Table_5_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营养与健康促进物质，在石榴（Punica granatum L.）果实的果皮与籽实中含量颇丰。然而不同石榴品种间的花青素含量差异显著。以色列发现的‘黑’石榴品种（P.G.127-28），其果皮花青素含量极高，相较其他石榴品种果皮的花青素水平可高出两个数量级。生化分析表明，该‘黑’品种果皮中飞燕草素（delphinidin）含量极高。‘黑’品种石榴果实发育过程中，果皮的花青素积累模式与其他品种截然不同：其在整个发育阶段均维持较高的花青素水平，且该过程并不依赖光照。对分离出‘黑’表型的F₂群体开展遗传分析，结果表明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）标记对该F₂群体进行遗传定位，筛选到数个与‘黑’表型紧密连锁的分子标记。通过对F₂及F₃群体的重组分析，将‘黑’性状的定位区间缩小至石榴品种‘大笨子’草图基因组序列上的178.5 kb区域内。该区域内存在一个推定的花青素还原酶（anthocyanidin reductase, ANR）基因。仅携带‘黑’性状的石榴品种，在该基因末端存在一处碱基缺失，该突变引发移码效应，最终生成截短的蛋白质。本研究推测，ANR基因的该突变正是导致‘黑’石榴品种花青素组成改变与含量显著升高的原因。