A down-regulated epi-allele of the genomes uncoupled 4 gene generates a xantha marker trait in rice.

key messageA γ-ray-induced xantha trait is epigenetically controlled by the genomes uncoupled 4 gene with enhanced promoter segment methylation and down-regulated expression in rice. For easy testing and to increase varietal purity, a xantha mutation (xnt), which turns plants yellow and makes them visually distinguishable from normal green rice, has been generated and bred into male sterile lines for hybrid rice production. The xnt locus was previously fine mapped to a ~100-kb interval on chromosome 11, but its identity was unknown. In this study, xnt was further narrowed down to a 57-kb fragment carrying eight opening reading frames (ORFs). All eight ORFs had identical genomic sequences and all but ORF2 (g enomes uncoupled 4, OsGUN4) had similar transcript abundance in the xantha mutant Huangyu B (HYB) and its parental variety Longtefu B (LTB). The expression of OsGUN4, however, was significantly reduced in HYB compared with LTB in terms of both transcript abundance (0.2% that of LTB) and expressed protein level (barely detectable in HYB but greater than the heat shock protein reference in LTB). Therefore, OsGUN4 was identified as the candidate gene underlying the xantha trait. The function of OsGUN4 in the xantha phenotype was confirmed by identification and characterization of new allelic OsGUN4 mutations. Comparative bisulfite genomic sequencing of OsGUN4 revealed increased methylation in a promoter region in the mutant, and the correlation between increased methylation and the xantha phenotype was further verified by demethylation treatment. In summary, we have identified an epi-allele of OsGUN4 as the causal gene of the xantha marker trait and revealed that enhanced methylation in its promoter down-regulated its expression in rice.