Ma1纯合型‘秦冠’苹果低酸性状的遗传分析及基因标签开发

Acids have crucial impacts on the overall organoleptic quality of apple fruits. The Ma1 controlling fruit acidity on chromosome 16 accounted for 12 – 42% of the observed phenotypic variation. Apple accession bearing homozygous Ma1 (Ma1Ma1), shows a high acidity in fruits. However, the apple cultivar ‘Qinguan’, which contained homozygous Ma1, shows low acidity in fruits and a major QTL for fruit acidity was identified on chromosome 8 in population of ‘Qinguan’ × ‘Honeycrisp’, the genetic mechanism regarding to variation of fruit acidity is unclear. In order to find out which genes controlled fruit acidity in QTL interval on chromosome 8, we will further develop molecular markers and perform fine mapping of fruit acidity associated QTL. Combine with bulked segregant analysis (BSA) and transcriptome analysis, the fine mapping was used to identify candidate gene(s) for fruit acidity on chromosome 8. The core allelic variation(s) of candidate gene(s) will be identify via compare the sequence difference between high and low acidity apple individuals. It will be clarified that the genetic contribution of the core allelic variation(s) of candidate gene(s) and their contribution to fruit acidity in apple. The target gene for fruit acidity will be identified, and the gene tag will be developed base on the core allelic variation(s) of target gene. The biological function of the target gene will be also verify..These research results possess a theoretical significance in expanding our knowledge of genetic mechanism regarding fruit acidity in apple, and will also provide gene tag useful for genetic improvement for apple fruit quality.

酸度是影响苹果内在品质的因素之一。研究表明，16号染色体上的Ma1基因能够解释苹果果实酸度表型变异的12%至42%，其中Ma1纯合型一般表现为高酸。但项目组前期研究发现，Ma1纯合型‘秦冠’表现为低酸性状，且在‘秦冠’ב蜜脆’杂交群体的8号染色体上检测到控制果实酸度的主效QTL位点，该位点导致果实酸度变异的遗传机制尚不清楚。为进一步挖掘该QTL区间控制苹果果实酸度的基因，本项目拟以‘秦冠’ב蜜脆’杂交群体为试材，进一步开发分子标记，进行QTL位点精细定位；结合群组分离分析法（BSA）以及转录组测序结果，筛选QTL区间控制果实酸度的候选基因；分析候选基因的核心变异序列对果实酸度的遗传效应，筛选控制果实酸度的目的基因；验证目的基因的功能，开发基因标签。研究结果不仅能深入了解影响苹果果实酸度的遗传机制，而且可获得用于苹果果实品质遗传改良的基因标签，具有重要的理论意义和应用价值。

本项目主要以‘秦冠’和‘蜜脆’杂交群体为研究材料，筛选调控‘秦冠’苹果中果实低酸性状的关键基因。结果表明：（1）构建正向遗传分析体系，为酸度控制基因的遗传分析提供条件，同时发现苹果栽培种中柠檬酸的含量很低（或者说液泡中不储存柠檬酸）这种现象可能是由于进化过程中栽培苹果祖先种新疆野苹果和森林苹果中柠檬酸含量极低的缘故，属于一种遗传效应，而非人工选择的原因；（2）筛选出控制苹果果实酸度相关质子泵PEPP基因，番茄和苹果愈伤组织中过表达该基因能够显著增加果实中有机酸的含量；（3）对‘秦冠’和‘蜜脆’成熟时期果实甲基化及转录组进行分析，筛选出位于液泡膜上控制果实酸度基因MdMa11，该基因能够介导H+的跨膜转运，启动子区甲基化差异从而导致在‘秦冠’和‘蜜脆’成熟时期果实中表达不同，瞬时转化该基因能够显著降低液泡中的pH; （4）利用蛋白质组学研究方法，在蛋白质水平上发现酸合成相关基因以及H+转运体相关的基因的高表达是导致苹果果实中有机酸含量较高的主要原因；（5）利用计算生物学的方法，鉴定出苹果中苹果酸脱氢酶（MDH）一个家族成员在进化过程中发生了正选择，且该基因的在苹果果实发育过程中表达模式跟苹果果实中苹果酸的积累呈正相关。.本项目采用多种研究方法及手段初步筛选出控制苹果果实酸度性状的关键基因，并初步验证了关键基因的生物学功能，这为进一步深入阐明苹果果实酸度形成分子机制及酸度的遗传改良提供了科学依据