Positional cloning of the parthenocarpic fruit (pat) mutant gene and identification of new parthenocarpic sources in tomato (Solanum lycopersicum, L.)

摘要

In this work we characterized two parthenocarpic sources in tomato by positionallycloning the gene responsible of the parthenocarpic fruit (pat) phenotype and by TILLINGthe SlIAA9 gene, a major member of the ovary repressor machinery before pollination.The importance of parthenocarpic mutations is due to their possible use in breedingprograms, as well as in studies aimed to the comprehension of mechanisms underlying thefruit set. An understanding of the molecular events underlying parthenocarpy, in fact,would provide information on factors regulating fruit and seed formation, and thus opennew perspectives for yield improvements by biotechnological means.The pat mutation (Bianchi and Soressi, 1969), induces parthenocarpy with strongexpressivity along with other pleiotropic effects such as short anthers and aberrant ovules(Mazzucato et al., 1998). Through Bulk Segregant Analysis the Pat gene was mapped, byusing two segregating populations, on the long arm of chromosome 3 between twoconserved ortholog set (COS) markers (COSes; Fulton et al., 2002), T0796 and T1143(Beraldi et al., 2004), previously anchored on the genetic tomato map (EXPEN 2000,www.sgn.cornell.edu).We developed and mapped novel PCR-derived COS markers inside the target window forthe Pat gene by pursuing the microsynteny between tomato and Arabidopsis (Fulton et al.,2002). Through genetic and physical mapping, the genetic region spanning 1.2 cMbetween COSes T0796 and T1143, was refined with new anchor-points and the targetinterval for the Pat locus was restricted to less than 0.2 cM between markers named T17and T20. The small size of the new target region and the recent publication of the tomatogenome sequence (SGN, www.sgn.cornell.edu) allowed us to carry out a candidate geneapproach with the aim to clone the gene responsible for the pat phenotype. Four of thenine potential candidate genes mapping in the T17-T20 genomic window, were amplifiedand sequenced from WT (Chico III) and pat lines. A point mutation in the SlHB15 gene, atranscriptor factor belonging to the HD-Zip III family, was found and proposed asresponsible for the pat phenotype. In order to confirm this hypothesis a complementationexperiment with the HB15 WT gene will be necessary. HD-Zip III and KANADI, twoantagonistic gene families, were included in a new model that would explain the patphenotype and the molecular pathway that triggers the fruit set.Abstract2In the second part of this work, a TILLING approach has been undertaken in orderto identify tomato genotypes carrying mutations in the SlIAA9 coding sequence. Asdemonstrated in recent publications, TILLING shows promise as a non-transgenic tool toimprove domesticated crops by introducing and identifying novel genetic variation in genesthat affect key traits. SlIAA9, a member of the Aux/IAA family of transcription factors intomato, has been described as playing a major role in the ovary repressor machinery andplants silenced by antisense showed several IAA-related developmental defects and aparthenocarpic behavior (Wang et al., 2005). The analysis of M3 families yielded threelines carrying a genetic lesion in the coding sequence of SlIAA9, two showing a pointmutation leading to amino acidic substitution and the third showing a single-base deletionleading to a frame-shift and a premature stop codon. Characterization of the former lines,showed some of the expected phenotypes, albeit with low penetrance and expressivity(occurrence of polycots, abnormal growth of axillary shoots, low number of seeds per fruitor seedlessness). Characterization of the latter line showed severe phenotypes, inagreement with those expected, that mainly consisted in an obvious loss of leafcompoundness and parthenocarpy. This observation suggest the interest of this line instudying the role of the IAA9 transcription factor in reproductive development, although itspartial sterility may hamper its employment in breeding parthenocarpic tomato varieties.