Genetic Diversity in Tomato (Solanum lycopersicum) and Its Wild Relatives

Tomato, ranking 1st in the world for vegetables, accounts for 14% of world vegetable production (over 100 million metric tons/year $ 1.6 billion market; (Food and Agriculture Organisation [FAO] 2010). Tomato is a rich source of micronutrients for human diet. It is also an acknowledged model species for research on fruit development and metabolite accumulation. The major goals of tomato breeders (higher productivity, better tolerance to biotic and abiotic stresses and increased sensory and health value of the fruit) require a good comprehension and management of tomato genetic resources diversity.

Many studies were conducted on evolutionary aspects of the lycopersicon clade.The mating system was extensively studied, using the clade as a model to study its effects on species variation (Bedinger, Chetelat et al. 2011).Mating system has played a key role in evolution of wild tomatoes, varying from allogamous self-incompatible, to facultative allogamous, to autogamous and self-compatible (Table 1).Flower stigma exertion and gametophytic incompatibility system contribute in greater outcrossing and genetic diversity.All the species of the clade are intercrossable (Table 1), but with a variable success rate (Rick, Fobes et al. 1977a;Rick, Fobes et al. 1979).Fruit color discriminate the wild relative species.Most of the latter carry green fruits, with the exception of the two species from the Galapagos (with yellow and orange fruits) and S. pimpinellifolium, which is the only wild relative species with red fruits.S. pimpinellifolium fruits are round, small, weighing only few grams.These fruits are edible and the species referred as the currant tomato.The plant presents a reduced apical dominance and prostrate growth habit resulting in a large shrub with inflorescence carrying many flowers and fruits (Paran and van der Knaap 2007).S. pimpinellifolium undergone bottleneck only recently with a drastic reduction of its natural habitats and is now an endangered species (Biodiversity-International 2006).S. lycopersicum var cerasiforme fruit is larger than S. pimpinellifolium and is commonly round and red.This subspecies of tomato is referred to as the "cherry tomato".It has been proposed as the direct ancestor of cultivated tomato because of its diversity, its wide spread occurrence in central America and its close genetic relationship with cultivated tomato (Rick and Chetelat 1995).The modern cultivated tomato, S. lycopersicum, is cosmopolite.It has spread all around the world and is now cultivated under a broad range of environments and conditions.

Tomato domestication in South America
Domestication is a special type of species diversification, distinct from species divergence through natural selection in the wild (Darwin and Wallace 1858).Domesticated species differ from wild and relative species for a set of traits known as the domestication syndrome (Doebley, Gaut et al. 2006).Domestication is often controlled by a limited number of chromosomal regions with major phenotypic effect (Purugganan and Fuller 2009).In tomato, edible fruits, attractive red color and fruit size increase are characterizing this process.
The domestication time of tomato is unclear.It is supposed to be due to a recent divergence from S. pimpinellifolium.The first hypothesis supports Peru as the center of origin and domestication (de Candolle 1882).This hypothesis gives emphasis on botanical evidences and has been complemented by botanical, linguistic and historical aspects.It was further supported by other colleagues (Müller 1940a;Müller 1940b;Luckwill 1943) and recent molecular studies (Nesbitt and Tanksley 2002).Nevertheless, very little and unclear archeological evidences are available to clearly support this hypothesis (McMeekin 1992).The second hypothesis supports that domestication occurred primarily in Mexico in the Vera Cruz Puebla area (Jenkins 1948), as there is no evidence for pre-Colombian cultivation of tomato in South America but good evidences in Mexico.Referring to Guilandini (1572), Jenkins also argued that tomato name comes most probably from the Mexican Nahua people word "Tomatl" that described "plants bearing globous and juicy fruit" (Sahagún 1988).Based on its Table 1.Principal features of the lycopersicon subsection (Solanum sect.Lycopersicon) Data are compiled from Peralta et al. 2007, Moyle et al. 2008, Grandillo et al. 2011 center theory, Harlan suggested that biloculed domesticated forms found in south Mexico and Guatemala are the oldest cultivated types (Harlan 1971).Quoting Sahagun, Diez argued that tomato was totally "integrated" in the Aztec civilization food consumption in XVI century, contrary to South American Incas (Diez and Nuez 2008).Nevertheless, two authors identified Quechua names possibly referring to tomato: "pirca" (Horkheimer 1973) and "pesco-tomate" (Yakovleff 1935).However, botanists consider the origins of tomato domestication as unsolved (Peralta and Spooner 2007b).These authors mention recent evidences showing that the Mexican hypothesis is not supported by comparative data, as South American and Mexican tomato accessions share similar isozymes (Rick and Fobes 1975) as well as molecular markers (Villand, Skroch et al. 1998).So far, no evidence appears to be enough conclusive and tomatoes may have been domesticated independently in both areas.To go further a more extensive analysis of molecular polymorphism in the wild and cultivated tomatoes is needed.This would allow investigating demographic scenarios and estimating the parameters of these scenarios (bottleneck intensity, ancestral population size, migration rates) using Markovian model implemented in tools such as IM * program (Hey and Nielsen 2004) or ABC1 methodology (Beaumont, Zhang et al. 2002;Lopes and Beaumont 2010).Very recently, this approach has been implemented to infer past demography and ecological parameters of two tomato wild relatives, S. chilense and S. peruvianum (Tellier, Laurent et al. 2011).
Many authors consider S. lycopersicum var.cerasiforme as ancestral form of the cultivated tomato.It is present in both Mexico and Peru, on the contrary to S. pimpinellifolium which is absent from Mexico.If we assume that S.l.cerasiforme results from direct domestication from S. pimpinellifolium, a consequence of this domestication is that S.l.cerasiforme suffered a decrease of its population effective size during domestication (Bai and Lindhout 2007).Subsequent changes occurred for domestication traits such as growth habit, mating system, gigantism and fruit morphological diversity.Notably a change from exerted to inserted stigmas is responsible for the change from partial allogamy to strict autogamy.Selection for self-pollinating as well as shortening of the stigma compared to close wild relatives such as S. pimpinellifolium has allowed a yield increase (Rick 1977b).This "selfing syndrome" (Sicard and Lenhard 2011) is striking in tomato where a mutation in gene controlling stigma length has been identified in cultivated germplasm (Chen, Cong et al. 2007).

Early cultivation in Europe and in the world
Probably only a few tomato seeds were brought back from Mexico to Europe, leading, after domestication, to a new genetic bottleneck.George McCue has extensively reviewed the history of tomato diverse uses, tracking back the first references by country upon the time (McCue 1952).Most remote reference available comes from Petrus Matthiolus, an Italian Physician (1544).Due to its botanical closeness with toxic Solanum species common in Europe (Mandrake, Belladonna), tomato was for long mostly used as an ornamental.Two centuries later, it was referred as a cultivated plant in Italy by Saccardo (1769).Southern Europe was precursor in use of tomato for human consumption.In France, Bois at first mentions it as ornamental (1760).The same author reported it as vegetable seeds sold in the catalogue of the seeds of the "Maison grainière Andrieux Vilmorin" (1778).Lamarck mentioned it in 1798.Extensive consumption in Spain is described by Quer (1784).
Progressively, following South-North gradient, tomato consumption reached Northern Europe (Sabine 1819).Similarly in USA, Bartram (1766) reported tomatoes being used as food plants.Boyd (1784) mentioned that The David Landreth Seed Co. started to sell tomato seeds for vegetable consumption.Selection for diverse fruit shapes and local adaptation probably rapidly occurred through bulk selection.The crop gained in economic importance by the end of XIX th century with the establishment of tomato breeding programs.Most of the plant material at that time can be considered as landraces: selected for subsistence agriculture environments, producing low but relatively stable yield.At the end of the XIX th century, tomato cultivars (nowadays called landraces or heirlooms) were open pollinated from which seeds were saved by the farmers from a year to the other.Selection of new genotypes within heterogeneous cultivars (or selection of chance variance) resulted from spontaneous mutations, natural outcrossing or recombination of pre-existing genetic variation.Thus, S. lycopersicum found in Europe a secondary centre for diversification (García-Martínez, Andreani et al. 2006).In the XIX th century, establishment of commercial routes and colonies contributed to spread the species worldwide (Diez and Nuez 2008).In United States, prior to 1850 and "Trophy" the first commercially successful variety, no breeding programs were effective (Smith 2000).On an evolutionary perspective, domestication and implementation of breeding programs induced physiological changes.Artificial selection has reduced the genetic diversity of the crop which suffered a new bottleneck.

Tomato breeding in the XXth century: Seeking for diversity and intensive production
After domestication and adaptation to North hemisphere growing conditions for two centuries, the crop started the XX th century with benefits of two major scientific discoveries: The rediscovery of Mendel pioneering work to set up the basis of experimental methods on the use of plant hybridization (Mendel 1866).Second are established domestication concept (Darwin and Wallace 1858) and selection theory (Darwin 1859).
This context has seen the emergence in public institutes of plant germplasm banks, starting point for collecting existing genetic diversity, preserving and valorizing it, following the pioneer work of Nikolai Vavilov (1887-1943) (Kurlovich, Rep'ev et al. 2000).Later on, he was followed by Charles Rick (1915Rick ( -2002) ) who dedicated his life to discover, collect and characterize exotic tomato germplasm (Tanksley and Khush 2002).Today, more than 83,000 tomato accessions are stored in seed banks worldwide, ranking 1 st among vegetable species collected (FAO 2010).The main collections in the world are: In USA, the Tomato Genetic Resources Center in California (TGRC), (www.tgrc.ucdavis.edu ) and the USDA2 collection (www.ars.usda.gov), the World Vegetable Center in Taiwan (www.avrdc.org)and several Europeans collections.The establishment of tomato resource collections made major contributions to understand the distribution of its diversity around the world.Nevertheless the lack of coordination and conflicting passport data is a pitfall for an efficient tomato germplasm management.Efforts are now made to coordinate national initiatives in global or regional approaches.Since 2007, The European Cooperative Programme for Plant Genetic Resources (www.ecpgr.cgiar.org) is a collaborative project between most European countries for long-term conservation and utilization of plant genetic resources in Europe.
This project is based on large network of national centers for tomato genetic resources including COMAV3 (Spain), CGN4 (Netherlands), INRA (France), IPK5 (Germany), Vavilov Institute (Russia) and others.These institutions share their germplasm informations through a database (http://documents.plant.wur.nl/cgn/pgr/tomato/).More recently, in the context of a European Solanaceae project (EU-SOL, www.eu-sol.wur.nl), a collection of more than 6,000 domesticated tomato accessions was established and phenotyped, accompanied by an ad hoc database (Finkers, de Weerd et al. 2011).Finally, since 2008, a world initiative, is conducted under the Plant Biodiversity Inventories (www.nhm.ac.uk/researchcuration/projects/solanaceaesource/).Project aim is to produce a worldwide taxonomic monograph of the species occurring within the plant genus Solanum.As well, tomato is part of long term collection of plant species project, launched by the Svalbard Global Seed Vault initiative (Food 2008).
Due to its broad use for food consumption and adaptation to many environmental conditions (from Alaska summers to tropical conditions) and different crop systems, tomato experienced an important phenotypic diversification.Hundreds of past and present cultivated varieties are now available.Cultivars are dedicated to two main markets, processing and fresh market.Processing tomatoes are cultivated as a field crop, whereas fresh market tomatoes are grown outdoor or indoor (heated and non-heated greenhouses).Breeding objectives have evolved over time, with the evolution of production systems.Nevertheless, three main objectives remain: adaptation to growth constraints, disease and pest resistances and fruit productivity and quality.Wild species were first used as source of adaptation to biotic stress.Disease resistance selection started in United States early XX th century.The first fusarium wilt -resistant cultivar "Tennessee red" was released in 1912.Early 1920's breeders used hybridization with selection in segregating generations.By the mid-30's, plant breeders developed technical procedures to improve selection, such as pedigree selection.Later, existing or emerging private companies enhanced their development with the release of F1 hybrid varieties.Selection for disease resistance was successful as dominant resistance genes were found in the wild relatives for most of the diseases and pests.Modern cultivars can cumulate up to 12 different disease resistance genes which all derive from wild species.Wild germplasm has been primarily used as a source of major resistances.
Processing tomato industry was developed to provide North American and European households canned tomato, tomato paste and ketchup.Processing tomato varieties differ from fresh market ones in their pulp volume.Their growing conditions are dramatically different from fresh tomatoes (open field, mechanical harvesting).Thus the main criteria for processing tomatoes are fruit firmness, plant type with short fruit set period to produce a high percentage of ripe fruits simultaneously.Compact fruit set was obtained from a natural mutation discovered at the beginning of the XX th century, named sp (for self-pruning), conferring to the plant a determinate growth.This mutation was introduced in the well known "Roma" variety, whose long fruit type became a specific trait in processing tomatoes.In the 60's, VF145 was the first variety mechanically harvested.This cultivar has been the major cultivar for tomato ketchup industry for more than a decade in California.Apart from sp, several other mutations (detected in cultivated tomato or through interspecific hybridization) were used in tomato breeding.For example, the jointless (Szymkowiak and Irish 2005;Quinet, Kinet et al. 2011) j2 allele was introgressed from S. Cheesmanii.In combination with sp, j2 has been bred into many processing varieties, allowing a large scale mechanical harvest of tomato fruits.Major genes used in tomato breeding are listed in Table2.
Today, after a rapid evolution towards very firm fruits and long shelf life varieties (with the major success of the variety Daniela, which carried the spontaneous rin mutation), consumers request more diverse texture and tastes (Causse, Friguet et al. 2010).The fresh tomato market faces rapid developments and diversification (Navez 2011).New products and varieties are emerging always faster and their life cycle gets shorter, 5 years in average (Bai and Lindhout 2007).Consumption trend is for broader and diverse choice of fruit types.After the development of truss and cherry type tomatoes, new cultivars resembling to old heirloom varieties are developed.If access to allelic diversity is a must to improve fruit quality, choices in breeding objectives are critical to maintain organoleptic fruit values.Strong associations are often made by consumers between morphology and sensory values.Association of "Oxheart" or "Marmande" fruit shape with a pleasant texture is a good example.This link can be lost through modern breeding (Casals, Pascual et al. 2011).Improved content in potential health beneficial components such as anti-oxidants (lycopene, vitamin C) is also promoted.This can be obtained thanks to specific mutations like hp (Lieberman, Segev et al. 2004) or, again, by the introgression of genes from wild relatives.
Farmers and breeders have shaped diversity over years in an ever-evolving process that is hard to track and to record.Intensive breeding of crop varieties by modern science has increased the genetic erosion which started with domestication.Nevertheless the introgression from wild relatives allowed major progress and introduced a new source of diversity.Charles Rick observed that crosses between wild and cultivated species generated a large diversity of novel phenotypic diversity.Rick's work represents milestones for the modern use of genetic diversity in tomato.It led to uncover positive transgressive variation within interspecific progenies.This encouraged a greater use of exotic germplasm and thus larger gene pools to unlock causal polymorphism.

Biotechnology as a source of new diversity
Many natural mutants discovered in cultivated tomato have been extensively studied and characterized (http://tgrc.ucdavis.edu/),but their amount is limited.Thus reverse genetic techniques were developed aiming to discover gene function by analyzing the phenotypic effects of specific variants of targeted gene sequence.This approach is complementary to classical ("forwards") approaches, as they allow silencing or promoting the expression of targeted gene.They can also be used to generate genetic diversity within DNA sequences.
A delayed ripening tomato, named Flavr-savr® tomato, with a reduced expression of a cell wall protein (a polygalacturonase), obtained by an antisense construction, was the first transgenic plant released on the fresh market (Kramer and Redenbaugh 1994;Sanders and Hiatt 2005).This transformation significantly improved fruit shelf life and storage quality.Nevertheless, it was a commercial failure.Few years later, consumer's concerns about GMO 6 , Table 2. List of genes characterized through molecular techniques with their related function and germplasm origin www.intechopen.comGenetic Diversity in Tomato (Solanum lycopersicum) and Its Wild Relatives 143 as well as the high engineering cost, stopped further commercial developments.In the research field, transformation with Agrobacterium thumefaciens is still widely used for the functional characterization of specific genes.For instance, transformed tomato plants were produced to enable study of endotoxins genes (Zhang, Buehner et al. 2006) plant disease resistance genes, abiotic stress genes or to produce molecules useful in human medicine (Sharma, Singh et al. 2008).
TILLING (Target Induced Local Lesion In Genomes), a mutagenesis technique, has experienced important development (Comai and Henikoff 2006).Early days of this technique were in the 50's (Rick 1991).It is now widely used for reverse genetics to generate and identify induced point mutations in genomes.A chemical reagent (Ethylmethane Sulphonate) is used to induce genetic mutations.Collections of tomatoes carrying artificially induced genetic variants, called mutant libraries are currently available (Menda, Semel et al. 2004;Minoia et al. 2010) or under development (Okabe, Asamizu et al. 2011).In contrast to transgenic methods, mutagenesis is random, cost effective and is not submitted to GMO regulation.TILLING allows generating variants in cultivated genetic background (Piron, Nicolaï et al. 2010) and thus transfer rapidly interesting mutations into cultivars (Gady, Hermans et al. 2009).Application of TILLING technique to screen for natural variation within tomato germplasm collection is now performed (Rigola, van Oeveren et al. 2009).

Molecular markers offer a new vision of tomato diversity
Natural genetic diversity is the fuel of evolution.No evolutive forces or adaptation to environment changes can apply without it (Alonso-Blanco, Aarts et al. 2009).Consequently it is a vital characteristic for species adaptation in general and for crop breeding in particular.Genetic variation occurs both within cultivated tomato (intraspecific) and between wild species (interspecific).Tomato breeding for adaptation to specific growing areas is in progress for more than two centuries now (Stevens and Rick 1986).Since the early days of quantitative genetics, initiatives were developed to improve the understanding of trait inheritance.Attempts to construct genetic maps based on interspecific crosses (S. pimpinellifolium x S. lycoperpersicum) and to map disease resistance genes are performed for years (Langford 1937).A linkage map showing the distribution of agronomic trait with Mendelian inheritance, based on linkage between two or three mutations, was proposed (Butler 1952).Nevertheless, the lack of polymorphic and neutral markers was strongly limitant.Development of isozymes allowed a first evaluation of wild germplasm (Rick and Fobes 1975) and introgression diagnostic (Tanksley, Medina-Filho et al. 1981), but isozyme marker scarcity and their low polymorphism was still limitant.This limitation was progressively overcome since the 80's thanks to the discovery of several molecular marker types.

Ecological and evolution in wild tomato related species
Molecular studies provide important clues into ecological and evolutionary questions in wild tomatoes species.In speciation process, hybrid sterility is frequently due to dysfunctional interactions between loci that accumulate between different lineages.A "snowballing effect" characterizes loci controlling such reproductive barrier and hybrid sterility that should accumulate faster than linearly with time.Such "snowballing" effect has been recently described within distinct populations derived from crosses of S. lycopersicum with S. pennellii, S. habrochaites and S. lycopersicoides (Moyle and Nakazato 2010).However, further investigations are suggested to confirm these results (Stadler, Florez-Rueda et al. 2011).
Tellier and colleagues quantified the number of adaptive and deleterious mutations and the distribution of fitness effects of new mutations within housekeeping genes in 4 species, S. arcanum, S. chilense, S. habrochaites and S. peruvianum.Little evidence for adaptive mutations was shown but strong purifying selection in coding regions was detected (Tellier, Fischer et al. 2011).This suggests that closely related species with similar genetic backgrounds but contrasted environments differ in the frequency of deleterious fitness effects.
The west coastal area between the Andes and the ocean, from Ecuador to Chile is widely recognized as the center of origin of the species from the Solanum sect.lycopersicon.This area covers a wide range of geographical conditions.Complex geography and ecology of Andes had a major impact in species divergence and hybridization between S. pimpinellifolium and S. lycopersicum (Nakazato and Housworth 2011).The two species present a distinct lineage, separated by the Andes.They hybridize extensively in north and central Ecuador.Nakazato and colleagues demonstrated using molecular markers and geographic information system (GIS) data that S. lycopersicum has likely experienced a severe population bottleneck during the colonization of the eastern Andes followed by a rapid population expansion.In plant, resistance genes and homologs (RGA) tend to be highly variable.Caicedo et al (2004) studied the geographic distribution of a RGA family Cf-2 (see Table 2) within and among plant populations of S. pimpinellifolium.They underlined that the geographical distribution of RGA diversity has been primarily shaped by demographic factors and selective pressure (Caicedo and Schaal 2004;Caicedo 2008).The authors underlined the reduction of natural habitat.This phenomenon is also observed on Galapagos Islands.The endemic species S. cheesmanii shows a reduction of its population due to human activity.Differentiation within S. cheesmanii was also observed (Nuez, Prohens et al. 2004) as well as hybridization with the two introduced species S. lycopersicum and S. pimpinellifolium (Darwin, Knapp et al. 2003).

Diversity analysis among wild and cultivated germplasm
Allelic richness (number of different alleles segregating in the population) is used to measure the genetic diversity and is considered as a key parameter for genetic resources management.It reveals past fluctuations in population size (Nei, Maruyama et al. 1975).Molecular differences between more than 200 Peruvian and Ecuadorian S. pimpinellifolium accessions were highlighted by Zuriaga and colleagues.Climate and genetic data were highly correlated.Thus the non-uniform nature of climates between the two countries is shown to be an important factor.Highest diversity was found in North Peru, lowest on Galapagos Islands.Authors stressed the fact that interspecific variation between S. pimpinellifolium and S. lycopersicum was indicating a very close relatedness between the two species (Zuriaga, Blanca et al. 2009).
Cherry tomato accessions show typically a large genetic diversity and an intermediate fruit size between S. pimpinellifolium and large cultivated ones.Botanists postulate that cherry tomato accessions are feral plants (also called revertant) or a possible genetic admixture of wild and cultivated germplasm (Rick and Holle 1990;Peralta et al. 2007a).Recently molecular analysis of the structure of a large set of accessions of wild S. pimpinellifolium, cherry tomato and cultivated accessions showed that domesticated and wild tomatoes have evolved as a species complex with intensive hybridization.This highlighted the admixture position of S. lycopersicum var.cerasiforme (Ranc, Muños et al. 2008) which is illustrated on figure 2 using a data from Ranc et al (2010) and analyzed using Structure 2.0 (Pritchard et al. 2000) output data.Accessions display clustering patterns (circled) following two phenotypic traits: fruit size and stigma insertion.Structuration effect of those domestication traits can be observed.The emergence of molecular markers has allowed quantifying with accuracy the diversity within germplasm material.The first molecular diversity studies on cultivated tomato revealed the very low polymorphism compared to wild species, whether it was based on RFLP 7 (Miller and Tanksley 1990), SSR 8 (Bredemeijer, Cooke et al. 2002;He, Poysa et al. 2003) AFLP 9 (Park, West et al. 2004;Berloo, Zhu et al. 2008), SSAP 10 (Tam, Mhiri et al. 2005) or SNP 11 (Yang, Bai et al. 2004;Labate and Baldo 2005).However, Bredemeijer et al (2002) characterized 500 cultivated lines from European lines and showed that it was possible to distinguish them all from each other using a set of 20 SSR markers.When comparing old varieties (or landraces) to modern hybrids, a higher level of molecular diversity in landraces is usually observed (Mazzucato, Papa et al. 2008;van Berloo, Zhu et al. 2008).If interspecific populations for genetic analyses and diversity studies answered to many questions, it has left a void in the understanding of genotypic variation within tomato breeding programs which focus on intra-specific populations (Van Deynze, Stoffel et al. 2007).The recent discovery of SNP markers, first detected in EST (expressed sequenced tag) sequences (Van Deynze et al. 2007;Jimenez-Gomez and Maloof 2009) then in non-coding sequences (Labate et al. 2009) provided access to a higher level of polymorphism.Labate and colleagues estimated parameters of diversity among S. lycopersium accessions, first using the SNP detected in 50 loci that were resequenced in a diversity panel of 31 accessions.In a second investigation, multilocus estimates of polymorphism were obtained and led to rejection of the neutral equilibrium model of evolution within the studied collection (Labate, Robertson et al. 2009).Public germplasm are potential allele mining sources for crop improvement as illustrated by previous authors who sampled among US seed banks 30 accessions from the five continents.The study confirmed that history of crossing with wild tomato species and distribution among different environments across the world has spread allelic variation (Labate, Sheffer et al. 2011).
Molecular markers have proven their efficiency in sampling and maximizing allelic richness (Schoen and Brown 1993) through the development of nested core collections (McKhann, Camilleri et al. 2004).Such nested core collections (from 8 to 96 accessions) were constructed in tomato, capturing most of the molecular and phenotypic variation present in a set of 360 constituted of wild, feral and cultivated accessions (Ranc, Muños et al. 2008).

Use of molecular diversity to dissect phenotypes
Molecular markers allowed the construction of high density genetic maps of the tomato genome (Tanksley, Ganal et al. 1992).This permitted the dissection of quantitative traits into Mendelian factors or QTL (Quantitative Trait Loci) (Paterson, Lander et al. 1988;Tanksley et al. 1992).This strategy also opened the way to investigate physical mapping and molecular cloning of genetic factors underlying quantitative traits (Paterson, Damon et al. 1991).Moreover, Lycopersicon varieties and related species are all diploid and chromosomally collinear, making genetic dissection straightforward.The first gene cloned by positional cloning was the Pto gene, confering resistance to Pseudomonas syringae (Martin, Brommonschenkel et al. 1993).Since then, interspecific crosses with each wild species were performed.Due to the low genetic diversity within the cultivated compartment (Miller and Tanksley 1990), most of the mapping populations are based on interspecific crosses between a cultivar and related wild species from the lycopersicon group (as reviewed by Foolad (2007) ; Labate, Grandillo et al.(2007); Grandillo et al (2011)) or from lycopersicoides (Pertuzé, Ji et al. 2002) and juglandifolia group (Albrecht, Escobar et al. 2010).However, maps based on intraspecific crosses have proved their interest notably on fruit quality aspects (Saliba-Colombani, Causse et al. 2001).All those populations allowed discovering and/or characterizing a myriad of major genes (Table 2) and QTLs involved in various traits.
Rapidly, molecular breeding strategies were set up and implemented to "pyramid" genes of interest for agronomical traits, notably using Advanced Backcross QTL method (AB-QTL) (Tanksley, Grandillo et al. 1996).Using this approach with a S. lycopersicum x S. pimpinellifolium progeny, in which agronomical favorable QTL alleles were detected, Grandillo and colleagues showed how a wild species could contribute to improve cultivated tomato (Tanksley, Grandillo et al., 1996).Introgression Lines (IL) derived from interspecific crosses allowed to dissect the effect of chromosome fragments from a donor (usually from a wild relative) introgressed into a recurrent elite line.IL offer the possibility to evaluate the agronomic performance of a specific set of QTL (Paran, Goldman et al. 1995).IL was used as a base for fine mapping and positional cloning of several genes and QTL of interest.The first IL library was developed between S. pennellii and S. lycopersicum (Eshed and Zamir 1995;Zamir 2001).QTL mapping power was increased compared to biallelic QTL mapping population, and was again improved by the constitution of sub-IL set with smaller introgressed fragments.This progeny was successful in identifying QTLs for fruit traits (Causse, Duffe et al 2004); anti-oxidants (Rousseaux, Jones et al. 2005), vitamin C (Stevens, Buret et al. 2007) and volatile aromas (Tadmor, Fridman et al. 2002).The introgression of a QTL identified in these IL has allowed plant breeders to boost the level of soluble solids (brix) in commercial varieties and largely increased tomato yield in California (Fridman, Carrari et al. 2004).Such exotic libraries were thus designed with several species, involving S. pimpinellifolium (Doganlar, Frary et al. 2002), S. habrochaites (Monforte and Tanksley 2000;Finkers, van Heusden et al. 2007) and S. lycopersicoides (Canady, Meglic et al. 2005).
Introgression lines were also used to dissect the genetic basis of heterosis (Eshed and Zamir 1995).Heterosis refers to phenomenon where hybrids between distant varieties or crosses between related species exhibit greater biomass, speed of development, and fertility than both parents (Birchler, Yao et al. 2010).Heterosis involves genome-wide dominance complementation and inheritance model such as locus-specific overdominance (Lippman and Zamir 2007).Heterotic QTL for several trait were identified in tomato IL (Semel and Nissenbaum, 2006).A unique QTL was shown to display at the heterozygous level improved harvest index, earliness and metabolite content (sugars and amino acids) in processing tomatoes (Gur, Osorio et al. 2010;Gur, Semel et al. 2011) Furthermore, a natural mutation in the SFT gene, involved in flowering (Shalit, Rozman et al. 2009), was shown to correspond to a single overdominant gene increasing yield in hybrids of processing tomato (Krieger, Lippman et al. 2010).
Metabolite detection is an approach of choice to identify compounds involved in fruits quality traits.Metabolite QTL (mQTL) can be now identified for non-volatile metabolites like sugars, pigments or volatiles compounds (Bovy, Schijlen et al. 2007).This was done on several interspecific populations, notably on S. lycopersicum x S. Chmielewskii (Do, Prudent et al. 2010) and intraspecific crosses (Saliba-Colombani et al. 2001;Causse, Saliba-Colombani et al. 2002;Zanor, Rambla et al. 2009).Recent technologies allowed screening for diversity in a wide range of components on whole genomes.This can be done in a targeted way to better characterize known metabolites (Tieman, Taylor et al. 2006) or untargeted manner to identify new metabolites (Tikunov, Lommen et al., 2005).Further than identify and quantify compounds, metabolomics can be of great help to decipher biosynthetic pathways (Keurentjes 2009).Metabolome studies can be combined to transcriptomic data to identify the key factors (Mounet, Moing et al. 2009;Do, Prudent et al. 2010).Metabolomics has an important role to play in characterization of natural diversity in tomato (Schauer, Zamir et al. 2004;Fernie et al. 2011).As well, it can boost the biochemical understanding of fruit content and be an enhancer for quality breeding (Fernie and Schauer 2009; de Vos, Hall et al. 2011).

Dissection of the molecular bases of domestication and diversification
Product of human domestication and later diversification of fruit types, led to a large morphological diversity in tomato fruit (with small to large, round, blocky, elongated, pear shaped fruits, with color ranging from red to green, white, black, pink, orange or yellow).On the contrary, wild tomato species carry small, round red or green fruits, with a low intraspecific phenotypic diversity.This has drawn scientist attention on the inheritance and development of fruit size and shape in the tomato (Yeager 1937).Influence of chromosome 2 in fruit morphology was noticed (Butler 1964).Thus, using available molecular techniques, fruit traits genetic control has been widely dissected (Grandillo, Ku et al. 1999;Lippman and Tanksley 2001;Barrero and Tanksley 2004).The first QTL, fw2.2, controlling fruit weight variation was cloned (Frary, Nesbitt et al 2000).It has been suggested that diversity of fruit shape in cultivated germplasm can be explained to a large extent by four genes (Rodriguez, Muños et al. 2011).The study established a model for fruit shape evolution in tomato.This model includes four major mutations recently identified: FAS which increases locule number, fruit fasciation and size (Cong, Barrero et al. 2008), LC which increases locule number and fruit size (Muños, Ranc et al. 2011), OVATE which gives ovoid fruit shape (Liu, van Eck et al 2002) and SUN which gives an elongated fruit shape (van der Knaap, Lippman et al. 2002;Xiao, Jiang et al. 2008) or the oxheart shape when associated to LC and FAS.The allelic distribution of the four genes was associated with morphologic, geographical and historical data in a collection of diverse cultivated accessions.This study established that the selection occurred in distinct chronologic and historic periods: LC arose first, followed by OVATE, both in S.l.cerasiforme background but in distinct populations.FAS arose later in a LC background.Presence of those three mutations in Latin American germplasm suggests Pre-Columbian mutations.Combined with fw2.2, they must have strongly contributed to the increase in fruit size during tomato domestication.On the contrary, SUN mutation is not carried by any Latin American material tested, suggesting that SUN mutation appeared post domestication in European material (probably in Italy).This study also showed that the selection for fruit shape is strongly responsible for the underlying genetic structure in tomato cultivars.The recent discoveries of the molecular events shaping tomato fruit indicate that the germplasm is frequently more diverse phenotypically than the wild related germplasm but not necessarily showing a similar pattern at the molecular level."The irony of all this," says Steve Tanksley (geneticist at Cornell University, and precursor of all these studies) "is all that diversity of heirlooms can be accounted for by a handful of genes.There are probably no more than 10 mutant genes that create the diversity of heirlooms you see" (Borrell 2009).Tomato selection and spread worldwide has led to the immense diversity of varieties that characterizes many domesticated plant species (Purugganan and Fuller 2009).

Association genetics: New valorization of natural diversity
Recent advance in molecular genetics and computation has allowed the emergence of association mapping (Myles, Peiffer et al. 2009).Association mapping takes advantage of historical recombination events and natural genetic diversity.By using large numbers of lines and molecular markers over the whole genome, the resolution of Genome Wide Association studies (GWAS) is much higher than in conventional segregating populations.Such approach requires an accurate estimate of the genetic structure of the sample studied (Price, Zaitlen et al. 2010) and linkage disequilibrium (LD) extend among loci.Yu and colleagues (2005) proposed a unified mixed model taking into account the genetic structure of the sample, based on single locus analysis.This model is being updated by integrating a multi-locus analysis (Ayers et al. 2010).In autogamous crops, it is expected that large extent of LD will reduce the resolution and risks to lead to false positive associations.Nevertheless, successful results have been obtained in selfing crops (Atwell, Huang et al. 2010;Ramsay, Comadran et al. 2011).
In tomato, several studies revealed contrasted results according to the samples studied.First studies of the linkage disequilibrium revealed large LD in cultivated tomatoes (Mazzucato, Papa et al, 2008;van Berloo, Zhu et al, 2008;Robbins, Sim et al, 2010).Van Berloo and colleagues performed association mapping within a collection of 94 accessions containing both old and elite (hybrids) European germplasm and about 300 markers (AFLP).Structure coinciding with fruit size was identified allowing grouping between cherry tomato and round-beef types, extensive LD was observed (15 cM average).Robbins and colleagues investigated the population structure among 70 tomato cultivars (modern and vintage, from fresh and processing market).The STRUCTURE analysis (Pritchard, Stephens et al. 2000) revealed groups predefined by market niche and age into distinct subpopulations.Furthermore, they detected two subpopulations within the processing varieties, corresponding to historical patterns of breeding conducted for specific production environments.They found no subpopulation within fresh-market varieties.High levels of admixture were shown in several varieties representing a transition in the demarcation between processing and fresh-market.Mapping and LD analysis on a genome wide level was performed (Robbins, Sim et al. 2010).Using a panel of 102 accessions including 95 cultivars (heirloom, fresh and processing cultivars) and 9 wild species), effect of selection on genome variation was studied using 340 markers (SNP, SSR, and INDEL12 ).LD value varied from 6-8 cM (all accessions) up to 3-16 cM (fresh market cultivars).Inter-chromosomal LD appeared to be population dependent, suggesting cautious approach for association mapping.Notably, a genetic divergence between fresh market and processing types was also shown.On the contrary, the use of cherry tomato allowed the construction of core collection with a reduced structure and lower LD (Ranc et al, 2008;2010).In a pilot study on chromosome 2, using markers distant from several cM to few kb, Ranc (2010) showed that LD varied strongly from one region to the other.A few distant markers remained in strong LD, but could be removed from the analysis.
The first association study was performed by Nesbitt and Tanksley (2002) to identify the SNP responsible for FW2.2 gene they had cloned.They failed to find any association between fruit size and genomic sequence of the fw2.2 region in a collection of 39 cherry tomato accessions.Ranc and colleagues (2010) identified significant association in the promoter region, thanks to a larger and more representative sample.From a breeding point of view, the admixture mapping between the cultivated tomato and its closest relative is a method of choice for allele mining in wild germplasm.Muños and colleagues (2011) used this approach to identify causal polymorphism of QTL controlling locule number on chromosome 2. New SNP arrays are now available thanks to Next Generation Sequencing technologies (NGS), as the genotyping array developed under the Solanaceae Coordinated Agriculture Project (SolCAP) initiative carrying 7,000 effective SNP (SolCAP 2008).These tools will be very useful to scan the whole genome for associations.

Conclusion: Toward a change in the way to manage and use diversity
Crossing wild and cultivated species can reveal alleles left behind during the domestication process.Molecular markers strongly helped to reinforce the use of wild relatives (Zamir 2008).Interfacing genetic resources management and plant breeding, pre-breeding is now recognized as an important adjunct to plant breeding, as a way to introduce new traits from non-adapted populations and wild relatives, notably for abiotic stress (FAO 2010).Nevertheless, the extensive use of this genetic richness contained in seed banks and germplasm collection faces limits.The difficulty to introgress accurately the targeted allele (with favorable effect) without unfavorable ones, carried on by "linkage drag", remains.
With the emergence of bioinformatics and nanotechnologies -so called "post-genomics" erathe last decade has opened high throughput sequencing era.Now, conducting large intraspecific studies becomes a reality in tomato, allowing a better characterization of its genetic diversity.With the completion of its genome sequence (Mueller, Lankhorst et al. 2009) and rich annotation as well as a large number of tools available via SGN (SOL Genome Network; http://solgenomics.net/organism/solanum_lycopersicum/genome)platform (Bombarely, Menda et al. 2010), tomato and its relatives is the most advanced vegetable crop.A draft of the genome sequence of S. pimpinellifolium LA1589 is also released by D. Ware, W. R. McCombie, and Z. B. Lippman at Cold Spring Harbor Laboratory allowing a detailed comparison of both species.The genome sequences of tomato provide clues for understanding the Solanum clade evolutive history and identify genes involved in fleshy fruit development.
Progress in sequencing technologies has reached the point where genotyping by sequencing (GBS) is now possible (Davey, Hohenlohe et al. 2011;Elshire, Glaubitz et al. 2011).This opens new perspectives in terms of genetic diversity management, notably toward conservation and survey of large populations.In a near future, techniques such as GBS may allow breeders and scientists of the tomato community to determine population characteristics prior concretely establishing genome or nucleotide diversity.GBS opens ways to a global and quantitative management of diversity, and let foresee an a priori genetic resource management.It also opens perspectives in allele based breeding called genomic selection (Hamblin, Buckler et al. 2011).
If ex situ germplasm conservation is well developed and will benefit of these developments, in situ conservation of tomato and its wild relatives is becoming critical due to major ecological changes in its area origin.Efforts on in situ conservation and participatory approaches as proposed by Jarvis, Brown et al (2008) and (Thomas, Dawson et al. 2011) could be very useful to maintain the adaptive potential of tomato genetic resources.Nuez and colleagues proposed to use S. cheesmanii accessions now stored in germplasm banks to reinstate some extinct populations in Galapagos Islands (Nuez et al. 2004).This could help avoiding the present paradox: the more knowledge we gain on tomato diversity and its evolutive history, the less available those genetic resources are available in the wild.