【作者】 曹亚萍；

【导师】 陈佩度；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 簇毛麦是小麦的一个野生近缘种,抗白粉病、锈病、全蚀病、眼斑病及梭条花叶病毒病等多种病害,还兼有抗寒耐旱、分蘖力强、密穗多花、籽粒蛋白质含量高等特性,是小麦遗传改良的优良基因源。抗白粉病基因Pm21和抗梭条花叶病基因Wss1已分别以整臂易位6VS·6AL和4VS·4DL的形式被成功转移到普通小麦背景中。为了定位、转移和利用簇毛麦其它有益基因,本研究以硬粒小麦-簇毛麦双倍体为基础材料,用花粉辐射诱导小麦-簇毛麦属间染色体易位,研究辐射诱导效应和易位染色体的传递行为,并用筛选的EST-STS分子标记对普通小麦背景中的簇毛麦染色体或染色体片段的身份进行鉴定。1小麦-簇毛麦染色体易位的高效诱导与传递为了诱导更多小麦-簇毛麦属间染色体易位,采用60Co-γ射线800 rad、1200 rad、1600 rad三种剂量照射硬粒小麦-簇毛麦双倍体即将成熟的花粉,分别在照射后第1、2、3天从辐射处理过的麦穗上取新鲜花粉给已去雄的普通小麦中国春授粉。用基因组原位杂交方法,在M1代检测小麦-簇毛麦属间染色体易位,分别在BC1、BC2、BC3代考查易位染色体的传递行为。结果表明：这3种辐射剂量都可以高效诱导小麦-簇毛麦染色体易位,并且M1代种子发芽正常。在800～1600rad剂量范围内,随着辐射剂量的提高,易位诱导频率和染色体臂内断裂融合频率增加。辐射处理后第1天采集的花粉,授粉后产生的M1植株易位诱导频率较高。M1代检测到的小麦-簇毛麦易位染色体有70%以上可通过雌配子传递给BC1代,在BC1代重现的易位染色体在随后的世代中绝大多数都能检测到。各种类型易位染色体在不同遗传背景中的传递率具有相对稳定性,通过雌、雄配子的传递率均为整臂易位染色体>外源小片段易位染色体>外源大片段易位染色体。易位染色体通过雌配子的传递率通常高于通过雄配子的传递率。2基于EST-PCR的簇毛麦染色体特异分子标记筛选及应用通过用普通小麦中国春对易位诱导群体连续回交,已得到普通小麦背景中只包含单条簇毛麦染色体或单条小麦-簇毛麦易位染色体的一些单株。为了鉴定这批材料中的簇毛麦染色体身份,根据小麦、水稻的EST序列合成了240对STS引物,其中34对引物在普通小麦中国春与簇毛麦间存在多态性；进一步对亲本及簇毛麦二体异附加系进行PCR扩增分析,标记CINAU32-300可追踪簇毛麦1V染色体,标记CINAU33-280 CINAU34-510、CINAU35-1100和CINAU37-400可追踪簇毛麦2V染色体,标记CINAU38-250可追踪簇毛麦3V染色体,标记CINAU39-950和CINAU40-800可追踪簇毛麦4V染色体,标记CINAU41-745、CINAU42.1050和CINAU43-245可追踪簇毛麦5V染色体,标记CINAU44-765和CINAU45-495可追踪簇毛麦7V染色体。加上本研究室已开发的2个6V染色体特异标记,用这些簇毛麦染色体特异分子标记鉴定辐射诱导材料的部分回交后代,选育出小麦背景中只包含单条簇毛麦染色体的整套1V～7V染色体系,同时有18条易位染色体的簇毛麦身份得到确定,表明这些标记可以用来快速检测普通小麦背景中的簇毛麦染色体或染色体片段。3普通小麦-簇毛麦1V染色体系的选育与鉴定簇毛麦1V染色体长臂具有编码高分子量谷蛋白亚基的位点Glu-V1,短臂具有编码低分子量谷蛋白亚基的位点Glu-V3和ω、γ醇溶蛋白位点Gli-V1,小麦-簇毛麦1V附加系和代换系的蛋白质含量和沉降值均高,将簇毛麦1V染色体的优质基因导入普通小麦,进一步创造小麦-簇毛麦1V染色体易位系是小麦品质改良的有效途径。为转移和利用簇毛麦1V染色体上的优质基因,在(中国春/硬粒小麦-簇毛麦双倍体(60Co-γ射线照射花粉)//中国春)回交后代中,综合运用染色体C-分带、荧光原位杂交、高分子量谷蛋白亚基分析和分子标记分析,从BC1F1-BC1F3代中检测到1V染色体系,在BC1F3、BC2F1代中选育出分别涉及簇毛麦1V染色体长臂和短臂的四种染色体结构变异系,包括1VS·W易位系、W·1VL易位系、1VS单端体系和1VL端二体系,为小麦育种创造了新的种质资源。更多还原

【Abstract】 Haynaldia villosa （L.） Schur, a wild relative of common wheat, possesses many unique useful traits, such as resistances to powdery mildew, rust, take-all, eyespot, wheat streak mosaic virus, as well as drought and frost tolerance, good tiller ability, dense spike, polyanthus, and high protein content in kernels. Therefore, it is a potential gene resource for wheat improvement. Powdery mildew resistance gene Pm21 and wheat streak mosaic virus resistance gene Wssl have been transferred from H. villosa into common wheat. To localize, transfer and utilize other interested genes of H, villosa, the pollens of T. durum-H. villosa amphiploid were irradiated by 60Co-γ-rays and a mass of wheat-H, villosa intergeneric chromosomal translocations were induced in the present research. Effects of irradiation induction and transmission activities of different translocation types in different genetic backgrounds were investigated.chromosome or chromosome segments of H. villosa in common wheat background were identified by EST-STS molecular markers.1 Induction and transmission of wheat-H. villosa intergeneric chromosomal translocations To induce as many as wheat-H. villosa intergeneric translocation chromosomes involved in different chromosomes and chromosome segments of H. villosa, T. durum-H. villosa amphiploid was irradiated with 800,1200 and 1600 rad 60Co-y-rays, and pollens collected from the spikes 1,2 and 3 days after irradiation was pollinated to emasculated common wheat c.v. Chinese Spring. Wheat-H. villosa chromosome translocations were detected using genomic in situ hybridization technique in the M1 and their transmission was analyzed in BC1, BC2 and BC3 generations. The results showed that all the three dosages of irradiation treatments were highly efficient for inducing wheat-H. villosa translocations, and the produced M1 seeds were viable to germinate. The translocation induction efficiency and interstitial chromosome breakage-fusion frequency was increased as the irradiation dosage increased from 800 rad to 1 600 rad. Minor increase of translocation induction frequency was observed when using the pollen collected from the spikes one day than those 2 or 3 days after irradiation. More than 70%of the translations detected in the M1 generation could be transmitted to the BC1 through female gametes. Most of the translocations recovered in the BC1 generation could be recovered in the following generations. Various types of translocations showed relatively stable transmission ability in different genetic backgrounds with an order of ’whole arm translocation> small alien segment translocation> large alien segment translocation’, either through male or female gametes. Transmission ability through female gametes was generally higher than that through male gametes.2 Screening and Application of EST-based PCR Markers Specific to Individual Chromosomes of H villosaAll plants involved in wheat-H villosa translocation chromosomes were backcrossed with Chinese Spring, and some plants involved in single chromosome or chromosome segment of H. villosa in common wheat background have been developed. To identify H. villosa chromosomes or chromosome segments in these materials,240 STS primer pairs were designed based on the EST sequences of wheat and rice, and 34 of them could amplify specific polymorphic bands between Chinese Spring and H. villosa. These 34 STS primer pairs were further used for screening markers specific to individual chromosomes of H. villosa using 1V-7V disomic addition lines and their parents. The results showed that the marker CINAU32₃oo could be used for tracing chromosomeⅣ, CINAU33₂8o, CINAU34.510, CINAU35-1100 and CINAU37-400 for 2V, CINAU38-250 for 3V, CINAU39.950 and CINAU40.800 for 4V, CINAU41.745, CINAU42-1050 and CINAU43-245 for 5V, CINAU44-765 and CINAU45-495 for 7V. These EST-STS markers combined with the two 6V specific markers developed in our institute were used to identify the H. villosa chromosome and chromosome segments of backcrossed generations derived from pollen irradiation. A set of 1V-7V addition lines and 18 translocation chromosomes involved in different H. villosa chromosomes have been identified. Therefore, these chromosome-specific EST-STS markers could be used to detect chromosomes and chromosome segments of H. villosa in common wheat background.3 Development and characterization of Wheat-H villosa alien Chromosome Lines involved inⅣGlu-Vl loci coding for high molecular weight glutenin subunits is located on long arm, and Glu-V3 loci coding for low molecular weight glutenin subunits and Gli-Vl loci coding gliadin （co-type and y-type） are located on short arm of chromosome IV. Higher seed protein concentration and SDS-sedimentation value were found in addition or substitution lines of 1V in Chinese Spring background than those in Chinese Spring. The development of wheat-H.villosa alien chromosome lines involved in chromosome 1V is an efficient method for introducing high quality genes from 1V into common wheat and provide potential genetic resources for wheat quality improvement. Chromosome C-banding, fluorescence in situ hybridization, high molecular weight glutenin subunits combined with molecular marker analysis were applied to detect chromosome 1V and its structure aberrants in backcrossed generations derived from （Chinese Spring/T. durum-H. villosa amphiploid （irradiated by 60Co-γ-rays）//Chinese Spring）. Alien chromosome lines involved in 1V were identified from BC1F1 to BC1F3. Four variants with chromosome structure changes of 1V were identified in BC1F3 and BC2F1, including whole arm translocation 1VS·W, whole arm translocation W·1VL, monotelosomic 1VS and ditelosomic 1VL, they would be new germplasms for wheat breeding.更多还原