A Chromosome-level Assembly of the Japanese Eel Genome, Insights into Gene Duplication and Chromosomal Reorganization

  1. Hongbo Wang
  2. Hin Ting Wan
  3. Bin Wu
  4. Jianbo Jian
  5. Alice HM Ng
  6. Claire Yik-Lok Chung
  7. Eugene Yui-Ching Chow
  8. Jizhou Zhang
  9. Anderson OL Wong
  10. Keng Po Lai
  11. Ting Fung Chan
  12. Eric Lu Zhang
  13. Chris Kong-Chu Wong

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Abstract

Japanese eels ( Anguilla japonica ) are commercially important species that have been harvested extensively for foods. Currently, this and related species (American and European eels) are difficult to breed on a commercial basis. Wild stock is used for aquaculture. Due to pollution, overfishing, and international trafficking, eel populations are declining. The International Union for Conservation of Nature lists Japanese eels as critically endangered and on its red list. Here we presented a high-quality genome assembly for Japanese eels and demonstrated that large chromosome reorganizations occurred in the events of third-round whole-genome duplications (3R-WRD). Following multiple chromosomal fusion and fission rearrangement, the Anguilla lineage has reduced the haploid chromosomal number of 19 from the ancestral proto-chromosomal number of 25. Phylogenetic analysis of expanded gene families showed the gene families of olfactory receptors and voltage-gated Ca 2+ -channel expanded significantly. The expansion of olfactory receptors (group δ and ζ genes) and voltage-gated Ca 2+ -channel gene families are important for olfaction and neurophysiological functions. Following 3R-WGD, additional tandem (TD) and proximal (PD) duplications occurred to acquire immune-related genes for adaptation. The Japanese eel assembly presented here can be used to study other Anguilla species that are related to evolution and conservation.

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  1. GigaScience

    Japanese eels

    This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giac120), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows: **Reviewer 1：Christiaan Henkel ** This paper describes a new chromosome-level assembly of the Japanese eel, which could finally supersede the various more fragmented assemblies. The assembly process is perhaps overly complex (many data sources and assembly steps, suppl. figure 3), but the result in general appears to be of high quality, as demonstrated by BUSCO (twice) and alignment to a closely related genome (Anguilla anguilla, suppl. figure 4). Figures 1 and 2, however, contain some inconsistencies:

    Figure 1: track B (nanopore coverage) shows a clear bimodal signal, with large blocks of high (double) coverage. These appear possibly correlated with areas low in gene content (track E). Are these possibly collapsed duplicate regions? That would have a strong effect on the analyses of genome duplication. Do other somewhat comparable data sources, for example PacBio CLR, show this feature?

    Figure 2, right panel: the new A. japonica assembly appears to have many unclustered genes (brown), similar to the fragmented draft assembly of A. rostrata and unlike the other included chromosome-level assemblies. This appears to be related to the annotation process? Or are there other problems that preclude orthology assignment for these genes? And how does A. rostrata get its gain of 11756 genes in this analysis? (By the way, line 323 has genus Anguilla as +919/-531, the figure +919/-631).

    Some other questions and comments I would like the authors to address:

    The discussion of previous and current eel sequencing efforts in the Introduction is not complete. For example, I miss the assemblies by Kai et al (2014) and Nakamura et al (2017) of the Japanese eel genome. In addition, the Introduction and Discussion (lines 415-417) present the current assembly as the first chromosome-scale Anguilla genome, which is not the case. At least two high-quality assemblies of Anguilla anguilla (European eel) are available, and should be acknowledged: one is by the Vertebrate Genome Project, and this assembly is even used in the manuscript for comparative purposes (line 199). The other has been described in a preprint (Parey et al 2022). Some of the mentioned papers include similar analyses (mostly on evolution after genome duplication and ancestral genome reconstruction, see figure 5).

    Kai et al (2014) A ddRAD-based genetic map and its integration with the genome assembly of Japanese eel (Anguilla japonica) provides insights into genome evolution after the teleost-specific genome duplication. BMC Genomics 15, 233. https://doi.org/10.1186/1471-2164-15-233 Nakamura et al (2017) Rhodopsin gene copies in Japanese eel originated in a teleost-specific genome duplication. Zoological Lett 3, 18. https://doi.org/10.1186/s40851-017-0079-2 Parey et al. (2022) Genome structures resolve the early diversification of teleost fishes. BioRxiv https://doi.org/10.1101/2022.04.07.487469 The different statistics listed for each alternative assembly in the Introduction make comparisons difficult.

    The statement in line 79, that eels as the most basal teleost group are 'close' to non-teleosts, is incorrect. They are just as close to non-teleosts as any other teleost. (The rest of the sentence, up to line 82, could use rephrasing).

    The statement in line 307 that 'Japanese eels are phylogenetically closer to American than European eels' contradicts the phylogeny presented (fig. 2), or is this based on some additional analysis (a density plot not shown), or even on figure 2 right panel (see comment earlier)? Even if they are incrementally 'closer' by some metric, I would not interpret this a phylogenetic distance, given the inferred divergence dates. In any case, the American eel assembly is still highly fragmented, and not the best basis for inferences which otherwise rely on chromosome-scale assemblies.

    Similarly, the statements on divergence between teleost groups in lines 495-500 need rephrasing. Anguilla species did not diverge from Megalops etc.

    Figure 2 & lines 205-213/310-313: These divergence times are calibrated using a few intervals taken from TimeTree.org (red dots). I wonder how reliable this is, as I get quite different intervals when checking now: for Anguilla-Megalops it is 162.2-197.3 (the paper has 179.3-219.3). Also TimeTree appears to have arowana (Scleropages) as the most basal branch among the teleosts, the paper has a combined Osteoglossomorpha(arowana)/Elopomorpha(eels) branch. Has the phylogenetic tree topology been inferred or imposed? Why have the specific calibration points been chosen? The early branching among teleosts (see line 310-312) is somewhat controversial, see the preprint by Parey et al.

    Line 346-348: This uses the eel genome size (~1 Gbp) and the further (4R) duplicated salmon genome (3 Gbp) to argue against a such further genome duplication in eels. Although I agree that the eel 4R probably did not occur, comparing genome sizes presents no evidence in this case. Genome size changes by other processes as well, and more dramatically (e.g. transposon proliferation). In addition, salmon and eel are not closely related, at all. Compare this to the genomes of the (much more closely related) common carp and zebrafish, both ~1.5 Gbp: the carp genome, but not zebrafish, has experienced an additional duplication, but the zebrafish genome contains a higher transposon density.

    The second argument against 4R (lines 352-356, figure 4b) also does not really work. With 8 Hox clusters, the eel genome appears duplicated with respect to the gar (4 clusters), and not quadruplicated. However, with 8 clusters and 70+ genes, eels actually have more than all established 3R teleost genomes (max. 7 clusters, 42-50 genes). So the question is then whether these 8 clusters form nice 3R WGD ohnolog pairs, or if some clusters have been lost (as in nearly all other teleosts) and re-duplicated. The former hypothesis is consistent with the high level of retained WGD genes (line 369), the latter with the inferred high level of local duplication (line 363). The observation of duplicate eel Hox clusters goes back to the initial European eel genome assembly (Henkel et al 2012), but there the draft status precluded confident assignment to 3R for some clusters.

    The eel olfactory receptors have previously been identified using an assembled transcriptome (Churcher et al. 2015, not cited). How do the analyses of line 214-229/324-333/420-434/figure 3 compare?

    Churcher et al (2015) Deep sequencing of the olfactory epithelium reveals specific chemosensory receptors are expressed at sexual maturity in the European eel Anguilla anguilla. Molecular Ecology 24, 822-834. https://doi.org/10.1111/mec.13065 Lines 460-467 state eels have retained duplicates of immune genes, which have been under positive selec tion. So how does this translate to a (very recent) negative effect on eel fitness (line 460-462)?

    The discussion of line 482-502 on chromosome numbers invokes ecological explanations (freshwater vs. marine habitats, 482-489), but subsequently does not translate this to the low Anguilla chromosome numbers. As these ecological factors are highly applicable to Anguillidae, this connection should be explored here - including their evolutionary history (e.g. Inoue et al, 2010, Deep-ocean origin of the freshwater eels. Biology Letters 6, https://doi.org/10.1098/rsbl.2009.0989)

    In this discussion: how do the numbers of line 482/3 (modal 2n 54/48 chromosomes in fish) correspond to those of line 492 (peak chromosome number n = 24/25 in extant teleosts)?

    The supplementary figures/tables lack legends (just mentions in the main text).

    Line 109: which ONT flowcell, kit, and basecaller versions have been used? In the M&M, please list software versions.

    **Reviewer 2: Zhong Li ** This manuscript by WANG et al. titled "A Chromosome-level Assembly of the Japanese Eel Genome, Insights into Gene Duplication and Chromosomal Reorganization " provides a high quality genome assembly of Japanese Eel, and economically important fish. The authors have used for kinds of sequencing technologies, and assembling strategies, and provided well annotated genomes. This genome provides useful information for the genome organization and evolution and other fields of this species.

    Overall, the manuscript is sufficiently descriptive and easy to follow. I have three major concerns:

    The genome annotation rely on the transcriptome. No detailed information was given the the method section. The analyses do not include command lines or software versions and thus are not repeatable easily. A document that include these information is higly recommended included as a supplementary file. The genome assembly seems has not been released on NCBI database (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA852364). Besides, the gene models (nucleotide, protein, and GFF files) should also be made available and included in the Data Availability section when the manuscript is accepted.

  3. Version published to 10.1101/2022.06.28.497880v1 on bioRxiv