DNA content, morphometric and molecular marker analyses of Citrus limonimedica, C. limon and C. medica for the determination of their variability and genetic relationships within the genus Citrus

This work investigated the fingerprinting and phenotyping of Citrus germplasm; species selected were of historical importance belonging to Citrus limonimedica Lush. and its supposed ancestors, along with some other species of the Citrus genus. An integrated approach based on the exploitation of nuclear DNA content, morphological traits and molecular markers, such as RAPD fingerprints and ITS-based SNPs, was employed. We studied a core collection of 54 distinct accessions, including 43 genotypes of the Citrus species (18 species or supposed species) and 11 genotypes of the Poncirus genus, which was used as the reference outgroup. Morphological trait analysis and statistical analysis of DNA content and markers were useful for reconstructing a Citrus phylogeny. In particular, our experiments aimed at estimating the genetic variation within and the genetic relatedness among C limon (L.) Burm., C. limonimedica and C. medica L. to shed light on the hybrid origin hypothesis of C. limonimedica. The results of the multidisciplinary analyses allowed us to confirm a remarkable differentiation between Poncirus and Citrus genera and to highlight a close relationship among the three investigated Citrus species but a distinct difference between these three species and other species in the Citrus genus. RAPD fingerprints and ITS polymorphisms enabled us to point out a variation gradient between lemon and citron, with C. limonimedica as a possible intermediate species. Some accessions of C. medica and C. limonimedica that deviate from such a trend suggest recurrent introgression and/or hybridisation with other species of Citrus.

► In this study we investigate the fingerprinting and phenotyping of Citrus germplasm belonging to Citrus limonimedica Lush. and its supposed ancestors, along with some other species of the Citrus genus.
► We used an integrated approach based on the exploitation of nuclear DNA content, morphological traits and molecular markers, such as RAPD fingerprints and ITS-based SNPs.
► Morphological trait analysis and statistical analysis of DNA content and markers were useful for reconstructing a Citrus phylogeny.
► RAPD fingerprints and ITS polymorphisms enabled us to point out a variation gradient between lemon and citron, with C. limonimedica as a possible intermediate species.
► In this study, numeric taxonomy and molecular marker methods have been exploited as an integrated tool to shed light on the hybrid origin of the historical C. limonimedica species from C. limon and C. medica.