Reverse Genetics in Rice Using Tos17

Transposon of Oryza sativa 17 (Tos17 ), a Ty 1-Copia Class I retroelement, is one of the few active retroelements identified in rice, the main cereal crop of human consumption and the model genome for cereals. Tos17 exists in two copies in the standard Nipponbare japonica genome (n = 12 and 379 Mb). Tos17 copies are inactive in the plant grown under normal conditions. However, the copy located on chromosome 7 can be activated upon tissue culture. Plants regenerated from 3- and 5-month-old tissue cultures harbor, respectively, an average of 3.5 and 8 newly transposed copies that are stably inserted at new positions in the genome. Due to its favorable features, Tos17 has been extensively used for insertion mutagenesis of the model genome and 31,403 sequence indexed inserts harbored by regenerants/T-DNA plants are available in the databases. The corresponding seed stocks can be ordered from the laboratories which generated them. Both forward genetics and reverse genetics approaches using these lines have allowed the deciphering of gene function in rice. We report here two protocols for ascertaining the presence of a Tos17 insertion in a gene of interest among R2/T2 seeds received from Tos17 mutant stock centers: The first protocol is PCR-based and allows the identification of azygous, heterozygous and homozygous plants among progenies segregating the insertion. The second protocol is based on DNA blot analysis and can be used to identify homozygous plants carrying the Tos17 copy responsible for gene disruption while cleaning the mutant background from other unwitting mutagen inserts.