Characterization And Candidate Gene Analysis Of Abnormally Developed Spikelet Mutants Del And Mg1 In Rice

Rice（Oryza sativa L.）is an important food crop and a representative monocotyledonous plant.The development of floral organs is the key factor affecting rice yield.Among the traits affecting rice yield,such as number of panicles per unit area,number of grains per panicle,seed setting rate and grain weight,the number of grains per panicle is the key factor affecting rice yield,while other traits are important factors affecting grain number per panicle.The number of grains per panicle is determined by the flower development,so the study of flower development has very crucial theoretical significance and application value for increasing and stabilizing rice yield.Two spikelets and floral organ abnormally developed mutants del（degenerated lemma）and mg1（multi-grains 1）were identified from an ethyl methane sulfonate-treated population of the rice xian-type（Indica）maintainer line‘Xinong 1B’.In the present study,the two mutants were analyzed for phenotype and agronomic traits,histological and cytological observation,early spikelet development morphological observation,expression analysis of floral organ identity genes,gene mapping and candidate gene analysis.The resultes are shown below:1.Phenotype analysis of del and mg1 mutants at flowering and mature stagesIn the del mutant,spikelets developed a degenerated lemma.53%of these spikelets had extremely degenerated lemma,which transformed into rod-like lemma,and the palea and lemma cannot close to cover the inner floret organs.In the mg1 mutant,64.15%of spikelets grown an extra lemma-like organ.In the mg1 spikelets with extra lemma-like organs,58.82%had degenerated palea.At the mature stage,56%of the spikelets in the mg1 mutant produced multi-grains.2.Histological and cytological observationComparing with the wild type,the four-layer cell structure of the lemma still exists,but the number and volume of them decreased significantly in the del mutant.In these spikelets,the lemma was extremely degenerated,which transformed into awn-like organs.There was only one midvein left in the lemma.The silicified cells that should have existed on the surface of the lemma were now covered with the rod-like lemma.The extra lemma organs of the mg1 mutant were distributed with a large number of silicified cells and a small number of burrs,and their anatomical structure was similar to that of the wild-type lemma,containing five vascular bundles and four cell layers.The mrp（marginal regions of the palea）regions and bop（body of the palea）region of the degenerate lemma were similar in structure to the wild-type lemma,and the degenerated lemma contained two vascular bundles.In addition,the degenerated lemma contained an abnormal number of inner flower organs,but the characteristics remained unchanged.3.Early spikelet development morphological observationDuring early spikelet development,there was no difference for del mutant compared with the wild type in the Sp4 stage.The slight degenerated lemma primordia were noticeably shorter and narrower than the wild type,and the cell differentiation on both side of the rod-like lemma remained slow and developed into a rod-like structure gradually.The mg1 mutation induced two floral meristems compared with the wild type in the Sp4 stage.The two florets developed within a single spikelet and underwent a further stage of development in the Sp4 to Sp8 stage.In summary,the abnormal development of spikelets in the del and mg1 mutants begins at the early spikelet development.4.Expression analysis of floral organ identity genes in the del and mg1 mutantsTo determine the identity of the organs in the floret organ of the del mutant and the second florets of the mg1 mutant,quantitative real-time PCR（RT-q PCR）was used to investigate the expression patterns of known genes associated with floral organ identity.DL was detected predominantly in the slight degenerated lemma of the del.OsMADS1,OsMADS14,and OsMADS15 were detected significantly in the slight degenerated lemma,rod-like lemma,and palea of the del mutant.We also detected transcripts of OsMADS2,OsMADS3,and OsMADS4 and each gene was expressed significantly in the stamens and pistil of the del mutant.The above expression levels indicated that the slight degenerated lemma of the del mutant still had the characteristics of lemma,and the formation of the rod-like lemma may be due to a decrease in DL expression.Generally,these consequences suggested that the del mutation played a critical role in the regulation of DL for lemma identity.OsMADS1,OsMADS14,and OsMADS15 were detected significantly in the lemma,palea-like organs and extra lemma-like organs of the mg1 mutant.DL was detected mainly in the lemma and extra lemma-like organs of the mg1 mutant.OsMADS6 was detected in the palea-like organs of the mg1 mutant.OsMADS2,OsMADS3,and OsMADS4 were expressed significantly in the stamens and pistil of the mg1 mutant.Taken together,these consequences suggested that the palea,lemma,and extra lemma-like organs in the mg1 mutant all had corresponding characteristics,while the organs of palea were degenerated palea,and the pistil and stamens were normally developed.These results suggested that the two-floret spikelets produced in the mg1mutant may result from a deficiency in the contribution of MG1 to spikelet determinacy.5.Genetic analysisThe del mutant was crossed with‘56S’（Oryza sativa L.subsp.indica）and mg1mutant was crossed with‘ZH11’（Oryza sativa L.subsp.japonica）to obtain the F₁generation.All F₁ plants exhibited a normal phenotype.In the F₂ population,the segregation ratio of normal phenotype to mutant phenotype was 3:1(del:1091 normal plants and 358 mutant plants;χ²=0.26<χ²_0.05=3.84)(mg1:2198 normal plants and676 mutant plants;χ²=0.75<χ²_0.05=3.84).These consequences showed that the target mutant performance of both del and mg1 were seperately controlled by a single recessive nucleic gene.6.Gene mappingThe DEL gene was mapped to the interval between In Del markers In1-18.94 and In1-19.08 on chromosome 1 within a physical region of 140-kb,and there were 15annotated genes in this region.The MG1 gene was mapped to the interval between SSR markers RM20754 and RM494 on chromosome 6 within a physical region of 121.57-kb,and there were 17 annotated genes in this region.7.Candidate gene analysisSequencing analysis identified a single-nucleotide substitution from T to A within Os01g0527600（OsRDR6）,causing an amino acid mutation of Leu-34 to His-34 in the del mutant.This finding suggests that DEL may be a novel allele of OsRDR6.Sequencing analysis identified a single-nucleotide substitution from G to A within Os06g0717200（FON1）,causing an amino acid mutation of Gly-869 to Asp-869 in the mg1 mutant.This finding suggests that MG1 may be a novel allele of FON1.