Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing.

We explored genetic variation by sequencing a selection of 84 tomato accessions and related wild species representative of the Lycopersicon, Arcanum, Eriopersicon and Neolycopersicon groups, which has yielded a huge amount of precious data on sequence diversity in the tomato clade. Three new reference genomes were reconstructed to support our comparative genome analyses. Comparative sequence alignment revealed group-, species- and accession-specific polymorphisms, explaining characteristic fruit traits and growth habits in the various cultivars. Using gene models from the annotated Heinz 1706 reference genome, we observed differences in the ratio between non-synonymous and synonymous SNPs (dN/dS) in fruit diversification and plant growth genes compared to a random set of genes, indicating positive selection and differences in selection pressure between crop accessions and wild species. In wild species, the number of single-nucleotide polymorphisms (SNPs) exceeds 10 million, i.e. 20-fold higher than found in most of the crop accessions, indicating dramatic genetic erosion of crop and heirloom tomatoes. In addition, the highest levels of heterozygosity were found for allogamous self-incompatible wild species, while facultative and autogamous self-compatible species display a lower heterozygosity level. Using whole-genome SNP information for maximum-likelihood analysis, we achieved complete tree resolution, whereas maximum-likelihood trees based on SNPs from ten fruit and growth genes show incomplete resolution for the crop accessions, partly due to the effect of heterozygous SNPs. Finally, results suggest that phylogenetic relationships are correlated with habitat, indicating the occurrence of geographical races within these groups, which is of practical importance for Solanum genome evolution studies.

Reverse breeding: a novel breeding approach based on engineered meiosis.

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity. Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome.

The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation.

PsEND1 is a pea anther-specific gene that displays very early expression in the anther primordium cells. Later on, PsEND1 expression becomes restricted to the epidermis, connective, endothecium and middle layer, but it is never observed in tapetal cells or microsporocytes. We fused the PsEND1 promoter region to the cytotoxic barnase gene to induce specific ablation of the cell layers where the PsEND1 is expressed and consequently to produce male-sterile plants. Expression of the chimaeric PsEND1::barnase gene in two Solanaceae (Nicotiana tabacum and Solanum lycopersicon) and two Brassicaceae (Arabidopsis thaliana and Brassica napus) species, impairs anther development from very early stages and produces complete male-sterile plants. The PsEND1::barnase gene is quite different to other chimaeric genes previously used in similar approaches to obtain male-sterile plants. The novelty resides in the use of the PsEND1 promoter, instead of a tapetum-specific promoter, to produce the ablation of specific cell lines during the first steps of the anther development. This chimaeric construct arrests the microsporogenesis before differentiation of the microspore mother cells and no viable pollen grains are produced. This strategy represents an excellent alternative to generate genetically engineered male-sterile plants, which have proved useful in breeding programmes for the production of hybrid seeds. The PsEND1 promoter also has high potential to prevent undesirable horizontal gene flow in many plant species.

Silencing of DELLA induces facultative parthenocarpy in tomato fruits.

DELLA proteins are plant nuclear factors that restrain growth and proliferation in response to hormonal signals. The effects of the manipulation of the DELLA pathway in the making of a berry-like fruit were investigated. The expression of the Arabidopsis thaliana gain-of-function DELLA allele Atgai (del) in tomato (Solanum lycopersicum L.) produced partially sterile dwarf plants and compacted influorescences, as expected for a constitutively activated growth repressor. In contrast, antisense silencing of the single endogenous tomato DELLA gene homologue (SlDELLA) produced slender-like plants with elongated flower trusses. Interestingly, the depletion of SlDELLA in tomato was sufficient to overcome the growth arrest normally imposed on the ovary at anthesis, resulting in parthenocarpic fruits in the absence of pollination. Antisense SlDELLA-engineered fruits were smaller in size and elongated in shape compared with wild type. Cell number estimations showed that fruit set, resulting from reduced SlDELLA expression, arose from activated cell elongation at the longitudinal and lateral axes of the fruit pericarp, bypassing phase-II (post-pollination) cell divisions. Parthenocarpy caused by SlDELLA depletion is facultative, as hand pollination restored wild-type fruit phenotype. This indicates that fertilization-associated SlDELLA-independent signals are operational in ovary-fruit transitions. SlDELLA was also found to restrain growth in other reproductive structures, affecting style elongation, stylar hair primordial growth and stigma development.

Intra- and interspecific variation in DNA content in Cistus (Cistaceae).

Flow cytometry, using propidium iodide and 4',6-diamidano-2-phenylindole staining, was used to estimate the nuclear DNA content (2C) and the proportion of A-T base pairs in 16 species of the Mediterranean genus Cistus. Genome sizes were shown to be constant within species, since no significant intraspecific variation in 2C DNA content was detected. At the genus level, up to about 1.5-fold differences in absolute DNA amounts were observed, ranging from 3.92 pg in C. crispus to 5.88 pg in C. monspeliensis. The (AT) : (GC) ratio was close to 1, and was similar for all species examined, ranging from 47.87% A-T content in C clusii, to 50.67% in C. populifolius. Pink-flowered species (subgenus Cistus) had lower DNA amounts than white-flowered species (subgenera Leucocistus and Halimioides). However, the distribution of DNA amounts in Cistus appeared to be continuous and did not permit a clear separation of infra-generic ranks in the genus.