【作者】 张永生；

【导师】 张举仁；

【作者基本信息】 山东大学 ， 细胞生物学， 2005， 博士

【摘要】 玉米（Zea mays L.）是世界上重要的粮食兼饲料作物,产量居农作物之首,也是重要的遗传模式植物。我国是世界上仅次于美国的第二大玉米生产国,玉米生产在国民经济中占有非常重要的地位。因此,玉米的基础研究和遗传改良具有重大的理论和现实意义。近十余年来,玉米的基因组学和遗传转化一直是国内外研究的热点。 构建遗传连锁图谱是玉米基因组学研究的主要内容之一。目前已利用多种分子标记构建了高密度的玉米遗传连锁图谱。由于不同种质存在着基因组多样性及染色体结构变异等现象,利用其它作图群体构建的遗传图谱对特定种质进行基因定位、基因克隆等研究会产生较大偏差,甚至误入歧途。因此,以不同种质材料构建玉米遗传连锁图谱仍具有重要意义,是玉米基因组学研究的基础性工作。本工作利用自交系掖478×90110的F₂群体构建玉米分子标记连锁图,为我国玉米优良基因源的深入开发和基因组学研究奠定基础。自交系掖478是我国选育的一个优良玉米自交系,具有高配合力、高产、抗倒及抗病性强等优点,为我国玉米生产作出了突出贡献,但该自交系对玉米病毒病的抗性较差。自交系90110具有抗玉米粗缩病、高产、高配合力等优良特性,是我国一个新的玉米类群—P类群的重要成员,该类群种质遗传基础丰富,是对原有杂种优势群的有效补充。 本工作检测了317个分子标记在双亲（掖478和90110）间的多态性,这些分子标记覆盖大部分玉米基因组。双亲间表现多态性的分子标记有215个,频率为67.8%。其中,双亲间多态性的RFLP标记有59个,占筛选RFLP标记的68.6%。利用五种限制酶酶切基因组DNA,发现在双亲间表现多态性的“RFLP探针/酶”组合有145个,占总组合数的33.7%。双亲间多态性的SSR标记有156个,占筛选的SSR标记的67.5%。根据Southern杂交或SSR电泳带谱,挑选149个双亲间多态性条带清晰的分子标记进行F₂群体作图,群体样本由150个随机F₂单株组成。经x²检验,13个分子标记在F₂群体中表现偏分离,这些偏分离标记位子5条不同染色体上,其中在3、4和10号染色体上较多。其余分子标记的分离数据按要求建立文件(共更多还原

【Abstract】 Maize （Zea may L.） is an economically important crop in the world. Maize is also the best-studied and most tractable genetic system among the cereals, making it the premier model system for studying this important group of crops. Maize yield in China is next below that in the United States, which plays an important role in national economy. So both basic research and genetic improvement of maize have great theoretic and practical significance.For any crop plants, a genetic map serves as the foundation of numerous studies. Up to now, high-resolution maize genetic maps have been generated using many groups of molecular markers. However, the maize genome variations may trouble the work of gene mapping or cloning with special maize lines, if the basic genetic map was not constructed with the same parental lines. So it is necessary to construct genetic map with different maize lines, which is the fundamental step in maize genomics research. In this study, we constructed a molecular linkage map for maize with the F₂ population of Ye478×90110. Inbred line Ye478, with the characteristics of high combining ability, high yield and lodging-resistance, accounted much for maize production in China. However, this line is susceptible to maize virus disease. Inbred line 90110, with the characteristics of high combining ability, high yield and high resistance to MRDV （Maize Rough Dwarf Virus）, is a member of P group, which is a new maize group and an important supplement to the old maize heterosis groups in China.The polymorphisms between parental lines （Ye478 and 90110） were detected with 317 markers covering almost whole maize genome. Of these, 215 （67.8%） showed polymorphisms between the two parents, including 59 RFLP markers （68.6% of total detected RFLP markers） and 156 SSR markers （67.5% of total detected SSR markers）. RFLP analysis presented 145 combinations （33.7% of total detected combinations） of "probe/enzyme" showing polymorphisms between parents with five restriction enzymes. According to band patterns, 149 optimal markers were chosen to test 150 random individuals of a Ye478×90110 F₂ population. Through Chi-square test, 13 markers weredetected segregation distortion among five different chromosomes. We used MAPMAKERP/EXP3.0 to construct the maize linkage map. The map consisted of 134 molecular markers and spanned maize genome about 2201.1cM with the average distance of 16.4cM between markers, which formed the frame molecular linkage map of Ye478><90110. On this map, locations of most markers were identical with that on the IBM （MaizeGDB） map. Relative to IBM map, the linkage map located several markers on different positions, inclulding: umcl841 （bin7.03, IBM） was located on chromosome 2; bnlg381 （bin2.04, IBM） was located on chromosome 5; umc36 （bin2.09, IBM） was located on bin2.07; umclO4 （bin5.08, IBM） was located on bin5.03⁵.04; umc76b （bin9.03, IBM） and umcl733 （bin9.08, IBM） were located on bin9.05 9.07 respectively.The genetic diversity of parental lines was analyzed among different chromosomes and special chromosome regions. The genetic similarity （GS） of parents was 0.682, which indicated the close hereditary relation between parental lines, even if they belong to different maize groups. Furthermore, the GS of chromosomes varied from 0.564 to 0.801, and that of special chromosome regions varied from 0.145 to 0.920. Among them, the chromosome regions with higher GS include binl.01 （0.920）, bin3.05 （0.916） and bin8.06 （0.894）, and that with lower GS include bin9.07 （0.145）, bin3.04 （0.189） and binl.06 （0.372）.Maize rough dwarf disease （MRDD） is severe in recent years. Inbred line Ye478 is highly susceptible to MRDV. However, inbred line 90110 is highly resistant to MRDV. Here we not only constructed the linkage map, but also built a segregated population over generations from the cross of Ye478x90110. The population could serve as the plant materials for studying the resistance to MRDV.The segregated population was generated from 60 original F2 plants by successive self-pollination up to F6 generation, with 3⁴ plants self-pollinated in each F3 or later-generation line. During 1999-2003, the segregated population was evaluated the resistance to MRDV by natural infection. In 1999 and 2000, the resistance of parental lines, Fi, F2 and F3 populations were evaluated. The symptom or’Ye478 showed severe with the disease index of 61.1 （1999） and 71.1 （2000）, whereas the 90110 line and Fi displayed completely resistant to MRDV. The mean resistances of F2 （0.5, 1999; 0.57,2000） and F3 population （0.65, 2000） were between that of parents and biased to the resistant-parent Within F2 and F3 populations, the resistant plants were significantly more than the susceptible ones, indicating major genes may control the resistance to MRDV.The genetics of resistance was analyzed with the parental lines, Fi, F2 and F3 populations. Through the single-generation and multiple-generation scale tests, the resistance was confirmed fitting for the additive-dominant genetic model. The genetic parameters, nu [d] and [h], were estimated as 0.9236, -1.1001 and -0.9794 by weighted least square method respectively. So both additive and dominant effects were detected remarkable, and the additive factor was predominant. Moreover, the broad heritability were estimated with F2 （86%, 1999; 85%, 2000） and F3 （83.3%, 2000） populations respectively.For the disease pressure reduced from 1999 to 2003, we scored the resistance scales of higher-generation lines with the relative index of disease. Along with successive self-pollination, the mean resistance tended to reduce with the populations. In F5 or F6 generation, we found the lines showing completely resistant or susceptible to MRDV, and that segregating for MRDV-resistance following Mendelian fashion. These lines could be used to map the resistant genes to MRDV.The two principal maize transformation methods are particle bombardment and Agrobacteriurn-mediated transformation. The pollen-tube pathway is a transformation method developed by Chinese scholar, which has been successfully used in many crops. In this study, we introduced the als gene, a herbicide-resistant gene from Arabidopsis thaliana, into maize inbred line Qi319 by pollen-tube pathway, and analyzed the inheritance of transgene up to T3 generation. Moreover, the characteristics of als inheritance were compared over generations among the transgenic maize plants that generated by pollen-tube pathway, particle bombardment and Agrobacterium-mediated transformation respectively.The maize transformation via pollen-tube pathway was performed by method A （applying plasmid DNA solution on the severed styles after self-pollination） and method B （applying plasmid DNA solution on the severed just prior to self-pollination）. A total of 44 ears were treated, and 28 of them set seeds. 1200 treated seeds were sown in flowerpots for screening the TO plants. Through herbicide screening, 6.3% of the TOplants were herbicide resistant. By PCR and southern blot analysis, 17 TO plants were confirmed transgenic, in which 16 were obtained following transformation method B. The B-3 procedure, that is, applying 200ul plasmid DNA solution （120u.g/ml） on the severed styles prior to self-pollination, obtained the maximal positive rate （6.9%）. The inheritance of als was analyzed up to T3 generation. 12 out of 16 Tl lines included the positive plants. But none of them segregated the als gene according to expected Mendelian ratio of 3:1. Out of 65 T2 lines, 47 showed the plants including als gene. Through Chi-square test, only line QP8-3 segregated the als gene according to expected Mendelian ratio. In T3 generation, 88 out of 115 lines presented the als gene in a portion of plants. Through Chi-square test, lines QP4-3-1, QP4-3-3 and QP4-4-1 segregated the als gene following the 3:1 Mendelian ratio. All plants of seven lines from QP8-3 were detected with als gene, so line QP8-3 was confirmed as a stably inherited line of transgene. Moreover, the loss of als gene was found in higher rate with pollen-tube pathway method.In this study, we compared the inheritance of als gene transformed by pollen-tube pathway, particle bombardment and Agrobacterium-mediated transformation respectively. As a result, the als gene transformed by pollen-tube pathway was not transmitted according to Mendelian fashion in most cases, especially in early generations. Otherwise, the als gene delivered by particle bombardment and Agrobacterium-mediated transformation was inherited generally according to Mendelian fashion. Moreover, the als gene was introduced into maize genome with single or lower copies （general 1-2） by Agrobacterium-mediated transformation, but that introduced by particle bombardment tended to be more copies, resulting in distortion segregation of transgene sometimes. Therefore, the Agrobacterium-mediated transformation has advantages over pollen-tube pathway and particle bombardment method in maize transformation.更多还原