BackgroundIn the last decade, the availability of gene sequences of many plant species, including tomato, has encouraged the development of strategies that do not rely on genetic transformation techniques (GMOs) for imparting desired traits in crops. One of these new emerging technology is TILLING (Targeting Induced Local Lesions In Genomes), a reverse genetics tool, which is proving to be very valuable in creating new traits in different crop species.ResultsTo apply TILLING to tomato, a new mutant collection was generated in the genetic background of the processing tomato cultivar Red Setter by treating seeds with two different ethylemethane sulfonate doses (0.7% and 1%). An associated phenotype database, LycoTILL, was developed and a TILLING platform was also established. The interactive and evolving database is available online to the community for phenotypic alteration inquiries. To validate the Red Setter TILLING platform, induced point mutations were searched in 7 tomato genes with the mismatch-specific ENDO1 nuclease. In total 9.5 kb of tomato genome were screened and 66 nucleotide substitutions were identified. The overall mutation density was estimated and it resulted to be 1/322 kb and 1/574 kb for the 1% EMS and 0.7% EMS treatment respectively.ConclusionsThe mutation density estimated in our collection and its comparison with other TILLING populations demonstrate that the Red Setter genetic resource is suitable for use in high-throughput mutation discovery. The Red Setter TILLING platform is open to the research community and is publicly available via web for requesting mutation screening services.

A collection of about 8000 expressed sequence tags (ESTs) deriving from a ÔtotipotentÕ cDNA library constructed in the durum wheat cv. ÔOfantoÕ (Patent n. WO2005003344) were screened to search for simple sequence repeat (SSR) motifs. Five hundred twenty-three SSRs were identified in 460 EST sequences with an average frequency of one SSR per 6.96 kb. Out of 523 microsatellites, 364 were successfully used to design specific primer pairs, while for the remaining ones the available sequence information did not allow the identification of suitable primers. Among the EST-derived SSRs, the trinucleotides were the most frequent (49.1%), followed by dinucleotides (26%) and mononucleotides (21.4%), while motif size of five or six nucleotides were rare, 0.6% and 0.2%, respectively. A total of 125 EST-derived SSRs chosen among di-and tri-nucleotides were further characterized in a panel of 12 tetraploid wheat accessions. The average polymorphism level of the selected SSR was equal to 49.6%. Table S1 contains all the information on primer sequences, chromosome positions and putative function.

Totipotent cDNA libraries representative of all the potentially expressed sequences in a genome would be of great benefit to gene expression studies. Here, we report on an innovative method for creating such a library for durum wheat (Triticum turgidum L. var. durum) and its application for gene discovery. The use of suitable quantities of 5-azacytidine during the germination phase induced the demethylation of total DNA, and the resulting seedlings potentially express all of the genes present in the genome. A new wheat microarray consisting of 4925 unigenes was developed from the totipotent cDNA library and used to screen for genes that may contribute to differences in the disease resistance of two near-isogenic lines, the durum wheat cultivar Latino and the line 5BIL-42, which are respectively susceptible and resistant to powdery mildew. Fluorescently labeled cDNA was prepared from the RNA of seedlings of the two near-isogenic wheat lines after infection with a single powdery mildew isolate under controlled conditions in the greenhouse. Hybridization to the microarray identified six genes that were differently expressed in the two lines. Four of the sequences could be assigned putative functions based on their similarity to known genes in public databases. Physical mapping of the six genes localized them to two regions of the genome: the centromeric region of chromosome 5B, where the Pm36 resistance gene was previously localized, and chromosome 6B.

No abstract