Table_4_Fine Mapping of the “black” Peel Color in Pomegranate (Punica granatum L.) Strongly Suggests That a Mutation in the Anthocyanidin Reductase (ANR) Gene Is Responsible for the Trait.XLSX

Anthocyanins are important dietary and health-promoting substances present in high quantities in the peel and arils of the pomegranate (Punica granatum L.) fruit. Yet, there is a high variation in the content of anthocyanin among different pomegranate varieties. The ‘Black’ pomegranate variety (P.G.127-28) found in Israel contains exceptionally high levels of anthocyanins in its fruit peel which can reach up to two orders of magnitude higher content as compared to that of other pomegranate varieties’ peel anthocyanins. Biochemical analysis reveals that delphinidin is highly abundant in the peel of ‘Black’ variety. The pattern of anthocyanin accumulation in the fruit peel during fruit development of ‘Black’ variety differs from that of other pomegranates. High anthocyanin levels are maintained during all developmental stages. Moreover, the accumulation of anthocyanin in the fruit peel of ‘Black’ variety is not dependent on light. Genetic analysis of an F2 population segregating for the “black” phenotype reveals that it is determined by a single recessive gene. Genetic mapping of the F2 population using single nucleotide polymorphism (SNP) markers identified few markers tightly linked to the “black” phenotype. Recombination analysis of the F2 population and F3 populations narrowed the “black” trait to an area of 178.5 kb on the draft genome sequence of pomegranate cv. ‘Dabenzi.’ A putative anthocyanidin reductase (ANR) gene is located in this area. Only pomegranate varieties displaying the “black” trait carry a base pair deletion toward the end of the gene, causing a frame shift resulting in a shorter protein. We propose that this mutation in the ANR gene is responsible for the different anthocyanin composition and high anthocyanin levels of the “black” trait in pomegranate.

花青素是一类重要的膳食保健活性物质，在石榴（Punica granatum L.）果实的果皮与假种皮中含量较高。然而不同石榴品种间的花青素含量存在显著差异。在以色列发现的‘Black’石榴品种（编号P.G.127-28），其果皮中的花青素含量极高，较其他石榴品种果皮的花青素含量可高达两个数量级。生化分析表明，飞燕草素（delphinidin）在该‘Black’品种的果皮中含量尤为丰富。‘Black’品种在果实发育过程中，果皮花青素的积累模式与其他石榴品种截然不同：其在整个果实发育阶段均维持较高的花青素水平。此外，‘Black’品种果皮中花青素的积累并不依赖光照。对携带“黑皮”表型的F₂分离群体进行遗传分析发现，该性状由单个隐性基因调控。利用单核苷酸多态性（single nucleotide polymorphism, SNP）标记对F₂群体进行遗传定位，仅筛选到少数与“黑皮”表型紧密连锁的标记。通过对F₂及F₃群体的重组分析，将“黑皮”性状位点定位到石榴品种‘Dabenzi’的草图基因组（draft genome）的178.5 kb区域内。该区域内存在一个假定的花青素还原酶（anthocyanidin reductase, ANR）基因。仅携带“黑皮”性状的石榴品种，在该基因末端存在一处碱基缺失，这一突变导致移码突变，最终产生截短的蛋白质。我们推测，ANR基因的这一突变是导致‘Black’石榴品种花青素组成改变与花青素含量升高的根本原因。