I型红肉苹果“R6型白肉”变异性状的相关基因挖掘

The red-fleshed trait is a new good commercial character in apple, and it is controlled by MYB10 gene in type I red-fleshed apples. The R6 repeat in the promoter region of MYB10 is responsible for the increased anthocyanin level in flesh. Genotypes with R6 allele exhibit “red flesh and red leaf”, and without R6 allele exhibit “non-red flesh and green leaf”. However, we previously observed some “red leaf but white flesh” apples existing in natural hybrids of a type I red-fleshed apple ‘Hongxun NO.1’. These “red leaf but white flesh” progenies also contain R6 alleles, which are the same to their female parent. The current molecular mechanism regarding red-fleshed trait can’t explain this genetic variation. So the genetic factors controlling the “R6 allele with white flesh” variation are worth for exploring. In this study, with the general “red flesh and red leaf” apples as control, we will analysis the trends of anthocyanin accumulation and related enzymatic activities during fruit development of “R6 allele with white flesh” apples. In addition, transcriptome analysis will be performed between the general “red flesh and red leaf” apples and the variable “R6 allele with white flesh” apples to analyze the differentially expressed genes. Based on the anthocyanin related results, gene annotation, and the real time PCR confirmation, the candidate genes will be selected from the differentially expressed genes. Finally, we will design gene tags of these candidate genes, and perform the genetic test using a backcross population which was generated by a “R6 allele with white flesh” variable individual and its female parent ‘Hongxun NO.1’. This study will advance the current molecular mechanism regarding red-fleshed trait, as well as provide effective gene tags for the molecular assisted breeding of red-fleshed apple.

红色果肉是苹果新的优良商品性状。已知I型红肉苹果红肉性状的关键基因为MYB10，其启动子含R6序列时表现“红叶红肉”，不含R6序列时则为“绿叶白肉”。然而，本项目组新近研究发现：I型红肉苹果‘红勋一号’的子代中出现了“红叶白肉”变异个体，其基因型也为R6型。导致该“R6型白肉”遗传变异的原因尚不清楚，值得深入探索。本项目拟测定“R6型白肉”和“R6型红肉”个体各发育时期果肉的花色苷等酚类物质含量及相关酶的活性变化，以明确其花色苷积累规律的差异；其次，对两类果肉进行比较转录组分析，并结合基因功能注释、实时定量PCR验证和花色苷积累规律进行差异表达基因的筛选；最后，利用已构建的“R6型白肉”与其母本‘红勋一号’的回交群体，对候选基因进行正向遗传学验证，以确定与“R6型白肉”变异性状共分离的基因。本项目的开展，不但可推进苹果红肉性状的调控理论，还可将鉴定的基因在红肉苹果子代的筛除工作中予以应用。

红色果肉是苹果新的优良商品性状。已知 I 型红肉苹果红肉性状的关键基因为MYB10，其启动子含R6序列时表现“红叶红肉”，不含R6序列时则为“绿叶白肉”。对苹果属植物的I型红肉性状进行遗传多样性调查和基因型检测发现：大多数个体果肉红色性状由MYB10基因座控制，R6对其等位基因R1为完全显性（质量性状）；但同为R6型个体，其果肉花色苷含量不同，且存在少数个体表现为R6型白肉，说明还存在MYB10以外的其他基因座控制红肉性状的深浅（数量性状）。为了弄清R6型白肉个体的调控机制，选用“R6 型红肉”品种‘红勋一号’及其子代表现为“R6 型白肉”变异个体为研究对象，发现“R6 型白肉”子代变异个体红色性状表现出时空特异性。变异株系的花及幼叶均能正常积累花色苷，而其果肉在发育早期为红肉，中后期（花后50天开始）才表现为“白肉”。物质含量和基因表达检测发现，花色苷不能正常积累是由于“R6 型白肉”个体花色苷合成收到抑制引起的。通过基因表达检测筛选到目的基因MYB111，Luciferase试验证明MYB111蛋白可以结合MYB10转录因子，抑制后者激活花色苷合成基因DFR的表达，从而抑制果肉花色苷的积累。此外，对R6R1杂交群体（母本为‘长富二号’，白肉，基因型R1R1；父本为‘Red3’，红肉，基因型R6R6）进行SLAF测序，构建遗传连锁图谱，并对果肉花色苷含量与遗传图进行QTL定位。定位到位于16号连锁群顶部QTL区间marker2187260—marker 2173766。其区间Marker2175442最接近QTL位点峰，并且这个位点的基因型在分离群体中分为两组，含有ll基因型的纯合个体花色苷含量显著高于基因型为lm的杂合个体（P<0.05）。本项目的实施将为苹果色泽品质育种提供理论依据，丰富了苹果红肉性状的遗传与变异机制。