Candidate genes and quantitative trait loci affecting fruit ascorbic acid content in three tomato populations

Fresh fruit and vegetables are a major source of ascorbic acid (vitamin C), an important antioxidant for the human diet and also for plants. Ascorbic acid content in fruit exhibits a quantitative inheritance. Quantitative trait loci (QTL) for ascorbic acid content have been mapped in three tomato populations derived from crosses between cultivated tomato varieties (Solanum lycopersicum accessions) and three related wild species or subspecies. The first population consists of a set of introgression lines derived from Solanum pennellii, each containing a unique fragment of the wild species genome. The second population is an advanced backcross population derived from a cross between a cultivated tomato and a Solanum habrochaites (formerly Lycopersicum hirsutum) accession. The third population is a recombinant inbred line population derived from the cross between a cherry tomato line and a large fruited line. Common regions controlling ascorbic acid content have been identified on chromosomes 2, 8, 9, 10, and 12. In general, the wild alleles increased ascorbic acid content, but some improvement could also be provided by S. lycopersicum. Most QTLs appeared relatively stable over years and in different environments. Mapping of candidate genes involved in the metabolism of ascorbic acid has revealed a few colocations between genes and QTLs, notably in the case of a monodehydroascorbate reductase gene and a QTL present in two of the populations on chromosome 9 (bin 9-D), and a previously mapped GDP-mannose epimerase and a QTL on chromosome 9 (bin 9-J).     

Comparative genetics of hybrid incompatibility: sterility in two Solanum species crosses

The genetic basis of hybrid sterility can provide insight into the genetic and evolutionary origins of species barriers. We examine the genetics of hybrid incompatibility between two diploid plant species in the plant clade Solanum sect. Lycopersicon. Using a set of near-isogenic lines (NILs) representing the wild species Solanum pennellii (formerly Lycopersicon pennellii) in the genetic background of the cultivated tomato S. lycopersicum (formerly L. esculentum), we found that hybrid pollen and seed infertility are each based on a modest number of loci, male (pollen) and other (seed) incompatibility factors are roughly comparable in number, and seed-infertility QTL act additively or recessively. These findings are remarkably consistent with our previous analysis in a different species pair, S. lycopersicum x S. habrochaites. Data from both studies contrast strongly with data from Drosophila. Finally, QTL for pollen and seed sterility from the two Solanum studies were chromosomally colocalized, indicating a shared evolutionary history for these QTL, a nonrandom genomic distribution of loci causing sterility, and/or a proclivity of certain genes to be involved in hybrid sterility. We show that comparative mapping data can delimit the probable timing of evolution of detected QTL and discern which sterility loci likely evolved earliest among species.     

Evidence of cryptic introgression in tomato (Solanum lycopersicum L.) based on wild tomato species alleles

ABSTRACT: BACKGROUND: Many beneficial traits (e.g. disease or abiotic stress resistance) have been transferred into crops through crosses with their wild relatives. The 13 recognized species of tomato (Solanum section Lycopersicon) are closely related to each other and wild species genes have been extensively used for improvement of the crop, Solanum lycopersicum L. In addition, the lack of geographical barriers has permitted natural hybridization between S. lycopersicum and its closest wild relative Solanum pimpinellifolium in Ecuador, Peru and northern Chile. In order to better understand patterns of S. lycopersicum diversity, we sequenced 47 markers ranging in length from 130 to 1200 bp (total of 24 kb) in genotypes of S. lycopersicum and wild tomato species S. pimpinellifolium, Solanum arcanum, Solanum peruvianum, Solanum pennellii and Solanum habrochaites. Several of the markers had previously been hypothesized as carrying wild species alleles within S. lycopersicum, i.e., cryptic introgressions. RESULTS: Each marker was mapped with high confidence (e < 1 x 10-30) to a single genomic location using BLASTN against tomato whole genome shotgun chromosomes (SL2.40) database. Neighbor-joining trees showed high mean bootstrap support (86.8 plus or minus 2.34%) for distinguishing red-fruited from green-fruited taxa for 38 of the markers. Hybridization and parsimony splits networks, genomic map positions of markers relative to documented introgressions, and historical origins of accessions were used to interpret evolutionary patterns at nine markers with putatively introgressed alleles. CONCLUSION: Of the 47 genetic markers surveyed in this study, four were involved in linkage drag on chromosome 9 during introgression breeding, while alleles at five markers apparently originated from natural hybridization with S. pimpinellifolium and were associated with primitive genotypes of S. lycopersicum. The positive identification of introgressed genes within crop species such as S. lycopersicum will help inform conservation and utilization of crop germplasm diversity, for example, facilitating the purging of undesirable linkage drag or the exploitation of novel, favorable alleles.     

Quantitative resistance against Bemisia tabaci in Solanum pennellii: Genetics and metabolomics

The whitefly Bemisia tabaci is a serious threat in tomato cultivation worldwide as all varieties grown today are highly susceptible to this devastating herbivorous insect.Many accessions of the tomato wild relative Solanum pennellii show a high resistance towards B. tabaci. A mapping approach was used to elucidate the genetic background of whiteflyresistance related traits and associated biochemical traits in this species. Minor quantitative trait loci(QTLs) for whitefly adult survival(AS) and oviposition rate(OR) were identified and some were confirmed in an F2BC1 population, where they showed increased percentages of explained variance(more than 30%). Bulked segregant analyses on pools of whiteflyresistant and-susceptible F2 plants enabled the identification of metabolites that correlate either with resistance or susceptibility. Genetic mapping of these metabolites showed that a large number of them co-localize with whiteflyresistance QTLs. Some of these whitefly-resistance QTLs are hotspots for metabolite QTLs. Although a large number of metabolite QTLs correlated to whitefly resistance or susceptibility, most of them are yet unknown compounds and further studies are needed to identify the metabolic pathways and genes involved. The results indicate a direct genetic correlation between biochemical-based resistance characteristics and reduced whitefly incidence in S. pennellii.     

Development of a set of PCR-based anchor markers encompassing the tomato genome and evaluation of their usefulness for genetics and breeding experiments

Tomato and potato expressed sequence tag (EST) sequences contained in the solanaceae genomics network (SGN) database were screened for simple sequence repeat (SSR) motifs. A total of 609 SSRs were identified and assayed on Solanum lycopersicum LA925 (formerly Lycopersicon esculentum) and S. pennellii LA716 (formerly L. pennellii). The SSRs that did not amplify, gave multiple band products, or did not exhibit a polymorphism that could be readily detected on standard agarose gels in either of these species were eliminated. A set of 76 SSRs meeting these criteria was then placed on the S. lycopersicum (LA925) x S. pennellii (LA716) high-density map. A set of 76 selected cleaved amplified polymorphism (CAP) markers was also developed and mapped onto the same population. These 152 PCR-based anchor markers are uniformly distributed and encompass 95% of the genome with an average spacing of 10.0 cM. These PCR-based markers were further used to characterize S. pennellii introgression lines (Eshed and Zamir, Genetics 141:1147-1162, 1995) and should prove helpful in utilizing these stocks for high-resolution mapping experiments. The majority of these anchor markers also exhibit polymorphism between S. lycopersicum and two wild species commonly used as parents for mapping experiments, S. pimpinellifolium (formerly L. pimpinellifolium) and S. habrochaites (formerly L. hirsutum), indicating that they will be useful for mapping in other interspecific populations. Sixty of the mapped SSRs plus another 49 microsatellites were tested for polymorphism in seven tomato cultivars, four S. lycopersicum var. cerasiforme accessions and eight accessions of five different wild tomato species. Polymorphism information content values were highest among the wild accessions, with as many as 13 alleles detected per locus over all accessions. Most of the SSRs (90%) had accession-specific alleles, with the most unique alleles and heterozygotes usually found in accessions of self-incompatible species. The markers should be a useful resource for qualitative and quantitative trait mapping, marker-assisted selection, germplasm identification, and genetic diversity studies in tomato. The genetic map and marker information can be found on SGN (http://www.sgn.cornell.edu).     

Use of introgression lines and zonal mapping to identify RAPD markers linked to QTL

RAPD primers were identified as giving parent-specific bands when screened with a set of introgression lines containing introgressed regions of Lycopersicon pennellii that encompass 5 quantitative trait loci affiliated with the production and composition of acylsugars, compounds associated with insect resistance. Primers giving L. pennellii introgression specific bands were zonally mapped to identify bands affiliated with the quantitative trait target and flanking regions using subsets of 7 to 16 F2 individuals which contained small overlapping segments (zones) of the L. pennellii genome spanning those regions. Seventeen RAPD primers, agt-related primers, and an agt clone were then used in mapping the complete F2 population of 144 individuals. This work resulted in the identification of RAPD markers for three of the 5 quantitative trait loci and the construction of an integrated RAPD/RFLP genomic map for tomato (Lycopersicon esculentum x L. pennellii LA716) of 111 RAPD and 8 acylglucose transferase related markers added to a framework map of 150 RFLP markers.     

Mapping of loci from <Emphasis Type="Italic">Solanum lycopersicoides</Emphasis> conferring resistance or susceptibility to <Emphasis Type="Italic">Botrytis cinerea</Emphasis> in tomato

Cultivated tomato (Solanum lycopersicum, syn. Lycopersicon esculentum) is susceptible to the necrotrophic ascomycete and causal agent of gray mold, Botrytis cinerea. Resistance to this fungal pathogen is elevated in wild relatives of tomato, including Solanum lycopersicoides. An introgression line population (IL) containing chromosomal segments of S. lycopersicoides within the background of tomato cv. VF36 was used to screen the genome for foliar resistance and susceptibility to B. cinerea. Based on this screen, putative quantitative trait loci (QTL) were identified, five for resistance and two for susceptibility. Four resistance QTL decreased infection frequency while the fifth reduced lesion diameter. One susceptibility QTL increased infection frequency whereas the other increased lesion diameter. Overlapping chromosomal segments provided strong evidence for partial resistance on chromosomes 1 and 9 and for elevated susceptibility on chromosome 11. Segregation analysis confirmed the major resistance QTL on the long arm of chromosome 1 and susceptibility on chromosome 11. Linkage of partial resistance to chromosome 9 could not be confirmed. The usefulness of these data for resistance breeding and for map-based cloning of foliar resistance to B. cinerea is discussed.     

Fine mapping of fw3.2 controlling fruit weight in tomato

Tomato (Solanum lycopersicum) is an important crop in the Solanaceae family. One of the key traits selected during domestication is fruit mass which is controlled by many quantitative trait loci. The fruit weight locus fw3.2 is one of the major loci responsible for fruit mass in tomato. Identification of the underlying gene will improve our understanding of the molecular mechanism of fruit development while also providing insights into genes that were selected during domestication. We fine mapped fw3.2 to a 51.4-kb interval corresponding to a region comprising seven candidate genes. Gene action showed that the allele from cultivated tomato was additive to dominant in giving rise to an enlarged fruit. Fruit shape analysis indicated that fw3.2 primarily played a role in controlling fruit weight, with a minor effect on fruit shape. Gene expression and nucleotide diversity were investigated and the likelihood of the genes control fruit mass is discussed.     

Quantitative genetic analysis of flowering time in tomato.

Artificial selection of cultivated tomato (Solanum lycopersicum L.) has resulted in the generation of early-flowering, day-length-insensitive cultivars, despite its close relationship to other Solanum species that need more time and specific photoperiods to flower. To investigate the genetic mechanisms controlling flowering time in tomato and related species, we performed a quantitative trait locus (QTL) analysis for flowering time in an F2 mapping population derived from S. lycopersicum and its late-flowering wild relative S. chmielewskii. Flowering time was scored as the number of days from sowing to the opening of the first flower (days to flowering), and as the number of leaves under the first inflorescence (leaf number). QTL analyses detected 2 QTLs affecting days to flowering, which explained 55.3% of the total phenotypic variance, and 6 QTLs for leaf number, accounting for 66.7% of the corresponding phenotypic variance. Four of the leaf number QTLs had not previously been detected for this trait in tomato. Colocation of some QTLs with flowering-time genes included in the genetic map suggests PHYB2, FALSIFLORA, and a tomato FLC-like sequence as candidate genes that might have been targets of selection during the domestication of tomato.     

A Solanum neorickii introgression population providing a powerful complement to the extensively characterized Solanum pennellii population

We present a complementary resource for trait fine‐mapping in tomato to those based on the intra‐specific cross between cultivated tomato and the wild tomato species Solanum pennellii, which have been extensively used for quantitative genetics in tomato over the last 20 years. The current population of backcross inbred lines (BILs) is composed of 107 lines derived after three backcrosses of progeny of the wild species Solanum neorickii (LA2133) and cultivated tomato (cultivar TA209) and is freely available to the scientific community. These S. neorickii BILs were genotyped using the 10K SolCAP single nucleotide polymorphism chip, and 3111 polymorphic markers were used to map recombination break points relative to the physical map of Solanum lycopersicum. The BILs harbor on average 4.3 introgressions per line, with a mean introgression length of 34.7 Mbp, allowing partitioning of the genome into 340 bins and thereby facilitating rapid trait mapping. We demonstrate the power of using this resource in comparison with archival data from the S. pennellii resources by carrying out metabolic quantitative trait locus analysis following gas chromatography–mass spectrometry on fruits harvested from the S. neorickii BILs. The metabolic candidate genes phenylalanine ammonia‐lyase and cystathionine gamma‐lyase were then tested and validated in F₂ populations and via agroinfiltration‐based overexpression in order to exemplify the fidelity of this method in identifying the genes that drive tomato metabolic phenotypes.     

The SOL Genomics Network: a comparative resource for Solanaceae biology and beyond

The SOL Genomics Network (SGN; http://sgn.cornell.edu) is a rapidly evolving comparative resource for the plants of the Solanaceae family, which includes important crop and model plants such as potato (Solanum tuberosum), eggplant (Solanum melongena), pepper (Capsicum annuum), and tomato (Solanum lycopersicum). The aim of SGN is to relate these species to one another using a comparative genomics approach and to tie them to the other dicots through the fully sequenced genome of Arabidopsis (Arabidopsis thaliana). SGN currently houses map and marker data for Solanaceae species, a large expressed sequence tag collection with computationally derived unigene sets, an extensive database of phenotypic information for a mutagenized tomato population, and associated tools such as real-time quantitative trait loci. Recently, the International Solanaceae Project (SOL) was formed as an umbrella organization for Solanaceae research in over 30 countries to address important questions in plant biology. The first cornerstone of the SOL project is the sequencing of the entire euchromatic portion of the tomato genome. SGN is collaborating with other bioinformatics centers in building the bioinformatics infrastructure for the tomato sequencing project and implementing the bioinformatics strategy of the larger SOL project. The overarching goal of SGN is to make information available in an intuitive comparative format, thereby facilitating a systems approach to investigations into the basis of adaptation and phenotypic diversity in the Solanaceae family, other species in the Asterid clade such as coffee (Coffea arabica), Rubiaciae, and beyond.     

Identification of loci affecting flavour volatile emissions in tomato fruits

Fresh tomato fruit flavour is the sum of the interaction between sugars, acids, and a set of approximately 30 volatile compounds synthesized from a diverse set of precursors, including amino acids, lipids, and carotenoids. Some of these volatiles impart desirable qualities while others are negatively perceived. As a first step to identify the genes responsible for the synthesis of flavour-related chemicals, an attempt was made to identify loci that influence the chemical composition of ripe fruits. A genetically diverse but well-defined Solanum pennellii IL population was used. Because S. pennellii is a small green-fruited species, this population exhibits great biochemical diversity and is a rich source of genes affecting both fruit development and chemical composition. This population was used to identify multiple loci affecting the composition of chemicals related to flavour. Twenty-five loci were identified that are significantly altered in one or more of 23 different volatiles and four were altered in citric acid content. It was further shown that emissions of carotenoid-derived volatiles were directly correlated with the fruit carotenoid content. Linked molecular markers should be useful for breeding programmes aimed at improving fruit flavour. In the longer term, the genes responsible for controlling the levels of these chemicals will be important tools for understanding the complex interactions that ultimately integrate to provide the unique flavour of a tomato.     

Genetic dissection of vitamin E biosynthesis in tomato

Vegetables are critical for human health as they are a source of multiple vitamins including vitamin E (VTE). In plants, the synthesis of VTE compounds, tocopherol and tocotrienol, derives from precursors of the shikimate and methylerythritol phosphate pathways. Quantitative trait loci (QTL) for α-tocopherol content in ripe fruit have previously been determined in an Solanum pennellii tomato introgression line population. In this work, variations of tocopherol isoforms (α, β, γ, and δ) in ripe fruits of these lines were studied. In parallel all tomato genes structurally associated with VTE biosynthesis were identified and mapped. Previously identified VTE QTL on chromosomes 6 and 9 were confirmed whilst novel ones were identified on chromosomes 7 and 8. Integrated analysis at the metabolic, genetic and genomic levels allowed us to propose 16 candidate loci putatively affecting tocopherol content in tomato. A comparative analysis revealed polymorphisms at nucleotide and amino acid levels between Solanum lycopersicum and S. pennellii candidate alleles. Moreover, evolutionary analyses showed the presence of codons evolving under both neutral and positive selection, which may explain the phenotypic differences between species. These data represent an important step in understanding the genetic determinants of VTE natural variation in tomato fruit and as such in the ability to improve the content of this important nutriceutical.     

Introgression lines of Solanum sitiens,a wild nightshade of the Atacama Desert,in the genome of cultivated tomato

The wild tomato relative Solanum sitiens is a xerophyte endemic to the Atacama Desert of Chile and a potential source of genes for tolerance to drought, salinity and low‐temperature stresses. However, until recently, strong breeding barriers prevented its hybridization and introgression with cultivated tomato, Solanum lycopersicum L. We overcame these barriers using embryo rescue, bridging lines and allopolyploid hybrids, and synthesized a library of introgression lines (ILs) that captures the genome of S. sitiens in the background of cultivated tomato. The IL library consists of 56 overlapping introgressions that together represent about 93% of the S. sitiens genome: 65% in homozygous and 28% in heterozygous (segregating) ILs. The breakpoints of each segment and the gaps in genome coverage were mapped by single nucleotide polymorphism (SNP) genotyping using the SolCAP SNP array. Marker‐assisted selection was used to backcross selected introgressions into tomato, to recover a uniform genetic background, to isolate recombinant sub‐lines with shorter introgressions and to select homozygous genotypes. Each IL contains a single S. sitiens chromosome segment, defined by markers, in the genetic background of cv. NC 84173, a fresh market inbred line. Large differences were observed between the lines for both qualitative and quantitative morphological traits, suggesting that the ILs contain highly divergent allelic variation. Several loci contributing to unilateral incompatibility or hybrid necrosis were mapped with the lines. This IL population will facilitate studies of the S. sitiens genome and expands the range of genetic variation available for tomato breeding and research.     

Morphological variation in tomato: a comprehensive study of quantitative trait loci controlling fruit shape and development

Variation in fruit morphology is a prevalent characteristic among cultivated tomato. The genetic and developmental mechanisms underlying similarities and differences in shape between the fruit of two elongated tomato varieties were investigated. Fruit from two F2 populations constructed from either Solanum lycopersicum cv. Howard German or cv. Banana Legs crossed with S. pimpinellifolium accession LA1589, and one BC1 population constructed with S. lycopersicum Howard German as the recurrent parent, were analysed for shape by using a new software program Tomato Analyzer. Quantitative trait loci (QTLs) controlling 15 individual shape attributes were mapped by both single and multitrait composite interval mapping in each population. In addition, principal components analysis and canonical discriminant analysis were conducted on these shape attributes to determine the greatest sources of variation among and between the populations. Individual principal components and canonical variates were subjected to QTL analysis to map regions of the genome influencing fruit shape in the cultivars. Common and unique regions, as well as previously known and novel QTLs, underlying fruit morphology in tomato were identified. Four major loci were found to control multiple fruit shape traits, principal components, and canonical variates in the populations. In addition, QTLs associated with the principal components better revealed regions of the genome that varied among populations than did the QTL associated with canonical variates. The QTL identified can be compared across additional populations of tomato and other fruit-bearing crop species.     

Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs

Hybrid organisms may fail to develop, be sterile or they may be more vigorous than either of the parents. Examples of hybrid vigour or hybrid necrosis in the F1 are often not inherited stably in subsequent generations if they are associated with overdominance. There can also be transgressive phenotypes that are inherited stably in these later generations, but the underlying mechanisms are not well understood. Here we have investigated the possibility that stable transgressive phenotypes in the progeny of crosses between cultivated tomato (Solanum lycopersicum cv. M82) and a wild relative (Solanum pennellii, accession LA716) are associated with micro or small interfering(si) RNAs. We identified loci from which these small(s)RNAs were more abundant in hybrids than in either parent and we show that accumulation of such transgressive sRNAs correlated with suppression of the corresponding target genes. In one instance this effect was associated with hypermethylation of the corresponding genomic DNA. Our results illustrate a potential role of transgressive sRNAs in plant breeding and in natural evolution with wild plants.     

Investigation of tomato (Solanum lycopersicum) aminotransferases involved in biosynthesis of branched-chain-amino-acids

This study presents evidence for the role of BCAT3 and BCAT4 proteins in the synthesis of branched-chain-amino-acids in tomato Solanum lycopersicum. BCAT3 and BCAT4 genes were located on tomato chromosomal map by RFLP method (restriction fragment length polymorphism). Using confocal microscopy it was shown that BCAT3-GFP and BCAT4-GFP fusion proteins were localised in chloroplasts. It was observed that these aminotransferase isoforms exhibited distinct kinetic properties and a differential expression pattern of mRNA levels in various tomato tissues.