Previously reported maize, A. thaliana and O. sativa phosphate transporter proteins were used as queries against the maize genome database. After removal of overlapping sequences, the remaining genes were verified in the Pfam database (http://pfam.xfam.org/). The protein sequences were aligned by the ClustalX (Version 1.81) with default parameters. Then, the phylogenetic tree was constructed with the neighbor-joining method within MEGA 6, and the bootstrap was 1000 replicates in the mode of pairwise gap deletion [37 (link)]. The exon and intron structures of each ZmPHT1 gene were analyzed by GSDS (Gene Structure Display Server; http://gsds.cbi.pku.edu.cn/) through alignment of their CDS with corresponding genomic DNA sequences. Conserved motifs among the ZmPHT1 genes were examined via MEME software (Multiple Expectation Maximization for Motif Elicitation) [38 (link)], and the parameters were set as follows: the number of repetitions was choose any; the maximum number of motifs was 10; and the optimum motif width was set between 6 and 50 residues. Moreover, motif annotation was performed by the Pfam and SMART (http://smart.embl-heidelberg.de/) tools. Chromosome location was performed using the Map Inspect software [39 (link)]. The duplication pattern for each ZmPHT1 gene was analyzed by MCScanX software [40 (link)], and the synthesis map was drawn by Circos software (http://circos.ca/) [41 (link)]. Gene clusters that met the following specific clustering criteria were selected, i.e., genes arising from tandem duplication events on the same chromosome region, two or more homologous genes within a 200-kb region [42 (link)] and fragments derived from replication events [43 (link)]. The calculation of Ka, Ks was by DnaSPv5.0 [44 (link)]. The nonsynonymous substitution per synonymous site (Ka/Ks) ratio analysis was performed by sliding window analysis with the parameters as follows: window size, 150 bp; step size, 9 bp [42 (link)]. The gene expression data from Maize B73 transcriptomes were used to draw a heat map [28 (link)], including information from germinating seeds, primary roots, stems, SAMs (shoot apical meristem), leaves, endosperm, embryos and whole seeds. Promoters were analyzed by RSAT (http://floresta.eead.csic.es/rsat/) [45 (link)].
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