A comparative study of the genetic bases of natural variation in tomato leaf,sepal, and petal morphology

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 × L. pennellii). Thirty-six highly significant (P0.001) 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.     

Conservation of gene function in the solanaceae as revealed by comparative mapping of domestication traits in eggplant

Quantitative trait loci (QTL) for domestication-related traits were identified in an interspecific F(2) population of eggplant (Solanum linnaeanum x S. melongena). Although 62 quantitative trait loci (QTL) were identified in two locations, most of the dramatic phenotypic differences in fruit weight, shape, color, and plant prickliness that distinguish cultivated eggplant from its wild relative could be attributed to six loci with major effects. Comparison of the genomic locations of the eggplant fruit weight, fruit shape, and color QTL with the positions of similar loci in tomato, potato, and pepper revealed that 40% of the different loci have putative orthologous counterparts in at least one of these other crop species. Overall, the results suggest that domestication of the Solanaceae has been driven by mutations in a very limited number of target loci with major phenotypic effects, that selection pressures were exerted on the same loci despite the crops' independent domestications on different continents, and that the morphological diversity of these four crops can be explained by divergent mutations at these loci.     

Identification,analysis, and utilization of conserved ortholog set markers for comparative genomics in higher plants

We have screened a large tomato EST database against the Arabidopsis genomic sequence and report here the identification of a set of 1025 genes (referred to as a conserved ortholog set, or COS markers) that are single or low copy in both genomes (as determined by computational screens and DNA gel blot hybridization) and that have remained relatively stable in sequence since the early radiation of dicotyledonous plants. These genes were annotated, and a large portion could be assigned to putative functional categories associated with basic metabolic processes, such as energy-generating processes and the biosynthesis and degradation of cellular building blocks. We further demonstrate, through computational screens (e.g., against a Medicago truncatula database) and direct hybridization on genomic DNA of diverse plant species, that these COS markers also are conserved in the genomes of other plant families. Finally, we show that this gene set can be used for comparative mapping studies between highly divergent genomes such as those of tomato and Arabidopsis. This set of COS markers, identified computationally and experimentally, may further studies on comparative genomes and phylogenetics and elucidate the nature of genes conserved throughout plant evolution.     

The making of a bell pepper-shaped tomato fruit: identification of loci controlling fruit morphology in Yellow Stuffer tomato

The heirloom tomato cultivar Yellow Stuffer produces fruit that are similar in shape and structure to fruit produced by the bell pepper varieties of garden pepper. To determine the genetic basis of this extreme fruit type in tomato, quantitative trait loci (QTL) analysis was performed on an F(2) population derived from a cross between Yellow Stuffer and the related species, Lycopersicon pimpinellifolium, which produces a small, round fruit typical of most wild species. F(2) plants were analyzed for both fruit size and the degree to which their fruit resembled the bell pepper. Three QTL were determined to influence bell pepper shape and seven QTL influenced fruit mass. The map positions of all three bell shape and six out of seven fruit size QTL appear to be allelic to components of fruit morphology analyzed in this population and to major fruit morphology QTL reported previously, adding support to the hypothesis that the majority of fruit size and shape variation in cultivated tomato is attributable to allelic variation at a limited number of loci. However, novel loci controlling components of fruit morphology, such as elongated fruit shape, bumpiness, number of seed per fruit and flowers per inflorescence were identified in this study as well. The three bell shape loci involved are: bell2.1, bell2.2 and bell8.1, and appear to correspond to locule number2.1 ( lcn2.1) and fruit weight 2.2 ( fw2.2) and fruit shape 8.1 ( fs8.1), respectively. The Yellow Stuffer alleles at lcn2.1 and fw2.2 increase locule number and fruit size, respectively, hence contributing to the overall bell pepper shape. The Yellow Stuffer allele at fs8.1 causes convex locule walls, giving the extended, bumpy shape characteristic of bell peppers. In addition, most fruit size QTL correspond to loci controlling number of flowers per inflorescence and/or stem-end blockiness. Comparisons among previously identified fruit morphology loci in tomato, eggplant and pepper suggest that loci affecting several aspects of fruit morphology may be due to pleiotrophic effects of the same, orthologous loci in these species. Moreover, it appears that the evolution of bell pepper-shaped tomato fruit may have proceeded through mutations of some of the same genes that led to bell pepper-type fruit in garden pepper.     

Selective mapping: a strategy for optimizing the construction of high-density linkage maps

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.     

Identification of trait-improving quantitative trait loci alleles from a wild rice relative,Oryza rufipogon.

Wild species are valued as a unique source of genetic variation, but they have rarely been used for the genetic improvement of quantitative traits. To identify trait-improving quantitative trait loci (QTL) alleles from exotic species, an accession of Oryza rufipogon, a relative of cultivated rice, was chosen on the basis of a genetic diversity study. An interspecific BC2 testcross population (V20A/O. rufipogon//V20B///V20B////Ce64) consisting of 300 families was evaluated for 12 agronomically important quantitative traits. The O. rufipogon accession was phenotypically inferior for all 12 traits. However, transgressive segregants that outperformed the original elite hybrid variety, V20A/Ce64, were observed for all traits examined. A set of 122 RFLP and microsatellite markers was used to identify QTL. A total of 68 significant QTL were identified, and of these, 35 (51%) had beneficial alleles derived from the phenotypically inferior O. rufipogon parent. Nineteen (54%) of these beneficial QTL alleles were free of deleterious effects on other characters. O. rufipogon alleles at two QTL on chromosomes 1 and 2 were associated with an 18 and 17% increase in grain yield per plant, respectively, without delaying maturity or increasing plant height. This discovery suggests that the innovative use of molecular maps and markers can alter the way geneticists utilize wild and exotic germplasm.     

A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

The interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development.     

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).