Identification, Association of Natural Variation and Expression Analysis of <i>ZmNAC9</i> Gene Response to Low Phosphorus in Maize Seedling Stage

Phosphorus (P) is an essential macroelement supporting maize productivity and low-P stress is a limiting factor of maize growth and yield. Improving maize plant tolerance to low P through molecular breeding is an effective alternative to increase crop productivity. In this study, a total of 111 diverse maize inbred lines were used to identify the favorable alleles and nucleotide diversity of candidate <i>ZmNAC9</i>, which plays an important role in response to low P and regulation in root architecture. A significant difference was found under low- and sufficient-P conditions for each of the 22 seedling traits, and a total of 41 polymorphic sites including 32 single nucleotide polymorphisms (SNPs) and 9 insertion and deletions (InDels) were detected in <i>ZmNAC9</i> among 111 inbred lines. Among the 41 polymorphic studied sites, a total of 39 polymorphic sites were associated with 20 traits except for the dry weight of shoots and forks, of which six sites were highly significantly associated with a diverse number of low-P tolerant root trait index values by using a mixed linear model (MLM) at −log10 P = 3.61. In addition, 29 polymorphic sites under P-sufficient and 32 polymorphic sites under P-deficient conditions were significantly associated with a diverse number of seedling traits, of which five polymorphic sites (position S327, S513, S514, S520, and S827) were strongly significantly associated with multiple seedling traits under low-P and normal-P conditions. Among highly significant sites, most of the sites were associated with root traits under low-P, normal-P, and low-P trait index values. Linkage disequilibrium (LD) was strong at (r2 > 1.0) in 111 inbred lines. Furthermore, the effect of five significant sites was verified for haplotypes in 111 lines and the favorable allele S520 showed a positive effect on the dry weight of roots under the low-P condition. Furthermore, the expression pattern confirmed that <i>ZmNAC9</i> was highly induced by low P in the roots of the P-tolerant 178 inbred line. Moreover, the subcellular localization of <i>ZmNAC9</i> encoded by protein was located in the cytoplasm and nucleus. Haplotypes carrying more favorable alleles exhibited superior effects on phenotypic variation and could be helpful in developing molecular markers in maize molecular breeding programs. Taken together, the finding of this study might lead to further functions of <i>ZmNAC9</i> and genes that might be involved in responses to low-P stress in maize.