The Phenotypic Analysis And Map-based Cloning Of Nitrogen Fixing Deficient Mutants In Medicago Truncatula

The symbiotic nitrogen fixation system formed by the interaction between legumes and rhizobium in soil will change the way of obtaining agricultural nitrogen sources in the future.Symbiotic nitrogen fixation takes place in nodules,new organs formed in the roots of legumes.Plant hosts regulate nodule formation and development,rhizobium differentiation and survival,carbon-nitrogen exchange,and so on,through an extensive and sophisticated signaling network.However,the key genes for these processes and their functions need to be further explored.In this study,we tried to find new genes involved in the regulation of nitrogen fixation symbiosis through forward genetic methods.Through mutant screening,map-based cloning,candidate genes and phenotypic analysis of 15 possible fast neutron bombardment nitrogen fixation mutants in Medicago truncatula,we identified several potential key regulators of the signal exchanges between plant hosts and rhizobium,which can provide new directions for further analysis of the symbiotic nitrogen fixation system.The specific research results are as follows:1.Finding four nitrogen-fixing-deficient mutants and one forming clusters of nodules mutant.Phenotypic screening of 15 candidate mutants inoculated with rhizobia ABS7 showed that 7 mutants had a nitrogen-fixing-deficient phenotype and 1mutant could form clusters of nodules.PCR method was used to detect the reported genes,and it was found that fn21384,fn21454 and fn21589 were caused by deletion mutation of Mt DNF7,Mt SEN1 and Mt DNF4,respectively.2.Map-based cloning analysis of 5 candidate mutants.Based on the rough mapping and fine mapping analysis of the mutants outcross F2 generation population,they were found that the mutation site of fn21433 was mapped between the molecular markers h2?175h23a and h2?2p16d on chromosome 6,the mutation site of fn21613 was mapped between the molecular markers h2?2p16d and 005D01 on chromosome 6,the mutation site of fn21693 was mapped between the molecular markers h2?8g20b and 005D07 on chromosome 7,the mutation site of fn21399 was mapped two regions,one between molecular markers h2?35g18c and h2?146b13a on chromosome 4,and the other between molecular markers 001G03 and h2?145c3d on chromosome 8,the mutation site of fn6168 was mapped below the molecular marker h2?102a8b on chromosome 6.Sequencing analysis of the reported genes in the mapping region revealed that fn21693 and fn6168 were caused by the deletion of the Mt EIN2 and Mt SST1 coding region,respectively.3.Candidate gene analysis and phenotypic analysis of fn21433.The whole genome sequencing analysis revealed that the mutant had 164.088 kb deletion in the region 16359247 to 16523335 on the upper arm of chromosome 6,and there were two nodule-specific highly expressed genes NCR341 and NCR343,so they were selected as the candidate genes of fn21433.Phenotypic analysis of nodules inoculated with different reporter genes showed that there was only a circle of differentiated rhizobium close to the plasma membrane in the nodule cells of fn21433 at two weeks.At three weeks,there was no accumulation of pink leghemoglobin in the nodules of fn21433.In the nitrogen-fixing zone,the green fluorescence of the rhizobia was weak,the expression of Lac Z representing the survival of rhizobium was weak,and the GUS activity representing the ability of nitrogen fixing was not detected,indicating that fn21433 was closely related to nodule formation and nitrogen fixation.4.Candidate gene analysis and phenotypic analysis of fn21613.The whole genome sequencing analysis showed that the mutant had 25.342 kb deletion in the region 30184976 to 30210318 on the lower arm of chromosome 6,and there was a nodule-specific highly expressed gene NCR001,so it was selected as a candidate gene of fn21613.Phenotypic analysis of the nodules at 3 weeks post inoculation with different reporter genes showed that the nitrogen-fixing zone of the nodule of fn21613 was brownish.In the nitrogen-fixing zone,the green fluorescence of the rhizobia was weak,the expression of Lac Z representing the survival of rhizobium was weak,and the GUS activity representing the ability of nitrogen fixing was not detected,indicating that fn21613 was closely related to nodule formation and nitrogen fixation.In this study,we successfully identified NCR genes that may affect nitrogen fixation symbiosis by map-based cloning and whole genome sequencing,providing new directions for the study of plant host cell regulating bacteroid differentiation and the study of NCR gene family with more than 600 members.