Determination by SSCP markers of the allelic diversity of candidate genes for tolerance to iron chlorosis in Citrus germplasm

Iron chlorosis is one of the main abiotic constraints for Mediterranean citriculture and the development of marker assisted selection (MAS) for this trait would be a great aid for rootstock breeding. We performed SSCP (Single Stranded Conformation Polymorphism) analysis in order to discover allelic diversity of candidate genes for iron chlorosis tolerance in citrus. Two iron chlorosis tolerance candidate genes were selected from existing Citrus ESTs databases and Arabidopsis thaliana genome databases. Iron-Sulfur assembly protein (Fe_S=AT2G16710) and root iron transporter (IRT1=AT4G19690) candidate gene sequences were used to define primers in conserved regions. Six Citrus genotypes from the basic taxon of Citrus where used to identify polymorphic areas in the genes. Direct sequencing of amplified DNA fragments of candidate genes was performed and SNPs (single-nucleotide polymorphisms) and Indels where searched after sequence alignment. A total of 2215 bp DNA fragments were sequenced and 56 SNPs and 2 Indels were determined. New primers were defined, in conserved areas flanking polymorphic ones, for Single Strand Conformation Polymorphism (SSCP) analysis for further diversity and routine genotyping. Two pairs of primers were defined for each gene. SSCP-PCR analysis was performed with twenty-five Citrus genotypes. The neighbor joining method was used for cluster analysis. Poncirus trifoliate genotypes and their hybrids known to be sensitive to iron chlorosis clustered together and mandarins showed high diversity for both genes. Differences were found among sour orange genotypes known to have differential tolerance behavior to iron chlorosis.