Abstract
Although the availability of genetic and genomic resources for Cucurbita pepo has increased significantly, functional genomic resources are still limited for this crop. In this direction, we have developed a high throughput reverse genetic tool: the first TILLING (Targeting Induced Local Lesions IN Genomes) resource for this species. Additionally, we have used this resource to demonstrate that the previous EMS mutant population we developed has the highest mutation density compared with other cucurbits mutant populations. The overall mutation density in this first C. pepo TILLING platform was estimated to be 1/133 Kb by screening five additional genes. In total, 58 mutations confirmed by sequencing were identified in the five targeted genes, thirteen of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was studied in a peroxidase gene, revealing that the phenotype of seedling homozygous for one of the isolated mutant alleles was albino. These results indicate that the TILLING approach in this species was successful at providing new mutations and can address the major challenge of linking sequence information to biological function and also the identification of novel variation for crop breeding.
Highlights
Cucurbita pepo is the main species of the genus Cucurbita and represents one of the most important vegetable crops worldwide in terms of food consumption
The TILLING platform validated here is the first developed in Cucurbita pepo, probably because the low fertility that the mutants of this species have shown in previous attempts, even using different varieties [32], has hampered the perpetuation of the populations
The density of mutations that we discovered in this assay appears to be much higher than the obtained for other cucurbit species such as Cucumis melo [13], [16] or Cucumis sativus [17] and if we compare this with other species, we find only five populations of diploid species with higher mutation densities: Arabidopsis thaliana [21], Brassica rapa [33], Oryza sativa [34], Linum usitatissimum [35] and a diploid wheat (Triticum monococcum) [36]
Summary
Cucurbita pepo is the main species of the genus Cucurbita and represents one of the most important vegetable crops worldwide in terms of food consumption. Of the eight existing morphotypes in this species, the morphotype Zucchini is the most economically valuable It was recently developed and displays low levels of genetic variation [1] but it is commonly cultivated. Apart from the intraspecific resources, grouped in three subespecies, only a few crossable Cucurbita species can be used as sources of agriculturally useful alleles and new genotypes. In this way, the development of new genetic resources along with the application of new molecular techniques in breeding would facilitate the improvement of characters, mainly those with more complex regulation, allowing breeders to face new challenges in this species
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