Positional cloning of the parthenocarpic fruit (pat) mutant gene and identification of new parthenocarpic sources in tomato (Solanum lycopersicum, L.)
Author(s)
Selleri, Luigi
Date Issued
March 21, 2011
Type
Doctoral Thesis
Abstract
In this work we characterized two parthenocarpic sources in tomato by positionally
cloning the gene responsible of the parthenocarpic fruit (pat) phenotype and by TILLING
the SlIAA9 gene, a major member of the ovary repressor machinery before pollination.
The importance of parthenocarpic mutations is due to their possible use in breeding
programs, as well as in studies aimed to the comprehension of mechanisms underlying the
fruit set. An understanding of the molecular events underlying parthenocarpy, in fact,
would provide information on factors regulating fruit and seed formation, and thus open
new perspectives for yield improvements by biotechnological means.
The pat mutation (Bianchi and Soressi, 1969), induces parthenocarpy with strong
expressivity 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, by
using two segregating populations, on the long arm of chromosome 3 between two
conserved 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 for
the 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 cM
between COSes T0796 and T1143, was refined with new anchor-points and the target
interval for the Pat locus was restricted to less than 0.2 cM between markers named T17
and T20. The small size of the new target region and the recent publication of the tomato
genome sequence (SGN, www.sgn.cornell.edu) allowed us to carry out a candidate gene
approach with the aim to clone the gene responsible for the pat phenotype. Four of the
nine potential candidate genes mapping in the T17-T20 genomic window, were amplified
and sequenced from WT (Chico III) and pat lines. A point mutation in the SlHB15 gene, a
transcriptor factor belonging to the HD-Zip III family, was found and proposed as
responsible for the pat phenotype. In order to confirm this hypothesis a complementation
experiment with the HB15 WT gene will be necessary. HD-Zip III and KANADI, two
antagonistic gene families, were included in a new model that would explain the pat
phenotype and the molecular pathway that triggers the fruit set.
Abstract
2
In the second part of this work, a TILLING approach has been undertaken in order
to identify tomato genotypes carrying mutations in the SlIAA9 coding sequence. As
demonstrated in recent publications, TILLING shows promise as a non-transgenic tool to
improve domesticated crops by introducing and identifying novel genetic variation in genes
that affect key traits. SlIAA9, a member of the Aux/IAA family of transcription factors in
tomato, has been described as playing a major role in the ovary repressor machinery and
plants silenced by antisense showed several IAA-related developmental defects and a
parthenocarpic behavior (Wang et al., 2005). The analysis of M3 families yielded three
lines carrying a genetic lesion in the coding sequence of SlIAA9, two showing a point
mutation leading to amino acidic substitution and the third showing a single-base deletion
leading 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 fruit
or seedlessness). Characterization of the latter line showed severe phenotypes, in
agreement with those expected, that mainly consisted in an obvious loss of leaf
compoundness and parthenocarpy. This observation suggest the interest of this line in
studying the role of the IAA9 transcription factor in reproductive development, although its
partial sterility may hamper its employment in breeding parthenocarpic tomato varieties.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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