Cotton fiber is one of the main raw materials for the textile industry. In recent years, many cotton fiber quality QTL have been identified, but few were applied in breeding. In this study, a genome wide association study (GWAS) of fiber-quality traits in 265 upland cotton breeding intermediate lines (GhBreeding), combined with genome-wide selective sweep analysis (GSSA) and genomic selection (GS), revealed 25 QTL. Most of these QTL were ignored by only using GWAS. The CRISPR/Cas9 mutants of GhMYB_D13 had shorter fiber, which indicates the credibility of QTL to a certain extent. Then these QTL were verified in other cotton natural populations, 5 stable QTL were found having broad potential for application in breeding. Additionally, among these 5 stable QTL, superior genotypes of 4 showed an enrichment in most improved new varieties widely cultivated currently. These findings provide insights for how to identify more QTL through combined multiple genomic analysis to apply in breeding.

microRNA 160 (miR160), targeting auxin response factors (ARFs), plays many roles in plant development. We investigated the role of the miR160/ARF axis in regulation of cotton seed size. Suppressing miR160 activity, specifically in the seed coat, led to smaller seeds and less fiber production owing to attenuated growth of the maternal integument. Scanning electron microscopy and histology showed that expansion of cells in the integument was retarded in miR160-suppressed lines. Four GhARF genes were targeted by miR160 and were upregulated in miR160-suppressed lines, indicating that a miR160/ARF axis is present in cotton. Five genes (Ghir_A05G003740, Scaffold1878G000010, Ghir_D09G024980, Ghir_A11G010730, and Ghir_A05G041590), associated with reduced seed development were downregulated in miR160-suppressed lines. Our results suggest that the miR160/ARF axis controls maternal integument growth to influence seed size by directly or indirectly regulating seed development-associated genes.