Abstract
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.
Highlights
Breeding for genes controlling key agronomic traits is an important goal of rice genetic improvement
We described the characterization of FN60–4, a mutant altered in grain shape, which was identified upon visual inspection of M3 plants derived from 1000 whole-genome sequenced (WGS) M2 Kitaake mutants (Li et al 2016, 2017)
We divided the BC2F2 population into three groups based on grain shape (L/W): wild-type group (WT group), length-to-width ratio (L/W) > 2.00; heterozygous type group (H group), 1.85 < L/W ≤ 2.00; and mutant group (M group), L/W ≤ 1.75
Summary
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. With advances of nextgeneration sequencing (NGS) and comparative genomic analyses, gene cloning is more efficient Such an approach is suitable for lethal mutants or mutants defective in seed setting which cannot be isolated using traditional map-based cloning approaches. Whole-genome sequencing (WGS) via NGS has been successfully applied to isolate genes from diverse species including the Caenorhabditis elegans neuronal cell fate decision gene laterally symmetric-12 (Sarin et al 2008), the Drosophila melanogaster encore (enc) gene that controls the egg morphology (Irvine et al 2009), the Schizosaccharomyces pombe E2 ubiquitin ligase gene ubiquitin conjugating enzyme 4 (ubc4) (Irvine et al 2009), the Bacillus subtilis stringent response mediator gene (relA) (Srivatsan et al 2008), the A. thaliana clock mutant early bird gene (ebi-1) (Ashelford et al 2011), and the O. sativa the male sterility gene (MER3) (Chen et al 2014)
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