Gene Map-based Cloning Of Rice Floral Organ Mutants(cy15 And Cy10)and Gene Functional Analysis Of Cy15

Rice is one of the most important food crops in the grass family(Poaceae).The normal development of the rice flower is an important prerequisite to ensure the rice yield The "ABCDE" model,which explains the mechanism of plant flower development,is based on the study of the dicotyledonous model plants(Arabidopsis thaliana and Antirrhinum majus).In recent years,the model of Arabidopsis flower development has been proved to be basically sultable for the rice.However,whether the riee lemma/palea is homologous with Arabidopsis sepals has been controversial,and the development mechanism of three extermal structures of riee spikelets(rudimentary glumes,empty glume,and lemma/palea)is not clear compared with the three intermal organs(lodicules,stamens and pistils).Besides,other regulatory mechanisms are not clear except for the MADS-box genes involved in the regulation of rice floral organ morphogenesis.So,we need to identify more flower mutants to figure out the regulation mechanism of rice floral organ development and establish a suitable model for the rice flower development.In this study,we identified two rice flower mutants(cy10 and cy15)from M2 mutant library mutagenized by 60Co irradiation treatment of an indica rice variety 93-11.Phenotype characterization,gene map-based cloning and function analysis were earried out.The main results are as follows:1.cy15 had shrunken palea and slightly elongated spikelets but without any changes in inner three organs.Compared to the wild type,the lengths of panicle and grain of cy15 were increased,while the 1000-grain weight and seed setting rate were decreased.Paraffin sections showed that the defective phenotype of mutant palea was caused by underdeveloped body of the palea.The development of cy15 palea primordia was dramatically delayed compared with that of the wild type.For defective cy15 palea,the bulges on the bop could not be recognized easily,and the length of bulges became much longer than that of wide-type.In addition,the size of bulges on the cy15 lemma surface became much smaller and the bulge length was shorter than that of the wide-type.The mutation in cy15 is controlled by a single recessive nuclear gene.237 mutant individuals were chosen from the F2 progeny of a cross between cy15 and DJY,and the mutation locus was narrowed down to a 156-Kb region on the short arm of chromosome 3,including 16 open reading frames(ORFs).Of them,the ORF6 encoded a typical floral organ identity OsMADS1 protein.Genome sequencing revealed a 22 base deletion in the seventh exon of cy15 OsMADS1.The deletion resulted in a truncated protein due to a frame shift and premature stop which abolished a number of highly conserved amino acids.Transgenic results show that the defective phenotype of cy15 was caused by the mutation in the C domain of OsMADS1.2.Subcellular localization showed that the mutant OsMADS1 protein was located in the nucleus like the normal protein,but the mutant OsMADS1 protein was distributed with dots unlike the uniform distribution of normal OsMADS1 protein.Quantitative real-time PCR showed that OsMADS1 was specifically expressed in spikelets and the expression of rice floral organ genes were affected in cy10.Yeast two hybrid analysis demonstrated that the mutant OsMADS1 protein could not interact with OsMADS14 and itself,resulting in the failure in the formation of normal homodimer and heterodimer in cy15.3.In the vegetative stage,the cy10 mutant resembled the wild-type;whereas,the defective morphology of cy10 spikelet emerged after heading.Compared with the wild type,the cy10 spikelet missed a glume,lemma significantly reduced or distorted,lodicules transformed into glume-lodicule mosaic structuresl,the number of stamens decreased with occasional stamen-pistil mosaic organ,the number of pistils increased.Scanning eletron microspcopy(SEM)observation showed that the defects occurred during the development of flower primordia.Additionally,typical bulges disappeared on the outer surface of mutant lemma and trichomes became short.4.Genetic analysis revealed cy10 was controlled by a single recessive nuclear gene.Using map-based cloning,the candidate region was narrowed down to an 85-Kb region on the long arm of chromosome 8,including 12 ORFs.Genome sequencing revealed a 15 base deletion in the ninth ORF.In addition,Sasanishiki/Habataki backcross inbred lines(BILs)were used in the correlation analysis and QTL mapping for rice quality traits in previous studies.In this study,the genetic basis of the correlation among different quality traits was dissected and 5 new QTLs were identified.