Variability of the phenolic profiles in the fruits from old, recent and new apple cultivars cultivated in Belgium

The roles of flavonoids in plant defence against pathogens, herbivores, and environmental stress are reviewed and their significant contribution to plant resistance is discussed. The induction of flavonoids is of particular interest for gathering evidence of their roles. Tools are mentioned which may enhance flavonoid biosynthesis and accumulation. These include metabolic engineering and UV light. The induction of defence-related flavonoids is modified by other determining factors and competition between growth and secondary metabolism may exist. In an evolutionary context, stress-related oxidative pressure may have been a major trigger for the distribution and abundance of flavonoids. UV protection is one of their most significant, or even the most significant, functional role for flavonoids. The multi-functionality of these compounds, however, often complicates the interpretation of experimental results but, overall, it supports the importance of flavonoids.

Introduction Domestication is a process of increasing codependence between plants and animals on the one hand, and human societies on the other [1], [2]. The key questions relating to the evolutionary processes underlying domestication concern the identity and geographic origin of the wild progenitors of domesticated species [3], the nature of the genetic changes underlying domestication [4], [5], the tempo and mode of domestication (e.g., rapid transition versus protracted domestication) [6] and the consequences of domestication for the genetic diversity of the domesticated species [7], [8], [9], [10]. An understanding of the domestication process provides insight into the general mechanisms of adaptation and the history of human civilization, but can also guide modern breeding programs aiming to improve crops or livestock species further [11], [12]. Plant domestication has mostly been studied in seed-propagated annual crops, in which strong domestication bottlenecks have often been inferred, especially in selfing annuals, such as foxtail millet, wheat and barley [11], [13], [14], [15], [16], [17]. Genetic data have suggested that domestication or the spread of domesticated traits has been fairly rapid in some annual species (e.g, maize or sunflower), with limited numbers of populations or species contributing to current diversity [10], [18], [19], [20], [21], [22]. In contrast, a combination of genetics and archaeology suggested a protracted model of domestication for other annual crops, and in particular for the origin of wheat or barley in the Fertile Crescent [11], [23]. However, the genetic consequences of domestication have been little investigated in long-lived perennials, such as fruit trees [24], [25], [26]. Trees have several biological features that make them fascinating and original models for investigating domestication: they are outcrossers with a long lifespan and a long juvenile phase, and tree populations are often large and connected by high levels of gene flow [27], [28]. Differences in life-history traits probably result in marked differences in the mode and speed of evolution between trees and seed-propagated selfing annuals [27], [28], [29]. For example, outcrossing may tend to make domestication more difficult, in part because the probability of fixing selected alleles is lower than in selfing crops [6], [13]. The combination of self-incompatibility and a long juvenile phase also results in highly variable progenies, making breeding a slow and expensive process, and rendering crop improvement difficult. The development of vegetative propagation based on cuttings or grafting has been a key element in the domestication of long-lived perennials, allowing the maintenance and spread of superior individuals despite self-incompatibility [30]. However, the use of such techniques has further decreased the number of sexual cycles in tree crops since the initial domestication event, adding to the effect of long juvenile phases in limiting the genetic divergence between cultivated trees and their wild progenitors [30], [31], [32], [33]. Thus, domestication can generally be considered more recent, at least in terms of the number of generations, in fruit tree crops than in seed-propagated selfing annuals. Given the slow process of selection and the limited number of generations in which humans could exert selection, the protracted nature of the domestication process in trees has probably resulted in limited bottlenecks [25], [31] and in a weaker domestication syndrome [34] than in seed-propagated annuals. Nevertheless, many cultivated fruit trees clearly display morphological, phenotypic and physiological features typical of a domestication syndrome, such as large fruits and high sugar or oil content [32], [35]. Many aspects of fruit tree domestication have been little studied [25]. Consequently, most of the hypotheses concerning the consequences of particular features of trees for their domestication/diversification remain to be tested. Recent studies on grapevines, almond and olive trees have provided illuminating insights, such as the importance of outcrossing and interspecific hybridization [36], [37], [38], but additional studies of other species are required to draw more general conclusions. Here, we investigated the origins of the domesticated apple Malus domestica Borkh., one of the most emblematic and widespread fruit crops in temperate regions [35]. A form of apple corresponding to extant domestic apples appeared in the Near East around 4,000 years ago [39], at a time corresponding to the first recorded uses of grafting. The domesticated apple was then introduced into Europe and North Africa by the Greeks and Romans and subsequently spread worldwide [35]. While the ancestral progenitor has been clearly identified as being M. sieversii, the identity and relative contributions of other wild species present along the Silk route that have contributed to the genetic makeup of apple cultivars remain largely unknown. This is surprising given the potential importance of this knowledge for plant breeding and for our understanding of the process of domestication in fruit trees. The wild Central Asian species M. sieversii (Ldb.) M. Roem has been identified as the main contributor to the M. domestica genepool based on similarities in fruit and tree morphology, and genetic data [40], [41], [42], [43]. The Tian Shan forests were identified as the geographic area in which the apple was first domesticated, on the basis of the considerable intraspecific morphological variability of wild apple populations in this region [44], [45]. Nucleotide variation for 23 DNA fragments even suggested that M. sieversii and M. domestica belonged to a single genepool (which would be called M. pumila Mill.), with phylogenetic networks showing an intermingling of individuals from the two taxa [43]. Some authors have also suggested possible contributions of additional wild species present along the Silk Route: M. baccata (L.) Borkh, which is native to Siberia, M. orientalis Uglitz., a Caucasian species present along western sections of the ancient trade routes, and M. sylvestris Mill. (European crabapple), a species native to Europe [46], [47], [48], [49]. These hypotheses were based on the history of human migration and trade, the lack of phylogenetic resolution between M. domestica and these four wild species [41], [42], genetic evidence of hybridization at a local scale between domesticated apple and M. sylvestris [40], and the recent finding of sequence haplotype sharing between M. sylvestris and M. domestica [50]. However, such secondary contributions remain a matter of debate, mostly due to the difficulty of distinguishing introgression from incomplete lineage sorting [43], [50], [51]. The three wild species occurring along the Silk Route all bear small, astringent, tart fruits. None of these species has the fruit quality of M. sieversii, but they may have contributed other valuable horticultural traits, such as later flowering, resistance to pests and diseases, capacity for longer storage or climate adaptation. The organoleptic properties of the fruits of these wild species may also have been selected during domestication, for the preparation of apple-based beverages, such as ciders [46], [52]. Cider apples are indeed smaller, bitter and more astringent than dessert apples and bear some similarity to M. sylvestris apples. There is also evidence to suggest that Neolithic and Bronze Age Europeans were already making use of M. sylvestris [39]. In this study, we used a comprehensive set of apple accessions sampled across Eurasia (839 accessions from China to Spain; Figure 1 and Figure S1; Table S1) and 26 microsatellite markers distributed evenly across the genome to investigate the following questions: 1) Is there evidence for population subdivision within and between the five taxa M. domestica, M. baccata, M. orientalis, M. sieversii and M. sylvestris? 2) How large is the contribution of wild species other than the main progenitor, M. sieversii, to the genome of M. domestica? 3) Does M. domestica have a genetic structure associated with its different possible uses (i.e., differences between cider and dessert apples)? 4) What consequences have domestication, subsequent crop improvement and vegetative propagation by grafting had for genetic variation in cultivated apples? Most of our samples of M. domestica corresponded to cultivars from Western Europe (Figure 1 and Figure S1), as almost all the cultivars available in modern collections (including American, Australasian cultivars) are of European ancestry and this region is therefore the most relevant area for the detection of possible secondary introgression from the European crabapple. 10.1371/journal.pgen.1002703.g001 Figure 1 Geographic origins of the samples of the four wild Malus species used: M. sylvestris (blue), M. orientalis (yellow), M. baccata (purple), and M. sieversii (red). Samples of unknown origin (N = 28) were not projected onto the map. Results High diversity and low deviations from random mating expectations within species Our sampling scheme (Figure 1 and Figure S1), based on the collection of a single tree for each apple variety, was designed to avoid the sampling of clones. However, there may still be some clonality if some varieties differing by only a few mutations were propagated by grafting. We corrected for this potential clonality, using the clonal assignment procedures implemented in GENODIVE [53]. We found no pair of samples assigned to the same clonal lineage unless using a threshold of 22 pairwise differences between multilocus genotypes, indicating that our samples did not include any clonal genotypes (the threshold corresponds to the maximum genetic distance allowed between genotypes deemed to belong to the same clonal lineage). Many apple cultivars, including modern cultivars in particular, share recent common ancestors, and siblings or clones of wild species can also be collected unintentionally in the field. Because these features could result in a spurious genetic structure due to the presence of closely related individuals in the dataset, we checked for the presence of groups of related individuals in our dataset between M. domestica cultivars and between the individuals of each wild species. The percentage of pairs with a pairwise relatedness (rxy ) greater than 0.5 (i.e., full sibs) was: 0.4% in M. domestica (N = 168 pairs), 0.3% in M. sieversii (N = 79), 0.004% in M. orientalis (N = 20), and 0.7% in M. baccata (N = 40). For M. sylvestris, no individual pair with rxy >0.5 was identified. However, the distribution of pairwise relatedness rxy among M. domestica cultivars did not deviate significantly from a Gaussian distribution centred on 0 and with a low variance (Fisher's exact test, P≈1, standard deviation = 0.11, Figure S2). This suggests that closely related cultivars are unlikely to have biased subsequent analyses of population structure. We also checked for the limited effect of relatedness on our conclusions by performing all analyses of population subdivision on both the full dataset and a pruned dataset excluding related individuals (see below). We tested the null hypothesis of random mating within each species by calculating FIS , which measures inbreeding. All five Malus species had relatively low values of FIS , although all were significantly different from zero (Table 1), suggesting that each species corresponded to an almost random mating unit. This is consistent with the self-incompatibility system of these species and indicates a lack of widespread groups of related individuals in M. domestica. Low FIS values at species level also indicate a lack of population structure within species. The higher values of FIS observed in M. baccata probably resulted from the occurrence of null alleles, as the microsatellite markers were developed in M. domestica, to which M. baccata is the most distantly related (Table 2). The lowest FIS value was that obtained for M. domestica, reflecting outcrossing between dissimilar parents in breeding programs, or that selection targeted higher levels of heterozygosity [54]. 10.1371/journal.pgen.1002703.t001 Table 1 Summary of genetic variation in the five Malus species. HO HE FIS Ar Ap Ap* PNA M. domestica 0.81 0.83 0.02*** 8.0 0.8 1.2 0.02 M. sieversii 0.77 0.82 0.07*** 8.0 1.1 1.2 0.03 M. sylvestris 0.75 0.87 0.14*** 9.9 1.7 2.5 0.02 M. orientalis 0.79 0.84 0.06*** 8.8 2.1 1.9 0.03 M. baccata 0.56 0.75 0.24*** 7.8 1.4 2.1 0.12 HO and HE : observed and expected heterozygosity, respectively, FIS : inbreeding coefficient, Ar and A p : allelic richness and private allele richness averaged across loci, respectively, estimated by rarefaction using a standardized sample size of 22, A p *: private allele richness averaged across loci using the pruned dataset without hybrids in both wild and cultivated species, estimated by rarefaction using a standardized sample size of 12; PNA : proportion of null alleles, ***: : P-value 0.95). We therefore detected no signal of a demographic bottleneck associated with the domestication of apples. Variable recent contributions of wild relative species to the M. domestica genepool, with the strongest introgression from M. sylvestris We used the admixture coefficients estimated by STRUCTURE to assess the recent contribution of the various wild species to the M. domestica genepool. STRUCTURE analyses of the full dataset showed some admixture among Malus species for the minor mode separating the five species at K = 5. Admixture coefficients were higher between M. domestica and M. sylvestris (α = 0.23) than between M. domestica and respectively M. sieversii (α = 0.06), M. orientalis (α = 0.034) and M. baccata (α = 0.032). We further analysed the contribution of each wild species to the genome of M. domestica by running STRUCTURE separately on each pair of species including M. domestica (Figure 3; Table 3 and Table S2). Malus domestica genotypes with membership coefficients ≥0.20 in a wild species genepool were considered to display introgression. Using this somehow arbitrary cut-off value, STRUCTURE analyses revealed that 26% of M. domestica cultivars displayed introgression from the European crabapple, M. sylvestris (Table 3 and Table S2). By contrast, only 2%, 3% and 0.02% of the M. domestica genotypes displayed introgression from M. sieversii, M. orientalis and M. baccata, respectively (Table 3 and Table S2). The M. domestica cultivars displaying admixture with the M. sylvestris genepool were mostly Russian (e.g., “Antonovka”, “Antonovka kamenicka”, “Novosibirski Sweet”, “Yellow transparent”), French (e.g., “Blanche de St Anne”, “St Jean”, “Api” and “Michelin”) and English (e.g., “Worcester Pearmain” and “Fiesta”). The M9 dwarf apple cultivar (“Paradis jaune de Metz”, [59]) commonly used as a rootstock also appeared to display introgression from the European crabapple (proportion of ancestry in the M. domestica genepool: 0.28; Table S2). When French cultivars were removed from the dataset (N = 89) and pairwise STRUCTURE analyses were repeated for all species pairs including M. domestica, 18% of cultivars displayed introgression from M. sylvestris, including commercial cultivars such as Granny Smith, Michelin, Antonovka and Ajmi (Figure S3) with a mean membership coefficient of M. sylvestris into M. domestica genepool of 47%. Malus sylvestris thus appears to have made a significant contribution to the M. domestica genepool through recent introgression, building on the more ancient contribution (see below) of the Asian wild species M. sieversii. We also note that a few M. domestica individuals appeared to display introgression from several wild species (Table S2), and that M. baccata ornamental cultivars, such as M. baccata flexilis, M. baccata Hansen's and M. baccata gracilis, were partially or even mostly assigned (from 32% to >80%) to the M. domestica genepool (Table S3). 10.1371/journal.pgen.1002703.g003 Figure 3 Proportions of ancestry in two ancestral genepools inferred with the STRUCTURE program, based on datasets including M. domestica (green, N = 299) and each of the four wild Malus species (red). The x-axis is not to scale (details in Table S2). 10.1371/journal.pgen.1002703.t003 Table 3 Mean proportions of assignment to each of the two species in species pair comparisons (K = 2) including M. domestica (Genepool 1) and each of the four wild Malus species (Genepool 2). Species pairs Genepool 1 Genepool 2 M. domestica 0.841 0.159 M. sylvestris 0.119 0.881 M. domestica 0.993 0.007 M. baccata 0.104 0.896 M. domestica 0.980 0.020 M. orientalis 0.030 0.970 M. domestica 0.981 0.019 M. sieversii 0.231 0.769 Wild Central Asian apple origin of the M. domestica genepool Previous studies [43], [50], [60] identified the Central Asian wild apple M. sieversii as the main progenitor of M. domestica on the basis of DNA sequences. Due to the large contribution by M. sylvestris detected in our dataset, corresponding mostly to Western European cultivars, M. domestica and M. sylvestris appeared to be the most closely related pair of species in our analyses of microsatellite markers. We investigated the more ancient contribution of M. sieversii to the M. domestica genepool, by reassessing the genetic differentiation between species in analyses restricted to “pure” individuals (i.e., assigned at ≥0.9 to their respective genepools) from both wild and cultivated species. All FST values were highly significant (P<0.001), but the ranking of FST values between M. domestica and the various wild species was affected: the highest differentiation was still observed between M. domestica and M. baccata (FST  = 0.22), but the lowest differentiation was observed between M. domestica and M. sieversii (FST  = 0.11). Regarding the differentiation between M. sylvestris and M. domestica, we observed the opposite of what was found with the full dataset: M. sylvestris appeared to be more strongly differentiated (FST  = 0.14) from M. domestica than M. sieversii. Thus, by removing signals of recent introgression between cultivated and wild species we were able to confirm that M. sieversii was the initial progenitor of M. domestica. Recent introgression from M. domestica into wild species The finding of a significant level of introgression from wild species into cultivated apple suggested that gene flow might also have occurred in the opposite direction. STRUCTURE analyses of pairs of species confirmed this hypothesis (Figure 3), revealing possible introgression of genetic material into M. sylvestris, M. baccata, M. orientalis and M. sieversii from M. domestica (mean proportions of ancestry in the M. domestica genepool of 0.12, 0.10, 0.03 and 0.23, respectively; Table 3). Considering genotypes with membership coefficients ≥0.9 in the M. domestica genepool as misclassified, we found a total of N = 31 misclassified wild Malus individuals. These results suggest gene flow from the domesticated apple genepool could significantly affect the genetic integrity of wild apple relatives, their future evolution and, possibly, their use as resources for crop improvement. Inference of demographic history Model-based Bayesian clustering algorithms, such as that implemented in STRUCTURE, have a high level of power only for the detection of recent introgression events [55], [61], [62]. We therefore investigated the contributions of M. sylvestris and M. orientalis to the M. domestica genepool using approximate Bayesian computation (ABC) methods that offer a more historical perspective on gene flow [63]. We used a demographic model implementing admixture events [64]. We compared several admixture models to infer what species pairs underwent introgression events and to estimate introgression rates [64]. Malus baccata was not included in these analyses because of its high level of divergence from M. domestica. We assumed, as suggested by previous studies, that M. domestica derived originally from M. sieversii. The most complex model simulated sequential admixtures between M. domestica and all wild species. Other models sequentially removed introgression with each wild species, the order being based on FST values and admixture rates inferred by STRUCTURE. The compared models were the following: (i) the model a assumed that M. domestica was derived from M. sieversii and that the ancestral M. domestica population was involved in reciprocal introgression events with M. orientalis and M. sylvestris, and subsequently introgressed back into M. sieversii (Figure 4a), (ii) model b was similar to the model a, but without introgression events from M. domestica into wild species (Figure 4b), (iii) the model c included a single introgression event, from M. sylvestris into M. domestica (Figure 4c), and (iv) the model d simulated no admixture (Figure 4d). The number of parameters estimated in the model was limited by fixing the times of admixture with M. orientalis, M. sylvestris and M. sieversii at 600, 200 and 13 generations before the present, respectively. We used the following underlying hypotheses: (i) as the juvenile period of Malus lasts five to 10 years, we assumed a generation time of 7.5 years, (ii) admixture between ancestral M. domestica and M. orientalis in the Caucasus occurred approximately 4,500 years ago, shortly before the appearance of sweet apples in the Middle East (4,000 years ago), (iii) admixture between ancestral M. domestica and M. sylvestris in Europe occurred approximately 1,500 years ago, soon after the introduction of domesticated apples into Europe by the Greeks and Romans (iv) back-introgression into M. sieversii from M. domestica occurred approximately 100 years ago, when the cultivation of modern varieties reached Central Asia. 10.1371/journal.pgen.1002703.g004 Figure 4 Admixture models compared in approximate Bayesian computations. Model a assumes that M. domestica is derived from M. sieversii and that the ancestral M. domestica population was involved in reciprocal introgression events with M. orientalis and M. sylvestris, and subsequently introgressed back into M. sieversii. Model b assumes no introgression from M. domestica into wild species, model c assumes the only admixture event is from M. sylvestris into M. domestica, and model d assumes no admixture. Admixture times between M. domestica and the three wild species were fixed (see text). Abbreviations: Nk, effective population sizes; Tk, divergence times; r1, r3, r4 introgression from M. domestica into M. sieversii, M. sylvestris, and M. orientalis respectively; r2, r5 introgression from M. sylvestris and M. orientalis, respectively, into M. domestica. The relative posterior probabilities computed for each model provided strongest statistical support for model c, which assumed a single introgression event, from M. sylvestris into M. domestica (Table 4; posterior probability [p] = 0.67, 95% confidence interval: 0.63–0.72). Note that the model without admixture (model d) had the lowest relative posterior probability (Table 4). In analyses under alternative admixture models (models a and b), the posterior distributions were flat for introgression between M. domestica and M. orientalis and highly skewed towards low values for introgression into M. sylvestris and M. sieversii (not shown), which is consistent with statistical support being highest for model c. 10.1371/journal.pgen.1002703.t004 Table 4 Relative posterior probabilities (p) for the four historical models compared using approximate Bayesian computations. Model p CI2.5 CI97.5 a 0.0349 0.0253 0.0445 b 0.2819 0.2509 0.3130 c 0.6832 0.6504 0.7159 d 0.0000 0.0000 0.0000 Models are described in Figure 4. CI2.5 and CI97.5 are boundaries of the 95% confidence intervals. Given that the model c was clearly favoured, parameter estimates are shown below only for this model (Table 5; prior distributions in Table S4). The contribution of M. sylvestris to the M. domestica genepool was estimated at about 61% (95% credibility interval [95% CI]: 50–68%). We obtained estimates of effective population sizes of 3,520 (95% CI: 2,090–5,680) for M. domestica, 13,200 (95% CI: 6,920–19,300) for M. sieversii, 34,600 (95% CI: 15,100–48,000) for M. sylvestris, and 28,300 (95% CI: 11,700–64,000) for M. orientalis. Using a generation time of 7.5 years, the divergence between M. domestica and M. sieversii (T3) was estimated to have occurred 17,700 years ago (95% CI: 6,225–25,200), which is earlier than previously thought, but we note that the credibility interval is quite large. We estimated that M. sylvestris and M. sieversii diverged about 83,250 years ago (T1, 95% CI: 40,575–334,500), with M. orientalis and M. sieversii diverging about 20,775 years ago (T2, 95% CI: 9,900–47,775). 10.1371/journal.pgen.1002703.t005 Table 5 Demographic and mutation parameters estimated using approximate Bayesian computation for model c. Parameter Mode CI2.5 CI97.5 N1 (M. dom) 3,520 2,090 5,680 N2 (M. ori) 28,300 11,700 64,000 N3 (M. siev) 13,200 6,920 19,300 N4 (M. sylv) 34,600 15,100 48,000 T1 (M. siev - M.sylv) 11,100 5,410 44,600 T2 (M. siev - M. ori) 2,770 1,320 6,370 T3 (M. siev - M. dom) 2,360 830 3,360 r2 (introgr. by M. sylv into M. dom) 0.61 0.50 0.68 μ 2.0.10−4 1.1.10−4 6.9.10−4 p 0.3 0.1 0.3 μSNI 3.0.10−8 5.0.10−8 5.9.10−5 θ1 ( = 4N1μ) 0.7 0.5 2.5 θ2 ( = 4N2μ) 6.1 3.1 23.6 θ3 ( = 4N3μ) 2.8 1.9 7.4 θ4 ( = 4N4μ) 6.8 4.4 18.1 τ1 ( = μT1) 2.28 1.30 16.10 τ2 ( = μT2) 0.54 0.31 2.38 τ3 ( = μT3) 0.41 0.19 1.55 Posterior distributions are summarized as the mode and boundaries of the 95% credibility intervals (CI2.5 and CI97.5). Demographic parameters are introduced in Figure 4 (note that admixture times are fixed in these analyses). Composite parameters scaled by the mutation rate are also shown. The mutation parameters are μ (mean mutation rate), p (mean value of the geometric distribution parameter that governs the number of repeated motifs that increase or decrease the length of the locus during mutation events), μSNI (mean single nucleotide indel mutation rate). Species names are abbreviated. The results above were obtained using the full dataset. We checked the validity of our inferences by conducting analyses on the dataset without admixed and misclassified individuals and using different times of admixture, by assessing the goodness-of-fit of models to data, and by checking that sufficient power was achieved to discriminate among competing models (Text S1; Tables S5, S6, S7). Overall, ABC analyses all provided clear support for a model with contribution of the European crabapple into the domesticates, although the estimated value of the actual contribution of M. sylvestris is probably overestimated here, and should therefore be treated with caution. Indeed, the simulation of a single introgression event hundreds of years ago most likely demanded higher rates of introgression to account for the actual genetic contribution of M. sylvestris into M. domestica than would be needed under continuous gene flow over a long period. Weak genetic structure within M. domestica: linked to cultivar use or geography? As cider cultivars produce apples that are smaller, more bitter and astringent than dessert cultivars, we expected to observe genetic differentiation between these two groups of cultivars and a closer genetic proximity of cider cultivars to M. sylvestris [35], [65]. Neither hypothesis was supported by our data. The classification of apples into “dessert” and “cider” varieties as prior information for STRUCTURE (Locprior model) revealed a very weak tendency of cider and dessert cultivars to be assigned to different clusters at K = 2 (Figure 5), but increasing K did not further result in clearer differentiation between the two types of cultivars. At K = 2, M. domestica cider genotypes had a mean membership of 94.7%, and M. domestica dessert genotypes had a mean membership of 52.5%. However, STRUCTURE analyses without this prior information gave essentially the same clustering patterns at K = 2 (G′ = 0.95 similarity to analyses using classification to assist clustering). The weak differentiation between cider and dessert cultivars (FST  = 0.02) and their high level of admixture in STRUCTURE analyses (Figure 5) indicated a shallow subdivision of the M. domestica genepool. Analyses on a pruned dataset from which closely related individuals had been removed (i.e., pairs of genotypes with rxy ≥0.5; N = 172) revealed the same pattern, confirming that the presence of related cultivars in the dataset did not bias clustering analyses. STRUCTURE was also run on a dataset including all M. sylvestris genotypes, to test the hypothesis that cider cultivars would display a higher level of introgression from the European crabapple. However, the opposite pattern was observed: the proportion of genotypes displaying introgression from M. sylvestris was actually significantly higher in dessert than in cider cultivars (36.4% and 15.5% respectively, χ2  = 16.9, P = 4×10−5). Finally, little genetic differentiation was observed between groups of cultivars of different geographic origins (95% CI: −0.8–0.6, Table S8). 10.1371/journal.pgen.1002703.g005 Figure 5 Proportions of ancestry of M. domestica genotypes (cider and dessert apples) in two ancestral genepools inferred with the STRUCTURE program. Discussion The apple is so deeply rooted in the culture of human populations from temperate regions that it is often not recognized as an exotic plant of unclear origin. We show here that the evolution of the domesticated apple involved more than one geographically restricted wild species. The domesticated apple did not arise from a single event over a short period of time, but from evolution extending over thousands of years. The genepool of the current domesticated apple varieties has been enriched by the contribution of at least two wild species. Malus species have a self-incompatibility system; apple domestication and traditional variety improvement have therefore been based mostly on the selection of the best phenotypes grown from open-pollinated seeds. This breeding strategy has probably favoured the incorporation of genetic material from multiple wild sources and the maintenance of high levels of genetic variation in domesticated apples, despite the extensive use of large-scale vegetative propagation of superior individuals by grafting. Our results are consistent with those reported for the few other woody perennials studied to date, such as grape [37], red mombin [26] and olive trees [36], and support the view that domestication in long-lived plants differs in many respects from the scenarios described for seed-propagated annuals. Weak differentiation from wild progenitors and the Central Asian origin of M. domestica Malus sieversii was previously identified as the main contributor to the M. domestica genome on the basis of morphological and sequence data [41], [43]. The flanks of the Tian Shan mountains have been identified as a likely initial site of domestication, based on the high morphological variability of the wild apples growing in this region, and their similarity to sweet dessert apples [44], [45]. We show here, using a set of rapidly evolving genetic markers distributed throughout the genome and a large sampling, that M. domestica now forms a distinct, random mating group, surprisingly well separated from M. sieversii, with no difference in levels of genetic variation between the domesticate and its wild progenitor. This contrasts with the pattern previously reported, based on a twenty three-gene phylogenetic network [43], where domesticated varieties of apple appeared nested within M. sieversii. After the removal of individuals showing signs of recent admixture, M. sieversii and M. domestica nevertheless appeared to be the pair of species most closely related genetically, confirming their progenitor-descendant relationship. Lack of a domestication bottleneck Apple breeding methods (grafting and “chance seedling” selection), life-history traits specific to trees and/or the genetic architecture of selected traits have likely played a role in the conservation of levels of genetic diversity in cultivated apples similar to those in wild apples. Some factors, such as “chance seedling” selection [66], may even have increased genetic diversity, by favouring outcrossing events among domesticates and introgression from wild species [39]. The low inbreeding coefficients inferred in domesticated apples and the low level of differentiation between cultivated and wild apple populations [40], [54], [67], [68] indicate a high frequency of crosses between individuals of M. domestica, M. sieversii and other wild relatives hailing from diverse geographic origins. Such a high level of gene flow has likely contributed to maintenance of a high level of genetic diversity in domesticated apples. The grafting technique, which was probably developed around 3,000 years ago, has made it possible to propagate superior individuals clonally. The spread of grafting, together with the lengthy juvenile phase (5–10 years) and the long lifespan of apples, may have imposed strong limits on the intensity of the domestication bottleneck thereby limiting the loss of genetic diversity [27], [28], [31]. By decreasing the number of generations since domestication, these factors have probably also helped to restrict the differentiation between domesticates and wild relatives. In theory, grafting may have limited the size of the apple germplasm dispersed early on to a few very popular genotypes, thereby provoking a sudden shrink in effective population size and a loss of diversity. However, we found no evidence that the clonal propagation of apples resulted in a long-lasting decrease in population size or clonal population structure. We can speculate that this may be due to a combination of various factors such as: gene flow with wild species, small-scale propagation (many farmers producing a few grafts each), a large variation in preferences for taste and other quality characteristics between farmers and cultures, large differences in growth conditions leading to the adoption of different sets of genotypes in different regions or the typical behaviour of hobby breeders, who tend to spot particular differences and multiply them. Similarly, for grape, there are huge numbers of old varieties and as much genetic variation in cultivated varieties as in wild-relative progenitors [37]. A major secondary contribution from the European crabapple There has been a long-running debate concerning the possible contribution of other wild species present along the Silk Route to the genetic makeup of M. domestica [40], [46], [47], [65], [69]. Our results clearly show that interspecific hybridization has been a potent force in the evolution of domesticated apple varieties. Apple thus provides a rare example of the evolution of a domesticated crop over a long period of time and involving at least two wild species (see also the cases of olive tree and avocado [24], [26], [37], [70]). A recent study argued that introgression from M. sylvestris into the M. domestica genepool was the most parsimonious explanations for shared gene sequence polymorphisms between the two species [50]. Using an unprecedentedly large dataset, more numerous and more rapidly evolving markers and a combination of inferential methods, we provide a comprehensive view of the history of domestication in apple. We confirm that M. sieversii was the initial progenitor and show that the wild European crabapple M. sylvestris has been a major secondary contributor to the diversity of apples, resulting in current varieties of M. domestica being more closely related to M. sylvestris than to their central Asian progenitor. This situation is reminiscent of that for maize, in which the cultivated crop Zea mays is genetically more closely related to current-day highland landraces than to lowland Z. mays ssp. parviglumis from which the crop was domesticated [71]. This pattern has been attributed to large-scale gene flow from a secondary source, a second subspecies of teosinte, Z. mays ssp. mexicana, into highland maize populations [71]. The usefulness of wild relatives for improving elite cultivated crop genepools has long been recognised and the exploitation of wild resources is now considered a strategic priority in breeding and conservation programs for most crops [11], [12], [44]. Domesticated apples are unusual in that the contribution of wild relatives probably occurred early and unintentionally in the domestication process, preceding even the use of controlled crosses. The use of genetic markers with lower mutation rates than our set of microsatellites might also make it possible to investigate the contribution of more phylogenetically distant apple species growing in areas away from the Silk Route to the diversification of modern apple cultivars. The Romans introduced sweet apples into Europe at a time at which the Europeans were undoubtedly already making cider from the tannin-rich fruits of the native M. sylvestris [35], [72]. Cider is not typical of Asia [35], but it was widespread in Europe by the time of Charlemagne (9th century, [73]). Large numbers of apple trees were planted for cider production in France and Spain from the 10th century onwards [48], [52]. The very high degree of stringency of cider apples (often to the extent that they are inedible) led to the suggestion that cider cultivars arose from hybridization between M. sylvestris and sweet apples [35], [46], [65]. We show here that the genetic structure within the cultivated apple genepool is very weak, with poor differentiation between cider and dessert apples. Cider cultivars thus appear to be no more closely genetically related to M. sylvestris than dessert cultivars. As wild Asian apples are known to cover the full range of tastes [44], [46], it is possible that fruits with the specific characteristics required for cider production were in fact initially selected in Central Asia and subsequently brought into Europe. There is a long-standing tradition of cider production in some parts of Turkey [35], for instance, which is potentially consistent with an Eastern origin of cider cultivars. However, the low level of genetic differentiation between dessert and cider apples indicates that, even if different types of apples were domesticated in Asia and brought to Europe, they have not diverged into independent genepools. Concluding remarks This study settles a long-running debate by confirming that 1) M. domestica was initially domesticated from M. sieversii, and 2) M. domestica subsequently received a significant genetic contribution from M. sylvestris, much larger than previously suspected [35], at least in Western Europe, where originated most of our samples and most cultivar diversity. The higher level of introgression of the European crabapple into the domesticated apple in this study than in previous studies [43], [50], [51] may be attributed to the use of a larger and more representative set of M. domestica genotypes coupled with the genotyping of numerous and rapidly evolving markers known to trace back more recent events. Our inferences also have important implications for breeding programs and for the conservation of wild species of apple. The major contribution of the various wild species to the M. domestica genepool highlights the need to invest efforts into the conservation of these species, which may contain unused genetic resources that could further improve the domesticated apple germplasm [74], such as disease resistance genes or genes encoding specific organoleptic features. Materials and Methods Sample collection and DNA extraction Leaf material was retrieved from the collections of various institutes (INRA Angers, France; USDA - ARS, Plant Genetic Resources Unit, Geneva, NY; ILVO Melle, Belgium) and from a private apple germplasm repository in Brittany for M. domestica (N = 368, Figure S1 including only diploid cultivars N = 299) and from forests for the four wild species (Figure 1; Table S1). Malus sieversii (N = 168) material was collected from 2007 to 2010 in the Chinese Xinjiang province (N = 26), Kyrgyzstan (N = 5), Uzbekistan (N = 1), Tajikistan (N = 1) and Kazakhstan (N = 114). Malus orientalis (N = 215) was sampled in 2009 in Armenia (N = 203), Turkey (N = 5) and Russia (N = 5). Malus sylvestris (N = 40) samples were obtained from 15 European countries. Malus baccata (N = 48) was sampled in 2010 in Russia. The origins of M. domestica cultivars were: France (N = 266), Great Britain (N = 12), USA (N = 12), Russia (N = 7), the Netherlands (N = 6), Australia (N = 4), Belgium (N = 4), Germany (N = 4), Japan (N = 3), Ukraine (N = 3), Tunisia (N = 2), Switzerland (N = 2), Spain (N = 2), New Zealand (N = 2), Israel (N = 1), Ireland (N = 1), Canada (N = 1), Armenia (N = 2) and unknown/debated (N = 34). Genomic DNA was extracted with the Nucleo Spin plant DNA extraction kit II (Macherey & Nagel, Düren, Germany) according to the manufacturer's instructions. Microsatellite markers and polymerase chain reaction (PCR) amplification Microsatellites were amplified by multiplex PCR, with the Multiplex PCR Kit (QIAGEN, Inc.). We used 26 microsatellites spread across the 17 chromosomes (one to three microsatellites per chromosome), in 10 different multiplexes previously optimised on a large set of genetically related progenies of M. domestica [75]. The four multiplexes (MP01, MP02, MP03, MP04; Table S9; Lasserre P. unpublished data) were performed in a final reaction volume of 15 µl (7.5 µl of QIAGEN Multiplex Master Mix, 10–20 µM of each primer, with the forward primer labelled with a fluorescent dye and 10 ng of template DNA). We used a touch-down PCR program (initial annealing temperature of 60°C, decreasing by 1°C per cycle down to 55°C). Six other multiplex reactions (Hi6, Hi4ab, Hi5-10, Hi13a, Hi13b, Hi4b) were performed using previously described protocols [75]. Genotyping was performed on an ABI PRISM X3730XL, with 2 µl of GS500LIZ size standard (Applied Biosystems). Alleles were scored with GENEMAPPER 4.0 software (Applied Biosystems). We retained only multilocus genotypes presenting less than 30% missing data. Suitability of microsatellites for population genetic analyses We checked the suitability of the markers for population genetic analyses. None of the 26 microsatellite markers deviated significantly from a neutral equilibrium model, as shown by the non significant P-values obtained in Ewen-Watterson tests [76], and no pair of markers was found to be in significant linkage disequilibrium in any of the species [77], [78]. The markers could therefore be considered unlinked and neutral. Analyses of genetic variation and differentiation between the five species Apple cultivars may be polyploid [79]. We therefore first checked for the presence of polyploidy individuals of M. domestica within our dataset. Individuals presenting multiple peaks on electrophoregrams were first re-extracted to eliminate contamination as a possible source of apparent polyploidy. We then checked whether they had been reported to be polyploidy in previous studies [79]. After completion of this checking procedure, we removed 69 polyploids (of the 368 samples) from subsequent analyses. We tested for the occurrence of null alleles at each locus with MICROCHECKER 2.2.3 software [80]. Allelic richness and private allele frequencies were calculated with ADZE software [81], for a sample size of 22. Heterozygosity (expected (HE ) and observed (HO )), Weir & Cockerham F-statistics, deviation from Hardy-Weinberg equilibrium and genotypic linkage disequilibrium were estimated with GENEPOP 4.0 [77], [78]. The significance of differences between FST values was assessed in exact tests carried out with GENEPOP 4.0 [77], [78]. Individuals were assigned to clonal lineages with GENODIVE [53]. We estimated relatedness between pairs of cultivars and between pairs of individuals within each species, by calculating the rxy of Ritland and Lynch [82] with RE-RAT online software [83]. We tested whether the distributions of rxy deviated significantly from a Gaussian distribution with a mean of zero and a standard deviation equal to the observed standard deviation, by comparing observed and simulated distributions in Fisher's exact test (R Development Core Team, URL http://www.R-project.org). Assessing bottlenecks during apple domestication and diversification We tested for the occurrence of a bottleneck during apple domestication with the method implemented in BOTTLENECK [58], [84]. The tests were performed under the stepwise-mutation model (SMM) and under a two-phase model (TPM) allowing for 30% multistep changes. We used Wilcoxon signed rank tests to determine whether a population had a significant number of loci with excess genetic diversity. Analyses of population subdivision We used the individual-based Bayesian clustering method implemented in STRUCTURE 2.3.3 [55], [85], [86] to investigate species delimitation, intraspecific population structure and admixture. This method is based on Markov Chain Monte Carlo (MCMC) simulations and is used to infer the proportion of ancestry of genotypes in K distinct predefined clusters. The algorithm attempts to minimize deviations from Hardy–Weinberg and linkage equilibrium within clusters. Analyses were carried out without the use of prior information, except for analyses of population subdivision within the M. domestica genepool for which the “cider”/“dessert” classification of cultivars was used as prior information to assist clustering. K ranged from 1 to 8 for analyses of the five-species dataset and the M. domestica dataset, and was fixed at K = 2 for analyses of pairs of species including M. domestica and each of the wild species. Ten independent runs were carried out for each K and we used 500,000 MCMC iterations after a burn-in of 50,000 steps. We used CLUMPP v1.1.2 (Greedy algorithm) [87] to look for distinct modes among the 10 replicated runs of each K. STRUCTURE analyses were run for the full dataset (N = 839) and for two pruned datasets excluding non-pure individuals (i.e., genotypes with <0.9 membership of their species' genepool) and related individuals (rxy ≥0.5). Inference of demographic history We used the DIYABC program [88] to compare different admixture models and infer historical parameters. We simulated microsatellite datasets for 14 loci (Ch01h01, Ch01h10, Ch02c06, Ch02d08, Ch05f06, Ch01f02, Hi02c07, Ch02c09, Ch03d07, Ch04c07, Ch02b03b, MS06g03, Ch04e03, Ch02g01) previously reported to be of the perfect repeat type [89], [90], [91]. In total, we generated 5×105 simulated datasets for each model. A generalized stepwise model (GSM) was used as the mutational model. The model had two parameters: the mean mutation rate (μ) and the mean parameter (P) of the geometric distribution used to model the length of mutation events (in numbers of repeats). As no experimental estimate of microsatellite mutation rate is available for Malus, the mean mutation rate was drawn from a uniform distribution by extreme values of 10−4 and 10−3, and the mutation rate of each locus was drawn independently from a Gamma distribution (mean = μ; shape = 2). The parameter P ranged from 0.1 to 0.3. Each locus L had a possible range of 40 contiguous allelic states (44 for CH02C06, 42 for CH04E03) and was characterized by individual values for mutation rate (μL ) and the parameter of the geometric distribution (PL ); μL and PL were drawn from Gamma distributions with the following parameter sets: mean = μ, shape = 2, range = 5×10−5–5×10−2 for μL , and mean = P, shape = 2, range = 0.01–0.9 for PL . As not all allele lengths were multiples of motif length, we also included single-nucleotide insertion-deletion mutations in the model, with a mean mutation rate (μSNI ) and locus-specific rates drawn from a Gamma distribution (mean = μSNI ; shape = 2). The summary statistics used were: mean number of alleles per locus, mean genetic diversity [92], genetic differentiation between pairwise groups (FST ; [93]), genetic distances (δμ)2 [94]. We used a polychotomous logistic regression procedure [95] to estimate the relative posterior probability of each model, based on the 1% of simulated data sets closest to the observed data. Confidence intervals for the posterior probabilities were computed using the limiting distribution of the maximum likelihood estimators [64]. Once the most likely model was identified, we used a local linear regression to estimate the posterior distributions of parameters under this model [96]. The 1% simulated datasets most closely resembling the observed data were used for the regression, after the application of a logit transformation to parameter values. Supporting Information Figure S1 Geographic origins of diploid M. domestica cultivars (N = 299). See details in Table S1. (TIF) Click here for additional data file. Figure S2 Distribution of pairwise relatedness coefficients [82] among the M. domestica cultivars. rxy values among cultivars are normally distributed around a mean of zero, with a low variance between pairs of cultivars (Fisher's exact test, P≈1). (TIF) Click here for additional data file. Figure S3 Proportions of ancestry in two ancestral genepools inferred with the STRUCTURE program from datasets including M. domestica (green, N = 89) and each of the wild Malus species (red) except M. baccata. The x-axis is not at scale. (TIF) Click here for additional data file. Table S1 Description of the Malus species accessions analysed, with their geographic origin and providers. (DOC) Click here for additional data file. Table S2 Malus domestica cultivars used in the study, with their use, provider, geographic putative origin. Details of the STRUCTURE analysis summarized in Table 3 are also provided. (DOC) Click here for additional data file. Table S3 Membership coefficients inferred from the STRUCTURE analysis for M. baccata individuals. (DOC) Click here for additional data file. Table S4 Prior distributions used in approximate Bayesian computations. Prior distributions are uniform between lower and upper bound. Parameters are introduced in Figure 4 and Table 5. Species names are abbreviated. (DOC) Click here for additional data file. Table S5 Relative posterior probabilities (p) for the four historical models compared using approximate Bayesian computations. Models are described in Figure 4. CI2.5 and CI97.5 are boundaries of the 95% confidence intervals. (A) Analyses on a pruned dataset with misclassified wild individuals and individuals with a recent admixed ancestry removed. (B) Analyses on the full dataset, assuming that admixture between ancestral M. domestica and M. sylvestris was more recent (67 generations–500 ybp) than in original analyses (200 generations–1,500 ybp). (DOC) Click here for additional data file. Table S6 Demographic and mutation parameters estimated using approximate Bayesian computation for model c. Posterior distributions are summarized as the mode and boundaries of the 95% credibility intervals (CI2.5 and CI97.5). Demographic parameters are introduced in Figure 4 (note that admixture times are fixed in these analyses). Composite parameters scaled by the mutation rate are also shown. The mutation parameters are μ (mean mutation rate), p (mean value of the geometric distribution parameter that governs the number of repeated motifs that increase or decrease the length of the locus during mutation events), μSNI (mean single nucleotide indel mutation rate). Species names are abbreviated. (A) Analyses on a pruned dataset with misclassified wild individuals and individuals with a recent admixed ancestry removed. (B) Analyses on the full dataset, assuming that admixture between ancestral M. domestica and M. sylvestris was more recent (67 generations–500 ybp) than in original analyses (200 generations–1,500 ybp). (DOC) Click here for additional data file. Table S7 Model checking based on comparisons of test quantities between observed data and 100 pseudo-observed datasets generated using parameter values drawn from posterior distributions. (A) Analyses on the full dataset, (B) Analyses on a pruned dataset with misclassified wild individuals and individuals with a recent admixed ancestry removed. (C) Analyses on the full dataset, assuming that admixture between ancestral Malus domestica and M. sylvestris was more recent (67 generations–500 ybp) than in (A). (DOC) Click here for additional data file. Table S8 Genetic differentiation (FST ) between cultivars of different geographic origins (N = 266). Cultivars of unknown origin have been removed. (DOC) Click here for additional data file. Table S9 Description of the Multiplex PCRs (MP01, MP02, MP03, MP04) used for microsatellite amplification. (DOC) Click here for additional data file. Text S1 Method used for approximate Bayesian computations on alternative datasets/admixture times. (DOC) Click here for additional data file.

Mass peak alignment (ion-wise alignment) has recently become a popular method for unsupervised data analysis in untargeted metabolic profiling. Here we present MSClust—a software tool for analysis GC–MS and LC–MS datasets derived from untargeted profiling. MSClust performs data reduction using unsupervised clustering and extraction of putative metabolite mass spectra from ion-wise chromatographic alignment data. The algorithm is based on the subtractive fuzzy clustering method that allows unsupervised determination of a number of metabolites in a data set and can deal with uncertain memberships of mass peaks in overlapping mass spectra. This approach is based purely on the actual information present in the data and does not require any prior metabolite knowledge. MSClust can be applied for both GC–MS and LC–MS alignment data sets. Electronic supplementary material The online version of this article (doi:10.1007/s11306-011-0368-2) contains supplementary material, which is available to authorized users.