Direct cloning of mutant genes by long-read sequencing

Map-based gene cloning has been used to identify many new genes, greatly contributing to gene function studies in modern biological research. However, the traditional approach is labor-intensive and time consuming. Here, we report an ultra-fast gene cloning strategy of directly sequencing phenotypic sorted mutants. A high-quality reference genome of rice wildtype ZH11 (Oryza sativa L. Geng var. Zhonghua 11) was assembled from PacBio long-read sequences, and used to identify mutations in ten humidity-sensitive genic male sterility (HGMS) mutants induced by sodium azide. We developed a stepwise filtering pipeline for identification of genuine effective mutations, and successfully identified four allelic mutations in the previously cloned gene OsOSC12 (hgms1) and a new HGMS gene (hgms2) containing three allelic mutations. hgms2 encodes a member (OsUGT701A1) of uncharacterized UGT702 glycosyltransferase family, and expression of OsUGT701A1 restores the fertility of hgms2 plants.