Evaluation of maize inbred lines and topcross progeny for resistance to pre-harvest aflatoxin contamination

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Available online at www.sciencedirect.com

ScienceDirect

Short communication

Evaluation of maize inbred lines and topcross progeny for resistance to pre-harvest aflatoxin contamination Jake C. Fountaina,b , Hamed K. Abbasc , Brian T. Scullyd , Hong Lie , Robert D. Leef , Robert C. Kemeraitb , Baozhu Guoa,⁎ a

USDA-ARS Crop Protection and Management Research Unit, Tifton, GA, USA Department of Plant Pathology, University of Georgia, Tifton, GA, USA c USDA-ARS Biological Control of Pests Research Unit, Stoneville, MS, USA d USDA-ARS National Horticultural Research Laboratory, Fort Pierce, FL, USA e Shanxi Academy of Agricultural Sciences, Millet Research Institute, Changzhi, Shanxi, China f Department of Crop and Soil Sciences, University of Georgia, Tifton, GA, USA b

AR TIC LE I N FO

ABS TR ACT

Article history:

Pre-harvest aflatoxin contamination occurs in maize following kernel colonization by Aspergillus

Received 29 May 2018

flavus. Aflatoxin contamination resistance is a highly desired trait in maize breeding programs.

Received in revised

The identification of novel sources of resistance to pre-harvest aflatoxin contamination is a

form 24 September 2018

major focus in germplasm screening efforts. Here, we performed a field evaluation of 64 inbred

Accepted 3 November 2018

lines over two years for pre-harvest aflatoxin contamination. Topcrosses were also performed

Available online 30 November 2018

with two testers, B73 and Mo17, to generate 128 F1 hybrids which were also evaluated over two years. Hybrid performance was used to calculate both general combining ability (GCA) of the

Keywords:

inbreds, and observed heterosis for aflatoxin resistance. Over both years of the study, aflatoxin

Aspergillus flavus

concentrations ranged from 80 ± 47 to 17,617 ± 8816 μg kg−1 for inbreds, and from 58 ± 39 to

Pre-harvest

2771 ± 780 μg kg−1 for hybrids with significant variation between years and lines. The inbred

Aflatoxin

lines CML52, CML69, CML247, GT-603, GEMS-0005, Hi63, Hp301, and M37 W showed <1000 μg kg−1

Maize

of aflatoxin accumulation in both years of the study and less than the resistant check, Mp313E, in

Topcross

at least one season. Among these, CML52, GT-603, and Hi63 also showed significant GCA with the testers in hybrid progeny. CML52, GT-603, and M37 W also showed heterotic effects of −13.64%, −12.47%, and − 24.50%, respectively, with B73 resulting in reduced aflatoxin contamination. GT603 also showed a similar heterotic effect for aflatoxin contamination, −13.11%, with Mo17 indicating that this line may serve as a versatile source of aflatoxin contamination resistance in breeding programs. © 2018 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

⁎ Corresponding author. E-mail address: [email protected]. (B. Guo). Peer review under responsibility of Crop Science Society of China and Institute of Crop Science, CAAS.

https://doi.org/10.1016/j.cj.2018.10.001 2214-5141 © 2018 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

TH E C ROP J O U R NA L 7 (2 0 1 9) 11 8 –1 2 5

1. Introduction Aspergillus flavus Link is a facultative pathogen of maize (Zea mays L.) and produces potent mycotoxins collectively known as aflatoxins [1]. These mycotoxins are highly carcinogenic and are also acutely toxic in sufficient quantities resulting in increased likelihood of developing hepatitis, cirrhosis, hepatocellular carcinoma, and birth defects [2,3]. Due to the hazards associated with acute and chronic aflatoxin exposure, the US Food and Drug Administration (FDA) currently regulates aflatoxin content in maize products for human consumption at 20 μg kg−1 [4]. It is estimated that aflatoxin contamination of maize results in losses of over $225 million per year in the United States alone [5], but models estimate losses of $1.68 billion in years where conditions are highly conducive for aflatoxin contamination [6]. Given the potential losses, both human and economic, the development of commercial varieties with aflatoxin contamination resistance is a major focus of breeding efforts. Numerous breeding approaches have been applied since the mid-1970s following a severe outbreak of aflatoxin contamination that reached the Midwestern US. This underlined the importance of pre-harvest aflatoxin contamination resistance, and stimulated research in a new direction considering this was previously considered primarily a postharvest storage issue [7–9]. Traditional breeding approaches have focused on the identification of resistant germplasm using field evaluations and laboratory kernel assays in combination [10,11]. These efforts have resulted in the identification of several aflatoxin resistant inbred lines including Mp420 and Mp313E [12,13], African and tropical germplasm such as TZAR101-TZAR106 developed by the International Institute of Tropical Agriculture (IITA) in collaboration with other institutions [14], and southern-adapted inbred lines such as GT-601, GT-602, GT-603, Mp715, Mp717, Mp718, and Mp719 [15–19]. Recent advances in DNA marker technologies and genotyping capabilities have also allowed for the application of molecular breeding techniques to this issue. Several quantitative trait loci (QTL) for aflatoxin resistance have been identified using both traditional bi-parental mapping populations and using diverse germplasm collections with a genome-wide association study (GWAS) method. For example, Dhakal et al. [20] utilized an F2:3 mapping population derived from Mp715 and B73 to identify seven QTL for aflatoxin contamination resistance explaining <10% phenotypic variance (PVE). Recent genome wide-association study (GWAS) has also been used to identify QTL. For example, Farfan et al. [21] used 346 inbred lines testcrossed to Tx714 for GWAS resulting in six minor QTL with PVE of approximately 5%, and Zhuang et al. [22] used a combination of GWAS using 437 diverse lines and a recombinant inbred line (RIL) population with 228 individuals derived from RA × M53 to identify QTL and markers with 6.7% to 26.8% PVE. These molecular studies also detected significant genotype × environment interactions, which, along with the relatively low PVEs obtained for detected QTL, are major confounding factors for aflatoxin resistance breeding. To account for this and improve cultivar selection, multi-location germplasm

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selection trials such as the Southeast Regional Aflatoxin Test (SERAT) have been performed to examine germplasm performance across multiple environments [23]. Another confounding factor is the poor and/or inconsistent agronomic performance of inbred lines observed due to inbreeding depression. In order to compensate for this, F1 topcross hybrids can be used to screen inbreds as potential sources of aflatoxin contamination resistance. This has been employed both in standard testcross designs, and in diallel designs to examine both general and specific combining ability for aflatoxin resistance in addition to other relevant agronomic traits [24–27]. Given the potential for more consistent agronomic performance in hybrids, and the possibility of effects from heterosis and/or hybrid performance on aflatoxin resistance, the objective of this study was to evaluate breeding germplasm available in our breeding program in Tifton, Georgia for aflatoxin contamination resistance in both inbreds and in F1 topcross hybrids. Both GCA and heterotic effects on aflatoxin contamination observed in the generated hybrids were also investigated. By identifying novel sources of resistance, these lines can be used in breeding commercial varieties with enhanced aflatoxin resistance.

2. Materials and methods 2.1. Plant materials Sixty-four maize inbred lines were selected from among diverse germplasm available in our breeding program in Tifton, Georgia, and were cultivated as described by Guo et al. [28]. Briefly, these inbred lines were grown at the USDA-ARS Crop Protection and Management Research Unit (CPMRU) Belflower Research Farm, Tifton, GA, in 2014 and 2015. The inbreds were planted in a randomized complete block design with three replicates. Each inbred was planted in two row plots 3.0 m in length with 0.6 m row spacing, 1.0 m alleys between plots, and seed spacing of 15.2 cm. Irrigation was applied as needed and recommended management practices were employed for all plots. F1 topcross hybrids were generated at the Belflower Farm, USDA-CPMRU, and the University of Georgia Gibbs Farm, Tifton, GA in 2014 and 2015. Hybrids were generated using the 64 inbred lines planted in crossing blocks in isolation from other maize plots and were open pollinated following inbred line emasculation prior with a tester line, B73 or Mo17. Each tester was used at a different location to avoid cross-contamination. The testers were also included with the other 64 inbreds for inoculation in 2014 and 2015. The 128 F1 hybrids generated (64 inbreds × B73 and 64 inbreds × Mo17) were then grown in 2015 and 2016 at the Belflower Farm as previously described for the inbreds. Two commercial hybrids, Dekalb DKC69–43 and Dupont-Pioneer P2023HR, were also included in 2016 as controls.

2.2. A. flavus inoculation The A. flavus isolate NRRL3357 was used for all field inoculations. The isolate was cultured on V8 agar (20% V8 juice, 1% CaCO3, and 2% agar) at 37 °C for 5–7 days. Conidia were then harvested into 0.01% (v/v) Tween 20 buffer and

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quantified using a hemocytometer. Inoculum concentration was then adjusted to 4.0 × 106 conidia/mL and refrigerated at 4 °C until use. Both the inbred and the hybrid plants were inoculated using a side-needle inoculation method as previously described [28,29]. Briefly, at 14 days after 50% silk emergence (DAS) within each individual plot, an Idico treemarking gun outfitted with a 14-guage hypodermic needle was used to inoculate maize ears by inserting the needle into the husk and injecting 3.0 mL of inoculum. Only the uppermost ear on each plant was inoculated. In total, five plants per row in each two-row plot were inoculated, and bulk harvested at 45–55 days after inoculation (DAI). In 2014, inbreds were harvested at multiple times corresponding to a range of 45–50 DAI for each plot. In 2015 and 2016, harvest was conducted as a single event relative to the median inoculation date for each year and test. This was done since ~80% of plots had inoculation dates within one week of the median corresponding to a single harvesting event in both years. Late-flowering plots with inoculation dates more than one week later than the median were harvested individually at 45–50 DAI. Ears from each bulk harvested row were dried and shelled, all kernels were ground together, and a 50.0 g subsample was taken for aflatoxin analyses.

2.3. Aflatoxin quantification Aflatoxin quantification was performed as previously described [28]. Ground subsamples from both the inbred lines and the hybrids were tested for total aflatoxin content using a Neogen Veratox for Aflatoxin ELISA kit (Neogen, Lansing, MI, USA) according to the manufacturer's instructions. Initially, 20.0 g of ground kernel tissue was placed into an 8.0 oz. opaque container with a lid, and aflatoxin was extracted in 100 mL 70% (v/v) methanol with gentle shaking for 3 min. The mixture was then allowed to stand for several minutes before an aliquot of liquid extract was transferred to a 1.5 mL microcentrifuge tube and stored at 4 °C until use in ELISA. Spectrometric analysis using a Veratox ELISA reader (Neogen) was then performed to quantify total aflatoxin content.

2.4. Statistical analysis The normality of the obtained aflatoxin data was assessed based on skewness (Sk) and kurtosis (K) using PROC Univariate using SAS v9.2 (SAS Institute, 2003). Given that the original data were highly skewed and not normally distributed, the data were log2(y + 1) transformed (Fig. S1). These transformed aflatoxin data for both inbred and hybrid samples were used for analysis of variance (ANOVA) based on the model yijk = μ + yeari + linej + (year × line)ij + eijk, where both year and line were considered fixed effects using PROC GLM followed by Tukey's post-hoc analysis for pairwise comparisons within inbreds and hybrids using SAS v9.2 (SAS Institute, 2003) and R (v.3.3.0). Data presented in the text and tables are means and standard deviations calculated from the original data prior to transformation. General combining ability (GCA) for each inbred was calculated using line × tester analysis using the Analysis of Genetic Designs with R for Windows (AGD-R, v.4.0) software package [30]. For all statistical analyses, significance was defined based on α = 0.05. Hybrid vigor (heterosis) was

calculated as described by Fehr [31] using the equation heterosis (%) = [(F1 – MP)/(MP)] × 100 where F1 is the average F1 hybrid performance and MP is the average performance of the parent inbreds.

3. Results and discussion Over the course of two years of replicated analyses, 64 maize inbred lines and 128 F1 hybrids generated from topcrosses of the inbreds with testers B73 and Mo17 were evaluated for aflatoxin contamination resistance. All of the field evaluated maize samples contained total aflatoxin in concentrations exceeding the FDA limit of 20.0 μg kg−1 [4]. For the inbreds, an overall average of 2275 ± 2323 μg kg−1 ranging from 201 ± 186 to 5000 ± 4876 μg kg−1 in 2014 and from 80 ± 47 to 17,617 ± 8816 μg kg−1 in 2015 were measured. Averages of 1258 ± 1196 μg kg−1 (coefficient of variation (CV) = 0.9507) and 2941 ± 3784 μg kg−1 (CV = 1.2866) were observed for the inbreds in 2014 and 2015, respectively. For the hybrids, an overall average of 1045 ± 784 μg kg−1 ranging from 58 ± 39 to 2387 ± 430 μg kg−1 in 2015 and from 157 ± 135 to 2771 ± 780 μg kg−1 in 2016 were measured. Averages of 666 ± 811 μg kg−1 (CV = 1.2177) and 1230 ± 855 μg kg−1 (CV = 0.6951) were observed for the hybrids in 2015 and 2016, respectively. Interestingly, 2015 showed higher degrees of variation compared to the other years possibly indicating a greater seasonal effect in that year on aflatoxin accumulation. Bulk harvesting at a single time point may also result in elevated aflatoxin in inbreds with earlier anthesis or less in lines with later anthesis. While no apparent trend was observed in the data, this may contribute to higher aflatoxin levels and variation observed in inbreds compared to hybrids and warrants further study. Overall, the hybrid progeny showed lower and less variable total aflatoxin content than the inbreds. The high standard deviations observed were also likely due to the skewed distribution of the raw aflatoxin data across the experiment necessitating data transformation for further analyses. Therefore, aflatoxin content for both inbreds and hybrids was log2(y + 1) transformed for downstream analyses (Fig. S1). Analysis of variance (ANOVA) showed that there was a significant year effect and variety × year interaction for both inbred lines and hybrids (Table S1). This interaction is representative of genotype × environment interactions classically reported for aflatoxin contamination resistance in maize [32]. The overall range of aflatoxin content observed in the hybrids, however, does suggest that the magnitude of this variation may indeed be reduced due to more consistent agronomic performance within and across environments compared to the inbred lines although variability between hybrids as indicated by CVs was similar to that seen in the inbreds in 2015. Given this significant interaction, inbred line and hybrid effects were analyzed separately by year, both of which resulted in significant effects on aflatoxin content in both inbred and hybrid samples in all years (Table S1). Statistical examination for differences in aflatoxin accumulation among the inbreds showed very little mean separation in post-hoc analysis (Table 1). No significant differences were observed between the resistant control Mp313E and the

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Table 1 – Mean aflatoxin contamination and general combining ability (GCA) for the inbred lines. Line

Mean concentrations of aflatoxin (μg kg−1) 2014

Grace E-5 (E-1) LH 51 Coy 05 AT 709 B37 NC350 GP 280 Lo 1016 Tx303 Coy 05 AT 805 Oh43 LH 132 Ky21 Mo18W Va35 GT A2 R CY 1 Coy 05 AT 819 CY 4 Lo 964 Coy 05 AT 823 CML333 CY 3 CML322 10 GEM06845-1B-1B FAW 1430 NC358 CY 5 B97 GP 282 Mo17 F54 Hi31–1B-1B-1B Tun 88-1B-B ZM 521 E-1 B73 Tun 85-1B-B TX 732 NC290A-B Tzi8 GT A554 GT A638 A638/MP313E//MP313E/f6–35-B6 GT A641(SP) CML277 HBA-1-B MS71 EP M6 GT A661(SP) CML228 M37 W CML103–2 MP313E I114H GT 603 Ki11 Syn AM 1 P43 GEMS 0005-1-1-1-1B-1B Hp301 CML52 CY 2 Hi 63-1B-1 GEM 0028-2-1-B-B-B-1B-1B

2909 ± 1046 785 ± 981 4416 ± 2821 1129 ± 785 201 ± 186 3074 ± 2795 1632 ± 1648 1175 ± 1628 2733 ± 1109 1429 ± 1535 1901 ± 824 1857 ± 1970 381 ± 646 481 ± 740 1307 ± 1603 1930 ± 1104 1188 ± 1116 665 ± 800 1918 ± 1544 1823 ± 1378 937 ± 475 2613 ± 1783 503 ± 612 4489 ± 4687 1304 ± 1115 1015 ± 1202 2759 ± 3097 2559 ± 860 2588 ± 1383 759 ± 654 1687 ± 1364 907 ± 738 969 ± 708 1576 ± 956 1824 ± 1091 516 ± 597 1530 ± 689 912 ± 1094 2428 ± 3138 4446 ± 1991 2473 ± 949 792 ± 1060 N/A 518 ± 520 945 ± 800 841 ± 751 2112 ± 1205 5000 ± 4876 1116 ± 667 547 ± 602 2525 ± 1071 N/A N/A 639 ± 616 1303 ± 843 1406 ± 1654 253 ± 189 464 ± 638 388 ± 423 1283 ± 1644 714 ± 793 1156 ± 1207

GCA

2015 ab abc ab abc abc abc abc abc ab abc ab abc abc bc abc ab abc bc abc abc abc abc abc abc abc abc abc ab ab abc abc abc abc abc abc abc abc abc abc a ab abc abc abc abc abc abc abc abc ab

abc abc abc abc abc abc abc abc abc

17,617 ± 8816 13,513 ± 17,716 8245 ± 3353 6832 ± 3223 6825 ± 2129 6267 ± 3286 5578 ± 3823 5553 ± 1218 4723 ± 2409 4345 ± 3923 4287 ± 871 4287 ± 4641 4213 ± 2242 4197 ± 2962 4074 ± 4832 3957 ± 2340 3680 ± 632 3603 ± 1383 3493 ± 1926 3490 ± 702 3367 ± 1002 3300 ± 2217 3293 ± 1743 3240 ± 1234 3220 ± 1157 3213 ± 2913 3160 ± 1299 3137 ± 741 2700 ± 142 2671 ± 713 2450 ± 1387 2300 ± 1814 2217 ± 1448 2177 ± 1712 2170 ± 519 2107 ± 900 1960 ± 166 1860 ± 1042 1820 ± 972 1710 ± 442 1660 ± 1912 1600 ± 737 1479 ± 1114 1430 ± 1679 1359 ± 1055 1326 ± 1301 1215 ± 1764 1128 ± 1652 1114 ± 1248 967 ± 687 878 ± 531 765 ± 420 750 ± 779 747 ± 240 743 ± 49 731 ± 807 439 ± 128 393 ± 346 322 ± 87 301 ± 305 282 ± 166 258 ± 92

a ab ab ab ab abc a-d abc a-d a-e a-d a-e a-e a-e a-e a-e a-e a-e a-e a-e a-e a-e a-e a-e a-e a-e a-e a-e a-e a-e a-e a-f a-e a-f a-e a-e a-e a-f a-f a-f b-f a-f a-f a-f a-f b-f b-f b-f a-f a-f a-f a-f b-f a-f a-f b-f b-f def b-f def c-f c-f

2015

2016

−0.02 0.23 0.14 N/A −0.34 0.28 0.42 0.59 0.01 0.22 −0.01 0.29 −0.37 0.10 0.13 0.24 −0.10 0.26 0.67 0.23 −0.12 0.34 N/A −0.12 −0.32 −0.14 0.17 0.51 0.69 0.39 0.25 0.02 −0.05 0.01 −0.39 0.27 −0.20 −0.03 −0.24 −0.08 −0.16 0.02 −0.12 −0.16 0.12 −0.04 0.48 −0.01 0.07 −0.64 −0.44 N/A N/A −0.32 −0.45 N/A −0.07 N/A −0.21 −0.16 −0.40 −0.60

0.34 0.14 0.36 −0.19 −0.42 0.15 −0.06 0.33 −0.08 −0.25 −0.03 0.29 −0.15 0.07 0.17 0.48 0.45 −0.02 −0.06 −0.42 0.24 −0.08 0.10 0.17 0.28 0.10 0.18 0.38 0.12 0.00 0.14 −0.13 −0.09 0.38 0.00 0.47 −0.17 −0.41 −0.05 0.14 0.04 0.26 −0.08 0.04 0.37 −0.21 −0.25 −0.08 0.27 −0.30 0.15 0.04 0.35 −1.04 −0.08 0.05 −0.05 −0.59 −0.86 −0.09 −0.63 −0.11

(continued on next page)

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Table 1 (continued) Mean concentrations of aflatoxin (μg kg−1)

Line

2014 GT-888 CML69 CML247 GT A1R TP Yellow E-1

880 758 245 1264

± ± ± ±

595 738 541 1403

GCA

2015 abc abc c abc

231 ± 187 180 ± 97 80 ± 47 N/A

ef ef f

2015

2016

−0.12 −0.38 −0.45 0.09

0.36 0.19 −0.38 −0.25

Data are means of all replications within the indicated year. Concentrations of total aflatoxin (μg kg−1) are means ± SD. N/A, not available. Means with the same letters are not significantly different at P < 0.05 (Tukey's post-hoc analysis). HSD Critical values: 5.87 and 5.97 in 2014 and 2015 at α = 0.05; 5.57 and 5.65 in 2014 and 2015 at α = 0.10. The GCA data were log2(y + 1) transformed.

other lines in 2015. This was the case at both α = 0.05 and 0.10 which is reflective of the high degree of variability in aflatoxin contamination existing within each line, and significant plot to plot variability within the field environments. The only inbred line found to significantly differ from B73 and Mo17 in the post-hoc analysis in 2015, though not in 2014, was CML247 which had the lowest consistent level of aflatoxin across both years (Table 1). Numerically, the inbred lines CML52, CML69, CML247, GT603, GEMS-0005, Hi63, Hp301, and M37 W showed <1000 μg kg−1 of aflatoxin accumulation in both years of the study (Table 1). This is consistent with previous reports on the observed resistance of these inbred lines in other environments. For example, Mideros et al. [33] found that CML52, CML247, and M37 W accumulated levels of aflatoxin and A. flavus mycelial growth comparable to Mp313E in Mississippi field conditions. CML69 was also found to be resistant to aflatoxin contamination in Mississippi and Texas field environments in previous evaluations [34]. GT-603 was derived from the GT-MAS:gk population, and has been shown to exhibit resistance in Georgia and other Southern US environments [35]. GEMS-0005 was developed as a part of the Germplasm Enhancement of Maize (GEM) cooperative effort to enhance maize resistance to disease, and hybrids of GEMS-0005 have been shown to exhibit resistance to aflatoxin contamination across multiple environments in the Southern US [23,27]. Hi63 has no previously reported resistance to aflatoxin contamination, but has been reported to possess resistance to Puccinia polysora [36]. Hp301 has been found to possess an active ZmLOX5 gene which has been linked to aflatoxin contamination resistance in previous QTL studies [37]. This indicates that these inbred lines may serve as novel sources of resistance for use in breeding programs and warrant additional investigation. This reduction in aflatoxin content in the hybrids is consistent with the observed GCA (Table 1) and heterotic (Table S2) effects observed for these lines. While no significant GCA effects were observed for the 2015 hybrid samples, in 2016, CML52, GT-603, and Hi63 showed significantly negative GCA effects (P < 0.05) toward reduced aflatoxin content in their hybrids with B73 and Mo17. Previous studies have also found that GCA effects for inbred lines in diallel crosses are negatively correlated with observed aflatoxin contamination in hybrids. For example, Williams and Windham [27] found that during examination of a diallel cross between 10 inbred lines with contrasting aflatoxin resistance GCA effects observed for susceptible lines (i.e., B73, PHW79, T173, and Va35) tended to be significantly positive while for lines with less contamination (i.e., Mp313E, Mp494, Mp717, and Mp719) GCA

effects were significantly negative. The results in the present study also reflect this negative correlation, though few significant GCA effects were observed, indicating that inbred and hybrid performance may be correlated. While the number of testers used in this experiment do not allow for a complete evaluation of the potential GCA of these lines, the lines with significant GCA here may be useful in diallel crosses and broader topcrosses to investigate their GCA and specific combining ability (SCA) in future studies. Testcross hybrids generated in also exhibited varying levels of resistance to aflatoxin contamination, though as with the inbreds little means separation was observed in the post-hoc analysis (Table 2). In particular, very few differences were observed in 2015 with only M37 W × B73 being significantly lower compared to the other hybrids at both α = 0.05 and 0.10. This may be due either to both plot-to-plot variation as mentioned for the inbreds, or lack of significant GCA effects detected between the inbreds and the testers in 2015. In 2016, a greater number of significant differences could be observed thought the hybrid with the resistant check, Mp313E × B73, showed no significant differences with any other hybrid except GT-603 × Mo17 which accumulated the least aflatoxin in the 2016 study (Table 2). Along with GT-603 × Mo17, the hybrids CML52 × B73, Hp301 × Mo17, and M37 W × B73 also showed significantly less aflatoxin accumulation compared to the most heavily contaminated hybrids (Table 2). These additional hybrids were also not significantly different from Mp313E × B73. Numerically, hybrids of several of the most resistant inbred lines including CML247, CML52, GT-603, and Hi63 showed among the lower levels of aflatoxin contamination observed when crossed with both B73 or Mo17 (Table 2). CML52, GT-603, and Hi63 also had the only significant GCA effects detected in 2016 (Table 1). M37 W × B73 was also the most consistently resistant F1 hybrid identified in the study with an average of 200 ± 180 μg kg−1 across both years of the study. In contrast to these lines, the inbred lines used as testers in the topcrosses, B73 and Mo17, showed higher levels of aflatoxin contamination with an average of 1824 ± 1091 and 759 ± 654, respectively (Table 1). These hybrids also exhibited lower aflatoxin contamination than observed in the two commercial hybrids included as checks in 2016, Dekalb DKC69–43 and Dupont-Pioneer P-2023HR, which accumulated an average of 764 ± 661 and 1404 ± 962 μg kg−1, respectively. When examining mean parent heterosis (Table S2), CML52 and M37 W show heterotic effects across years on aflatoxin content of −24.50% and − 13.64% when crossed with B73. In

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Table 2 – Mean aflatoxin contamination for F1 topcross hybrids. Line

2015 ×B73

GP 282 Tx303 Lo 964 Grace E-5 (E-1) NC290A-B F54 B97 Ky21 CML69 Lo 1016 HBA-1-B Tun 85-1B-B CML228 Oh43 MS71 Va35 GP 280 GEM 0028-2-1-B-B-B-1B-1B CML52 GT A1R TP Yellow E-1 CY 5 Tun 88-1B-B 10 GEM06845-1B-1B CY 1 Hi31–1B-1B-1B GT A2 R GEMS 0005-1-1-1-1B-1B Hi 63-1B-1 Tzi8 GT A554 ZM 521 E-1 Mo18W CML103–2 GT 603 NC358 CML333 A638/MP313E//MP313E/f6–35-B6 GT-888 NC350 CML247 Ki11 M37 W I114H EP M6 Coy 05 AT 823 CY 3 CY 4 LH 51 LH 132 Coy 05 AT 709 GT A661(SP) Coy 05 AT 819 TX 732 Coy 05 AT 805 GT A641(SP) CY 2 GT A638 CML277 FAW 1430 Syn AM 1 P43

2387 ± 430 2277 ± 2082 2217 ± 2404 N/A 1392 ± 2260 1075 ± 1208 1047 ± 1435 874 ± 900 822 ± 1030 821 ± 814 651 ± 892 611 ± 249 562 ± 433 544 ± 218 467 ± 528 435 ± 312 414 ± 227 399 ± 511 398 ± 441 388 ± 283 388 ± 49 369 ± 354 362 ± 299 350 ± 108 298 ± 44 251 ± 135 246 ± 117 245 ± 30 245 ± 277 222 ± 29 220 ± 116 210 ± 130 207 ± 78 199 ± 237 195 ± 52 191 ± 96 187 ± 188 169 ± 117 152 ± 122 117 ± 101 98 ± 62 58 ± 39 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A

2016 ×Mo17

a ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab b

614 ± 195 N/A 1435 ± 1164 551 ± 365 N/A 476 ± 177 1790 ± 1001 913 ± 165 166 ± 196 1027 ± 564 782 ± 552 N/A 488 ± 170 N/A N/A N/A 977 ± 250 99 ± 72 713 ± 998 815 ± 716 657 ± 268 N/A N/A 1100 ± 687 N/A 1642 ± 1318 N/A 151 ± 63 N/A 573 ± 562 1065 ± 808 226 ± 50 493 ± 752 586 ± 662 N/A 528 ± 133 1100 ± 257 853 ± 640 N/A 344 ± 143 406 ± 219 N/A N/A 2290 ± 1736 1801 ± 2425 1437 ± 1084 1020 ± 410 1007 ± 543 941 ± 1232 758 ± 409 626 ± 487 516 ± 510 516 ± 417 497 ± 116 433 ± 346 409 ± 291 382 ± 230 318 ± 59 222 ± 100 N/A

×B73 ab ab ab ab ab ab ab ab ab ab

ab ab ab ab ab

ab ab ab ab ab ab ab ab ab ab ab ab ab

ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab

1647 ± 1265 1289 ± 809 1585 ± 1473 1311 ± 872 913 ± 70 855 ± 518 1737 ± 833 1431 ± 949 1116 ± 710 799 ± 765 2382 ± 1138 2321 ± 895 1374 ± 1022 1086 ± 1250 1236 ± 916 1319 ± 691 911 ± 519 653 ± 475 293 ± 159 595 ± 217 620 ± 301 773 ± 362 1079 ± 715 2588 ± 1358 841 ± 1039 1842 ± 1132 898 ± 822 715 ± 697 1076 ± 848 1789 ± 1051 2051 ± 1168 N/A 1095 ± 980 591 ± 596 1073 ± 636 1622 ± 694 1670 ± 533 2771 ± 780 520 ± 220 944 ± 729 1210 ± 824 270 ± 182 1439 ± 938 1367 ± 1306 1031 ± 834 892 ± 626 851 ± 389 954 ± 452 1552 ± 935 2104 ± 791 N/A 2404 ± 1218 793 ± 220 993 ± 510 1108 ± 702 1370 ± 708 1220 ± 810 1351 ± 858 1452 ± 799 1151 ± 502

×Mo17 a-e a-e a-e a-e a-e a-f a-e a-e a-e a-f a-d abc a-e a-f a-f a-e a-f a-f def a-f a-f a-f a-f abc a-f a-e a-f a-f a-f a-e a-d a-f a-f a-e a-e a-e a a-f a-f a-e ef a-e a-f a-f a-f a-f a-f a-e abc a-e a-f a-e a-e a-e a-e a-e a-e a-e

1144 ± 816 2121 ± 835 1030 ± 1169 2073 ± 743 447 ± 333 1889 ± 564 1808 ± 845 1804 ± 1065 1518 ± 458 1816 ± 1278 1409 ± 632 1596 ± 874 1722 ± 722 1190 ± 695 668 ± 346 990 ± 585 1744 ± 869 1396 ± 800 404 ± 302 961 ± 880 2238 ± 481 989 ± 465 1689 ± 511 1621 ± 923 1123 ± 383 1055 ± 615 1290 ± 746 449 ± 280 1040 ± 763 1822 ± 1165 1365 ± 467 745 ± 473 1586 ± 591 157 ± 135 1269 ± 532 1194 ± 602 1260 ± 685 1038 ± 589 636 ± 324 665 ± 513 781 ± 567 1812 ± 786 1908 ± 829 747 ± 162 415 ± 117 901 ± 290 1290 ± 1032 1623 ± 1025 701 ± 450 1462 ± 1216 912 ± 534 1925 ± 572 987 ± 629 1119 ± 950 980 ± 819 671 ± 513 1518 ± 947 955 ± 506 1582 ± 796 888 ± 275

a-f abc a-f abc c-f a-d a-e a-e a-e a-e a-e a-e a-e a-e a-f a-f a-e a-e b-f a-f ab a-e a-e a-e a-e a-e a-e b-f a-f a-f a-e a-f a-e f a-e a-e a-e a-e a-f a-f a-f a-d a-d a-f a-f a-f a-e a-e a-f a-e a-f a-d a-f a-f a-f a-f a-e a-f a-e a-f

(continued on next page)

124

TH E CR OP J OUR NA L 7 ( 2 0 19 ) 11 8 –1 2 5

Table 2 (continued) Line

2015 ×B73

CML322 Hp301 B37 MP313E

N/A N/A N/A N/A

2016 ×Mo17 N/A N/A N/A N/A

×B73 1123 ± 552 N/A ± N/A 531 ± 231 1210 ± 790

×Mo17 a-e a-f a-e

1178 ± 504 460 ± 325 1089 ± 351 N/A

a-e def a-e

Data are means of all replications within the indicated year. Concentrations of total aflatoxin (μg kg−1) are means ± SD. N/A, not available. Means with the same letters are not significantly different at P < 0.05 (Tukey's post-hoc analysis). HSD Critical Values: 6.12 and 6.25 in 2015 and 2016 at α = 0.05; 5.81 and 5.98 in 2015 and 2016 at α = 0.10.

contrast, if crossed with Mo17, these lines showed effects of 13.98% and − 0.68%, respectively. This indicates that these lines show greater heterotic effects for aflatoxin contamination resistance with the stiff-stalk tester B73. For GT-603 and Hi63, negatively heterotic effects across years were observed with both testers with −12.47% and − 8.84%, respectively, with B73 and − 13.11 and − 9.90%, respectively, with Mo17. This suggests that these lines may serve as versatile sources of resistance in breeding programs. In addition, the greatest heterotic effect was observed with Mo18W which showed a heterotic effect across years of −29.23% with B73 and − 11.38% with Mo17. These results are consistent with previous observations of heterosis having an effect on aflatoxin contamination through unique genetic combinations, or through heterotic increases in hybrid yield relative to the parental inbred lines [23,24,26,34]. In this case, however, these effects are more likely due to the more consistent and superior agronomic performance of hybrids compared to inbred lines given that elevated heterotic effects were observed for crosses within the same heterotic groups in this study. Regardless, these inbred lines and hybrid combinations can be useful sources of aflatoxin resistance in breeding programs and potentially useful in commercial applications for the development of resistant germplasm.

4. Conclusions Aflatoxin contamination of maize and other staple food crops is a serious threat to food safety and security, particularly in developing countries. Since the discovery of pre-harvest aflatoxin contamination, breeding efforts have been focused on the discovery of novel sources of resistance, and the investigation of the interactions of maize resistance and the environment toward the development of high yielding varieties with stable aflatoxin contamination resistance across multiple environments. We evaluated the resistance of 64 maize inbred lines from our breeding program, and 128 topcross hybrids with two testers, B73 and Mo17 across two years in a field environment. Eight inbred lines, i.e., CML52, CML69, CML247, GT-603, GEMS-0005, Hi63, Hp301, and M37 W, were found to have aflatoxin contamination levels <1000 μg kg−1 which were less than or comparable to the resistant check, Mp313E. In particular, CML247 significantly accumulated less aflatoxin compared to the susceptible tester lines in both years of the study. Significant GCA effects were also detected for CML-52, GT-603, and Hi63. F1 hybrids of these

lines also possessed reduced aflatoxin contamination, and heterotic effects demonstrating their potential use in breeding programs for the development of resistant cultivars. Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.10.001.

Acknowledgments We thank Billy Wilson and Hui Wang for technical assistance in the field. This work is partially supported by the U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS), the Georgia Agricultural Commodity Commission for Corn, and AMCOE (Aflatoxin Mitigation Center of Excellence, Chesterfield, MO, USA). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity provider and employer.

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