Dataset Information

EcoTILLING in Capsicum species: searching for new virus resistances.

ABSTRACT: BACKGROUND: The EcoTILLING technique allows polymorphisms in target genes of natural populations to be quickly analysed or identified and facilitates the screening of genebank collections for desired traits. We have developed an EcoTILLING platform to exploit Capsicum genetic resources. A perfect example of the utility of this EcoTILLING platform is its application in searching for new virus-resistant alleles in Capsicum genus. Mutations in translation initiation factors (eIF4E, eIF(iso)4E, eIF4G and eIF(iso)4G) break the cycle of several RNA viruses without affecting the plant life cycle, which makes these genes potential targets to screen for resistant germplasm. RESULTS: We developed and assayed a cDNA-based EcoTILLING platform with 233 cultivated accessions of the genus Capsicum. High variability in the coding sequences of the eIF4E and eIF(iso)4E genes was detected using the cDNA platform. After sequencing, 36 nucleotide changes were detected in the CDS of eIF4E and 26 in eIF(iso)4E. A total of 21 eIF4E haplotypes and 15 eIF(iso)4E haplotypes were identified. To evaluate the functional relevance of this variability, 31 possible eIF4E/eIF(iso)4E combinations were tested against Potato virus Y. The results showed that five new eIF4E variants (pvr2(10), pvr2(11), pvr2(12), pvr2(13) and pvr2(14)) were related to PVY-resistance responses. CONCLUSIONS: EcoTILLING was optimised in different Capsicum species to detect allelic variants of target genes. This work is the first to use cDNA instead of genomic DNA in EcoTILLING. This approach avoids intronic sequence problems and reduces the number of reactions. A high level of polymorphism has been identified for initiation factors, showing the high genetic variability present in our collection and its potential use for other traits, such as genes related to biotic or abiotic stresses, quality or production. Moreover, the new eIF4E and eIF(iso)4E alleles are an excellent collection for searching for new resistance against other RNA viruses.

SUBMITTER: Ibiza VP

PROVIDER: S-EPMC3091766 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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EcoTILLING in Capsicum species: searching for new virus resistances.

Ibiza Vicente P VP Cañizares Joaquín J Nuez Fernando F

BMC genomics 20101112

<h4>Background</h4>The EcoTILLING technique allows polymorphisms in target genes of natural populations to be quickly analysed or identified and facilitates the screening of genebank collections for desired traits. We have developed an EcoTILLING platform to exploit Capsicum genetic resources. A perfect example of the utility of this EcoTILLING platform is its application in searching for new virus-resistant alleles in Capsicum genus. Mutations in translation initiation factors (eIF4E, eIF(iso)4 ...[more]

PMID: 21073716

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Project description:BackgroundInformation on polymorphic DNA in organelle genomes is essential for evolutionary and ecological studies. However, it is challenging to perform high-throughput investigations of chloroplast and mitochondrial DNA polymorphisms. In recent years, EcoTILLING stands out as one of the most universal, low-cost, and high-throughput reverse genetic methods, and the identification of natural genetic variants can provide much information about gene function, association mapping and linkage disequilibrium analysis and species evolution. Until now, no report exists on whether this method is applicable to organelle genomes and to what extent it can be used.Methodology/principal findingsTo address this problem, we adapted the CEL I-based heteroduplex cleavage strategy used in Targeting Induced Local Lesions in Genomes (TILLING) for the discovery of nucleotide polymorphisms in organelle genomes. To assess the applicability and accuracy of this technology, designated ORG-EcoTILLING, at different taxonomic levels, we sampled two sets of taxa representing accessions from the Brassicaceae with three chloroplast genes (accD, matK and rbcL) and one mitochondrial gene (atp6). The method successfully detected nine, six and one mutation sites in the accD, matK and rbcL genes, respectively, in 96 Brassica accessions. These mutations were confirmed by DNA sequencing, with 100% accuracy at both inter- and intraspecific levels. We also detected 44 putative mutations in accD in 91 accessions from 45 species and 29 genera of seven tribes. Compared with DNA sequencing results, the false negative rate was 36%. However, 17 SNPs detected in atp6 were completely identical to the sequencing results.Conclusions/significanceThese results suggest that ORG-EcoTILLING is a powerful and cost-effective alternative method for high-throughput genome-wide assessment of inter- and intraspecific chloroplast and mitochondrial DNA polymorphisms. It will play an important role in evolutionary and ecological biology studies, in identification of related genes associated with agronomic importance such as high yield and improved cytoplasmic quality, and for identifying mitochondrial point mutations responsible for diseases in humans and other animals.

Project description:BackgroundCapsicum chlorosis virus (CaCV) is an emerging pathogen of capsicum, tomato and peanut crops in Australia and South-East Asia. Commercial capsicum cultivars with CaCV resistance are not yet available, but CaCV resistance identified in Capsicum chinense is being introgressed into commercial Bell capsicum. However, our knowledge of the molecular mechanisms leading to the resistance response to CaCV infection is limited. Therefore, transcriptome and expression profiling data provide an important resource to better understand CaCV resistance mechanisms.Methodology/principal findingsWe assembled capsicum transcriptomes and analysed gene expression using Illumina HiSeq platform combined with a tag-based digital gene expression system. Total RNA extracted from CaCV/mock inoculated CaCV resistant (R) and susceptible (S) capsicum at the time point when R line showed a strong hypersensitive response to CaCV infection was used in transcriptome assembly. Gene expression profiles of R and S capsicum in CaCV- and buffer-inoculated conditions were compared. None of the genes were differentially expressed (DE) between R and S cultivars when mock-inoculated, while 2484 genes were DE when inoculated with CaCV. Functional classification revealed that the most highly up-regulated DE genes in R capsicum included pathogenesis-related genes, cell death-associated genes, genes associated with hormone-mediated signalling pathways and genes encoding enzymes involved in synthesis of defense-related secondary metabolites. We selected 15 genes to confirm DE expression levels by real-time quantitative PCR.Conclusion/significanceDE transcript profiling data provided comprehensive gene expression information to gain an understanding of the underlying CaCV resistance mechanisms. Further, we identified candidate CaCV resistance genes in the CaCV-resistant C. annuum x C. chinense breeding line. This knowledge will be useful in future for fine mapping of the CaCV resistance locus and potential genetic engineering of resistance into CaCV-susceptible crops.

Dataset Information

EcoTILLING in Capsicum species: searching for new virus resistances.

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EcoTILLING in Capsicum species: searching for new virus resistances.

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