Introduction

Mexico serves as a significant reservoir of corn diversity, boasting 59 breeds (^{Sanchez et al., 2000}). All these breeds feature pigmented kernel variants, ranging in color from black to pale pink. However, the most prevalent colors include blue/purple, black, and red (^{Salinas et al., 2012}b). Pigmented kernel corn is rich in anthocyanins, flavonoid pigments linked to the prevention or development of chronic degenerative diseases such as cancer (^{Bello et al., 2016}; ^{Alegría et al., 2020}), and exhibiting anticancer, anti-neurodegenerative, and anti-inflammatory properties (^{Kraft, 2008}; Bello et al., 2016). Typically, these pigments are found in the pericarp, aleurone, or both kernel structures (^{González et al., 1999}; ^{Agama et al., 2011}).

According to ^{Salinas et al. (2013}b), the quantity and type of anthocyanins in corn kernels vary based on the kernel color and the concentration of the pigment in different structures; magenta-red kernels concentrate anthocyanins in both the pericarp and aleurone layer and can possess up to 10 times more anthocyanins than blue-purple kernels, which only concentrate anthocyanins in the aleurone layer. Furthermore, if the pigment is predominantly found in the aleurone, the kernel is suitable for nixtamalization, whereas if it accumulates adequately in the pericarp, the kernel could be utilized for pigment extraction. The significance of genetic enhancement for the development and utilization of varieties with high anthocyanin content stems from two primary reasons: first, their impact on the sensory characteristics of foods, which can influence their technological performance during food processing, and second, their implications for human health through several pathways (^{Aguilera et al., 2011}).

The anthocyanin content in pigmented corn ranges, according to one study, between 276.8 and 904.0 μg g^-1 (^{Salinas et al., 2012}a), while another study found values between 271.01 and 1,989 μg g^-1 (^{Hernández et al., 2017}), where the highest values were found in samples originating from Tlaxcala. In breeding work carried out by ^{Ballesteros et al. (2019}) to improve the nutraceutical properties in western corn, the anthocyanin content was reduced with respect to the original populations, which justifies the importance of taking advantage of and characterizing native varieties in their original state, as well as continuing breeding to increase or maintain the highest anthocyanins contents and other nutraceutical compounds, as well as other desirable characteristics.

The corn used for nixtamalization must comply with certain physical characteristics according to Mexican standards NMX-FF-034/1-SCFI-2020 and NMX-FF-034/1-SCFI-2002; for white corn, the maximum percentage of floating grains must be 10 to 50 % (flotation index to measure hardness and cooking time), i.e., hard and very hard grains are preferred (Secretaría de Economía-México 2002). Kernel width should exceed 4.76 mm, (Secretaría de Economía-México 2020). Since pigmented corn undergoes nutraceutical compound extraction processes, the above standards are not necessarily applicable to pigmented corn but could be useful as a standard for quality traits.

The objective of this research was to characterize 300 accessions of pigmented maize from the Mexican Republic, according to the physical characteristics of the grain and the total anthocyanin content, to use them in genetic improvement programs.

Material and Methods

The methodology described below was designed to achieve the general objective by fulfilling the following specific objectives:

Results and Discussion

Table 2 presents the average values of total anthocyanin content for the 31 breeds of pigmented corn. The breed with the highest total anthocyanin content was Tabloncillo Perla, with a value of 511.74 µg g^-1, while the breed with the lowest value was Pepitilla, averaging 248.7 µg g^-1 of total anthocyanins. Breeds such as Elotes de Sinaloa, Mushito, Reventador, Tabloncillo, and Tepecintle fall within the average values.

In terms of total anthocyanin content, the HIDA 250 accession recorded the highest value at 723.9 μg g^-1, while the HIDA 247 accession had the lowest value at 136.53 μg g^-1 (Table 2). These values fall below those reported by ^{Salinas et al. (2012}b), with a range from 276.8 to 904.0 μg g^-1, and Hernández et al. (2017), averaging 281.01 μg g^-1 of total anthocyanins. Remarkably, variability was observed within the samples analyzed, with high values of 1,989 μg g^-1 reported in samples from Tlaxcala. Additionally, these values were lower than those reported by ^{Arellano et al. (2021}), who obtained 774.7 µg g^-1 of anthocyanins.

The flotation index results indicate that 62 accessions exhibited very hard kernels, including accessions HIDA 250, PUEB 509, and CHIS 1053. Furthermore, 80 accessions had hard kernels, 62 accessions had intermediate hardness, 59 accessions had soft kernels, and 37 accessions had very soft kernels. This implies that 142 of the 300 accessions (47.3%) possess a desirable degree of hardness for the nixtamal industry, while a total of 204 (68%) have an acceptable degree, according to the white corn guidelines required by NMX-FF-034/1-SCFI-2020 (Secretaría de Economía-México, 2020).

The lowest kernel size value was found in accession HIDA 74 and the highest value was obtained in accession OAXA 783 (data not shown). According to ^{Salinas et al. (2012}b), kernel size affects the concentration of anthocyanins, influenced by a dilution effect of anthocyanins in the flour of the kernel used for quantification, where a large kernel will have a greater dilution effect than a small kernel. On the other hand, 100 % of the accessions showed an average width greater than 4.76 mm, conforming to NMX-FF-034/1-SCFI-2020 (Secretaría de Economía-México 2020), the VERA 87 accession showed the lowest width value and DURA 163 the highest, 5.6 and 11.7 mm respectively (data not shown).

Accession CHIS 1089 obtained the lowest thousand-seed weight, 239 g, while accession MICH 86 obtained the highest value, 532 g (Data not shown). These values are similar to those obtained by ^{Maldonado et al. (2021)} who report values between 223 g and 522 g for thousand-seed weight and ^{Cieza et al. (2020}) who when evaluating 21 maize hybrids obtained results for thousand-seed weight between 297 and 381 g. Standard NMX-FF-034/1-SCFI-2020 (Secretaría de Economía-México 2020), does not specify minimum or optimum values for this variable, but it is useful in the characterization of varieties.

The accessions showed variable coloration, with 70 % of blue kernels, 20 % of red kernels, and 10 % of absent coloration; 88 % of the kernels showed coloration in the aleurone, 11 % in aleurone and pericarp, and 1 % in pericarp. In Chalqueño accessions, coloration in the aleurone layer predominated (78 %) followed by coloration in aleurone and pericarp (19 %), and finally, in pericarp (3 %); ^{Salinas et al. (2012}b) found that anthocyanins were located only in the aleurone layer in blue-purple grain samples of that race, thus making evident the association of coloration localization with the presence of anthocyanins.

There was a negative correlation between the percentage of grains floating (inverse of grain hardness according to NMX-FF-034/1-SCFI-2020), and length and size (p ≤ 0.05, Table 3), thus it is evident that the larger the size, the greater the hardness (fewer grains floating). Anthocyanin content was not correlated with any of the physical characteristics (Table 3).

Table 4 shows the characteristics of the accessions with the highest anthocyanin content, those corresponding to the two highest intervals in Table 2. These accessions, in addition, could be considered with potential for the nixtamalization industry, since: the coloration is predominantly located in the aleurone, which is preferable according to ^{Salinas et al. (2013}a), although if it accumulates in the pericarp in sufficient quantity it could be used for pigment extraction. The flotation rate of 10 to 50 % predominates, which is desirable according to NMXFF-034/1-SCFI-2002 (Secretaría de Economía-México 2020). In nixtamalization, it could be desirable that the coloration is located in the aleurone, because if it is located in the pericarp, it is more likely that the anthocyanins are lost when they are detached during the process together with the pericarp.

Exclusive coloration in the pericarp was observed in accessions GUER GP36 (95%) [294.64 μg g^-1], PUEB 589 (100%) [274.8 μg g^-1], SNLP (35%), MEXI 64 (20%), PUEB 461 (20%), and others with percentages equal to or less than 10% (CHIH 403, CHIH 421, GUAN 146, PUEB 233, NAYA 191, MORE 52, MICH 414). These accessions may be suitable solely for pigment extraction, although their total anthocyanin content is lower than the average (323.17 μg g^-1) and falls below the median range: (356.8275, 503.6925). GUER GP36 and PUEB 589 accessions exhibited blue (100%) and red (100%) coloration, respectively.

The results of the principal component (PC) analysis indicate that the first three components accounted for 66.35% of the proportion of variance (Table 5). The most relevant variables in PC1 were 100-kernel weight, length, and thickness. CP2 highlighted anthocyanin content, hardness, and kernel width as the most relevant variables. Figure 1 depicts the biplot of the first two principal components, illustrating the dispersion of the 300 pigmented maize accessions. Previous studies have also demonstrated the relevance of variables related to kernel and ear dimensions for the characterization of maize varieties, as shown in the study by ^{Rocandio et al. (2014}).

The most relevant variables for PC1 were the weight of a thousand seeds and grain length, and for PC2, were anthocyanin content and grain width.

Figure 1 Dispersion of 300 corn accessions from 31 races.  

The dendrogram generated from the physical characteristics and total anthocyanin content of the 31 corn breeds (Figure 2) shows four groups at a Euclidean distance of 5: The first group comprises Dulce, Cristalino de Chihuahua, San Marceno, Negro de Tierra Caliente, Perla, and Pepitilla. The second group includes Olotillo, Arrocillo, Nal-tel, Cónico, Tuxpeño, Bolita, Arrocillo Amarillo, Tepecintle, Elotes de Sinaloa, Cónico Norteño, Chalqueño, Olotón, Elotes Occidentales, Elotes Cónicos, Ancho, Tabloncillo, Mushito, Cacahuacintle, Bofo, Azul, San Juan, Reventador, and Celaya. The third group consists of Negro Chimaltenango, and the fourth group comprises Tabloncillo Perla. A greater affinity was observed between the races Cónico Norteño and Chalqueño, as well as a notable similarity between the races Elotes Occidentales and Elotes Cónicos. ^{Torres et al. (2022}) analyzed the morphological diversity of seven maize races, identifying four groups in the dendrogram at an Euclidean distance of 7.8 units. Similarly, in the study of genetic diversity of 10 accessions, four groups were delineated from an Euclidean distance of 5.16 (^{Sanchez et al., 2019}).

Figure 2 Dendrogram of 31 corn races, considering the variables: total anthocyanins, one hundred grain weight, hardness, length, width, and grain thickness. 

Conclusions

The variability uncovered in this research could significantly contribute to the selection of maize accessions offering high total anthocyanin content. Accessions such as HIDA 250 (Tepecintle), PUE 509 (Elotes Occidentales), CHIS 1053 (Olotón), CHIS 1044 (Olotón), MICH 119 (Cónico), and VERA 161 (Tuxpeño) exhibited total anthocyanin content exceeding 600 μg g^-1. These accessions demonstrate substantial potential for utilization in breeding programs due to their alignment with the requirements of the nixtamalization industry. Furthermore, most of these accessions display coloration predominantly in the aleurone layer, reducing the likelihood of its release during nixtamalization compared to those with coloration in the pericarp. The breeds were grouped into four categories based on the characteristics under study.