【作者】 王海平；

【导师】 李锡香；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2011， 博士

【摘要】 大蒜(Allium sativum L.)为百合科(Liliaceae)葱属(Allium)重要的蔬菜兼药用植物。中国是世界上最大的大蒜生产国和出口国。然而,我国大蒜研究基础薄弱,对种质资源遗传背景和群体遗传结构尚不明确;无性繁殖大蒜不能人工杂交构建常规作图群体的难题也阻碍了大蒜辣素等重要性状相关基因的挖掘。随着国际市场对大蒜商品品质和营养品质,尤其对大蒜辣素含量要求的提高,迫切需要开展相关方面研究,以促进我国大蒜产业的可持续发展,保持我国在大蒜国际贸易上的优势。本文通过对我国大蒜种质资源表型性状及AFLP、SSR和InDel三种分子标记鉴定数据的分析,明确了我国大蒜资源遗传背景和群体遗传结构,为关联作图奠定了理论基础;建立了大蒜辣素的超高效液相色谱(UPLC)检测技术体系,对212份资源大蒜辣素含量进行了检测;通过TAIL -PCR、RT-PCR技术获得了大蒜辣素合成途径中的关键基因-蒜氨酸酶基因的编码区,对其结构和基因多样性进行了分析;根据蒜氨酸酶基因内含子2序列开发了一个InDel标记,并对其与大蒜辣素含量的关联性进行了探讨。主要结果如下:1.我国大蒜种质资源的表型多样性与分类。对资源圃保存的212份大蒜种质资源的表型性状进行了系统鉴定,统计分析表明我国大蒜种质资源在表型变异很大,据此提出了21个数量性状的5级分级标准。为了避免质量性状在种质评价中的主导作用,对与产量相关的鳞茎数量性状进行了重点分析,结果表明,构成产量的数量性状均表现较大变异。主成分分析显示,前三个主成分累积贡献率达74.83%,第一主成分中鳞茎重、鳞茎直径和鳞芽数是影响产量的主要因子。鳞茎重和鳞茎直径与产量相关性最大。通过主坐标排序,将所有资源分为6类。通过综合评价,将大蒜鳞茎产量分为6个级别,筛选出单产大于15×103 kg ha-1的资源3份。2.基于AFLP、SSR和InDel标记的大蒜种质资源群体遗传结构和聚类分析。利用三种分子标记技术在212份种质中扩增出502个位点,多态性位点为492个。群体遗传结构与聚类分析均将所有资源划分为5个类群体。划分的类别基本一致。然而,群体遗传结构分析划分的5个群体,群体内的遗传信息多样性指数较小。对212份种质鳞茎大蒜辣素和21个数量性状与群体遗传结构进行线型模型分析,结果表明包括大蒜辣素含量在内的多个数量性状受群体遗传结构的影响较小,说明我国资源圃中保存的大蒜种质资源遗传背景丰富,适合进行相关分子标记与表型性状的关联作图分析。3.大蒜辣素超高效液相色谱(UPLC)检测技术体系的建立和应用。通过对样品处理、提取方法和检测方法的研究,首次建立了完善的大蒜辣素超高效液相色谱(UPLC)检测方法:使用UPLC BEH C18色谱柱,流动相为甲醇:水=1:1,检测波长为254 nm,进样体积为1 uL,流速为0.3 mL min-1。大蒜辣素在2.04～510 mg L-1范围内呈良好线性关系(R2 = 0.9991)。研究了大蒜辣素水提取液对酸碱溶液、温度及光照的稳定性,结果显示大蒜辣素对温度和强酸强碱敏感,对光照不敏感。在室温下,大蒜辣素在pH=6.0左右时较稳定,5d内未发现明显降解。但在pH低于1.5或高于11.0的强酸或强碱条件下,大蒜辣素降解迅速。当温度高于40℃时,降解速度加快,高于70℃时降解明显加速。提取液浓度较高,稳定性便较强。利用建立的方法对212份大蒜鳞茎的大蒜辣素含量进行检测,发现资源圃中大蒜资源的大蒜辣素含量差异显著,含量分布在0.82～3.01%之间,最高值与最低值相差近4倍。大蒜辣素含量与植株对蒜蛆抗性的关系分析表明,大蒜辣素含量越高,大蒜对蒜蛆的抗性越强,进一步说明了大蒜辣素抗菌、杀虫的生物活性。4.蒜氨酸酶基因编码区的结构变异与品种演化分析。通过TAIL-PCR、RT-PCR技术获得蒜氨酸酶基因编码区,从基因组水平及cDNA水平上研究了大蒜蒜氨酶基因编码区的基因结构,发现蒜氨酸酶基因家族基因结构复杂,由5个外显子和4个内含子组成。内含子差异明显,外显子相对保守。4个内含子长度和序列均有较大差异,存在很多InDel和重复序列。其中内含子2多样性最大,而在外显子1、3和5发现了InDel现象和单个碱基变异。通过对外显子和内含子分析,将蒜氨酸酶基因家族编码区划分为10个基因模式。其中内含子2包括了所有基因模式。对来源不同的3份大蒜资源E1, GH1和CN313(E1来自乌孜别克的野生资源,GH1来自美国,CN313来自的中国)的蒜氨酸酶基因的基因组序列进行聚类分析,在一定程度上证明了大蒜起源于中亚,而后逐渐向西方传播,最后传入东方的观点。5. InDel标记的开发和应用。基于不同种质蒜氨酸酶基因内含子2序列,首次开发了InDel标记,并对212份大蒜种质进行了检测,在所有资源中共检测到10个基因位点,多态性为100%。基于InDel标记将212份大蒜划分为5类,说明开发的蒜氨酸酶基因InDel标记对资源遗传多样性及分类具有研究价值。6.大蒜辣素含量与蒜氨酸酶基因变异的关联分析。首次对大蒜分子标记(InDel标记)和表现型(大蒜辣素含量)的相互关系进行了研究,结果表明,InDel基因型不同的大蒜资源之间大蒜辣素含量有显著差异。基因型C与高大蒜辣素含量相关联,基因变异位点分别为335和327bp,基因型I与低大辣素含量相关联,基因变异位点分别343、340和323bp。说明开发的InDel标记可以作为辅助分子标记,筛选高大蒜辣素含量资源。更多还原

【Abstract】 Garlic (Allium sativum L.), an asexually propagated crop, is grown as an important vegetable and medicinal plant. China is the biggest garlic producer in the world. However, little is known about the genetic background of garlic germplasm resources in China. The characteristics of vegetative propagation of garlic makes it very difficult to construct mapping populations for research on elite genes. With the increasing requirement for nutritional quality, especially for high allicin content, it is urgent to carry out relative research for sustainable development of garlic industry to keep China at the present prosperous position in international garlic trade.In this study, the genetic diversity and the genetic structure of garlic germplasm in China were evaluated based on the morphological traits and three kinds of molecular markers (AFLP,SSR and InDel ); an efficient UPLC detection method for allicin content in garlic bulb was developed and used to detect the allicin content among 212 accessions of garlic germplasm; the gene structure and diversity of alliinase genes from garlic within encoding region were studied by TAIL-PCR and RT-PCR technology; a novel InDel marker was developed according to the sequences of intron 2 of alliinase genes; association mapping method was used to study the relationship between allicin content and InDel marker. The major results are as follows:1. Phenotypic diversity and classification of garlic germplasm in China were studied based on morphological traits by methods of clustering, principal component analysis, and correlation analysis. The results showed that the garlic germplasm in China presented a wide diversity from all traits. The five-grade classification method for each quantitative trait was brought forward. The results of clustering and principle component analysis indicated that clustering factors were mainly from qualitative traits. To avoid the overrating effect from qualitative traits, eight important quantitative traits related to bulb yield were evaluated. Principal component analysis showed the cumulative contribution proportion of the first three components was up to 74.83% and three traits (bulb weight, bulb diameter and clove number) were included in the first components which could be the most important traits affecting bulb yield. Correlation analysis suggested that bulb yield was significantly related with seven of eight characteristics except clove back width, and the most highly related with bulb diameter and bulb weight. All accessions of garlic germplasm were divided into 6 groups according to yield and the analysis of principal coordinates. Moreover, three accessions yielded above 15×103 kg ha-1 were selected out.2. Analysis of population genetic structure and clustering analysis of garlic germplasm was performed based on AFLP, SSR and InDel. Totally, 502 allels were amplified by AFLP, SSR and InDel primers, 492 of them were polymorphic among 212 accessions of garlic. All accessions were divided into 5 groups by both structure analysis and neighbor-joining clustering. However, the Shannon’s index of each group assumed by genetic structure analysis was smaller than that assumed by neighbor-joining clustering, which indicated the genetic structure analysis could interpret genetic relationship among the individual accessions in more details. Most of traits including allicin content were slightly affected by population structure, which indicated the germplasm in this study was acceptable to be the populations for association mapping.3. An efficient UPLC method for quantitative determination of allicin in garlic bulb was developed and used to detect the allicin content among 212 accessions of garlic germplasm. In the UPLC method,methanol/water (50:50, v/v) as a mobile phase which ran through at a constant flow rate of 0.3 mL min-1 was performed onUPLCTM BEH C18 column. The stability of allicin extracted with water from garlic bulb powder was evaluated in the following conditions: phosphate buffer , pH value from 1.50 to 11.0; temperatures, from -20 to 85℃, and light in absence or presence. Results indicated that allicin in solution was sensitive to pH and temperature of storage, but not to light. At room temperature, allicin was most stable with pH 5.0 to 6.0, but it degraded quickly at lower or higher pH. but it degraded quickly when the pH was lower than 1.5 or higher than 11.0. Allicin degraded more quickly when temperature was higher than 40℃, and especially when temperature was higher than 70℃. At room temperature (25℃), allicin in water could be stored for 5 days without obvious degradation. Higher the concentrations of allicin in solution were somewhat more stable than low concentrations. Allicin content of garlic germplasm ranging from 0.82 to 3.01% was significantly different. Based on investigation of 52 accessions, the accessions with the higher allicin content were more resistant against the Delia antique Meigen.4. The gene structure and its diversity of alliinase genes from garlic within coding region were studied by TAIL-PCR and RT-PCR technology. Results confirmed that the alliinase genes possess 5 exons and 4 introns. Extraordinary diversity was found among these alliinase genes, especially within introns. Besides, some InDels and couples of candidate SNPs positions were found within exons as well. Combining the introns and exons, 10 types of alliinase genes were predicted. Three garlic germplasm E1, GH1and CN313, from Uzbekistan (Central Asia), USA (West), and China (East) respectively, were used for analyisis of diversity and evolvement of alliinase genes. Cluster tree based on the alliinase genes from genomic sequences supported the idea that garlic was spread from the original center (central Asia) to the west countries and then to the east.5. A novel alliinase gene specific InDel-primer flanking the intron 2 was designed according to the alliinase sequences from the three accessions of garlics. The InDel primer produced 10 polymorphic alleles and divided 212 accessions of garlic into 5 genotype groups. The results indicated that the InDel maker was valuable for the diversity and classification analysis of garlic germplasm.6. The association analysis based on candidate gene method was used to study on the relationship between the InDel marker and allicin content. Results showed the high allicin content was significantly linked to the genotype C with bands of 335 and 327 bp, and low allicin content was significantly linked to genotype I with bands of 343, 340 and 323 bp. The InDel maker could be an assisted marker to select high allicin content variety from garlic germplasm.更多还原