Use of a self-compatible diploid potato for mutagenesis and forward genetic studies.

Lead Research Organisation: Earlham Institute
Department Name: Research Faculty

Abstract

Studies of the genetics of many plants have benefited greatly from the use of mutants, either spontaneous or artificially induced, in which the function of one or more genes has been lost or modified. Mutagenesis can be achieved by use of mutagenic chemicals or other means (e.g. ionizing radiation), and of course all naturally occurring genetic is due to mutation occurring over long time periods. Mendel's famous experiments with peas utilized naturally occurring mutants differing from the 'wild type' in highly visual, single gene traits. Many crop varieties are themselves mutants. The barley variety Golden Promise is a mutant of an older variety Maythorpe. Mutations can be genetically mapped and various approaches can be used to identify the mutated gene, providing useful information, which can be applied to further research and to plant breeding. The crop plant potato is naturally an outbreeding tetraploid, which makes it extremely difficult to generate the sort of mutational variation that has been so useful in other crop plants. This is because potato has four copies of each gene, and to generate a plant that has a mutated version of all four copies of any one gene is virtually impossible. The same is true to a lesser extent in diploid potatoes which have only two copies of each gene (diploids). For this and other reasons genetic studies in potato have lagged behind other plants, and genetic analysis has had to rely on naturally occurring variation, most of which is manifest as continuous or 'non-discrete' variation. In this project we plan to address this by utilizing a species of potato (Solanum verrucosum) that is both diploid and which is a natural inbreeder, allowing the construction of 'homozygous' genotypes, where both copies of each gene are identical. In pilot experiments we have shown that, using S. verrucosum, it is feasible to generate populations of mutant plants that can greatly benefit genetic studies, which will have downstream impact on potato improvement. The mutant panel will be assessed for variation in traits relevant to potato breeding: tuber characteristics, plant architecture traits, tuber sprouting etc and a panel of ~100 interesting mutants will be selected for further study. In pilot experiments a few interesting mutants have been identified and these will be studied within this project. Some of these mutants including one having a clear 'dwarfing' phenotype will used to test a novel approach for isolating the gene that has been mutated. This will also entail generating a draft genome sequence of the genotype used for the mutagenesis. The dwarf mutant will also be tested for its fertility to see whether it is feasible to use this in future mutagenesis studies.
The outcomes of this project will be (1) The establishment of the first ever mutant collection of potato. (2) The elucidation of a draft genome sequence of the 'base genotype' of Solanum verrucosum. (3) The identification of candidate genes and mutated alleles for one or more of the mutants identified.

Technical Summary

Potato, one of the world's major crop plants, has not been subjected to the types of classical genetic analysis that have been employed in other plants. Being a polyploid outbreeder it has been impossible to generate mutational variation, that in other systems, has played such a critical role in elucidating the mechanisms and processes, which underlie plant developmental and related traits. Genetical studies in potato have been limited to the study of relatively few qualitative traits as well as a large number of continuously varying traits. In this project we will address this issue by using a self-compatible Mexican diploid tuber-bearing species of potato Solanum verrucosum to generate a population of EMS mutagenized plants, with a particular focus on identification of mutations that affect plant development, architecture and tuberization. Pilot experiments conducted at JHI have demonstrated the feasibility of this goal, and a few mutants with drastically altered plant development have been obtained and mapping populations for these are under construction. These mutants will be subjected to further genetic and sequence analysis within this project. To this end we will use a novel method, employing next generation sequencing of mutant and wild type bulks to localize a small number of mutants (up to 5) to a small region of the genome. We will also generate draft genome sequence from the progenitor S. verrucosum genotype (VER54-GW1), which will be mapped to the published DM potato genome assembly, as well as assembled de novo. This will provide a template for mutant mapping and isolation, and in addition, it will provide a valuable set of data for comparative genome analysis in potato, and which will contribute to the 'SOL100' initiative aimed at sequencing 100 Solanaceous plant genomes. One of the existing mutants that has a dwarf phenotype will be tested for its fertility to see whether it is feasible to use this in future mutagenesis studies.

Planned Impact

Who will benefit from this research?
The proposal is directly relevant to the BBSRC priority area in Crop Science/Food Security and Living with Environmental Change. The project also builds on BBSRC investment in sequencing the potato genome by generating a draft sequence of a strategically important diploid wild species with unusual biological properties, and to generate a scientific first for potato - a panel of genetic mutants that can be mapped and used to dissect potato biology.
Potato researchers will thus benefit directly. In the longer term, potato breeders and eventually farmers stand to benefit.
The staff employed on the project will benefit by gaining experience in a completely new set of activities. While TGAC staff have extensive experience of carrying out genome assemblies this will be their first potato genome sequencing project, and it will be interesting to see how well the large families of closely related genes and pseudogenes e.g NB-LRR genes assemble. Again while sequence variation analysis is routine, and TGAC have used the SHOREMAP software before, this was on test Arabidopsis data, real life data from a larger genome species will be an interesting test of the technique.

How will they benefit from this research?
TGAC staff will benefit from using new informatics tools and tackling new biological problems. They will have to meet project deadlines, and become more connected with the potato researchers and learn more about the underlying potato biology and genetics.
Potato researchers will benefit by having access to the new genome sequence, plus the mutant resources generated and the methods used to produce and clone them. The increased focus towards 'monogenic trait biology' will also be a benefit to potato scientists who have customarily worked on the analysis of quantitative traits, which has allowed few opportunities for experimental science. For such scientists it is often quite frustrating to find that variation in a complex trait is controlled by several loci of small effect, and analysis ends at the QTL mapping stage! Having the opportunity to work with discontinuous trait variation would open up new scientific vistas that would enable real experimental science to be performed.
Potato researchers, breeders and eventually farmers will be aided by an improved understanding of the potato biology derived from this and future projects. We believe that this project forms an excellent pilot for future potato species and variety resequencing projects which will allow a better understanding of potato biology: plant shape, tuberisation, yield, ability to thrive in a variety of environments and survive pathogens e.g. blight.

The project will provide multi-disciplinary training activities for all staff employed on the project, delivering scientists with a high level of scientific and communication skills. The nature of the work will entail the TGAC researchers acquiring new skills such as using the latest genome assembly tools to combine short and long read technologies, and using bulk segregant methods to clone forward genetic mutants. Because we place great importance on excellent communication between teams, and true understanding of how best to meld genetics and genomics together to solve biological problems, TGAC will host the JHI PDRA for two months, training them in informatics skills to use the genome sequence and identify candidate SNPs from the SHOREMAP technique. This exchange of knowledge and skills is bi-directional, the PDRA will share their deep knowledge of the existing potato genome, genetics and biology to highlight the best genome assembly and how to make it more useful for potato researchers. For example the PDRA will use the genome browser and other tools to highlight areas of the genome that are difficult to assemble or particularly important e.g. the NB-LRR genes and also genome browser tracks that can be used to better highlight features for biologists.

Publications

10 25 50
 
Description Most genome assembly methods are tested on microbial (small genomes) or human (not representative of metazoans let alone eukaryotes). Most of these perform more poorly on plants because 1) high quality plant DNA is much harder to extract 2) Plant genomes are often more complex than human in terms of size, repetitiveness, heterozygosity or even polyploidy. We have compared numerous genome assembly methods to find the best for our plant Solanum verrucosum, a wilf potato species.
Exploitation Route Understanding the impacts of different plant genome assembly methods and using that to tune their own projects. The availability of the genome, plus forward and reverse genetic mutant resources will aid in understanding potato genetic and tuber development.
Sectors Agriculture, Food and Drink,Environment

URL https://doi.org/10.1101/201830
 
Description By other groups interested in genome assembly, including plant breeding companies e.g. KeyGene and commercial sequence providers e.g. GATC and providers of sequencing equipment e.g. BiNano Genomics
First Year Of Impact 2016
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Advances in Genome Biology and Technology conference (Florida) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A poster describing the BioNano scaffolding for potato with other BioNano activities, presented by Kirsten McLay.
Year(s) Of Engagement Activity 2016
URL http://www.agbt.org/meetings/agbt-general-meeting/
 
Description Illumina users group meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation: "Plant genomics - assembling genomies and understanding haplotypes"
Year(s) Of Engagement Activity 2018
 
Description Mathematical and Statistical Aspects of Molecular Biology conference (Helsinki) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation in an international conference, describing our newest sequencing technologies and results. Informed the mathematics and statistics people of the technology development of the datatypes that will come their way soon.
Year(s) Of Engagement Activity 2015
URL http://masamb2015.hiit.fi
 
Description PAG conference 2016 - S. verrucosum, a Wild Mexican Potato As a Model Species for a Plant Genome Assembly Project 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation on 10 January 2016 at the International Plant and Animal Genome Conference - San Diego, CA, USA - on the Wild Mexican Potato as a potential model system
Year(s) Of Engagement Activity 2016
URL https://pag.confex.com/pag/xxiv/webprogram/Paper20356.html
 
Description Plant and Animal Genomes Conference (California) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I have a talk in a workshop about Solanaceae (tomato, potato, eggplant, pepper) session of the Plant and Animal Genomics conference. The session was attended by researchers and breeders for these species and they learnt about the wonderful development of genomic resources we do at TGAC, and learnt how to do similar things themselves.
Year(s) Of Engagement Activity 2016
URL https://pag.confex.com/pag/xxiv/webprogram/Paper20356.html
 
Description Poster at AGBT 2015 - Low Cost Assemblies To Transform Comparative Genomics 
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 Poster to Advances in Genome Biology and Technology - Marco Island, FL, USA - 1 March 2015

Opportunity to discuss with peers and colleagues the approach being taken within TGAC
Year(s) Of Engagement Activity 2015
 
Description Poster to Mathematical and Statistical Aspects of Molecular Biology - New Technologies for Plant Genome Assembly 
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 Poster at the conference - Mathematical and Statistical Aspects of Molecular Biology - University of Helsinki - 16 April 2015

Opportunity to discuss with peers the S Verrucosum assembly and the ways we are attacking it using new technologies
Year(s) Of Engagement Activity 2015
URL http://masamb2015.hiit.fi/
 
Description Presentation to AGBT - Low Cost Assemblies To Transform Comparative Genomics 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation to the Advances in Genome Biology and Technology conference - Marco Island, FL, USA

Opportunity to present and discuss with peers the process developed within TGAC
Year(s) Of Engagement Activity 2015
 
Description Throwing the kitchen sink at Solanum verrucosum: genome assemblies integrating PacBio, Bionano and other tools 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact I was invited by BioNano Genomics to present on our work comparing different genome assembly methods in plant genomics
Year(s) Of Engagement Activity 2016