Phylogenetic and taxonomic status of Citrus halimii B.C. Stone determined by genotyping complemented by chemical analysis of leaf and fruit rind essential oils

Citrus phylogeny is currently based on genome analysis using molecular markers and sequencing. The 7 pure genetic groups that gave rise to all cultivated citrus underlie the diversity of citrus accessions originating from Asia. However, there are wild citrus forms whose phylogenetic position is unknown, such as mountain citron (Citrus halimii B.C. Stone) that was discovered in Malaysia in the early 1970s. We sought to elucidate its status by determining its genetic profile with 30 SSR and InDel markers distributed on the 9 chromosomes of the citrus reference genome as compared to those of the 7 pure genetic groups represented by 4 or 5 varieties each. The genetic study was supplemented by a comparison of the composition of essential oils obtained by fruit peel and leaf hydrodistillation to those of the citrus fruits used for genotyping. The genetic study demonstrated that C. halimii is not an interspecific hybrid (low heterozygosity) but rather a true species that shares a common ancestor with kumquats (Fortunella sp.), which would have evolved separately. The fruit aromatic profiles confirmed this kumquat/mountain citron relationship but also highlighted the uniqueness of C. halimii due to the presence of high proportions of compounds that have never been observed in other citrus fruits, such as germacrene d-8-one (accounting for 8.7% of the leaf essential oil).