【作者】 李森；

【导师】 邢国明； Yiqun Weng；

【作者基本信息】 山西农业大学 ， 蔬菜学， 2015， 博士

【摘要】 众所周知,黄瓜(Cucumis sativus L.)是世界上最重要的蔬菜作物之一。黄瓜的遗传基础较为狭窄,栽培种间的多态性仅为3-9%。长期的人为选择进一步降低了黄瓜育种材料的多样性,导致现有黄瓜栽培品种的优异基因来源单一,对各种不利环境条件和病虫害的抵抗能力逐渐下降。为了发掘优异的黄瓜种质资源,并利用关联分析高效挖掘黄瓜优异基因,从而为黄瓜育种提供候选种质和黄瓜分子辅助选择育种提供理论依据,本论文从以下几方面开展了研究：1.以不同地理分布和生态类型的具有代表性的203份黄瓜自交系为材料构建了黄瓜关联分析群体。选择在黄瓜基因组中均匀分布,并多态性较高的215对SSR引物对203份黄瓜材料进行基因型分型,共检测到1165个等位基因,每个位点的等位基因数(Na)为2-16,平均为5.42个。采用Structure v2.3.4软件中的贝叶斯(Bayesian)聚类方法对黄瓜种质资源的遗传结构进行分析,设置群体分组范围K值为1到10,选择不含任何种群来源信息的混合祖先模型(Admixture model)和等位变异发生频率相关模型(Allele frequency correlated model)来进行分析,设定不作数迭代(Length of burn-in period)和不作数迭代后的马尔可夫链MCMC (Markov’s Chain Monte Carlo)分别为50000和100000,各20次重复运算。根据运算结果,判断K=2时接近真实K值,将203份黄瓜材料分为2个类群,一个为欧美黄瓜类群,共114份黄瓜材料,主要包括北美加工型、北美鲜食型和欧洲温室型黄瓜。另一个为亚洲黄瓜类群,共99份黄瓜材料,主要包括华北型、华南型、日本型、印度型、野生黄瓜(Cucumis sativus var. hardwickii)和西双版纳黄瓜。当K=3时,亚洲黄瓜类群被划分印度黄瓜类群和东亚黄瓜类群。当选择K=4时,欧美黄瓜类群被划分为北美型黄瓜和欧洲温室型黄瓜。2.以WI 2757(3心皮)和True Lemon(5心皮)作为亲本,对控制黄瓜心皮数基因Cn进行了定位及克隆研究。对110个F3家系进行表型调查,通过遗传分析发现,3心皮性状对5心皮性状是显性,F2群体的分离比例符合1：2：1,说明该性状是简单质量性状。初定位结果显示Cn位点位于黄瓜1号染色体的长臂,位于2个SSR标记SSR01816和UW083720之间,且分别位于Gy14的Scaffold03078和Scaffold00464。在连锁分析初定位的基础上,利用自然群体对心皮数性状进行了关联分析。以Gy14基因组序列为参考序列,利用BWA程序对5心皮和3心皮黄瓜材料的基因组重测序数据进行了SNP频度分析,结果表明在Scaffold03078的270337bp至286040bp大约16Kb区间内有45个SNP位点与心皮数性状高度相关。FGENESH程序在此区段预测到5个基因,其中CLAVATA3为控制心皮数的最优候选基因。在此候选基因内有5个与心皮数相关的SNP,与122份不同的黄瓜材料基因组重测序数据进行比对后,结合已知的部分黄瓜材料的心皮数表型数据,判断位置为271311bp的SNP与黄瓜心皮数高度关联。对该位点SNP设计了1个dCAPS标记,并在分离群体中进行了验证。利用qPCR对CLAVATA3进行了表达分析,结果表明该基因在双亲WI 2757和True Lemon的同一组织间表达差异显著：CLAVATA3在WI 2757幼叶、未开花的子房和顶芽中的表达量分别是True Lemon的2.84,3.17和3.74倍。推测位于271311bp的碱基由“T”变为“C”后,抑制了CLAVATA3基因的表达,WUX基因表达量增加,导致心皮数增加。根据以上研究结果,克隆了CLAVATA3基因在WI 2757和True Lemon中基因组序列。更多还原

【Abstract】 As known by everyone, cucumber (Cucumis sativus L.) is one of the most important vegetable crops in the world. The genetic background is narrow, and the polymorphism between the cultivars is only 3%-9%. During the artificial selection in the long term, the polymorphism of cucumber lines were reduced more, the less sources of excellent gene were caused, and the resistance was declined for adverse environment, disease and insects.In order to explore the excellent cucumber germplasm, and detect the excellent cucumber gene by association, furthermore supply the candidate germplasm for cucumber breeding and the theoretical basis for cucumber molecular assistant selection, the research was conducted from follow aspects.1. The association population was constructed with 203 cucumber lines which were selected from the different geographic distribution and biology type. According to the Gy14 Scaffold physical map, the higher polymorphic 215 pair primers were selected homogeneously which used for genotyping the 203 cucumber lines.Averagely, every 3.29 cM distance distributed with 1 SSR primer. Totally,1165 alleles were detected, the number of detected alleles (Na) varied from 2 to 16, with an average of 5.42 alleles per locus.The genetic structure was analyzed by Structure v2.3.4 software with Bayesian cluster method. The parameter was set as K=1 to 10, Admixture model, Allele frequency correlated model, Length of burn-in period 50 000, MCMC (Markov’s Chain Monte Carlo) 100000, and 20 times iteration. The result showed that K=2 was the true K value, and 203 cucumber lines were clustered to 2 groups. One is America-Europe group with 114 cucumber lines, and the other one is Asian group with 99 cucumber lines. When K=3, the Asian group was divided to India subgroup and East-Asian subgroup. When K=4, the America-Europe group was divided to North-America subgroup and Europe Greenhouse subgroup.2. The mapping and clone of cucumber carpel number (Cn) gene were conducted with two inbred lines WI2757 (3 carpels, few 4 carpels) and True Lemon (5 carpels, few 4 carpels). The phenotypes of 110 F3 families were investigated, and the genetic analysis showed that the 3-carpel is dominant for the 5-carpel and the segregation ratio in F2 family was consistent with 1:2:1. This trait was a simply inherent trait.The Cn gene was initially mapped between two markers of SSR01816 and UW083720 which were belong to two different Gy14 Scaffolds of 03078 and 00464 separately in the long arm of cucumber chromosome 1.Based on the result of linkage analysis, the association mapping of carpel number were conducted with natural population. With Gy14 genome sequence as reference, the re-sequenced genome data of 5-carpels and 3-carpels were aligned by BWA. The results showed that the candidate region for Cn gene is 15703bp genomic DNA region with 45 SNPs between 270337 and 286040 in Scaffold03078. Gene annotation of this 16 Kb region identified 5 genes include a CLAVATA3 gene. There were 5 SNPs in this gene region, which was aligned with 121 re-sequenced lines by Reference Mapper. According to the known phenotype data of the 121 cucumber lines, the SNP in 271311bp of Scaffold03078 was associated with cucumber carpels number. The dCAPs marker for this SNP was developed and confirmed with segregation population. Quantitative RT-PCR revealed lower level of expression of this CLAVATA3 gene in True Lemon than in WI2757 in ovary, younger leaf and terminal bud. The 271311bp position SNP which is "T" in the lines of 3 carpels and "C" in the lines of 5 carpels down regulated the expression of CLAVATA3, the WUX expression was higher, and the carpel number increased.According to these result, the CLAVATA3 genome sequence in the parents were cloned.更多还原