A single gene underlies the dynamic evolution of poplar sex determination

Abstract

Although hundreds of plant lineages have independently evolved dioecy (that is, separation of the sexes), the underlying genetic basis remains largely elusive1. Here we show that diverse poplar species carry partial duplicates of the ARABIDOPSIS RESPONSE REGULATOR 17 (ARR17) orthologue in the male-specific region of the Y chromosome. These duplicates give rise to small RNAs apparently causing male-specific DNA methylation and silencing of the ARR17 gene. CRISPR–Cas9-induced mutations demonstrate that ARR17 functions as a sex switch, triggering female development when on and male development when off. Despite repeated turnover events, including a transition from the XY system to a ZW system, the sex-specific regulation of ARR17 is conserved across the poplar genus and probably beyond. Our data reveal how a single-gene-based mechanism of dioecy can enable highly dynamic sex-linked regions and contribute to maintaining recombination and integrity of sex chromosomes.

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Fig. 1: Male-specific partial ARR17 duplicates arranged as inverted repeats are present in distantly related poplar species.
Fig. 2: ARR17 represents a sex switch that triggers female development when on and male development when off.
Fig. 3: The ARR17 gene is deleted in white poplar males, causing a switch from an XY system to a ZW system.
Fig. 4: Independently evolved SDRs regulate ARR17 expression across the Populus genus.

Data availability

The DNA- and RNA-seq data have been deposited in NCBI’s SRA under the accession number PRJNA542603. The genome assemblies have been deposited at ftp://plantgenie.org/Publications/Muller2019/.

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Acknowledgements

We thank members of the Thünen Institute of Forest Genetics and S. DiFazio, M. Olson and G. Tuskan for helpful comments and discussions; J. Mank and T. Brügmann for critical reading of the manuscript; S. Müller for language editing; and K. Groppe, A. Eikhof, D. Ebbinghaus, G. Wiemann, D. Boedecker, M. Wellern, A. Worm, M. Spauszus, L. Lierke and J. Lüneburg for technical assistance. We acknowledge funding from grants of the Deutsche Forschungsgemeinschaft to N.A.M. (DFG grant no. MU 4357/1-1) and M.F. (DFG grant no. FL 263/15-1). N.R.S., N.M., Z.C.L., V.K. and K.M.R. acknowledge funding from Trees for the Future (T4F), the Knut and Alice Wallenberg Foundation, the Umeå Plant Science Centre Berzelii Centre, the Stiftelsen för Strategisk Forskning Centre for Plant Developmental Biology, the Kempe Foundation and the Swedish Research Council Vetenskapsrådet. We thank the Swedish National Genomics Infrastructure hosted at SciLifeLab, the National Bioinformatics Infrastructure Sweden (NBIS), for providing computational assistance and the Uppsala Multidisciplinary Center for Advanced Computational Science for providing computational infrastructure. We further acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) to K.B. (grant no. RGPIN-2017-06552) and Q.C. (grant no. RGPIN-2019-04041).

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N.A.M., B.K., Q.C., N.R.S. and M.F. conceptualized the project. N.A.M., B.K., A.P.L.M., N.M., C.B., K.B., Z.C.L., M.M., B.P., K.M.R., N.R.S. and M.F. conceived the methodology. N.A.M., B.K., A.P.L.M., N.M., C.B., K.B., Z.C.L., H.H., V.K., B.P. and N.R.S. performed experiments and collected the data. N.A.M., B.K., A.P.L.M., N.M., C.B., K.B., M.M., K.M.R., P.K.I. and N.R.S. analysed the data. M.S. and C.V. provided resources. N.A.M., B.K., N.R.S. and M.F. acquired the funding. N.A.M., B.K., P.K.I., Q.C., N.R.S. and M.F. supervised the project. N.A.M. and N.R.S. validated the data. N.A.M., N.M., K.B., K.M.R. and P.K.I. conducted the visualization. N.A.M. wrote the paper, and all authors reviewed and edited it.

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Correspondence to Niels A. Müller or Matthias Fladung.

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Peer review information Nature Plants thanks Roberta Bergero, 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 Male-specific long non-coding RNAs span TOZ19 and the ARR17 inverted repeat.

a, mRNA-seq data mapped against the new male P. tremula genome assembly (Methods) for seven female and five male biologically independent aspen samples. TOZ19 is represented by the left rightward pointing arrow, the ARR17 inverted repeat by the two small arrows pointing towards each other. Coverage on the forward strand is indicated by lines above zero and coverage on the reverse strand by lines below zero. b, Integrative Genomics Viewer (IGV)70 screenshot of the same RNA-seq data shown in (a). In males, transcription starts from the TOZ19 transcriptional start site. The ARR17 inverted repeat mainly behaves as an intron. The few reads mapping directly to the inverted repeat may represent alternative transcript isoforms. No reads map to the ARR17 gene (data not shown). c, Repeats identified by the CENSOR webtool69 with an alignment score > 2,000. The right part of the ‘TOZ19/ARR17 inverted repeat transcript’ is generated from a sequence with partial identity to an En-Spm3 DNA transposon.

Extended Data Fig. 2 Size distribution of the small RNAs mapping to the ARR17 inverted repeat.

Summed number of alignments of female (n = 11 biologically independent samples) and male (n = 7 biologically independent samples) aspen small RNAs mapping to the ARR17 inverted repeat region of the new male P. tremula genome assembly (Methods). Most reads map to 1-5 mapping locations, which corresponds to the total number of ARR17 sequences: the ARR17 gene, the two arms of the ARR17 inverted repeat and two additional male-specific partial ARR17 fragments. All multi-mapping reads > 18 bp mapped exclusively to these five ARR17 sequence features. Data are from two independent experiments.

Extended Data Fig. 3 The ARR17 locus exhibits male-specific methylation coinciding with the Y-chromosomal duplicated part.

a, Mean percent methylation along the ARR17 genomic region of the P. trichocarpa genome based on bisulfite-sequencing of 9 female (magenta) and 12 male (cyan) biologically independent P. balsamifera samples18. The location of ARR17 is indicated by an arrow. Colored shading marks the standard error. Intron 4, which represents a Copia/LTR sequence (Supplementary Note 2), is highly methylated in both sexes. The first half of ARR17 is only methylated in males. b, Percent methylation along the ARR17 genomic region of the P. tremula genome assembly based on bisulfite-sequencing of two female (Asp201 and Asp044) and three male (Asp005, Asp113 and Asp116) biologically independent P. tremula samples. The location of ARR17 is indicated by an arrow. Again the first half of ARR17 is only methylated in males.

Extended Data Fig. 4 P. tremula flower buds exhibit female-specific ARR17 expression.

Relative ARR17 expression as determined by qRT-PCR for n = 3 biologically independent flower buds/young catkins of each of eight field-grown P. tremula genotypes (females: Brauna11, Tio13-67, W7 and W97; males: Bliz153, C44, Rend14-67 and W52). UBQ (Potri.001G418500) was used as a reference gene for normalization. Boxplots show upper and lower quartiles (box limits), the median (center line) and the 1.5* interquartile range (whiskers).

Extended Data Fig. 5 Masculinized arr17 flowers develop viable pollen.

a, Dehiscing anthers of a representative flower of an early-flowering arr17 mutant line release white pollen grains. Photo shows 40 magnification. The experiment was repeated twice with similar results. b, Fluorescein diacetate (FDA) staining indicates pollen viability42. Photo shows 200 magnification of stained pollen grains.

Extended Data Fig. 6 Pool-sequencing reveals a single region of chromosome 19 exhibiting marked female-specific coverage in P. alba.

a, The number of 1 kbp regions with sex-specific coverage (exceeding at least 50% of the expected haploid coverage), indicative of hemizygosity, for a pool of seven female (magenta) and seven male (cyan) white poplar (P. alba) individuals in sliding windows (window = 100 kbp, step=25 kbp). DNA-seq data of the pools were mapped against the chromosome-level P. tremula v2.2 genome assembly44. b, DNA-seq coverage for the two P. alba pools, zooming into the major hemizygous region on chromosome 19. The location of the ARR17 gene is indicated by a gray arrow.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7 and Notes 1 and 2.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–8.

Supplementary Data 1

Contig of a P. deltoides genome assembly (Methods) containing part of the MSY harbouring the male-specific partial ARR17 duplicates used to infer the sketch in Fig. 1c.

Supplementary Data 2

Multifasta file of all ARR17 and ARR17 inverted repeat sequences used for the phylogenetic analysis shown in Fig. 4.

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Müller, N.A., Kersten, B., Leite Montalvão, A.P. et al. A single gene underlies the dynamic evolution of poplar sex determination. Nat. Plants 6, 630–637 (2020). https://doi.org/10.1038/s41477-020-0672-9

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