Fine Mapping And Functional Analysis Of Defective Glume Gene DG1 And Grain Shape Gene Grain Roundness GR5 In Rice

Rice is one of the world’s most important food crops and provides the staple food for more than half of the world’s population.As farmland declines and the global population increases,there is an urgent need to ensure food production.Therefore,increasing grain yield has been the main target of the current rice breeding programmers.Rice vegetative organs and flower organs are the important agricultural organs to determine the grain yield and quality.The typical floral organs of eukaryotes consists of four whorls structures,which are followed by sepals,petals,stamens and pistil.Rice floral organ,the model monocotyledonous plant,from the outside to the inside is a pair of sterile lemma,lemma,palea,two lodicules,six stamens and a pistil(including two stigmas).In cereal crops,vegetative and spikelet development play an important role in grain yield and quality,but the genetic mechanisms of controlling vegetative and spikelet development in rice are still poorly understood.In this study,a new rice mutant dg1 was obtained by treating the cultivar ZH11 with ethyl methanesulfonate(EMS).The mutant showed dwarfing and rolling leaf features due to smaller cells and more bulliform cells.The dg1 mutant also showed a larger leaf angle and defect in brassinosteroid signaling.In the mutant,the rudimentary glume and sterile lemma were converted into lemma-like organs,and the dg1 mutant produces thin and long seeds.Further analysis showed that DG1 affected the grain size by regulating cell proliferation and cell expansion.The DG1 gene was located in a 31-kb region containing eight open reading frames(ORFs)by fine mapping.DNA sequencing and expression analysis of these loci did not determine the candidate gene.Therefore,the cloning and functional analysis of DG1 will require more work,and further study of DG1 will help us understand the molecular mechanism of plant growth in rice.Grain shape is a typical breeding target.Over the past decade,researchers have conducted extensive researches on the genetic control of this trait.In this study,the gr5-1 mutant was obtained by EMS mutagenesis of Japonica rice cultivar Yundao 32.The investigation of agronomic traits showed that the gr5-1 mutant had a higher1000-grain weight and a larger grain width,but a shorter grain length than the wild type.Scanning electron microscopy showed that the number of longitudinal cells did not change,but the number of cells per unit area was increased than that in wild type.Therefore,it may be speculated that the grain length is shorter than that of the wild type due to the smaller cells.The paraffin section of the hull at heading stage showed that the lemma and palea of gr5-1 was longer than the wild type,and the number of cells per length is more than that of the wild type.It is possible to conclude that the more cells in gr5-1 mutant may lead to a wider seed than that of the wild-type YD32.By the map-based cloning,the GR5 gene was located on chromosome 5 and encoded a predicted AP2 domain transcription factor.DNA sequencing revealed that the mutation of C to G on the fifth intron terminal resulted in abnormal RNA splicing,and thus resulted in altered protein sequence.Genomic complementation test revealed that the gr5-1 phenotypes were completely rescued in transgenic lines.Further,we used the CRISPR/Cas9 gene editing to knock out GR5 gene in the YD32 wild-type plants.The Cas9-GR5 mutant produced the wider and shorter grains,and increased 1000-grain weight,resembling the gr5-1 phenotypes.Together,these data confirmed that LOC_Os05g32270 is the target gene of GR5.