Evolutionary strata on the X chromosomes of the dioecious plant Silene latifolia: evidence from new sex-linked genes

Abstract

Despite its recent evolutionary origin, the sex chromosome system of the plant Silene latifolia shows signs of progressive suppression of recombination having created evolutionary strata of different X-Y divergence on sex chromosomes. However, even after 8 years of effort, this result is based on analyses of five sex-linked gene sequences, and the maximum divergence (and thus the age of this plant's sex chromosome system) has remained uncertain. More genes are therefore needed. Here, by segregation analysis of intron size variants (ISVS) and single nucleotide polymorphisms (SNPs), we identify three new Y-linked genes, one being duplicated on the Y chromosome, and test for evolutionary strata. All the new genes have homologs on the X and Y chromosomes. Synonymous divergence estimated between the X and Y homolog pairs is within the range of those already reported. Genetic mapping of the new X-linked loci shows that the map is the same in all three families that have been studied so far and that X-Y divergence increases with genetic distance from the pseudoautosomal region. We can now conclude that the divergence value is saturated, confirming the cessation of X-Y recombination in the evolution of the sex chromosomes at approximately 10-20 MYA.

Figure 1.—

Fixed intron-size differences (in base pairs) among three S. latifolia sex-linked homologous genes: SlX1/SlY1, SlSSX/SlSSY, and DD44X/DD44Y. Intron-size differences exceeding the y-axis scale are given in boxes.

Figure 2.—

The ISVS method. Primers (shown as narrow bars) were designed in exonic regions close to intron boundaries. The forward (specific) primer contains a 5′ 20-bp tail complementary to a universally labeled primer and is added to the PCR reaction at limiting concentrations. This guarantees the incorporation of the universally labeled primer after a few PCR cycles. If the PCR product was >500 bp, restriction with a panel of restriction enzymes was performed before capillary electrophoresis analysis.

Figure 3.—

Inference of X and Y linkage for locus SlCypX/SlCypY. (a) Intron 3 variants. All S. latifolia individuals from several natural populations yielded a 447-bp band, and male plants have an extra 438-bp variant. (b) Segregation of intron 2 variants in the H2005-1 family after restriction with HaeIII. The male parent has 257- and 259-bp variants, whereas the female parent is homozygous for a 260-bp variant. The segregation of these variants follows a clear X linkage pattern in the F₁. All males inherit the 260-bp variant from the mother, while all females are heterozygous, inheriting both maternal and paternal variants. Moreover, because only the F₁ males inherit the 257-bp variant, this must correspond to the Y-linked intron variant.

Figure 4.—

Inference of X and Y linkage for locus SlX6a/SlY6a. Forward and reverse primers were designed within exon 1 and intron 2, respectively. Segregation of four amplicons (510 and 730 bp from the maternal plant and 590 and 1830 bp from the paternal plant) in the H2005-1 family is clearly consistent with X and Y linkage of the PCR size variants. All F₁ males, and no F₁ female, inherited the Y-linked 1830 bp from the parental male. All F₁ female plants inherited the 590-bp band from the father and either the 510- or the 730-bp band from the mother. Conversely, all F₁ males did inherit either the 510- or the 730-bp band from the mother, and not the 590-bp band from the father. An extra faint band observed in the maternal plant (size 610 bp) cosegregated with the 730-bp band in F₁ males. This probably represents an X-linked paralogous locus.

Figure 5.—

Inference of X linkage for locus SlX7. X linkage was inferred on the basis of an SNP causing a nonsynonymous substitution (I → S) at position +434 in H2005-1 family. The maternal plant was homozygous for T whereas the paternal plant was hemizygous for G. All male plants in the F₁ progeny were hemizygous for T, inheriting the maternal SNP, whereas all females belonging to the F₁ progeny were heterozygous (G/T), inheriting both the paternal and the maternal SNPs.

Figure 6.—

Gene structures of the new X- and Y-linked gene pairs. (a) SlCypX/SlCypY. (b) SlX6a/SlY6a. (c) SlX6b/SlY6b. (d) SlX7/SlY7. For the SlX6a/SlY6a pair, only partial genomic sequences are known (almost to the end of exon 3 of SlX6a and of exon 4 of SlY6a). The X and Y homologs exhibit strict conservation in gene structure, but the intron sizes often differ, with some large indels. Intronic insertions >3 bp are represented by triangles. Sizes in base pairs of exons and intronic insertions are given. Solid boxes represent the 5′ and 3′ untranslated regions.

Figure 7.—

Genetic map of the S. latifolia X chromosome (bottom), and the relationship with synonymous site divergence (K_s) of X-linked loci from their Y counterparts (top). The regression is highly significant (P = 0.001 for a linear regression or 0.012 with a Wilcoxon signed rank test). Map positions, in centimorgans, from the pseudoautosomal marker (OPA) are indicated. The markers shown below the diagram of the X chromosome are the new ones, and the previously mapped markers are shown above the diagram. Two of the three previously described markers, OPA, SlX3, and SlX4, were also mapped in our family, while the SlSSX, DD44X, and SlX1 map positions are based on previously published maps.