Dataset Information

First TILLING platform in Cucurbita pepo: a new mutant resource for gene function and crop improvement.

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.

SUBMITTER: Vicente-Dolera N

PROVIDER: S-EPMC4227871 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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First TILLING platform in Cucurbita pepo: a new mutant resource for gene function and crop improvement.

Vicente-Dólera Nelly N Troadec Christelle C Moya Manuel M del Río-Celestino Mercedes M Pomares-Viciana Teresa T Bendahmane Abdelhafid A Picó Belén B Román Belén B Gómez Pedro P

PloS one 20141111 11

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 cu ...[more]

PMID: 25386735

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Project description:The Cucurbita pepo genome comprises 263 Mb and 34,240 gene models organized in 20 different chromosomes. To improve our understanding of gene function we have generated an EMS mutant platform, consisting of 3,751 independent M2 families. The quality of the collection has been evaluated based on phenotyping and whole-genome re-sequencing (WGS) results. The phenotypic evaluation of the whole platform at seedling stage has demonstrated that the rate of variation for easily observable traits is more than 10%. The percentage of families with albino or chlorotic seedlings exceeded 3%, similar or higher to that found in other EMS collections of cucurbit crops. A rapid screening of the library for triple ethylene response in etiolated seedlings allowed the identification of four ethylene-insensitive mutants, that were found to be semidominant (ein1, ein2, and ein3) or dominant (EIN4). By evaluating 4 adult plants from 300 independent families more than 28% of apparent mutations were found for vegetative and reproductive traits, including plant vigor, leaf size and shape, sex expression and sex determination, and fruit set and development. Two pools of genomic DNA derived from 20 plants of two mutant families were subjected to WGS by using NGS methodology, estimating the density, spectrum, distribution and impact of EMS induced mutation. The number of EMS mutations in the genomes of families L1 and L2 was 1,704 and 859, respectively, which represents a density of 11.8 and 6 mutations per Mb, respectively. As expected, the predominant EMS induced mutations were C > T and G > A transitions (80.3% in L1, and 61% L2), that were found to be randomly distributed along the 20 chromosomes of C. pepo. The mutations were mostly affecting intergenic regions, but 7.9 and 6% of the identified EMS mutations in L1 and L2, respectively, were located in the exome, and 0.4 and 0.2% had a moderate and high putative impact on gene functions. These results provide information regarding the potential use of the obtained mutant platform in the discovery of novel alleles for both functional genomics and Cucurbita breeding by using direct- or reverse-genetic approaches.

Project description:Cotton is cultivated worldwide for its white fiber, of which around 90% is tetraploid upland cotton (Gossypium hirsutum L.) carrying both A and D genome. Since centuries, yield increasing efforts for the cotton crop by conventional breeding approaches have caused an extensive erosion of natural genetic variability. Mutation based improvement strategies provide an effective way of creating new allelic variations. Targeting Induced Local Lesions IN Genomes (TILLING) provides a mutation based reverse genetic strategy to create and evaluate induced genetic variability at DNA level. Here, we report development and testing of TILLING populations of allotetraploid cotton (G. hirsutum) for functional genomic studies and mutation based enrichment of cotton genetic resources. Seed of two cotton cultivars "PB-899 and PB-900" were mutagenized with 0.3 and 0.2% (v/v) ethyl methanesulfonate, respectively. The phenotyping of M1 and M2 populations presented numerous mutants regarding the branching pattern, leaf morphology, disease resistance, photosynthetic lesions and flower sterility. Molecular screening for point mutations was performed by TILLING PCR aided CEL1 mismatch cleavage. To estimate the mutation frequency in the mutant genomes, five gene classes were TILLed in 8000 M2 plants of each var. "PB-899" and "PB-900." These include actin (GhACT), Pectin Methyl Esterase (GhPME), sucrose synthase (GhSUS), resistance gene analog, and defense response gene (DRGs). The var. PB-899 was harboring 47% higher mutation induction rate than PB-900. The highest rate of mutation frequency was identified for NAC-TF5 (EU706348) of DRGs class, ranging from 1/58 kb in PB-899 to 1/105 kb in PB-900. The mutation screening assay revealed the presence of significant proportion of induced mutations in cotton TILLING populations such as 1/153 kb and 1/326 kb in var. "PB-899" and "PB-900," respectively. The establishment of a cotton TILLING platform (COTIP) and data obtained from the resource TILLING population suggest its effectiveness in widening the genetic bases of cotton for improvement and utilizing it for subsequent reverse genetic studies of various genes.

Dataset Information

First TILLING platform in Cucurbita pepo: a new mutant resource for gene function and crop improvement.

Publications

First TILLING platform in Cucurbita pepo: a new mutant resource for gene function and crop improvement.

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