Genetic Analysis And Fine Mapping Of The Rachis Abnormal Development Mutant Radin Rice

Rice is one of the most important grain foods in the word,and its spikelet traits are the crucial study contents in rice breeding.We found a spikelet mutant named rachis abnormal development(rad)derived from Nipponbare by using EMS mutagenesis in 2010.In this paper,the mutant rad was used as material to study on the morphological and anatomical traits,genetic analysis and gene mapping.The main results are as follows:1.Morphological traits of the radComparing the characters such as growth period,plant height,leaf age,spike length,uppermost internode length,panicle emersion,branch number,grain number per spike,secondary spikelet number and bract number etc,between rad and wild-type,we found that,the mutant was similar with wild-type in the vegetative growth stage.But after heading,the rad showed significantly less plant height,secondary branch number and grains per spike than wildtype.So was the uppermost internode length which caused enclosed panicle and one to three bracts in the uppermost node or neck-panicle node,which was accompanied by one to two secondary spikelets.However,seed setting of rad is normal.From the microscopic observation of resin section of the young spikelet of the mutant and wild-type,we found that rad has a more branching point located at the base of spikelet.2.The genetic analysis of the radThe F1 plants crossed rad with wild-type Nipponbare resulted normal,and its F2 population showed a 3:1 ratio segregation.The F1 plants of rad crossing with Indica ZaoR974 is also normal phenotype,and BC1F1 population(ZaoR974/rad//rad)and the ratio of normal strains against mutants is 1:1,which indicates that the mutant character is controlled by a single recessive nuclear gene named ARD(t)tentatively.3.Mapping of ARD(t)We used mutantrad and wild-type Niponbare as parents to build population for sequencing.Using BSA-seq method,we found the geneLOC_Os05g50270(coding protein containing GATA zinc finger domain)located on chromosome 5,generated frameshift mutation in 177th amino acid in the mutant,causing the bases CAA turned to CA and amino acid from asparaginate into threonine.Therefore,this gene was the target gene,and we designed primers flanking it.Using the mutant rad and ZaoR974 as parents to build F2population for mapping to test we found seven pairs of primers which showed polymorphism between parents and use these to analyze the F2 population.The result indicated that all of the seven primers were closely linked with RAD(t)gene.The gene was located between InDel 534 and InDel 540and showed co-segregation with InDel 531 and InDel 532.The physical distance to the gene LOC_Os05g50270are 22kb toInDel 534,53.7kbto InDel 540,lkb toInDel 531 and 6.4kbto InDel 532.Moreover,we designed primers to amplify gene and sequenced gene to determine the annotation gene.Sequencing analyze indicated that the sequence of wild-type,normal DNA pool and the geneLOC_Os05g50270 are the same,but mutant and mutant DNA pool had delete in 531th base of the coding region(CAA mutate to CA),producing frameshift mutation.This result conformed the resequencing analysis.The aforesaid analysis showed that,gene LOC_Os05g50270 might be the candidate gene of RAD(t)gene.