【作者】 孟蕊；

【导师】 赵政阳；

【作者基本信息】 西北农林科技大学 ， 果树学， 2017， 博士

【摘要】 果实色泽是苹果果实重要的外观质量指标,很大程度上决定着果实的商品价值。果实的着色程度与果皮花青苷含量密切相关,同时花青苷也是果实重要的功能营养成分。研究苹果果皮色泽的遗传特性及调控机制,改善果实色泽和提高果皮花青苷含量是苹果育种及相关领域研究关注的热点。本研究以“粉红女士×富士”、“秦冠×富士”、“嘎拉×富士”等杂交组合的F₁群体为材料,对苹果果皮色泽的遗传趋势进行了田间调查分析;以杂交后代典型个体及不同色泽类型代表性品种为试材,以基于色差计的果实色泽参数为基础,初步建立了苹果果实色泽的量化评价方法。同时,以‘红星’、‘金冠’、‘秦冠’和‘澳洲青苹’等品种为材料,对与果皮花青苷合成相关的调控基因及结构基因进行了鉴定和表达分析。旨在进一步探究苹果果皮色泽的形成及其调控机理,为苹果色泽育种和改善果实色泽的栽培技术提供理论依据。取得的主要研究结果如下:（1）以“粉红女士×富士”、“秦冠×富士”、“嘎拉×富士”3个杂交组合后代群体为试材,对其果实表色和底色的色泽类型进行了调查,并对“粉红女士×富士”杂交后代部分植株的果皮色素含量做了测定。结果发现,果实的表色遗传与底色遗传相互独立,果皮红色对非红色为显性。表色遗传并不遵循单基因控制的质量性状的分离特点,也不遵循多基因控制的数量性状的分离特点;表色分离在3个杂交组合中差别较大,同一组合在不同的成熟期,红色与非红色植株的分离比存在显著差异,成熟期越晚,红色株系出现的比率越高,这些结果表明基因与环境因素相互作用,共同决定了果实表色的表现。果实底色的表现型同样也呈现由基因型和环境因素共同作用的特点。F₁代植株的底色类型和程度,主要受亲本底色类型影响,同时受成熟期影响也较大,同一组合内成熟期越晚的后代,其绿色型底色出现的频率越高。此外,果皮类胡萝卜素和叶绿素含量显著相关,说明果实底色类型（黄色或绿色）可能并不是完全独立的。从对杂种后代的分析看,果皮类胡萝卜素和叶绿素含量的频率分布均接近正态分布,表现出多基因控制的数量性状的特点。（2）选取杂交后代典型个体及不同色泽类型代表性品种,利用色差计法对不同色泽类型果实的色泽参数进行了测定,并做了相关性分析和主成分分析。结果表明,果实表色评分与果实表面a*值的变化呈显著正相关,而与L*、b*和h^o值呈显著负相关（P<0.01）;果实底色评分与果实表面L*和a*值呈显著正相关（P<0.01）,果实色泽参数L*、a*、b*、h^o之间也存在不同的相关性。主成分分析发现,果面L*值、b*值、h^o等主要决定了果实表色评分,而a*值、b*值、L*值、h^o均不同程度地参与了果实底色评分的构成。依据主成分分析的结果,选用L*值和h^o值两个指标作为定义果实色泽最基础的参数,同时将a*值、b*值也作为辅助的参考指标,定义了各色泽描述符所对应的L*、h^o、a*、b*值范围,最终提出了利用色差计对苹果果实着色面积、底色、表色进行评判的基本方法。（3）对‘红星’、‘金冠’、‘秦冠’、‘澳洲青苹’4个品种果实不同发育阶段的果皮色泽参数、花青苷含量和其它通过苯丙烷代谢途径合成的多酚类物质含量进行了测定分析。结果表明,苹果果实发育过程中色泽参数L*、a*、b*值的变化与果实颜色变化的趋势基本一致,果面L*值总是伴随着果实红色加深呈下降趋势,而a*值则呈上升趋势。果皮花青苷在果实发育过程中有两个合成高峰期——幼果期和成熟期,不同品种果皮花青苷的主要成分均是矢车菊素-3-半乳糖苷,但同一时期不同品种间果皮花青苷含量存在一定差异。幼果期‘秦冠’和‘金冠’果皮中花青苷的含量显著高于‘红星’和‘澳洲青苹’,而果实成熟期花青苷的合成仅发生在红色品种‘秦冠’和‘红星’中。果皮中二氢查尔酮、黄酮醇和黄烷醇的含量在幼果中最高,与花青苷一样,在盛花期后20 d或30 d达到峰值后开始迅速下降,在果实膨大过程中基本保持稳定。在果实接近成熟时,果皮中黄酮醇的含量有所上升,但果皮原花青素的含量仅在红色品种果皮中有明显的上调。（4）利用已经发表的与果实花青苷合成相关的MYB和bHLH基因,以及苹果和梨的UFGT基因,在苹果基因组中鉴定出了2个MYB1基因,2个bHLH3基因,3个bHLH33基因和4个UFGT基因,对它们在不同色泽类型苹果品种果实花青苷合成的两个高峰期的表达进行了分析。结果表明,MdbHLH3-2、MdbHLH33-2、MdbHLH33-3、MdUFGT1和MdUFGT3表达水平的变化与果皮花青苷积累没有明显的相关性。MdbHLH3-1和MdbHLH33-1的转录水平不论是在幼果期还是成熟期的果实着色过程中与果皮花青苷积累始终呈正相关。但是,MYB1和UFGT家族的表达模式在不同品种间和果实发育的不同时期都有所差别。MdMYB1a和MdUFGT2的表达水平在深红色果皮的幼果中要高于着色浅的幼果,而MdMYB1b和MdUFGT4在红色品种成熟过程中表达水平的变化与花青苷合成正相关。虽然MdMYB1a、MdMYB1b、MdbHLH3-1、MdbHLH33-1、MdUFGT2和MdUFGT4都参与了苹果果皮的花青苷合成,但是它们在果实发育的不同时期可能扮演了不同的角色。（5）对‘红星’、‘金冠’、‘秦冠’、‘澳洲青苹’果实进行套袋处理,测定了果实解袋后的着色过程中上述与花青苷合成相关的基因的表达水平。结果表明,套袋抑制了果实的花青苷合成,解袋后,果皮迅速开始花青苷的合成,与花青苷合成相关的结构基因和调节基因的表达水平在果实解袋后迅速上调,但不同品种间果皮花青苷含量和相关基因的表达模式各不相同。两个红色品种‘红星’和‘秦冠’果皮中花青苷含量最高,绿色品种‘澳洲青苹’果皮也积累了一定水平的花青苷,而黄色品种‘金冠’果皮中花青苷水平最低。MdMYB1a、MdMYB1b、MdbHLH3-1和MdbHLH33-1等调节基因的表达水平在4个品种解袋后的果实中均有不同程度的上调,其中在‘红星’和‘澳洲青苹’果实中表达高峰出现得最早。MdUFGT2仅在‘金冠’、‘澳洲青苹’和‘秦冠’解袋果实中表达,而MdUFGT4的表达水平仅在红色品种果实解袋后有所上调。更多还原

【Abstract】 Fruit color is one of the most important exterior qualities for apple,which greatly influenced the market value of the fruits.Anthocyanins in fruit skin not only contribute to fruit coloration,but also affect fruits’nutritional value.Therefore,apple breeders and researchers have paid a lot of attentions on inheritance of fruit color and its regulation,as well as improvement of fruit color and anthocyanin level in fruit skin.To explore the inheritance of apple fruit color,in this study,we investigated the fruit color in the F₁ progeny of three crosses,‘Pink Lady’בFuji’,‘Qinguan’בFuji’,‘Gala’בFuji’,and using the color-typical fruits,we established a method to evaluate apple fruit color quantitatively based on color parameters.Then,to further our knowledge about genetics of apple fruit color,we identifies members of several regulatory gene families and structural gene families involved in the anthocyanin pathway and analyzed their expression during fruit development in apple skin of‘Starkrimson’,‘Golden Delicious’,‘Qinguan’and‘Granny Smith’.Our research furthers our knowledge about apple fruit color development and its regulation,which has implications for improving fruit color through breeding and cultivation.Main results in this study are as follows:（1）We investigated over color and ground color in three cross populations,‘Pink Lady’בFuji’,‘Qinguan’בFuji’,‘Gala’בFuji’,and main pigments concentrations in fruit skin of progeny of‘Pink Lady’בFuji’,our results suggested that inheritance of fruit over color and ground color in apple were independent from each other and red was dominant and yellow or green recessive.The segregation ration showed that fruit over color was not controlled by either a single gene or multiple genes.The over color segregation differed greatly between the three populations,and even between different harvest date in the same population,the percentage of red individuals increased as the maturity season delayed.It was indicated that apple fruit over color was determined by both genetic effect and interaction between genotype and environment.It was also indicated that fruit ground color in apple were also controlled by genotype and interaction of genotype and environments.Ground color of F₁ progeny was remarkably influenced by their parents and maturity season.Percentage of individuals with greenish ground color was higher at late-season than at early season.Furthermore,carotenoid concentrations in fruit skin were significantly correlated with chlorophyll concentrations,suggesting that inheritance of yellow or green ground color was not completely independent.Chlorophyll a concentrations and carotenoid concentrations in apple skin both showed a typical normal distribution,indicating that the chlorophyll and carotenoid synthesis in apple skin were controlled by polygenes.（2）Fruit color,L*,a*,b*,h^oand C of the selected apples were measured by colorimeter,then correlation analysis and principal component analysis were initially applied to the rheological data.Fruit over color grade was positively correlated with a*value,while it was negatively correlated with L*,b*and h^o（P<0.01）.Fruit ground color grade was positively correlated with L*and a*values（P<0.01）.There were significant correlations between color parameters,too.The data by principal component analysis showed that over color grade was primarily determined by L*,b*and h^o,while L*,a*,b*and h^owere all involved in grading ground color.Thus,L*and h^o were chosen to be the basic parameters for evaluating fruit color,and a*and b*ancillary,a method to evaluate fruit over color intense,over color and ground color with clorimeter were developed after scoping L*,a*,b*and h^ovalues for each color descriptors.（3）Fruit color parameters,as well as concentrations of anthocyanin and other phenolic compounds that were synthesized by the phenylpropanoid pathway in apple skin of‘Starkrimson’,‘Golden Delicious’,‘Qinguan’and‘Granny Smith’during fruit development were determined.Values of L*,a*and b*changed during fruit development,which followed a similar pattern to fruit color,as red deepening,L*values decreased and a*values increased.There are two peaks of anthocyanin accumulation during apple fruit development,one at fruitlet stage,and the other one at ripening fruit stage.Cyanidin-3-galactoside was dominant in both young fruit and ripen fruit,but there were variations among cultivars at both fruitlet and ripening fruit stage.Anthocyanin concentrations in fruitlet skin were significantly higher in‘Qinguan’and‘Golden Delicious’than in‘Strakrimson’and‘Granny Smith’,while at ripening fruit stage,anthcoyanin was not detected in‘Golden Delicious’and‘Granny Smith’.Similar to anthocyanins,concentrations of dihydrochalcone,flavonols and favanols in fruit skin were highest in young fruit,then remained stable at fruit enlargement stage after a sudden drop from 20d DAFB or 30d DAFB.Before maturity,flavonols concentrations in fruit skin increased,but proanthocaynidins concentrations were up-regulated only in red-skin cultivars.However,further research is needed to find out whether proanthocyanidins synthesis was co-regulated with anthocyanins synthesis in apple skin.（4）All previously reported MYB and bHLH proteins related to the anthocyanin pathway and UFGT from apple or pear were used as Blastp queries to search candidate sequences in apple published genome,two MYB1,two bHLH3,three bHLH33 and four UFGT were identified.Transcript levels of all the identied genes were analyzed in the four cultivars during their two peak of anthocyanin accumulation.Results showed that the transcripts of MdbHLH3-2,MdbHLH33-2,MdbHLH33-3,MdUFGT1 and MdUFGT3 were not involved in anthocyanin synthesis in apple skin.Transcript levels of MdbHLH3-1 and MdbHLH33-1 were positively correlated with anthocyanin concentrations in fruit skin of both young fruit and ripen fruit.However,there were differences in expression pattern among cultivars at different stages.At fruitlet stage,transcript levels of MdMYB1a and MdUFGT2 were higher in dark red skin than in pale red skin,while transcript levels of MdMYB1b and MdUFGT4 were positively correlated with anthocyanin accumulation at ripening fruit stage.MdMYB1a,MdMYB1b,MdbHLH3-1,MdbHLH33-1,MdUFGT2 and MdUFGT4 were all involved in anthocyanin pathway in apple skin,but they might play different roles at different fruit development stages.（5）Expression of MdMYB1a,MdMYB1b,MdbHLH3-1,MdbHLH33-1,MdUFGT2 and MdUFGT4 were analyzed in apple skin of‘Starkrimson’,‘Golden Delicious’,‘Qinguan’and‘Granny Smith’after bag removal.Our data showed that anthocyanin synthesis in apple skin were inhibited by fruit bagging,and these genes expression was up-regulated after bag removal,accompanied with anthocyanin accumulation.However,there were variations in anthocyanin concentrations and expression pattern of the selected genes among different cultivars.Anthocyanin contents were highest in fruit skin of two red-skinned cultivars,‘Starkrimson’and‘Qinguan’,the second highest in fruit skin of the green-skinned cultivars,‘Granny Smith’,the lowest in fruit skin of‘Golden Delicious’.Transcript levels of most regulatory genes,MdMYB1a,MdMYB1b,MdbHLH3-1 and MdbHLH33-1,increased in fruit skin after bag removal,and their peak of transcripts occurred earlier in‘Starkrimson’and‘Granny Smith’than in‘Qinguan’and‘Golden Delicious’.MdUFGT2 were expressed only in fruit skin of‘Golden Delicious’,‘Granny Smith’and‘Qinguan’,yet MdUFGT2 transcripts were active only in red-skinned fruits,indicating that UFGT might be key gene in the anthocyanin pathway in apple.更多还原