Aflatoxin Accumulation in a Maize Diallel Cross Containing Inbred Lines with Expired Plant Variety Protection

Abstract

In-field infection of maize (Zea mays L.) ears by the fungus Aspergillus flavus Link:Fr causes pre-harvest aflatoxin contamination of maize grain. Germplasm lines with host-plant resistance to aflatoxin accumulation are available to breeders, but these lines often possess undesirable agronomic characteristics. Commercial lines with expired plant variety protection (ex-PVP lines) are a potential source of elite germplasm available to public maize breeders. A diallel cross containing three aflatoxin-accumulation-resistant germplasm lines and seven ex-PVP lines were evaluated in replicated trials for aflatoxin contamination after artificial inoculation and for yield. The resistant germplasm lines Mp313E, Mp715, and Mp717 were the only lines with significant general combining ability (GCA) for reduced aflatoxin accumulation. Of the ex-PVP lines evaluated, the Stiff-Stalk line F118 was the most promising line to use in breeding crosses. Based on its GCA, it was the only ex-PVP line that did not significantly increase aflatoxin and the only ex-PVP line that significantly increased yield. Second-cycle breeding lines derived from crosses between F118 and the resistant donor lines will be valuable if they combine the donor lines’ disease resistance with F118’s earlier maturity while introgressing the disease resistance into a genetic background that aligns with the industry’s well-defined heterotic groups.

1. Introduction

Aflatoxins are carcinogenic mycotoxins produced by Aspergillus spp. [1,2]. Aflatoxins are produced by many species of Aspergillus [3], but pre-harvest aflatoxin contamination of maize (Zea mays L.) grain is primarily caused by in-field infection of maize ears by Aspergillus flavus Link:Fr [4,5]. A. flavus is an opportunistic pathogen of maize, and increased infection is associated with insect damage and stress from heat and drought [6,7]. Aflatoxin contamination is a chronic problem in the Southern United States due to the prevalence of these biotic and abiotic stresses in the south, but climate change is likely to increase future risks in the midwestern Corn Belt [8].

Maize host-plant resistance to A. flavus infection and subsequent aflatoxin accumulation is heritable but it is a complexly inherited quantitative trait [9]. Several inbred maize lines with decreased susceptibility to aflatoxin accumulation have been released by public maize breeding programs, e.g., [10,11,12,13]. Despite possessing quantitative disease resistance, many of these germplasm lines possess unfavorable agronomic characteristics such as late flowering, slow grain dry down, and low combining ability for yield, which complicate their use in breeding [14]. Second-cycle breeding lines that combine the germplasm lines’ quantitative disease resistance with more favorable agronomic characteristics are valuable to breeders working to introgress A. flavus resistance into parental inbred lines, e.g., [15].

Proprietary inbreds with expired plant variety protection (ex-PVP) are a potentially important source of elite germplasm available to public maize breeders [16,17]. Commercial inbred lines are protected for 20 years under the U.S. Plant Variety Protection Act (PVPA) [18]. The PVPA requires the seed of PVP-protected inbred lines to be deposited in the U.S. National Plant Germplasm System (NPGS). These lines are available to the entire corn breeding industry once that protection expires and seed is distributed through the NPGS [18].

This study evaluated seven ex-PVP maize inbred lines along with three germplasm lines with resistance to aflatoxin accumulation in a diallel cross. The cross was evaluated for aflatoxin accumulation after artificial inoculation over two years and yield in one year. We aimed to identify ex-PVP lines that showed promise as breeding material in a public breeding program focused on developing inbred lines well-adapted to the southeast and resistant to aflatoxin accumulation. Ideally, ex-PVP lines that possess superior agronomic characteristics and a degree of resistance to A. flavus infection could be identified.

2. Materials and Methods

A diallel was produced by crossing ten maize inbreds: three aflatoxin accumulation resistant germplasm lines (Mp313E, Mp715, and Mp717) and seven ex-PVP lines (F118, LH195, LH197, PHW79, PHN46, MM402A, and HBA1) (

Table 1. Inbred lines included in diallel.

Inbred Line Organization	Origin a		Region Adapted b	Source c	PI Number d
Mp313E	USDA-ARS	MS		Reference [10]	PI 539859
Mp715	USDA-ARS	MS		Reference [11]	PI 614819
Mp717	USDA-ARS	MS		Reference [12]	PI 639919
F118	Dekalb			PVPA 9100248	PI 555462
LH195	Holden	IA, KY	Most Regions	PVPA 9000047	PI 537097
LH197	Holden	IA	Most Regions	PVPA 9200020	PI 557562
PHW79	Pioneer	TN	Southeast	PVPA 8800220	PI 601576
PHN46	Pioneer	IL	Northcentral	PVPA 9000249	PI 543844
MM402A	Dekalb			PVPA 9100222	PI 554615
HBA1	Dekalb	MO	Southcentral	PVPA 8500069	PI 601172

Notes: ^a Origin = the location of nursery rows (excluding winter nurseries) where lines were derived (MS = Mississippi; IA = Iowa; KY = Kentucky; TN = Tennessee; IL = Illinois; and MO = Missouri); ^b Region adapted = as indicated in the PVP application section “region best adapted in the U.S.A.”; ^c Source = germplasm registration or PVP application for each inbred; ^d PI Number = the plant introduction number assigned within the U.S. National Plant Germplasm System.

and

Table 2. Pedigrees, lineage background, and heterotic group for ten maize inbreds constituting a diallel.

Inbred Line	Pedigree	Lineage Background a	Heterotic Group
Mp313E	Direct selfing from Tuxpan	Tuxpan
Mp715	Direct selfing from Tuxpan	Tuxpan
Mp717	Mp420 × Tx601	Southern Open-Pollinated and Tuxpan
F118	B73 × T220	Stiff Stalk Synthetic	SS b
LH195	LH117 × LH132	Stiff Stalk Synthetic	SS
LH197	LH132 × B84	Stiff Stalk Synthetic	SS
PHW79	PHT90 × PH595	Oh07-Midland	NSS c
PHN46	PHZ51 × PHV78	Oh07-Midland	NSS
MM402A	LH38 × DKMANS	Oh43	NSS
HBA1	Pioneer Hybrids 3195 × 3199	Commercial Hybrid Derived	NSS

Notes: ^a The lineage background is based on the organization of germplasm into backgrounds presented in [15]; ^b SS = Stiff Stalk (female); ^c NSS = Non-Stiff Stalk (male).

). The diallel cross consisted of 45 crosses, with no inbred parents or reciprocals. The hybrids were planted on 3 May 2018 and 30 April 2019 at R.R. Foil Plant Science Research Center, Mississippi State, Mississippi (MS), USA. Yield trials and disease trials were planted separately. Disease trials were planted to one-row plots, and yield trials were planted to two-row plots. Plots were 4 m long and rows were spaced 0.97 m apart. Plots were thinned to 20 plants, 4 m row⁻¹. Trials were planted as randomized complete blocks with 3 replications. Plots were managed according to standard production practices.

In the disease trials, the top ear of each plant was inoculated by the side-needle technique with a 3.4 mL suspension containing 3 × 10⁸ conidia of A. flavus isolate NRRL 3357 [19,20]. Inoculated ears were hand-harvested approximately 63 days after silking and dried at 38 °C for 7 days. Ears were bulk-shelled and ground using a Romer subsampling mill (Romer Industries Inc., Union, MO, USA). Total aflatoxin concentration was determined on a 50 g subsample using a VICAM AflaTest (VICAM, Watertown, MA, USA) in compliance with USDA test protocol [21]. Yield trials were machine-harvested at maturity using a Kincaid 8-XP single plot combine (Kincaide, Haven, KS, USA). The yield was adjusted to 15.5% moisture for each plot.

Aflatoxin concentrations were log-transformed as ln[y + 1] to normalize the distribution prior to statistical analysis. Yield and transformed aflatoxin data were analyzed using SAS generalized linear model procedures in SAS 9.4 (SAS Institute, Cary, NC, USA). Diallel analysis was performed using DIALLEL-SAS05 [22]. The diallel was analyzed according to Griffing’s method 4 (no reciprocals and no inbred parents), model 1 [23,24].

3. Results

Regarding the aflatoxin data (Supplemental Table S1), the analysis of variance indicated a significant hybrid-by-year interaction (p = 0.0215) and significant differences between hybrids within both years (p < 0.0001;

Table 3. Diallel analysis of variance for aflatoxin contamination over and within years.

		Mean Squares
Source	df	2018	2019	Over Years
		ln[y+1] a
Years	1	-	-	0.19
Rep (Years)	2, 4 b	0.39	0.49	0.40
Hybrids	44	5.93 **	4.98 **	9.40 **
Hybrids × Years	44	-	-	1.48 **
GCA	9	24.96 **	17.42 **	41.18 **
SCA	35	1.03	1.76 **	1.27
GCA × Years	9	-	-	1.20
SCA × Years	35	-	-	1.52 **
Error	89, 175 c	0.74		1.13

Notes: ^a y = concentration (ng g⁻¹) of total aflatoxins in a grain sample; ^b df = 2 within years and combined over years; ^c df = 89 within years and 175 combined over years; ** significant at p < 0.05.

). There was no significant interaction between general combining ability (GCA) and years (p = 0.2494), but GCA, as the main effect, was a highly significant source of variance (p < 0.0001). The interaction between years and specific combining ability (SCA) was significant (p = 0.0293). Within individual years, GCA was a significant source of variation in both years (p < 0.0001), and SCA was significant in 2019 (p = 0.0493) but not in 2018 (p = 0.1103). The estimated GCA effects for each line, within and across years, are presented in

Table 4. Estimates of general combining ability (GCA) effects of 10 inbred maize lines evaluated for yield and aflatoxin accumulation in a diallel cross.

	GCA Effects
	Yield	Aflatoxin
Inbred Line	2018	2018	2019	Over Years
	Mg ha−1	ln[y+1] a
Mp313E	0.60 **	−1.54 **	−0.65 **	−1.10 **
Mp715	0.77 **	−0.97 **	−1.13 **	−1.05 **
Mp717	−0.56 **	−1.75 **	−1.66 **	−1.71 **
F118	0.52 **	0.30 *	0.15	0.22
LH195	−0.10	0.53 **	0.32	0.42 **
LH197	−1.26 **	0.67 **	0.64 **	0.66 **
PHW79	−0.48 **	0.80 **	0.64 **	0.73 **
PHN46	0.06	0.76 **	0.50 **	0.63 **
MM402A	0.20	0.94 **	0.94 **	0.94 **
HBA1	0.24	0.26	0.26	0.25 *

Notes: ^a y = concentration (ng g⁻¹) of total aflatoxins in a grain sample; * significant at p < 0.10; ** significant at p < 0.05.

. The mean aflatoxin accumulation and SCA effect within individual years for each hybrid are presented in

Table 5. Mean aflatoxin accumulation and estimates of specific combining ability (SCA) for F₁ hybrids comprising a diallel cross.

	2018			2019
Hybrid	Geometric Mean a	Mean b	SCA Effect	Geometric Mean	Mean	SCA Effect
	ng g−1	ln[y+1]		ng g−1	ln[y+1]
Mp313E × F118	40	3.69	−0.55	263	5.57	0.65
Mp313E × LH195	130	4.86	0.40	215	5.37	0.28
Mp313E × LH197	26	3.26	−1.35 **	420	6.04	0.63
Mp313E × PHW79	157	5.06	0.31	355	5.87	0.46
Mp313E × PHN46	212	5.36	0.66	63	4.14	−1.12 **
Mp313E × MM402A	133	4.89	0.01	553	6.32	0.60
Mp313E × HBA1	47	3.86	−0.34	112	4.72	−0.31
Mp313E × Mp715	31	3.43	0.46	5	1.69	−1.95
Mp313E × Mp717	13	2.58	0.39	47	3.85	0.74
Mp715 × F118	59	4.07	−0.74	193	5.26	0.82
Mp715 × LH195	380	5.94	0.91 **	77	4.35	−0.26
Mp715 × LH197	92	4.52	−0.66	106	4.66	−0.26
Mp715 × PHW79	310	5.74	0.42	407	6.01	1.08 *
Mp715 × PHN46	153	5.03	−0.23	151	5.02	0.23
Mp715 × MM402A	201	5.31	−0.14	645	6.47	1.24 *
Mp715 × HBA1	137	4.92	0.16	160	5.08	0.53
Mp715 × Mp717	13	2.59	−0.16	3	1.20	−1.43 **
Mp717 × F118	41	3.71	−0.31	14	2.64	−1.27 **
Mp717 × LH195	30	3.41	−0.84 *	41	3.71	−0.37
Mp717 × LH197	101	4.62	0.22	116	4.75	0.36
Mp717 × PHW79	116	4.75	0.23	56	4.03	−0.37
Mp717 × PHN46	40	3.69	−0.78 *	165	5.10	0.85
Mp717 × MM402A	379	5.94	1.28 **	209	5.34	0.64
Mp717 × HBA1	52	3.95	−0.03	129	4.86	0.84
F118 × LH195	1036	6.94	0.64	313	5.75	−0.15
F118 × LH197	965	6.87	0.42	457	6.12	−0.09
F118 × PHW79	1016	6.92	0.35	387	5.96	−0.26
F118 × PHN46	688	6.53	0.00	461	6.13	0.06
F118 × MM402A	1052	6.96	0.25	590	6.38	−0.14
F118 × HBA1	391	5.97	−0.06	487	6.19	0.36
LH195 × LH197	749	6.62	−0.06	497	6.21	−0.17
LH195 × PHW79	1059	6.96	0.16	616	6.42	0.04
LH195 × PHN46	623	6.43	−0.32	1220	7.11	0.87
LH195 × MM402A	462	6.14	−0.80 *	596	6.39	−0.30
LH195 × HBA1	476	6.17	−0.09	431	6.07	0.07
LH197 × PHW79	1258	7.14	0.18	743	6.61	−0.07
LH197 × PHN46	2063	7.63	0.73	466	6.14	−0.41
LH197 × MM402A	1144	7.04	−0.05	748	6.62	−0.39
LH197 × HBA1	1051	6.96	0.55	829	6.72	0.40
PHW79 × PHN46	675	6.51	−0.52	765	6.64	0.08
PHW79 × MM402A	687	6.53	−0.69	953	6.86	−0.15
PHW79 × HBA1	439	6.08	−0.45	251	5.53	−0.80
PHN46 × MM402A	1560	7.35	0.18	587	6.37	−0.49
PHN46 × HBA1	880	6.78	0.29	446	6.10	−0.07
MM402A × HBA1	757	6.63	−0.04	267	5.59	−1.03 *

Notes: ^a Geometric means were calculated by converting logarithmic means back to the original scale after analysis; ^b Logarithmic ln[y+1], where y = concentration (ng g⁻¹) of total aflatoxins in a grain sample; * significant at p < 0.10; ** significant at p < 0.05.

. The within-year SCA effects are presented since the SCA by year interaction was significant.

All three resistant germplasm lines (Mp313E, Mp715, and Mp717) had highly significant (p < 0.0001) and negative GCA effects for aflatoxin accumulation (Table 4). In this context, negative GCA effects indicated that the line reduced aflatoxin contamination in its hybrids relative to the mean of all hybrids. All seven ex-PVP lines had positive GCA effects. Positive GCA effects indicated that the line caused increased aflatoxin contamination in its hybrids. The positive GCA effects of four of the seven ex-PVP lines (LH197, PHW79, PHN46, and MM402A) were highly significant (p < 0.0001). The only ex-PVP lines without such highly significant positive GCA effects were LH195 (p = 0.0027), HBA1 (p = 0.0694), and F118 (p = 0.1092). The Stiff-Stalk (SS) line F118 and the Non-Stiff Stalk (NSS) line HBA1 were the only ex-PVPs whose GCA did not cause a significant increase in aflatoxin accumulation (α = 0.05). Although these lines did not decrease aflatoxin and would certainly not be described as resistant, they would be the most promising ex-PVPs of this set to use in breeding crosses if they possess favorable agronomic characteristics.

Additionally, the agronomic potential of the lines was assessed in a yield trial. Poor stands in 2019 resulted in poor-quality yield data and only the 2018 yield results are presented (Supplemental Table S2). Since the disease and yield trials were planted as distinct trials in both years, the 2019 aflatoxin data were not affected by the poor stands observed in the 2019 yield trials. There were highly significant differences between hybrids on yield (p < 0.0001), and both GCA and SCA were highly significant (p < 0.0001) sources of variation (

Table 6. Diallel analysis of variance for yield in 2018.

Source	df	Mean Square
Rep	2	2.74
Hybrid	44	4.93 **
GCA	9	10.07 **
SCA	35	3.61 **
Error	88	1.24

Notes: ** significant at p < 0.05.

). The ex-PVP lines were evaluated as potential sources of improved agronomic characteristics, but in this set of lines, F118 was the only ex-PVP line with a significant positive GCA effect on yield (Table 4). Two of the three germplasm lines (Mp313E and Mp715) had significant positive GCA effects and two of the seven ex-PVP lines (LH197 and PHW79) had significant negative GCA effects on yield (Table 4). Of the 45 hybrids, 11 were SS × NSS crosses, but based on the SCA for yield of these hybrids, no trend for SS × NSS heterosis was observed within this set of lines. Of the 11 SS × NSS hybrids (entries 27–30, 32, 33, and 35–39) only one, LH197 × HBA1, had a significant (p = 0.0004) and positive SCA on grain yield (

Table 7. Mean yield and estimates of specific combining ability (SCA) effects for F₁ hybrids comprising a diallel cross grown in 2018 and mean aflatoxin accumulation for the same F₁ hybrids combined over 2018 and 2019.

		Yield		Mean Aflatoxin over Years a
Entry	Hybrid	2018 Mean	SCA Effects
		Mg ha−1		ng g−1
1	Mp313E × F118	7.30	1.05 *	103
2	Mp313E × LH195	7.14	1.51 **	167
3	Mp313E × LH197	3.39	−1.08 *	105
4	Mp313E × PHW79	6.87	1.62 **	236
5	Mp313E × PHN46	6.12	0.32	116
6	Mp313E × MM402A	6.06	0.12	271
7	Mp313E × HBA1	4.34	−1.63 **	73
8	Mp313E × Mp715	4.74	−1.76 **	13
9	Mp313E × Mp717	5.02	−0.15	25
10	Mp715 × F118	5.96	−0.47	106
11	Mp715 × LH195	6.88	1.07	171
12	Mp715 × LH197	4.38	−0.26	99
13	Mp715 × PHW79	5.95	0.51	355
14	Mp715 × PHN46	7.50	1.53 **	152
15	Mp715 × MM402A	6.93	0.82	361
16	Mp715 × HBA1	6.32	0.17	148
17	Mp715 × Mp717	3.74	−1.61 **	7
18	Mp717 × F118	5.06	−0.04	24
19	Mp717 × LH195	3.91	−0.56	35
20	Mp717 × LH197	4.21	0.90	108
21	Mp717 × PHW79	4.33	0.23	81
22	Mp717 × PHN46	5.05	0.40	81
23	Mp717 × MM402A	6.43	1.64 **	282
24	Mp717 × HBA1	4.00	−0.82	82
25	F118 × LH195	5.77	0.22	569
26	F118 × LH197	4.51	0.12	664
27	F118 × PHW79	4.62	−0.56	627
28	F118 × PHN46	5.73	0.01	563
29	F118 × MM402A	4.63	−1.23 **	788
30	F118 × HBA1	6.79	0.90	436
31	LH195 × LH197	3.06	−0.71	610
32	LH195 × PHW79	4.60	0.05	808
33	LH195 × PHN46	4.26	−0.83	872
34	LH195 × MM402A	6.18	0.95 *	525
35	LH195 × HBA1	3.56	−1.71 **	453
36	LH197 × PHW79	2.40	−1.00 *	966
37	LH197 × PHN46	3.69	−0.24	980
38	LH197 × MM402A	4.23	0.16	925
38	LH197 × HBA1	6.21	2.10 **	933
40	PHW79 × PHN46	4.23	−0.49	718
41	PHW79 × MM402A	4.16	−0.70	809
42	PHW79 × HBA1	5.22	0.33	332
43	PHN46 × MM402A	3.85	−1.56 **	957
44	PHN46 × HBA1	6.28	0.85	627
45	MM402A × HBA1	5.37	−0.20	450

Notes: ^a Geometric means were calculated by converting logarithmic means (ln[y+1], where y = concentration (ng g⁻¹) of total aflatoxins in a grain sample) back to the original scale after analysis. The logarithmic mean was the adjusted mean of the data combined over years; * significant at p < 0.10; ** significant at p < 0.05.

).

Of the 45 hybrids, 24 contained at least one of the three resistant germplasm lines (entries 1−24). If the 45 hybrids were ranked by their mean aflatoxin over two years, all 24 of those hybrids would be within the lowest 25 (Table 7). The only ex-PVP by ex-PVP hybrid to accumulate less aflatoxin than any hybrid containing a resistant line was the NSS-by-NSS cross, PHW79 × HBA1 (entry 42). This hybrid only outperformed the crosses Mp715 × PHW79 and Mp715 × MM402A. Mp715 had the highest GCA effect for aflatoxin of the three resistant lines, and PHW79 and MM402A had the highest GCA effects of the ex-PVP lines (Table 4).

The cross F118 × HBA1 had the second lowest mean aflatoxin over years of any ex-PVP by ex-PVP cross. This hybrid was also the highest yielding ex-PVP by an ex-PVP hybrid (Table 7). Of the ex-PVP lines evaluated, the SS F118 had both the highest GCA for yield and the lowest GCA for aflatoxin (Table 4), making it the most promising ex-PVP included in the diallel. Similarly, of the ex-PVP lines, NSS HBA1 had the second lowest GCA for aflatoxin and the second highest GCA for yield.

4. Discussion

Being able to assign ex-PVP lines, based on their pedigrees, to heterotic groups prior to evaluation should facilitate their use in breeding programs. However, even when lines are organized into heterotic groups, identifying promising pairs that will produce superior hybrids remains a particularly challenging component of maize breeding [25]. The lack of SS × NSS heterosis within this set of seven ex-PVP lines points to this difficulty. The lack of heterosis among the ex-PVP lines may partially explain their low GCA for yield compared to the three germplasm lines. It is also likely that the relatively low plant density used in the yield trial biased yield results against the ex-PVP lines than if those lines were bred to maintain yield at higher population densities. The ability to maintain yield at increased plant densities (increasing yield potential per unit area of land, but not yield potential per plant) was a key source of genetic gains for yield in the private sector during the period in which these ex-PVP lines were developed [26].

Future research focused on utilizing ex-PVP lines to breed adapted, aflatoxin-accumulation-resistant inbreds will focus on: 1. selection within segregating breeding crosses between resistant donor lines and F118, the ex-PVP line identified as most promising in the diallel; and 2. further evaluation of more ex-PVP lines. The first objective can be accomplished by breeding second-cycle lines using the resistant germplasm line as a donor line and the ex-PVP as the source of improved agronomics. In this effort, maturity is one of the most important traits to target during selection. In a recent summer nursery, F118 silked 16−27 days (458−788 growing degree-days) earlier than Mp313E, Mp715, and Mp717, the resistant lines used in this study (

Table 8. Relative days to flowering of four inbred lines planted in summer nursery rows at Mississippi State, MS on 20 April 2021.

	Male Flowering			Female Flowering
Genotype	Date a	Days b	GDD c	Date d	Days	GDD
F118	26-Jun	67	1424	27-Jun	68	1454
Mp313E	10-Jul	81	1824	13-Jul	84	1912
Mp717	13-Jul	84	1912	14-Jul	85	1942
Mp715	22-Jul	93	2180	24-Jul	95	2242

Notes: ^a Date of male flowering is the date when ~50% of plants in a nursery row shed pollen; ^b Days = the number of days between planting (20 April 2021) and the date of flowering; ^c GDD = the number of growing degree days between planting and the date of flowering; ^d Date of female flowering is the date when silks emerged on ~50% of plants in a nursery row.

). If the disease resistance present in these germplasm lines is to be utilized by industry, it must be introgressed into lines that fit into the industry’s relative maturity classes.

In addition to producing germplasm acceptable to industry, maturity (measured as both flowering time and grain moisture at harvest) is also especially relevant to studying aflatoxin accumulation as a trait per se. The process of pre-harvest aflatoxin contamination is closely associated with the process of grain development [6]. Confounding factors are consequently introduced if this trait is studied in germplasm with extreme maturities. For the resistance to be validated, it must be expressed in earlier-maturing genetic backgrounds. Maturity is also relevant to studying tolerance to abiotic stress, which is important as increased susceptibility to aflatoxin accumulation is associated with plant stress. Abiotic stress tolerance is necessary for a stable resistance response. Similar to disease resistance, stress tolerance must be expressed in genetic backgrounds with appropriate maturity if it is to be properly studied and validated. If disease resistance and stress tolerance can be introgressed from late-maturing germplasm to earlier lines, such as F118, then the traits can be validated, are more likely to be utilized by industry, and can be more effectively studied.

In addition to targeting maturity, it is important that second-cycle lines align with the industry’s heterotic groups. An ex-PVP line’s heterotic group can be inferred from its pedigree [16] or marker analysis [27]. F118, for example, is derived from B73 (Table 2). B73 is, of course, a very significant line within the SS heterotric group [16]. A disease-resistant germplasm line with a B73 background would fit more easily into industry breeding programs than a line that was not specifically selected to fit into a well-defined heterotic group. It is likely that recovering the heterotic pattern of an ex-PVP would require selection to be practiced in backcross populations using the ex-PVP as the recurrent parent. At the same time, since resistance to aflatoxin accumulation is a quantitative trait, the donor line would need to contribute many alleles to the second cycle breeding line. Thus, there would likely be tradeoffs between recovering the quantitative disease resistance and stress tolerance of the donor line and the maturity and heterotic pattern of the recurrent parent. However, second-cycle germplasm lines that possess disease resistance and stress tolerance, but also align with the industry’s relative maturity classes and heterotic groups, are necessary for the traits present in the germplasm lines to be utilized.

The second objective for future research is to evaluate more ex-PVPs to identify more lines to use in the breeding objectives just described. Many of the ex-PVPs evaluated in the present study performed relatively poorly, but only seven ex-PVP lines were evaluated. These seven lines were chosen from a set of ex-PVP lines based on their relative adaptation as observed in nursery rows (data not shown). It is estimated that there are ~500 ex-PVPs currently available and that there will be >1200 by 2028 [17]. To adequately sample this growing number of lines, more efficient breeding systems than diallels should be used. Diallels were used to study heterosis in ex-PVPs, e.g., [17], but for the sake of evaluating promising germplasm, factorial breeding schemes (North Carolina Design II) would be more efficient since they require fewer crosses to evaluate the same number of lines, e.g., [28]. The ex-PVP lines can generally be classified as males (NSS) or females (SS) based on their pedigrees and this information can be used to organize the lines into a factorial breeding scheme prior to evaluating them. For most ex-PVP lines, the PVP certificate provides the location where the line was developed and lists the best-adapted region. This information should be used to select material adapted to a breeder’s target region.

5. Conclusions

Of the seven ex-PVP lines evaluated for yield and aflatoxin in a diallel cross, F118 was the only ex-PVP with a GCA that significantly increased yield and the only ex-PVP with a GCA that did not significantly increase aflatoxin. F118 should therefore be crossed with the resistant germplasm lines to produce second-cycle lines that combine disease resistance from the germplasm lines with F118’s early maturity. Introgressing the disease resistance into F118’s B73 background will facilitate its use by industry breeders. Many more ex-PVP lines are available than the relatively few lines included in this diallel. This growing set of materials needs to be sampled and further evaluated to identify even more promising lines.