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To explore the genetic robustness (canalization) of metabolism, we examined the levels of fruit metabolites in multiple harvests of a tomato introgression line (IL) population. The IL partitions the whole genome of the wild species Solanum pennellii in the background of the cultivated tomato (Solanum lycopersicum). We identified several metabolite quantitative trait loci that reduce variability for both primary and secondary metabolites, which we named canalization metabolite quantitative trait loci (cmQTL). We validated nine cmQTL using an independent population of backcross inbred lines, derived from the same parents, which allows increased resolution in mapping the QTL previously identified in the ILs. These cmQTL showed little overlap with QTL for the metabolite levels themselves. Moreover, the intervals they mapped to harbored few metabolism-associated genes, suggesting that the canalization of metabolism is largely controlled by regulatory genes.

Quantitative Trait Locus (QTL) mapping is a powerful technique for dissecting the genetic basis of traits and species differences. Established tomato mapping populations between domesticated tomato (Solanum lycopersicum) and its more distant interfertile relatives typically follow a near isogenic line (NIL) design, such as theSolanum pennelliiIntrogression Line (IL) population, with a single wild introgression per line in an otherwise domesticated genetic background. Here we report on a new advanced backcross QTL mapping resource for tomato, derived from a cross between the M82 tomato cultivar andS. pennelli. This so-called Backcrossed Inbred Line (BIL) population is comprised of a mix ofBC2andBC3lines, with domesticated tomato as the recurrent parent. The BIL population is complementary to the existingS. pennelliiIL population, with which it shares parents. Using the BILs we mapped traits for leaf complexity, leaflet shape, and flowering time. We demonstrate the utility of the BILs for fine-mapping QTL, particularly QTL initially mapped in the ILs, by fine-mapping several QTL to single or few candidate genes. Moreover, we confirm the value of a backcrossed population with multiple introgressions per line, such as the BILs, for epistatic QTL mapping. Our work was further enabled by the development of our own statistical inference and visualization tools, namely a heterogeneous Hidden Markov Model for genotyping the lines, and by using state of the art sparse regression techniques for QTL mapping.

Large-scale biology article; International audience; Updates in nanopore technology have made it possible to obtain gigabases of sequence data. Prior to this, nanopore sequencing technology was mainly used to analyze microbial samples. Here, we describe the generation of a comprehensive nanopore sequencing data set with a median read length of 11,979 bp for a self-compatible accession of the wild tomato species Solanum pennellii. We describe the assembly of its genome to a contig N50 of 2.5 MB. The assembly pipeline comprised initial read correction with Canu and assembly with SMARTdenovo. The resulting raw nanopore-based de novo genome is structurally highly similar to that of the reference S. pennellii LA716 accession but has a high error rate and was rich in homopolymer deletions. After polishing the assembly with Illumina reads, we obtained an error rate of

Correlation-based network analysis (CNA) of the metabolic profiles of seeds of a tomato introgression line mapping population revealed a clique of proteinogenic amino acids: Gly, Ile, Pro, Ser, Thr, and Val. Correlations between profiles of these amino acids exhibited a statistically significant average correlation coefficient of 0.84 as compared with an average correlation coefficient of 0.39 over the 16 119 other metabolite cliques containing six metabolites. In silico removal of cliques was used to quantify their importance in determining seminal network properties, highlighting the strong effects of the amino acid clique. Quantitative trait locus analysis revealed co-localization for the six amino acids on chromosome 2, 4 and 10. Sequence analysis identified a unique set of 10 genes on chromosome 2 only, which were associated with amino acid metabolism and specifically the metabolism of Ser-Gly and their conversion into branched-chain amino acids. Metabolite profiling of a set of sublines, with introgressions on chromosome 2, identified a significant change in the abundance of the six amino acids in comparison with M82. Expression analysis of candidate genes affecting Ser metabolism matched the observation from the metabolite data, suggesting a coordinated behavior of the level of these amino acids at the genetic level. Analysis of transcription factor binding sites in the promoter regions of the identified genes suggested combinatorial response to light and the circadian clock.

Over the past 20 years replicated field trials have been conducted on processing tomato varieties by the California Tomato Research Institute and the Israeli Ministry of Agriculture with the objective of identifying superior varieties. Because common check varieties were included in consecutive years, environmental effects could be factored out allowing an estimate of the genetic improvement due to breeding. The results indicate an average genetic gain for yield of 1.54%/yr for California and of 0.4%/yr for Israel. No significant genetic gain in brix was found for California, whereas for Israel, the data indicate an average increase of 0.53%/yr. For the derived trait brix × yield, the overall genetic gain ranged from 0.9%/yr in Israel to 1.5%/yr in California. A significant genetic gain of 1.15%/yr was determined for fruit color in California for the period 1977–87 and of 2.73%/yr for Israel during the years 1985–1995. The improvement in yield is partially due to the increase in the proportion of hybrids in the trials starting from the mid 1980's. The implications of the results for future tomato breeding are discussed.

Improved-processing tomato lines were produced by the molecular breeding strategy of advanced backcross QTL (AB-QTL) analysis. These near-isogenic lines (NILs) contained unique introgressions of wild alleles originating from two donor wild species, Lycopersicon hirsutum (LA1777) and L. pimpinellifolium (LA1589). Wild alleles targeted for trait improvement were selected on the basis of previously published replicated QTL data obtained from advanced backcross populations for a battery of important agronomic traits. Twenty three NILs were developed for 15 genomic regions which were predicted to contain 25 quantitative trait factors for the improvement of seven agronomic traits: total yield, red yield, soluble solids, brix×red yield, viscosity, fruit color, and fruit firmness. An evaluation of the agronomic performance of the NILs in five locations worldwide revealed that 22 out of the 25 (88%) quantitative factors showed the phenotypic improvement predicted by QTL analysis of the BC3 populations, as NILs in at least one location. Per-location gains over the elite control ranged from 9% to 59% for brix×red yield; 14% to 33% for fruit color; 17% to 34% for fruit firmness; 6% to 22% for soluble-solids content; 7% to 22% for viscosity; 15% to 48% for red yield, and 20% to 28% for total yield. The inheritance of QTLs, the implementation of the AB-QTL methodology for characterizing unadapted germplasm and the applicability of this method to other crops are discussed.

Vegetative and reproductive phases alternate regularly during sympodial growth in tomato. In wild-type ‘indeterminate’ plants, inflorescences are separated by three vegetative nodes. In ‘determinate’ plants homozygous for the recessive allele of the SELF-PRUNING (SP) gene, sympodial segments develop progressively fewer nodes until the shoot is terminated by two consecutive inflorescences. We show here that the SP gene is the tomato ortholog of CENTRORADIALIS and TERMINAL FLOWER1, genes which maintain the indeterminate state of inflorescence meristems in Antirrhinum and Arabidopsis respectively. The sp mutation results in a single amino acid change (P76L), and the mutant phenotype is mimicked by overexpressing the SP antisense RNA. Ectopic and overexpression of the SP and CEN transgenes in tomato rescues the ‘indeterminate’ phenotype, conditions the replacement of flowers by leaves in the inflorescence and suppresses the transition of the vegetative apex to a reproductive shoot. The SELF-PRUNING gene is expressed in shoot apices and leaves from very early stages, and later in inflorescence and floral primordia as well. This expression pattern is similar to that displayed by the tomato ortholog LEAFY and FLORICAULA. Comparison of the sympodial, day-neutral shoot system of tomato and the monopodial, photoperiod-sensitive systems of Arabidopsis and Antirrhinum suggests that flowering genes that are required for the processing of floral induction signals in Arabidopsis and Antirrhinum are required in tomato to regulate the alternation between vegetative and reproductive cycles in sympodial meristems.

A pair of processing tomato (Lycopersicon esculentum Mill.) lines, nearly isogenic for the Tm-2a gene for resistance to tobacco mosaic virus, were grown in replicated trials under commercial production conditions in five locations worldwide. The lines were evaluated for 17 processing traits including fruit yield, size, soluble solids concentration, color, firmness, and viscosity. Eight of those traits differed significantly among the nearly-isogenic lines (NILs). Most notably, the NIL heterozygous for Tm-2a yielded, on average, 16% more than the NIL homozygous for the susceptible allele and 33% more than the NIL homozygous for Tm-2a. Viscosity was lower in the heterozygous NIL and the homozygous and heterozygous resistant NILs had softer fruit with larger stem scars compared to the homozygous susceptible NIL. These results indicate the presence of the Tm-2a locus affects many traits of importance for processing tomatoes and may be used, in the heterozygous state, to significantly increase yield. Whether the observed effects are due to the Tm-2a gene itself or genes associated via linkage drag could not be determined.

The Curl (Cu) and Mouse-ear (Me) mutations of tomato cause two seemingly unrelated developmental syndromes with a wide range of pleiotropic phenotypes. Yet, the distinct morphogenic alterations in shoots, leaves, and inflorescences conferred by the two mutations appear to be caused by unchecked meristematic activity that characterizes dominant mutations in Knotted1 (Kn1)-like genes of monocot plants. We have been unable to separate the two closely linked Cu and Me mutations, and they may lie in the same gene. A homeobox-containing class I Kn1-like gene, TKn2, also maps to the same location. Significantly, the dominant mutations are associated with two aberrant modes of TKn2 transcription. Overexpression of the two in-frame wild-type transcripts of TKn2 is associated with the Cu mutation, whereas misexpression of an abundant and oversized fusion mRNA is associated with the Me mutation. Available molecular evidence strongly suggests that the defective Me-TKn2 transcript is generated via a novel splicing event that merges transcripts of two closely linked genes. The translated fusion product is comprised of most of the 5' end of the adjacent PPi-dependent fructose 6-phosphate phosphotransferase (PFP) transcript spliced in-frame to coding position 64 of the TKn2 transcript, leaving the TKn2 homeobox intact. We suggest that class I Kn1-like genes were selected early during evolution to regulate basic programs of aerial meristems and that subtle alterations in their function may be the basis for the wide diversity in growth parameters of shoot systems, leaves, and inflorescences among plant species.

Variation in the branching of plant inflorescences determines flower number and, consequently, reproductive success and crop yield. Nightshade (Solanaceae) species are models for a widespread, yet poorly understood, program of eudicot growth, where short side branches are initiated upon floral termination. This “sympodial” program produces the few-flowered tomato inflorescence, but the classical mutants compound inflorescence (s) and anantha (an) are highly branched, and s bears hundreds of flowers. Here we show that S and AN, which encode a homeobox transcription factor and an F-box protein, respectively, control inflorescence architecture by promoting successive stages in the progression of an inflorescence meristem to floral specification. S and AN are sequentially expressed during this gradual phase transition, and the loss of either gene delays flower formation, resulting in additional branching. Independently arisen alleles of s account for inflorescence variation among domesticated tomatoes, and an stimulates branching in pepper plants that normally have solitary flowers. Our results suggest that variation of Solanaceae inflorescences is modulated through temporal changes in the acquisition of floral fate, providing a flexible evolutionary mechanism to elaborate sympodial inflorescence shoots., Author Summary Among the most distinguishing features of plants are the flower-bearing shoots, called inflorescences. Despite a solid understanding of flower development, the molecular mechanisms that control inflorescence architecture remain obscure. We have explored this question in tomato, where mutations in two genes, ANANTHA (AN) and COMPOUND INFLORESCENCE (S), transform the well-known tomato “vine” into a highly branched structure with hundreds of flowers. We find that AN encodes an F-box protein ortholog of a gene called UNUSUAL FLORAL ORGANS that controls the identity of floral organs (petals, sepals, and so on), whereas S encodes a transcription factor related to a gene called WUSCHEL HOMEOBOX 9 that is involved in patterning the embryo within the plant seed. (F-box proteins are known for marking other proteins for degradation, but they can also function in hormone regulation and transcriptional activation) Interestingly, these genes have little or no effect on branching in inflorescences that grow continuously (so-called “indeterminate” shoots), as in Arabidopsis. However, we find that transient sequential expression of S followed by AN promotes branch termination and flower formation in plants where meristem growth ends with inflorescence and flower production (“determinate” shoots). We show that mutant alleles of s dramatically increase branch and flower number and have probably been selected for by breeders during modern cultivation. Moreover, the single-flower inflorescence of pepper (a species related to tomato, within the same Solanaceae family) can be converted to a compound inflorescence upon mutating its AN ortholog. Our results suggest a new developmental mechanism whereby inflorescence elaboration can be controlled through temporal regulation of floral fate., Plant flower production is largely determined by the number of inflorescences, the branches produced on flower stems. Two genes identified in tomato reveal a new phase transition that may explain the mechanism of evolution of compound inflorescences in the Solanaceae family.

The isozyme pattern of superoxide dismutase (SOD) in tomato consists of two Cu,Zn isozymes located, respectively, in the chloroplast and in the cytosol, as well as additional isozymes of the Mn or Fe SOD type. We have shown that SOD-1 is the chloroplastic Cu,Zn SOD and is related to cDNA clone T10. Restriction fragment length polymorphism (RFLP) analysis was performed with two cDNA clones representing tomato Cu,Zn-superoxide dismutases. T10, coding for the chloroplast isozyme, was thus mapped to chromosome 11, between marker TG46 and TG108, while clone P31, coding for the cytosolic Cu,Zn SOD isozyme, was mapped to chromosome 1 between TG24 and TG81. SOD is associated with the response of plants to various environmental stresses; the mapping information presented here would permit the demonstration of this association by genetic analysis.

Humanity depends on fewer than a dozen of the approximately 300,000 species of flowering plants for 80% of its caloric intake. And we capitalize on only a fraction of the genetic diversity that resides within each of these species. This is not enough to support our food system in the future. Food availability must double in the next 25 years to keep pace with population and income growth around the world. Already, food-production systems are precarious in the face of intensifying demand, climate change, soil degradation and water and land shortages. Farmers have saved the seeds of hundreds of crop species and hundreds of thousands of ‘primitive’ varieties (local domesticates called landraces), as well as the wild relatives of crop species and modern varieties no longer in use. These are stored in more than 1,700 gene banks worldwide. Maintaining the 11 international gene-bank collections alone costs about US$18 million a year.

The tomato yellow leaf curl virus (TYLCV) gene that encodes the capsid protein (V1) was placed under transcriptional control of the cauliflower mosaic virus 35S promoter and cloned into an Agrobacterium Ti-derived plasmid and used to transform plants from an interspecific tomato hybrid, Lycopersicon esculentum X L. pennellii (F1), sensitive to the TYLCV disease. When transgenic F1 plants, expressing the V1 gene, were inoculated with TYLCV using whiteflies fed on TYLCV-infected plants, they responded either as untransformed tomato or showed expression of delayed disease symptoms and recovery from the disease with increasingly more resistance upon repeated inoculation. Transformed plants that were as sensitive to inoculation as untransformed controls expressed the V1 gene at the RNA level only. All the transformed plants that recovered from disease expressed the TYLCV capsid protein.

The dominant gene I 2 confers on tomato (Lycopersicon esculentum) resistance against the fungus Fusarium oxysporum f. sp. lycopersici race 2. A restriction fragment length polymorphism (RFLP) marker, TG105, has recently been found to be tightly linked to I 2. The potential for cloning this gene by a reverse genetics approach prompted us to describe in both genetic and physical detail the region surrounding the I 2 locus on chromosome 11. We have analyzed patterns of segregation of RFLP markers on chromosome 11 and Fusarium resistance in 140 F2 plants from a cross between Fusarium-resistant and susceptible parental lines. Marker TG105 mapped 0.4 centi-Morgan (CM) from I 2. Physical analysis of TG105 and its flanking RFLP markers, TG26 and TG36, by pulsed field gradient gel electrophoresis (PFGE) yielded a restriction map for this region encompassing at least 620 kb of the tomato genome. TG105 and TG26 hybridized to the same 175 kb MluI-NruI restriction fragment. We have therefore linked two genetically distinct RFLP markers. Based on the 4.1 cM distance between them, we have assigned a mean value of 43 kb for each cM recombination distance in the vicinity of I 2. This local ratio between physical and genetic distances is more than 10-fold below the average for the tomato genome. It should therefore be possible to clone I 2 by chromosome walking from TG105.

Chloroplast DNA was purified from 12 accessions that represent most of the species diversity in the genus Lycopersicon (family Solanaceae) and from 3 closely related species in the genus Solanum . Fragment patterns produced by digestion of these DNAs with 25 different restriction endonucleases were analyzed by agarose gel electrophoresis. In all 15 DNAs, a total of only 39 restriction site mutations were detected among 484 restriction sites surveyed, representing 2,800 base pairs of sequence information. This low rate of base sequence change is paralleled by an extremely low rate of convergent change in restriction sites; only 1 of the 39 mutations appears to have occurred independently in two different lineages. Parsimony analysis of shared mutations has allowed the construction of a maternal phylogeny for the 15 accessions. This phylogeny is generally consistent with relationships based on morphology and crossability but provides more detailed resolution at several places. All accessions within Lycopersicon form a coherent group, with two of the three species of Solanum as outside reference points. Chloroplast DNA analysis places S. pennellii firmly within Lycopersicon , confirming recent studies that have removed it from Solanum . Red-orange fruit color is shown to be a monophyletic trait in three species of Lycopersicon , including the cultivated tomato, L. esculentum . Analysis of six accessions within L. peruvianum reveals a limited amount of intraspecific polymorphism which, however, encompasses all the variation observed in L. chilense and L. chmielewskii . It is suggested that these latter two accessions be relegated to positions within the L. peruvianum complex.

Electrophoretic analysis of 26 enzyme coding genes was conducted on accessions of threeCitrullus species and the relatedPraecitrullus fistulosus andAcanthosicyos naudinianus. The isozyme phylogeny of the genusCitrullus and the related species was constructed based on pairwise measurements of the respective genetic distances between the species and races.P. fistulosus andA. naudinianus form two distinct outgroups toCitrullus which is characterized by two main clusters: The first includes twoC. colocynthis races and the second,C. lanatus andC. lanatus var.citroides, which are more closely related to each other than they are toC. ecirrhosus. The isozyme phylogeny is consistent with the variability in six seed protein bands and with the crossability relations among the examined species.

Anthers of 15 non-allelic male-sterile tomato mutants and their isogenic fertile lines were cultured in vitro. Anthers of one mutant ( ms 10 35 ) readily produced callus containing haploid cells and cells of higher ploidy levels. Conditions for the regeneration of plantlets from this callus were identified. Anthers of the remaining mutants and all the fertile isolines degenerated rapidly in culture. Role of the stage of microsporogenesis in successful anther culture is discussed.

The genusLens includes 5 taxonomic species:L. culinaris is cultivated andL. orientalis, L. odemensis, L. ervoides, andL. nigricans are wild. All the species are annual and almost exlusively selfers. The wild lentils are distributed over a large geographical area and form small disjunct populations which are composed of a small number of plants. 67Lens populations were assayed electrophoretically for 9 enzyme systems; 15 enzymic genes with 37 alleles were identified. The genetic distances (D) measured between the pairs of populations indicated a significantly greater similarity between populations belonging to the same taxonomic species. Assuming the populations represent a random sample of the variability in each of the species the genetic distances (D) between the 5 taxa were calculated. The shortest genetic distance was found betweenL. orientalis andL. culinaris. Another significant feature of the data is the apparent isolation ofL. nigricans from the other 4 species. The genetic distances between theLens species are compared to the patterns of crossability barriers between them.

Pollen grains were harvested from an interspecific F1 hybrid between the cultivated tomato, Lycopersicon esculentum Mill., and its wild relative Lycopersicon hirsutum Humb. & Bonpl., a low temperature tolerant accession originating from an altitude of 3200 m in the Peruvian Andes. The two species differ for electrophoretically-detectable loci that mark six (possibly seven) of the 12 tomato chromosomes. Isozyme analysis of the BC1 populations derived from controlled pollinations at normal and low temperatures indicates a significant skewing of allelic frequencies favoring two independent chromosome segments of L. hirsutum at low temperatures. The results demonstrate that gametophytic selection for low temperature tolerance of tomato pollen is determined, at least in part, by genes expressed in the haploid pollen.

The Tm-2a gene of tomato confers resistance to the viral pathogen, tobacco mosaic virus. Like many economically important plant genes, Tm-2a has been characterized phenotypically and by classical linkage analysis, yet nothing is known about its gene product. We report here the isolation of two DNA clones which are very tightly linked to the Tm-2a gene. These clones were identified by testing 122 genomic clones as hybridization probes against Southern blots consisting of DNA from pairs of nearly isogenic lines with or without the Tm-2a gene. Screening such a large number of clones in a short period of time was facilitated by co-labeling and simultaneous probing of sets of up to 10 random genomic clones. Tightly linked clones were distinguished by the fact that they exhibited one or more restriction fragment length polymorphisms between the nearly isogenic lines. Tight linkage of the clones with Tm-2a was verified in a segregating F(2) population. Both mapped to the same locus 0.4 +/- 0.4 centimorgans away from Tm-2a and may provide starting points for a genomic ;;walk'' to this gene. Due to the availability of isogenic lines in many plant species, the strategy outlined in this paper should be widely applicable for selecting DNA clones tightly linked to genes of interest.

Electrophoretic and morphological variation was studied in 13 cultivars ofC. lanatus and 31 accessions ofC. colocynthis from Israel. Twelve enzyme systems were assayed, representing 19 loci. We found 12 commercially grown cultivars to be monomorphic at all loci. OneC. lanatus accession collected from Israel is highly polymorphic and carries alleles ofC. colocynthis; this accession is probably a representative of a locally cultivated land race grown by Bedouins for animal feed. Over a range of 500 km two forms ofC. colocynthis were identified: one which grows along the coastal plains of the Mediterranean and the other in the arid Negev and Sinal deserts. A high level of electrophoretic and morphological divergence was found between plants of the two regions, whereas within the ecotypes little variation was observed.

Plants of the salt-sensitiveL. esculentum, the halophytic wild speciesL. pennellii, their F1 hybrids and the interspecific F2 generation were grown in Hoagland's liquid culture containing 100 mM NaCl and 6 mM K+. Analysis of the Na+, Cl- and K+ ions contents of the leaves showed, as observed also in previous studies, that the cultivated parent accumlated more K+ and less Na+ than the wild parent. A total of 117 F2 plants were assayed for 15 electrophoretically detectable isozyme markers which map to nine of the twelve tomato chromosomes. Four loci, all with a similar quantitative effect on Na+ and Cl- uptake, were identified by virtue of their linkage to isozyme markers. Two other loci were found to affect K+ uptake. This study demonstrates the potential value of using genetic markers in order to gain a better understanding of the genetic basis of quantitative traits associated with the response of plants to salinity stress.

Soluble enzymes were extracted from 23 geographically diverse isolates of Trichoderma harzianum and compared by electrophoresis. Seven enzyme systems were assayed and the isolates were grouped into five different types according to their isozymic profiles. The results indicate that enzyme electrophoresis can be used to distinguish between T. harzianum isolates. The potential of this system for T. harzianum breeding is discussed.

Male gametophyte (pollen) isozyme profiles were compared with those of the sporophyte for nine enzyme systems. Sixty percent of the structural genes coding for these enzymes in the sporophyte were also found to be expressed by the gametophyte. All the genes tested were found to be expressed after meiosis, apparently transcribed and translated in the haploid gametophytes.