Table_1_Phylogenetic Origin of Primary and Secondary Metabolic Pathway Genes Revealed by C. maxima and C. reticulata Diagnostic SNPs.docx

Modern cultivated Citrus species and varieties result from interspecific hybridization between four ancestral taxa. Among them, Citrus maxima and Citrus reticulata, closely associated with the pummelo and mandarin horticultural groups, respectively, were particularly important as the progenitors of sour and sweet oranges (Citrus aurantium and Citrus sinensis), grapefruits (Citrus paradisi), and hybrid types resulting from modern breeding programs (tangors, tangelos, and orangelos). The differentiation between the four ancestral taxa and the phylogenomic structure of modern varieties widely drive the phenotypic diversity’s organization. In particular, strong phenotypic differences exist in the coloration and sweetness and represent important criteria for breeders. In this context, focusing on the genes of the sugar, carotenoid, and chlorophyll biosynthesis pathways, the aim of this work was to develop a set of diagnostic single-nucleotide polymorphism (SNP) markers to distinguish the ancestral haplotypes of C. maxima and C. reticulata and to provide information at the intraspecific diversity level (within C. reticulata or C. maxima). In silico analysis allowed the identification of 3,347 SNPs from selected genes. Among them, 1,024 were detected as potential differentiation markers between C. reticulata and C. maxima. A total of 115 SNPs were successfully developed using a competitive PCR technology. Their transferability among all Citrus species and the true citrus genera was very good, with only 0.87% of missing data. The ancestral alleles of the SNPs were identified, and we validated the usefulness of the developed markers for tracing the ancestral haplotype in large germplasm collections and sexually recombined progeny issued from the C. reticulata/C. maxima admixture gene pool. These markers will pave the way for targeted association studies based on ancestral haplotypes.

现代栽培的柑橘物种和品种，源自四种原始类群间的种间杂交。其中，与柚子和橘柑园艺组紧密相关的柚橘（Citrus maxima）和橘柑（Citrus reticulata），作为酸橙（Citrus aurantium）和甜橙（Citrus sinensis）、葡萄柚（Citrus paradisi）以及现代育种计划中产生的杂交类型（如柚橘、橘柚和橙橘）的先祖，尤为重要。四种原始类群之间的分化以及现代品种的进化树结构，广泛驱动了表型多样性的组织结构。特别是，在色泽和甜度方面存在显著的表型差异，这成为育种者的重要评判标准。在此背景下，聚焦于糖、类胡萝卜素和叶绿素生物合成途径的基因，本研究的目的是开发一套用于区分柚橘和橘柑祖先单倍型的诊断性单核苷酸多态性（SNP）标记，并提供在种内多样性水平（柚橘或橘柑内部）的信息。计算机模拟分析从选定的基因中识别出3,347个SNP。其中，1,024个被检测为柚橘和橘柑之间潜在的分型标记。总共成功开发出115个SNP，采用竞争性PCR技术。这些标记在所有柑橘物种和真实柑橘属中的可转移性极好，仅有0.87%的数据缺失。确定了SNP的祖先等位基因，并验证了所开发标记在追踪大型种质库中的祖先单倍型和由柚橘/橘柑杂交基因池产生的性重组后代的有用性。这些标记将为基于祖先单倍型的针对性关联研究铺平道路。