The genomic landscape of transposable elements in yeast hybrids is shaped by structural variation and genotype-specific modulation of transposition rate

Reviewer #1 (Public Review):

Henault et al build on their own previous work investigating the longstanding hypothesis that hybridization between divergent populations can activate transposable element mobilization (transposition). Previously they created crosses of increasing sequence divergence, using both intra- and inter-species hybrids, and passaged them neutrally for hundreds of generations. Their previous work showed that neither hybrids isolated from natural environments nor hybrids from their mutation accumulation lines showed consistent evidence of increased transposable element content. Here, they sequence and assemble long-read genomes of 127 of their mutation-accumulation lines and annotate all existing and de novo transposable elements. They find only a handful of de novo transposition events, and instead demonstrate that structural variation (ploidy, aneuploidy, loss of heterozygosity) plays a much larger role in the transposable element load in a given strain. They then created transposable element reporter constructs using two different Ty1 elements from S. paradoxus lineages and measured the transposition rate in a number of intraspecific crosses. They demonstrate that the transposition rate is dependent on both the Ty1 sequence and the copy number of genomic transposable elements, the latter of which is consistent with what has been observed in the literature on transposable element copy number control in Saccharomyces. To my knowledge, others have not directly tested the effect of Ty1 sequence itself (have not created diverse Ty1 reporter constructs), and so this is an interesting advance. Finally, the authors show that mitotype has a moderate effect on transposition rate, which is an intriguing finding that will be interesting to explore in future work.

This study represents a large effort to investigate how genetic background can influence transposable element load and transposition rate. The long read sequencing, assembly, and annotation, and the creation of these reporter constructs are non-trivial. Their results are straightforward, well supported, and a nice addition to the literature.

The authors state that the results from their current work support results taken from their previous study using short-read sequencing data of the same lines. The argument that follows is whether the authors gained anything novel from long-read sequencing. I would like to see the authors make a stronger argument for why this new work was necessary, and a more detailed view of similarities or differences from their previous study (when should others choose to do long read vs. short read of evolved lines?). Relatedly, the authors should report the rates of structural variants that they observe. How are these results similar/different from other mutation-accumulation work in S. cerevisiae?

Since the authors show a small, but consistent influence of mitotype on transposition rates, adding further evidence for the role of mtDNA in regulating transposition, I'm curious what the transposition rate of a p0 strain is. I think including these results could make this observation more compelling.

Reviewer #2 (Public Review):

This is an interesting follow-up study that uses long-read sequencing to examine previously constructed mutation accumulation lines between wild populations of S. cerevisiae and S. paradoxus. They also complement this work with reporter assays in hybrid backgrounds. The authors are attempting to test the hypothesis that hybridization leads to genome shock and unrestrained transposition. The paper largely confirms previous results (suggesting hybridization does not increase transposition) that are well cited and discussed in the paper, both from this group and from the Smukowski Heil/Dunham group but extends them to a new set of species/hybrids and with some additional resolution via the long read sequencing. The paper is well written and clear and I have no serious complaints.

In the abstract, the authors make three primary claims:

Structural variation plays a strong role in TE load.
Transposition plays only a minor role in shaping the TE landscape in MA lines.
Transposition rates are not increased by hybridization but are affected by genotype-specific factors.

I found all three claims supported, albeit with some minor questions below:

Structural variation plays a strong role in TE load.
Convinced of this result. However:
Line 185-187/Figure 3C: I'm curious given that the changes in Ty count are so often linked to changes in gross DNA sequence whether the count per total DNA sequence is actually changing on average in these genomes. Ie., does hybridization tend to increase TE count via CNV or does hybridization tend to increase DNA content in the MA lines and TEs come along for the ride?

One question about ploidy (lines 175-177):

Both aneuploidy and triploidy seem easy to call from this data. A 3:1 tetraploidy as well. However, in Figure 2B there are tetraploids that are around the 1:1 line. How are the authors calling ploidy for these strains? This was not clear to me from the text.

Reviewer #3 (Public Review):

Henault et al. address the important open question of whether hybridization could trigger TE mobilization. To do this they analysed MA lines derived from crosses of Saccharomyces paradoxus and Saccharomyces cerevisiae using long-read sequencing. These MA lines were already analysed in a previous publication using Illumina short-read data but the novelty of this work is the long-read sequencing data, which may reveal previously missed information. It is an interesting message of this study that hybridization between the two species did not lead to much TE activity. Due to this low activity, the authors performed an additional TE activity assay in vivo to measure transposition rates in hybrid backgrounds. The study is well written and I cannot spot any major problems. The study provides some important messages (like the influence of the genotype and mitochondrial DNA on transposition rates).

Major comments
- What I miss the most in this work is the perspective of the host defence against TEs in Saccharmoces. Based on such a mechanistic perspective, why do the authors think that hybridization could lead to a TE reactivation? For example, in Drosophila small RNAs important for the defence against a TE, are solely maternally transmitted. Hybrid offspring will thus solely have small-RNAs complementary to the TEs of the mother but not to the TEs of the father, therefore a reactivation of the paternal TEs may be expected. I was thus wondering, what is the situation in yeast. Why would we expect an upregulation of TEs? Without such a mechanistic explanation the hypothesis that TEs should be upregulated in hybrids is a bit vague, based on a hunch.