Changes in gene expression during sex chromosome evolution in the dioecious plant Silene latifolia

We propose studies of expression levels of genes on the sex chromosomes of the plant Silene latifolia. This plant is one of the minority of plant species that is not hermaphroditic, but has separate sexed individuals, and an X/Y sex chromosomal sex-determination system like that in mammals, including humans. It is well known that the Y (male-determining) chromosomes evolved from an ancestral chromosome that was similar to other chromosomes, and the X chromosomes in female mammals are still not very different from other chromosomes, though they have some distinctive features that probably reflect their evolution as sex chromosomes. The Y chromosomes, however, are extremely strange in both mammals and in the other very well studied system, the fruitfly, Drosophila. In both cases, the Y chromosomes carry only very few genes, and only a small proportion of them are present on the X (in Drosophila melanogaster, no genes appear to be shared between the X and Y chromosomes, and in mammals there is a handful of such genes); in both cases the Y has acquired genes not present on the X. How these strange genetic properties evolved is an interesting question. Theories for the process of 'genetic erosion' (or 'degeneration') of Y chromosomes from an initial state in which most loci were shared with the X chromosome, suggest that genes on Y chromosomes lose their former functions and can then be deleted, and that this occurs because the X and Y chromosomes do not cross-over and undergo genetic recombination like other chromosomes. Population genetic models of regions of genome without genetic recombination predict that advantageous genes are less likely to be able to spread, and that natural selection will be unable to resist spread of disadvantageous genes. To study this genetic degeneration process, we need genes that are located on the sex chromosomes of a species in which degeneration is occurring. Because mammalian and fruitfly sex chromosomes evolved long ago, their Y have already lost most genes present on the X, so they are not well suited for such studies. Some plant species are known to have evolved sex chromosomes much more recently than these animals, and these are of interest for studying Y chromosome genetic erosion. We propose to work on the sex chromosomes of the plant Silene latifolia, the white campion. Its sex chromosomes have been studied cytologically since the early days of genetics, and in recent years a few genes on the sex chromosomes have been sequenced. Our project is to test in a direct manner for genetic degeneration by testing whether Y chromosomal copies of several genes are expressed at lower levels than X-linked copies of the same genes. This is now possible because we have developed a straightforward approach to identifying sex-linked genes (allowing us to discover some new genes on the sex chromosomes); thus there are now enough loci to gain a general picture of whether Y copies have low expression levels, and more genes on the sex chromosomes can readily be found. The project includes tests to show whether any differences found are due to low Y expression , versus the less likely, but also interesting, possibility of an increase in expression of X copies since the species separated from its hermaphrodite relative, the bladder campion, S. vulgaris. The expression experiments are possible because the X and Y copies are similar enough that short sequences of both can be amplified simultaneously in experiments, yet their sequences differ enough that expression of each of the two different alleles can be measured.

We propose to compare expression of S. latifolia Y-linked genes with those of X-linked homologues. SNP variants will be used to estimate expression levels by Pyrosequencing, a highly accurate method that is particularly suited for estimating relative expression levels of alleles without the need for 'control' genes or quantification of total RNA recovery. Short portions of two alleles are amplified. Each dNTP incorporated releases pyrophosphate (PPi) leading, via an enzymatic reaction, to production of light in proportion to the amount of nucleotide incorporated. Using two primers, two allelic types can be quantified. The equipment is available in the Ashworth laboratory sequencing centre. RB has experience with gene expression estimation using this equipment, including all aspects of primer design and testing. Sequence variants suitable for pyrosequencing assays are readily found between the Y and X homologues. RNA will be extracted from male whole seedlings (sexed using Y-specific markers) and from leaves and flower buds of adult male plants, to detect tissue-specific expression differences. X and Y chromosomes originating from different natural populations will be included. The Pyrosequencing assays will be replicated using RNA extracted from independent male plants, to test the null hypothesis of a 1:1 ratio of transcript abundances from the X and Y alleles. To test whether Y-linked genes have evolved lower expression or X-linked expression has increased, we will compare expression of the same genes in mixed samples of S.vulgaris/S. latifolia males and females. To obtain further sex-linked loci, we will sequence three thousands ESTs, select as many single- or low copy number genes as possible (based on by BLAST searches of other plant genomes) and test for sex-linkage. 5'- and 3'-RACE reactions will be used to obtain complete coding sequences of the new genes. The new genes will also be genetically mapped using a family already created.