【作者】 亓晓蕾；

【导师】 王洪刚；

【作者基本信息】 山东农业大学 ， 作物遗传育种， 2014， 博士

【摘要】 中间偃麦草（Thinopyrum intermedium，2n=6x=42，EeEeEbEbStSt或JJJSJSStSt）是进行小麦遗传改良的重要基因资源之一，利用其与普通小麦杂交育成的各种小偃麦种质系是转移偃麦草优异基因的重要桥梁材料。本研究利用细胞学、基因组原位杂交和分子标记分析的方法，对八倍体小偃麦混合染色体组的形成机理进行了探讨；综合利用形态学、细胞学、特异分子标记和基因组原位杂交技术，对选育的具有优良性状特点的小偃麦种质系鉴定，明确其主要的性状特点、细胞学特点和染色体构成特点。获得以下主要研究结果：1.利用细胞学和基因组原位杂交技术（GISH），对本实验室选育的10个八倍体小偃麦的根尖细胞（RTC）染色体数目、花粉母细胞减数分裂中期I（PMC MI）染色体构型及其外源染色体的构成特点等进行了分析。结果表明，八倍体小偃麦山农TE256、山农TE259、山农TE261、山农TE262、山农TE263、山农TE265、山农TE266、山农TE267-1、山农TE270和山农TE274等均在普通小麦全套染色体的基础上附加了14条中间偃麦草染色体，其外源染色体构成分别为2St+8JS+2J+2J-St、2St+8JS+4J、2St+8JS+2J+2J-St、2St+8JS+2J+2J-St、2St+8JS+2J+2J-St、6St+4JS+2J+2J-St、4St+6JS+2J+2J-St、2St+8JS+4J、2St+8JS+4J和4St+6JS+4J，10个八倍体小偃麦附加的外源染色体组均由来自于中间偃麦草的不同染色体组的染色体构成的混合基因组，且多数为JS（Ee）和J（Eb）基因组的混合。2.选用本实验室现有的5760对G-SSR、EST-SSR、STS和RAPD引物在中间偃麦草、二倍体长穗偃麦草、披萨偃麦草和拟鹅观草中进行中间偃麦草的多态性引物的筛选，并利用获得的中间偃麦草的多态性引物进一步在中间偃麦草、烟农15和三个供体种中进行全基因组扫描，筛选中间偃麦草各基因组（Ee、Eb和St基因组）的特异分子标记，分别得到440、430和430个中间偃麦草Ee、Eb和St的特异标记。利用全套中国春-长穗偃麦草双体异附加系和中国春-披萨偃麦草双体异附加系进行中间偃麦草Ee基因组和Eb基因组的特异分子标记的染色体定位，将142个和119个中间偃麦草Ee基因组和Eb基因组的特异分子标记定位在特定染色体上。获得的中间偃麦草基因组和染色体特异分子标记可用于基因定位、中间偃麦草染色体追踪、基因的转移和种质系的鉴定。3.利用筛选到的中间偃麦草Ee（JS）、Eb（J）和St基因组的特异分子标记，对中间偃麦草、烟农15和10个八倍体小偃麦进行分析，筛选出八倍体小偃麦中的中间偃麦草染色体组（染色体）的特异分子标记，发现多数标记来自中间偃麦草的Ee和Eb基因组。进一步证明八倍体小偃麦中附加的中间偃麦草染色体为混合基因组，且多数为Ee和Eb基因组的混合，与原位杂交结果一致。结合基因组原位杂交和特异分子标记分析结果，明确了山农TE256、山农TE259、山农TE261、山农TE262、山农TE263、山农TE267-1和山农TE270中附加的中间偃麦草染色体为2Ee、4Ee、5Ee、6Ee、7Ee和5Eb；山农TE265中附加的中间偃麦草染色体为2Ee、5Ee、7Ee和5Eb；山农TE266中附加的中间偃麦草染色体为2Ee、4Ee、5Ee、7Ee和5Eb；山农TE274中附加的中间偃麦草染色体为2Ee、5Ee、6Ee、7Ee和5Eb。4.对从中间偃麦草与小麦杂种后代选育的171份小偃麦种质系进行了鉴定，明确了63份小偃麦种质系的细胞学特点和主要性状特点，为其后续研究和利用提供了参考依据。在63份小偃麦种质系中，八倍体小偃麦45份，附加系2份、代换系16份。5.对八倍体小偃麦山农TE256、山农TE265和山农TE267-1苗期的抗白粉病、抗旱和耐盐性鉴定结果证明，3个八倍体小偃麦具有抗白粉病、抗旱和耐盐等优良特性，推测其抗性来自于中间偃麦草。6.利用基因组原位杂交和分子标记技术，鉴定明确了SN100109为小麦-中间偃麦草的双体异附加系，附加染色体为2Ee染色体，并将其携带的白粉病抗性基因定位到2Ee染色体上；SN0946为小麦-中间偃麦草的双体异代换系，其一对小麦2D染色体被1对2Ee染色体代换，并将其携带的白粉病抗性基因定位到2Ee染色体上。更多还原

【Abstract】 Thinopyrum intermedium (2n=6x=42, EeEeEbEbStSt or JJJSJSStSt) is one of theimportant genetic resources for wheat genetic improvement, many kinds of Trititrigiagermplasm lines which were developed from the progeny of common wheat and Th.intermedium are an important bridge materials for transferring excellent Thinopyrum gene tocommon wheat, are also basis materials of gene discovery and genetic research. In the presentstudy, molecular markers, genomic in situ hybridization and genome comparison analysiswere used to study the formation mechanism of mixed genomes of octoploid Trititrigia, inorder to analyse the genetic basis of the octoploid Trititrigia species and types; And using themethods of morphology, cytology, molecular markers and genomic in situ hybridization, theTrititrigia germplasm lines with good characters and features were identified and analysed, inorder to clarify its main characteristics, characters of cytological features and chromosomestructure. The main research results were as following:1. By methods of cytology and genomic in situ hybridization (GISH), ten octoploidTrititrigia were analysed on chromosome number of root tip cells (RTC), chromosomeconfiguration of mediumterm I pollen mother cell meiosis (PMC MI) and constitution of alienchromosomes. The octoploid Trititrigia shannong TE256, shannong TE259, shannong TE261,shannong TE262, shannong TE263, shannong TE265, shannong TE266, shannong TE267-1,shannong TE270and shannong TE274all had intact wheat genome chromosomes plus14Th.intermedium chromosomes, and their constitutions of alien chromosomes were2St+8JS+2J+2J-St,2St+8JS+4J,2St+8JS+2J+2J-St,2St+8JS+2J+2J-St,2St+8JS+2J+2J-St,6St+4JS+2J+2J-St,4St+6JS+2J+2J-St,2St+8JS+4J,2St+8JS+4J and4St+6JS+4J. The alien chromosomes in each octoploid Trititrigia were all from differentgenomes of Th. intermedium, and mostly consisting of JS(Ee) and J (Eb) genomes.2. In total5760primers were used to amplify the genomic DNA of Th. intermedium, Th.elongatum, Th. bessarabicum and Pseudoroegneria strigosa. The obtained polymorphicprimers of Th. intermedium were used to amplify the genomic DNA of Th. intermedium, Th.elongatum, Th. bessarabicum, Pseudoroegneria strigosa and common wheat cultivarYannong15. The results showed that440,430, and430special molecular markers wereeffectively mapped to the Ee, Eb, and St genomes, respectively. Using two sets of disomicaddition lines (DA1Ee-DA7Eeand DA1Eb-DA7Eb),142and119special molecular markers were effectively mapped to the specific Eeand Ebchromosomes, respectively. These genomicspecific molecular markers will be useful in comparative gene mapping, chromosomeevolutionary analysis, taxonomic studies, gene introgression, and cultivar identification.3. Using the Ee(JS), Eb(J) and St genome-specific molecular markers of Th. intermedium,10octoploid Trititrigia, Th. intermedium and yannong15were analysed in order to obtain thegenome or chromosome-specific molecular markers of Th. intermedium in octoploidTrititrigia. The results showed that the special molecular markers of each octoploid Trititrigiawere mostly from Eeand Ebgenomes. Further, the added Th. intermedium chromosomes inthe ten octoploid Trititrigia were all confirmed to be mixed genomes, and mostly were fromEeand Ebgenomes, consistenting with the results of GISH. According to the results ofspecific molecular marker and GISH, the added alien chromosomes of Shannong TE256,Shannong TE259, Shannong TE261, Shannong TE262, Shannong TE263, Shannong TE267-1and Shannong TE270were2Ee,4Ee,5Ee,6Ee,7Eeand5Eb; Shannong TE265were2Ee,5Ee,7Eeand5Eb; TE266were2Ee,4Ee,5Ee,7Eeand5Eb; Shannong TE274were2Ee,5Ee,6Ee,7Eeand5Eb.4. By methods of morphology and cytology,171Trititrigia germplasm lines developedfrom the progenies between Th. intermedium and common wheat Yannong15had beenidentified. Sixty-three Trititrigia germplasm lines of major characteristics and cytologicalcharacteristic were obtained, providing references for the subsequent use. The63Trititrigiagermplasm lines included45octoploid Trititrigia,2disomic alien addition lines and16disomic substitution lines.5. By resistance identification of powdery mildew resistance in seedling and adult,drought resistance in seedling and salt tolerence in seedling, octoploid TrititrigiaShannongTE256, ShannongTE265and ShannongTE267-1were analysed. The results werethat they were all good resistance to powdery mildew, drought and tolerance to salt, and theresistance of them were inferred from Th. intermedium.6. Combining the methods of molecular markers and GISH, SN100109was a wheat-Th.intermedium disomic addition line, containing a pair of2Eechromosomes from Th.intermedium, and the powdery mildew resistance gene was located on the2Eechromosomes.SN0946was a wheat-Th. intermedium disomic substitution line, with a pair of chromosomesfrom common wheat substituted by a pair of2Eechromosomes from Th. intermedium, and thepowdery mildew resistance gene was located on the2Eechromosomes.更多还原