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. 2019 Jun;32(6):619-628.
doi: 10.1111/jeb.13443. Epub 2019 Apr 10.

How Clonal Are Clones? A Quest for Loss of Heterozygosity During Asexual Reproduction in Daphnia Magna

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Free PMC article

How Clonal Are Clones? A Quest for Loss of Heterozygosity During Asexual Reproduction in Daphnia Magna

Marinela Dukić et al. J Evol Biol. .
Free PMC article

Abstract

Due to the lack of recombination, asexual organisms are predicted to accumulate mutations and show high levels of within-individual allelic divergence (heterozygosity); however, empirical evidence for this prediction is largely missing. Instead, evidence of genome homogenization during asexual reproduction is accumulating. Ameiotic crossover recombination is a mechanism that could lead to long genomic stretches of loss of heterozygosity (LOH) and unmasking of mutations that have little or no effect in heterozygous state. Therefore, LOH might be an important force for inducing variation among asexual offspring and may contribute to the limited longevity of asexual lineages. To investigate the genetic consequences of asexuality, here we used high-throughput sequencing of Daphnia magna for assessing the rate of LOH over a single generation of asexual reproduction. Comparing parthenogenetic daughters with their mothers at several thousand genetic markers generated by restriction site-associated DNA (RAD) sequencing resulted in high LOH rate estimation that largely overlapped with our estimates for the error rate. To distinguish these two, we Sanger re-sequenced the top 17 candidate RAD-loci for LOH, and all of them proved to be false positives. Hence, even though we cannot exclude the possibility that short stretches of LOH occur in genomic regions not covered by our markers, we conclude that LOH does not occur frequently during asexual reproduction in D. magna and ameiotic crossovers are very rare or absent. This finding suggests that clonal lineages of D. magna will remain genetically homogeneous at least over time periods typically relevant for experimental work.

Keywords: RAD-sequencing; ameiotic recombination; apomixis; loss of complementation.

Conflict of interest statement

All authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Schematic diagram of the experimental design and sampling procedure. The experiment was replicated for four different clonal lines of Daphnia magna and encompassed three generations of asexual reproduction (F0–F2). Empty arrows indicate generational transition and the production of all‐female clutches. Circles with black arrows indicate five randomly chosen asexual daughters from the first asexual generation (F1), produced by young or old stem mother (2nd or 12th clutch, n = 5) that were subsequently screened for loss of heterozygosity (LOH). A pool of nine F1 females from other clutches was used for genotyping the stem mother (F0). A pool of nine F2 females was used for genotyping each of the F1 asexual daughter (F1)
Figure 2
Figure 2
Examples of falsely annotated loss of heterozygosity (LOH) events. Detected Illumina short‐reads and the corresponding Sanger sequence for the stem mother (IXF1_mother) and the daughters showing LOH (IXF1_d21, IXF1_d22 and IXF1_d24) are depicted. For simplicity, only the heterozygous consensus sequence for one of two stem mother samples (see text) is shown, whereas all short‐read variants detected are shown for daughter individuals and the homozygous consensus sequence that passed our bioinformatic check is marked with the red frame. Maternal SNPs are labelled with yellow and blue squares, whereas the grey squares are marking the unrelated (sequencing error) nucleotide changes. Green squares are marking polymorphic sites detected in Sanger sequences. Numbers on the right are denoting the coverage for a locus/variant in each depicted individual. Upper panel is showing Illumina reads summary and Sanger sequences for the restriction site‐associated DNA (RAD)‐locus Scaffold00687_215401 indicating LOH in two parthenogenetic daughters (IXF1_d21 and IXF1_d22), whereas the lower panel is showing the same summary for the RAD‐locus Scaffold01005_615383 that appeared homozygous in one daughter (IXF1_d24) but presents a different type of erroneous call since the second maternal variant was not even sequenced at the low coverage

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