An efficient method for producing an indexed, insertional-mutant library in rice

Generation of an indexed, saturated, insertional-mutant library is an aid to understanding the functions of genes in an organism. However, 10 years of work by many investigators have not yet yielded such a library in rice. The major reason is that determining the chromosomal locations of a very large number of random insertion mutants by flanking sequence analysis is highly labor intensive, and therefore, libraries that do exist have not been indexed. We report here an efficient procedure to construct an indexed, region-specific, insertional-mutant library of rice. The procedure makes use of efficient long-PCR-based high-throughput indexing, coupled with a random but anchored population of Ds transposants. Long-PCR indexing allows rapid and simultaneous determination of the chromosomal locations of a large number of mutants that surround a particular anchor line, thus converting a random library into an indexed one. Such a library can be used directly, without the need to screen a large random library for a desired mutant plant.