Discovery of chemically induced mutations in rice by TILLING.
BACKGROUND
Rice is both a food source for a majority of the world's population and an important model system. Available functional genomics resources include targeted insertion mutagenesis and transgenic tools. While these can be powerful, a non-transgenic, unbiased targeted mutagenesis method that can generate a range of allele types would add considerably to the analysis of the rice genome. TILLING (Targeting Induced Local Lesions in Genomes), a general reverse genetic technique that combines traditional mutagenesis with high throughput methods for mutation discovery, is such a method.


RESULTS
To apply TILLING to rice, we developed two mutagenized rice populations. One population was developed by treatment with the chemical mutagen ethyl methanesulphonate (EMS), and the other with a combination of sodium azide plus methyl-nitrosourea (Az-MNU). To find induced mutations, target regions of 0.7-1.5 kilobases were PCR amplified using gene specific primers labeled with fluorescent dyes. Heteroduplexes were formed through denaturation and annealing of PCR products, mismatches digested with a crude preparation of CEL I nuclease and cleaved fragments visualized using denaturing polyacrylamide gel electrophoresis. In 10 target genes screened, we identified 27 nucleotide changes in the EMS-treated population and 30 in the Az-MNU population.


CONCLUSIONS
We estimate that the density of induced mutations is two- to threefold higher than previously reported rice populations (about 1/300 kb). By comparison to other plants used in public TILLING services, we conclude that the populations described here would be suitable for use in a large scale TILLING project.
Till
Bradley J
BJ
Fred Hutchinson Cancer Research Center, Seattle, WA 98107, USA. btill@fhcrc.org
Cooper
Jennifer
J
Tai
Thomas H
TH
Colowit
Peter
P
Greene
Elizabeth A
EA
Henikoff
Steven
S
Comai
Luca
L
D000383:Agriculture; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification; D005020:Ethyl Methanesulfonate / Q000494:pharmacology*; D008770:Methylnitrosourea / Q000494:pharmacology*; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation; D012275:Oryza / Q000187:drug effects / Q000235:genetics*; D019810:Sodium Azide / Q000494:pharmacology*
D018744:DNA, Plant; D009153:Mutagens; D008770:Methylnitrosourea; D019810:Sodium Azide; D005020:Ethyl Methanesulfonate