## 4 Discussion  
The flowering time of tropical maize is known to be more dependent on photoperiods than temperate maize lines [47]. The reference genome B73 is a temperate line that was developed under long-day conditions, whereas Ki11 is a tropical line of maize and has adapted to short-day conditions. Ki11 is a drought-tolerant cultivar; its ASI is about 2.22 days under normal conditions [24] and experiences small changes under drought [51; 53]. The ASI of tropical maize is increased by drought stress, resulting in a delay in silking [61]. As in previous studies, the ASI in this study increased by about 4 days under drought conditions, manifesting as a delay in silking (Table 1). To determine whether changes in the ASI were due to changes in the expression of drought-responsive genes, we analyzed genetic changes through RNA-seq and bioinformatics. To reveal candidate genes that may cause changes in the ASI under drought, we analyzed previous reports on drought and flowering-time genes, including homologs in other plants [27], expression profiles of drought stress-responsive genes at each stage and in each tissue [19], and changes in the ASI under drought and short-day conditions [30]. We compared the DEGs of Ki11 to those of B73 under the same conditions. A total of 130 genes with changes in expression in response to drought were found to be common to both cultivars. Among DEGs, five genes (GRMZM2G012717, GRAMZM2G142718, GRMZM2G135013, GRMZM2G094990, and GRMZM2G104549) appear to have the potential to change the ASI. Four of these genes (excluding GRMZM2G142718) were expressed in Ki11, and GRMZM2G142718 was found to be expressed in two homologous forms (contig312 and contig477). GRMZM2G104549 was upregulated under drought in B73 but downregulated in Ki11, and other genes showed the same expression pattern. Thus, few flowering-time genes showed similar patterns between B73 and Ki11 under drought and  
Figure 4: Validation of drought-responsive genes related to flowering time by qRT-PCR. Relative expression values of qRT-PCR and relative expression changes from RNA-seq are presented with the average SD of three biological replicates. SD conditions, suggesting that the flowering mechanisms of tropical and temperate lines are independent. Furthermore, we compared the flowering-time genes of rice and maize under SD. Our results suggest that changes in the expression of florigens may cause a delay in flowering times. _ID1_ (GRMZM2G011357), which promotes the expression of _ZCN8_, is reported to be a regulator of flowering time; the overexpression of _ZCN8_ causes early flowering, while its downregulation results in late flowering [31; 44; 75; 76]. Our data show that the _ID1_ gene was upregulated under drought. Other major genes related to flowering time were downregulated by drought. Two _ZCN8_ genes (GRMZM2G179264 and GRMZM2G103666), which are crucial factors for flowering time, were downregulated under drought conditions. The expression of _ZCN8_ has been reported to increase at the reproductive stage and interact with _DELAYED FLOWERING 1_ (_ZmDLF1_) under SD conditions in tropical maize [31; 45], but _ZmDLF_ family genes were not detected among DEGs. Another flowering-time gene, _Hd3a_ (contig6971, homolog of GRMZM2G373928), was also downregulated under drought. _Hd3a_ is known to be activated by _HD1_ and to accelerate flowering time under SD [77]. Similarly, _Dof_ genes were mostly downregulated under drought. _Dof_ is a repressor of _CONSTANS_ and _CO_-like transcription factor, a regulator of photoperiodic flowering [78; 79]. These transcriptome changes due to drought stress may delay silking and increase the ASI of Ki11. However, the detailed flowering time mechanism in SD conditions remains unclear, so we could not perform a more detailed analysis. Based on our results and the known flowering pathway under SD conditions, a decrease in the expression level of _Ehd1_ and _CONZ1_ may lead to a decrease in the expression level of _ZNC8_, which may cause a delay in the flowering time.