【作者】 樊红科；

【导师】 王飞；

【作者基本信息】 西北农林科技大学 ， 果树学， 2011， 博士

【摘要】 苹果新品种选育以常规杂交育种为主,杂交后代选择研究是苹果育种研究的一个重要方面。分子标记技术作为一种新兴的研究手段,在苹果育种研究中能进一步补充与充实常规方法的研究内容与结果。WRKY转录因子是近年来在植物体内发现的特有转录调控因子,能够调控多个下游功能基因的表达,从而使转基因植物获得综合抗病抗逆能力,克隆和转化这些转录因子具有较高的应用价值。苹果早期落叶病是渭北黄土高原发生比较严重的一种苹果真菌性病害,已经成为苹果生长发育和产量形成的重要限制因子。因此克隆与此相关抗病基因并对这些基因进行表达特性分析及功能鉴定具有重要意义。本文以短枝富士和粉红女士杂交的216株F1代群体为试材,应用数学方法,对杂交后代果实性状选择研究进行了探索。应用简单重复序列(Simple Sequence Repeat,SSR)技术,对果实酸度基因(Ma)进行了分子标记研究。同时,对苹果斑点落叶病的综合评价及与其抗性相关MdWRKY基因功能进行了研究。获得的主要研究结果如下:1.通过对富士、粉红女士及216株杂交F1代果实大小、风味、颜色等性状进行了统计分析,明确了双亲各果实性状的差异大小。同时对杂交后代果实性状遗传研究表明苹果果皮表色遗传表现为简单的质量遗传,红色遗传表现为多基因控制的数量遗传。果形表现了明显的趋中变异现象,果实大小、可溶性固形物(Soluble Soild Content, SSC)及含糖量(Total Sugar Content,TSC)呈退化的负向优势,后代群体有劣变趋势。杂交后代果实分别呈现肉质变粗、质地变硬、果汁减少的趋势。2.以富士和粉红女士多年的果实性状平均表现和预期育种目标为基础,制订了杂交后代选择标准。以果色、果型、风味作为选择的主要依据,确定了单果重、果形指数、果实硬度、糖酸比等12个适宜性评价指标,采用5级评分制对各指标进行量化分级,运用层次分析法和熵值法确定了各指标的综合权重,提出了杂交F1代果实性状综合评价模型;运用改进列联表法,建立了杂交F1代果实丰产性状综合选择模式。3.利用SSR标记,结合集群分类分析法(Bulked Segregation Analysis, BSA)进行了苹果酸度基因(Ma)分子标记研究。通过对102对SSR引物的筛选,获得了与果实酸性状紧密连锁的分子标记CH03d12-104和CH03d12-118,连锁距离分别为3.24cM和2.31cM。分子遗传表明果实含酸量受一对主基因和一对加性基因控制, Ma对ma表现为完全显性。将获得的标记在40个栽培品种中做了进一步验证,基因型与表型性状符合率为97.5%。证明了该标记在苹果酸性状验证的准确性,进一步讨论了该标记在苹果品质育种辅助选择中的应用。4.苹果杂交后代斑点落叶病抗性生理指标的综合评价及预测。以富士与粉红女士杂交F1代为试材,将苹果各生理指标的抗斑点落叶病系数,作为衡量苹果单项抗病能力大小的指标,用主成分分析法将各单项抗病系数综合成几个新的相互独立的综合指标,再利用隶属函数求出每一单株抗病性的综合评价值,以此评价各杂交F1代的抗病性。同时建立了植株抗病性综合评价值与单项指标抗斑点落叶病系数间的最优回归方程。杂交F1代单株抗病性综合评价值越高,抗病性越强。叶片净光合速率、绿原酸含量、苯丙氨酸解氨酶含量和百叶厚4个指标,可以作为苹果抗斑点落叶病的鉴定指标。回归分析表明,所建方程相关性显著,方程预测值的聚类分析结果与各株抗病性强弱顺序一致,所建回归方程能够预测苹果杂交后代个体的抗病性。5.苹果抗斑点落叶病MdWRKY基因功能研究。采用反转录聚合酶链式反应(Reverse Transcriptase Polymerase Chain Reaction, RT-PCR)方法从抗苹果斑点落叶病的秦冠苹果中克隆到一个WRKY基因,全长1224bp,推断其编码331个氨基酸,命名为MdWRKY,GenBank登录号为HM859901。亚细胞定位分析MdWRKY蛋白分布在细胞核内,属于核蛋白。酵母体内的转录激活实验表明,MdWRKY在酵母细胞中具有转录激活功能。实时定量分析结果显示,MdWRKY基因受苹果斑点落叶病菌的诱导,表明其参与植物与病原菌的互作。半定量分析结果显示,MdWRKY被水杨酸(Salicylic Acid,SA)和茉莉酸甲酯(Methyl Jasmonate,MeJA)转录诱导,表明MdWRKY基因参与了SA和MeJA在抗病防御反应中的信号传导。MdWRKY基因在烟草中的过表达能够提高转基因烟草抗烟草黑胫病能力,表明MdWRKY基因具有一定的抗病功能。更多还原

【Abstract】 Cross breeding was still main methods for breeding new apple cultivars. Genetic evaluation of main characters can provide basic genetic theory for selecting parents and improving efficiency of apple breeding. Molecular marker as a newly appeared technique can enrich the results of normal methods,and supply molecular basics for breeding program. WRKY transcription factors play important roles in plant defense signaling network. Alternaria leaf spot is considered one of the most important limiting factors with apple growth and yield in Weibei loess plateau. Clone and identification a gene related to apple Alternaria f. Mail resistance can provide application in apple genetic engineering. The experiment carried out the evaluation of parental breeding values with Fuji and PinkLady, the genetic variation and selection of fruit traits in apple hybrids by applying of mathematical and statistical methods, the molecular markers on fruit acid / low acid trait by SSR, the comprehensive evaluation and the function of MdWRKY gene with resistance to apple Alternaria f. Mail. The main results were as follows:1. Analysis on the fruit traits of parents and hybrid progenys. The genetic variation of fruit traits were analyzed in 216 hybrids of Fuji×PinkLady. The results showed that Fuji and PinkLady had differences in the fruit traits. The inheritance of fruit skin color was simple qualitative heredity and the red color showed quantitative heredity which was controlled by multi genes. The inheritance trends of fruit shape appeared distinct central variation. The fruit size、SSC and TSC tended to be less than parental mean, which causing the fruit quality of progenies was poor. The hybrids showed coarser flesh, firmer texture, less juice and smaller core size.2. Study on comprehensive selection of fruit traits in apple progenys. Based on the several year average traits and expected breeding goals of Fuji and PinkLady, the selective standards of each trait were drawn up. A set of twelve traits for selection such as single weight, fruit shape, firmness and so on, were formulated as the suitability indicators of hybrids. A five-graded method was adopted to quantitatively evaluate each indicator of hybrid and hierarchic analysis as well as adopted to determine weight of each index. A set of indices as well as quantitative model for evaluating the fruit traits of hybrids were developed. At the same time, according to the improved contingency table method, a model of comprehensive selection of apple traits for high yield was also proposed. The practice proved that two model was useful for apple breeding.3. SSR markers linked to the Ma gene for fruit acidity in apple. A molecular marker linked to the acid gene (Ma) in apple was explored based on the 216 progenies of Fuji×PinkLady by BSA and SSR technique. A total of 102 primers were screened and SSR markers (CH03d12-104+ CH03d12-118) closely linked to Ma gene was identified, and the linking distance of two markers respectively is 3.24cM and 2.31cM. The SSR marker analysis showed that the fruit acid trait was governed by one major gene and one additive gene. Ma was completely dominant to ma. The CH03d12-104 + CH03d12-118 marker was further confirmed in 40 apple varieties which showed concordant rate between the genotype and the phenotype were 97.5%.Simultaneously the potential application of two markers in a marker-assisted quality breeding programme is further discussed.4. Comprehensive evaluation and forecast on physiological indices of Alternaira alternata f. sp. mali (AaM) resistance in F1 hybrids of apple.Based on F1 hybrids of Fuji and PinkLady, the physiological indices of resistance to AaM were comprehensively evaluated and predicted, which were used to value the F1 hybrids’single capacity of AaM resistance to some new independent comprehensive indexes by principal component analysis. The comprehensive value of different F1 hybrids AaM resistance was obtained by using subor-dinative function,So the AaM resistance of different F1 hybrids can be evaluated. Meanwhile, the regression were analyzed between the comprehensive value of AaM resistance and the value of single index to AaM resistance.The results showed that the comprehensive value were higher, the F1 hybrids AaM resistance were better. The net photosynthetic rate, chlorogenic acid content、phenylalanine ammoniumlyase content in apple leaves and thickness of hundred leaves would be used as indexes of evaluating AaM resistance in apple. The analyzed regression indicated the function’s square were prominent.The results of Cluster analysis show that strength of the order was the same. The regression predicted Apple hybrids individual resistance.5. Function of MdWRKY gene with resistance to apple Alternaira leaf spot. One MdWRKY was isolated from Qinguan apple, which is resistant to Alternaria leaf spotusing RT-PCR approaches. MdWRKY was 1224bp and encoded a 331 amino-acid protein. Its GenBank accession number is HM859901. The MdWRKY protein was targeted to the nucleus and activated the expression of a reporter gene, consistent with the functioning of a transcription factor. When plants were infected with the pathogen Alternaria alternata f. sp. mali, MdWRKY wasinduced dramatically. Similarly, treatment with hormones SA and MeJA increased transcription significantly. Overexpression in tobacco also enhanced resistance to Phytophthora parasitica var. nicotianae Tucker. These results suggest that MdWRKY is a positive regulator of the defense response in higher plants.更多还原