Abstract
Targeting Induced Local Lesions in Genomes (TILLING) provides a nontransgenic method for reverse genetics that is widely applicable, even in species where other functional resources are missing or expensive to build. The efficiency of TILLING, however, is greatly facilitated by high mutation density. Species vary in the number of mutations induced by comparable mutagenic treatments, suggesting that genetic background may affect the response. Allopolyploid species have often yielded higher mutation density than diploids. To examine the effect of ploidy, we autotetraploidized the Arabidopsis (Arabidopsis thaliana) ecotype Columbia, whose diploid has been used for TILLING extensively, and mutagenized it with 50 mm ethylmethane sulfonate. While the same treatment sterilized diploid Columbia, the tetraploid M1 plants produced good seed. To determine the mutation density, we searched 528 individuals for induced mutations in 15 genes for which few or no knockout alleles were previously available. We constructed tridimensional pools from the genomic DNA of M2 plants, amplified target DNA, and subjected them to Illumina sequencing. The results were analyzed with an improved version of the mutation detection software CAMBa that accepts any pooling scheme. This small population provided a rich resource with approximately 25 mutations per queried 1.5-kb fragment, including on average four severe missense and 1.3 truncation mutations. The overall mutation density of 19.4 mutations Mb(-1) is 4 times that achieved in the corresponding diploid accession, indicating that genomic redundancy engenders tolerance to high mutation density. Polyploidization of diploids will allow the production of small populations, such as less than 2,000, that provide allelic series from knockout to mild loss of function for virtually all genes.
Highlights
The growing availability of whole-genome sequences is spurring functional gene studies in species where specific tools for reverse genetics are not available
Mutagenesis is usually applied by the treatment of seed with a chemical mutagen in a manner that produces tolerable lethality and sterility of the treated individuals, the M1 plants, while allowing sufficient production of fertile M2 plants
One to four dead embryos occurred in 35% of M1 plants
Summary
The growing availability of whole-genome sequences is spurring functional gene studies in species where specific tools for reverse genetics are not available. TILLING efficiency (i.e. the cost of obtaining informative mutations per gene) depends on the characteristics of the population used and on the mutation density (Comai and Henikoff, 2006). High-Efficiency TILLING Population at least in part on the intensity used to mutagenize the target species and on its response. Since similar treatments yield different mutation densities in different species, cellular or developmental characteristics depending on the genetic background must play a role in the outcome. There is no information on the molecular mechanisms underlying variable mutation yields in plants Such knowledge should be useful for improving TILLING populations. Polyploids may be physiologically more tolerant of genotoxic treatments or more susceptible to mutagenesis Such changes, for example, could result from adaptive changes affecting DNA repair and genome maintenance taking place after polyploidization
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