【作者】 王红梅；

【导师】 王志强； 李国怀；

【作者基本信息】 华中农业大学 ， 果树学， 2024， 博士

【摘要】 桃(Prunus persica L.Batsch)在我国栽培历史悠久,其果实味道鲜美,营养价值丰富,深受广大消费者的喜爱。桃果实质地是影响果实品质的重要因素。随着人们生活水平的提高,对桃品质及种类多样性需求越来越高,现有品种已无法满足市场需求,急需培育丰富多样的肉质类型新品种来满足桃产业发展需求。在桃新品种选育过程中,我们发现溶质桃和硬质桃中都存在粉质桃,且桃粉质性状具有遗传和品种特异性,厘清桃粉质性状形成机理,可以充分利用粉质桃种质资源来丰富桃品种类型,为选育出更多满足不同消费者需求的优异新品种提供理论依据。本研究以硬质和溶质两种类型的桃为材料,对其进行细胞显微结构观察、细胞壁结构及生理生化特性分析,明确粉质桃的生理变化特点;通过转录组比较分析,筛选调控粉质性状的关键候选基因;运用群体遗传分析、BSA定位,开发分子标记;通过转基因番茄验证关键基因的功能;并利用酵母单杂交和双荧光素酶等实验进一步明确粉质的形成机制,为桃果实肉质改良奠定理论基础。主要研究结果如下:1.PpPG1高表达可介导硬质桃粉质性状的发生粉质桃具有特征性细胞显微结构,与非粉质桃细胞显微结构存在明显差异。硬质桃中粉质桃‘中桃14’(CP14)和‘中桃白玉2号’(CPWJ2)的果肉组织细胞都有共同的显微结构特征,细胞形态完整且分离,单独或成团释放,细胞间隙较大,相邻细胞间粘附性丧失,排列不规则。硬质桃非粉质品种‘中桃13’(CP13)、‘中桃9号’(CP9)的细胞结构完整,排列规整。硬质桃细胞壁甲基化程度都很高,但粉质桃果胶降解程度更高。硬质桃的细胞壁中性糖Gal,Gal-UA,Ara和Xyl的含量在粉质桃和非粉质桃中差异显著,表明果胶降解的差异是引起硬质桃粉质的主要原因。转录组分析发现,PpPG1在硬质型粉质桃中显著高表达。自然群体杂交回补PpPG1基因验证基因功能,表明PpPG1为调控硬质桃粉质的关键基因,PpPG1高表达可引起硬质桃粉质。2.PG和PME活性失衡介导的细胞壁代谢紊乱是溶质桃粉质产生的主要原因过表达PpPG1可促进溶质桃粉质,但溶质桃粉质调控机理与硬质桃不同。在溶质桃中,粉质桃‘6-10-34’(RM)细胞结构分离,非粉质桃‘春美’(CM)细胞破裂,细胞结构崩塌。粉质桃‘RM’的PG与PME的活性和基因表达之间变化趋势失衡是引起粉质的主要原因。转录组分析筛选出PpPG1、PpPG2、PpEGase2、PpEXP1、PpEXP3、PpAGP2、IAA4和ABA2可作为调控溶质桃果实粉质的候选基因,且转录因子PpBL(Pure.5G006200,NAC)对PpPG1启动子的激活作用可能是引起早熟离核红肉桃为粉质的关键因素。3.粉质区间精细定位及PpPG1功能验证构建溶质桃‘CN8’ב6-10-34’和‘陇油1号’ב6-10-16’,通过BSA及精细定位,将粉质候选区间定位到Pp01:34908512-35908513的1M范围内和Pp05:868372-1868444的1M范围内。开发的Indel标记p01-7,在‘陇油1号’ב6-10-16’群体中准确率为75%;Pp05-1,在‘陇油1号’ב6-10-16’群体中准确率为73%,可作为候选分子标记,与粘离核标记(F1)结合使用,用于分子标记辅助育种。Indel标记P和Chr05:689647处开发的Kasp标记也可准确区分红肉离核粉质单株。对候选基因PpPG1进行转基因番茄功能验证,发现过表达PpPG1可促进转基因番茄的果实提前成熟软化,乙烯释放峰也略有提前,且PpPG1的启动子活性具有组织部位特异性,在种子周围显著高表达。Y1H和双荧光素酶验证实验证明,PpBL对PpPG1的激活作用,也解释了红肉群体后代中早熟离核红肉桃均为粉质的原因。综上所述,本文明确了粉质桃的细胞结构特点及生理变化规律;对粉质性状进行BSA定位,并开发了候选分子标记,可应用于粉质桃的选育;进一步鉴定了PpPG1的功能,验证PpBL转录因子对PpPG1启动子的激活作用,解释了红肉群体后代中早熟离核红肉桃粉质的原因,为深入研究桃粉质性状的分子机制及桃肉质改良提供理论基础。更多还原

【Abstract】 Peach(Prunus persica L.Batsch)has a long history of cultivation in China.Its fruits are delicious and nutritious,and they are popular with customers.The fruit texture is an important trait affecting the quality of peach fruit.The need for peach quality and variety diversity is growing as people’s levels of life rise.Existing cultivars are no longer capable of meeting market demands.There is an urgent need to cultivate new cultivars with rich and different flesh types to meet the development needs of the peach industry.During the breeding process of new peach cultivars,we found that both melting peaches and stony hard peaches have mealy flesh types,and the mealy trait of peaches has genetic and cultivar characteristics.Clarifying the trait characteristics of peach mealiness can provide a sufficient basis for improving and enriching the peach texture,utilizing peach variety resources to cultivate more cultivars that meet the needs of different consumers,enriching peach cultivars,and providing a theoretical basis.This study uses two types of peaches,stony hard peach and melting flesh peach materials and conducts cell microstructure observation,cell wall structure,and physiological and biochemical characteristics to clarify the physiological changes of mealy peach;through transcriptome analysis,selecting key candidate genes that regulate mealy traits;using population genetic analysis,BSA sequencing,and transcriptome selecting to screen mealy-related genes;verifying the functions of key genes through experiments such as transgenic tomatoes,Y1 H,and dual-luciferase,to further clarifying the regulatory mechanism of mealiness and laying a theoretical foundation for peach fruit texture improvement.The major findings are the following:1.High expression of PpPG1 can cause stony hard peach mealinessMealy peaches have a characteristic cell microstructure that is significantly different from that of non-mealy peaches.The flesh tissue cells of mealy peaches ‘Zhongtao14’(CP14)and ‘Zhongtao White Jade 2’(CPWJ2)have common microstructural characteristics: complete and separated cell morphology,release as intact cells,singly or in clusters,large intercellular gaps,loss of adhesion between adjacent cells,and irregular arrangement.The cell structure of non-mealy SH peaches ‘Zhongtao13’(CP13)and‘Zhongtao9’(CP9)is complete and regularly arranged.The cell walls of stony-hard peaches all have a high degree of methylation,but the degree of pectin degradation in mealy peaches is higher.There were significant differences in the content of neutral sugars Gal,Gal-UA,and Ara and Xyl in the cell walls of ‘CP14’ and ‘CP13’ in the mealy stage.It shows that the main factor that promote stony hard peach mealiness are differences in pectin metabolism,which impact cell wall composition.Transcriptome analysis was performed to investigate the molecular mechanism of the mealiness process.Six up-regulated cell wall-related genes(PpPG1,PpPG2,PpAGP1,PpAGP2,PpEXT1,and PpEXP1)and one down-regulated cell wall-related gene(PpXET2)were identified as being involved in the mealiness process during stony hard fruit ripening.Natural population hybridization of the PpPG1 gene verified the gene function,indicating that PpPG1 is a crucial gene in regulating stony hard peach mealiness and that high expression of PpPG1 can cause stony hard peach mealiness.2.In melting flesh peaches,the imbalance of PG and PME activities leads to cell wall metabolism disorders and mealinessOverexpression of PpPG1 enhances melting flesh peach mealiness,although the mealy regulatory mechanism differs from that of stony hard peach.In melting flesh peach,the mealy peach ‘6-10-34’(RM)cell structure separates,while the non-mealy peach‘Chunmei’(CM)cell ruptures and the cell structure breakdowns.The fluctuations in polygalacturonase(PG)and pectin methylesterase(PME)activities between mealy and non-mealy peaches were the main factor contributing to mealiness.Transcriptome analysis found that PpPG1,PpPG2,PpEGase2,PpEXP1,PpEXP3,PpAGP2,IAA4,and ABA2 can be used as candidate genes for regulating peach fruit mealiness,and the transcription factor PpBL(Pure.5G006200,NAC)may be responsible for causing early-ripening freestone blood peach mealiness.3.Fine mapping of the gene controlling the mealiness of peach and functional verification of PpPG1Construction of two hybrid populations of solubilized peach ‘CN8’ב6-10-34’ and‘Longyou1’ב6-10-16’.Through BSA and fine mapping,the mealy candidate interval was located within the 1M range of Pp01: 34908512-35908513 and the 1M range of Pp05:868372-1868444.In the ‘Longyou1’ב6-10-16’ progeny groups,the developed Indel marker Pp01-7 has a coincidence rate of 75%,and Pp05-1 has a coincidence rate of 73%.It can be a molecular candidate marker of mealiness and combined with F/f markers(F1)for molecular marker-assisted breeding.Indel marker P and the Kasp marker developed at Chr05:689647 can also accurately distinguish the mealy peaches of freestone blood flesh peaches.The candidate gene PpPG1 was functionally tested in transgenic tomatoes.Overexpression of PpPG1 has been shown to promote early fruit ripening and softening in transgenic tomatoes,and the ethylene release peak is also slightly advanced.Moreover,the promoter activity of PpPG1 is tissue-site-specific and is significantly highly expressed around the seeds.Y1 H and dual-luciferase verification experiments demonstrated that PpBL activates PpPG1,which also explains why early-maturing blood flesh peaches of freestone have a mealy texture in the blood flesh progeny groups.In conclusion,this article clarified the cell structure characteristics and physiological changes of mealy peaches;BSA localized the mealy traits and developed candidate molecular markers that can be applied to the breeding of mealy peaches;further identified PpPG1 function,verifying the activation effect of the PpBL transcription factor on PpPG1;explained the cause of mealiness in early-maturing freestone blood flesh peaches of the blood flesh progeny groups;and provided a theoretical basis for an in-depth study of the molecular mechanism of peach mealy traits and the improvement of peach flesh quality.更多还原