Genetic characterization of Ocimum genus using flow cytometry and inter-simple sequence repeat markers

Abstract

The taxonomy of the Ocimum genus is difficult and still unclear, mainly due to the existence of numerous, morphologically similar species, varieties and forms, many synonymous names, together with the occurrence of the interspecific hybridization, polyploidy, aneuploidy and other changes in the genome structure, which makes the identification as well as characterization of the Ocimum species/varieties very problematical. Molecular identification by estimation of genome size and ISSR markers proved to be effective in the Ocimum species and cytotypes identification, and in establishing a relationship between them. Genome size estimation revealed that Ocimum species possessed very small, small and intermediate genomes. Furthermore, within one species different values of genome size occurred, which indicated the presence of polyploids and aneuploids. Among O. americanum accessions three different values of genome size we detected: 2.3, 4.4 and 7.4pg/2C, while within O. basilicum accessions most of the genotypes possessed genome with 4.4–4.9pg/2C DNA content and two accessions, from India and Iran, with 7.3 and 7.4pg/2C, respectively. Similarly, in O.×citriodorum genomes with 4.6, 6.9 and 7.5pg/2C DNA content were observed. Very small genomes were detected in O. campechianum (1.2pg/2C) and in O. gratissimum (1.8–2.3pg/2C), while small genome in O. selloi (3.1pg/2C). Within O. tenuiflorum accessions with 0.9, 1.9 and 4.5pg/2C DNA content were identified. Genome size estimation allowed to distinguish some species and cytotypes, and can be used as a first step in species identification. However, more precise and detailed characterization was provided by ISSR markers. Using one of the following primers (GACA)4T, (AG)8YA and (CA)6AC, it was possible to identify species, cytotypes, and also some varieties/cultivars. The relationships within tested accessions reflected by genetic distance estimation and dendrogram construction revealed the existence of three main clusters and one accession (O.×citriodorum, USA) being not clustered to any of the created groups. The first cluster grouped all accessions of O. basilicum and two of O.×citriodorum, the second cluster–all accessions of O. americanum, O. tenuiflorum with small genome size and one accession of O.×citriodorum. The third cluster was the most diversified since it contained O. gratissimum, O. campechianum, O. selloi and O. tenuiflorum (accessions with very small genomes). The identification system composed of flow cytometry and ISSR-PCR techniques demonstrated to be useful for genotypes in the Ocimum genus.