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

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.

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.

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 sequencing 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. Even a modest number of novel mutants will have a large effect, as there are very few available at the current time. The sequencing of another potato genome from a wild species will start to inform scientists about the overall level of variation in this diverse group of species, and will generate a huge number of new SNP markers, not to mention candidate sequence variants for various mutations. Of course, by comparing S. verrucosum genome with the published DM genome, a very timely structural comparison between quite divergent potato genomes can be performed.
The staff employed on the project will benefit by gaining experience in a completely new set of activities that have not been performed in potato before. The technician has done all pilot work but a much larger project will provide a major organisational challenge. The PDRA will benefit in a similar way but will also gain experience in analysing large volumes of sequence data, and in designing a 'first generation vocabulary' for classifying potato mutants. The potato research community will benefit from this research by having access, for the first time, to mutant phenotypes. Additionally, the project will be of interest for plant scientists working on other species (e.g. Arabidopsis, tomato) as the new resources generated here will open up potato as a more tractable system for studying basic plant processes, some of which will impact on the applied aspects of potato research.
If we can gain insights into the mechanisms of tuber development and for how plant architecture impacts on yield and other aspects of agronomic performance, there will be longer term benefits for the potato industry.

How will they benefit from this research?
JHI staff working directly on the project will benefit from acquiring new skills, having to meet project deadlines, and become more connected with the external scientific environment as seed and mutants are provided to other scientists. Scientists will benefit by having access to both the mutant resources generated and the methods/seed used to produce 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.

Development of staff skills
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 researchers learning to detect and classify mutational variation in large populations of potato plants. This will be a completely different type of project to the more typical genetic mapping analysis that JHI scientists have been involved in previously, and thus will broaden the relevant staff's scientific expertise. The PDRA will visit TGAC for bioinformatics training to empower them to deal with large datasets, a key skill set in this age of next generation sequencing, and absolutely invaluable at JHI where such resources are already in demand and increasingly limiting.