Genetic and chemical diversity of citron (Citrus medica L.) based on nuclear and cytoplasmic markers and leaf essential oil composition
Graphical abstract
The genetic and chemical diversity of the Mediterranean citron was examined with regard to the multiplication and dissemination practices that were related to its uses.
Highlights
► Twenty four citron varieties were studied. ► Study of the polymorphisms of 27 nuclear and cytoplasmic genetic markers. ► Composition of leaf essential oils was determined. ► Relationships were established between varieties, genetics and chemistry.
Introduction
The citrus is believed to have originated in southeast Asia (Swingle and Reece, 1967, Tanaka, 1961). However, the parentage and taxonomy of this genus are confusing, due to the wide sexual compatibility between Citrus and related genera, the high frequency of bud mutations, its long history of cultivation, and the occurrence of polyembryony in most species (Handa et al., 1986, Nicolosi et al., 2000, Ollitrault et al., 2003). Citron (Citrus medica), mandarin (Citrus reticulata), and pummelo (Citrus maxima) are considered by modern citrus taxonomists to be true Citrus species or the most similar to ancestors of modern cultivated species (Barrett and Rhodes, 1976, Gmitter and Hu, 1990, Scora, 1975). Other citrus types (orange, grapefruit, lemon, and lime) are believed to have originated from one or more generations of hybridization between these ancestral species (Barkley et al., 2006, Fang and Roose, 1997, Jena et al., 2009, Li and Xie, 2010, Nicolosi et al., 2000, Uzun et al., 2009). Most cultivars of orange, grapefruit, and lemon are thought to have originated from nucellar seedlings or bud sports. Consequently, the genetic diversity in these groups is relatively low (Luro et al., 2001). Conversely, mandarin, pummelo, and citron harbor higher levels of genetic diversity, because many of their cultivars arose through sexual hybridization. Nevertheless, as evidenced by isozyme genotyping, the citron group is characterized by low intraspecific polymorphism rates and is relatively homozygous (Ollitrault et al., 2003, Torres et al., 1978). The contribution of the citron to the development of several important cultivated genotypes is recognized by all taxonomists. Combined with sour orange (Citrus aurantium), citron lies putatively at the origin of lemon (Citrus limon) and bergamot (Citrus bergamia), and with Citrus micrantha, it spawned lime (Citrus aurantifolia) (Barkley et al., 2006, Li and Xie, 2010, Moore, 2001, Ollitrault et al., 2003). Rough lemon (Citrus jambhiri) could be a hybrid between citron and mandarin (Li and Xie, 2010). Jena et al. (2009) demonstrated by chloroplast DNA analysis the strong genetic relation between Citrus indica (Indian Wild Orange) and C. medica supporting a putative commune ancestor. Uzun et al. (2009) reported that Citrus webberii, assumed to be hybrid of Citrus macroptera and mandarin (Swingle and Reece, 1967), was clustered with the citron group. All these systematics studies highlight the major role of citron in the phylogeny of several varieties of the Citrus genus, always as pollinator parent. Moreover, citron group has a very significant genetic distance with respect to the two other ancestral species. Although the phylogeny of the genus Citrus is relatively well described at the level of interspecific diversity, the relationships and mechanism within species diversification are still little known.
Considered indigenous to northeastern India, Myanmar, and Yunnan province of China, the citron was the first citrus tree to be introduced in the Mediterranean basin. The establishment of the citron in Persia appears to have occurred no later than the first half of the first millennium BC (Webber et al., 1967). The citron tree was then disseminated throughout the Near East by Greek and later Jewish settlers and became acclimatized around the eastern Mediterranean region by the beginning of the Christian era. The citron could be the only citrus that was known by Europeans until the Middle Ages, when Arabs extended the culture of sour orange and lemon trees in northern Africa, Spain, and Sardinia (Webber et al., 1967).
However, few authors supposed that lemon and oranges were already known by Romans and Greeks at the beginning of Christian era (Tolkowsky, 1938, Nicolosi, 2007) on the basis of ancient mosaic paintings discovered in Roman villa. The citron has often been described by several Greek botanists from antiquity as Theophrastus (371–287 BC), Dioscoride (40–90), Galien (131–201) or Athenee (third century) and by some Latin poets such as Ovide (43 BC–17 AD) and Virgile (70–19 BC) (Loret, 1997). Apart the citron, these authors do not mention other citrus varieties in their writings. So, there is uncertainty about the date of introduction of other citrus varieties in the Mediterranean area but it is likely that the expansion of lemon and sour orange cultivation in the western Mediterranean area has been conducted by the Arabs around the tenth century.
The citron’s highly perfumed skin is attributed to its external vesicles, which contain essential oils, rather than its pulp, which is hardly consumed. For centuries, the use of citron was essentially ornamental, with little practical exploitation for human benefit. Initially, in the Mediterranean area, the citron was traditionally used as drugs with antiseptic and digestive properties and as a repellent of moths and other insects (Loret, 1997). Its skin is thick, and its white inner tissue, called albedo, was consumed but usually after being processed into jam or candied fruit.
In the nineteenth century, Corsica was the largest producer and exporter of citron for the candied fruit industry, using the Corsican variety, which had less acidic pulp and sweet albedo. If the technical process of extracting essential oils was known around the eleventh century, its industrial exploitation by the perfume industry began in Sicily between 1500 and 1600 (Ruberto, 2002). The citron is an important religious symbol in Judaism, used during the Feast of Tabernacles (Loret, 1997, Nicolosi et al., 2005).
The past several decades have witnessed the development of biochemical and molecular markers that have tremendous potential in managing germplasm collections, including characterizing germplasms and establishing systematic relationships (Bretting and Widrlechner, 1995). These markers have a distinct advantage over morphologically based phenotypic characterizations, because they are generally unaffected by environmental factors that influence plants and organs.
Several groups have examined citrus diversity. Barkley et al. (2006) reported an extensive survey of citrus germplasm holdings using simple sequence repeat (SSR) markers, suggesting that certain accessions that are assigned to the citron group are actually hybrids or hybrid derivatives. Of the citrus groups that are believed to be true Citrus species, citron has the lowest observed heterozygosity. Very few of the citron varieties that were studied, however, exist in Mediterranean countries. Using random amplified polymorphic DNA (RAPD) and cleaved amplified polymorphic sequence (CAPS) markers, another study (Nicolosi et al., 2005) determined that the diversity of 12 citron varieties that are dedicated to the Jewish Feast of Tabernacles was relatively high and was devoid of polymorphisms in the cytoplasmic genome. An integrated approach combining DNA content, morphometric and molecular markers analysis was conducted to study the inter-varietal diversity and to verify the relationship between cultivars of Citrus limonimedica and its supposed ancestors, C. medica and C. limon (Pessina et al., 2011). Eight citron accessions were investigated in this study. A high polymorphism was observed between citron accessions with RAPD (Random Amplified Polymorphic DNA) markers and nuclear ITS (Internal Transcribed Sequence) nucleotide variation. During evolution, introgressions and/or hybridizations with other citrus species were suggested by the authors to explain the diversity of these three species.
Regardless of its ornamental, medicinal, alimentary, cosmetic, and religious use, the notoriety of the citron is based primarily on the aromatic properties of its essential oils. We hypothesize that in the Mediterranean area, humans have selected for the aromatic properties of citron peel (zest or albedo) over the last 2 millennia. Thus, we recently compared essential oil compositions in a chemiotaxonomic analysis of 17 citron varieties of the fruit peel and petitgrain oil (Venturini et al., 2010), demonstrating a divergence in structure between fruit peel oil composition and petitgrain oil. Three and four chemiotypes were observed, with disparate repartitions, particularly for the Diamante, Corsican, Florence, Poncire, and Etrog varieties.
Leaf oil is used for characterizing variety and specie and evaluating the environmental impact on chemical variability (Lota et al., 1999, Lota et al., 2002, Tomi et al., 2008), for which citrus leaf is more advantageous than fruit because it is available all year round, and its oil composition is more diverse and is not dominated by limonene or limonene/γ-terpinene, which constitutes more than 70% of fruit peel oil.
The objective of this study was to examine citron diversity at the molecular and chemical levels and determine the phylogenic relationships among citron varieties that have been cultivated in the Mediterranean area for 2 millennia and other indigenous citrons.
Section snippets
Genetic diversity
All the molecular genotyping results expressed in terms of amplified DNA length (nucleotides) are described in supplementary material (Table S1).
Cytoplasmic diversity
Three polymorphic mitochondrial markers were used to detect polymorphisms between citron mitochondrial genomes and other Citrus species, identifying eight alleles (seven among citrons) and differentiating five mitotypes. Thirteen citron cultivars that shared profiles were identified from other basic species, such as lime, pummelo, and mandarin (Fig. 1
Conclusions
Biochemical markers (essential oil components) and molecular markers (SSR polymorphism) can be used to study diversity and determine the impact of human selection and reproductive mechanisms in the history of the citron in the Mediterranean area. Diversity, as estimated by leaf oil composition, does not necessarily agree with the molecular diversity and is unsuitable for intraspecific phylogenic studies. Nevertheless, chemical composition is informative in characterizing specific phenotypes.
Plant material
Twenty-four citron accessions were used (Table 3). Twenty three of which are maintained in the INRA CIRAD citrus depository at San Giuliano (Corsica, France); although the Diamante variety was represented by an accession from Greece, we added a second sample of this variety using Diamante leaves that were cultivated in Italy (Consorzio Vivaistico pugliese from Bari). Citron trees that were maintained in the citrus germplasm at San Giuliano were grafted onto Volkamer lemon (Citrus limonia Osb.)
Acknowledgements
We thank Dr. Luigi Catalano Direttore CO.VI.P. - Consorzio Vivaistico pugliese from Bari (Italy) for the kindling sending of “Diamante” citron leaves. This work was partially supported by the 2007-2013 Interreg IVA program “France-Italie maritime” on the PYRGI Project (Project B5H10000000006).
References (57)
- et al.
Molecular phylogeny in Indian Citrus L (Rutaceae) inferred through PCR-RFLP and trnL-trnF sequence data of chloroplast DNA
Sci. Hort.
(2009) - et al.
Chemical variability of peel and leaf essential oils of 15 species of mandarins
Biochem. Syst. Ecol.
(2001) Oranges and lemons: clues to the taxonomy of Citrus from molecular markers
Trends in Genetics
(2001)- et al.
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
Sci. Hort.
(2011) - et al.
Genetic diversity and relationships within Citrus and related genera based on sequence related amplified polymorphism markers (SRAPs)
Sci. Hort.
(2009) Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy
(2001)- et al.
Assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs)
Theor. Appl. Genet.
(2006) - et al.
A numerical taxonomic study of the affinity relationships in cultivated Citrus and its close relatives
Syst. Bot.
(1976) - Belkhir, K., Borsa, P., Chikhi, L., Raufaste, N., Bonhomme, F., 2002. Genetix 4.04, logiciel sous Windows TM pour la...
- et al.
Genetic markers and plant genetic resource management
Plant Breed. Rev.
(1995)
Polymorphic simple sequence repeat markers in chloroplast genomes of Solanaceous plants
Theor. Appl. Genet.
Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion
Plant Cell Rep.
Silver staining and recovery of AFLP amplification products on large denaturing polyacrylamide gels
Bio. Tech.
A set of primers for analyzing chloroplast DNA diversity in Citrus and related genera
Tree Physiol.
Measure of the amount of ecologic association between species
Ecology
A rapid DNA isolation procedure for small quantities of fresh leaf tissue
Phytochem. Bull.
Identification of closely related citrus cultivars with inter-simple sequence repeat markers
Theor. Appl. Genet.
Characterization of microsatellite markers in Citrus reticulata Blanco
Mol. Ecol. Res.
New universal mitochondrial PCR markers reveal new information on maternal citrus phylogeny
Tree Genet. Genomes
The possible role of Yunnan, China, in the origin of contemporary Citrus species (Rutaceae)
Econ. Bot.
Linkage information content of polymorphic genetic markers
Hum. Hered.
Phylogenic study of Fraction I protein in the genus Citrus and its close related genera
Japan. J. Genet.
The chemical composition of the leaf essential oils from 110 citrus species, cultivars, hybrids and varieties of Chinese origin
Perfum. Flavor.
Les huiles essentielles d’agrumes
Fruits
Chemical composition of some Citrus oils from Malaysia
J. Essent. Oil Res.
Terpenoids and Related Constituents of Essential Oils. Library of MassFinder 2.1
Lime Oil
Cited by (39)
Phylogenetic and taxonomic status of Citrus halimii B.C. Stone determined by genotyping complemented by chemical analysis of leaf and fruit rind essential oils
2022, Scientia HorticulturaeCitation Excerpt :Heterozygosity between sweet orange (C. sinensis), sour orange (C. aurantium), lemon (C. limon), and grapefruit (C. paradisi) ranged from 0.36 to 0.82 depending on the study and the markers used. As the heterozygosity of C. halimii was found to be low (0.13) and equivalent to that of C. medica and the high homozygosity of citron was favored by cleistogamic fertilization (Luro et al., 2012; Curk et al., 2016), it is therefore unlikely that C. halimii is an interspecific hybrid. It is hence quite likely that C. halimii is a member of a true species with a distant common ancestor with kumquat.
Citrus taxonomy
2020, The Genus CitrusDomestication and history
2020, The Genus CitrusGenetic, morphological and chemical investigations reveal the genetic origin of Pompia (C. medica tuberosa Risso & Poiteau) – An old endemic Sardinian citrus fruit
2019, PhytochemistryCitation Excerpt :Poncire commun and Diamante citrons both originated from Italy. Common Poncire may have generated the ‘Corsican’ variety by self-fertilization (Luro et al., 2012). These two citrons were commonly cropped in Italy and Sardinia.
Finding Noemi: The Transcription Factor Mutations Underlying Trait Differentiation Amongst Citrus
2019, Trends in Plant Science