The Primula S locus: gene function and the maintenance and breakdown of heterostyly

Pollination is important, ~70% of our food results directly from pollination as seeds, grains, fruits and berries. It is important to plants too for reproduction, which not only produces the next generation but creates variation upon which natural selection can act. This variation enables plants to adapt to changing environments and colonize new habitats. Most plants produce hermaphrodite flowers, but plants cannot move or actively choose a partner, instead they have evolved intriguing strategies to prevent self-pollination and promote cross-pollination. One of the most remarkable of these strategies is heterostyly, which uses insect pollinators (hetero=different; style=female structure). Charles Darwin observed that Primroses have two forms of flower, pin with a long style and short stamens, and thrum with a short style and longs stamens. These reciprocal positions facilitate pollen transfer by insect visitors between each flower type. A group of genes, known collectively as the S locus, controls development of the two forms of flower. A rich history of scientific research on Primroses by early botanists and geneticist made heterostyly an important textbook example of a plant breeding system. Our study will add to this work by using the latest research tools to explore how heterostyly arose, how it is controlled and why it occasionally goes wrong.

We sequenced the Primrose (Primula vulgaris) genome and identified a group of five genes that control development of its two flower types. These genes are found only in thrum and are absent in pin plants. Only those individuals that inherit this gene cluster can produce thrum flowers. Our studies show the gene which we believe is responsible for elongating stamens in thrum flowers arose ~51.7million years ago in an ancestor of all modern Primula species. We have also identified the gene responsible for controlling style length. The gene cluster contains a further three genes and in this study we will characterize their function to determine how they work together to control the floral architectures of pin and thrum flowers that facilitate insect-mediated pollination in the Primrose. Over the past 51.7 million years, 30 different species of Primula have lost the ability to produce two types of flower, they all have long styles and long stamens and never produce short styled flowers. These homostyles (Homo = same) do not use insect pollinators, but self-fertilize. Our studies suggest the gene that controls style length has been lost or damaged in these species.

These homostyle species provide an exciting opportunity to explore how one gene has, over time, on multiple separate occasions, been lost or damaged during the evolution of the different Primula species. This is important because understanding how development has gone wrong can help explain how it normally works. We will use our Primrose genome sequence to identify and characterize the corresponding S locus gene clusters in these different Primula species to discover the gene mutation that has resulted in the loss of heterostyly. Most plant and animal genes are present in two copies, one set from the mother, one from the father; this is useful, if one copy is damaged, the second copy acts as backup so function is retained, it also enables repair of the damaged copy using the other as a template. This arrangement provides genetic stability from generation to generation. One very surprising finding from our studies of genes controlling heterostyly in Primula is that they are present only as a single copy, there is no back up copy. It is therefore surprising that heterostyly is stable as any genetic damage to the key genes cannot be repaired. We do not yet have an explanation for this conundrum but our project will initiate studies to seek an explanation.

Heterostyly facilitates insect-mediated pollination, it is found in ~430 Primula species which have two forms of flower, pin (long style, low anthers) and thrum (short style, high anthers); each form is self-incompatible (SI). We sequenced the Primula genome, identified the S locus as a cluster of 5 genes, CCMT, GLOT, CYPT, PUMT and KFBT, found only in thrums and absent in pins, and dated its origin to ~51.7 MYA before speciation in the Primulaceae. We characterised 2 independent long homostyle (LH) mutants (long style, high anthers) and showed that CYPT controls style length, and a short homostyle (SH) mutant (short style, low anthers) that suggests GLOT controls anther elevation. We will analyse 2 new SH mutants to confirm this role for GLOT. There are no mutants for the other 3 genes; we will use gain and loss of function transgenics to define their role in heterostyly and SI. Analysis of S locus gene expression in LH and SH mutants suggests CYPT and GLOT cross-regulate other S locus genes. We will define this regulation using transgenics and transcriptomics to reveal how di-morphic flower development is controlled by the S locus. Since its single origin, Primula heterostyly has broken down ~30 times leading to species with self-fertile homostyle flowers (high anthers, long styles). Our data show the S locus is hemizygous, not heterozygous; heterostyles must therefore arise by mutation of S locus genes. Hemizygosity raises a conundrum: how is the S locus stable if there is no opportunity for recombination repair of such mutants? We will use oligo-select capture of the S locus to define sequence variation in different heterostyle populations of P. vulgaris, and to analyse different homostyle species to determine whether independent loss of heterostyly is due to mutation in CYPT. We will also identify ancestral S locus architecture in out-groups. These studies will explore the origin, maintenance and breakdown of this pollination syndrome.

This project focuses on one of the most successful pollination syndromes involving insect-mediated pollination. Heterostyly evolved in the Primulaceae ~45 million years ago before speciation and widespread global distribution across five continents. Understanding the control of plant pollinator interactions is of direct relevance to food security and biodiversity in a changing world. ~70% of our food is the direct product of pollination and as climates change, the balance of plant pollinator interactions in crops and native species will also change; this imbalance, coupled to global pollinator decline, means that understanding the mechanisms controlling insect-mediated pollination will identify opportunities to adapt and modify genetic systems from model species into crop species.

Importantly, this highly adapted pollination system has, over the past 45 million years, broken down at 30 independent times, leading to self fertility and loss of reliance on insect pollinators. Studying how a system breaks is valuable in understanding how it works and therefore how it can be exploited. Following Darwin's discovery of this adaptation, historical studies in Primula mark landmark events in the history of genetics and the neoDarwinian synthesis. In combination, this project will have academic, technological and societal impact and will provide tools and resources that could be developed by others to have economic impact and support the knowledge economy.

Who will benefit and how?

Academic community: Academic beneficiaries include plant scientists working on reproduction and pollination, evolutionary biologists focused on gene and genome evolution, speciation and co-adaptation, developmental geneticists exploring plant architecture and cell-cell signalling, and ecologists studying plant-insect interactions, habitat loss and colonization, plus science historians interested in the contribution of this breeding system to modern scientific methods. Knowledge and insight from studies in one species are not limited to that species and can facilitate step change discoveries in unrelated areas.

Informed amateurs: Amateur gardening in the UK has an estimated economic value of at least £89 billion with the average adult spending 2-3 hours per week gardening; as a nation we spend £5bn a year on gardening. Public access to science and the understanding of important scientific advances need to be presented in a stimulating way that people can relate to. The huge audience of informed amateurs and enthusiastic gardeners will benefit from the accessibility of complex scientific knowledge through the vehicle of the familiar common primrose.

Future scientists: Engaging young audiences in the value of science and scientific discovery needs simple accessible stories. The sex life of the common primrose provides an example through which a spectrum of biological concepts can be presented in simple and appealing ways. Understanding the relevance and significance of such concepts through accessible and intriguing stores is an ideal way to engage, educate and enthuse future young scientists.

Agbiotech and horticulture industries: Primula is a key northern-clime horticultural crop, estimated EU annual market value ~400M Euro. Development of molecular genetic tools such as plant regeneration and transformation and genome resources in Primula has value and applications. Understanding the mechanisms that control floral reproductive architecture, and its impact on plant pollinator interactions, has the potential to identify genes and pathways that may be of value in engineering floral traits to increase reproductive productivity, develop responses to pollinator decline, or even prevent self pollination to facilitate hybrid seed production. This project has the potential for economic and food security impact by providing tools and resources that could be exploited and applied for commercial benefit.