【作者】 Hassan Hamza Abdel-halim Mostafa；

【导师】 李锡香；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2014， 博士

【摘要】 大蒜(Allium sativum L.)可作为蔬菜、调味品和药物,是一种十分重要的作物。中国和埃及是全世界大蒜的主要生产国。然而,有限的自然变异并不能满足遗传改良的要求,同时由于大蒜不能进行有性繁殖,因此至今在选育高产、优质和抗生物和非生物逆境品种方面开展的工作十分有限。另外,通过传统的方法很难准确测定次生代谢组分含量和评价遗传变异。因此,挖掘优异种质、创造新的变异型显得十分重要。超高效液相色谱(UPLC)是测定大蒜素的一种十分有效的方法。SSR标记是一种共显性标记,在鉴定基因变异方面具有很多优势。基于组织培养技术的无性系诱变是无性繁殖植物遗传改良的一种十分有效的方法。基于以上原因,我们进行了下面三方面的研究：首先,评估遗传和环境因素对埃及和中国大蒜种质大蒜蒜素含量的影响；第二,利用形态学和新的SSR标记评价大蒜种质资源的遗传多样性；最后,基于大蒜愈伤组织的伽玛辐射和甲基磺酸乙酯(EMS)处理诱变技术研究。试验结果总结如下：1.遗传和环境因素对大蒜种质大蒜辣素含量的影响利用超高效液相色谱测定了104份大蒜种质(83份来源于中国,21份来源于埃及)的大蒜辣素含量。根据大蒜辣素含量高低,将所有种质分成4组。大蒜辣素含量最高的种质分在A组,包含埃及和中国种质各一个。聚类分析表明在这些大蒜种质间大蒜辣素含量的变异范围广,可以在育种中通过选择利用这些优选种质来改良大蒜品质。我们还发现选择获得的无性系的大蒜辣素含量高于其亲本,这表明从自然群体中筛选变异无性系不失为大蒜遗传改良的有效途径。基因型、地理位置及其交互作用对埃及大蒜种质的大蒜辣素含量影响均达极显著水平(P<0.001)。种植在中国的埃及大蒜比种植在埃及的同样的大蒜种质有较低的大蒜辣素含量。在这两个不同的地理位置种植的相同大蒜种质中,其大蒜辣素含量的降低幅度为16%-63.7%。这一发现证实了对于大蒜产业来说,大蒜种植区的选择对提高大蒜辣素含量是至关重要的。2.基于形态性状和SSR标记的大蒜遗传多样性分析利用19个表型性状和基于转录组测序结果开发的16对SSR引物分析了中国(83份)和埃及(21份)104份大蒜种质资源的遗传多样性。这16对SSR引物在所有材料中共检测出了44个等位基因,每对引物检测到1-4个等位基因,平均每个基因座有2.75个多态性位点。在104份种质中,等位基因频率在0.5048-0.9615之间,基因多样性为0.0740-0.5000,遗传杂合度为0.0481-0.9904,平均多态信息含量(PIC)为0.2225。基于形态学标记获得的聚类树将104份大蒜种质资源分为2个主要类群,而以16对SSR标记构建的104份大蒜种质系统发育树将其分为3个主要类群,但两种方法构建的系统树与大蒜的地理分布基本一致。利用12对SSR引物比较选择的无性系及其亲本,证实选择的大蒜无性系后代和亲本之间存在遗传变异,说γ明无性系选择在大蒜品种改良中仍是一种有效的方法。3.利用γ射线处理大蒜愈伤组织的诱变技术研究对2个中国大蒜品种(‘永年’和‘中牟’)的愈伤组织分别用1,3,5,7戈瑞(Gray)的γ射线进行诱变处理,发现‘永年’大蒜对高剂量的γ射线敏感,而‘中牟’大蒜在高剂量射线处理后各项生长指标表现相对较好。具体表现为：用1Gray的γ射线处理‘永年’大蒜的愈伤组织,其生芽愈伤组织的平均数、每个愈伤组织的生芽数、总芽数、成苗芽数和株高分别为33.33,2.37,296.00,170.00和13.32cm,均高于其他剂量处理。然而,对于‘中牟’大蒜的愈伤组织,用7Gray的剂量处理,上述指标高于其他剂量处理,分别为10.33,3.92,78和17.10cm。此外,‘中牟’大蒜的愈伤组织在4Gray的辐射处理下出芽所需时间最短,为154d；而‘永年’大蒜的愈伤组织在1Gray的辐射处理下,出芽需129.7d。利用16对SSR引物检测诱变材料,其中2对引物检测到了遗传变异,即分别在经过5Gray和7Gray剂量Y射线辐照处理的‘永年’和‘中牟’大蒜愈伤组织产生的组培苗中获得了2个和1个突变体。4.EMS处理对大蒜愈伤组织、再生芽和再生植株的影响调查结果显示,不同大蒜基因型对EMS的反应不同。一定浓度的EMS处理一段时间后,可促使ZM基因型大蒜愈伤组织分化出芽并促进芽生长。而YN基因型大蒜愈伤组织在所有的处理组及对照组中都未能成功诱导出芽,且处理组中的愈伤组织发生褐化。ZM基因型大蒜愈伤组织诱变研究的主要结果显示,随着EMS浓度的提高和处理时间的延长,平均每个愈伤组织产生的芽数、总芽数和由芽形成的植株数、植株长度和根数会减少。具体来讲,以0.2%的EMS处理1h,平均每个愈伤组织产生的再生芽数、总芽数和由芽形成的再生植株数、植株长度和根数达最大值,而后随着EMS浓度和处理时间的增加而减少。但是,再生植株的最大根长出现是在EMS为0.2%和处理时间为2h的处理中；0.4%和2h的EMS处理叶片数最多；在0.5%和3h的EMS处理条件下,形成的鳞茎最大。从表型看,存在潜在的遗传变异。更多还原

【Abstract】 Garlic (Allium sativum) is one of the most important plants used as vegetable, spice and flavoring agent for foods and medicine. China is the main producer of garlic in the world, with the production accounting for77.4%of the world. Additionally, Egypt is the fourth producers with the highest average yield of25.287ton/ha recorded. However, limited natural variation can not meet the requirement for genetic improvement and little breeding work for higher yield, good quality and resistance to biotic and a biotic stress has been done so far since garlic does not reproduce sexually. In addition, it is difficult to measure the secondary metabolic components and characterize the genetic variation accurately by conventional methods. So, it was important to discover elite germplasm and create new variants. UPLC as an effective method has developed for allicin quantification. SSR markers as a co-dominant marker have many advantages in identifying genetic variation. Mutagenesis based on tissue culture techniques may offer an effective method in improving vegetative propagated plants. For these reasons, three experiments were carried out:Firstly, assessment of impact of genetic and environment factors on allicin content in garlic germplasm from Egypt and China, Secondly, estimation of genetic diversity among garlic germplasms based on morphological traits and new SSR primers, lastly, somaclonal mutagenesis technology by gamma radiation and ethyl methane sulphonate (EMS) treatment on garlic callus. The main results are summarized as follows:1. Impact of genetic and environment factors on allicin content of garlic germplasmAllicin contents were quantified among104garlic germplasms (83accessions from China and21from Egypt) by UPLC. All germplasm were clustered into four groups based on allicin content. The germplasms with the highest allicin content clustered into Group A, which comprises one germplasm from Egypt and one from China. Cluster analysis revealed a wide range of variation in allicin content among these garlic germplasms, which can be exploited by selection in garlic breeding programs to improve garlic quality. It was found that some selected clones had higher allicin content than their parents, which shows that clone selection from a natural population can be effective for genetic improvement in garlic. The effects of germplasm, geographical location, and the interaction between germplasm and geographical location on allicin content (P<0.001) of Egyptian garlic germplasm were all highly significant. Egyptian garlic germplasms grown in China had lower allicin content than that grown in Egypt. The reduction in allicin content of the same germplasm grown in these two locations ranged from16%to63.7%. This finding confirmed that the choice of garlic growing region is of critical importance for improving allicin content for the garlic industry. 2. Estimation of garlic genetic diversity based on morphological traits and new microsatellite markersGenetic diversity of Chinese (83accession) and Egyptians (21accession) garlic germplasms have been analyzed using19morphological characters and16new microsatellites (SSR) primers developed upon transcriptome sequencing. These designed16new microsatellites primers generated44alleles among all accessions and the numbers of alleles per primers pair ranged from1to4with average polymorphic alleles per locus were2.75. The allele frequency ranged from0.5048to0.9615. The gene diversity ranged from0.0740to0.5000. Moreover, the hetrozygosity ranged from0.0481to0.9904and the average of PIC was0.2225. The obtained cluster tree based on morphological characters separated the germplasms into two major groups. However, dendrogram constructed based on SSR data using16novel microsatellites, divided the104garlic germplasm into three main clusters. Furthermore, there is a notice that both of dendrograms were almost in accordance with geographical location. In addition,12primers of16novel SSR primers confirmed the genetic variation between the clonal offspring selected and its parent, which proved that clonal selection could be effective method for garlic improvement.3. Study of mutation induction technology from garlic callus by gamma raysCallus of two Chinese garlic varieties (’Yong Nian’ and ’Zhong Mou’) were exposed to1,3,5and7Gray of gamma radiation. The obtained results revealed that YN was sensitive to high dose of gamma ray, while the variety ZM was the better performing variety for all growth parameters at higher gamma ray doses. More specifically, the average number of callus producing sprouts, sprout number per callus, total number of sprouts, the number of sprouts forming plantlets and plantlet’s length (which were33.33,2.37,296.00,170.00and13.32cm, respectively) were higher when callus was treated with1Gy in variety YN compared with other dosages. Whereas, in the former phenotype,10.33,3.00,92.00,78.00and17.10cm for the same parameters, respectively were higher in variety ZM at7Gy. Furthermore, days for bud emergence in ZM were shorter (154d) at4Gy irradiation treatment. However,129.7days were needed for variety YN callus treated with1Gy. SSR analysis showed that two primers out of16novel primers detected out genetic variation among treated materials. Two mutants were obtained from ’Yong Nian’when callus were treated with5Gray while one mutant was created in ’Zhong Mou’callus treated with7Gray of gamma rays. It could be found5-7gray of gamma ray could be the effective dosage for induction of genetic variations (mutation).4. Effect of EMS treatments on performance of garlic callus, regenerated sprouts and formed plantlets.In this investigation, it was observed that different genotypes responded to EMS in different ways. Certain concentration and exposure time of EMS were effective to initiate the callus to differentiate and enhance the sprout to growth only on ZM callus. However, all treatment combinations even control failed to induce sprouts in YN callus and the treated callus turned to browning.The main results from ZM showed that the reduction in the average number of sprouts per callus, total number of sprouts, number of sprouts forming plantlets, plantlet length and root number were observed with the increase in the concentration of EMS and its exposure duration. More specifically, the maximum value for previous most parameters above were recorded when the callus was treated with lowest EMS concentration (0.2%) for short duration (1h). However, the maximum root length was observed in the plantlet formed from callus subjected to0.2%of EMS for2h. Moreover, the most leave number was obtained from0.4%of EMS for2hour. Additionally, the treated callus with0.5%for3h gave the biggest bulblet diameter. From phenotypes, there are potential genetic variations.更多还原