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BackgroundBreeding for genes controlling key agronomic traits is an important goal of rice genetic improvement. To gain insight into genes controlling grain morphology, we screened M3 plants derived from 1,000 whole-genome sequenced (WGS) M2 Kitaake mutants to identify lines with altered grain size.ResultsIn this study, we isolated a mutant, named fast-neutron (FN) 60-4, which exhibits a significant reduction in grain size. We crossed FN60-4 with the parental line Kitaake and analyzed the resulting backcross population. Segregation analysis of 113 lines from the BC2F2 population revealed that the mutant phenotype is controlled by a single semi-dominant locus. Mutant FN60-4 is reduced 20% in plant height and 8.8% in 1000-grain weight compared with Kitaake. FN60-4 also exhibits an 8% reduction in cell number and a 9% reduction in cell length along the vertical axis of the glume. We carried out whole-genome sequencing of DNA pools extracted from segregants with long grains or short grains, and revealed that one gene, LOC_Os09g02650, cosegregated with the grain size phenotype in the BC1F2 and BC2F2 populations. This mutant allele was named grain shape 9-1 (gs9-1). gs9-1 carries a 3-bp deletion that affects two amino acids. This locus is a new allele of the BC12/GDD1/MTD1 gene that encodes a kinesin-like protein involved in cell-cycle progression, cellulose microfibril deposition and gibberellic acid (GA) biosynthesis. The GA biosynthesis-related gene KO2 is down-regulated in gs9-1. The dwarf phenotype of gs9-1 can be rescued by adding exogenous GA3. In contrast to the phenotypes for the other alleles, the gs9-1 is less severe, consistent with the nature of the mutation, which does not disrupt the open reading frame as observed for the other alleles.ConclusionsIn this study, we isolated a mutant, which exhibits altered grain shape and identified the mutated gene, gs9-1. Our study reveals that gs9-1 is a semi-dominant gene that carries a two-amino acid mutation. gs9-1 is allelic to the BC12/GDD1/MTD1 gene involved in GA biosynthesis. These results demonstrate the efficiency and convenience of cloning genes from the whole-genome sequenced Kitaake mutant population to advance investigations into genes controlling key agronomic traits in rice.

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa ssp japonica), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations.

Grain weight is an important characteristic of grain shape and a key contributing factor to the grain yield in rice. Here, we report that gw2.1 , a new allele of the Grain Width and Weight 2 (GW2) gene, regulates grain size and grain weight. A single nucleotide substitution in the coding sequence (CDS) of gw2.1 resulted in the change of glutamate to lysine (E128K) in GW2.1 protein. Complementation tests and GW2 overexpression experiments demonstrated that the missense mutation in gw2.1 was responsible for the phenotype of enlarged grain size in the mutant line jf42. The large grain trait of the near-isogenic line NIL- gw2.1 was found to result from increased cell proliferation during flower development. Meanwhile, NIL- gw2.1 was shown to increase grain yield without compromising the grain quality. The GW2 protein was localized to the cell nucleus and membrane, and interacted with CHB705, a subunit of the chromatin remodeling complex. Finally, the F 1 hybrids from crosses of NIL- gw2.1 with 7 cytoplasmic male-sterile lines exhibited large grains and desirable grain appearance. Thus, gw2.1 is a promising allele that could be applied to improve grain yield and grain appearance in rice. The datasets generated and/or analyzed in the study are available from the corresponding author on reasonable request. • gw2.1 was a new GW2 allele that influences grain size and grain weight. • NIL- gw2.1 exhibits a larger grain size and increased grain weight without reducing rice appearance quality. • gw2.1 affects glume shape by regulating cell proliferation. • GW2 is located in the cell nucleus and membrane and interacts with CHB705. • The application of gw2.1 improves grain yield and appearance quality in hybrid rice combinations. [ABSTRACT FROM AUTHOR]