Can Cyclin Dependent Kinase Activity be manipulated to control chromosome pairing and recombination in plants?

Lead Research Organisation: Aberystwyth University
Department Name: IBERS

Abstract

This project will address an important problem, which has hampered the efficient exploitation of the genetic diversity held within wild relatives of wheat. Domestication resulted in a significant genetic bottleneck with the result that breadwheat is much less diverse than its wild relatives. Being able to work with wild relatives so that beneficial characteristics can be introduced into commercial wheat will be a major scientific achievement and dramatically improve the way breeders can generate new varieties of wheat with increased performance.

Some wild relatives are adapted to thrive under different climatic conditions to that of domestic wheat, or they carry natural resistance to important diseases and/or carry other important characteristics, which could influence yield. What we want to do is to develop approaches that will enable us to exploit this diversity effectively so as to introduce these favourable characteristics into wheat. In doing so we will be enable wheat breeders, amongst others, to improve wheat performance in a sustainable way, increase yield, and introduce disease resistance and drought tolerance.

What stops these wild relatives being used efficiently? Ideally, the wild relative and the wheat chromosomes should align and efficiently exchange (recombine) during meiosis but this does not occur effectively. Without recombination, there isn't the opportunity to introduce the genetic diversity of wild relatives into wheat. A genetic element called Ph1 controls this process. Ph1 has a positive effect in wheat itself, by stabilizing wheat as a polyploid during meiosis, but Ph1 does this by substantially reducing recombination between wild relative and wheat chromosomes or between segments of these chromosomes. Ph1 even reduces recombination between chromosomes derived from wheat landraces where they are significantly diverged. This makes gene transfer by recombination during meiosis difficult in the case of wild relatives, or inefficient in the case of landraces. Deletion of Ph1 enhances recombination but is very deleterious because it perturbs stability of the polyploid genome.

So how can we overcome this problem? In wheat and its hybrids, Ph1 regulates recombination. Understanding this regulation provides an insight into this process. It will provide us with an understanding of how the recombination process can be altered and tailored for specific needs, thus enabling us manipulate it for plant breeding. In understanding how to enhance recombination, the project will also identify approaches which prevent homoeologous recombination between chromosomes in wheat itself, and so stabilize it as a polyploid.

Our research focuses on the role of particular kinase-like genes found within the Ph1 locus. Kinases regulate or control the function of other proteins by means of transferring a phosphate group from ATP to particular amino acids within the target protein. They are highly sensitive to a range of compounds, including ATP analogues, that have been developed for biomedical purposes particularly in cancer biology and medicine. Therefore, there is a tremendous resource available for testing and identifying compounds that would be efficacious in the modulation of these related plant kinases. Moreover, we have recently identified Ph1 related kinases in the experimentally amenable model plant, Arabidopsis, that are phylogenetically conserved across all species, from plants to animals including humans.

In this project, we aim to investigate the role of kinase activity in chromosome pairing and recombination with the view to developing chemically mediated methods to modulate the activity of these in meiosis,. Such chemical tools would be tremendously useful in not only wheat breeding but potentially for other species as well.

Technical Summary

Most related chromosomes of wild relatives of wheat exhibit extensive gene synteny along their chromosome length. The genes on these related chromosomes exhibit more than 95% homology at the sequence level. Despite this level of similarity, there is little recombination between wild relative and wheat chromosomes at meiosis due the presence of the Ph1 locus. Ph1 even reduces (homologous) recombination between chromosomes derived from wheat landraces where they are significantly diverged. Deletion of the Ph1 locus allows the chromosomes to behave more like homologous chromosomes and recombine. Recombination involves the initiation of double strand breaks within genic regions and then repair of these breaks. Recent data indicates that whether Ph1 is present or absent, homoeologues pair and the recombinational machinery is loaded. The final marker for crossing over MLH1 is even loaded, but the process then stalls unless Cdk activity is increased or Ph1 is deleted.

Recently a Ph1 like kinase, CDKG, has been identified in Arabidopsis, and it also affects pairing and recombination. This opens up the possibility of exploiting this system for dissecting the regulation of kinase activity during meiosis, including the development of methodology for modulating its function.

To understand the function of these kinases, we will first produce tools such as specific antibodies that will allow their isolation and manipulation. Using these tools, we will assess the behaviour of these kinases and their molecular function, and their interaction with selected classes of small molecules. Finally, we will develop a reliable and robust system for delivering these compounds into wheat and wheat-wild relative spikes at various stages of meiosis, either in the presence of Ph1 or not, and assessing the promotion of either homologous chromosome pairing in wheat itself or homoeologous pairing in the wheat-wild relative hybrid.

Planned Impact

During the next 30 years, as much wheat grain will be required as has been produced since the beginning of agriculture. This will require a step change in breeding strategies. Breeders will need a combination of strategies including exploiting wild relatives of wheat carrying useful traits. One of the limiting factors highlighted by a number of high-level reviews (including by BBSRC) of such strategies is the ability to regulate chromosome exchange or recombination in wheat. The issue is further complicated by constraints placed on chromosome exchange resulting from the presence of related chromosomes. There are examples where wild relatives have been successfully exploited as a novel source of genetic variation for traits in wheat breeding programmes, e.g., the transfer by Sears of leaf rust resistance of Aegilops umbellulata to common wheat saved the US economy billions of dollars.

However, the introgression of genes from wild relatives into wheat is very time consuming and inefficient and therefore fell out of favour. Recently, international breeding centres have again started to exploit wild relatives to generate synthetic hexaploid Triticum aestivum, creating a "synthetic wheat" breeding programme. Some 25% of elite lines of wheat generated by CIMMYT are derived from crosses to these synthetic wheat genotypes. Having exploited this approach successfully, many argued that to increase yield production in wheat it is imperative to revisit the exploitation of genetic variation available in the wild relatives in breeding programmes. As a result of this, a number of private sector breeders encouraged the reestablishment of wheat alien introgression in the UK public sector, as part of BBSRC's wheat pre-breeding programme.

To facilitate the transfer of genetic variation via wheat/alien introgression, research is required to increase the speed and enhance the efficiency of the process. In brief, wheat/alien introgression involves hybridisation with a wild relative followed by repeated backcrossing to generate lines of wheat carrying an alien chromosome on which a target gene is located. A series of further crosses to specific genotypes/mutant lines are then required before the chromosome of the alien species can recombine with those of wheat, allowing the transfer of the target gene to wheat without linked deleterious effects.

The expected outcome of this project will be the creation of tools to allow breeders to better exploit the genetic potential of wild wheat relatives as well as enhancing "homologous" recombination between more divergent wheat chromosomes. It will provide an indication of how the level and distribution of recombination may be altered in wheat. The immediate beneficiaries will be those involved directly in wheat pre-breeding. The programme will also allow the development of a skills base, particularly in cytogenetics, a dying art in the UK which can be utilised by the next generation of scientists involved in wheat breeding and the larger wheat breeding community.

This research will make a contribution to food security and sustainable agriculture, key objectives of BBSRC's strategic plan. Wheat has been identified as a key crop by BBSRC and it has financially supported a new pre-breeding programme. Ultimately, the farming industry will benefit by having high-yielding, durable wheat cultivars tailored made to suit particular abiotic and biotic conditions. This in turn will have major societal benefits through the production of reasonably priced food produced in an environmentally sustainable manner.

Publications

10 25 50
 
Description - What were the most significant achievements from the award?

Our results

1. Show that CDKG, and in particular its protein kinase activity (the ability to add phosphate groups to other proteins) is necessary for normal meiotic crossing over (where the sister chromosomes exchange genetic material). CDKG1, however, is not directly required for chromosome pairing, which can be restored if crossovers are increased via other pathways. If CDKG1 is absent, the crossing over and pairing process can become less stringent (less fussy) and may even lead to recombination between non-homologous chromosomes. This has implications for plant breeding, particularly when making crosses between different species (Plant Cell submitted)
2. indicate that the kinase transcript undergoes temperature sensitive alternative splicing. This means that the transcript can make more than one protein and that the identity of these proteins differ at high and low temperature, perhaps explaining why temperature affects the extent to which chromosomes recombine with each other (Plant J 2018)
3. the different protein isoforms have been produced as epitope-tagged versions and these show different subcellular localisation patterns and stability, similar to mammalian CDK11
4. using a homologous recombination assay, we have demonstrated that the role for CDKG in recombination extends to somatic cells as well as meiocytes. This demonstrates that the kinase has a wider role in DNA repair /recombination ( Plant Cell submitted) than previously suspected and supports the observations that the Ph1 locus from wheat also has effects in somatic tissues.
5. using a genetics approach, we have placed CDKG upstream of the class I crossover pathway (Plant Cell 2020). These findings are reminiscent of aspects of the Ph1 phenotype in wheat where inappropriate pairing between non-homologous chromosomes leads to genomic instability.
6. other processes affected in a temperature-sensitive manner include flowering and perhaps seed germination (Frontiers in Plant Sci)
Exploitation Route How might the findings be taken forward and by whom?

1.potential targets for small molecule modulation of crossing over have been identified and compounds are being screened for effectiveness in planta (EU MC CoFund project)
2. transcripts subject to alternative splicing were identified in a collaboration with Barta lab, some of which are subject to modulation by CDKG and/or tempature. A search for possible meiosis related targets will be undertaken in a future project.
Sectors Agriculture, Food and Drink,Education,Pharmaceuticals and Medical Biotechnology

 
Description Partnering Award
Amount £30,000 (GBP)
Funding ID BB/R02118X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2018 
End 02/2021
 
Description PlantRecombination
Amount £158,882 (GBP)
Funding ID 663830-AU-110 
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start 02/2018 
End 02/2021
 
Description Allternative Splicing 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution we have supplied candidate gene lists with likely targets subject to alternative splicing
Collaborator Contribution they have screened these gene lists against their datasets for those transcripts showing temperature dependant alternative splicing
Impact No outputs yet. A grant application is planned
Start Year 2018
 
Description Allternative Splicing modulation by CDKG 
Organisation University of Vienna
Country Austria 
Sector Academic/University 
PI Contribution Provision of cdkg alleles
Collaborator Contribution alternative splicing expertise
Impact Paper published in Plant J
Start Year 2016
 
Description Community Science Project 
Organisation U.S. Department of Energy
Department Genome-Enabled Plant Biology for Determination of Gene Function
Country United States 
Sector Public 
PI Contribution we provided samples from grass to DOE
Collaborator Contribution they provided sequence information on genomes and transcriptomes
Impact manuscript in advanced draft
Start Year 2017
 
Description HARMONIA: Polish Research project "CDKG/Ph1: czy istnieje uniwersalny mechanizm regulujacy stabilnosc genomu u traw?" 
Organisation Faculty of Biology and Environmental Protection
Country Poland 
Sector Academic/University 
PI Contribution We provide the molecular biology and cell biology expertise to the Polish group. John Doonan is named foriegn collaborator on the grant (equivalent to responsive mode BBSRC grant) and the PDRA/students will spend significant periods of time in IBERS
Collaborator Contribution They are expert in molecular cytology and ploidy variants in grasses
Impact VOGEL JP, GORDON S, STEINWAND M, BRAGG JN, YOUNG H, BARRY K, SCHMUTZ J, CATALAN P, CONTRERAS B, HASTEROK R, DOONAN J, MUR L, BUDAK H, CHALHOUB B (2013) The genus Brachypodim as a tool to study perenniality and polyploidy. Invited lecture on the 1st International Brachypodium Conference, 19-21 June, Modena, Italy. Abstract Book, pp. 15 Visit to Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, United Kingdom (3 weeks - 2015; 3 weeks - 2014)
Start Year 2015
 
Description John Innes Centre 
Organisation John Innes Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution Led a joint responsive mode grant on control of chromosome pairing in model and crop plants
Collaborator Contribution wheat genetics and molecular probes
Impact n/a
Start Year 2015
 
Description John Innes Centre 
Organisation John Innes Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide phenotyping expertise for a funded BBSRC LoLa project on Brassicas
Collaborator Contribution They provide a variety of other expertises and germ plasm
Impact Funded BBSRC sLoLa; links to stakeholders in industry and policy
Start Year 2017
 
Description Trageted knock out of CDKG in crops 
Organisation John Innes Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided information to the BRACT transformation facility at JIC to allow the design of gene targeting constructs and verfied the targeting of the appropriate genes
Collaborator Contribution The BRACT transformation facility has provided several lines of Brassica with targetted genes; two putative Knock Outs have been identified
Impact on-going
Start Year 2018
 
Title microCT_grain_analyser 
Description The pipeline provides a simple method to segment and extract grain traits from 3D models 
Type Of Technology Software 
Year Produced 2018 
Open Source License? Yes  
Impact at least 2 other papers, one published and one accepted subject to modifications, will result from this software, in addition to on going collaborative grants 
 
Description Fascination of plants, Aberaeron 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact NPPC staff ran a stall, aimed at introducing plant biology and crop breeding to the general public. The exhibits were "hands on" and illustrated bio-diversity, adaptation as well as the use of tachnology to study and improve plants/crops
Year(s) Of Engagement Activity 2017
 
Description Hosting the 11th British Meiosis Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The 2019 British Meiosis Meeting will gather UK and international researchers with interests in meiosis, with presentations predominantly by postdocs and students, with informal discussions between the talks. The meeting will cover all aspects of meiosis, ranging from research into the molecular mechanisms that promote pairing, recombination and chromosome segregation in model organisms, to how these events impact human fertility and crop breeding. The two-day event provides a forum for PhD students and Postdocs to present their research
Year(s) Of Engagement Activity 2019
URL https://www.aber.ac.uk/en/ibers/news/events/britishmeiosismeeting/
 
Description NPPC website and data portal 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A general website including information on activities at NPPC, platforms available, resources generated in terms of data and software. A secure access portal is provided for transfer of unpublished data between the Centre and users.
Year(s) Of Engagement Activity 2015,2016,2017,2018,2019
URL https://www.plant-phenomics.ac.uk/
 
Description RWS, provided centre piece of IBERS marquee 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact NPPC provided rhizatrons and other phenotyping equipment as the centre piece in the IBERS marqee, with staff to explain the role of new computer assisted technologies in plant improvement and food security
Year(s) Of Engagement Activity 2018
 
Description Ystwyth Valley Farmers 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact This was one of many farming groups, including Young Farmers groups from across the UK, that have visited NPPC/IBERS over the past several years. The visitors are shown the breeding and phenotyping technologies and other science related topics such as climate change, GM, food science and environmental issues are often discussed.
Year(s) Of Engagement Activity 2019
 
Description visit Lord Bourne Parliamentary Under Secretary of State at the Wales Office 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Tour and discussion of the role of the NPPC as a National Facility, and its role in training
Year(s) Of Engagement Activity 2018