【作者】 王玉海；

【导师】 王洪刚；

【作者基本信息】 山东农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 在小麦的近缘植物中,山羊草属(Aegilops L.)与小麦(Triticum L.)的亲缘关系最近,含有丰富的优良基因,在小麦的遗传改良中具有重要的利用价值。SDAU18是本实验室利用偏凸山羊草(Aegilops ventricosa, 2n = 28, DDNN)和柱穗山羊草(Aegilops cylindrica, 2n = 28, DDCC)杂交人工合成的新型双二倍体。本研究综合采用细胞遗传学、A-PAGE、SDS-PAGE、SSR、GISH和抗性接种鉴定等方法对其进行了鉴定;对SDAU18及其双亲与普通小麦的杂交亲和性、杂种F1的育性、SDAU18与普通小麦不同杂种世代的染色体及主要农艺性状的分离特点进行了分析;对从SDAU18与普通小麦杂交后代中选出的部分种质系进行了初步鉴定。获得以下主要研究结果:1.细胞学观察的结果表明,SDAU18的根尖细胞染色体数目变异范围为52-56,在多数染色体数目为56的SDAU18减数分裂中期I花粉母细胞(PMC MI)内可观察到28个二价体,在部分细胞中可观察到一定频率的单价体、三价体和四价体,平均染色体构型为2n = 56 = 3.21I + 19.78IIRing + 6.50IIRod + 0.01III + 0.04IVRing + 0.01IVRod;但在部分PMCs中出现了不同程度的染色体丢失现象。种子贮藏蛋白电泳分析发现,在SDAU18种子贮藏蛋白电泳图谱中,亲本偏凸山羊草和柱穗山羊草的多数特异带能够出现,SDAU18高分子量麦谷蛋白亚基图谱中既出现双亲的亚基谱带,也观察到新型亚基谱带。分别利用偏凸山羊草和柱穗山羊草基因组总DNA作探针,另一亲本基因组总DNA作封阻,对SDAU18根尖细胞制片进行染色体原位杂交,在SDAU18的56条染色体中分别有14条出现绿色杂交信号。上述结果证明,SDAU18是偏凸山羊草和柱穗山羊草的双二倍体。2. SSR标记分析结果表明,在SDAU18基因组中存在着广泛的遗传变异。利用定位在普通小麦染色体上的89对SDAU18进行扩增分析,在SSR引物扩增图谱中,除了双亲的特异谱带外,还观察到亲本谱带缺失和新型谱带。其中,柱穗山羊草缺失谱带的频率约为偏凸山羊草的2.4倍,这表明在SDAU18中,双亲遗传信息的丢失或基因的沉默可能不是随机的。这可能反映了双亲基因组在新合成的双二倍体SDAU18中存在着复杂的互作。3.对SDAU18及其双亲与普通小麦的杂交亲和性、杂种F1的育性调查结果表明,SDAU18与普通小麦的杂交亲和性以及它与普通小麦杂种F1的育性显著高于其双亲偏凸山羊草和柱穗山羊草。因此,SDAU18可以作为转移偏凸山羊草和柱穗山羊草优良遗传物质的桥梁材料,用于小麦遗传改良。4.抗病性鉴定结果表明,SDAU18对小麦白粉病和条锈病均表现高抗至免疫。对SDAU18和普通小麦不同自交和回交世代染色体和性状分离特点的研究结果表明,随自交和以烟农15为轮回亲本回交世代的增加,染色体数目逐渐减少,回交比自交能使后代的染色体数目更快趋近普通小麦的42条,至F5和BC3F1代,染色体数目为42的植株已分别达93.9%和92.0%。与自交世代相比,回交后代减数第一分裂中期花粉母细胞的染色体构型较为简单,回交次数过多不利于外源染色体与普通小麦染色体发生重组,一般应以回交2~3次为宜;随自交和回交世代的增进,杂种的育性提高,至F3和BC2F1代育性基本恢复正常,且较为稳定。在不同杂种世代可分离出具有矮杆、大穗、大粒、对白粉病、条锈病免疫或高抗和外观品质优良的变异类型,其中以F3和BC1F1代的变异类型最丰富。在通过杂交将偏凸山羊草和柱穗山羊草遗传物质导入普通小麦中SDAU18具有重要利用价值。5.运用形态学、细胞学和抗性接种鉴定相结合的方法,从烟农15与SDAU18杂交后代中选育出ZH-1、ZH-2、ZH-3、ZH-4等优良种质材料,并对其进行了初步鉴定,明确了它们的主要农艺性状和细胞学特点。在上述种质材料中,ZH-1的突出特点是“矮杆”,株高为35.5cm,染色体数目为2n=40-41,其形态学及细胞学特征尚不稳定。ZH-2的突出特点是“抗白粉病”,分小种鉴定结果表明,它对E09生理小种表现免疫,其细胞学及形态特征基本稳定。ZH-3和ZH-4的突出特点是“大穗多花”,二者在细胞学及形态学上基本稳定。更多还原

【Abstract】 Among the related species of wheat, Aegilops L. is the closest relative, conferring many valuable genes for wheat, and is of great interest as potential resources of new genetic material to improve wheat. SDAU18, a novel amphiploid of Ae.ventricosa (2n=28, DDNN) with Ae.cylindrica (2n=28, DDCC), was identified by cytological analysis,A-PAGE, SDS-PAGE, SSR, GISH and inoculation assessment, and so on; The crossability and the F1 fertility of SDAU18, Ae.ventricosa and Ae.cylindrica with common wheat were compared, and the segregation of chromosomes and traits of hybrid generations derived from the cross between Triticum aestivum and SDAU18 was studied; Several new germplasms derived from the progenies of common wheat Yannong15 and SDAU18 were preliminarily characterized.The main results were as follows:1. The chromosome number of root tip cells ( RTCs ) of SDAU18 plants varied from 52 to 56. 28 bivalents were observed in most PMCs MI of SDAU18 with 56 chromosomes, meanwhile, a few univalents, multivalents also existed in some PMCs MI, and the average chromosome configuration was 2n = 56 = 3.21I + 19.78IIRing + 6.50IIRod + 0.01III + 0.04IVRing + 0.01IVRod. However, an abnormal cytology of chromosome loss occurred in partial PMCs MI of SDAU18 with 56 chromosomes. There were both Ae.ventricosa-specific bands and Ae.cylindrica-specific bands in the seed storage protein electrophoretogram of SDAU18, furthermore, SDAU18 had one novel HMW-GS not found in the parents and two novel ones not found in common wheats. By labeling the total genomic DNA of Ae.ventricosa and Ae.cylindrica as probes respectively, and using that of another parent as block, GISH of RTCs spread of SDAU18 was carried out. The green hybridization signal was observed in 14 chromosomes respectively, within 56 ones in RTCs of SDAU18. The results above suggested that SDAU18 was an amphiploid of Ae.ventricosa with Ae.cylindrica. and had very important significance in wheat breeding and genetic improvement.2. Extensive genomic changes in SDAU18 with 56 chromosomes were detected by microsatellite analysis using 89 pairs of primers of common wheat. The changes mainly involved loss of parental SSR fragments and gaining of novel ones. The frequency of losing parental fragments was much higher than that of gaining novel fragments. The percentage of sequence losing in Ae. cylindrica was about 2.4 times as high as in the Ae. ventricosa. The data above indicated that the parental SSR losing of SDAU18 was not random, and that complex interaction perhaps existed between parental genomes in SDAU18.3. The comparison of crossability and the hybrids F1 fertility of SDAU18, Ae.ventricosa and Ae.cylindrica with common wheat indicated that the crossability and the hybrids F1 fertility of SDAU18 with common wheat were significantly higher than that of its parents, Ae.ventricosa and Ae.cylindrica, with common wheat. Therefore, SDAU18 could be used as a bridge parent to transfer good genes from Ae.ventricosa or Ae.cylindrica or both into common wheat.4. Inoculation testing suggested that SDAU18 was highly resistant or immune to powdery mildew and stripe rusts. The analysis on segregation of chromosomes and traits of hybrid generations derived from the cross between Triticum aestivum and SDAU18 indicated that in higher generations of selfing and backcross, the chromosome number gradually decreased and eventually tended to 42, which was the same as common wheat. Backcrossing was able to fasten the process than selfing. In the F5 and BC3F1 generations, plants with 42 chromosomes were accounted for 93.9% and 92.0%, respectively. Chromosome configuration in PMCs MI was simpler in backcross generations than in selfing ones. Compared with the BC1F1 and BC2F1 generations, BC3F1 showed less diversity in chromosome configuration, indicating that excessive backcross resulted in less chromosomes recombination between SDAU18 and common wheat. Two or three rounds of backcross were feasible. With the increasing generation of selfing and backcross, fertility of the hybrid was improved till the stable status in F3 and BC2F1 generations. In various generations, variant plants with excellent traits were found, such as dwarf plant, huge spike, large grain, high resistance or immunity to powdery mildew and stripe rust, and good appearance of grain. In particular, the F3 and BC1F1 generations had the most variation types. SDAU18 was of important significance in transferring genetic materials from Aegilops ventricosa and Aegilops cylindrica to common wheats by using it as bridge parent to cross with common wheats.5. Morphological and cytogenetic methods were used to identify new germplasms ZH-1, ZH-2, ZH-3, ZH-4,and so on, which was derived from the progenies of Yannong15 and SDAU18.The main agronomic characters, chromosome number and the genetic stability of these new germplasms were determined.ZH-1, with the most distinguishing characteristics of“dwarf plant of 35.5 cm”, was morphologically and cytologically unstable, and the chromosome number of ZH-1 plants varied from 41 to 42.ZH-2, with the most prominent characteristics of“excellent resistance to powdery mildew”, was basically staible based on morphological and cytological data. Inoculation testing revealed that it was immune to the strain E09.The most outstanding trait of ZH-3 and ZH-4 was both“huge spike and multi-florets”.Morphologically and cytologically, they were both basically stable.更多还原