在小麦第七同源群染色体上的EST引物,在AGE-PAGE-SSCP分离扩增产物,筛选出中间偃麦草抗黄矮病7XL染色体特异标记14对(比率是18.7%),标记开发结果表明,利用小麦已定位EST开发小麦近缘种特异的EST-PCR标记是可行的。而且这些EST-PCR标记不同于中间偃麦草2Ai-2染色体抗黄矮病基因,14个EST-PCR标记与Bdv2相连锁的那条特异带已经克隆测序并通过序列分析,结果指出这些特异带的序列信息显示了很高的同源性与原始wEST的序列,BLAST结果也显示了这些序列分别编码蛋白激酶P450,中心蛋白(RCA),转导蛋白(G-蛋白亚基)和假定的蛋白。因携带Bdv2的中间偃麦草染色体片段与小麦部分同源染色体亲缘关系远、很难发生交换分离,难以精细定位Bdv2与分子标记之间的遗传距离,分离抗黄矮病基因非常困难。然而,创造不同类型的抗黄矮病基因Bdv2感病突变体,可为Bdv2、分子标记精细定位、Bdv2基因分离和研究Bdv2介导的抗黄矮病机制提供坚实的材料基础。本试验采用两种诱变方法获得突变群体,一种是化学诱变剂甲基磺酸乙酯(Eethyl methane sulfonate)EMS诱变处理小麦HW642种子,另一种60Co-γ辐射诱变小麦HW642雌配子成熟期(开花前2~3天)进行成株辐射,后与普通小麦杂交,将获得的M2代材料接种毒蚜BYDV-GAV,进行了生物学性状与一些农艺学性状的鉴定,并对M2代接种进行表型感病鉴定,EMS表型发病变异率约为13.7%,在分子水平上,检测突变频率是7.3%;60Co-γ表型发病变异率约为10.7%,在分子水平上,检测突变频率是58.3%。部分感病突变体通过基因组原位杂交得到了进一步验证,本研究所构建的两个突变体库有望有效的应用于小麦抗病功能基因研究和小麦遗传改良中。更多还原

【Abstract】 Wheat yellow dwarf disease (WYD) is one of the most serious diseases of cereals worldwide.It is caused by barley yellow dwarf virus, which was spreaded by aphids and often causes serious yield loss. Thinopyrum intermedium, a wheatgrass, shows a high level of resistance to BYDV The wheatgrass possesses three resistance genes which locate on the 7St. 7E and 2Ai-2 chromosomes in Thirropyrum intermedium. A resistance gene from 7St chromosome was designated as Bdv2. Researchers crossed the wheatgrass to wheat to obtain a series of virus-resistant wheat translocation lines which carries Bdv2, for example, HW642. In order to detection and trackingWild-hybridization t introgression into wheat bankgroud alive chromosome, used disomic addition/ translocation lines and the parental materials which contain alive chromosome as template, On the basis of the BLAST information on 90 wESTs mapped on the bin 7DL3-0.82-1.00,development of the specific EST-PCR markers for alive wheat chromosome , To effectively tracking and detection of homologous with wheat seventh linked to the yellow dwarf marker genes, in this study we designed 75 pairs of EST primers which derived from ESTs allocated on wheat group 7 chromosomes. 75 wheat EST sequences located in the distal region of 7DL were explored to identify specific PCR markers for the Bdv2 region on the basis of the homoeologous relationship between wheat chromosome 7D and Th.intermedium chromosome7Ai#1.These markers show polymorphism in 3 levels: AGE-PAGE-SSCP and could be classified into 2 types: simple polymorphism and Acomplex polymorphism. amplified successfully and 14 (with a yield rate18.7%)7XL specific EST-PCR markers. These EST-PCR markers could distinguish Bdv2 from another BYDV-resistance gene located on Th.intermedium chromosome 2Ai-2. These specific bands for the Bdv2 region were further cloned and sequenced. The sequencing analysis indicated that the specific sequences for the Bdv2 region were highly homologous with the original wheat EST sequences that were used to design primers, and encode respectively a protein kinase, P450, centrin, transducin, and a hypothetical protein.However, the results showed that the alien Bdv2 region segment could not recombine with the homoeologous wheat chromosome segment in the F2 population and all the EST-PCR markers co-segregated with the Bdv2 region, it is necessary for genetic mapping of Bdv2 to develop an idea mapping population, in which some recombinants can occur. This test method is obtained by using two kinds of induced mutations in this paper , one kinds is EMS was used as chemical mutagen to build the mutant library with wheat translocation lines HW642 Another kind of 60Co-γradiation mutagenesis HW642 wheat before flowering period female gamete (2 ~ 3 days) after radiation with common wheat hybrid, Aphids infected with the BYDV-GAV serotype were used to inoculate the wheat M2 generation.mutant seedlings in the field, Biological characters and agronomic investigated in M2 generation. Some of susceptible mutants identified from M2 plants will be validated , The rate of EMS M2 phenotype mutation was 13.7%,on the molecular level , The rate of M2 detection mutation was 7.3%, thus ,the rate of 60Co-γM2 phenotype mutation was 10.7%,on the molecular level , The rate of M2 detection mutation was 58.3%。The two libraries of induced with EMS and 60Co-γare ideal mutant library and it will be efficiently used in wheat functional genomic research and wheat improvement.更多还原