Maize inbred lines: B73, Lo1016, Mo17, Mp313E, A638, Tex6, and Lo964 were grown in the field along with two controls, GTP2 and GTP27 [2], at Belflower Farm, Tifton, GA, USA, in a Tifton loamy sand soil. Peanut and corn were previously rotated biannually. The field trials were designed as a randomized complete block with 6 replications for aflatoxin analysis. Experiment plots were 6.0 m long and spaced 0.76 m apart with 2.4-m alleys. The ear shoots were bagged before silk emergence, and ears were self-pollinated. The pinbar method was used for the inoculation with A. flavus spores at 21 days after pollination (DAP). Inoculated ears were hand harvested at maturity for aflatoxin analysis with ELISA and HPLC methods as described by Abbas et al. [12].

To enhance gene expression analysis, the seven inbred lines were grown in field rain-out shelters with clear plastic cover with drought stress imposed by moving the shelter over the plots at V5 stage. Ears were self-pollinated, drought stress conditions were then initiated by the cessation of irrigation at 25 DAP in the rain-out shelters while normal irrigation continued in control shelters. The intensity of drought stress was monitored by measuring photosynthesis efficiency of the leaf at or near the top ear. Ear samples were collected at 35 DAP. Analyses of three sub-samples per replicate were performed for each sample. Immediately upon collection samples were frozen in liquid nitrogen and subsequently stored at −80 °C until analysis.

In the previous work [1,13], 299 kernel stress related genes with known function were discovered by microarray analysis, and 94 of these genes were selected for further scrutiny (Table 1). Gene expression levels among different maize germplam were analyzed by qRT-PCR. Total RNA was extracted from kernels of a single ear of each inbred line selected from harvested samples of each drought stress treatment using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and performed in accordance with the manufacturer’s instructions. Isolated total RNA was then treated with DNase (Qiagen, Valencia, CA, USA) and purified using an RNeasy Cleanup Kit (Qiagen). Purified total RNA was then checked for quality and quantity using a Nano-Drop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

One-step qPCR was performed using a QuantiTect SYBR Green RT-PCR kit (Qiagen) on DNA Engine Opticon Continuous Fluorescence Detection System (MJ Research, Inc.) according to the manufacturer’s instructions. A total reaction volume of 25 µL containing 300 ng total RNA and 25 µM of each primer was used in this study. The expression level of maize glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Accession No: U45856), amplified with primer pair G-F (5′-ACTGTTCATGCCATCACTGC-3′) and G-R (5′-GAGGACAGGAAGCACTTTGC-3′), was used as an internal control.

Amplification curves were generated from the real-time qPCR data and the cycle threshold (C_T) was calculated based on a fluorescence threshold of 0.01, where C_T was defined as the threshold cycle of PCR at which an amplified product was first detected. Subsequently, the ΔC_T for each sample was determined using the equation ΔC_T = C_T target gene—C_T reference gene to calculate the relative expression of each gene to the internal reference control. This was accomplished via a modification of the original equation to relative expression = 2^−ΔCt for both the control and treatment samples [14,15]. The hierarchical cluster of maize inbred lines based gene expression level was performed using Gene Cluster 3.0, and the graphical representation of the tree was obtained by TreeView 1.60.

Previous research has indicated that Tex6 and Mp313E consistently exhibit resistance to aflatoxin contamination, while Mo17 and B73 were susceptible to aflatoxin contamination [3,4]; Lo964 is drought tolerant with a very intensive root system, while Lo1016 is drought susceptible with a superficial and extensive root system [16], and previously unknown for their aflatoxin reaction; A638 showed a higher resistance to stalk rot in contrast to B73 [17] and had drought tolerance. Aflatoxin concentrations in these inoculated lines ranged from 291 ppb to 964 ppb (Table 1); Tex6, Lo964 and Mp313E exhibited relatively lower levels of aflatoxin contamination, whereas B73, Lo1016, A638 and Mo17 exhibited relatively higher aflatoxin contamination (Table 2). The two controls, GTP2 and GTP27, had higher levels of aflatoxin but GTP27 had aflatoxin levels similar to Mo17. In other studies, GTP27 was consistently susceptible to Aspergillus infection and higher aflatoxin contamination [2].

The expression levels of the 94 stress-related genes among seven maize inbred lines revealed the diversity patterns of gene expression in different lines. The same gene was expressed differently in the tested lines, and each maize inbred line had differential patterns of different gene expressions (Table 1). Based on the relative-expression levels of the 94 genes, the inbred lines clustered into two different groups (Figure 1), which correlated well with the aflatoxin levels (Table 2). Group 1 (susceptible group) includes B73, Lo1016 and Mo17, and Group 2 (resistant group) included low-aflatoxin Tex6, Mp313E, Lo964 and A638. By comparing the expression of these 94 stress-relative genes in these two groups, there were more genes expressed higher than average (up-regulated genes) in the resistant Group 2 maize lines with lower-aflatoxin than in the susceptible Group 1 maize lines with higher-aflatoxin (Table 1 and Figure 2). In contrast, there were more genes expressed lower (down-regulated) in the susceptible Group 1 than in the resistant Group 2 (Figure 2).

By comparison of the expression pattern (up-regulated, down-regulated, or unchanged) of each gene in different maize lines, there were a total of 34 genes with the same expression patterns in the susceptible Group 1 and 14 genes in the resistant Group 2 (Table 1, Figure 3). However, there were only six genes expressed differently in these two groups (Table 1, Figure 3): down-regulated in the susceptible Group 1 and up-regulated in the resistant group 2. The thirty-four genes in Group 1 included two genes with higher expression, dehydrin DHN1 (TC248912) and LEA14-A (TC258769), but these two genes were expressed differently in Group 2 (Table 1). The 14 genes in Group 2 were all highly expressed in all maize lines in this resistant group.

During drought stress 49% and 62% of the 94 genes were up-regulated in B73 and Tex6, respectively, while 42% and 30% were down-regulated in B73 and Tex6 (Figure 2). However, there were 31 genes with same expressed patterns in both lines, including 25 up-regulated genes and six down-regulated genes. This study supported the previous studies reporting that B73 and Tex6 were different in resistance to drought stress and aflatoxin contamination [1,6,13].