Oak ribosomal DNA: characterization by FISH and polymorphism assessed by IGS PCR–RFLP

Abstract

Oaks taxonomy has been revised over the years, demanding expeditious and cost-effective tools for DNA fingerprinting and taxonomic discrimination. We focused on the characterization of the ribosomal DNA (rDNA) of 22 Quercus species, belonging to four infrageneric groups, using silver nitrate staining, fluorescence in situ hybridization (FISH) and IGS PCR–RFLP markers (produced by the digestion of the rDNA intergenic spacer, IGS). A chromosome complement of 2n = 2x = 24 was confirmed in 21 species, and ascribed to Quercus phellos. Silver nitrate staining detected one or two nucleoli per nucleus, and two nucleolar organizer regions (NORs) per prometaphase cells. FISH performed with rDNA probes revealed two 5S and four 35S rDNA loci in nine species and confirmed their location in the remaining. Since no cytogenetic polymorphisms were detected, the IGS was studied. A single amplicon with ca. 2 kb was amplified in all oaks, and digested with six restriction enzymes, that produced a total of 125 IGS PCR–RFLP fragments with 99.2 % of polymorphism. A monomorphic HaeIII fragment with 225 bp was found. Among the infrageneric groups, 11 monomorphic bands were observed and 6 were considered group-specific. Based on the pool of molecular data different genetic analyses were performed. The UPGMA dendrogram clustered most of the oaks per infrageneric group, and was corroborated by the principal coordinates analysis (PCoA). The genetic structure also matched the assumed taxonomy. Globally, IGS PCR–RFLP proved their usefulness for DNA fingerprinting, evaluation of phylogenies and genetic structure, proving to be an adequate complementary tool for rDNA based studies in genus Quercus.