Genomic profiling of plastid DNA variation in the Mediterranean olive tree

Guillaume Besnard Pilar Hernndez Bouchaib Khadari Gabriel Dorado Vincent Savolainen - DNA variation Mediterranean Besnard et al. Open Access Genomic profiling of plastid DNA variation in the Mediterranean olive tree Background: Characterisation of plastid genome (or cpDNA) polymorphisms is commonly used for phylogeographic, population genetic and forensic analyses in plants, but detecting cpDNA variation is sometimes challenging, limiting the applications of such an approach. In the present study, we screened cpDNA polymorphism in the olive tree (Olea europaea L.) by sequencing the complete plastid genome of trees with a distinct cpDNA lineage. Our objective was to develop new markers for a rapid genomic profiling (by Multiplex PCRs) of cpDNA haplotypes in the Mediterranean olive tree. Results: Eight complete cpDNA genomes of Olea were sequenced de novo. The nucleotide divergence between olive cpDNA lineages was low and not exceeding 0.07%. Based on these sequences, markers were developed for studying two single nucleotide substitutions and length polymorphism of 62 regions (with variable microsatellite motifs or other indels). They were then used to genotype the cpDNA variation in cultivated and wild Mediterranean olive trees (315 individuals). Forty polymorphic loci were detected on this sample, allowing the distinction of 22 haplotypes belonging to the three Mediterranean cpDNA lineages known as E1, E2 and E3. The discriminating power of cpDNA variation was particularly low for the cultivated olive tree with one predominating haplotype, but more diversity was detected in wild populations. Conclusions: We propose a method for a rapid characterisation of the Mediterranean olive germplasm. The low variation in the cultivated olive tree indicated that the utility of cpDNA variation for forensic analyses is limited to rare haplotypes. In contrast, the high cpDNA variation in wild populations demonstrated that our markers may be useful for phylogeographic and populations genetic studies in O. europaea. Background In the last decades, major technical innovations have allowed a rapid development of various methods for genomic analysis. These have led to applications ranging from phylogeographical reconstructions to forensic analyses and species identification [1,2]. In plants, many studies have focused on the organelle genomes (i.e., plastid DNA - cpDNA - and mitochondrial DNA mtDNA) for six major reasons: (i) these genomes are usually uniparentally inherited (either from the mother or the father) and thus allow for investigations of gene dispersal by seeds or pollen without recombination effect [3]; (ii) their haploid nature facilitates their sequencing and usually does not require cloning; (iii) such genomes are more prone to stochastic events because their effective population size is half that of diploid genomes, allowing a more accurate detection of evolutionary events such as a long persistence of relict populations in refuge zones during last glaciations [4]. In addition the dispersion of maternally inherited genomes (due to the seed dissemination only) occurs at shorter geographic distances than for nuclear genomes. The consequence of a reduced gene dispersal and high genetic drift in organelle genomes is a generally pronounced geographic structure, which facilitates phylogeographic analyses as well as tracing the origins of cultivated species or invasive populations [3]; (iv) they exhibit a high number of identical copies per cell [5], which may represent a significant advantage for forensic analyses; (v) they are circular and protected by a doublemembrane envelope, which makes them resistant to exonucleases and less prone to endonuclease degradation (another advantage for forensics; [6]); and (vi) they exhibit a lower mutation rate than nuclear genomes [7,8], and such stability is generally required for traceability analyses (although see below). The olive tree (Olea europaea, Oleaceae) is among the oldest woody crops, and nowadays represents one of the major cultivated species in the Mediterranean area [9]. The origins of this species have been recently investigated using different molecular techniques, including looking at organelle variation [10-15]. These previous studies allowed the detection of seven main cpDNA lineages in the O. europaea complex (for the olive tree classification see [16]): lineage E1 was detected in the Mediterranean area and Saharan Mountains, lineages E2 and E3 were specific to the Western Mediterranean area, lineage M was only detected in Macaronesia, lineages C1 and C2 were observed from Southern Asia to Eastern Africa, and lineage A was characteristic of Tropical African olives [15]. One limitation encountered during these studies was the particularly low level of cpDNA and mtDNA polymorphism in the Mediterranean olive tree. Until now only seven haplotypes have been detected with different combinations of loci [17,18]. These haplotypes belong to lineages E1, E2 and E3 (i.e., two or three haplotypes per lineage [15]). Recently, the first olive plastid genome (cpDNA) was released [18]. For detecting polymorphism in the cultivated olive tree, Mariotti and co-workers analysed sequence variation in 21 cpDNA fragments [18]. Variable microsatellites (also known as simple sequence repeats; SSR), insertions/deletions (indels) in repeated or non-repeated regions, and single nucleotide polymorphisms (SNPs) were identified and allowed for the identification of six cpDNA haplotypes (or chlorotypes) on a set of 30 cultivated olive trees, but they did not find new variants compared to previous studies [17]. The low cpDNA variation detected in the Mediterranean lineages hampered any applications of these markers, particularly for traceability or authenticity of olive oils [17]. Such a low level of cpDNA polymorphism has already been observed for other cultivated woody species such as Prunus avium [19], Vitis vinifera [20] and Pinus pinea [21]. This is probably due to human dispersal of cultivated genotypes originating from a reduced gene pool. In addition, low cpDNA polymorphism has also been reported in forest trees and this may also stem from low mutation rate in long-living organisms [22-24]. However, higher cpDNA variation has been detected in wild olives than in cultivars, and this allowed some population genetic analyses, for instance in the laperrinei and guanchica subspecies from Saharan Mountains and Canary Islands, respectively [25-27]. Additional investigations are needed to maximise the cpDNA haplotype identification in olive trees by testing new markers (especially multiallelic microsatellites [28]) on representatives of both cultivated and wild pools. Here, we address this challenge. Firstly, we sequenced the complete plastid genomes of seven O. europaea accessions, including one Spanish cultivar (Manzanilla de Sevilla) and six wild olive trees. These taxa were chosen to represent the seven lineages previously reported in (...truncated)