title: 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.
authors.affiliation: Instituto de Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), Universidad Politécnica de Valencia, 8E CPI, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Vicente P
authors.initials: VP
authors.lastname: Ibiza
authors.forename: Joaquín
authors.initials: J
authors.lastname: Cañizares
authors.forename: Fernando
authors.initials: F
authors.lastname: Nuez
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002212:Capsicum / Q000235:genetics* / Q000276:immunology / Q000821:virology*; D018076:DNA, Complementary / Q000235:genetics; D017753:Ecosystem; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics; D006239:Haplotypes / Q000235:genetics; D007113:Immunity, Innate / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D010935:Plant Diseases / Q000235:genetics / Q000276:immunology* / Q000821:virology*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics; D017800:Potyvirus / Q000302:isolation & purification / Q000502:physiology*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013045:Species Specificity
chemical_list: D018076:DNA, Complementary; D039561:Eukaryotic Initiation Factor-4E; D010940:Plant Proteins