| Phosphorus（Pi）is an essential element for plant growth and development,as well as a component of some important compounds such as nucleic acid,phospholipids and ATP,et al.It is involved in various important physiological metabolic precesses.Most Pi in fertilizer is fixed in the soil or is lost with soil erosion,resulting in insufficient bioavailability of Pi.And due to the low utilization of Pi for crops,it leads to environment pollution and resource depletion of phosphate rock.Improving plant Pi utilization efficiency is a foundamental approach to solve this problem.Metabolites acting as intermediates between genes and phenotypes can directly reflect the microstate of plants in response to low phosphorus stress.So some genes that control phenotype through metabolic pathways in response to low-Pi stress can be mined by combining genome-wide association study and metabolome profile.In this way,we can characterize the molecular mechanisms of Pi response versatilely and provide important theoretical foundations for developing varieties with high utilization efficiency of Pi.This study evaluated the low-Pi tolerance degree of a set of maize association panel which are assembled by 337 diverse inbred lines from the current Southwest China breeding program and the genome-wide association study of maize seedling phenotypes was also conducted in this association panel.Metabolite profiles for the screened six low-Pi-sensitive lines and six low-Pi-tolerant lines under Pi-sufficient and Pi-deficient conditions to identify the metabolic changes and the corresponding genes.Then five low-Pi-responding consensus genes associated with morphological traits and simultaneously involved in metabolic pathways were mined by combining genome-wide association study and metabolites profiles.The consensus genes induced by Pi deficiency in maize seedlings were also validated by reversetranscription quantitative polymerase chain reaction（RT-q PCR）.Moreover,these genes were further validated in a recombinant inbred line（RIL）population.The main results are as follows:（1）22 seedling phenotypes of the association panel were measured under Pi-sufficient and Pi-deficient conditions respectively.Except the crown root fresh weight,the remaining 21morphological traits differed significantly between low-and normal-Pi conditions.And the whole association panel were divided into Pi-sensitive,Pi-moderate and Pi-resistant inbred lines via dynamic low-Pi tolerant cut-off.Among the 80 Pi-sensitive lines,the temperate inbred lines predominate among the Pi-sensitive lines and 47 tempetate inbred lines are Pi sensitive in tatal.Among the 19 Pi-resistant lines,14 of them are tropical lines.This result indicated that there existed genotype differences in maize low-phosphorus response mechanisms and the tropical maize inbred lines possess high resistant to low-Pi stress.（2）Combining a set of maize 56K SNPs with 22 seedling phenotypes to conduct genome-wide association study.A total of 178 SNP markers were found to be significantly associated with a variable number of traits with low-Pi-tolerance index values（LPTI=TPD/TPS）,using the mixed linear model（MLM）with a threshold of P=2.16×10-5.The range of phenotype variation explained by these SNPs is 5.55%-16.50%.In addition,15 SNP markers were significantly associated with multiple traits under Pi-sufficient conditions and eight SNPs were significantly associated with multiple traits under Pi-deficient conditions,using the same calculation method and threshold.The range of phenotype variation explained by these SNPs are 5.58%-7.81%and 5.58%-6.67%,respectively.According to the physical position of these SNP markers and average linkage disequilibrium（LD）decay distance,a total of 1062 candidate genes were identified near these significantly associated SNP markers.These candidate genes are mainly distributed on chromosomes 1,2,4 and 8.（3）Metabolite profiles were applied on the screened six low-Pi-sensitive lines and six low-Pi-tolerant lines under Pi-sufficient and Pi-deficient conditions.Results showed that under Pi deprivation the concentrations of nucleic acids,organic acids and sugars were increased,but that the concentrations of phosphorylated metabolites,certain amino acids and nitrogenous compounds were decreased.These results probably reflect compensatory mechanisms to maintain metabolism under Pi deficiency.The levels of secondary metabolites involved in plant immune reactions were significantly different in plants grown under Pi-deficient conditions,especially the benzoxazinoids and flavonoids.This study speculated that certain immune-related secondary metabolites responding to low Pi levels may provide clues on plant Pi-response mechanisms.（4）60 metabolic markers in total were filtered to distinguish the stable metabolic differences between the two extreme groups of genotypes and between the two levels of Pi-treatments with the screening condition of CV<15%in both pairwise groups and differing significantly between pairwise groups.Isoleucine and alanine were the most stable metabolites for the identification of Pi-sensitive and Pi-resistant maize inbred lines.In both Pi-sensitive and Pi-resistant leaves,as the metabolic markers to discriminate the Pi response state,isoleucine and nonanoic acid were stable and differed significantly between Pi-deficient and Pi-sufficient conditions.In roots,the nine most stable metabolites showed significant difference between Pi-deficient and Pi-sufficient conditions.（5）Associations between the 22 related traits and the identified metabolites were investigated using Pearson correlation tests.In total,159 metabolites were strongly correlated with at least one morphological phenotype under Pi-deficient conditions and 77 metabolites were strongly correlated with at least one morphological phenotype under Pi-sufficient conditions.The results of Pearson correlation analysis showed strong correlations between morpho-physiological phenotypes under Pi-deficient and Pi-sufficient conditions,which provides the theoretical basis for combining the GWAS results with the genes associated with significantly different metabolites to screen low-phosphorus response genes involved in metabolic pathways.Then,by combining the GWAS results with the genes associated with significantly different metabolites,five consensus genes were identified.（6）The RT-q PCR results showed that the transcript abundance of the five consensus genes was induced by Pi deficiency in maize seedlings.On the other hand,the fragments of three genes（GRMZM2G039588,GRMZM5G841893,and GRMZM2G051806）with great differences between line 178 and line 9782 were studied in recombinant inbred line（RIL）families.GRMZM2G039588（glucose-6-phosphate-1-epimerase encoding gene）not only affects the cob diameter,ear length and even the shoot phosphorus state in maize,but also mediates ear diameter and row number per ear in response to the availability of phosphorus.Some yield components were also significantly different between the separating genotypes of GRMZM5G841893 and GRMZM2G051806;however,there were no consistent and stable differences,possibly because of the minor effect of these two genes on yieldcorrelated traits. |
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