Micronutrient deficiency, resulting from inadequate intake of essential minerals such as zinc (Zn), is an increasingly serious food-related health problem [1
]. Approximately 20% of the world’s population suffers from Zn deficiency, with the highest risks for young children and pregnant women in sub-Saharan Africa and South Asia [2
]. Approaches to mitigate Zn deficiency include diet supplementation, industrial fortification, and food diversification. However, at a large scale, the impact of these interventions remains limited, especially in low-income countries, due to recurrent costs, poor infrastructure, and delivery systems [3
]. Therefore, development of Zn-enriched staple crops through breeding may complement those options [5
Maize is one of the major crops grown and consumed in regions where Zn deficiency is prevalent [8
]. For instance, in sub-Saharan Africa, 80% of the maize is consumed directly as food, providing at least 30% of the total calories [10
]. However, maize improvement programs have primarily focused on developing high-yielding varieties able to tolerate various biotic and abiotic stress factors in different agro-ecologies [13
]. Therefore, the production of micronutrient-rich varieties has lagged behind the improvement of other traits.
The physiological processes by which Zn accumulates in the maize kernels have not been completely described. A maize plant acquires Zn through the roots with uptake mediated by Zn-regulated transporters [14
]. Then, Zn is transferred to the vascular bundles for transport to the shoot [15
] and remobilized from the leaves into the kernels during grain-filling [16
]. In kernels, higher concentrations of Zn are observed in the aleurone and embryo than in the endosperm [17
The plant’s ability to accumulate Zn in the kernels can also be influenced by environmental conditions and soil properties. For example, an increase in soil pH decreases the uptake of Zn from the soil and reduces its availability to the plant [21
]. Low soil moisture, organic matter content, and temperature impairs Zn diffusion to the roots causing reductions in uptake and translocation into the shoot [23
]. Consequently, the genetic capacity of a plant to absorb Zn from the soil and accumulate it in the kernels for optimal nutritional benefit may not be fully realized.
The successful identification of desirable hybrid combinations depends on the combining ability of the parents and the gene effects involved in the expression of a trait. Several genetic studies involving mating designs documented that for kernel Zn general combining ability (GCA) effects were greater than specific combining ability (SCA) [26
]. Significant GCA effects indicate the preponderance of additive gene action for kernel Zn inheritance, implying that genetic gains can be realized from selection.
Kernel Zn has been investigated in several analyses of quantitative trait loci (QTL), which have shown that Zn accumulation is under the control of several loci, from 4 to 20 per population [30
]. Additionally, genomic regions associated with important QTLs for kernel Zn have been reported on chromosomes 2 and 6 [34
]. Consistent with mating designs, additive gene effects predominantly controlled kernel Zn concentration in the QTL studies. The QTL studies, however, were conducted with populations of inbred progeny created from parents unadapted to tropical environments [31
]. So, the relevance of those studies for hybrid breeding for tropical environments may be limited.
In maize, nutritional-related research has emphasized quality protein maize (QPM) to address protein malnutrition [36
]. QPM inbred lines are bred to be homozygous for a recessive allele at the opaque-2 locus, with elevated levels of amino acids lysine and tryptophan, and a hard endosperm in elite genetic backgrounds. Inbred lines with high-Zn have been identified among QPM [18
]. The high-Zn values suggest a possible influence of opaque2 (o2
) locus or possibly other genetic factors present in the QPM lines [44
]. A key hypothesis of this study is that some genetic effects for increased levels of zinc in some QPM inbred lines will be observed in their hybrid progeny. However, QPM maize with relatively low levels of Zn have been observed, suggesting that although o2
may play an important role, there might be other favorable loci unrelated to o2
that are required for the enhancement of Zn [18
Significant differences in concentration of Zn have also been documented among non-QPM inbred lines [28
]. The variability for kernel Zn among the inbred lines suggests a possibility to enhance the Zn content in maize [4
]. In the present study, groups of QPM (high-Zn QPM and low-Zn QPM) and non-QPM (high-Zn non-QPM and low-Zn non-QPM) inbred lines adapted to tropical environments were mated to produce hybrids using a modified mating design. The objectives of this study were (i) to estimate the combining ability of elite QPM and non-QPM inbred lines for kernel Zn, (ii) to explore the potential of developing high-Zn hybrids using QPM, non-QPM, and/or a combination of QPM and non-QPM inbred lines, (iii) to investigate the relationship between kernel Zn and other traits of agronomic importance, and (iv) to evaluate the relative importance of additive and non-additive genetic effects for Zn.
Genotypes (hybrids) and genotype × environment (G × E) variance components were significantly different (p
< 0.001) for Zn and grain yield (Table 2
). For Zn, the variance component for genotypes was larger (~3-fold) compared to the variance due to G × E and the heritability (H2
) estimate was 0.85. For grain yield, the variance due to genotypes was 5-fold larger than the G × E variance component and the H2
estimate was 0.91. The heritability for grain yield, while numerically higher than that of Zn, is substantially equivalent. The estimates of heritability for both traits suggest environmental sources of variation were relatively low in this experiment.
Averages for Zn and grain yield of all hybrids for each environment and across environments were estimated (Table 2
). The highest mean for Zn (26.51 µg/g) was observed in Tlaltizapan 2016 and the lowest was in Tlaltizapan 2015 (22.47 μg/g). Tlaltizapan 2016 was an exceptional environment in which 21 hybrids accumulated ≥30 µg/g of Zn (Table S3
). The mean grain yield across environments was 7.07 t ha−1
with range of 8.75 t ha−1
for Tlaltizapan 2015 to 4.87 t ha−1
for Cotaxtla 2015 (Table 2
The Pearson correlation coefficient values between pairs of traits ranged from −0.14 for Zn and plant height to 0.20 between Zn and days to anthesis (Table S3
). Correlation values between Zn and flowering dates (anthesis and silking date) were low (0.16–0.20) but significantly different from zero at p
-value <0.05 in each environment. Across environments, there was no significant correlation between Zn and any other trait.
The lack of an association between Zn and other traits is promising for maize breeding. Across environments, 15 hybrids were ranked in the top 10% for Zn (Table 2
). Those hybrids involved at least one inbred from the high-Zn group (QPM or non-QPM), had 12%–27% Zn content above mean of all hybrids in all environments (24.70 μg/g), and were produced from 13 inbred parents. Five inbreds were from the high-Zn QPM group and four inbred lines each were from the high-Zn non-QPM and low-Zn QPM groups. Six of the 15 hybrids were exclusively produced from QPM inbred lines, while nine were from crosses between QPM and non-QPM inbred lines. Inbred 2 from the high-Zn QPM group and inbred 20 from the high-Zn non-QPM group were parents to four hybrids each.
Among the top 10% of hybrids with high-Zn across environments, high-yielding hybrids with 7.80–9.40 t ha−1
of grain were identified (Table 2
). Inbred lines 1, 2, 3, and 4 from the high-Zn QPM group and 16, 17, 19, and 20 from the high-Zn non-QPM group were parents to those hybrids. However, despite the lack of correlation between grain yield and Zn, some of the hybrids that showed high-Zn concentration across environments were low-yielding. Overall, grain yield averages of the top 10% hybrids for Zn were 8%–13% lower compared to the averages for all hybrids in each environment and across environments.
The average values of Zn for each inbred line as measured in their hybrids were estimated for each environment and across environments (Table 3
). Based on those hybrids, average values for Zn among the four groups of inbred lines (A: high-Zn QPM, B: low-Zn QPM, C: low-Zn non-QPM, and D: high-Zn non-QPM) ranged from 21.15 to 27.97 μg/g. In all environments, the highest average value for Zn corresponded to high-Zn QPM inbreds (26.00 μg/g), while the lowest average value of Zn was recorded for low-Zn non-QPM inbreds (22.96 μg/g).
The genetic potential of the inbreds to serve as parents was assessed exclusively on the basis of their hybrid progenies (Table 3
). The top five mean values for Zn in each environment and across environments involved inbred lines 1 and 2. The highest mean value for grain yield was observed for 14 hybrids, which had inbred 13 as one of the parents. Genotypes with higher levels of Zn and grain yield were evident based on the performance of hybrids across the environments. Hybrids 51 and 60 were among the top 10 hybrids with high Zn and grain yield (Table S3
). Based on the average grain yield for inbreds as assessed in hybrid combinations, inbreds 1, 2, 16, and 20, which were parents to hybrids 51 and 60, attained grain yields of ≥7 tons ha−1
Analyses of means for Zn and grain yield were conducted for the hybrids across sets (Table 4
). Values for average Zn ranged between 19.72 µg/g for low-Zn (QPM × non-QPM) hybrids to 29.72 µg/g for high-Zn (QPM × non-QPM) hybrids. The set of hybrids formed from high-Zn inbreds (QPM × non-QPM) had the highest mean for Zn while the set formed from low-Zn inbreds (QPM × non-QPM) attained the lowest mean for Zn. The average values for grain yield across the sets of hybrids ranged from 3.35 t ha−1
for high-Zn QPM × low-Zn non-QPM to 10.57 t ha−1
for high-Zn QPM × non-QPM hybrids (Table 4
). Similarly, the set of hybrids formed from high-Zn inbreds (QPM × non-QPM) had the highest mean for grain yield, while hybrids produced from QPM inbreds (high-Zn × low-Zn) had the lowest mean for grain yield.
Variances of general combining ability (GCA) effects, i.e., female, male, or both, and specific combining ability (SCA) effects, i.e., female × male, differed among the six sets of hybrids for all traits (Table 5
). For Zn, significant variances due to GCA (female, male, or both) were observed in five of the six sets. The SCA effects were significant only in set four (low-Zn QPM × non-QPM). Partitioning the variances in each set and across the four environments, GCA (GCAm
) accounted for 76% to 96% of the variation observed in Zn (Table S5
Six inbred lines showed positive GCA effects for Zn (Table 6
). Among the six, two inbred lines, 2 and 8, were QPM, and four, 11, 13, 16, and 20, were non-QPM. Inbred lines 1, 2, 16, and 20 were parents to hybrids that attained ≥30 μg/g of Zn across environments (Table S3
). The significant and positive GCA effects indicated the inbreds would contribute favorable alleles for Zn in a breeding program if used as males or females, irrespectively. Of the two QPM inbred lines, line 2 was a member of the high zinc group. Of the four non-QPM lines, 16 and 20 were from the high zinc group. Inbred line 16 showed positive GCA for Zn and zero or negative GCA for grain yield (Table 6
). Forty-one hybrids had positive SCA for kernel Zn (Table S7
The inbreds’ phenotype may provide useful information for creating hybrids with elevated levels of Zn in the kernel. In this study, hybrids with a Zn content ≥30 μg/g across environments were produced exclusively from inbred lines classified as high-Zn parents, such as inbred 1 and 2 from the high-Zn QPM group and 16 and 20 from the high-Zn non-QPM group. Similar observations were reported in maize [58
] and pearl millet [59
]. However, the Zn levels were lower for all hybrids derived from high-Zn lines compared to the respective values observed in their parental inbred lines. This is consistent with previous studies in maize [58
] and pearl millet [60
], which reported significantly lower Zn in hybrids compared to their parental inbred lines. Therefore, an additional criterion, evaluation in hybrid combinations, should be considered when selecting inbred lines for use as parents of hybrids with higher Zn content.
Nutritional improvement in crop plants, including Zn-enriched maize hybrids, may result in a yield penalty [8
]. However, previous studies have reported that yield and nutritional traits, such as kernel Zn, could be improved simultaneously [64
]. In this study, grain yield was not correlated with Zn (r = 0.02). Similar observations were reported in previous studies of maize [27
] and pearl millet [61
]. Lack of correlation between grain yield and Zn suggested the possibility of improving maize for Zn concentration without reducing the grain yield potential of the hybrids. Consistent with previous studies, hybrids with elevated Zn and grain yield have been reported [7
Hybrids developed from mating high-Zn (QPM × non-QPM) inbreds had enhanced levels of Zn concentration. Increased levels of Zn have been reported for QPM germplasm compared to non-QPM germplasm [18
]. The dominant, wild-type allele of the o2
locus codes for a transcriptional factor that regulates the synthesis of zeins [70
]. In genotypes homozygous recessive at the o2
locus, there is a decrease in α-zein [71
] with a proportional increase of non-zeins such as albumins, glutelins, and globulins [72
]. Those non-zeins are known to bind Zn in the endosperm [73
]. Thus, in QPM inbreds, and possibly in some of their hybrids, the elevated levels of Zn could be attributed to reduced levels of zeins and relatively higher levels of other Zn-binding proteins [40
Higher levels of Zn have also been reported in non-QPM inbred lines [43
]. For such inbreds, and perhaps in their hybrids, higher Zn levels could be attributed to genetic factors unrelated to the o2
locus. Therefore, it might be helpful to explore other mechanisms that can potentially account for high-Zn in those genotypes. During grain filling, metal-binding proteins such as metallothioneins, phytochelatins, and nicotianamine are thought to bind Zn in large amounts [75
]. The storage capacity of those binding proteins could possibly be associated with the amount of Zn that accumulates in a maize kernel. Genotypes with a high capacity for Zn storage may possess more Zn-binding proteins. Consequently, enhanced levels of Zn may be achieved in genotypes with more Zn-binding proteins than genotypes with fewer Zn-binding proteins [40
]. Instead, enhanced levels of Zn in those hybrids may be attributed to the increase in Zn-binding capacity because of the metal-binding proteins.
In addition, other possible sources of higher levels of Zn in kernels may be attributable to disproportionate growth of the endosperm and embryo. In maize, approximately 49% of the total kernel Zn is in the embryo and the remainder is in the endosperm [19
]. If either tissue grows in an unexpected and disproportionate manner, the total amount of Zn in the kernel could be affected. Inbred lines and their hybrid progeny often display different phenology and durations of developmental stage. For example, it is well-known that inbred lines flower later and are shorter than their hybrid progeny. Thus, in addition to the possibility of Zn-binding proteins, the relative proportions of embryo and endosperm in the hybrid progeny should be considered in future investigations.
Understanding the nature of gene action responsible for Zn accumulation in maize kernels could be important in designing an effective breeding strategy for hybrids with increased Zn. The GCA effects accounted for ≥70% of the total variability, suggesting that the accumulation of Zn in maize kernels is predominantly governed by additive gene effects. Similar results were reported in maize [26
], pearl millet [59
], rice [77
], sorghum [79
], and wheat [80
]. With predominance of variance due to GCA, hybrids with enhanced Zn levels can be obtained by crossing parents with positive GCA effects [81
Among the 10 inbred lines that were originally classified as high-Zn parents, only three inbreds, namely, inbred 2 from the high Zn-QPM group, and 16 and 20 from the high-Zn non-QPM group, had positive GCA effects. This observation was contrary to an earlier study involving 14 inbred lines in which positive GCA effects were observed for all high-Zn parents (seven), while significantly negative GCA effects were detected for the low-Zn lines [28
]. In addition, positive GCA effects for Zn were detected for inbreds 8, a low-Zn QPM, and 11 and 13, both from the low-Zn non-QPM group. The positive GCA for Zn observed for inbred lines 2, 8, 11, 13, 16, and 20 suggest the possibility of transmitting favorable alleles from these parental lines to their hybrids and could be useful for breeding to improve Zn content.
Kernel Zn is a phenotype determined late in the development of a maize crop, subject to environmental influences, requiring extensive sample preparation, trained analysts, and costly equipment. Therefore, it could be helpful to identify a secondary trait that can potentially be used for indirect selection of Zn. During growth and development of a maize plant, the vegetative parts serves as a primary source of Zn for kernels. Consequently, plant height could conceivably be used as a secondary trait associated with Zn in kernels. More Zn may be remobilized to the kernels of taller plants than kernels of shorter plants. However, in this study, there was no correlation between plant height and Zn concentration as noted in previous research [68