桃红肉位点DBF和bf的精细定位和关键基因解析

Nutrition and health benefits contained in daily fruit were highlighted by consumers. Some blood-flesh peach accessions were identified to have very high antioxidant capacity which is comparable to blueberry. Due to its health benefits and attractive colour, the blood-flesh traits are emphasized in peach research nowadays. Two types of blood-flesh traits, dominant blood-flesh (DBF) and recessive blood-flesh (bf), were found in peach genetic resources. In previous study, we mapped DBF and bf loci on linkage 5 and linkage 4, respectively. Bioinformatics analysis in the DBF and bf region predicted a novel mechanism underlining blood-flesh traits comparing to other Rosaceae family. This program was designed (1) to fine map DBF and bf loci by using large and multiple populations, respectively; (2) to search polymorphic loci between blood genotypes and non-blood ones in the mapping regions by whole genome re-sequencing of the parents and typical blood-flesh accessions; (3) to discover the key genes or regulators controlling blood-flesh by transcriptome analysis and RT-qPCR analysis; (4) to design co-dominant markers for marker assisted selection (MAS), and apply them in identifying germplasm collections and in breeding practice. By running the project, the molecular mechanism underlying the anthocyanin accumulation in each blood-flesh genotypes will be discovered, effective markers for MAS will be developed, and theoretical informations supporting utilization and innovation of blood-flesh peach germplasm will be obtained.

水果的营养与保健价值越来越受关注，一些红肉桃果实抗氧化能力可与蓝莓相媲美，红肉性状既能迎合消费需求又能丰富桃果类型，成为研究热点。现有研究已证实桃红肉性状存在显性遗传（DBF）和隐性遗传（bf）两种类型。项目前期研究已分别将DBF和bf初定位于第5和第4连锁群，生物信息学预测桃红肉的分子机制可能完全不同于其他蔷薇科果树。本项目拟开展的主要研究：⑴利用大群体和多群体，分别完成DBF和bf的精细定位；⑵通过亲本及典型资源的全基因组重测序，搜索精细定位区段内的差异位点，并在种质资源群体中发掘与红肉性状相关的位点；(3)通过转录组分析和RT-qPCR验证，锁定DBF和bf关键基因或调控元件；(4)设计共显性的MAS标记，用于种质资源的基因型鉴定和红肉桃分子育种。本项目的实施将能：揭示红肉桃花色苷代谢的分子机制；将MAS技术首次应用于红肉桃育种；为红肉桃种质资源挖掘与创新利用提供理论支撑。

本项开展了基于多组合的红肉性状遗传规律分析，红肉桃果实发育过程中抗氧化组分的积累，DBF和bf位点的精细定位研究，以及区间内的MAS标记筛选、候选基因分析和初步功能验证。主要结果如下：.（1）通过8个DBF_类型红肉桃组合，进一步验证了其受单基因控制的显性遗传的特点，并分析了红肉性状与其他性状的遗传倾向。.（2）通过两种基因型红肉桃果实发育过程中果肉原花青素单体儿茶素和表儿茶素的积累以及相关基因的表达特征研究，发现：两种基因型红肉桃儿茶素和表儿茶素存在明显差异，且与相关酶的活性调控相关。.（3）通过bHLH和MYB基因家族全基因组生物信息学分析，明确了桃全基因组水平：含有95个成员bHLH基因家族，被分成19个亚家族；含有256个MYB基因家族成员，分成6个家族。.（4）将DBF位点精细定位在Prupe_5 (638367-694555)的区间，区间大小为56188 bp，是初定位区间的11.1%；将bf位点定位于Prupe_4 (4327498_4405353) 的区间，区间大小为77856 bp，为初定位区间的25.01%。.（5）在DBF区间开发出能够区分DBFDBF、DBFdbf和dbfdbf三种基因型的共线性标记12个；在bf区间开发出能够区分bfbf基因型和非bfbf基因型的共显性标记3个。 .（6）精细定位区间内含控制DBF类型的候选基因6个，bfbf类型的候选基因8个，转录水平发现：Prupe.5G006100、Prupe.5G006000、Prupe.5G005600可能是控制DBF类型红肉性状的关键基因；Prupe.4G088200、Prupe.4G088300可能是控制bfbf类型红肉性状的关键基因。.（7）构建了11个候选基因的植物表达载体，遗传转化到烟草中，并明确了其亚细胞定位，有待进一步开展功能验证研究；开展了11个候选基因的VIGS基因沉默研究，但基因瞬时沉默的结果不稳定。