Identifying sex-determining genes in the dioecious plant Silene latifolia – PlantGenomYX
Sequencing of the giant Y chromosome and identification of the sex-determining genes in the dioecious plant Silene latifolia
In 1923, Kathleen Blackburn discovered the existence of sex chromosomes in plants by studying Silene latifolia under the microscope. A century later, an international consortium led by Gabriel Marais and supported by the ANR has sequenced the genome of this plant, including its giant Y chromosome (550 Mb, ten times the size of the human Y), and successfully identified the sex-determining genes using an original approach.
Bridging the knowledge gap on sex determination in plants. Focus on Silene latifolia
Most flowering plants are hermaphroditic, with bisexual flowers that contain both male and female organs. However, about 15,600 species (5–6% of all flowering plants) are dioecious, with individuals bearing either female flowers (= females) or male flowers (= males). Dioecious plants are overrepresented among crops, and those that are dioecious (e.g., papaya, date palm, asparagus, cannabis, hop) or derived from a wild dioecious ancestor (e.g., strawberry, grapevine, hemp) account for approximately 13% of all cultivated species. The ancestor of all flowering plants was most likely hermaphroditic, and dioecy is a derived trait that has evolved recently and independently on numerous occasions—several hundred to several thousand times—among flowering plants. Unlike in animals, where sex determination is known for about 90% of species, this information is available for only about a hundred plant species, representing just a few percent of all dioecious species. Candidate genes responsible for sex determination have recently been identified in a few dioecious plants, such as persimmon, asparagus, strawberry, date palm, kiwifruit, and grapevine. Several population genetics models for the evolution of dioecy have been proposed, but there is still a lack of data to test them. Despite recent advances, sex determination in plants remains poorly understood. This limits (1) our understanding of the origin and evolutionary causes of separate sexes in plants, and (2) the development of biotechnological tools to improve the management of dioecious crop species. In this project, we identified the sex-determining genes in the dioecious plant Silene latifolia (Caryophyllaceae). We sequenced the male genome of S. latifolia, including its giant Y chromosome of approximately 550 Mb. This is the first time a Y chromosome of such size has been sequenced.
We followed the strategy below:
1. Obtain a high-quality male (XY) genome using long-read sequencing (ONT) combined with Hi-C/optical mapping approaches.
2. Identify candidate genes based on data from mutants carrying deletions on the Y chromosome and transcriptomic analyses comparing males, females, and natural hermaphrodites.
Task 1 – High-quality male genome
A male genome, essential for this project, was obtained by combining long-read sequencing, short-read sequencing (for error correction), Hi-C/optical mapping data, and a genetic map (for assembly). The assembly was optimized to produce separate sequences for the X and Y chromosomes. To identify the pseudo-molecules corresponding to the sex chromosomes, we used our genetic map and all previously known sets of sex-linked genes in S. latifolia.
Task 2 – Identification of candidate genes
We used two complementary approaches. The first relied on the analysis of mutants with altered sexual phenotypes carrying deletions on the Y chromosome: gene presence/absence profiles allowed us to pinpoint critical regions. The second relied on the functional annotation of genes in the assembled genome and on a transcriptomic study comparing male and female flowers at different stages of floral development (stages 5 and 8, which are critical for sex determination). By combining these two approaches, only a few candidates with an excellent profile for sex-determining genes were obtained.
All project results were published in an article in Science (Moraga et al. 2025).
We assembled the sex chromosomes of Silene latifolia at high resolution. The obtained X chromosome is 346 Mb long, while the Y chromosome reaches 485 Mb (excluding the pseudoautosomal region, or PAR). The sequences show that the Y chromosome is highly rearranged and degenerated, having lost more than half of its original genes, with a massive accumulation of repetitive sequences and a very high density of retrotransposons, much greater than that observed on autosomes.
Comparative analysis of X–Y gene pairs (gametologs) revealed three successive evolutionary strata of recombination suppression. The oldest one (S1) dates back to approximately 11 million years ago, corresponding to the estimated origin of the species’ sex chromosomes. Two more recent strata (S2 and S3), about 5 million years old, differ in their degree of rearrangement and synteny. The enlargement of both X and Y chromosomes coincides with the formation of S2 and with the accumulation of transposable elements.
Functional analysis of mutants carrying deletions on the Y chromosome allowed us to identify several candidate genes potentially acting as sex determinants. Three main regions are involved: (i) a factor inhibiting gynoecium development (GSF), (ii) a factor promoting stamen formation (SPF), and (iii) a male fertility factor (MFF). Among these, the gene slCLV3-Y appears to be a strong GSF candidate, while slCyp704B1-Y and slTHI1-Y are associated with pollen production (MFF). Another candidate, slSCL4-Y, may play a role through the expression of a non-coding RNA (SPF).
These results demonstrate that sex determination in S. latifolia results from the accumulation of mutations in several regions of the Y chromosome, combined with inversions that progressively suppressed recombination.
Our work represents a double breakthrough: (1) the sequencing of complex sex chromosomes using long-read technologies, and (2) a deeper understanding of plant sex chromosomes and the mechanisms of sex determination.
Our original approach is transferable to any dioecious plant, including cultivated species. It can enable the reliable identification of sex-determining genes in dioecious crops, particularly those with complex sex chromosomes. This knowledge will, in turn, make it possible to control the reproduction and sexual system of these plants.
Dioecy makes it possible to control crosses and is particularly valuable when breeding new varieties. Hermaphroditism, on the other hand, is very useful for maximizing yields and ensuring that 100% of the plants are productive (e.g., fruit-bearing).
About 15000 flowering plants are dioecious (= sex is carried by different individuals). These plants are over-represented among crops (~17%). Understanding sex determination in dioecious plants is thus important for both fundamental and applied research. However, despite recent advances on this question, very little data is available. The objective of this proposal is to identify the sex-determining genes in Silene latifolia, one the best studied dioecious plants. We will produce a high-quality XY male genome (thanks to long-read data), combine different approaches (mutant collection, transcriptomic data) to identify candidate genes, and validate and characterize those candidates (thanks to Crispr-cas9). Here are joined two partners (LBBE Lyon et IPS2 Saclay) with a strong expertise in genomics, bioinformatics, functional and evolutionary studies, which is needed for this proposal.
Project coordination
Gabriel Marais (BIOMÉTRIE ET BIOLOGIE EVOLUTIVE)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partnership
LBBE BIOMÉTRIE ET BIOLOGIE EVOLUTIVE
IPS2 Institut des Sciences des Plantes de Paris Saclay
Help of the ANR 572,168 euros
Beginning and duration of the scientific project:
October 2020
- 48 Months
