Genomic analysis and characterisation of the Primula S locus.

Lead Research Organisation: University of East Anglia
Department Name: Biological Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/H019278/1 01/01/2011 01/09/2011 £382,414
BB/H019278/2 Transfer BB/H019278/1 02/09/2011 30/06/2014 £297,981
 
Description We have pursued our search for the genes located at the Primula S locus which control floral heteromorphy The sequence of a BAC contig spanning the S locus has been completed and several new S locus linked genes identified, these will form the basis of further studies. We have also completed a de novo sequence assembly of the Primula genome. We subsequently identified and characterized the gene cluster that controls heterostyly and identified the five genes that control Primula flower development as described by Charles Darwin.
Exploitation Route We have published four research papers and a review, a further manuscript describing the Primula vulgaris genome sequence is in preparation. We have also started discussions with a commercial Primula breeder about the opportunities for exploiting the genome sequence for Primula breeding, we have established a collaboration with a group of three Japanese laboratories. We also submitted a further research grant to BBSRC that builds on the data obtained during this award but it was not funded, a subsequent BBSRC grant application is under review.
Sectors Agriculture, Food and Drink,Education
 
Description We have published four research papers and a review, a further manuscript describing the Primula vulgaris genome sequence is in preparation. We have also started discussions with a commercial Primula breeder about the opportunities for exploiting the genome sequence for Primula breeding, we have established a collaboration with a group of three Japanese laboratories. We also submitted a further research grant to BBSRC that builds on the data obtained during this award but it was not funded, a subsequent BBSRC grant application is under review. The work has also had extensive coverage in Social media which has brought it to a very wide non scientific audience.
Sector Agriculture, Food and Drink,Education
Impact Types Cultural,Societal
 
Description Establishment of a transformation system for Primula vulgaris
Amount £32,815 (GBP)
Organisation Gatsby Charitable Foundation 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 01/2016 
End 06/2016
 
Description Studies on floral heteromorphy in Primula
Amount £80,000 (GBP)
Organisation University of East Anglia 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2015 
End 09/2019
 
Title Primula genome sequence 
Description Complete de novo assembly of the Primula vulgaris genome including annotation with transcriptome data 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact A new resource of a novel genome sequence and gene expression data that will lead to a further research publication 
 
Description Japanese collabrorative partnership 
Organisation Kobe University
Country Japan 
Sector Academic/University 
PI Contribution I initiated a collaborative partnership with Prof. Ryo Ohsawa (Tsukuba University), Prof Yasuo Yasui (Kyoto University) and Dr Yasuko Yoshida (Kobe University). Dr Yoshoda has applied for University sabbatical leave to join my laboratory in Norwich for a year, Dr Jonathan Cocker, a UEA-funded PhD student who worked on this project has applied for JSPS funding to visit our collaborating partners in Japan for 3 months.
Collaborator Contribution Our partners have provided plant material, DNA and expertise to enable development of a collaborative research program, Dr Yoshida has applied for funding to visit our lab and Prof. Ohsawa has agreed to host Dr Cocker from my lab.
Impact None yet
Start Year 2016
 
Description Japanese collabrorative partnership 
Organisation University of Kyoto
Country Japan 
Sector Academic/University 
PI Contribution I initiated a collaborative partnership with Prof. Ryo Ohsawa (Tsukuba University), Prof Yasuo Yasui (Kyoto University) and Dr Yasuko Yoshida (Kobe University). Dr Yoshoda has applied for University sabbatical leave to join my laboratory in Norwich for a year, Dr Jonathan Cocker, a UEA-funded PhD student who worked on this project has applied for JSPS funding to visit our collaborating partners in Japan for 3 months.
Collaborator Contribution Our partners have provided plant material, DNA and expertise to enable development of a collaborative research program, Dr Yoshida has applied for funding to visit our lab and Prof. Ohsawa has agreed to host Dr Cocker from my lab.
Impact None yet
Start Year 2016
 
Description Japanese collabrorative partnership 
Organisation University of Tsukuba
Country Japan 
Sector Academic/University 
PI Contribution I initiated a collaborative partnership with Prof. Ryo Ohsawa (Tsukuba University), Prof Yasuo Yasui (Kyoto University) and Dr Yasuko Yoshida (Kobe University). Dr Yoshoda has applied for University sabbatical leave to join my laboratory in Norwich for a year, Dr Jonathan Cocker, a UEA-funded PhD student who worked on this project has applied for JSPS funding to visit our collaborating partners in Japan for 3 months.
Collaborator Contribution Our partners have provided plant material, DNA and expertise to enable development of a collaborative research program, Dr Yoshida has applied for funding to visit our lab and Prof. Ohsawa has agreed to host Dr Cocker from my lab.
Impact None yet
Start Year 2016
 
Description Alumni News Letter article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Article in UEA alumni news letter on research undertaken on flower development in Primula
Year(s) Of Engagement Activity 2015
 
Description Fascination of Plants school event at JIC 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Fascination of Plants School engagement event organised at John Inness Centre
Year(s) Of Engagement Activity 2014,2015
 
Description JIC Youth STEMM Conference 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact STEMM engagement event with Schools
Year(s) Of Engagement Activity 2017
 
Description Observer on Sunday article 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Full page article in the Observer on Sunday that was picked up and reported in international media and on line
Year(s) Of Engagement Activity 2015
 
Description Public Lecture at Oxford Botanic gardens 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public Lecture at Oxford Botanic Gardens
Year(s) Of Engagement Activity 2016
 
Description Public engagement (JIC) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public engagement on research with Friends of John Innes which involved small group presentations and discussion on research project with the general public
Year(s) Of Engagement Activity 2014
 
Description Public engagement (Norwich) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Science in Norwich day a the City Forum where an audience of schools and general public engage with research activity.
Year(s) Of Engagement Activity 2015
 
Description Scientific Cafe event 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Scientific Cafe discussion event, captured by resident artist and broad social media coverage
Year(s) Of Engagement Activity 2017
 
Description Social media coverage of our 2016 Nature Plants paper 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Extensive social media coverage following press release and publication of Nature Plants journal article (LI, J., Cocker, J.M., Wright, J., Webster, M.A., McMullan, M., Dyer, S., Swarbreck, D., Caccamo, M., van Oosterhout, C. & Gilmartin, P.M. (2016) On the genetic and molecular architecture of the Primula S locus supergene. Nature Plants 2; Article number 16188). The press release was the second most trended news item on www.Phys.org that week. Altmetrics for the published paper show 17 news article around the world, 177 tweets, 2 blogs, 4 Facebook pages, with an Altmetric score of 239 which place it in and place it in the top 1% of the 219148 tracked articles of a similar age in all journals. There was other extensive social media and mainstream media coverage of the press release (as opposed to the published paper Altmetrics data) on Facebook and Twitter for the published paper). Tweeted <250 times by people with a cumulative following of 1.99M followers, and re-tweeted a further ~250 times by people with 2.46M followers. Although it is not possible to know how many people received the message it multiple times, the number of received tweets totals 4.45M within the first few weeks. The social media coverage alos generated a press release by the Seed Company Thompson and Morgan and an item in the RHS Plantsman Journal.
Year(s) Of Engagement Activity 2016,2017
URL https://www.uea.ac.uk/about/-/biologists-unlock-51-7-million-year-old-genetic-secret-to-landmark-dar...