Next generation sequencing to analyse rDNA complexity in Nicotiana and a model that proposes homogenisation is driven by unit amplification at novel loci
Tandemly arranged nuclear ribosomal DNA (rDNA), encoding 18S-5.8S-26S ribosomal RNA (rRNA), evolves by concerted evolution, a process that homogenizes rDNA arrays. However detailed studies of rDNA complexity have been targeted at eukaryotes with relatively short arrays (<a few hundred units). Here study rDNA complexity in species with arrays thousands of units in length.Methods: We examined homogeneity of genic (18S) and non-coding internal transcribed spacer (ITS1) regions of rDNA using Roche 454 and Illumina platforms in four angiosperm species, Nicotiana sylvestris, N. tomentosiformis, N. otophora and N. kawakamii. We compared the data with the structure of intergenic spacer sequences (IGS) and the distribution of rDNA loci.    Results:  In all four species the intragenomic homogeneity of the 18S gene was high, evidenced by > 90% of genes likely formed by a single ribotype.  However increased heterogeneity was observed in the ITS1 region, particularly in species with two or more rDNA loci. Southern hybridisation showed that IGS heterogeneity was high in all species.  Conclusions:  The increased number of ITS1 ribotypes compared with 18S may reflect incomplete homogenisation, followed by selection for functional genic regions coupled with tolerance of alternative ITS1 variants. Moreover there is a correlation between the number of ITS1 ribotypes and the number of rDNA loci and we propose a ‘move-and-select’ model where rDNA amplification at novel loci may be rapid and drive the homogenisation of alternative variants at these sites, increasing overall genomic diversity of rDNA.