【作者】 吴坤；

【导师】 马正强；

【作者基本信息】 南京农业大学 ， 应用植物基因组学， 2011， 博士

【摘要】 突变体的构建与分析是功能基因组研究的重要内容,为基因功能分析提供了最直接有力的工具。本研究利用快中子(FN)和甲基磺酸乙酯(EMS)处理四倍体小麦Khapli,创建了一个突变体库。通过M2世代筛选及M3、M4、M5代的表型鉴定,得到443个EMS突变体和61个FN突变体。这些突变涉及到小麦生长的大部分时期,多数在营养生长早期就有表现。根据这些突变体的表型特征,可按熟期、叶片、株型、穗、育性、根、抗病抗逆性等分为13个主要类型和104个次级类型。许多突变体表现出多个性状的变异。70%以上的突变体表现出株型或株高变异,60%以上突变体表现出生长势或叶片形态变异,38%左右的突变体存在穗形变异。在突变体筛选中,还鉴定出了8个盐胁迫相关突变体和13个感白粉病突变体,以及1个对BR不敏感的叶直立突变体。为了研究小麦产量形成的机制,对EMS诱变的产量相关性状突变体Meh0239进行了鉴定。通过形态学、生理学和组织学分析,发现该突变体相对于野生型具有以下主要特征：籽粒短圆、千粒重低、籽粒萌发慢、穗短且扭曲、穗密度大、穗柄及穗轴脆而易断,株型矮,叶片短宽,叶尖钝圆,旗叶叶片中ABA含量显著高于野生型,IAA、ZR、MeJA含量明显低于野生型,叶表皮细胞在轴向上明显短于野生型,且上表皮泡状细胞和长细胞形态极不规则等。遗传分析表明这些性状均受同一个隐性突变基因控制,该突变基因被定位于小麦7DS染色体末端,与标记Xwmc506相距3.1cM。该基因被命名为Yield trait related 1(Yt1)。本实验室在栽培一粒小麦TA2026中鉴定到一个隐性抗白粉病新基因pm2026,并将其初步定位到5A染色体长臂上。为了图位克隆该基因,本研究对其进行了精细定位。利用pm2026所在小麦5AL染色体区域与水稻、短柄草染色体的共线性关系开发了3对STS-CAPS标记MAG5222-MspⅠ、MAG5067-MspⅠ和MAG5066-AluⅠ,在小群体中它们均与pm2026共分离。在一个由4000个家系组成的F2:3群体中,利用pm2026的旁侧标记MAG1491和MAG4586筛选到44个抗病重组体。利用上述开发的STS标记分析这些重组体,将pm2026定位在0.2cM的遗传距离内,与Xmag5067和Xmag5066共分离。利用pm2026两侧紧密连锁标记MAG5222和MAG2170筛选TA2026基因组BAC文库,得到四个阳性克隆27L13、438013、330P9和178016。通过酶切指纹图谱、测序及标记分析,最终将pm2026定位到15kb的物理区域内。更多还原

【Abstract】 To establish mutant libraries for wheat functional genomics, the tetraploid wheat accession Khapli was treated with fast neutron (FN) and ethylmethane sulfonate (EMS).443 mutants involved in most of the phenotypes from EMS-treatment and 61 from FN-treatment were identified by phenotypic evaluation of M2 plants and M3, M4, M5 progenies. These mutants distributed all through the whole growth period of tetraploid wheat, and most mutant phenotype appeared since their early vegetative growth. According to phenotype characters, these mutants could be classified into 13 major category including maturity, leaf, plant type, spike, sterility, root, response to stress and so on, and even 104 subcategory. Most mutants had changed in more than one character. More than 70%of them showed plant height or type variation. And more than 60%were growth vigour or leaf morphology related. Spike morphology related mutants accounted for about 38%. In this mutant pool, eight salinity tolerance related mutants had been identificated. There were also thirteen mutants showing variable degree of susceptibility to powder mildew. In addition, a BR-insensitive mutant showing erect leaf over its life was also identified.Characterization of yield trait mutants is important to understand the regulation of grain yield formation of staple food crops. Meh0239 is a yield trait-related mutant that created by EMS treatment of dry seeds of common wheat cultivar Wangshuibai. To shed some light on the nature of this mutation, it was investigated morphologically, physiologically, anatomically and genetically. The mutant plant shows obvious phenotypical difference from the wild type starting at the seedling stage. It has a reduced plant height, wider and shorter leaves, shortened spikes, spikelets and grains, and a more compact spikelet distribution. Seeds produced in the mutant germinated slower. Meh0239 contains a significantly higher level of abscisic acid (ABA) but a lower level of indole-3-acetic acid (IAA), methyl jasmonate (MeJA) or zeatin riboside (ZR) in flag leaves. Cells of all types in the leaf epidermis appeared shorter along the axial direction. The bulliform cells and long cells on the adaxial leaf surface were abnormal in shape. Genetic analysis using two F2 segregating populations indicated that a single recessive mutation on wheat chromosome 7DS, about 3.1cM distal from Xwmc506, causes these variations. Because of the pleiotropic nature of this gene and its relations with yield trait formation, we named it Ytl for yield trait-related 1.A documented recessive powdery mildew resistance gene pm2026 had been identified in einkorn accession TA2026, and had been located on 5AmL by primary mapping. For fine mapping and cloning of pm2026, three more closely linked STS-CAPS markers of MAG5222-MspI, MAG5067-MspI and MAG5066-AluI, were developed based on the colinearity of wheat, rice and Brachypodium in this chromosome region. And an enlarged F2:3 segregating population containing about 4000 lines was constructed.44 resistant recombinants were identified by flanking markers of MAG 1491 and MAG4586. Through these recombinants and those developed STS-CAPS markers, pm2026 was located in a genetic region of 0.2cM finally and was consegrated with Xmag5066 and Xmag5067. The genomic BAC library of TA2026 was screened by MAG5222 and MAG2170 through PCR, and four positive clones of 27L13,438013,330P9 and 178016 had been identified. Then the restriction enzyme finger printing, sequencing of positive BAC clones and genetic analysis by markers derived from BAC sequence in these recombinants had been done alternately. Finally, pm2026 was restricted to a physical range of 15kb.更多还原