Molecular evidence for recent divergence of X- and Y-linked gene pairs in Spinacia oleracea L.

Dioecy has evolved recently and independently from cosexual populations in many angiosperm lineages, providing opportunities to understand the evolutionary process underlying this transition. Spinach (Spinacia oleracea) is a dioecious plant with homomorphic sex chromosomes (XY). Although most of the spinach Y chromosome recombines with the X chromosome, a region around the male-determining locus on Y does not recombine with its X counterpart, suggesting that this region might be related to the evolution of dioecy in the species. To identify genes located in the non-recombining region (MSY, male-specific region of Y), RNA-seq analysis of male and female progeny plants (eight each) from a sib-cross of a dioecious line was performed. We discovered only 354 sex-chromosomal SNPs in 219 transcript sequences (genes). We randomly selected 39 sex-chromosomal genes to examine the reproducibility of the RNA-seq results and observed tight linkage to the male-determining locus in a spinach segregating population (140 individuals). Further analysis using a large-scale population (>1400) and over 100 spinach germplasm accessions and cultivars showed that SNPs in at least 12 genes are fully linked to the male-determining locus, suggesting that the genes reside in the spinach MSY. Synonymous substitution rates of the MSY genes and X homologues predict a recent divergence (0.40 ± 0.08 Mya). Furthermore, synonymous divergence between spinach and its wild relative (S. tetrandra), whose sex chromosomes (XY) originated from a common ancestral chromosome, predicted that the species diverged around 5.7 Mya. Assuming that dioecy in Spinacia evolved before speciation within the genus and has a monophyletic origin, our data suggest that recombination around the spinach sex-determining locus might have stopped significantly later than the evolution of dioecy in Spinacia.