Authors: YILDIZ KAÇAR, ÖZHAN ŞİMŞEK, DİCLE DÖNMEZ, MELDA BONCUK, TURGUT YEŞİLOĞLU, PATRICK OLLITRAULT
Abstract: Iron is one of the most important elements in plant mineral nutrition. Fe deficiency is a critical abiotic stress factor for Mediterranean citriculture; the development of marker-assisted selection for this trait would greatly enhance rootstock breeding. In this study, DNA sequencing and single-stranded conformation polymorphism (SSCP) analyses were performed to determine the allelic diversity of genes associated with tolerance to iron chlorosis in citrus. Two candidate iron chlorosis tolerance genes were selected from existing Citrus EST databases and Arabidopsis thaliana genome databases. Ferritin-3 chloroplast precursor and putative membrane transporter candidate gene sequences were used to define primers in conserved regions. Six citrus genotypes from the basic taxon of Citrus were used to identify polymorphic regions in the genes. Direct sequencing of the amplified DNA fragments from the candidate genes was performed, and single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified after sequence alignment. Based on the DNA sequencing analysis, a total of 6840 nucleotides of DNA were sequenced to identify SNPs and indels. In total, 263 SNPs and 15 indels were identified for both genes. We detected 38.45 SNPs and 2.19 indels for each 1000 b on average from the DNA sequencing results. New primers were designed in conserved areas flanking polymorphic ones for SSCP analysis. SSCP-PCR analysis was performed with 25 citrus genotypes. The neighbor-joining method was used for cluster analysis. Trifoliate genotypes and their hybrids (known to be sensitive to iron chlorosis) clustered together, whereas genotypes tolerant to iron chlorosis were more spread out on the dendrogram. Mandarins also showed high diversity for both genes according to SSCP results. Differences were found among sour orange genotypes known to have differential tolerance behavior to iron chlorosis.
Keywords: Single-stranded conformation polymorphism, DNA sequencing, polymorphism, DNA
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