EcoTILLING in Capsicum species: searching for new virus resistances

Abstract

BackgroundThe 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.ResultsWe 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 (pvr210, pvr211, pvr212, pvr213 and pvr214) were related to PVY-resistance responses.ConclusionsEcoTILLING 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.

Tilling and Eco-Tilling - A Reverse Genetic Approach for Crop Improvement

“…This technique is now applied to rice, maize, lotus, poplar , Brassica, zebrafish, Drosophila, Caenorhabditis and human , indicating its broad applicability. The implication of ECO-TILLING to identify potential novel functional alleles in the known and candidate genes/transcription factors (TFs) regulating qualitative and quantitative agronomic traits by association/genetic mapping is well-documented for expediting the genetic enhancement of crop plants (Mejlhede et al, 2006;Barkley and Wang, 2008;Ibiza et al, 2010;Negrao et al, 2011;Yu et al, 2012;Frerichmann et al, 2013). ECO-TILLING usually employs a mismatchspecific CEL-I nuclease to cleave the PCR amplified fragments at the site of heteroduplex formation involving nucleotide (SNP-allelic) polymorphism.…”

Biotech Potatoes in the 21st Century: 20 Years Since the First Biotech Potato

“…One such example is the eIF4E gene that has been found to be associated with virus resistance in many plant species (Nicaise et al 2003;Gao et al 2004;Yoshii et al 2004;Kang et al 2005;Kanyuka et al 2005;Stein et al 2005;Nieto et al 2006Nieto et al , 2007Ibiza et al 2010;Naderpour et al 2010;Piron et al 2010), including resistance to potato virus Y in potato, tomato, and pepper (Ruffel et al 2002(Ruffel et al , 2005Cavatorta et al 2011;Duan et al 2012). Variants of eIF4E confer resistance to PVY in the potato wild species relatives S. chacoense, S. demissum, and S. etuberosum , permitting the eventual use of this gene in future biotech potato varieties.…”

Targeted mutation breeding of horticultural plants

“…For clarity in this review, TILLING will refer to detection through mismatchspecific nuclease cleavage, which was used for all TILLING screens shown in Table 1. EcoTILLING is a technically similar approach that has been used to identify natural polymorphisms in target genes of melon, bean, sunflower, and peppers (Nieto et al, 2007;Galeano et al, 2009;Fusari et al, 2010;Ibiza et al, 2010).…”

“…An EcoTILLING analysis of 80 banana accessions identified 870 novel alleles in 14 genes (Till et al, 2010). Variants in eIF4E that conferred virus resistance were discovered by the genetic screening of 113 melon accessions and 233 pepper accessions (Nieto et al, 2007;Ibiza et al, 2010). High-throughput sequencing of eIF4E from 92 tomato lines identified six allelic variants, although virus resistance was not reported (Rigola et al, 2009).…”