Is it a mammal thing? Determining when the change in rDNA unit size occurred in amniotes

Abstract

The ribosomal RNA genes in eukaryotes are organised into long arrays of tandem repeats, collectively termed ribosomal DNA (rDNA). Each rDNA repeat unit comprises a rRNA coding region and an intergenic spacer (IGS). Eukaryotes exhibit remarkably little variation in rDNA unit size, with the majority ranging between 9 - 15 kb in length. Eutherian mammals, though, present a striking exception to this rule, with rDNA unit sizes of ~45 kb. However, rDNA unit size is poorly characterized in amniotes, of which eutherian mammals are a member, partly due to the rDNA failing to assemble from short-read whole genome sequences. Thus, it is unclear where in amniote evolution this rDNA unit size increase occurred, or why. I utilised Oxford Nanopore datasets and PacBio assemblies to determine if long rDNA unit sizes are limited to eutherian mammals or are also found in other lineages of the amniote clade. Nanopore reads were interrogated to identify reads with at least two rDNA units, and the rDNA unit size was then determined by calculating the average distance between adjacent rDNA units. Where no nanopore sequencing data was available, rDNA unit size was determined using PacBio assembly approaches. The results revealed that representatives of the bird, snake, lizard, turtle and tuatara lineages all have short rDNA unit sizes. In contrast, representatives of the marsupial and monotreme lineages have long rDNA unit sizes, indicating the increase in rDNA unit size occurred after the divergence of Mammalia from Sauropsida. This Mammalia increase in size occurred primarily as a result of expansion of the IGS. However, comparison of the “long” rDNA unit IGS from representative Mammalia showed a lack of homology in the IGS sequences and transposon profiles, implying that the long rDNA unit size is maintained in this lineage despite high sequence turnover. These results raise questions regarding what drove the rDNA unit size increase in a Mammalian ancestor and why this increased size has been maintained since.