Mapping QTL For Maize Ear Leaf Stay-green And Analysis Of Candidate Genes Under Low Nitrogen Conditions

The ear leaf of maize is the most important source of carbohydrates for grain.Insufficient nitrogen application significantly reduced the chlorophyll content of spike leaf and eventually led to a reduction in yield.Therefore,it is significant to improve the stay-green retention of maize ear leaf under low nitrogen condition to improve the nitrogen use efficiency of maize.The two-year field experiment in the early stage screened out a single segment substitution line(SSSL)1314 which contained the stay-green QTL of ear leaf in maize under low nitrogen.And 1314 was also detected biomass,nitrogen content,nitrogen accumulation and root traits QTL under low nitrogen.On this basis,the SSSL 1314 and recurrent parent Xu 178 were used in this study to analyze the ear leaf stay-green,photosynthesis and enzymes related to carbon and nitrogen metabolism of parents and F1.F2 isolation populations of 1314 and Xu 178 were constructed and the ear leaf greenness phenotype of F2 population were constructed under low nitrogen condition.100 individual plants with extreme high SPAD or low SPAD phonotype were screened,respectively,to construct DNA mixing pools for BSA analysis to map the ear leaf stay-green QTL under low nitrogen condition.The functions of genes in the candidate fragments was predicted based on transcriptome data obtained in the previous research.The main results are as follows:1?Field phenotypic results showed that the yellowing area of lower leaf of SSSL 1314 was higher than that of allowable Xu 178 and F1 under high and low nitrogen condition.The chlorophyll content of panicle leaf of 1314 was significantly lower than that of recurrent parents Xu 178 and Fi 10 days after silking,which suggested that the substitution fragment of 1314 contained alleles controlling the stay-green of panicle leaf.The photosynthetic rate of panicle leaf of parents showed that the photosynthetic rate,the activities of key enzymes GOGAT and GDH in panicle leaf of SSSL 1314 were significantly lower than those of Xu 178 and F1 under low nitrogen condition.However,there was no significant difference in the activity of PEPC between parents.The results showed that the persistent stay-green QTL contained in 1314 was closely related to nitrogen metabolism.Overall,the phenotypic values of SSSL 1314 were close to those of donor parent Zhong 3,and the phenotypic values of Fi were close to those of recurrent parent Xu 178.2?Statistical results of SPAD values of 4890 F2 plants under low nitrogen condition showed that the population phenotype distribution was normal,and the parents phenotype was on both sides of the peak value,which met the requirements of BSA-seq gene mapping.100 extreme phenotypic plants(high SPAD value and low SPAD value)were selected to construct extreme mixing pools.BSA sequencing analysis was carried out to locate the genes related to ear leaf stay-green in maize under low nitrogen condition.3?Whole genome resequencing was carried out on four samples of parents Xu 178 and 1314,as well as high-phenotype single-plant DNA mixing pool and low-phenotype single-plant DNA mixing pool.The sequence depth of parents was 20× and that of mixed-pool was 30×.A total of 247.29 Gbp data were obtained.The filtered data were 243.50 Gbp,Q30 was more than 93%,the average sequence depth was 28.15x and the average comparison efficiency with reference genome B73 v4 was 99.44%.The data quality was reliable and could be used for further BSA analysis.4?Sequencing result was analyzed by SNP and InDel correlation analysis.The genes related to stay-green in ear and leaf of maize were located in 168720000-171110000 fragment of chromosome 6 under low nitrogen condition.The fragment size was 2.39 Mb and contained 322 genes.5?322 genes in the candidate fragment were analyzed by GO and KEGG annotation.There are 290 genes annotated by GO.Peroxisome(GO:0005777)and ubiquitin ligase complex(G0:0000151)in cell components,ATP binding(GO:0005524)and protein kinase activity(GO:0004672)in molecular functions,protein enters peroxisome matrix(GO:0016558)and protein phosphorylation(G0:0006468)were significantly enriched.There are 83 genes annotated by KEGG.Amino sugar and nucleotide sugar metabolism,pyrimidine metabolism,ribosome biogenesis,terpenoid skeleton synthesis were significantly enriched.6?Based on the transcriptome data of Xu 178 under high and low nitrogen conditions,the analysis result of 322 candidate genes showed that under low nitrogen conditions,eight genes were up-regulated in the candidate regions of Xu 178 leaves,namely Zm00001d039110(related to calcium binding),Zm00001d039037(involved in intracellular signal transduction functions),Zm00001d039141(involved in protein phosphorylation and regulating enzyme activity,etc.),Zm00001d039102(involved in oxidation-reduction process),Zm00001d039016(involved in intracellular protein transport and signal transduction,etc.),Zm00001d039090(involved in protein entering nucleus),Zm00001d039142(related to photosynthesis),and Zm00001d039052(involved in transmembrane transport of carbohydrates).The role of these genes in the stay-green of ear leaf after silking in maize will be further analyzed in the later stage.