Natural antisense transcripts ( NATs) are commonly observed in eukaryotic genomes, but only a limited number of such genes have been identified as being involved in gene regulation in plants. In this research, we investigated the function of small RNA derived from a NAT in fiber cell development., Using a map-based cloning strategy for the first time in tetraploid cotton, we cloned a naked seed mutant gene ( N 1) encoding a MYBMIXTA-like transcription factor 3 ( MML3)/ Gh MYB25-like in chromosome A12, Gh MML3_A12, that is associated with fuzz fiber development., The extremely low expression of Gh MML3_A12 in N1 is associated with NAT production, driven by its 3′ antisense promoter, as indicated by the promoter-driven histochemical staining assay. In addition, small RNA deep sequencing analysis suggested that the bidirectional transcriptions of Gh MML3_A12 form double-stranded RNAs and generate 21-22 nt small RNAs. Therefore, in a fiber-specific manner, small RNA derived from the Gh MML3_A12 locus can mediate Gh MML3_A12 mRNA self-cleavage and result in the production of naked seeds followed by lint fiber inhibition in N1 plants., The present research reports the first observation of gene-mediated NATs and si RNA directly controlling fiber development in cotton. [ABSTRACT FROM AUTHOR]