【作者】 李灵；

【导师】 高平；

【作者基本信息】 四川大学 ， 生物学—植物学， 2023， 硕士

【摘要】 猕猴桃（Actinidia chinensis Planch.）是一种可食用型水果,其果肉颜色主要为黄肉、绿肉、红肉这三种类型。猕猴桃口感鲜甜、风味独特、营养价值极高。本研究以不同颜色猕猴桃为材料,对其进行了果实品质分析,遗传多样性分析以及采摘前后风味代谢物分析,探究了不同颜色猕猴桃的品质差异以及采摘前后的风味转变特征,旨在为研究猕猴桃的遗传多样性和亲缘关系提供理论依据,也为不同颜色猕猴桃的的品质评价及适宜采收和存储提供理论支持。主要研究结果如下:（1）不同颜色猕猴桃果实多种生理指标的测定。本研究以不同颜色猕猴桃种质资源共29个供试材料,测定了果实色差、硬度、可溶性固形物、总糖、淀粉、可滴定酸、维生素C和类胡萝卜素等指标。结果表明,不同颜色的果实之间存在显著差异,红肉猕猴桃整体可溶性固形物高于黄肉和绿肉;维生素C含量的变异系数最高,为49.01%。随后,使用相关性分析探究不同生理指标之间的关系,色差、类胡萝卜素、可滴定酸之间存在显著或极显著的相关性。初步评价了不同颜色猕猴桃的果实品质差异。（2）猕猴桃ISSR-PCR扩增反应体系的优化。首先对选出的12条引物进行温度梯度试验,得到合适退火温度,接着对DNA浓度、引物浓度、Mg²⁺浓度等主要因素进行单因素实验,利用直观分析法对其结果进行评分。结果显示,反应总体系为20μL,模板浓度70 ng/μL、引物浓度1.0μmol/L,Mg²⁺浓度2 m M,mix体积为10μL时为最佳组合,利用该参数获得了清晰明确的扩增产物。该研究结果对猕猴桃ISSR-PCR扩增反应的优化和应用具有重要的参考价值。（3）猕猴桃的遗传多样性分析。利用12条ISSR引物对42份猕猴桃材料进行扩增分析,对结果进行遗传多样性研究。结果显示,这些引物共扩增出105条条带,多态性比例高达96.19%。随后,聚类分析表明,42份材料被分为了两大类,Ⅰ类全是杂交F1代,Ⅱ类属于其他品种（系）材料。Ⅰ类和Ⅱ类分别将所有品种中的黄肉、绿肉、红肉猕猴桃聚到一起。该研究结果初步表明了ISSR标记可用于猕猴桃果肉颜色分类。（4）猕猴桃采摘前后风味代谢物的高通量检测。采用顶空固相微萃取与气相-质谱联用技术（HS-SPME-GC-MS）和超高效液相-质谱联用技术（UHPLC-MS）检测猕猴桃代谢化合物种类。结果显示GC-MS分析共鉴定485种挥发性化合物,分为14类,以萜类、酯类、杂环化合物、醇类为主;LC-MS分析共鉴定834种非挥发性化合物,分为13类,以脂质和类脂类、苯丙类和聚酮类、生物碱类为主。（5）不同颜色猕猴桃采摘前后代谢物变化特点。结合主成分分析和K均值聚类结果,发现不同颜色猕猴桃在萜类、酯类和杂环类化合物存在较大的代谢模式差异。猕猴桃采后储存一段时间后,差异代谢物最多,不同颜色猕猴桃在采摘后储藏的过程中代谢模式差异显著。通过KEGG通路富集分析,不同颜色猕猴桃在酪氨酸代谢、α-亚麻酸代谢、丁酸甲酯代谢、柠檬酸循环、单萜类生物合成通路上差异显著。比较不同类别代谢物的含量变化,红肉在采后常温储存的过程中积累酯类和醇类化合物比其他品种更明显,与其他颜色的代谢物模式差异最大,这可能导致了红肉猕猴桃香气比其他品种更浓郁。该研究结果表明不同颜色猕猴桃具有各自的采摘特性,以及不同的成熟特性和风味,可为猕猴桃品质评价及适宜采收和存储提供数据支持。更多还原

【Abstract】 Kiwifruit（Actinidia chinensis Planch.）is an edible fruit.The flesh of kiwifruit is commonly classified into three types:yellow,green,and red.Kiwifruit is characterized by its fresh and sweet taste,unique flavor,and high nutritional value.In this study,we analyzed the fruit quality,genetic diversity,and flavor metabolites of kiwifruits with different colors,and explored the differences in quality and flavor changes before and after harvest.The purpose of this study is to provide theoretical basis for the study of genetic diversity and genetic relationship of kiwifruit,as well as to provide theoretical support for the quality evaluation,suitable harvest,and storage of kiwi fruits with different colors.The main research results are as follows:（1）Determination of multiple physiological indicators in kiwifruit with different colors.In this study,29 kiwifruit with different colors were used to measure color difference,hardness,soluble solids,total sugar,starch,titratable acidity,vitamin C,and carotenoids.The results showed significant differences among various physiological indicators.The overall soluble solids in red-fleshed kiwifruit were higher than those in yellow-fleshed and green-fleshed kiwifruit.The coefficient of variation of vitamin C content was the highest,reaching 49.01%.Subsequently,correlation analysis was used to explore the relationship between different physiological indicators,and there was a certain correlation between color difference,carotenoids,and titratable acidity.The quality and differences of kiwifruit with different colors were preliminarily evaluated.（2）Optimization of ISSR-PCR amplification system for kiwifruit.Firstly,a temperature gradient was used to select the optimal annealing temperature for the 12selected primers,and then the single factor experiment was carried out on the main factors such as DNA concentration,primer concentration and Mg²⁺concentration.The results showed that the optimal combination was achieved with a reaction volume of20μL,template concentration of 70 ng/μL,primer concentration of 1.0 umol/L,Mg²⁺concentration of 2 m M,and mix volume of 10μL,which produced clear and specific amplification products.This study provides a reference for the optimization and application of the ISSR-PCR system for kiwifruit.（3）Genetic diversity of kiwifruit.A total of 42 kiwifruit materials were analyzed using 12 ISSR primers for amplification,and the genetic diversity was analyzed.The results showed that 105 bands were amplified by these primers,and the proportion of polymorphism was up to 96.19%.Subsequently,cluster analysis showed that the 42materials were divided into two main categories,with Category I consisting of all hybrid F1 materials,and Category II consisting of other variety（strain）materials.The yellow meat,green meat and red meat kiwifruit of all varieties were grouped together in classⅠand ClassⅡrespectively.This study preliminarily demonstrated that ISSR markers can be used for kiwifruit flesh color classification.（4）High-throughput detection of flavor metabolites in kiwifruit before and after harvesting.Headspace solid phase microextraction（HS-SPME）combined with gas Chromatography-mass Spectrometry（GC-MS）and ultra-high performance liquid chromatography-mass spectrometry（UHPLC-MS）were used to detect the types of kiwifruit metabolites.The results showed that a total of 485 volatile compounds were detected by GC-MS analysis,divided into 14 categories,mainly terpenes,esters,heterocyclic compounds,and alcohols.834 non-volatile compounds were detected at LC-MS analysis,divided into 13 categories,mainly lipids and lipid-like compounds,phenylpropanoids and polyketones,and alkaloids.（5）The characteristics of metabolite changes before and after harvest of kiwifruit of different colors were studied.Combined with principal component analysis and K-means results,it was found that there were significant differences in the metabolic patterns of terpenes,esters,and heterocyclic compounds among kiwifruit of different colors.After kiwifruit was stored for a period of time after harvest,the number of different metabolites with differential accumulation was highest,and the metabolic pattern differences among kiwifruit of different colors during storage after harvest were significant.Through KEGG pathway enrichment analysis,significant differences in tyrosine metabolism,α-linolenic acid metabolism,methylbutyrate metabolism,citric acid cycle,and monoterpenoid biosynthesis pathways were observed among kiwifruit of different colors.Comparing the changes in the content of different categories of metabolites,the accumulation of esters and alcohols in red-fleshed kiwifruit during storage at room temperature after harvest was more significant than other varieties,and the difference in metabolic patterns between red-fleshed kiwifruit and other colors was the largest.This may result in a stronger aroma of red-fleshed kiwifruit compared to other varieties.The results of this study indicate that kiwifruit of different colors have their own harvest characteristics,as well as different maturity and flavor characteristics,which can provide data support for kiwifruit quality evaluation,suitable harvest,and storage.更多还原