S. latifolia sex-linked genes, p. 1 Evolutionary strata on the X chromosomes of the dioecious plant Silene latifolia: evidence from new sex-linked genes

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 eight 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 homologues on the X and Y chromosomes. Synonymous divergence estimated between the X and Y homologue pairs are 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 so far been studied, and that X-Y divergence increases with genetic distance from the pseudo-autosomal 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 about 10-20 MYA. Introduction Silene latifolia is a model system for the study of the evolution of plant sex chromosomes. The sex chromosomes of dioecious Silene species have several striking similarities with those of animals, including mammals (FILATOV 2005c; GUTTMAN and CHARLESWORTH 1998; NICOLAS et al. 2005), but they evolved independently and much more recently. The recent origin in a largely hermaphroditic plant genus, and the evidence of synteny of sex-linked genes and their orthologues in the hermaphroditic species S. vulgaris (FILATOV 2005a), show clearly that, like mammalian sex chromosomes, those in S. latifolia evolved from a pair of ordinary chromosomes. Due to its recent origin, the S. latifolia Y chromosome is probably in an early stage of degeneration. It is large in size, and it has been suggested that this reflects a large gene content (NEGRUTIU et al. 2001). However, this must be tested; an alternative is that the large size of the Y might reflect a high repetitive DNA content, suggesting a stage in the degeneration process when repetitive sequences, including S. latifolia sex-linked genes, p. 3 transposable elements, have probably accumulated, but before the stage in which most individual genes have lost function and can be deleted. The Y chromosome indeed appears to have accumulated chloroplast sequences (KEJNOVSKY et al. 2006), and there is also evidence of repetitive sequences and transposons in the S. latifolia genome (PRITHAM et al. 2003), but the extent of male-specific (Y-linked) sequence accumulation is not yet clear, though Yspecific sequences certainly exist (DONNISON and GRANT 1999). Similarly, the small Y-chromosome-like region surrounding the sex-determining region in papaya (which may possibly have evolved more recently than the S. latifolia sex chromosomes) has a higher repetitive sequence content (and thus a lower gene density) than the genome as a whole (LIU et al. 2004). To make progress in understanding sex chromosome evolution and organization in plants, and to test for genetic degeneration of Y chromosomes, sex-linked genetic markers are required. Several kinds of Y-linked genetic markers have been developed in S. latifolia, including anonymous markers such as AFLP and RAPDs (DI STILIO et al. 1998; NAKAO et al. 2002; OBARA et al. 2002). Although it is straightforward to develop such anonymous markers, and these are useful for obtaining genetic maps of the sex chromosomes (LEBEL-HARDENACK et al. 2002; MOORE et al. 2003; SCOTTI and DELPH 2006; ZLUVOVA et al. 2005), they provide no information about the age of the sex chromosome system, or the times since recombination between the X and Y stopped in different regions of these chromosomes, nor about whether the Y chromosome is genetically degenerated or degenerating. Genic markers are thus potentially much more valuable than anonymous ones. Such markers provide access to the genes’ coding sequences, containing synonymous and non-synonymous sites which are subject to different selective constraints in functional copies (GILLESPIE 1991; OHTA 1995), so that it becomes possible to estimate the divergence time between the X and Y copies, and to test for genetic degeneration of Y-linked copies (GUTTMAN and CHARLESWORTH 1998; NICOLAS et al. 2005). Such studies are progressing rapidly for the neo-Y chromosome of Drosophila miranda (BACHTROG 2003; BACHTROG 2004). Only seven genes on the Y chromosome of Silene latifolia have been described after almost a decade of work (ATANASSOV et al. 2001; DELICHERE et al. 1999; FILATOV 2005c; GUTTMAN and CHARLESWORTH 1998; MATSUNAGA et al. 2003; MOORE et al. 2003; NICOLAS et al. 2005). One of these has no X counterpart, being S. latifolia sex-linked genes, p. 4 duplicated from an autosomal gene (MATSUNAGA et al. 2003), and only one is degenerated (GUTTMAN and CHARLESWORTH 1998). The five X-linked genes so far mapped are arranged along a gradient of X-Y synonymous divergence (FILATOV 2005a), increasing with distance from the pseudo-autosomal region (FILATOV 2005a; NICOLAS et al. 2005), though neither family mapped all these genes. These findings suggest progressive steps in the cessation of recombination between the X and Y chromosomes, thus creating “evolutionary strata” on the sex-chromosomes, similar to those described in mammalian X and Y chromosomes (LAHN and PAGE 1999). In the S. latifolia sex chromosomes, three divergence levels have been suggested. The two genes, SlX3/Y3 and SlX4/Y4, with the highest divergence have synonymous site divergence (Ks) above 15%, while, for the least diverged pair, SlX1/Y1, Ks is only 3.6% (and intron divergence about 2%), and two gene pairs, DD44X/DD44Y and SlSSX/SlSSY, have intermediate divergence (Ks ~ 7-8%). With only five loci, discrete groupings of Ks values cannot be statistically significant, and the number is too low to formally test the ordering along the X chromosome of genes with different X-Y divergence, in “evolutionary strata”. To answer these questions, and to help understand the evolution of sex chromosomes, we use straightforward genetic approaches to identify sex-linked genes in S. latifolia, based on cDNA sequences obtained from this species. By using segregation analysis of intron size variants (ISVS) and single nucleotide polymorphisms (SNPs), we identify four new Y-linked loci with homologues on the X chromosome. Comparison of silent site divergence between pairs of X/Y homologues together with genetic mapping of the X-linked copies confirm the existence of a gradient in divergence (evolutionary strata) of genes in this sex chromosome system, which is much younger than the sex chromosomes of mammals or birds Materials and Methods Silene latifolia families and DNA samples: Male and female S. latifolia plants were grown from seeds collected from European natural populations (Table 1). Progeny from a total of four F1 families generated by between-population cross-pollination of females were used for segregation analyses. Genetic mapping (described in detail S. latifolia sex-linked genes, p. 5 below) was done using an F2 family of 92 plants generated by crossing two members of the F1 family H2005-1, a full sibship from crossing a female plant from France with a male from the Netherlands (Table 1). Genomic DNA from S. latifolia individuals was obtained from fresh leaves using the FastDNA kit (Q-biogene) following the manufacturer’s instructions.