Population structure and genetic differentiation associated with breeding history and selection in tomato (Solanum lycopersicum L.).

Tomato (Solanum lycopersicum L.) has undergone intensive selection during and following domestication. We investigated population structure and genetic differentiation within a collection of 70 tomato lines representing contemporary (processing and fresh-market) varieties, vintage varieties and landraces. The model-based Bayesian clustering software, STRUCTURE, was used to detect subpopulations. Six independent analyses were conducted using all marker data (173 markers) and five subsets of markers based on marker type (single-nucleotide polymorphisms, simple sequence repeats and insertion/deletions) and location (exon and intron sequences) within genes. All of these analyses consistently separated four groups predefined by market niche and age into distinct subpopulations. Furthermore, we detected at least two subpopulations within the processing varieties. These subpopulations correspond to historical patterns of breeding conducted for specific production environments. We found no subpopulation within fresh-market varieties, vintage varieties and landraces when using all marker data. High levels of admixture were shown in several varieties representing a transition in the demarcation between processing and fresh-market breeding. The genetic clustering detected by using the STRUCTURE software was confirmed by two statistics, pairwise F(st) (θ) and Nei's standard genetic distance. We also identified a total of 19 loci under positive selection between processing, fresh-market and vintage germplasm by using an F(st)-outlier method based on the deviation from the expected distribution of F(st) and heterozygosity. The markers and genome locations we identified are consistent with known patterns of selection and linkage to traits that differentiate the market classes. These results demonstrate how human selection through breeding has shaped genetic variation within cultivated tomato.

Construction of two BAC libraries from cucumber (Cucumis sativus L.) and identification of clones linked to yield component quantitative trait loci.

Two bacterial artificial chromosome (BAC) libraries were constructed from an inbred line derived from a cultivar of cucumber (Cucumis sativus L.). Intact nuclei were isolated and embedded in agarose plugs, and high-molecular-weight DNA was subsequently partially digested with BamHI or EcoRI. Ligation of double size-selected DNA fragments with the pECBAC1 vector yielded two libraries containing 23,040 BamHI and 18,432 EcoRI clones. The average BamHI and EcoRI insert sizes were estimated to be 107.0 kb and 100.8 kb, respectively, and BAC clones lacking inserts were 1.3% and 14.5% in the BamHI and EcoRI libraries, respectively. The two libraries together represent approximately 10.8 haploid cucumber genomes. Hybridization with a C(0)t-1 DNA probe revealed that approximately 36% of BAC clones likely carried repetitive sequence-enriched DNA. The frequencies of BAC clones that carry chloroplast or mitochondrial DNA range from 0.20% to 0.47%. Four sequence-characterized amplified region (SCAR), four simple sequence repeat, and an randomly amplified polymorphic DNA marker linked with yield component quantitative trait loci were used either as probes to hybridize high-density colony filters prepared from both libraries or as primers to screen an ordered array of pooled BAC DNA prepared from the BamHI library. Positive BAC clones were identified in predicted numbers, as screening by polymerase chain reaction amplification effectively overcame the problems associated with an overabundance of positives from hybridization with two SCAR markers. The BAC clones identified herein that are linked to the de (determinate habit) and F (gynoecy) locus will be useful for positional cloning of these economically important genes. These BAC libraries will also facilitate physical mapping of the cucumber genome and comparative genome analyses with other plant species.

Mechanism of escape of endogenous murine leukemia virus emv-14 from recognition by anti-AKR/Gross virus cytolytic T lymphocytes.

It was previously shown that spleen cells from endogenous ecotropic murine leukemia virus emv-14+ AKXL-5 mice fail to stimulate an anti-AKR/Gross virus cytolytic T-lymphocyte (CTL) response in a mixed lymphocyte culture with primed C57BL/6 responder spleen cells, whereas spleen cells from AKXL strains carrying the very similar emv-11 provirus do stimulate a response (Green and Graziano, Immunogenetics 23:106-110, 1986). We wished to determine whether the lack of response with AKXL-5 spleen cells was at the level of recognition between effector cell and target cell and whether the relevant mutation was within the emv-14 provirus. It is shown here that EMV-negative SC-1 fibroblast cells transfected with the major histocompatibility complex class I Kb gene and infected with virus isolated from the AKXL-5 strain (SC.Kb/5 cells) were not lysed by H-2b-restricted anti-AKR/Gross virus CTL. SC.Kb cells infected with virus isolated from emv-11+ strains, however, were efficiently lysed by anti-AKR/Gross virus CTL, indicating that there is nothing intrinsic to EMV-infected SC.Kb cells that would prevent them from being recognized and lysed efficiently by anti-AKR/Gross virus CTL. Analysis of virus expression for the infected SC.Kb cells by XC plaque assay and by flow cytometry indicated that emv-14 virus expression for SC.Kb/5 cells was not significantly different from that for emv-11-containing SC.Kb/9 or SC.Kb/21 cells. These data show that the mutation responsible for the lack of CTL recognition and lysis is at the level of recognition between target cell and effector cell. Furthermore, these data strongly suggest that the mutation is within the emv-14 genome. Flow cytometry experiments with monoclonal antibodies against a number of viral determinants indicated that there was no gross mutation detectable in the viral determinants analyzed. The data suggest that the relevant mutation may be a point mutation or a small insertion or deletion within a coding sequence that is critical for CTL recognition.

The CD28 and CTLA-4 receptors associate with the serine/threonine phosphatase PP2A.

CD28 and CTLA-4 are related members of a family of T lymphocyte cell surface receptors that function to regulate T cell activation. We have found that the cytoplasmic domains of both CTLA-4 and CD28 can associate with members of the PP2A family of serine/threonine phosphatases. The association of PP2A with CD28 was negatively regulated by tyrosine phosphorylation of the CD28 cytoplasmic domain. Inhibition of PP2A activity in Jurkat leukemia T cells by treatment with okadaic acid or by expression of a dominant-negative mutant enhanced T cell activation induced by CD28 engagement. Interactions between cell surface receptors such as CTLA-4 and CD28 and serine/threonine phosphatases may represent a novel mechanism for modulating the intracellular signal transduction pathways associated with cell activation.

Regulation of cytotoxic T lymphocyte-associated molecule-4 by Src kinases.

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a cell surface receptor expressed on activated T cells that can inhibit T cell responses induced by activation of the TCR and CD28. Studies with phosphorylated peptides based on the CTLA-4 intracellular domain have suggested that tyrosine phosphorylation of CTLA-4 may regulate its interactions with cytoplasmic proteins that could determine its intracellular trafficking and/or signal transduction. However, the kinase(s) that phosphorylate CTLA-4 remain uncharacterized. In this report, we show that CTLA-4 can associate with the Src kinases Fyn and Lck and that transfection of Fyn or Lck, but not the unrelated kinase ZAP70, can induce tyrosine phosphorylation of CTLA-4 on residues Y201 and Y218. A similar pattern of tyrosine phosphorylation was found in pervanadate-treated Jurkat T cells stably expressing CTLA-4. Phosphorylation of CTLA-4 Y201 in Jurkat cells correlated with cell surface accumulation of CTLA-4. CTLA-4 phosphorylation induced the association of CTLA-4 with the tyrosine phosphatase SHP-2, but not with phosphatidylinositol 3-kinase. In contrast, Lck-induced phosphorylation of CD28 resulted in the recruitment of phosphatidylinositol 3-kinase, but not SHP-2. These findings suggest that phosphorylation of CD28 and CTLA-4 by Lck activates distinct intracellular signaling pathways. The association of CTLA-4 with Src kinases and with SHP-2 results in the formation of a CTLA-4 complex with the potential to regulate T cell activation.