Natural epigenetic mutation of MLH3 affects seed-setting in rice

Seed-setting rate is a major determinant of grain yield and depends on the normal development of both male and female gametophytes. Although considerable progress has been made in understanding the genetic regulation of female fertility in rice, few naturally occurring epialleles associated with this process have been identified. Here, we identified a naturally occurring recessive epiallele in rice (Oryza sativa), designated Epi-mlh3, that causes a severe reduction in seed-setting rate owing to female gametophyte abortion. Epi-mlh3 exhibits no detectable DNA sequence variation but displays pronounced CG hypomethylation within the gene body of OsMLH3, accompanied by a marked reduction in full-length OsMLH3 transcripts and premature transcriptional termination. Bisulfite sequencing identified a 443-bp gene-body region containing nine differentially methylated CG sites associated with reduced OsMLH3 expression. CRISPR/Cas9-mediated knockout of OsMLH3 faithfully recapitulated the major phenotypes of Epi-mlh3, including reduced seed-setting rate, embryo sac abortion, and meiotic chromosome defects. Cytological and molecular analyses further demonstrated that reduced OsMLH3 expression was associated with impaired meiotic chromosome behavior and decreased chiasma frequency, consistent with impaired MutLγ-mediated crossover formation during meiosis. Collectively, our findings provide evidence that gene-body CG methylation is associated with stable OsMLH3 transcription and identify Epi-mlh3 as a naturally occurring functional epiallele that expands current understanding of the epigenetic regulation of female fertility in rice.

The online version contains supplementary material available at https://doi.org/10.1007/s11032-026-01724-y.