# Copy number and sequence variation in ribosomal DNA and the transposon, Pokey, in mutation accumulation lines of Daphnia obtusa

## TL;DR

The study tracks changes in ribosomal DNA copy number and a transposon called Pokey in Daphnia obtusa over generations.

## Contribution

The study provides novel insights into the dynamics of rDNA copy number and Pokey transposon variation in mutation accumulation lines.

## Key findings

- rDNA copy number declined overall but showed short-term fluctuations over generations.
- Pokey transposon numbers were positively correlated with 28S copy number and persisted even after large reductions.
- Recombination rates in rDNA were estimated at 0.094 events per generation.

## Abstract

Ribosomal DNA (rDNA) occurs as tandem arrays of a repeat unit containing the genes encoding 18S, 5.8S, and 28S rRNA separated by spacers. These rRNAs form the catalytic core of ribosomes and thus play a crucial role in protein synthesis. Due to its repetitive nature, rDNA copy number varies within and between eukaryotic species through recombination, which also results in homogenization of repeat sequences within species (concerted evolution). However, the recombination rate within rDNA has not been extensively estimated. Despite concerted evolution and strong selection to maintain the sequence of rRNA genes, some transposons insert into specific sequences in the 28S gene. We used short-read whole-genome sequences to examine the dynamics of change in rDNA copy number and sequence variation in 90 samples from clonally propagated Daphnia obtusa mutation accumulation (MA) lines over ∼95 generations. We also tracked the number of Pokey elements, a DNA transposon that inserts into the 28S gene of species in the subgenus Daphnia. We observed an overall decline in rDNA copy number across MA lines between generations 5 and ∼87, although both increases and decreases were observed over short intervals. The diploid 28S copy number ranged from 144 to 1,274, with a mean of 425.2. Diploid Pokey number varied from 65 to 537 and was significantly positively correlated with 28S copy number. Moreover, the element persisted in all lines even after large reductions in 28S copy number. We found that estimating rates of rDNA copy number change over long intervals resulted in substantial underestimates, as shorter intervals revealed that large copy number changes could occur in as few as 5 generations. We identified 5 rDNA haplotypes based on 58 single nucleotide polymorphisms (SNPs) that were distributed across the 18S and 28S genes, and the 3 non-repetitive intergenic spacer regions. We also identified 6 Pokey haplotypes based on 113 SNPs. The number of these haplotypes was strongly correlated with the number of the 3 most common rDNA haplotypes. By tracking changes in haplotype frequency and copy number within 4 MA lines over short time intervals, we estimated the mean rDNA recombination rate to be 0.094 events/generation. These results reveal that rapid changes can occur in rDNA over short timescales and show that Pokey transposon dynamics are tightly linked to rDNA structure.