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

Abstract

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.

Introduction

The sub-genus Citrus sensu Swingle (1967) belongs to the Rutaceae family and the Aurantioideae subfamily. A particular fruit called hesperidium, which is a specialised berry that has a leathery exocarp and mesocarp and a segmented and juicy endocarp, characterises this taxon. From a biogeographical point of view, the centre of origin and source of the subsequent spread of Citrus was an area of the Asian continent characterised by tropical or subtropical climates, such as India, southern China and Japan, Indonesia, and the Philippines (Calabrese, 1992).

The history of the cultivation of Citrus trees is long and extremely complex, with cultivation established by 4,000 BC in Persia and the Middle East and consisting of many species and cultivars (Calabrese, 1992, Webber, 1967). Cultivation of Citrus plants has spread over the world with diverse utilisations, such as for fruit growth, pharmacologic use, cosmetics, and ornamental plants (Lota et al., 1999, Lee et al., 2006).

Over time, hybridisation between species and related genera, bud mutations and apomixis (e.g. adventitious embryony) have played an important role in the evolution of the numerous Citrus species and cultivars (Federici et al., 1998). The origins of many species of Citrus are poorly known or often based only on historical information. For this reason, the scientific interest in Citrus has increased in recent years, especially with regard to its taxonomy and phylogeny, with the aim of characterising and conserving its germplasm (Nicolosi et al., 2000, Araujo et al., 2003, Mabberley, 2004, Moreira Novelli et al., 2004, Deng et al., 2007, Pang et al., 2007, Barkley et al., 2009, Bayer et al., 2009, Jena et al., 2009). The classification systems proposed by Swingle and Reece (1967) and Tanaka (1977) and further phylogenetic analyses, starting from those of Barrett and Rhodes (1976) by means of a numerical taxonomy approach, revealed that there were only three true species within the Citrus genus: C. medica L. (citron), C. reticulata Blanco (mandarin) and C. grandis (L.) Osb. (pummelo).

Within the genus, C. limonimedica Lush. has a very uncertain origin; it was described as a species by Lushington (1910), and the description was made exclusively considering the cultivated specimens. The few C. limonimedica cultivars known in Italy have been grown from the XVII century, during Medici's government in Florence, and have always been used as ornamental plants (Nati, 1674, Volkamer, 1708, Targioni-Tozzetti, 1853). Their great morphological variability, especially concerning fruits, has led to the hypothesis that C. limonimedica was a result of hybridisation between C. limon (L.) Burm. f. (lemon) and C. medica L. (citron) (Tanaka, 1954, Carpenter and Reece, 1969, Barcaccia et al., 2008). Some authors and/or Citrus official collections consider C. limonimedica to be identical to the famous Hebrew citron (Lota et al., 1999; http://www.plantnames.unimelb.edu.au/new/Citrus_2.html). Other classifications keep C. limonimedica and C. medica L. var. ethrog Engl. as distinct entities (Galeotti and Tintori, 2000; http://www.oscartintori.it/indice.pdf). Conversely, Calabrese (2002) hypothesised that C. limonimedica is likely a hybrid between citron and orange.

To investigate the genetic diversity and phylogenetic relationships in Citrus, several molecular marker systems were employed, such as ISSRs (inter simple sequence repeats), RAPD (random amplified polymorphic DNAs), SCARs (sequence-characterised amplified regions), and SNPs (single nucleotide polymorphisms), from either nuclear or chloroplast genomes (Fang and Roose, 1997, Federici et al., 1998, Nicolosi et al., 2000, Deng et al., 2007).

In the present study, the genetic diversity and the genetic differentiation and relationships among the considered Citrus species were inferred using an integrated approach based on the analysis of 17 morphological traits and molecular markers by means of genomic RAPD fingerprints and nuclear ITS (internal transcribed spacer)-derived SNPs. RAPD analysis has already been successfully used for population genetics in different plant species (Kump and Javornik, 1996, Barcaccia et al., 1997, Chan and Sun, 1997) and the nuclear marker ITS1 has been widely used for estimating genetic diversity (Baldwin et al., 1995) and parentage analysis within the Citrus genus (Xu et al., 2006).

The main objectives of this study were (i) to explore possible differences in the DNA content of Citrus and Poncirus genera and within Citrus accessions by flow cytometry analysis; (ii) to verify the relationships between Citrus and Poncirus genera and within the genus Citrus on the basis of phenotypic traits and molecular markers; (iii) to investigate the phylogenetic relationships among C. limonimedica and its supposed ancestors, C. limon and C. medica, by means of DNA polymorphisms.