Genome-Wide Association Analysis And Candidate Gene Mining For Traits Related To Low Nitrogen Tolerance In Rice Seedlings

Nitrogen is an essential macronutrient for plant growth and development,and it is important for the sustainable development of agriculture to explore low-nitrogen tolerance genes and improve the ability of rice to tolerate low nitrogen.In this study,root length,root diameter,leaf dry weight,root dry weight,root crown ratio,leaf nitrogen content,root nitrogen content,leaf nitrogen accumulation and root nitrogen accumulation of rice seedlings under low(8 ppm)and high(40 ppm)nitrogen conditions were used as phenotypes in a natural population of 295 japonica rice varieties.The genome-wide association analysis was carried out to locate QTL for low nitrogen tolerance and explore candidate genes.In this study,we also used Longjing 31(low-nitrogen tolerant variety)and Songjing 10(low-nitrogen sensitive variety)for transcriptome sequencing analysis,and combined the results of GWAS and RNA-seq to further explore and analyze the candidate genes for low-nitrogen tolerance at seedling stage,with a view to providing a theoretical basis for exploring new genes for low-nitrogen tolerance in rice and improving rice varieties through molecular marker-assisted selection.The main results of the study are as follows:1.The phenotypic values of root length(RL),root diameter(RD),leaf dry weight(YW),root dry weight(RW),root crown ratio(RSR),leaf nitrogen content(YN),root nitrogen content(RN),leaf nitrogen accumulation(YNAA)and root nitrogen accumulation(RNAA)under low nitrogen,phenotypic values under high nitrogen,and phenotypic relative values under high and low nitrogen of 295 japonica rice seedlings were The results showed that all the traits related to low nitrogen tolerance were normally distributed,and the population coefficient of variation varied from11.07% to 46.25%.The correlation analysis showed that there were significant or highly significant correlations between the traits related to low nitrogen tolerance.2.Eighty-two SNP loci were detected that were significantly associated with low nitrogen tolerance traits in rice,of which two SNP loci(Chr2＿25361672,Chr3＿13879513)were significantly associated with several traits.chr2＿25361672 and Chr3＿13879513 were significantly associated with root nitrogen content relative(RNR)and root nitrogen accumulation relative(RNAAR).There were 989 genes contained within the linkage disequilibrium decay distance interval of 82 association markers,of which 20 genes were jointly detected in the association analysis for different traits.Haplotype analysis revealed a total of 93 genes that differed significantly between haplotypes of the same trait.Therefore,these 93 genes are presumed to be candidate genes for low nitrogen tolerance.3.A total of 2976 differentially expressed genes associated with low nitrogen stress were detected in the transcriptome sequencing of Longjing 31(low-nitrogen tolerant variety)and Songjing 10(low-nitrogen sensitive variety)under low and high nitrogen conditions.Under low and high N conditions,2452 and 633 differentially expressed genes were identified in the comparative combinations of Longjing 31 and Songjing 10,respectively,containing 109 duplicate differentially expressed genes.The genes that were specifically expressed in the low-nitrogen tolerant variety(Longjing 31),as well as those that were specifically expressed in both low-nitrogen tolerant variety(Longjing 31)and low-nitrogen sensitive variety(Songjing 10),but at significantly higher levels of differential expression in the low-nitrogen tolerant variety(Longjing 31)than in the low-nitrogen sensitive variety(Songjing 10)[FC(Longjing 31/Songjing10)≥ 2],totalling 2381 genes as the low-nitrogen tolerant gene set.4.In this study,haplotype analysis was performed for the genes obtained from genome-wide association analysis,and the haplotype results were combined with the set of low nitrogen tolerance genes obtained from transcriptome sequencing screening.Finally,a total of 7 genes(Os07g0471300,Os11g0230400,Os11g0229300,Os11g0229400,Os11g0229333,Os11g0213800 and Os12g0472800)were screened as more valuable candidates for low nitrogen tolerance.