Next generation sequencing analysis reveals a relationship between rDNA unit diversity and locus number in Nicotiana diploids

Roman Matyášek,Richard Nichols,Marie-Angele Grandbastien,Jiří Macas,Jaroslav Fulneček,Simon Renny-Byfield,Aleš Kovařík,Andrew Leitch

doi:10.1186/1471-2164-13-722

Abstract

BackgroundTandemly arranged nuclear ribosomal DNA (rDNA), encoding 18S, 5.8S and 26S ribosomal RNA (rRNA), exhibit concerted evolution, a pattern thought to result from the homogenisation of rDNA arrays. However rDNA homogeneity at the single nucleotide polymorphism (SNP) level has not been detailed in organisms with more than a few hundred copies of the rDNA unit. Here we study rDNA complexity in species with arrays consisting of thousands of units.MethodsWe examined homogeneity of genic (18S) and non-coding internally transcribed spacer (ITS1) regions of rDNA using Roche 454 and/or Illumina platforms in four angiosperm species, Nicotiana sylvestris, N. tomentosiformis, N. otophora and N. kawakamii. We compared the data with Southern blot hybridisation revealing the structure of intergenic spacer (IGS) sequences and with the number and distribution of rDNA loci.Results and ConclusionsIn all four species the intragenomic homogeneity of the 18S gene was high; a single ribotype makes up over 90% of the genes. However greater variation was observed in the ITS1 region, particularly in species with two or more rDNA loci, where >55% of rDNA units were a single ribotype, with the second most abundant variant accounted for >18% of units. IGS heterogeneity was high in all species. The increased number of ribotypes in ITS1 compared with 18S sequences may reflect rounds of incomplete homogenisation with strong selection for functional genic regions and relaxed selection on ITS1 variants. The relationship between the number of ITS1 ribotypes and the number of rDNA loci leads us to propose that rDNA evolution and complexity is influenced by locus number and/or amplification of orphaned rDNA units at new chromosomal locations.

Highlights

Arranged nuclear ribosomal DNA, encoding 18S, 5.8S and 26S ribosomal RNA, exhibit concerted evolution, a pattern thought to result from the homogenisation of rDNA arrays
We analysed the nature of single nucleotide polymorphism (SNP) that define clusters and found C➝T/G➝A transitions were the most frequent polymorphisms in both 18S and ITS1 sequences, likely to be a consequence of high frequency mutations caused by deamination of methyl cytosine to thymine (Additional file 3)
We show that 18S sequences are more homogenous than ITS1; a pattern that could arise from incomplete rounds of homogenisation and stronger purifying selection acting on the 18S genic region

Summary

Introduction

Arranged nuclear ribosomal DNA (rDNA), encoding 18S, 5.8S and 26S ribosomal RNA (rRNA), exhibit concerted evolution, a pattern thought to result from the homogenisation of rDNA arrays. Of particular interest to evolutionary biologists is the pattern of divergence of the whole rDNA array known as concerted evolution, in which the units of the rDNA array are very similar within a species but diverge between species. This pattern suggests that the arrays are subject to homogenisation – so that novel variants arising by mutation spread relatively rapidly along the array within any one species [2,3,4]. It has been suggested that this model might explain the unusually high levels of ITS and IGS polymorphisms in some species (up to 40% in some cases) [9,10,11]

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Conclusion