Germplasm studied

In the spring-summer (SS) -2014 cycle, 12 producers from the municipality of Temoac and five from Ayala were selected. In the SS-2015 cycle, the same producers provided between 20 and 25 ears each year (Table 1).

In Ayala the climate is warm sub-humid and Temoac is temperate sub-humid. Based on the characteristics of the cobs, the race and the cross to which they belong were identified.

Physical and chemical characteristics of the grain

The hectoliter weight (PH), weight of one hundred grains (PCG), flotation index (IF) and percentages of pedicel (PED), pericarp (PER), germ (GER), mealy endosperm (EHA) and corneal (ECO) was evaluated according to the methodology described by ^{Salinas and Vázquez (2006)}. The chemical analysis was performed on whole grain flour obtained after milling in a cyclonic type mill (UDY, Mod.3010-080P^®), with 0.5 mm mesh. The content of oil was quantified following the method 920.85of the ^{AOAC (2000)}, protein by the Technicon Instruments method (^{Galicia et al., 2012}), tryptophan following the glyoxylic acid methodology (^{Nurit et al., 2009}) and the protein quality index (IQP) was calculated with the relationship proposed by ^{Twumasi-Afriyie et al. (2016)}: IQP= [(tryptophan (%)/protein (%))100].

The total anthocyanins were evaluated following the procedure described by ^{Salinas et al. (2013a)}. The anthocyanins of the red MNP were calculated with a chlorinated pelargonidin curve (^{Castañeda-Sánchez, 2011}) and are reported as mg equivalents of chlorinated pelargonidin per kilogram of dry matter (mg EPC kg^-1 MS). For blue MNP a cyanidin-3-glucoside curve was used, reported as mg equivalents of cyanidin-3-glucoside per kg dry matter (mg ECG kg^-1 MS) (^{Salinas et al., 2013a}).

Statistical analysis

The data corresponding to each of the variables evaluated in each production cycle were subjected to a variance analysis under a completely randomized design combined with two replications using the GLM procedure and the Tukey comparison test (α= 0.05). The Pearson correlation matrix was made. The statistical package SAS (Statistical Analysis System 9.0 for Windows) was used for all analyzes.

Physical and chemical characteristics of the grains

The analysis of variance showed a significant difference (p< 0.01) in the effects of crop cycle, population and the interaction between cycle x population in all the variables evaluated (Table 2).

In the two years of evaluation, the maizes were of large grains, corresponding to high values of PCG, reduced PH and high IF (Table 3).

The cycle x population interaction showed that the blue MNP were endosperm smoother than the red ones (Figure 2A and B) highlighted the populations ‘EOxPep9’ (Figure 2A), ‘AN18’, ‘EO3’, ‘PepxEO25’, ‘PepxAN26’ and ‘PepxEO5’ (Figure 2B), for the stability in the smoothness of its grain in the two evaluation cycles and for having been the softest grain, characteristic that was associated with PH < 62 kg hL^-1, PCG> 38 g and reduced percentage of PER (< 4.5%). Desirable attributes in the corn intended for consumption as a vegetable (corn) (^{Revilla and Ordaz, 2016}) and for the preparation of pozole, where the use of corn with a PH< 67 kg is recommended. hL^-1, PCG> 38 g, IF= 100 and pericarp <6% (^{Vázquez et al., 2016}).

Figure 2 Interaction of crop cycle by population in the flotation index of red (A) and blue (B) populations of pigmented native maize from the eastern of Morelos, Mexico.  

The greater size of the grains in the SS-2014, are attributed to a greater availability of water at the beginning of the crop and during the filling of grain (Figure 3), which favors a rapid accumulation of carbohydrates (^{Tanaka and Yamaguchi, 2014}).

Figure 3 Temperature (A) and precipitation (B) in Temoac and Ayala, Morelos in the years 2014 and 2015 (^{CONAGUA, 2016}).  

While the smaller size (<PCG) and IF, in the SS-2015 cycle are attributed to erratic and poorly distributed rain at the stage of grain filling (^{Ángeles-Gaspar et al., 2010}; ^{Bazinger et al., 2012}) (Figure 3), in this regard, ^{Zinselmeir et al. (1995)} noted that, in corn, low water potentials reduce embryonic growth, by decreasing the flow of sucrose and altering the metabolism of carbohydrates in the ovaries. The larger grain size was associated with a large germ, which in turn produced a greater amount of oil (r= 0.68, p< 0.01) (Figure 4). The populations of red color, where the race ‘EO’ predominated, were those with the highest percentage of germ and oil, with respect to the blue ones (Figure 4).

Figure 4 Oil content and proportion of germ in pigmented native maize from the state of Morelos, Mexico.  

Reduced PH values were correlated with high values of: IF (r= -0.54; p≤ 0.01) and mealy endosperm (EHA) (r= -0.61; p≤ 0.01), which corroborates the low density of these maize. In its chemical components, the populations of the PV-2014 cycle recorded higher amounts of ACE and less: PRO and ANT (Table 3).

The ACE values were between 4.46 and 5.88%, with an average value of 5.14%, similar results were reported by ^{Agama-Acevedo et al. (2011)} (4.85 to 5.98%) for 15 accessions of blue corn and were higher than what was declared for blue (4.1%) and red (4.2%) maize of Nayarit (^{Vidal et al., 2008}) and 50 accessions of Cuban maize (4.01 to 5.53%) (^{Martínez et al., 2009}), but inferior to that found by ^{Guzmán et al. (2015)} in native maize of Guanajuato.

In the MNP, the protein was between 7.6 and 11.5%, values that are within what was reported for Mexican native maize (^{Gaytan-Martínez et al. (2013)}, these authors reported an interval between 9.7 and 11.9%, while ^{Vázquez et al. (2010)} reported values from 6.9 to 12.5% of protein. In the MNP of Morelos it was observed that those with the lowest protein content were those with the milder grains, where the ‘starch granules’ that are mainly spherical are weakly packaged in a protein matrix that slightly surrounds the granules (^{Narvaez-Gonzáles et al., 2007}).

Another aspect that contributed to the protein content was the fertilization of the crop, where the producers reported that this occurred out of time, due to the absence of rain and in a reduced dose, added empirically (1 fist per bush). Some producers reported having incorporated the stubble from the previous crop and a small number of them, applied organic fertilizer from their draft animals. The practice of monoculture, the erratic rains and the limited application of fertilizer, are some of the causes that may have contributed to the low protein content of the populations investigated.

The crop cycle affected the content of total anthocyanins in 2015 was recorded a higher content than that of 2014 (Table 3); nevertheless, the mean values were numerically very similar in the two cycles, which could be attributed to the heritable character of the anthocyanins (^{Halbwirth et al., 2002}). The red grain populations had values from 24.79 to 623.2 mg EPC kg^-1 MS, with an average value of 151.4 mg EPC kg^-1 MS (Figure 5A).

Figure 5 Total anthocyanins in populations of red (A) and blue (B) maize in the eastern region of the state of Morelos in the 2014 and 2015 cycles.  

This variability was related to the color of the grains (results not shown), those with lighter shades (rose-lilac) were those with the lowest content of anthocyanins, while the population ‘EOxPep13’ (intense red or cherry) registered the higher content of these pigments (Figure 5A). The anthocyanins of the red populations of Morelos were superior to the values reported (62.3 and 154 mg ECG kg^-1 MS) for unidentified maize (red) breeds (^{López et al., 2009}) and similar to that reported by ^{Salinas-Moreno et al. (2012b)} (64.7 to 547.7 mg ECG kg^-1 MS) for four races (Olotillo, Tehua, Tuxpeño and Vandeño) of red grain from the state of Chiapas. The populations of blue grain registered a higher content of anthocyanins, with respect to the red ones (Figure 5A and 5B ). In the 2015 cycle, the following populations stood out: ‘Pep20’, ‘Pep16’, ‘EO6’, ‘PepxEO25’ due to its higher content of anthocyanins, while ‘EO22’, significantly reduced these pigments (Figure 5B).

In these populations the anthocyanins had between 312 and 646.4 mg ECG.kg^-1 MS, with an average value of 368.4 mg ECG kg^-1 MS (Figure 5B), results that were similar to those reported by ^{Salinas et al. (2013a)} for different breeds of blue native maizes, and lower than the range of: 579.4-1046.1 mg ECG kg^-1 MS reported by ^{Salinas-Moreno et al. (2012a)} for six accessions of Chalqueño corn, a breed adapted to heights above 2 000 masl, where low nocturnal temperatures and high luminosity prevail, which favors a greater synthesis of anthocyanins (^{Jin-Seng et al., 2006}; ^{Salinas et al., 2013a}). Although the genetics of the populations is important in the production of the anthocyanins, the hot sub-humid climates for the municipality of Ayala and temperate sub-humid for Temoac, could also have contributed.

Quality of the protein

The nutritional quality of corn as a food is determined mainly by the amino acid composition of its proteins. Tryptophan is one of the essential and limiting amino acids of corn (^{Vázquez et al., 2012}). Although there are no absolute values of lysine or tryptophan that define a corn with protein quality, ^{Twumasi-Afiyie et al. (2016)} suggested that maize with more than 0.075% tryptophan in whole grain samples, can be considered with protein quality. In the present study, the highest tryptophan content was found in the SS-2014 cycle (Table 2). Values were recorded from 0.064 to 0.09%, with an average value of 0.075%. According to ^{Twumasi-Afiyie et al. (2016)}, 62% of the populations (16 of 26) had a tryptophan content similar to that of maize with high protein quality (QPM) (Figure 6).

Figure 6 Tryptophan and protein quality index in populations of pigmented native maize from the state of Morelos, Mexico.  

The blue populations with the highest tryptophan content were: ‘PepxAN26’ (0.090%), ‘AN18’ (0.089%), ‘ECxPep15’ (0.085%), followed by pure populations of ‘Pep7’ and ‘EO22’, while the red ones were; ‘PepxEO19’ (0.086%) and ‘EOxPep9’ (0.081%) (Figure 6).

The populations with less tryptophan were ‘Ni21’, ‘ECxES11’, ‘ESxTAB1’, ‘EO10’ (red) and ‘PepxEO14’ (blue), this variability shows that tryptophan is a component that can be modified by the genetic information of each population (^{Krivanek et al., 2007}), but also due to environmental conditions and agronomic management (^{Vázquez et al., 2012}). The tryptophan content of the eastern populations of the state of Morelos was higher than that reported by ^{Vidal et al. (2008)} (0.048 and 0.051%) and lower than the results of ^{Martínez et al. (2009)} who found an interval from 0.053 to 0.097%.

It has been reported that the contents of tryptophan and lysine in corn are highly correlated (>0.9), so, with the quantification of tryptophan, it is possible to qualify the quality of the protein as an indicator of its nutritional value (^{Nurit et al., 2009}). Additionally, one of the most used criteria to identify a corn with protein quality is the protein quality index (IQP). In the MNP, the IQP presented values higher than 0.8, indicated as the limit to consider a corn with protein quality (^{Twumasi-Afiyie et al., 2016}), on the other hand, ^{Martínez et al. (2009)} proposed that the selection of maize with nutritional attributes should include the values of protein, tryptophan and IQP of: 8%, 0.075% and 0.8% respectively.

In the 2014 cycle, a higher tryptophan content was obtained (Table 3); however, in 2015 the populations ‘AN18’ ( X- = 0.093%), ‘ECxPep15’ ( X- = 0.092%) and ‘PepxAN26’ ( X- = 0.091%) were those with the highest TRI. In the general evaluation (the two study cycles), it shows that 90% of MNP populations had protein content above 8%. In the case of tryptophan, 15 populations were above 0.075% (Figure 6).

Finally, 61% (15/26) of the populations registered an IQP≥ 0.8 (Figure 6). According to these three variables, populations stood out; ‘Pep5’, ‘Pep20’, ‘EO22’ and ‘ANxEO4’ all blue (Table 4)

They were characterized by having very soft, large endosperm grains, with higher oil and tryptophan content and less protein (Table 4) than that of a commercial toothed corn ( X- = 9%) (^{Watson, 2003}).

Thus, the maize with the best protein quality, were not those with the highest protein, nor those with more tryptophan, but those that balance the values of these three variables (Table 4). MNP can be used to improve the nutritional conditions of rural families in conditions of malnutrition. The identified diversity can be explained by the changes that the local races have experienced over time, derived from the crossing with other races, as a result of the selection of the producers, which has been directed mainly to maintain the color and the special flavor that present this type of corn with respect to improved varieties and commercial hybrids. MNP from the eastern state of Morelos are a good option to produce them under variable climate conditions and to elaborate value-added foods and can contribute to address nutrition and health problems.