Fine Mapping And Functional Analysis Of OsGL And OsBLG Regulating Grain Shape And Quality In Rice (Oryza Sativa L.)

Rice(Oryza sativa L.)is mainly consumed by the majority of world communities,which is also the main food crop in China.Grain shapes are important traits that influence yield,while grains with different shapes are preferred by consumers in different geographical locations worldwide.Different grain sizes are selected by breeders,due to the preference for slender long-grain rice in many parts of the world.The characterizations of rice germplasm,gene mapping and functional analysis of the genes related to grain size are important,which would provide the new germplasm resources and important clues for rice breeding.In the current study,we isolated twograin shape mutants with ethyl methane sulfonate(EMS)in rice,and named them as gl and blg,respectively.The results are concluded as follows:1.Phenotypic evaluation of gl mutant and its wild-type showed contrasting difference,the plant architecture was upright with compact tillers in gl mutant.There was significant increase in plant height,grain length,and grain length-width ratio in gl mutant,whereas the panicle length,number of grains per panicle,and seed setting rate decreased compared to the WT.However,no noticeable difference in thousand-grain weight between the gl mutant and WT.2.To disclose the basis underlying cellular changes in grain size,scanning electron microscopy(SEM)observation was performed.We found that both grain length as well as grain width of the spikelet hull were longer in gl mutant compared with the WT.The outer parenchyma cells length and number of cells were noticeably increased in gl mutant compared with WT.In addition,the glume external and internal cells were enlarged in gl mutant than in WT.Taken together,the result indicated that the gl mutant regulated organ size and grain length by promoting cell division than cell expansion.3.Genetic analysis revealed that gl phenotype was controlled by a recessive nucleus gene on chromosome 3.The Os GL was mapped within 40 kb region between Indel marker R3M23 and M3MS1.There were five genes within the target region and all the five genes were sequenced and analyzed,the result showed no diversity in four genes with exception of Os03g0407400,we found single based substitution G-A which resulted in the substitution from amino acid Glycine to Argenine in PEBP-like domain in the fourth exon.Moreover,CAPS marker with Eco R I restriction enzymes site was developed.The lines with G allele (WT and male parent)lacked the restriction site,while the lines with A allele(the mutant and individuals with long grains in the F2 population)showed enzyme digestion of the Os GL gene.By checking the CAPS marker,we confirmed that the Os GLgene should be target gene regulating grain size.4.Furthermore,we evaluated the expression of genes that were involved in cell cycle,like CYCA2.1,CYCA2.2,CYCB2.2,CYCD4,CYCD7.1,CAK1,as well as H1.The relative expression of these genes was up-regulated in gl mutant,while genes regulating rice grain size due to cell expansion such as Os EXPA5,Os EXPA10,Os EXPB3,Os EXPB4 and Os EXPB7 were down-regulated.Thus,the result signified that the increase in grain length was due to cell proliferation of the spikelet hull in both latitudinal and longitudinal direction.5.Grain quality traits were compared between the gl mutant and the WT.There was a significant increase in alkali spreading value,protein content,and gel consistency in the gl mutant,whereas the degree of chalkiness and starch contents were significantly lower in gl than in the WT.There was no noticeable difference in amylose content between gl mutant and WT.The composite starch particles in the endosperm of gl mutant were closely arranged as regular polyhedron-like,while composite starch particles contained in the white,opaque parts of the WT endosperm was loosely arranged,and the shape change to spherical or ellipsoid.Rapid viscosity(RVA)analysis showed contrasting difference in breakdown viscosity and setback viscosity between gl mutant and WT.Final viscosity,setback and consistence viscosity were much higher in gl mutant than in the WT.The peak viscosity and breakdown viscosity in gl was lower compared to the WT.However,protein fractions were observed in gl mutant and WT.The result showed slightly high content of glutelin in gl mutant,whereas prolamin and globulin contents were considerably higher in gl mutant but the significance was not remarkable based on the t-test.Glutelin content in gl mutant was considerably decreased in WT by 0.984% compared with the WT.Comparative results for fatty acid between gl mutants and WT showed high content of palmitic acid,stearic acid and oleic in gl mutant,and high content of linoleic acid in WT.6.The blg mutant exhibited long grains with sandy beige color compared with its WT.And it was characterized by increase in grain length and weight with a decrease in grain number per panicle,whereas there was no significant difference in grain thickness,grain width and number of tillers between the blg mutant and WT.7.Genetic analysis revealed that blg phenotype was controlled by a recessive nucleus gene.The OsBLG gene was mapped within 54 kb region between RM16 and M3B15 on chromosome 3 and eight putative genes were predicted within the interval.The result showed no diversity in all the genes except for the LOC_Os03g44500,which had single A-T substitution in the seventeen exons resulting in an amino acid change from Tryptophan(Try)to Phenylalanine(Phe).The genomic sequence of the OsBLG gene consists of twentyone exons and one intron.The OsBLG encodedprotein phosphatase with kelch-like repeat domain,Serine/threonine phosphatase.9.The qRT-PCR analysis indicated that the expression level of OsBLG gene was relatively higher in panicle tissues than other organs i.e.(roots,stems,and leaves).Particularly,the expression was steadily increased in developing panicles with the highest expression when the panicle was 4-6 cm length.The relative expression of genes regulating cell cycle and cell expansion such as Os EXPA5,Os EXPA10,Os EXPB3,Os EXPB4 and Os EXPB7,were down-regulated compared with the bgl mutant,which failed to provide a clear clue about cell expansion.While cell cycle gene such as CYCA2:1;CYCA2:2;CYCB2:2;CAK1;CDCD7.1;and H1 were significantly up-regulated in blg mutant panicle compared with the WT.