Rice Genetics IV - IRRI books - International Rice Research Institute

cific PCR primers to screen a DNA pool of deletion lines. If the target gene has adeletion bracketed by the primers, a length polymorphism would be detected in thePCR products. More recently, McCallum et al (2000a,b) described a technique calledtargeting induced local lesions in genomes (TILLING) to detect base-pair mismatches.In this case, primers are designed to bracket the target gene. A single base-pair mismatchbetween the mutant and wild-type DNA sequences results in a heteroduplexthat can be detected by denaturing high-performance liquid chromatography (DHPLC)or by specific enzyme cleavage precisely at the 3’-end of the mismatch (Oleykowskiet al 1998, Colbert et al 2001). The TILLING procedure is attractive as it is wellsuited for high-throughput screening. Furthermore, the high density of mutations inducedby EMS makes it possible to cover the genome with relatively few individuals(approximately 10,000 lines). We have generated a collection of EMS-induced mutantsand DNA pools are being produced and subjected to TILLING. Successful applicationof a reverse genetics system to the IR64 mutants will greatly enhance thevalue of the collection of mutants and increase our ability to assign sequences to eachof the mutant lines.IR64 mutant databaseWe have systematically collected phenotypic information on the mutant stocks withthe goal of linking such information to genomic databases. As rice genome data becomeavailable, we envision that all mutant lines can be assigned to one or morephysical locations on the sequence map. Together with other collections of mutants, itis feasible to assign an allelic series of insertions, deletions, and point mutations toevery gene in the genome.As a first step, we have used the International Crop Information System (ICIS)database to document phenotypic and molecular data on the IR64 mutant stocks. Anontology of component traits of mutant phenotypes is being defined to allow structuredsearches of the database. The database of mutants has about 200 phenotypicallydescribed mutants. A prototype Web interface (www.cgiar.org/irri/genomics/index.htm)has been developed to allow users to search the database according to mutant phenotypes.The next phase of database development will involve linking phenotypic datato molecular characterization of the mutants to facilitate the identification of candidategenes.ConclusionsThe IR64 mutant collection provides a needed resource for gene discovery, particularlyfor indica varieties that contain many adaptive traits for rice grown in the tropics.Our data suggest that the IR64 mutants exhibit a wide range of variability usefulfor detecting conditional mutants. We will expand the mutant collection with a rangeof deletion sizes and point mutations such that an efficient reverse genetics systemcan be developed using high-throughput genotyping methods. Through database linkages,we will place these mutants onto the chromosomal regions by association withknown mutants or traits and by reverse genetics.Deletion mutants for functional genomics: . . . 249