偃麦草、大麦的着丝粒功能序列及其与小麦CENH3整合能力的比较研究

Wheat was hybridized with many wild relatives to transfer alien genes and useful traits. Serious of alien additions, substitutions were establisthed in wheat,and the donors cover most Trticeae genus and species. However, only species of Secale （rye）, Thinopyrum (wheatgrass), Aegilops and Dasypyrum were used succesfully in wheat breeding. Many other alien additions, substitutions just used as genetic stocks and never been used breeding because of unstability and hardness of homoeologous recombination. Variation of centromere function sequences between species may cause fail or partial fail of wheat CENH3 incorporating into the centromeres of the alien chromsomes,which leds wheat spindles can not fully attach centromeres of alien chromosomes. This probably causes unstability and delay of alien chromosomes in cell division. This also brings up extrem difficulty for homoeologous recombination between wheat and alien chromosomes.In this proposal, we will use additions and substitutions of wheat-Thinopyrum (relatively stable and easy for recombination) and wheat-barley (unstable and difficult for recombination) to testfy the above hypothesis. This research will shed light for wheat wide hybridization breeding and transfer of alien genes.

为了向小麦转移有用的重要基因或性状，科学家把小麦与大量的近缘种属植物杂交、回交，创造了大批中间材料，但育种中成功利用的却非常有限，基本局限于黑麦属、偃麦草属、山羊草属、簇毛麦属的个别物种，许多杂种后代因染色体不稳定、或部分同源重组极其困难而束之高阁。种属间着丝粒功能序列的差异，可能是引起小麦CENH3蛋白不能正常整合到外源染色体着丝粒区，造成小麦纺锤丝不能正常附着到外源染色体着丝粒区，引起染色体落后和丢失，也是造成"不稳定"和"重组难"的关键因素之一。本课题拟通过小麦、偃麦草和大麦三属间着丝粒功能序列的比较分析，以小麦与偃麦草及大麦杂交后代为材料，通过小麦CENH3 蛋白在偃麦草、大麦染色体着丝粒区整合能力差异分析，对以上假设进行检验，为外源基因的有效转移和利用提供科学依据。

种属间着丝粒功能序列的差异可能引起小麦CENH3蛋白不能正常整合到外源染色体着丝粒去，造成小麦纺锤丝不能正常附着到外源染色体着丝粒区，引起外源染色体落后或丢失。十倍体长穗偃麦草和六倍体中间偃麦草是小麦改良中利用最为成功的两个多年生种我们试图通过它们与小麦间着丝粒功能序列的异同揭示其属间杂种后代易发生易位重组的基础。为此我们构建了其基因组供体种Pseudoroegneiria stipifolia (StSt) 的大片段插入文库（BAC文库），同时还构建了十倍体长穗偃麦草的质粒文库，制备了小麦着丝粒蛋白CENH3的抗体，以谷类着丝粒基本序列CCS1为探针，筛选BAC文库，结合GISH分析，获得了偃麦草着丝粒相关的BAC克隆，通过阳性克隆的测序，发现了多个不同类型的反转录转座子和一个卫星DNA序列，通过细胞核免疫染色共定位、结合免疫沉淀测序（CHIP-Seq）和CHIP-PCR分析，证明偃麦草和小麦的着丝粒关键功能序列具有高度的同源性，即CRW、Quinta 和CentSt在小麦和偃麦草中均承担着着丝粒功能序列的角色，这为杂种后代中小麦CENH3整合到偃麦草染色体，形成功能着丝粒奠定了基础。此外对十倍体长穗偃麦草的质粒文库筛选，获得了一批偃麦草染色体特异的重复序列，为小麦-偃麦草杂交、回交后代中外源染色体片段的高效检测提供了工具。在项目资助下，培养博士2人，硕士2人。