Yeah, I know, it is a bad bumper sticker. But how often do you actually get to use it legitimately?? Recently I came across an article that made me think back to developmental biology in grad school and the amazing work that was coming out of the giant zebrafish mutant scans.
A Mother Lode of Mutant Fish (Science 1996, may require subscription) Researchers this month are announcing the first major findings from an international project to generate thousands of mutant zebrafish with telltale embryonic defects. The faulty fish are expected to yield fundamental insights into embryogenesis, the process by which an organism’s genetic blueprint unfolds into a body that can slither, swim, or saunter.
Of course, this followed on the heels of the giant Drosophila screens that yielded so much information. And preceded that Nobel prize for that work…
From the article: French Researchers Create Genetic Reference Collection of Pea Mutants
Senior author Abdelhafid Bendahmane, a researcher with the French National Institute for Agricultural Research’s Plant Genomics Research Unit, and his colleagues used an approach called Targeting Induced Local Lesions In Genomes, or TILLING — a targeted genetic modification based on ethane methyl sulfonate induced mutagenesis — to create a group of characterized pea mutants within one genetic background.
The work is reported in Genome Biology in February. Most of the first section of paper describes the benchwork processes of creation of the mutant lines, with EMS mutagenesis rather than the more common techniques in plants of insertional mutagenesis dues to some issues with that in peas, followed by the phenotyping strategies to locate variants. They developed a pea phenotype ontology to describe the mutants, describing visible characteristics in the parts above ground. They describe:
The most commonly observed phenotypes are related to stem size, leaf and plant architecture, followed by those related to cotyledons, stipules and seeds, with the least abundant phenotypes being related to flowers, plantlet architecture and petiole morphology.
They went hunting for the sequence lesions with a process called TILLING, which they describe as “TILLING (targeting induced local lesions in genomes) uses EMS mutagenesis coupled with gene-specific detection of single-nucleotide mutations”. Since the pea genome is not one that is available, they use comparisons to Medicago truncatula, which is underway. They used CODDLE (Codons Optimized to Discover Deleterious LEsions) and Primer3 to identify amplicons for sequencing. They found they had created an average of 34 alleles per “tilled” gene that they examined, with ratios of mutation of these types: “66.75% missense, 28.51% silent and 4.74% stop mutations”. With this in hand, they set up the database.
UTILLdb is the searchable database that contains this information. It includes information about the mutant lines, their phenotypes, sequences and mutations (when available). You can search with BLAST, or by keywords for the phenotypes. There may be images of the mutations in some cases. They have set up a way to order seeds as well. So yes, the world can have peas. Ahem. See, I got there eventually.
One of the other nice things about this paper is that they describe so many useful databases and analysis tools that they used for this project. If you want to explore some strategies for larger-scale mutational screening projects this provides some guidance. CODDLE offers the only submit button I have ever seen that allows you to “CODDLE Your Gene”. Primer3 is an old favorite of ours for primer identification. SIFT (Sorting Intolerant From Tolerant) we find used in a number of important projects to identify possibly damaging amino acid substitutions. They also used PARSESNP to align sequences and evaluate any SNPs they found.
There are a whole bunch of other plant databases that they describe in the paper that I’m going to investigate and will report back on later. Many of them were new to me. I’m developing a real fondness for plant research now that my major protein sources are plant based these days, I guess. I’m glad to have learned about the Grain Legumes Integrated Project from this paper as well.
Dalmais, M., Schmidt, J., Le Signor, C., Moussy, F., Burstin, J., Savois, V., Aubert, G., Brunaud, V., de Oliveira, Y., Guichard, C., Thompson, R., Bendahmane, A. (2008). UTILLdb, a Pisum sativum in silico forward and reverse genetics tool. Genome Biology, 9(2), R43. DOI: 10.1186/gb-2008-9-2-r43