Identification of a cytochrome P450 hydroxylase, CYP81A6, as the candidate for the bentazon and sulfonylurea herbicide resistance gene, Bel, in rice

Bentazon and sulfonylureas have been used for selective control of broadleaf weeds and sedges in rice fields for more than 20 years. A bentazon and sulfonylurea susceptible mutant, bel, was previously identified for the purpose of allowing these herbicides to be used for removing false hybrids from hybrid rice. While this mutation has been used successfully in rice breeding, the genetic nature of bel is not known. Using 1,776 susceptible plants from a population of 10,000 F₂ individuals, we constructed a fine map for the Bel locus and delimited it to a 36-kb DNA fragment between two restriction fragment length polymorphism markers, L5 and P17. Bioinformatic analysis indicated that there are five genes within this interval, an ethylene-responsive OsER33 gene and four tandem repeats of cytochrome P450 genes designated as CYP81A5, CYP81A6, CYP81A7, and CYP81A8. Comparative sequencing could not find any differences in the coding regions of the OsER33, CYP81A5, CYP81A7, and CYP81A8 genes between the mutant bel and its wild-type progenitor W6154S, but did identify a single base guanine deletion at position +1,332 bp downstream from the translation start codon of CYP81A6. This deletion introduces a premature stop codon and leads to the loss of the heme-binding motif, which is essential for cytochrome P450 function because it contains an absolutely conserved cysteine that serves as the fifth ligand to the heme iron. CYP81A6 presumably functions as a hydroxylase for the detoxification of bentazon and sulfonylurea herbicides in rice. A gene-specific cleaved amplified polymorphic sequence marker and tightly linked flanking markers were developed that will be very useful for selection of the bel allele when transferred to photoperiod-/thermo-sensitive genic male sterility and CMS lines in hybrid rice breeding programs.