Genome assembly of the basket willow, Salix viminalis, reveals earliest stages of sex chromosome expansion

Abstract

Background: Sex chromosomes have evolved independently multiple times in eukaryotes and are therefore considered a prime example of convergent genome evolution. Sex chromosomes are known to emerge after recombination is halted between a homologous pair of chromosomes, and this leads to a range of non-adaptive modifications causing gradual degeneration and gene loss on the sex-limited chromosome. However, the proximal causes of recombination suppression and the pace at which degeneration subsequently occurs remain unclear.

Results: Here, we use long- and short-read single-molecule sequencing approaches to assemble and annotate a draft genome of the basket willow, Salix viminalis, a species with a female heterogametic system at the earliest stages of sex chromosome emergence. Our single-molecule approach allowed us to phase the emerging Z and W haplotypes in a female, and we detected very low levels of Z/W single-nucleotide divergence in the non-recombining region. Linked-read sequencing of the same female and an additional male (ZZ) revealed the presence of two evolutionary strata supported by both divergence between the Z and W haplotypes and by haplotype phylogenetic trees. Gene order is still largely conserved between the Z and W homologs, although the W-linked region contains genes involved in cytokinin signaling regulation that are not syntenic with the Z homolog. Furthermore, we find no support across multiple lines of evidence for inversions, which have long been assumed to halt recombination between the sex chromosomes.

Conclusions: Our data suggest that selection against recombination is a more gradual process at the earliest stages of sex chromosome formation than would be expected from an inversion and may result instead from the accumulation of transposable elements. Our results present a cohesive understanding of the earliest genomic consequences of recombination suppression as well as valuable insights into the initial stages of sex chromosome formation and regulation of sex differentiation.

Keywords: Recombination suppression; Salix; Sex chromosomes; W-chromosome; Willow.

Fig. 1

Identification of two evolutionary strata in the sex-determining region (SDR) of S. viminalis. Chromosome positions for S. viminalis and P. trichocarpa are shown in Mb with the S. viminalis scaffold names shown on the top. The two identified strata are shown with different hues of pink and labeled above the plot. a Anchoring of S. viminalis scaffolds to the autosomal chromosome 15 of P. trichocarpa. Forward alignments are drawn in blue and reverse alignments are drawn in red. b Log₂ differences of normalized SNP density between S. viminalis females and males in non-overlapping windows of 10 kb. A moving average of 25 windows is shown in the black line. The gray shaded area corresponds to the bootstrap 95% confidence interval of the autosomal data. c Log₂ differences of normalized read coverage between females and males in non-overlapping windows of 10 kb. Moving average and bootstrap statistics are as in b. Values close to −1 indicate twice the coverage in males in comparison with females, thus potentially Z-linked

Fig. 2

Alignment between S. viminalis and S. purpurea SDR regions. One-to-one orthologous alignments between S. viminalis scaffolds and chromosome 15 of S. purpurea, with forward alignments drawn in blue and reverse alignments drawn in red. The SDR region of S. purpurea is delimited by the gray shaded area (10.7–15.3 Mb, from Zhou et al. [66]). S. viminalis scaffolds anchored to chromosome 15 of P. trichocarpa are highlighted in bold, those inferred to be part of the S. viminalis SDR are underlined, and scaffolds well anchored with several colinear markers on other chromosomes are marked with an asterisk

Fig. 3

Comparison of polymorphisms at synonymous (d_S) and non-synonymous (d_N) sites. a Boxplots of d_S estimates. b Boxplots of d_N estimates. d_S and d_N were calculated based on the coding sequence alignment of phased diploid haplotypes from one female and one male individuals in the genome (excluding chromosome 15), the pseudo-autosomal region (PAR), the sex-determining region (SDR), and the more divergent stratum I. The inset plots show the quartile distributions of d_S and d_N estimates without outliers. Significant values from the Mann-Whitney U test relative to the genome are indicated with asterisks: *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 4

Examples of phylogenetic trees between gametologous gene pairs in the basket willow SDR. a, b The W-linked copy of the female gametolog is more divergent and does not cluster with the other S. viminalis haplotypes, indicating that suppression of recombination in stratum I occurred prior to S. viminalis speciation. c, d The female W-linked copy clusters within the species’ branch suggesting that recombination has been halted more recently. Female S. viminalis gametologs are indicated with red squares, and male haplotypes are in blue. Trees were estimated by maximum likelihood. Bootstrap values > 75% are indicated with black dots on the respective nodes. The poplar (P. trichocarpa) ortholog was used to root the trees

Fig. 5

Synteny analyses of Z- and W-linked resolved haplotypes. a Circular plots showing that scaffolds 148 and 211 are W-linked and align to the SDR of chromosome 15. From the outside to the center, (I) depicts the heatmap of log₂ females:males read depth in non-overlapping windows of 5 kb, (II) shows the repeat proportion in non-overlapping windows of 10 kb, and (III) indicates the location of annotated genes. Links between genes were computed from the best BLASTP hits and are color coded relative to the BLASTP alignment percent identity, with percent identity > 80% in blue and > 90% in red. Positions are shown in kb. b, c A highly conserved synteny between Z- and W-linked scaffolds