Whole-Genome Sequencing Identifies a Rice Grain Shape Mutant, gs9-1

Abstract

Background: Breeding 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.

Results: In 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.

Conclusions: In 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.

Keywords: Fast-neutron-induced mutant population; Grain shape; Kitaake mutant database; Oryza sativa L.; Whole-genome sequencing.

Fig. 1

Grain length, grain width, grain length-to-width ratio, 1000-grain weight and plant height of the parental lines, mutant line, F₁ and the BC₂F₂ population. GL, grain length; GW, grain width; L/W, grain length-to-width ratio; KGW, 1000-grain weight, PH, plant height. a The mean value of grain length of X. Kitaake (X.Kit), Kitaake (Kit), gs9–1, F₁ (from the crossing between gs9–1 and Kit) and BC₁F₁ (from the crossing between F₁ and Kit). b-f Distribution of GL, GW, L/W, KGW and PH in the BC₂F₂ population. ** indicate the significant difference (P < 0.01) between the samples indicated using the unpaired Student’s t-test

Fig. 2

Grain, panicle and plant morphology of Kitaake and gs9–1 plants. a Grains of gs9–1 are shorter than those of Kitaake while grain width is slightly increased. b The culm brittle phenotype of gs9–1. c Plant stature of gs9–1 is shorter than that of Kitaake. d Panicles of gs9–1 are shorter than those of Kitaake. e Plant height. ** indicate the significant difference (P < 0.01) using the unpaired Student’s t-test

Fig. 3

Analysis of the epidermal cell size and number of the gs9–1 grain glume. a Cell length of grain on the horizontal axis and vertical axis. b Epidermal cell numbers of grain glume on the horizontal axis and vertical axis. c and d Epidermal cells of the Kitaake and gs9–1 grain glume under the scanning electron microscope (× 300). ^** indicates an extremely significant difference using the unpaired Student’s t-test

Fig. 4

Gene and protein structures, and the relative expression of GS9–1. a The genomic and amino acid changes of line gs9–1. BC₁F₂-W is the wild-type pool, and BC₁F₂-M is the gs9–1 mutant pool that carries a 3-base (ATC) deletion. The amino acid (AA) sequence alignment shows that amino acid lysine (K) in Kitaake is substituted by amino acids asparagine (N) and glutamine (Q) in gs9–1. b qRT-PCR assays of LOC_Os09g02650 in Kitaake and gs9–1 in roots and the 2nd leaf sheath at seeding stage, the lateral bud at tillering stage and the young panicle at the fifth stage of panicle differentiation. The unpaired Student’s t-test showed that there is no different expression level between Kitaake and gs9–1. c Gene structure of LOC_Os09g02650 and the mutated sites of different alleles. d Conserved protein domains in LOC_Os09g02650 and protein structures of different mutant alleles. Motor domain, kinesin motor domain; SMC_N, the N terminal domain of the structural maintenance of chromosomes (SMC) proteins; Leu zipper, Leucine residues conserved in the bZIP protein; Neuromodulin-N, Gap junction protein N-terminal region. Information on putative conserved domains is retrieved from NCBI. ‘AA’ indicates amino acids. The asterisk indicates a premature translation termination. e The two mutated amino acids are indicated by black triangles in gs9–1. The height of a letter indicates this amino acid’s relative frequency at the given position (x-axis) predicated using the MEME program

Fig. 5

Response of gs9–1 to GA₃ and qRT-PCR assays of gs9–1. Asterisks indicate significant differences using the unpaired Student’s t-test (*P < 0.05; ** P ≤ 0.01). a GA₃ response assays. Elongation of the second leaf sheath in gs9–1 and Kitaake in response to GA₃. b Relative length change of the 2nd leaf sheath, which was calculate by dividing the length of the 2nd leaf sheath stimulated with GA₃ by that without GA₃. c qRT-PCR assays of four genes involved in GA biosynthesis in the 2nd leaf sheath of gs9–1 and Kitaake. The expression level of each gene from Kitaake was set to 1. The actin gene was used as the internal control. Data are means ± SD (n = 3). d qRT-PCR assays of three genes involved in rice tillering in lateral buds of gs9–1 and Kitaake