Male sterility is a valuable trait for studying anther and pollen grain development, as well as for harnessing hybrid vigor in crops. Although a large number of genic male-sterility (GMS) genes have been reported in barley (Hordeum vulgare L.), only a few have been successfully cloned. Here, we cloned a barley GMS gene MsgN13910 using BSR-Seq combined with forward genetics methods.

The MsgN13910 locus was finally mapped to a 0.19 cM genetic interval, which corresponded to a 325 Kb physical region. According to the RNA-Seq data, only two genes in this region were specifically expressed in anthers. Compared with the wild type, one of the genes had a G to A mutation, which altered the normal splicing pattern and generated premature termination. CRISPR/Cas9-based knockout of TaMsgN13910 conferred complete male-sterile hexaploid wheat.

Histological and cytological analyses showed that the msgN13910 mutant exhibited delayed degradation of anther tapetum, abnormal ubisch body development, defective anther cuticle development, and loss of bacula of pollen wall, which eventually led to pollen abortion. Metabolomic analysis showed that the gene was mainly involved in the glycerophospholipid metabolic pathway, indicating that MsgN13910 controls pollen fertility by regulating lipid metabolism in anthers.

Yeast two-hybrid, bimolecular fluorescence and luciferase complementation assays showed that TaMsgN13910 interacted with NUS1 and cys protease in vivo. Yeast one‐hybrid and Dual‐luciferase reporter assays showed that MADS, Lim domain protein, PHD finger-like domain-containing protein and Heat shock transcription factor interact with the TaMsgN13910 promoter and repress TaMsgN13910 expression.

Taken together, this study cloned the barley male sterility gene MsgN13910 and preliminarily explored its male sterility mechanism, providing clues for further elucidating the function of MsgN13910 in another development.