Combining Ability and Heterosis for Agronomic Traits, Husk and Cob Pigment Concentration of Maize

: The objective of this study was to identify the maize inbred lines with good general combining ability (GCA), good specific combining ability (SCA), high heterosis for yield and phytochemicals, and the crosses with high yield of yellow kernels and high anthocyanin content in cobs and husk, which was probably related to the high antioxidant activity. The parental lines including ﬁve unpigmented females and ﬁve pigmented males were crossed in North Carolina design II. The parents, the resulting 25 hybrids, and 5 controls were evaluated at two locations in the dry season of 2016 / 2017. Additive and non-additive gene e ﬀ ects controlled the inheritance of grain yield, agronomic traits, and phytochemicals. KKU–PFC2 and KKU–PFC4 had the highest GCA e ﬀ ects for phytochemical traits in husk and cob, whereas Takfa1 and Takfa3 were good combiners for grain yield. F 1 hybrids had signiﬁcantly higher total anthocyanin content (TAC), total phenolic content (TPC), (2,2-diphenyl-1-picrylhydrazyl) (DPPH), and trolox equivalent antioxidant capacity (TEAC) in husk and cob than pigmented control cultivars. The hybrids superior for individual traits were identiﬁed, but the experiment was not able to identify superior hybrids for multiple traits. The Takfa3 × KKU–PFC5 and NakhonSuwan2 × KKU-PFC4 had the highest anthocyanin in husk and cobs, respectively. The breeding strategies to develop maize varieties with high anthocyanins and normal yellow kernels and utilization of the hybrids are discussed.


Introduction
Field corn is one of the most important cereal crops in the world, and it is used in human and animal diets [1]. Yellow corn is a source of provitamin A carotenoids required for growth, and it is used as a coloring agent for eggs and skin in poultry to better match the preference of customers [2]. Moreover, purple corn kernel is rich in anthocyanins and phenolic compounds [3][4][5], and these phytochemicals are also found at high concentrations in Husk [6,7] and cob [7,8]. Anthocyanins and phenolic compounds are known to have beneficial antioxidant properties [9]. The compounds help prevent several non-contagious diseases such as cancer [10,11], cardiovascular disease [12], obesity [13,14], and diabetes [15]. Recently, anthocyanin extracted from purple corn has been used ear height were measured on 10 randomly chosen plants in each plot after reproductive stage. Husk yield and cob yield were recorded as dry husk mass and cob mass per plot and converted to kg per hectare. The ears were shelled. Grain moisture was measured by a grain moisture tester (model EE-KU) developed by EE-KU Lab, Bangkok, Thailand according to the manufacturer's directions. Grain yield was expressed as kg ha −1 at 15% moisture content.

Sample Preparation and Extraction
Ten ears from each replication of each treatment were randomly harvested at physiological maturity (approximately 40 days after pollination for parental lines and approximately 50 days after pollination for hybrids) and oven-dried at 40 • C for 48 h. The anthocyanin extraction was performed as described in [36,37]. Husk and cobs were harvested from each replication and were ground into powder separately. The powdered samples of approximately 2 g were loaded into 100 mL flasks containing 20 mL of 100% methanol. The flasks were shaken on a multi-stirrer at 200 rpm for 1 h at room temperature. The samples were filtered through Whatman #1 filter paper. After filtration, the retentates were loaded again into 100-mL flasks containing 20 mL of 100% methanol, shaken on a platform shaker for 1 h, and again filtered through Whatman #1 filter paper. The two filtrates were combined and evaporated in a rotary evaporator at 40 • C to reduce the volume from 40 mL to 10 mL and the concentrated solution was stored at −20 • C.

Determination of Total Phenolic Content (TPC)
Total phenolic content in each sample was determined according to Folin-Ciocalteau's phenol reagent (FC reagent) procedure with minor modification [38]. The reaction was prepared by mixing 0.5 mL methanol extract, 2.5 mL water, and 0.5 mL FC reagent, which was pre-diluted from 2 M to 1 M with distilled water. The mixture was set aside at room temperature for eight minutes and 1.5 mL Na 2 CO 3 solution was added to the mixture. The solution was allowed to stand for 120 min at room temperature. Then, the absorbance was read at 765 nm using a UV-visible spectrophotometer. Gallic acid solutions (10-100 mg/L) were used as reference standards. The total phenolic content (TPC) was expressed as mg gallic acid equivalents/100 g dry weight of samples (mg GAE/100g DW).

Determination of Antioxidant Assay
The assay of DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical-scavenging activity was performed by measuring the capacity for bleaching a black-colored methanol solution of DPPH radicals as reported by [39]. Briefly, the reaction for each sample was prepared by mixing 4.5 mL methanolic solution of DPPH (0.065 mM) and 0.5 mL of solution extract or a standard solution. The reaction was conducted at room temperature for 30 min before the absorbance was recorded at 517 nm. The radical-scavenging activity of the extracts was calculated as follows; where Ao is the absorbance of the control solution (0.5 mL extraction solvent in 4.5 mL of DPPH solution), A1 is the absorbance of the extracts in DPPH solution, and As, which is a term for correction of errors arising from unequal color of the sample solutions, is the absorbance of the extract solution without DPPH. The value was expressed as percentage (%) of DPPH free radical-scavenging activity assay. The trolox equivalent antioxidant capacity assay (TEAC) for each sample was executed according to the method described by [39] with minor modifications. Briefly, ABTS+ radical cations were generated by a reaction of 7 mmol/l ABTS and 2.45 mmol/L potassium persulfate. The reaction mixture was allowed to stand in the dark at room temperature for 16-24 h before use and the mixture was used within 2 days. The ABTS+ solution was diluted with methanol to an absorbance of 0.700 ± 0.050 at 734 nm. The diluted extract of 50 microliters was mixed with 2.0 mL of diluted ABTS+ solution for 6 min at room temperature, and the absorbance was immediately recorded at 734 nm. Trolox solution (100-1000 µM) was used as a reference standard. The value was expressed as millimoles of trolox equivalents (TE) per 100 g of dry weight (mmol TE/100 g DW).

Statistical Analysis
Analysis of variance was performed separately for each location and error variances were tested for homogeneity [40]. Error variances were homogeneous, so the data from the two locations were combined. The following statistical model was used; where Y ijkd is the observed value in location d, replication k, male i, and female j; µ is the grand mean, L d is the location effect (d = 1,2), R k (L d ) is the effect of replicate k nested in location d (k = 1,2,3); m i is the male effect (i = 1,2,3,4,5); f j is the female effect (j = 1,2,3,4,5); m i × f j is the interaction between male and female; L d × m i is the interaction between location d and male i; L d × f j is the interaction between location d and female j; L d × m i × f j is the interaction between location d, female j and male i; and e ijkd is the pooled error effect. Calculations were performed with AGD-R [41]. Variances of hybrid effect were further partitioned into due to GCA and SCA, and GCA effect of parents and SCA effects of hybrids were calculated based on means of 25 hybrids for agronomic traits, total anthocyanin content, total phenolic content, and antioxidant activity to obtain estimates of SCA of the hybrids and GCA of the parents. Mid-parent heterosis (MPH) and high parent heterosis (HPH) of each hybrid for all traits were calculated and expressed in percentages using trait means of parents and hybrids across two locations. For each trait, the mid-parent value of a cross was calculated as the mean of the parental lines averaged across locations. Hence, MPH was computed as; Agriculture 2020, 10, 510 where F 1 is the mean performance of the cross; MP is the mid-parent value given by (P1 + P2)/2; P1 and P2 are the mean values of parent 1 and parent 2 averaged across locations, respectively. HPH was calculated as; where HP = the better parental mean across locations. The test for significance of MPH and HPH was done by comparing mean values of MP or HP to the hybrid value using Student's t test at 0.05 probability level.

Analysis of Variance
Locations were significantly different for most traits except for cob DPPH (Tables 2 and 3), indicating that the location was an important source of variations in agronomic traits and phytochemical content. Soil heterogeneity, temperature, and nutrient availability are the factors affecting anthocyanin pigment accumulation [42,43]. The effects of hybrids were also significant for all traits, suggesting that selection on the tested hybrids would be possible. Hybrid × location interactions were significant for most traits excluding cob weight and days to anthesis, demonstrating that hybrids responded differentially to environments although the magnitudes of interaction effects were small. These interaction effects, although small, could confound the selection of superior hybrids, and multi-location testing of the hybrids is still required.
The significance of GCA and SCA effects revealed the presence of both additive and non-additive gene effects for most traits. Additive gene effects were predominant for husk weight, anthesis day, plant height, ear height, husk TAY, husk TAC, husk TPC, husk DPPH, husk TEAC, cob TAY, cob TAC, cob TPC, and cob DPPH, whereas overwhelming non-additive gene effects were noticed for grain yield, cob weight, and cob TEAC.
Based on the results, three breeding strategies should be devised for the most effective selection programs. Because the interactions between genotype and environment were significant for yield, agronomic traits, and anthocyanin content, evaluation of breeding lines and hybrids in multi-location trials is required. As the purple color was expressed in the F 1 generation and gene expression for anthocyanins was additive, visual selection of colored plants using simple or modified mass selection would be effective for improving anthocyanins in husk and cob in early cycles of selection. Breeders could also perform visual selection for early flowering and lodging tolerance to fix the favorable alleles. In the latter selection cycles, when the colored plants are more uniform, chemical analysis of anthocyanins should be performed and selection for grain yield and cob weight should also be carried out to ensure cultivars with the greatest overall value are selected.
Female GCA effects were larger than male GCA effects for most traits shown in Table 4 except plant height and TAY in cob. Female GCA effects were larger than male GCA effects for all phytochemical traits in husk traits, but female GCA effects were smaller than male GCA effects for all phytochemical traits in cob (Table 5). This may reflect the difference in the genetic control of phytochemical accumulation in cob and husk tissues.

General Combining Ability Effects
The effects of general combining ability (GCA) are useful for identification of superior parents for direct use in breeding programs [33,44]. The selected inbred lines should have high GCA that is significantly different from zero and a high mean value to predict the best progeny based on GCA. The GCA effects of 10 parental lines for grain yield, agronomic traits, TAC, TPC, and antioxidant activity determined by the DPPH and TEAC methods across two locations are shown in Tables 4 and 5. The female lines had greater ranges of effects than the male lines for all agronomic traits (Table 4). This could be a property of the germplasm or it could be due to the direction of the cross. The cross of all possible combinations and reciprocal cross in diallel mating scheme might differentiate these possibilities.

Specific Combining Ability Effects and Heterosis
Specific combining ability (SCA) describes the performance of the crosses relative to the averaged performance of hybrids in the experiment. SCA is related to non-additive gene effects such as dominance and epistasis. The hybrids combinations that showed high and significant SCA effects may be valuable in a breeding programs [45][46][47].
An important objective of this research project is to find hybrids with high anthocyanin yield. This would be the combination of high anthocyanin concentration in husk and cob and high weights of husk and cob. In this study, superior hybrids for individual traits were identified. However, the study was not able to identify the superior hybrids for multiple traits such as grain yield, early maturity, and high anthocyanins. It may be helpful to implement a selection index in order to develop cultivars with optimal value considering both grain and phytochemical yield.
The F 1 hybrids in this study were crossed between female lines with unpigmented husk and cob and yellow kernels and male lines with purple husk and cob and purple, white, or yellow kernels, resulting in F 1 hybrids with purple husk and cob and yellow kernels (Figure 1). It has been observed that the P1 gene [48] affected the expression of color in husk, cob, and kernel in F 1 hybrids. The P1 gene has allelic diversity and is involved in the anthocyanin and phlobaphene biosynthetic pathways in plant leaf tissue, pericarp of kernel, and cob glumes [49,50]. The hybrids produced in this study have desirable coloration that meets the needs of the field corn yellow grain market and allows the cobs and husk to be used as feedstock for anthocyanin and phytochemical production. High and positive values of heterosis were recorded for all hybrids for grain yield, husk mass, and cob mass. This may be an indicator of genetic divergence between these female lines and male lines used. Similarly, high values of heterosis were reported for all hybrids of elite drought tolerant maize inbred lines possessing genes that are complimentary [27]. The values of heterosis for TAY in husk in some hybrids were higher than for other traits (up to 318.9%). In addition, some hybrids had negative heterosis values for TAY, TAC, TPC, DPPH, and TEAC in both husk and cob. Accumulation of pigments in husk and cob tissue depends on gene combination which may explain the observed heterosis.
The F1 hybrids in this study were crossed between female lines with unpigmented husk and cob and yellow kernels and male lines with purple husk and cob and purple, white, or yellow kernels, resulting in F1 hybrids with purple husk and cob and yellow kernels (Figure 1). It has been observed that the P1 gene [48] affected the expression of color in husk, cob, and kernel in F1 hybrids. The P1 gene has allelic diversity and is involved in the anthocyanin and phlobaphene biosynthetic pathways in plant leaf tissue, pericarp of kernel, and cob glumes [49,50]. The hybrids produced in this study have desirable coloration that meets the needs of the field corn yellow grain market and allows the cobs and husk to be used as feedstock for anthocyanin and phytochemical production.

Conclusions
The cross between female parents with normal yellow kernels and cob and male parents with pigmented purple husk and cob generated F1 hybrids with normal yellow kernels and purple husk and cob, and the resulting hybrids can be used for phytochemical production. Takfa3 × KKU-PFC5 and NakhonSuwan2 × KKU-PFC4 were identified as superior hybrids with high anthocyanins and antioxidant activity in husk and cob, respectively. These hybrids will be further evaluated for possible release. Based on GCA, SCA, and heterosis in this study, both additive genes and non-additive genes controlled the inheritance of agronomic traits and phytochemicals, and simultaneous improvement of traits agronomic traits and phytochemicals would be difficult. It may be possible to simultaneously select for agronomic traits and phytochemicals by using a selection index. However, a clear understanding on the value of the phytochemical traits is necessary for development of meaningful weights in the index.

Conclusions
The cross between female parents with normal yellow kernels and cob and male parents with pigmented purple husk and cob generated F 1 hybrids with normal yellow kernels and purple husk and cob, and the resulting hybrids can be used for phytochemical production. Takfa3 × KKU-PFC5 and NakhonSuwan2 × KKU-PFC4 were identified as superior hybrids with high anthocyanins and antioxidant activity in husk and cob, respectively. These hybrids will be further evaluated for possible release. Based on GCA, SCA, and heterosis in this study, both additive genes and non-additive genes controlled the inheritance of agronomic traits and phytochemicals, and simultaneous improvement of traits agronomic traits and phytochemicals would be difficult. It may be possible to simultaneously select for agronomic traits and phytochemicals by using a selection index. However, a clear understanding on the value of the phytochemical traits is necessary for development of meaningful weights in the index.

Conflicts of Interest:
The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.