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Evolution and functioning of an X–A balance sex-determining system in hops

Nature Plants volume 11, pages 1339–1352 (2025)Cite this article

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Abstract

Chromosomal sex-determining systems with male heterogamety include actively male-determining-Y and X–A balance systems, both of which are found in animals and plants. The sex-determining genes have been identified in several active-Y plant systems, but the evolution and functioning of X–A balance systems remains mysterious. Here we sequenced and compared the genomes of two hop species. The evolution of the hop X–A balance system involved an ancient recombination suppression event across a large X chromosome region shared by both species. In one species, an autosome fused to this ancestral sex chromosome, and recombination was subsequently suppressed again. The two evolutionary strata created in this neo-X have degenerated to different degrees and evolved correspondingly different dosage compensation levels that correlate with histone modification patterns. Finally, we identified an X-specific ETR1-like ethylene receptor in the ancestral X region. Its dosage may affect sex determination, as part of the counting mechanism of this X–A balance system.

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Fig. 1: Genome evolution in the family Cannabaceae.
Fig. 2: Structures of the sex chromosomes in the two Humulus species studied.
Fig. 3: Dosage compensation in H. lupulus and H. japonicus X chromosomes.
Fig. 4: Identification of an X-linked sex-determining candidate gene in the genus Humulus.
Fig. 5: Model for the evolution of sex chromosomes in the genus Humulus.

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Data availability

All of the assembled genome sequences and the raw sequencing data have been deposited in the DDBJ database (BioProject IDs PRJDB17941, PRJDB17942 and PRJDB18715). The annotated genome data have been deposited in the Plant Garden database (https://plantgarden.jp/en/index). The sequencing data for reduced representation genome libraries, DNA methylomes, ChIP-seq and RNA-seq have been deposited in the DDBJ database (BioProject IDs PRJDB19054, PRJDB19078, PRJDB19080 and PRJDB19097).

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Acknowledgements

We thank R. Terauchi (Kyoto University, Japan) and M. Suematsu, F. Sato, H. Kawashima, H. Matsubara, H. Shibata and all the members of Suntory Global Innovation Center Ltd for helpful suggestions and dedicated support. This work was supported by PRESTO from the Japan Science and Technology Agency (grant no. JPMJPR20D1), Grants-in-Aid for Transformative Research Areas (A) from JSPS (nos 22H05172 and 22H05173 to T.A., 22H05181 to K. Shirasawa and 22H02598 to T.I.) and the Czech Science Foundation (grant no. 22-00301S to R.H.).

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Author notes

  1. These authors contributed equally: Takashi Akagi, Tenta Segawa.

Authors and Affiliations

  1. Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan

    Takashi Akagi, Rika Uchida, Yuka Honniden, Cheng Zhang & Koichiro Ushijima

  2. Japan Science and Technology Agency, PRESTO, Kawaguchi-shi, Japan

    Takashi Akagi

  3. Suntory Global Innovation Center Ltd, Kyoto, Japan

    Tenta Segawa & Eiichiro Ono

  4. School of Life Science and Technology, Institute of Science Tokyo, Tokyo, Japan

    Hiroyuki Tanaka & Takehiko Itoh

  5. Kazusa DNA Research Institute, Kisarazu, Japan

    Kenta Shirasawa

  6. Faculty of Agriculture, Iwate University, Morioka, Japan

    Noriko Yamagishi & Hajime Yaegashi

  7. Iwate Biotechnology Research Center, Kitakami, Japan

    Satoshi Natsume, Hiroki Takagi & Akira Abe

  8. Division of Microbiology, Department of Infectious Medicine, Kurume University School of Medicine, Kurume, Japan

    Miki Okuno

  9. Advanced Genomics Center, National Institute of Genetics, Mishima, Japan

    Atsushi Toyoda

  10. Faculty of Science, Academic Assembly, University of Toyama, Gofuku, Japan

    Kyoko Sato

  11. Hop Research Institute Co. Ltd, Žatec, Czech Republic

    Josef Patzak

  12. Department of Plant Developmental Genetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic

    Lucie Horáková, Václav Bačovský & Roman Hobza

  13. Institute of Ecology and Evolution, University of Edinburgh, Edinburgh, UK

    Deborah Charlesworth

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  1. Takashi Akagi
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Contributions

T.A., T.I. and E.O. conceptualized the project. T.A., T.S., R.U., H. Tanaka, K. Shirasawa, N.Y., H.Y., S.N., H. Takagi, A.A., M.O., A.T., K. Sato, Y.H., C.Z., K.U., J.P., L.H., V.B. and R.H. devised the methodology. T.A., T.S., R.U., H. Tanaka, K. Shirasawa, N.Y., H.Y., S.N., A.A., M.O., K. Sato, C.Z., L.H. and V.B. conducted the investigation. T.A., T.S., H. Tanaka, K. Sato, C.Z., K.U. and T.I. visualized the data. T.A., K. Shirasawa, R.H., T.I. and E.O. acquired the funding. T.A., D.C., T.I. and E.O. were the project administrators. T.A., D.C. and E.O. supervised the project. T.A., D.C., T.I. and E.O. wrote the original draft of the paper. T.A., D.C., T.I. and E.O. reviewed and edited the paper.

Corresponding authors

Correspondence to Takashi Akagi, Takehiko Itoh or Eiichiro Ono.

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The authors declare no competing interests.

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Nature Plants thanks Dmitry Filatov, Susanne Renner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Organisation of the H. lupulus X and Y chromosomes and the PAR.

a, Sequence-based synteny dot plot between this species’ entire X and Y chromosomes. b, Only the approximately 20 Mbp recombinationally active region (see Supplementary Fig. 2), the likely PAR, exhibits clear long syntenic regions, but after the initial ~18 Mb, a long inversion is present. Raw divergence estimates for synonymous sites (dSXY values) are shown across these two parts. The left-hand sub-region of the non-inverted region (indicated by the blue box) includes predominantly high values (often up to 5%). Polymorphisms will contribute to these, as was observed in the Silene latifolia PAR15,37,91, and long-term balancing selection, or an inversion polymorphism maintained by balancing selection, might be responsible for this. However, differentiation between males and females (FST) in S. latifolia is high only close to the PAR boundary, and balancing selection maintains high diversity across only physically extremely small genome regions unless the recombination rate is very low, as discussed in Qiu et al.91,92, it is more likely that the effect is due to linkage disequilibrium in a region with an extremely low recombination rate. As the FST values in S. latifolia approach the maximum possible for sex linkage (like the values in H. lupulus, as mentioned in the main text), they support an extremely low recombination rate. Our interpretation in the main text is similar. The right-hand inverted (inverted, pink box) region has intermediate values that are consistently higher than those in the middle of the entire region, whose dSXY values are consistently very low, suggesting that only this smaller set of genes are PAR genes.

Extended Data Fig. 2 Population genetic quantities for regions in the left-hand terminal region of the H. lupulus X chromosome.

The values were estimated from GRAS-Di sequencing (see the Methods section of the main text). Data were obtained from 8 individuals (4 male and 4 female accessions). All quantities were estimated for all site types (not just synonymous sites), and were calculated using variants within the mapped regions; the results are plotted in 1-Mbp windows. The top row shows nucleotide diversity (Pi values), with males in green dots and females orange, and the difference between the male and female samples (Pi (male-female) is shown in the second row. The third and fourth rows show FST between the male and female samples, and Tajima’s D values in the whole population (both sexes). The results are consistent with the conclusions in the main text based on the dSXY values shown in Fig. 3, and with the recombination data for this species in Supplementary Fig. 2.

Extended Data Fig. 3 Relationships between X and Y1/Y2 chromosome sequences in H. japonicus.

The y axis shows dSXY values of inferred gametologous gene pairs (with genes annotated on both the X and an apparently Y-linked allele), with their X chromosome positions on the x axis. As expected, the Y-linked sequences of most such X-linked genes are on Y2 (derived from Hl3(j)) with only a few on Y1 (derived longer ago, from the ancestral X, whose Y is profoundly degenerated). However, the Hl3(j) genes are not confined to the Y2 as would be expected, and HlX(j) ones are not confined to the Y1; instead, gametologs from each set are found on both Y1 and Y2, with HlX(j)-derived genes in Strata 0 and 1 (shown in blue) found mainly on Y2, forming patches corresponding to 4 X assembly regions with low Y-X Ks values (indicating recent movements from either the ancestral X to Y2, or, more likely, from Y1 to Y2), and several Hl3(j)-derived genes (in yellow) in Stratum 2 (very unexpectedly) being found only in Y1. A few genes (indicated by the 4 magenta dots) are shared by both Y1 and Y2. The mosaic pattern observed in Y2 suggests rearrangements in which segments of Y1 moved to Y2 (or were perhaps duplicated onto chromosome 3 before it became Y2), while a few Y2 genes moved to Y1.

Extended Data Fig. 4 Frequent rearrangement in Stratum 2 in H. japonicus.

The male-hemizygous region in the right arm of the enlarged H. japonicus X exhibits clear synteny to the H. lupulus chromosome 3 (Extended Data Fig. 7). However, in the Stratum 2 region, synteny is observed only in discontinuous blocks that partially correspond to Y1 or Y2, with different dSXY values. Note that the dSXY values for individual genes have high variance, and the values fluctuate too highly for a change-point test to succeed. Blue and red bands respectively indicate the same and opposite directions in the two chromosome assemblies.

Extended Data Fig. 5 Genome coverage estimates of X-linked genes in males and females from 5 H. japonicus lines, showing the differences in inferred male-hemizigosity in the terminal regions.

a, Genomic read coverage in the X chromosome, standardized by the estimated genome-wide coverage; the results are shown as female/male ratios in 1-Mbp bins across the chromosome. In our whole genome-sequenced line, Seta-1 (indicated in magenta), coverage in the female is double that in the male at both ends of the chromosome, indicating male-hemizygosity, except a region with lower genomic coverage than the rest of the left-hand terminal region, suggesting retention of Y-gametologs in this part. Two other lines (Seta-2 and Otsu-2) also show coverage ratios indicating male-hemizygosity in both terminal regions. However, in two lines, Otsu-1 and Otsu-3, coverage is similar in both sexes, suggesting that these regions are either PARs, or, if Y-linkage has evolved, that some genes still have Y-linked gametologs. b, Close-up view of the coverage ratios between the sexes in the terminal regions, in 500-kbp bins. In all 5 lines analysed, both 2-Mb X chromosome tips include regions of up to 1-Mb with ratios mostly close to 1.

Extended Data Fig. 6 Estimates of synonymous site divergence between Y and X gametologs (dSXY) in the two terminal regions of the H. japonicus X chromosome.

Y-gametologs were ascertained by mapping deep genome sequencing data from a male Otsu-1 line to the Seta-1 line reference X chromosome sequences (Extended Data Fig. 5 shows coverage estimates from all lines of this species). The 10 and 5 Mbp termini at the left- and right-hand chromosome ends have only very low dSXY values (the medians for 1 Mbp windows, indicated by the magenta lines, are close to 0 for both ends), suggesting that these are PARs in the Otsu-1 line. More central regions have much higher dSXY values, up to 5%, similar to values in Stratum 2. We infer that these have ceased reccombining and are no longer pseudo-autosomal regions. The fluctuating and discountinuous decrease in the dSXY values may reflect rearrangements in the Y chromosomes as is the case of Stratum 2 (Extended Data Figs. 3–4).

Extended Data Fig. 7 Sex chromosome-specific rearrangements.

Synteny plots between H. lupulus and H. japonicus, in three representative autosomes and their X chromosomes. Highly disrupted synteny in the X chromosome (a, b), than in the autosomes (c: Chr. 1, d: Chr. 5, e: Chr. 7). The X chromosome in H. japonicus is consitituted of HlX(j) and Hl3(j), and Hl3(j) region showed less disrupted synetny (against Chr. 3, an autosome in H. lupulus) than HlX(j). This is presumably due to a comparison between X (in H. japonicus) and autosome (in H. lupulus), and also due to recent conversion from an autosome to a part of Chr. X in Hl3(j).

Extended Data Fig. 8 Enriched GO terms in compensated and non-compensated X-specific genes in the X chromosomes of H. lupulus and H. japonicus.

Significantly enriched GO terms (FDR < 0.05, against the whole genome background) in H. lupulus and H. japonicus. To assess compensation of individual X-specific genes in Strata 0-1 of H. lupulus and H. japonicus, we divided sex biases in expression into two categories, “compensated” (-0.5 < log2[Mexp/Fexp] < 0.5) and “non-compensated” (-0.5 > log2[Mexp/Fexp]). The compensated sets in both species were enriched for gene ontology (GO) terms relating to nucleoside or nucleotide phosphorylation/metabolism pathways, despite few genes being shared by these regions in the two species. These results suggest possible involvement in functions involving broadly expressed genes, such as energy supply (or ATP/ADP metabolic cycles) or DNA replication. The non-compensated genes shared no GO terms in common in H. lupulus and H. japonicus; many such genes are annotated with stress-response functions, and could (very speculatively) be involved in sexual dimorphism, including the slower growth rate and/or later flowering of hop species females.

Extended Data Fig. 9 Histone modification patterns in the Strata 0-1 in H. japonicus, and their correlations with expression levels in the whole genome.

Histone modification patterns in regions surrounding H. japonicus, using two criteria, (i) whole gene marked (not compensated), and (ii) Strata 0-1 (compensated). TSS: transcription start site, TTS: transcription termination site. The typical euchromatic marks, H3K9/27ac (a) and H3K4me1 (b), and also H3K9me3 (c) and H3K27me3 (d), which often repress gene expression, exhibited statistically significantly higher loading in males than females in Strata 0-1. The loadings correlate with the total H3 amount (e) in Strata 0-1 do not differ significantly between the sexes. Modification patterns of H3K9/27ac (f), H3K4me1 (g), H3K9me3 (h) and H3K27me3 (i) around genes in the H. japonicus genome. The genes’ expression levels (averaged values of 4 female leaves) are positively correlated with the loading amount in H3K9/27ac and H3K4me1, whereas neither positively nor negatively correlated in H3K9me3 and H3K27me3 contexts (in female).

Extended Data Fig. 10 Evolutionary topology of the ETR1/ERS1-clade ethylene receptors.

The topology of the ETR1/ERS1-clade ethylene receptors was estimated with the ML-method implemented in MEGA X, with ETR2/ERS2 clades as the outgroup. Bootstrap values (with 100 replicates) are shown on each branch. ETR1 and ERS1 clades are clearly separated with high statistical support (bootstrap values 96 and 100, repectively). The ETR1 clade is monophyletic, except for duplications within some lineages, and includes the EXER gene, a sex determining candidate in the genera Humulus and Cannabis. The prefixes used are as follows: Chr: avocado (Persea americana), GSMUA: banana (Musa accuminata), MELO: melon (Cucumis melo), Prupe: peach (Prunus persica), pycom: European pear (Pyrus commonis), Solyc: tomato (Solanum lycopersicum), Acc: kiwifruit (Actinidia chinensis), Dlo: Diospyros lotus, rna-XM: Cannabis sativa, 10_12: Humulus lupulus.

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Supplementary Texts 1 and 2, Figs. 1–16 and Tables 1–11.

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Akagi, T., Segawa, T., Uchida, R. et al. Evolution and functioning of an X–A balance sex-determining system in hops. Nat. Plants 11, 1339–1352 (2025). https://doi.org/10.1038/s41477-025-02017-6

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