Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development

Abstract

Background: Chromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution--some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 - 75 Gb, 12-74 Gb of which are lost from pre-somatic cell lineages at germline--soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown.

Results: Here, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes of Mesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome.

Conclusions: Our results suggest that germline genome expansion in M. edax reflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms.

Figure 1

Feulgen-stained nuclei of M. edax after and before chromatin diminution. A. Somatic cell nuclei in antennal segments contain ~3 Gb DNA. B. Whole anaphase figure during germline cell division. Germline genome in prediminuted embryo contains ~30 Gb DNA.

Figure 2

Germline and somatic genome content. Estimated Gb of sequence in the germline and somatic genomes annotated as known TEs; simple repeats; unknown, non-simple, non-tandem (within the length of a single read) repeats; and rRNAs. rRNA sequences were only a tiny fraction of both genomes (germline = 0.12%, soma = 0.02%).

Figure 3

Copy numbers of four repeat elements in germline and somatic genomes estimated using qPCR. In all cases, elements are more abundant in the germline than in the soma.

Figure 4

Sequence divergence distributions of repeats in the germline (red) and soma (green) of Mesocyclops edax. The y axis is the proportion of reads out of the total germline or somatic repeat dataset comprised of repeats of a given sequence divergence/age class, allowing comparisons of the distribution shapes between the two genomes. Sequence divergence distributions of both genomes are skewed towards younger (i.e. less divergent) elements. High proportions of repeats <1% diverged from the consensus demonstrate recent/ongoing repeat proliferation.

Figure 5

Maximum likelihood estimates of the relative frequencies of known repeat superfamilies in soma and germline. 95% confidence intervals are shown. Elements that exist at higher frequencies in the germline than in the soma (e.g. DNA-hAT, DNA-MuDR, LINE-L1) are disproportionately excised from the somatic genome during chromatin diminution. Elements that exist at higher frequency in the soma (e.g. LINE-CR1, LTR-ERV1, LTR-Gypsy) are disproportionately retained in the somatic genome.

Figure 6

Cumulative probability distributions of repeat element sequence divergence/age. Reference (dashed line) summarizes sequence divergences of elements present at equal frequencies in germline and somatic genomes (i.e. not disproportionately deleted from, or retained in, the somatic genome during chromatin diminution). Red line shows the distribution of elements present at significantly higher frequencies in the germline than soma; these elements are disproportionately deleted during chromatin diminution. They are significantly younger (less divergent) than the reference elements. Green line shows the distribution of elements present at significantly higher frequencies in the soma than in the germline; these elements are disproportionately retained in the somatic genome during chromatin diminution. They are significantly older (more divergent) than the reference elements. Thus, chromatin diminution disproportionately targets younger repeat elements.