Genetic Identification Of The Small Grain Mutant 8308 And Functional Verification Of The Premature Senescence Mutant Gene D475 In Rice

1.Identification of the small grain mutant 8308 in riceGrain shape is the key factor of rice yield.The study of granular mutant gene is of great beneficial to understanding the genetic pattern of grain and the regulation mechanism of grain growth,which supports the cultivation of high yield rice varieties.In this experiment,a small mutant 8308 was obtained by mutation of rice 188 R using chemical EMS.Then,phenotypic identification,genetic analysis and gene mapping of the 8308 mutant were performed.Main results were as follows:(1)The morphology and agronomic traits of mutant 8308:Self-intersected small grain mutant isolated the tiny,small and large grain.At maturity stage,compared with the wild-type,the small mutant showed that plant height decreased,the spike was more upright,the grain became smaller,and the shape of the glume was barbed.Different from the wild-type,plant height of the tiny grain decreased,blade became narrow,the spike was more upright,the grain was tiny and sterile,and the shape of the glume was barbed.In addition,we found that the plant height,spike length,number of spikelets per panicle,grain length,grain width,1000-grain weight and the seed yield of small grain mutant were decreased by 3.0 %,14.2 %,35.8 %,17.6 %,15.6 %,43.1 % and 15.7 %.On the other hand,the plant height,spike length,number of spikelets per panicle,grain length,grain width,and the aspect ratio of the tiny grain is decreased by 5.0 %、40.1 %、58.3 %、52.1 %、40.6 %、16.8 %.(2)Genetic analysis of mutant 8308.The(188R/8308)F1 population saparated into small and large grain.Self-intersected small grain obtained from(188R/8308)F1 population isolated the tiny,small and large grain.Statistical results show that small grain phenotype is controlled by a pair of incomplete dominance nuclear genes according to the chi-square test.(3)Gene mapping and selecting of mutant 8308.The F2 generations got from hybrid combinations of mutant 8308 and ―Dalixiang‖ were used as the mapping population.The gene was first located near RM348.According to high-throughput sequencing analysis,we found that the physical location of this gene is in chromosome 4.Combined with previous results,the mutant gene was mapped between In Del markers B2 and B3,whose genetic distance was 2.0 c M and 0.2 c M,physical distance was 592 kb.In addition,there were four SNP mutation sites in this area according to high throughput sequencing results.By sequencing and selecting,we know that the total length of gene was 7458 bp and it contains one mutation site: the 2317 th site,which has been changed from C to T,leading Leu to Phe.Because there is no report on this gene so far,it is speculated that this gene is a novel gene for rice small grain mutation.2.Functional verification of the premature senescence gene d475 in riceSenescence is the last step for life and essential for the integrity of growth.Nutrients of senescent organs are reused by other parts.Finally,cells died like a program.Besides,filling and maturation of the grain would be affected by plants premature senescence,which leads to the reduction of seed yield,1000-grain weight and production.The study of senescence mutant gene is of great importance to understand the regulation mechanism,which helps regulating the senescent process and increasing the yield of rice.In this experiment,the Nipponbare was mutated by EMS,so a premature senescence mutant d475 with stable heredity was obtained.We had screened out the candidate gene in previous study,which contains two mutation sites.That is to say,the 147 th sites has been changed from C to T,and the 287 th site has been changed from T to A.However,only 287 th site could change the amino acid.We continued to perform the functional verification of the candidate gene and main results were as follows:(1)The CDS sequence validation.Three databases have different predictions of the CDS sequence.To confirm the true sequence of c DNA,we used c DNA of wild type as a template to amplifing and sequencing.Finally,it was confirmed that the total length of the CDS was 3060 bp,which is different from the prediction of three databases.There is no report on this gene so far,it is speculated that the D475 is a novel gene for rice early senescence mutation(2)Functional verification and expression analysis of the candidate gene.In order to verify the function of the candidate gene,we constructed the functional complementary carrier.Gene d475 from wild type was transformed into mutant by agrobacterium-mediated,then we got the T0 generation transgenic plants.Transgenic plants were planted under natural conditon in Wen-jiang district,Chengdu city.It was observed that the phenotype of transgenic plants were consistent with the wild type.Besides,the results of PCR detection and sequencing confirmed that the gene had been transferred.In addition,tissue expression analysis indicated that it was expressed in various tissues of seedling stage and booting stage,especially the leaf,which was the highest.