Genomic analysis and characterisation of the Primula S locus.

The classic textbook example of cross-pollination and reproduction in plants is that of the common Primrose. Primroses, like their close relatives including cowslips, have evolved a specialised mechanism to prevent in breeding. Unlike animals, the majority of plants are hermaphrodite and produce both male and female reproductive structures within the same flower. This causes a problem, to which evolution has provided an ingenious solution in the case of Primroses and their close relatives. Charles Darwin was the first to document the natural history of this phenomenon. As Darwin observed, Primrose plants produce one of two forms of flower, known as pin and thrum. These flowers are essentially mirror images of each other in terms of the positioning of their male and female reproductive structures. The male structures, called anthers, produce pollen; the female structure that receives the pollen is called the stigma. Pin flowers are so called because the female reproductive structures resemble a dress-makers pin, with the stigma present at the mouth of the flower, and anthers hidden within the flower tube. Thrum flowers, develop anthers at the mouth of the flower, and are so called after an old weaving term because their appearance resembles a tuft of thread or thrum, the stigma in these flowers is hidden within the flower tube. In pin flowers the female structure are high and the male structure low, in thrum flowers male structures are high and female structures are low. Given the positioning of male and female reproductive structures in the two forms of flowers, self-pollination does not occur as male and female parts of the flower are physically separated. However this reciprocal positioning in the two forms of flower facilitates cross pollination by insects. A bee visiting a thrum flower will carry pollen on its body to a pin flower where the reciprocal geometry will result in its presentation to the awaiting stigma. Similarly the transfer of pollen will occur between low anthers of a pin flower and low stigma of a thrum. Darwin observed this phenomenon and documented the fact that pin plants only cross with thrums and thrums only with pins, however he could not explain the mechanisms that control this amazing phenomenon. Geneticists in the early part of the 20th century provided an explanation for the observed pattern of inheritance, and predicted the presence of specific genes that control anther position and stigma height. However, rather surprisingly, nothing is known about the genes or mechanisms that control this text book model. Several years ago we embarked on a project to provide an explanation for Darwin's observations using the latest molecular biology techniques. We are now at a point where we are close to identifying the key genes that control the development of the two forms of Primula flower that so fascinate Darwin. This project will lead to the identification of these genes. The majority of plant derived food products, with the exception of root vegetables, cabbages and the like, are the direct result of fertilisation; fruits, seeds, grains and cereals. Even carrots and cabbages start life as seeds. Some crops cannot self pollinate, and in other plants that do, the production of hybrids is complicated self seed setting Although the Primrose is not a food crop, understanding the mechanisms that control its ability to avoid self pollination and optimise the use of insects to transport its pollen are of significance and relevance to the constant need to increase crop productivity. Although this work is aimed at understanding a fundamental mechanism in plant pollination, it has potential future applications to understand and manipulate pollination in crop plants. This is an issue of extreme importance given the on-going decline in the numbers of bees and other insects that many crop species rely on for pollination and seed production, and that we rely on for food security.

Floral heteromorphy in Primula is a textbook example of a highly evolved out-breeding system; plants produce one of two forms flower, pin or thrum. Pins have long styles, have low anthers and produce small pollen. Thrum plants develop short styles and have elevated anthers that produce large pollen. This geometry is orchestrated by three genes which control style length (G), pollen size (P) and anther height (A). The co-adapted linkage group also contains an SI system and is known as the S locus; it has two haplotypes, S and s. Gene order is known from rare recombinants. Thrum plants are heterozygous Ss (GPA/gpa); pin plants are homozygous ss (gpa/gpa). Dominant G reduces cell elongation to produce a short style; dominant A increases cell division in the corolla to elevate anthers in thrums. We have used classical and molecular genetics to generate a linkage map of genes within and around the S locus with the intention of identifying the genes which control floral heteromorphy. Not only is this a fundamentally important problem from an historical perspective, but it offers the potential to identify key regulators of floral architecture, self incompatibility and reproductive development underpinning plant pollinator interactions. We will expand our background work to complete a BAC contig spanning the locus; 0.5 Mb has already been assembled. We will use 454 DNA sequencing alongside our unique BLAST searchable Primula EST database to identify S locus genes. We will use in silico comparative genomics to explore potential gene functions and experimental analysis to define gene expression and function. We will identify long and short homostyle mutants defective in G and A and use genetic approaches to establish associations between genes and phenotypes. This work will not only provide an explanation for a classical textbook model, but will yield novel genes that could aid manipulation of floral architecture and reproductive behaviour.

The Primrose has an established place not only in scientific publications but also in literature and the visual arts. The existence of its two types of flower and dependence upon insect pollinators to effect fertilisation between the reciprocal forms is familiar to the vast majority of biologists as well as many members of the general public. When I talk to individuals and non-specialist audiences about our work, many are already familiar with the two forms of flower. The striking visual nature of our model system, the link to Charles Darwin, and the extensive history of Elizabethan Primula mutants all contribute to create a familiar system through which to bring together Victorian natural history and modern molecular genetics. Communications and Engagement There are three constituencies with whom we recognise the need to communicate. These are the bioscience industry for development and exploitation of any discoveries of commercial value, and with whom interactions are covered under Exploitation and Application below. The General Public, with whom we see opportunities for interactions in terms of public engagement with science, as well as through the relevance of our system in popular culture and the arts. The third constituency, although part of the general public, are children and the early stage of their education, for whom science needs to be portrayed as an exciting and stimulating activity and our systems offers a visual and engaging example. Collaboration Well established academic collaborations already exist with Neil Hall (Liverpool), we have collaborated with him previously on two projects which are still underway.. We also have a long standing collaboration with Dave Westhead (Leeds) with three joint publications. We also have an ongoing collaboration with Pat Heslop-Harrison (Leicester) in which we are seeking to develop extended chromosome fibre FISH to the analysis of the S locus. This work will proceed in parallel to the current project and any data obtained will be integrated into the current work. We are, as is demonstrated in Track Record, keen to collaborate. We will when appropriate seek to develop new collaborations to gain maximal benefit form new expertise. Exploitation and Application The first step in exploitation and application is recognition of potential. The key outputs of potential commercial significance from this current project are quite simply genes and their defined functions. In characterising the Primula S locus we anticipate defining genes of novel function or expression profile which could have potential applications in overcoming existing self incompatibility mechanisms, manipulating floral architecture to prevent self pollination in normally self fertile species or modifying floral architecture to alter a pollination syndrome, for example to remove reliance on a declining insect pollinator by engineering a floral architecture or pollen characteristics better suited to wind pollination. Capability All members of the project will get involved in maximising impact, although as PI I will take the lead responsibility. We will work with the University Commercialisation Office as needed, the Durham Media Office to ensure maximal publicity, as well as external agencies such as Campus PR as appropriate, we will strengthen links with the Durham University Schools Liaison Office, and build on our established links with the friends of the Botanical Garden to maximise the opportunity for promoting the impact of our work to all three recognised constituencies.