Search

To determine if the desert tomato, Lycopersicon pennellii, possesses resistance to late blight, caused by Phytophthora infestans, two plant populations were analyzed. Resistance was identified through assessments of disease progress in an F2 mapping population (L. esculentum × L. pennellii) and in a series of introgression lines (L. pennellii into L. esculentum). Levels of resistance varied widely among individuals within each population. However, the response of individuals to different strains of P. infestans was consistent. In the mapping population, a quantitative trait locus (QTL) was detected near marker T1556 on chromosome 6. This QTL accounted for 25% of the phenotypic variance in the population. The occurrence of this QTL was confirmed from analysis of the introgression lines (ILs), where IL 6-2 (containing marker T1556) was the most resistant IL in 2002 and the second most resistant IL in 2001. The identification of an additional QTL for resistance to late blight in tomato will aid in the development of durable resistance to this devastating disease.

Using next-generation DNA sequencing, it was possible to clarify the genetic relationships of Avena species and deduce the likely pathway from which hexaploid oat was formed by sequential polyploidization events. A sequence-based diversity study was conducted on a representative sample of accessions from species in the genus Avena using genotyping-by-sequencing technology. The results show that all Avena taxa can be assigned to one of four major genetic clusters: Cluster 1 = all hexaploids including cultivated oat, Cluster 2 = AC genome tetraploids, Cluster 3 = C genome diploids, Cluster 4 = A genome diploid and tetraploids. No evidence was found for the existence of discrete B or D genomes. Through a series of experiments involving the creation of in silico polyploids, it was possible to deduce that hexaploid oat likely formed by the fusion of an ancestral diploid species from Cluster 3 (A. clauda, A. eriantha) with an ancestral diploid species from Cluster 4D (A. longiglumis, A. canariensis, A. wiestii) to create the ancestral tetraploid from Cluster 2 (A. magna, A. murphyi, A. insularis). Subsequently, that ancestral tetraploid fused again with another ancestral diploid from Cluster 4D to create hexaploid oat. Based on the geographic distribution of these species, it is hypothesized that both the tetraploidization and hexaploidization events may have occurred in the region of northwest Africa, followed by radiation of hexaploid oat to its current worldwide distribution. The results from this study shed light not only on the origins of this important grain crop, but also have implications for germplasm collection and utilization in oat breeding.

Researchers in the life sciences (i.e., healthcare and agriculture) commonly use heuristics to process and interpret the vast amount of available DNA sequence data. The application of discrete optimization techniques, such as mixed-integer programming (MIP), remains largely unexplored and has the potential to transform the field. This paper reports on the successful use of MIP to optimize experimental design in a practical genetics application. More generally, our results illustrate the potential benefits of using MIP for subset selection problems in genetics.

Using single-copy conserved ortholog set (COSII) and simple sequence repeat (SSR) markers, we have constructed two genetic maps for diploid Nicotiana species, N. tomentosiformis and N. acuminata, respectively. N. acuminata is phylogenetically closer to N. sylvestris than to N. tomentosiformis, the latter two of which are thought to contribute the S-genome and T-genome, respectively, to the allotetraploid tobacco (N. tabacum L., 2n = 48). A comparison of the two maps revealed a minimum of seven inversions and one translocation subsequent to the divergence of these two diploid species. Further, comparing the diploid maps with a dense tobacco map revealed that the tobacco genome experienced chromosomal rearrangements more frequently than its diploid relatives, supporting the notion of accelerated genome evolution in allotetraploids. Mapped COSII markers permitted the investigation of Nicotiana-tomato syntenic relationships. A minimum of 3 (and up to 10) inversions and 11 reciprocal translocations differentiate the tomato genome from that of the last common ancestor of N. tomentosiformis and N. acuminata. Nevertheless, the marker/gene order is well preserved in 25 conserved syntenic segments. Molecular dating based on COSII sequences suggested that tobacco was formed 1.0 MYA or later. In conclusion, these COSII and SSR markers link the cultivated tobacco map to those of wild diploid Nicotiana species and tomato, thus providing a platform for cross-reference of genetic and genomic information among them as well as other solanaceous species including potato, eggplant, pepper and the closely allied coffee (Rubiaceae). Therefore they will facilitate genetic research in the genus Nicotiana.

We report herein the mapping of 115 PCR-based orthologous markers, including 110 conserved ortholog set or COSII markers, on the reference RFLP map of eggplant. The result permitted inference of a detailed syntenic relationship between the eggplant and tomato genomes. Further, the position of additional 522 COSII markers was inferred in the eggplant map via eggplant-tomato synteny, bringing the total number of markers in the eggplant genome to 869. Since divergence from their last common ancestor approximately 12 million years ago, the eggplant and tomato genomes have become differentiated by a minimum number of 24 inversions and 5 chromosomal translocations, as well as a number of single gene transpositions possibly triggered by transposable elements. Nevertheless, the two genomes share 37 conserved syntenic segments (CSSs) within which gene/marker order is well preserved. The high-resolution COSII synteny map described herein provides a platform for cross-reference of genetic and genomic information (including the tomato genome sequence) between eggplant and tomato and therefore will facilitate both applied and basic research in eggplant.

Liriodendron tulipifera L. was selected by the Floral Genome Project for identification of new genes related to floral diversity in basal angiosperms. A large, non-normalized cDNA library was constructed from premeiotic and meiotic floral buds and sequenced to generate a database of 9,531 high-quality expressed sequence tags. These sequences clustered into 6,520 unigenes, of which 5,251 were singletons, and 1,269 were in contigs. Homologs of genes regulating many aspects of flower development were identified, including those for organ identity and development, cell and tissue differentiation, and cell-cycle control. Almost 5% of the transcriptome consisted of homologs to known floral gene families. Homologs of most of the genes involved in cell-wall construction were also recovered. This provides a new opportunity for comparative studies in lignin biosynthesis, a trait of key importance in the evolution of land plants and in the utilization of fiber from economically important tree species, such as Liriodendron. Also of note is that 1,089 unigenes did not match any sequence in the public databases, including the complete genomes of Arabidopsis, rice, and Populus. Some of these novel genes might be unique in basal angiosperm species and, when better characterized, may be informative for understanding the origins of diverged gene families. Thus, the Liriodendron expressed sequence tag database and library will help bridge our understanding of the mechanisms of flower initiation and development that are shared among basal angiosperms, eudicots, and monocots, and provide new opportunities for comparative analysis of gene families across angiosperm species.

Quantitative variation is prevalent in nature and in plant breeding. However, it wasn’t until the past two decades that it became technically feasible to begin probing the individual loci and genetic causes of natural, quantitative trait variation. Understanding the basis of quantitative variation is key not only to understanding evolution, but also in developing new insights and methodologies to improve traits such as yield, resistance to abiotic stress, and nutritional quality. In plants, tomato was one of the first species in which significant inroads were made into understanding the genetic and molecular bases of quantitative trait variation. Summarized herein are some of the lessons learned from tomato with regards to quantitative trait variation.

fw2.2 is one of the few QTLs thus far isolated from plants and the first one known to control fruit size. While it has been established that FW2.2 is a regulator (either directly or indirectly) of cell division, FW2.2 does not share sequence homology to any protein of known function (Frary et al. Science 289:85-88, 2000; Cong et al. Proc Natl Acad Sci USA 99:13606-13611, 2002; Liu et al. Plant Physiol 132:292-299, 2003). Thus, the mechanism by which FW2.2 mediates cell division in developing fruit is currently unknown. In an effort to remedy this situation, a combination of yeast two-hybrid screens, in vitro binding assays and cell bombardment studies were performed. The results provide strong evidence that FW2.2 physically interacts at or near the plasma membrane with the regulatory (beta) subunit of a CKII kinase. CKII kinases are well-studied in both yeast and animals where they form part of cell cycle related signaling pathway. Thus while FW2.2 is a plant-specific protein and regulates cell division in a specialized plant organ (fruit), it appears to participate in a cell-cycle control signal transduction pathway that predates the divergence of single- and multi-cellular organisms. These results thus provide a glimpse into how ancient and conserved regulatory processes can be co-opted in the evolution of novel organs such as fruit.

We develop codon-based models for simultaneously inferring the mutational effects of CpG and CpNpG methylation in coding regions. In a data set of 369 tomato genes, we show that there is very little effect of CpNpG methylation but a strong effect of CpG methylation affecting almost all genes. We further show that the CpNpG and CpG effects are largely uncorrelated. Our results suggest different roles of CpG and CpNpG methylation, with CpNpG methylation possibly playing a specialized role in defense against transposons and RNA viruses.

An EST database has been generated for coffee based on sequences from approximately 47,000 cDNA clones derived from five different stages/tissues, with a special focus on developing seeds. When computationally assembled, these sequences correspond to 13,175 unigenes, which were analyzed with respect to functional annotation, expression profile and evolution. Compared with Arabidopsis, the coffee unigenes encode a higher proportion of proteins related to protein modification/turnover and metabolism—an observation that may explain the high diversity of metabolites found in coffee and related species. Several gene families were found to be either expanded or unique to coffee when compared with Arabidopsis. A high proportion of these families encode proteins assigned to functions related to disease resistance. Such families may have expanded and evolved rapidly under the intense pathogen pressure experienced by a tropical, perennial species like coffee. Finally, the coffee gene repertoire was compared with that of Arabidopsis and Solanaceous species (e.g. tomato). Unlike Arabidopsis, tomato has a nearly perfect gene-for-gene match with coffee. These results are consistent with the facts that coffee and tomato have a similar genome size, chromosome karyotype (tomato, n=12; coffee n=11) and chromosome architecture. Moreover, both belong to the Asterid I clade of dicot plant families. Thus, the biology of coffee (family Rubiacaeae) and tomato (family Solanaceae) may be united into one common network of shared discoveries, resources and information.

Transcriptome profiling via cDNA microarray analysis identified 869 genes that are differentially expressed in developing tomato (Solanum lycopersicum) pericarp. Parallel phenotypic and targeted metabolite comparisons were employed to inform the expression analysis. Transcript accumulation in tomato fruit was observed to be extensively coordinated and often completely dependent on ethylene. Mutation of an ethylene receptor (Never-ripe [Nr]), which reduces ethylene sensitivity and inhibits ripening, alters the expression of 37% of these 869 genes. Nr also influences fruit morphology, seed number, ascorbate accumulation, carotenoid biosynthesis, ethylene evolution, and the expression of many genes during fruit maturation, indicating that ethylene governs multiple aspects of development both prior to and during fruit ripening in tomato. Of the 869 genes identified, 628 share homology (E-value ≤1 × 10−10) with known gene products or known protein domains. Of these 628 loci, 72 share homology with previously described signal transduction or transcription factors, suggesting complex regulatory control. These results demonstrate multiple points of ethylene regulatory control during tomato fruit development and provide new insights into the molecular basis of ethylene-mediated ripening.

Transcriptional profiling allows for the assessment and comparison of cross-species gene activity and function on a comprehensive scale. The Solanaceae is a large, diverse dicot family, with well-established genetic relationships between major crop species (tomato, potato, pepper, eggplant, and tobacco). Although Arabidopsis thaliana is often the model of choice for anchoring comparative studies, certain biological processes are better examined in other plants. The ripening of fleshy fruits is not tractable in Arabidopsis; however, it has received considerable attention in tomato. As a member of the Solanaceae, tomato provides a well-characterized system to anchor transcriptional profiles of fruit ripening and development in related species. By utilizing different stages of tomato, pepper, and eggplant fruit, the use of tomato microarrays for expression analysis has been demonstrated in closely related heterologous species, and groups of candidate expressed sequence tags, which are useful as orthologous markers, have been identified, as well as genes implicated in fruit ripening and development in the Solanaceae.

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) × 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 )., National Science Foundation (DBI-0116076), U.S. Department of Agriculture Plant Genome Program (9701552), Binational Agricultural Research and Development Fund (IS-3337-02)

There has recently been increased interest in the use of Markov Chain Monte Carlo (MCMC)-based Bayesian methods for estimating genetic maps. The advantage of these methods is that they can deal accurately with missing data and genotyping errors. Here we present an extension of the previous methods that makes the Bayesian method applicable to large data sets. We present an extensive simulation study examining the statistical properties of the method and comparing it with the likelihood method implemented in Mapmaker. We show that the Maximum A Posteriori (MAP) estimator of the genetic distances, corresponding to the maximum likelihood estimator, performs better than estimators based on the posterior expectation. We also show that while the performance is similar between Mapmaker and the MCMC-based method in the absence of genotyping errors, the MCMC-based method has a distinct advantage in the presence of genotyping errors. A similar advantage of the Bayesian method was not observed for missing data. We also re-analyse a recently published set of data from the eggplant and show that the use of the MCMC-based method leads to smaller estimates of genetic distances.

Comparative genome analysis can be a powerful tool to address questions pertaining to the evolution of the structure of genomes (Paterson and Bennetzen 2001). In plant families that include model taxa, such as the Brassicaceae, the Solanaceae, and the Poaceae, comparative mapping studies allow evolutionary inferences related to the nature and number of rearrangements that distinguish the genomes of the model species from those of their less well-studied relatives (Bonierbale et al. 1988; Ahn and Tanksley 1993; Prince et al. 1993; Chittendan et al. 1994; Kurata et al. 1994; Periera et al. 1994; Lagercrantz and Lydiate 1996; Gale and Devos 1998; Lagercrantz 1998; Doganlar et al. 2002). Not only do such inferences facilitate the transfer of knowledge from model taxa to their relatives, but they also provide insight into the process of genome restructuring that can lead to reproductive isolation and ultimately speciation (Rieseberg 2001; Hall et al. 2002). The larger the number of taxa analyzed within a family, the more comprehensive the information is for inferring the nature of ancestral genomes and the evolutionary history that led to the speciation of the members of that family. The Brassicaceae (or Cruciferae) is a dicot family divided into 13 tribes with a total of 360 genera (Al-Shehbaz 1973) including the agronomically important genus Brassica and the model species Arabidopsis thaliana. The near-complete annotation of the genomic sequence of A. thaliana, including detailed information of physical map position, copy number of genes and intergenic sequences, and location of duplicated chromosomal segments (The Arabidopsis Genome Initiative 2000; Blanc et al. 2000; Vision et al. 2000; Bowers et al. 2003; Ermolaeva et al. 2003; Raes et al. 2003) provides a solid foundation for comparative mapping studies within this family. A. thaliana, prized for its small genome, is actually an anomaly within the tribe Sisymbrieae of the Brassicaceae. A base chromosome number of 8 is inferred to be the ancestral state in the tribe (Koch et al. 1999), and reduction in chromosome number to n = 5–7 is assumed to be a derived state (Koch et al. 1999, 2000). A. thaliana, with its five chromosomes, is the single most extreme example of this genome reduction in the tribe. The nature and rate of occurrence of the chromosomal events that generated the reduced A. thaliana genome from the ancestral n = 8 genome are not well understood. As many as 90 rearrangements are estimated to differentiate Brassica nigra [n = 8] from A. thaliana (Lagercrantz 1998). Two recent mapping studies in which the A. thaliana genome was compared with the Capsella genome (n = 8; Boivin et al. 2004) and with the Arabidopsis lyrata ssp. petraea genome (A. l. petraea; n = 8; Kuittinen et al. 2004), suggest that genome reduction was the result of chromosomal fusion rather than chromosomal elimination. Whatever the nature of these rearrangements, they must have occurred ∼5 Mya, the estimated time of divergence (Koch et al. 2000, 2001; Kuittinen and Aguade 2000) between A. thaliana and its closest known relative A. lyrata (n = 8, genome ∼116%–123% larger than that of A. thaliana; Dart et al. 2004; Schmuths et al. 2004). Further insight into the evolutionary history of the A. thaliana genome is likely to be gained from a three-way comparison of the genomes of A. thaliana and its close relatives, A. lyrata and Capsella spp. In the study reported here, we used the wild, perennial, outcrossing Arabidopsis lyrata subspecies lyrata (hereafter A. l. lyrata; n = 8, diploid; Dart et al. 2004) which occurs in North America (in contrast to the A. l. petraea subspecies which occurs in Europe; Ramos-Onsins et al. 2004), to construct a low-density genetic map with 104 restriction fragment length polymorphism (RFLP) markers distributed on eight linkage groups, and we compared this map to the A. thaliana genome. We then performed comparative statistical analyses of rearrangements observed in A. thaliana versus A. l. lyrata and A. thaliana versus Capsella (based on data from Boivin et al. 2004), using an extension of the Bayesian method reported by Durrett et al. (2004). This method assigns probability estimates to the parsimony solutions as well as alternative scenarios, and is therefore more powerful than parsimony alone to infer the most probable events that underlie genome evolution. Based on these studies, the minimum number of major rearrangements that distinguish A. thaliana from the n = 8 genomes and their rate of occurrence are inferred.

The degree to which stigmas are exserted above the stamen in flowers is a key determinant of cross-pollination (and hence allogamy) in many plant species. Most species in the genus Lycopersicon are obligate or facultative outcrossers and bear flowers with highly exserted stigmas. In contrast, the cultivated tomato (Lycopersicon esculentum) bears flowers with flush or inserted stigmas promoting self-fertilization. It has been observed that a major QTL, se2.1, on chromosome 2 is responsible for a large portion of phenotypic variation for this trait and that mutation(s) at this locus were likely involved in the evolution from allogamy to autogamy in this genus. To understand the genetic and molecular basis of stigma exsertion, we have conducted a high-resolution mapping at the chromosome region harboring the se2.1 QTL. The results indicate that this is a compound locus, comprising at least five tightly linked genes, one controlling style length, three controlling stamen length, and the other affecting anther dehiscence, a taxonomic character used to distinguish Lycopersicon species from other solanaceous species. This cluster of genes may represent the vestiges of an ancient coadapted gene complex in controlling mating behavior.

International audience; Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit.

A large tomato expressed sequence tag (EST) dataset (152 635 total) was analyzed to gain insights into differential gene expression among diverse plant tissues representing a range of developmental programs and biological responses. These ESTs were clustered and assembled to a total of 31 012 unique gene sequences. To better understand tomato gene expression at a plant system level and to identify differentially expressed and tissue-specific genes, we developed and implemented a digital expression analysis protocol. By clustering genes according to their relative abundance in the various EST libraries, expression patterns of genes across various tissues were generated and genes with similar patterns were grouped. In addition, tissues themselves were clustered for relatedness based on relative gene expression as a means of validating the integrity of the EST data as representative of relative gene expression. Arabidopsis and grape EST collections were also characterized to facilitate cross-species comparisons where possible. Tomato fruit digital expression data was specifically compared with publicly available grape EST data to gain insight into molecular manifestation of ripening processes across diverse taxa and resulted in identification of common transcription factors not previously associated with ripening.

In an effort to better understand the dramatic differences in vegetative and floral morphology that differentiate species within the genus Lycopersicon, quantitative trait loci (QTL) for leaflet and perianth size and shape characters were mapped in an interspecific F2 population of tomato (Lycopersicon esculentum x L. pennellii). Thirty-six highly significant (Por = 0.001) QTL were associated with 18 separate traits. QTL for correlated traits were generally not colocalized in the genome unless there was a clear codependence between the traits (e.g., organ length and area). Little or no overlap in QTL positioning between different organs was observed, suggesting that the genes determining the size and shape of leaflets, sepals, and petals are organ specific. Thus, while leaves are considered the developmental and evolutionary precursors to floral organs, genes acting late in development to determine certain aspects of morphology (namely shape and size) must have specialized to exert control over individual organs. Five of the leaflet-trait QTL map to analogous regions in the genome of eggplant, and therefore it appears there has been some conservation in the genes controlling leaf morphology within the Solanaceae.

Despite their unsuitability for agricultural production, the wild relatives of crop species represent a largely untapped resource of novel QTLs potentially useful for crop plant improvement. In this regard, previous introgression studies, involving several different wild tomato species, have shown that the long arm of chromosome 4 contains QTLs for many horticulturally important traits including soluble solids content, fruit shape, lycopene content and biochemical composition. However, these earlier studies were unable to determine how many genes control these traits and whether genes affecting the same character from different wild species are allelic or not. In an effort to shed light on these issues, we have constructed a series of lines containing small, overlapping introgressions for portions of the long arm of chromosome 4 from L. peruvianum and L. hirsutum and tested these lines in replicated field trials. The results provide evidence for multiple, non-allelic loci controlling soluble solids and fruit weight. They also show that the loci controlling some traits (e.g. fruit shape, fruit weight, epidermal reticulation) co-localize to the same portions of chromosome 4, a result that may be attributed to pleiotropy and/or gene dense areas with lower than average recombination. The implications of these finding for molecular breeding and utilization of exotic germplasm are discussed., USDANational Research Initiative Cooperative Grants Program (No.9600558), The Binational Agricultural Research and Development Fund (No. IS-3009-98C)

It has been proposed that fw2.2 encodes a negative fruit-growth regulator that underlies natural fruit-size variation in tomato (Lycopersicon spp.) via heterochronic allelic variation of fw2.2 expression, rather than by variation in the structural protein itself. To further test the negative regulator and the transcriptional control hypotheses, a gene dosage series was constructed, which produced a wider range offw2.2 transcript accumulation than can be found in natural tomato populations. Fruit developmental analyses revealed thatfw2.2 transcript levels were highly correlated (negatively) with fruit mass, supporting the negative regulator and transcriptional regulation hypotheses. Further, the effect offw2.2 on fruit mass was mediated by repressing three- and two-dimensional cell division in placental and pericarp tissues, respectively. Finally, fw2.2 had little effect on fertility and seed size/number, indicating that fruit size effects offw2.2 are due largely to expression in the maternal tissues of developing fruit and not mediated through fertility or seed-setting-related processes.

Although tomato has been the subject of extensive quantitative trait loci (QTLs) mapping experiments, most of this work has been conducted on transient populations (e.g., F2or backcross) and few homozygous, permanent mapping populations are available. To help remedy this situation, we have developed a set of inbred backcross lines (IBLs) from the interspecific cross between Lycopersicon esculentum cv. E6203 and L. pimpinellifolium (LA1589). A total of 170 BC2F1plants were selfed for five generations to create a set of homozygous BC2F6lines by single-seed descent. These lines were then genotyped for 127 marker loci covering the entire tomato genome. These IBLs were evaluated for 22 quantitative traits. In all, 71 significant QTLs were identified, 15% (11/71) of which mapped to the same chromosomal positions as QTLs identified in earlier studies using the same cross. For 48% (34/71) of the detected QTLs, the wild allele was associated with improved agronomic performance. A number of new QTLs were identified including several of significant agronomic importance for tomato production: fruit shape, firmness, fruit color, scar size, seed and flower number, leaf curliness, plant growth, fertility, and flowering time. To improve the utility of the IBL population, a subset of 100 lines giving the most uniform genome coverage and map resolution was selected using a randomized greedy algorithm as implemented in the software package MapPop (http://www.bio.unc.edu/faculty/vision/lab/mappop/). The map, phenotypic data, and seeds for the IBL population are publicly available (http://soldb.cit.cornell.edu) and will provide tomato geneticists and breeders with a genetic resource for mapping, gene discovery, and breeding.Key words: tomato, Lycopersicon esculentum, IBLs, QTL, mapping.

A molecular genetic linkage map based on tomato cDNA, genomic DNA, and EST markers was constructed for eggplant, Solanum melongena. The map consists of 12 linkage groups, spans 1480 cM, and contains 233 markers. Comparison of the eggplant and tomato maps revealed conservation of large tracts of colinear markers, a common feature of genome evolution in the Solanaceae and other plant families. Overall, eggplant and tomato were differentiated by 28 rearrangements, which could be explained by 23 paracentric inversions and five translocations during evolution from the species' last common ancestor. No pericentric inversions were detected. Thus, it appears that paracentric inversion has been the primary mechanism for chromosome evolution in the Solanaceae. Comparison of relative distributions of the types of rearrangements that distinguish pairs of solanaceous species also indicates that the frequency of different chromosomal structural changes was not constant over evolutionary time. On the basis of the number of chromosomal disruptions and an approximate divergence time for Solanum, ∼0.19 rearrangements per chromosome per million years occurred during the evolution of eggplant and tomato from their last ancestor. This result suggests that genomes in Solanaceae, or at least in Solanum, are evolving at a moderate pace compared to other plant species., The U.S. Department of Agriculture National Research Initiative Cooperative Grants Program (no. 96-35300-3646); The Binational Agricultural Research and Development Fund (no. US 2427-94); The National Science Foundation (No. 9872617) to S.D.T.

fw2.2 is a quantitative trait locus responsible for approximately 30% of the difference in fruit size between large, domesticated tomatoes (Lycopersicon esculentum Mill.) and their small-fruited wild relatives. The gene underlying this quantitative trait locus was cloned recently and shown to be associated with altered cell division in ovaries (Frary et al., 2000). However, it was not known whether the change in fruit size is associated with other changes in plant morphology or overall fruit yield—changes that could potentially cause the fruit weight phenotype. To shed light on this issue, a detailed comparison was made between nearly isogenic lines differing for alleles at this locus to search for pleiotropic effects associated with fw2.2. Field observations show that although the small-fruited nearly isogenic line produced smaller ovaries and fruit as expected, this was compensated by a larger number of fruit—due mainly to a significantly greater number of inflorescences—but with no net change in total fruit mass yield. This strongly suggests that fw2.2 may have a pleiotropic effect on how the plant distributes photosynthate among fruit. In a flower removal experiment to control for differences in inflorescence size and number, fruit size remained significantly different between the nearly isogenic lines. These observations indicate that the primary effect of fw2.2 is in controlling ovary and fruit size, and that other associated phenotypic effects are secondary.

Cultivated tomatoes (Lycopersicon esculen- tum) encompass a wide range of fruit size and shape variants. This variation provides the basis for dissecting the genetic and molecular pathways of ovary and fruit development. One fruit shape variant is displayed by the cultivar Sun 1642 (TA491). TA491 has an elongated fruit phenotype, while the wild relative L. pimpinellifolium LA1589 produces fruit that are nearly perfect spheres, a shape typical of wild tomatoes. Developmental studies indicated that the differences in fruit shape between TA491 and LA1589 are determined by events occurring immediately after pollination and extending to 14 days post-pollination. Quantitative trait mapping revealed a single major locus on chromosome 7 (named sun) to be responsible for the differential development of TA491 and LA1589 fruit. Other fruit shape loci characterized in tomato (e.g. fs8.1 and ovate) exert their effects before anthesis and early in ovary development. sun is the first major locus identified in tomato controlling fruit shape through post-pollination events.

Large segmental duplications cover much of the Arabidopsis thaliana genome. Little is known about their origins. We show that they are primarily due to at least four different large-scale duplication events that occurred 100 to 200 million years ago, a formative period in the diversification of the angiosperms. A better understanding of the complex structural history of angiosperm genomes is necessary to make full use of Arabidopsis as a genetic model for other plant species.

Segregation analysis between Lysopersicon esculentum (cultivated tomato) and L. hirsutum (wild form) in conjunction with positional verification by using near-isogenic lines demonstrated that biosynthesis of two structurally different classes of sesquiterpenes in these species is controlled by loci on two different chromosomes. A locus on chromosome 6, Sesquiterpene synthase1 (Sst1), was identified for which the L. esculentum allele is associated with the biosynthesis of beta-caryophyllene and alpha-humulene. At this same locus, the L. hirsutum allele is associated with biosynthesis of germacrene B, germacrene D, and an unidentified sesquiterpene. Genomic mapping, cDNA isolation, and heterologous expression of putative sesquiterpene synthases from both L. esculentum and L. hirsutum revealed that Sst1 is composed of two gene clusters 24 centimorgans apart, Sst1-A and Sst1-B, and that only the genes in the Sst1-A cluster are responsible for accumulation of chromosome 6-associated sesquiterpenes. At a second locus, Sst2, on chromosome 8, the L. hirsutum allele specified accumulation of alpha-santalene, alpha-bergamotene, and beta-bergamotene. Surprisingly, the L. esculentum allele for Sst2 is not associated with the expression of any sesquiterpenes, which suggests that cultivated tomato may have a nonfunctional allele. Sesquiterpene synthase cDNA clones on chromosome 6 do not cross-hybridize on genomic DNA gel blots with putative sesquiterpene synthases on chromosome 8, an indication that the genes in Sst1 and Sst2 are highly diverged, each being responsible for the biosynthesis of structurally different sets of sesquiterpenes.

Historically, linkage mapping populations have consisted of large, randomly selected samples of progeny from a given pedigree or cell lines from a panel of radiation hybrids. We demonstrate that, to construct a map with high genome-wide marker density, it is neither necessary nor desirable to genotype all markers in every individual of a large mapping population. Instead, a reduced sample of individuals bearing complementary recombinational or radiation-induced breakpoints may be selected for genotyping subsequent markers from a large, but sparsely genotyped, mapping population. Choosing such a sample can be reduced to a discrete stochastic optimization problem for which the goal is a sample with breakpoints spaced evenly throughout the genome. We have developed several different methods for selecting such samples and have evaluated their performance on simulated and actual mapping populations, including the Lister and Dean Arabidopsis thaliana recombinant inbred population and the GeneBridge 4 human radiation hybrid panel. Our methods quickly and consistently find much-reduced samples with map resolution approaching that of the larger populations from which they are derived. This approach, which we have termed selective mapping, can facilitate the production of high-quality, high-density genome-wide linkage maps.

Tomato yellow leaf curl virus (TYLCV), a heterogeneous complex of whitefly-vectored geminiviruses, is a serious production constraint of tomato (Lycopersicon esculentum Mill.) in Asia, the Middle East, and the Americas. In this study we report on mapping of a DNA fragment introgressed into cultivated tomato presumably from the wild species L. hirsutum Humb. and Bonpl. and found to be associated with TYLCV resistance. To locate introgressions of wild tomato alleles in TYLCV-resistant tomato line H24, its DNA was digested with six restriction enzymes and probed with 90 RFLP markers evenly spaced throughout the genome. This polymorphism survey revealed the presence of one wild tomato introgression each on chromosomes 8 and 11. Plants of a F2 cross between H24 and a susceptible tomato line were probed with randomly amplified polymorphic DNA (RFLP) markers linked to the targeted regions and F3 families were developed by self-pollination of F2 plants that carried none, one, or both introgressions in either homozygous or heterozygous states. Plants of F3 families, parents, and control tomato line Ty52 (homozygous for the Ty-1 allele for TYLCV tolerance) were exposed to viruliferous whiteflies (Bemisia tabaci Gennadius) in greenhouses at the Asian Vegetable Research and Development Center, Taiwan, and the University of Agricultural Sciences, Bangalore, India. Results indicated that F3 families homozygous for the introgression on chromosome 11 were resistant to TYLCV at both locations. Additional probing showed that the chromosome 11 introgression spanned markers TG36 to TG393, covering a distance of at least 14.6 centimorgans. This is the first report of TYLCV resistance in tomato mapped to chromosome 11.

Carol M. Hamilton1,*,†, Anne Frary1,‡, Yimin Xu1,‡, Steven D. Tanksley2 and Hong-Bin Zhang3 1Plant Science Center, Center for Advanced Technology/ Biotechnology, Cornell University, Ithaca, New York 14853, USA , 2Department of Plant Breeding and Biometry, Cornell University, Ithaca, New York 14853, USA, and 3Department of Crop and Soil Sciences, Texas A & M University, College Station, Texas 77843, USA

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.

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.

Segregation of the Lycopersicon peruvianum genome was followed through three generations of backcrossing to the cultivated tomato L. esculentum cv ‘E6203’ using molecular markers. Thirteen BC1 plants were genotyped with 113 markers, 67 BC2 plants with 84 markers, and finally 241 BC3 plants were genotyped with 177 markers covering the entire genome and a BC3 map constructed. Several segments of the genome, including parts of chromosomes 3, 4, 6, and 10, quickly became fixed for esculentum alleles, possibly due to sterility problems encountered in the BC1. Observed overall heterozygosity and chromosome segment lengths at each generation were very near the expected theoretical values. Markers located near the top telomeric region of chromosome 9 showed segregation highly skewed towards the wild allele through all generations, suggesting the presence of a gamete promoter gene. One markers, TG9, mapped to a new position on chromosome 9, implying an intrachromosomal translocation event. Despite the great genetic distance between the two parents, overall recombination was only 25% less than that observed in a previous tomato cross, indicating that L. peruvianum genes may be more readily introgressed into cultivated germplasm than originally believed.

A BC 1 , population of the self-compatible tomato Lycopersicon esculentum and its wild self-incompatible relative L. hirsutum f. typicum was used for restriction fragment length polymorphism linkage analysis and quantitative trait loci (QTL) mapping of reproductive behavior and floral traits. The self-incompatibility locus, S , on chromosome 1 harbored the only QTL for self-incompatibility indicating that the transition to self-compatibility in the lineage leading to the cultivated tomato was primarily the result of mutations at the S locus. Moreover, the major QTL controlling unilateral incongruity also mapped to the S locus, supporting the hypothesis that self-incompatibility and unilateral incongruity are not independent mechanisms. The mating behavior of near-isogenic lines carrying the L. hirsutum allele for the S locus on chromosome 1 in an otherwise L. esculentum background support these conclusions. The S locus region of chromosome 1 also harbors most major QTL for several floral traits important to pollination biology ( e.g., number and size of flowers), suggesting a gene complex controlling both genetic and morphological mechanisms of reproduction control. Similar associations in other flowering plants suggest that such complex may have been conserved since early periods of plant evolution or else reflect a convergent evolutionary process.

Nearly a century has been spent collecting and preserving genetic diversity in plants. Germplasm banks—living seed collections that serve as repositories of genetic variation—have been established as a source of genes for improving agricultural crops. Genetic linkage maps have made it possible to study the chromosomal locations of genes for improving yield and other complex traits important to agriculture. The tools of genome research may finally unleash the genetic potential of our wild and cultivated germplasm resources for the benefit of society.

A high-resolution physical and genetic map of a major fruit weight quantitative trait locus (QTL),fw2.2, has been constructed for a region of tomato chromosome 2. Using an F2nearly isogenic line mapping population (3472 individuals) derived fromLycopersicon esculentum(domesticated tomato) ×Lycopersicon pennellii(wild tomato),fw2.2has been placed near TG91 and TG167, which have an interval distance of 0.13 ± 0.03 centimorgan. The physical distance between TG91 and TG167 was estimated to be ≤ 150 kb by pulsed-field gel electrophoresis of tomato DNA. A physical contig composed of six yeast artificial chromosomes (YACs) and encompassingfw2.2was isolated. No rearrangements or chimerisms were detected within the YAC contig based on restriction fragment length polymorphism analysis using YAC-end sequences and anchored molecular markers from the high-resolution map. Based on genetic recombination events,fw2.2could be narrowed down to a region less than 150 kb between molecular markers TG91 and HSF24 and included within two YACs: YAC264 (210 kb) and YAC355 (300 kb). This marks the first time, to our knowledge, that a QTL has been mapped with such precision and delimited to a segment of cloned DNA. The fact that the phenotypic effect of thefw2.2QTL can be mapped to a small interval suggests that the action of this QTL is likely due to a single gene. The development of the high-resolution genetic map, in combination with the physical YAC contig, suggests that the gene responsible for this QTL and other QTLs in plants can be isolated using a positional cloning strategy. The cloning offw2.2will likely lead to a better understanding of the molecular biology of fruit development and to the genetic engineering of fruit size characteristics.

The wild Bolivian potato, Solanum berthaultii Hawkes, has been used as a source of resistance to the Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, one of the most significant pests of potato. In this study, two reciprocal backcross S. tuberosum x S. berthaultii potato progenies, BCB and BCT, were mapped with RFLP markers and screened for resistance to CPB consumption, oviposition and defoliation. The genotypic and phenotypic data were combined and analysed to locate quantitative trait loci (QTLs) for resistance to CPB. Three QTLs on three chromosomes in BCB, and two QTLs on two chromosomes in BCT influenced resistance. The QTLs were generally additive but one instance of epistasis was noted. Each QTL accounted for 4-12% of the phenotypic variation observed in resistance. In the more resistant BCB population, a three QTL model explained ca. 20% of the variation in CPB oviposition. When alleles at the three QTLs were homozygous S. berthaultii, oviposition was reduced ca. 60% compared to the heterozygotes. The QTLs for resistance to CPB were compared to those previously identified for the type A and B glandular trichomes, which have been implicated in resistance in the same progenies. Generally, the QTLs for resistance to CPB coincided with loci associated with the glandular trichomes confirming the importance of the glandular trichomes in mediating resistance. However, a relatively strong and consistent QTL for insect resistance in both BCB and BCT on chromosome 1 was observed that was not associated with any trichome traits, suggesting the trichomes may not account for all of the resistance observed in these progenies.

In conjunction with an enhanced system for Agrobacterium-mediated plant transformation, a new binary bacterial artificial chromosome (BIBAC) vector has been developed that is capable of transferring at least 150 kb of foreign DNA into a plant nuclear genome. The transferred DNA appears to be intact in the majority of transformed tobacco plants analyzed and is faithfully inherited in the progeny. The ability to introduce high molecular weight DNA into plant chromosomes should accelerate gene identification and genetic engineering of plants and may lead to new approaches in studies of genome organization.

Estimation of genetic diversity present in gene pools is an important determination for breeding programs. This study was conducted to determine the level of variation in durum wheat cultivars (Triticum turgidum L. var durum) based on different measures. Genetic diversity in durum wheat was measured in a collection of 113 improved cultivars and landraces of diverse ecogeographical origin by means of restriction fragment length polymorphism (RFLP), morphophysiological traits, and coefficient of parentage (COP). Thirty-nine clones and a single restriction enzyme were used to measure the RFLP-based genetic distance. Average taxonomic distances were calculated for the morphophysiological traits evaluated in four location-years. Lower genetic distances were observed for both RFLP and morphophysiological traits for the improved cultivars and for some landraces from Morocco and Jordan, while genetic distances were larger for the rest of the landraces. Patterns of variation for morphophysiological traits were associated with days to heading, plant height, and harvest index. Landraces contained 99% of the total fragments observed in the pool of improved varieties and showed 13% unique fragments. Coefficient of parentage analysis revealed 15 ancestral lines that contributed 72% of the genetic make up of improved cultivars. Thirty-nine different ancestors contributed to the remaining 28%. Correlation of distances based on different measures was higher for average taxonomic distance and Nei's genetic distance (r = 0.47) while COP relationship with the other two measures was lower. Narrower genetic diversity in breeding lines based on the measures used, suggests the utilization of other sources of variation.

An improved procedure is presented to select clones from a tomato yeast artificial chromosome (YAC) library. The procedure is based exlcusively on the polymerase chain reaction (PCR). We combined DNA from approximately 36,000 YAC clones in pools containing 96-single YAC clones from one master plate and further in super pools representing 10 master plates. This pooling strategy allows the selection of single YAC clones homologous to a target sequence after three rounds of PCR using super pools, single pools, and single YAC clones as a template. Single YAC clones were spheroplasted prior to the third PCR round in order to omit the conventional radioactive colony hybridization step. To date, we applied this PCR-based selection strategy to isolate clones homologousto ten different sequence-tagged sites (STS) that are linked to genes targeted for map-based cloning. The selection of YAC clones can be readily accomplished within three days. The PCR-based screening strategy is easy to set up and contributes to a further acceleration of the construction of YAC contigs.

Approximately 170 BC2 plants from a cross between an elite processing inbred (recurrent parent) and the wild species Lycopersicon pimpinellifolium LA1589 (donor parent) were analyzed with segregating molecular markers covering the entire tomato genome. Marker data were used to identify QTLs controlling a battery of horticultural traits measured on BC2F1 and BC3 families derived from the BC2 individuals. Despite its overall inferior appearance, L. pimpinellifolium was shown to possess QTL alleles capable of enhancing most traits important in processing tomato production. QTL-NIL lines, containing specific QTLs modifying fruit size and shape, were subsequently constructed and shown to display the transgressive phenotypes predicted from the original BC2 QTL analysis. The potential of exploiting unadapted and wild germplasm via advanced backcross QTL analysis for the enhancement of elite crop varieties is discussed.

Tomato genomic libraries were screened for the presence of simple sequence repeats (SSRs) with seventeen synthetic oligonucleotide probes, consisting of 2- to 5-basepair motifs repeated in tandem. GAn and GTn sequences were found to occur most frequently in the tomato genome (every 1.2 Mb), followed by ATTn and GCCn (every 1.4 Mb and 1.5 Mb, respectively). In contrast, only ATn and GAn microsatellites (n > 7) were found to be frequent in the GenBank database, suggesting that other motifs may be preferentially located away from genes. Polymorphism of microsatellites was measured by PCR amplification of individual loci or by Southern hybridization, using a set of ten tomato cultivars. Surprisingly, only two of the nine microsatellite clones surveyed (five GTn, three GAn and one ATTn), showed length variation among these accessions. Polymorphism was also very limited betweenLycopersicon esculentum andL. pennelli, two distant species. Southern analysis using the seventeen oligonucleotide probes identified GATAn and GAAAn as useful motifs for the detection of multiple polymorphic fragments among tomato cultivars. To determine the structure of microsatellite loci, a GAn probe was used for hybridization at low stringency on a small insert genomic library, and randomly selected clones were analyzed. GAn based motifs of increasing complexity were found, indicating that simple dinucleotide sequences may have evolved into larger tandem repeats such as minisatellites as a result of basepair substitution, replication slippage, and possibly unequal crossing-over. Finally, we genetically mapped loci corresponding to two amplified microsatellites, as well as nine large hypervariable fragments detected by Southern hybridization with a GATA8 probe. All loci are located around putative tomato centromeres. This may contribute to understanding of the structure of centromeric regions in tomato.