Oaxacan Green Dent maize is not from Oaxaca

Abstract

Oaxacan Green Dent is a maize (Zea mays L.) cultivar marketed as an introduced Mexican heritage cultivar adapted to the higher latitudes of the United States. Its adaptation and appearance contradict an origin in Oaxaca, Mexico, however, and no indigenous cultivars in Oaxaca are known to have the unique kernel colors of Oaxacan Green Dent. We compared phenotypes and genotypes of Oaxacan Green Dent sampled from three different sources along with several Corn Belt cultivars and 15 landrace accessions collected from a wide range of geography, altitude, and cultural groups in Oaxaca. Multivariate analysis of 13 phenotypic traits measured in a field experiment suggested that Oaxacan Green Dent is more closely related to Corn Belt Dents than to Oaxacan cultivars. Genomic analysis from DNA sequencing demonstrated unambiguously that Oaxacan Green Dents are even more distantly related to Oaxacan cultivars than typical US Corn Belt Dent cultivars are. Phenotypic, genetic, and historical data indicate that Oaxacan Green Dent is almost certainly directly derived from Ernest Strubbe's Green Dent cultivar, which he developed in Minnesota from crosses between a Corn Belt Dent cultivar and an intensely colored popcorn cultivar, with no contribution from Oaxacan cultivars.

Plain Language Summary

Oaxacan Green Dent is an open-pollinated maize variety with unique green-colored kernels. Oaxacan Green Dent's origin is unclear; it does not match descriptions of known Oaxacan cultivars. Phenotypic, genetic, and historical data presented here demonstrate that Oaxacan Green Dent is not from Oaxaca. We show that Oaxacan Green Dent is a renamed version of Ernest Strubbe's Green Dent variety from Minnesota. We recommend seed dealers discontinue the use of “Oaxaca” in the variety name and instead refer to it as Ernest Strubbe's Green Dent.

Abbreviations

BLUE

best linear unbiased estimator

DTA

days to anthesis

DTS

days to silk

PC

principal component

SNP

single-nucleotide polymorphism

1 INTRODUCTION

Oaxacan Green Dent (sometimes Oaxacan Green) is a maize cultivar currently being sold as an introduced Mexican heritage cultivar in seed catalogs targeting home gardeners and small farmers in the United States. Oaxacan Green Dent is highly unusual among maize cultivars for its green-colored kernels due to green pigmentation in the aleurone (Stinard & Sachs, 2002) (Figure 1). There is growing interest in this cultivar because of its unusual and beautiful appearance combined with its good adaptation to northern United States environments, which is rare among Mexican cultivars, due to their photoperiod sensitivity (Castillo-Gonzalez & Goodman, 1989). Mexico is the center of origin and a center of diversity for maize and tropical maize germplasm, which holds promise as a source of unique and favorable alleles for temperate breeding programs (Samayoa et al., 2018). Despite their potential utility, Mexican maize cultivars have not been widely used in temperate breeding programs, in part because of their poor adaptation to temperate growing environments (Castillo-Gonzalez & Goodman, 1989). Adaptation is under complex polygenic control in maize, and breeding for adaptation can require numerous generations of selection in target growing environments (Choquette et al., 2023). A recently published study of the genetics of photoperiod sensitivity in maize flowering used Oaxacan Green as a representative Mexican maize to identify sensitivity to daylength in tropical maize (Zhong et al., 2021). Clarifying the origin of Oaxacan Green Dent and its genetic relationship to other Mexican cultivars and to Corn Belt Dent maize cultivars will aid interpretation of the genetics of adaptation in maize, help establish its potential utility as an exotic germplasm source, and possibly shed light on the source of its unusual seed coloration.

FIGURE 1

Representative ear of Oaxacan Green Dent sourced from seed savers exchange. Kernels vary for coloration, with some kernels expressing a green color in the aleurone.

We identified 23 companies and nonprofit organizations with seeds of this cultivar available for purchase (Table S1). Among these providers, 19 provided information on the origin of the cultivar. Eighteen of 19 providers indicate a Mexican origin of the cultivar, with some specifically indicating that it has been cultivated by the Zapotec people of Oaxaca “for centuries.” Thresh Seed Company indicated some uncertainty about this origin story: “reportedly originates from the Oaxacan region of Southern Mexico … Despite its origin, Oaxacan Green Dent's short stature and early maturity seem markedly more temperate in nature, and we've found it much easier to grow compared to other Mexican varieties.” (Thresh Seed Co., 2024). More pointedly, Nate Kleinman, cofounder of the Experimental Farm Network, wrote of Oaxacan Green Dent: “there seems to be no record of any corn like this actually growing in Oaxaca, nor are there credible stories of travelers encountering Oaxacan farmers growing a corn that resembles this one, or bringing back samples of any similar corn. This type of dent corn is most likely a modern strain related to (and possibly derived from) ‘Reid's Yellow Dent,’ developed in the US in mid-1800s … Finally, and most crucially, there is an almost identical corn in the USDA collection called ‘ES Green Dent’” (Experimental Farm Network, 2024).

Core Ideas

• Oaxacan Green Dent is an open-pollinated maize cultivar with unique green-colored kernels.
• Oaxacan Green Dent's origin is unclear; it does not match descriptions of known Oaxacan cultivars or native landraces.
• Phenotypic, genetic, and historical data presented here demonstrate that Oaxacan Green Dent is not from Oaxaca.
• We show that Oaxacan Green Dent is a renamed version of Ernest Strubbe's Green Dent cultivar from Minnesota.
• We recommend that seed dealers use Ernest Strubbe's Green Dent for the cultivar name.

To investigate the genetic relationship between Oaxacan Green Dent, ES Green Dent, US Corn Belt Dents, and Oaxacan maize cultivars, we searched for written documentation on the origin of Oaxacan Green Dent and other maize cultivars with green kernel color. We conducted a field study to measure morphological and phenological characteristics of samples of each of these groups of cultivars to quantify their multivariate phenotypic relationships. We also performed DNA sequencing of samples of these groups to directly measure their genetic relationships. Finally, we investigated the precise localization of green pigmentation in kernels of this cultivar. Our results confirm that Oaxacan Green Dent is closely related to ES Green Dent and more generally to US Corn Belt Dent cultivars. Oaxacan Green Dent and other Corn Belt Dents are distinct phenotypically and genetically from all Oaxacan landrace cultivars. We recommend that seed providers of this cultivar remove reference to Oaxaca in the cultivar name.

2 MATERIALS AND METHODS

2.1 Germplasm

The term “cultivar” is used throughout the paper as a generic and neutral term to refer to open-pollinated maize populations with distinct names. The genetic materials used in this study include open-pollinated maize populations that have been variously considered heirloom cultivars, heritage cultivars, farmer's varieties, or landraces (Curry, 2022; Preston et al., 2011).

We obtained seeds of Oaxacan Green Dent from Seed Savers Exchange, Restoration Seed Company, and Sand Hills Preservation Center. Seeds used in the field experiment were directly from the seeds obtained from suppliers, except for the Seed Savers Exchange source, which was increased by controlled cross-pollination among 100 plants in the field (Woore et al., 2024). We also obtained seeds of five US Corn Belt cultivars and 15 Oaxacan cultivars from the National Plant Germplasm System maize collection (Volk et al., 2023). The US cultivars included two populations of Reid's Yellow Dent and one population of Minnesota 13, which represent important germplasm sources that contributed to modern commercial corn hybrid pedigrees (Smith et al., 2022; Troyer, 1999). In addition, we included ES Green Dent and Black Beauty, a black popcorn cultivar donated to the Plant Introduction collection by Ernest Strubbe, which may have been a source of the kernel anthocyanin colors in ES Green Dent.

The 15 Oaxacan landrace accessions were selected to represent a range of previously described Mexican native maize groups, latitudes, and elevations within the Zapotec areas of Oaxaca, principally the Valles Centrales, the largest area of Zapotec culture, as well as the Sierra Norte where the Zapotecos de la Sierra live, and the area of the Istmo of Tehuantepec where the Zapotecos del Istmo are located (Table 1; Figure S1). Most of these collections were not assigned to landrace groups in the USDA Germplasm Resources Information Network, but the sampled cultivars represent at least Bolita, Negrito, Olotillo, Tepecintle, Tuxpeño, and Zapalote Chico groups (Table 1).

TABLE 1. Maize cultivars evaluated in field experiment.

Group	Cultivar	Seed source	Race	Collection site	Latitude	Longitude	Elevation
US Corn Belt	Black Beauty	PI 452061	Popcorn	Alberta, MN	45.575	−96.051	373
US Corn Belt	ES Green Dent	PI 452039	Corn Belt Dents	Alberta, MN	45.575	−96.051	373
US Corn Belt	Minnesota 13	Ames 23519	Corn Belt Dents	St. Paul, MN	44.991	−93.183	229
US Corn Belt	Reid's Yellow Dent	PI 213698	Corn Belt Dents	Springport, IN	40.050	−85.396	324
US Corn Belt	Reid's Yellow Dent	PI 213709	Corn Belt Dents	Clear Lake, IA	43.159	−93.393	377
Oaxacan Green	Oaxacan Green	Seed savers exchange
Oaxacan Green	Oaxacan Green	Restoration seeds
Oaxacan Green	Oaxacan Green	Sand hills
Oaxaca	Oaxaca 1	PI 645928	Tuxpeño	Loma Bonita, Oaxaca, Mexico	18.133	−95.933	70
Oaxaca	Oaxaca 17	PI 484842	Tuxpeño	Tuxtepec, Oaxaca, Mexico	18.069	−96.154	300
Oaxaca	Oaxaca 24	PI 484136		Tlacolula, Oaxaca, Mexico	16.956	−96.493	1610
Oaxaca	Oaxaca 26	PI 515390	Negrito	Etla, Oaxaca, Mexico	17.202	−96.806	1640
Oaxaca	Oaxaca 40	PI 484137	Bolita	Ocotlan, Oaxaca, Mexico	16.826	−96.676	1502
Oaxaca	Oaxaca 43	PI 628469		Etla, Oaxaca, Mexico	17.167	−96.783	1645
Oaxaca	Oaxaca 55	PI 628470	Zapalote Chico	Niltepec, Oaxaca, Mexico	16.583	−94.600	100
Oaxaca	Oaxaca 62	PI 645933	Bolita	Zaachila, Oaxaca, Mexico	16.950	−96.750	1500
Oaxaca	Oaxaca 210	PI 628479		San Antonio de la Cal, Oaxaca, Mexico	17.033	−96.700	1584
Oaxaca	Oaxaca 321	PI 645952		San Pedro Yolox, Oaxaca, Mexico	17.583	−96.533	1950
Oaxaca	Oaxaca 406	PI 645955		San Pablo Etla, Oaxaca, Mexico	17.167	−96.783	1600
Oaxaca	Oaxaca 425	PI 645957		Zimatlan, Oaxaca, Mexico	16.867	−96.783	1500
Oaxaca	Oaxaca 446	PI 645958		Ocotlan, Oaxaca, Mexico	16.783	−96.683	1500
Oaxaca	Benz 933	PI 705721	Olotillo/Tepecintle	Santiago Cuixtla, Oaxaca, Mexico	16.083	−97.183	500
Oaxaca	Benz 935	PI 705722	Tepecintle/Bolita	Ejutla, Oaxaca, Mexico	16.467	−96.800	1400

2.2 Field experiment

The 23 cultivars were evaluated in a randomized complete block design with two replications in Clayton, NC, in 2021. Experimental units were single 3.7-m rows, seeded at a rate of 25 seeds per plot with rows spaced 0.97 m apart. Seedlings were thinned as needed to ensure interplant distances of at least 0.1 m within rows. N was applied at 53.2 kg ha⁻¹ preplanting and then at 190.5 kg ha⁻¹ in a split application during the growing season. Irrigation was applied as needed up to 25 mm of water per week. Days to anthesis (DTA) and days to silk (DTS) were recorded as the number of days from planting to 50% of plants within a plot shedding pollen or with emerged silks, respectively. Anthesis–silk interval was computed as the number of days between anthesis and silk emergence. Five plants per plot were measured for ear height, plant height, ear position (ear height relative to plant height), number of primary tassel branches, and tassel branched part length (the length of the tassel between uppermost and lowest primary branches). Up to six plants per plot were measured for cob length, ear diameter, cob diameter, number of kernel rows, cob color, kernel width, and weight of 100 kernels. Details of most trait measurements are given in Woore et al. (2024). Traits measured were previously demonstrated to have high heritability and relatively low influence from environment and genotype-by-environment interaction effects (Sánchez G. et al., 1993; Woore et al., 2024), such that single-environment data on multiple traits are sufficient for measuring phenotypic relationships.

2.3 Trait data analysis

Traits with one measurement per plot were analyzed with the following linear mixed model:

$${Y_{ij}} = \mu + {R_i} + {C_j} + {\varepsilon _{ij}}$$, where $${Y_{ij}}$$ is the trait measurement on the ith replicate block and jth cultivar, $${R_i}$$ is the random effect of block, $${C_j}$$ is the fixed effect of cultivar, and $${\varepsilon _{ij}}$$ is the residual effect. Traits with more than one measurement per plot were analyzed with the following linear mixed model:

$${Y_{ijk}} = \mu + {R_i} + {C_j} + {\varepsilon _{ij}} + {w_{ijk}}$$, where terms are the same as the previous model except that $${Y_{ijk}}$$ is the trait measurement on the kth plant in the ith replicate block and jth cultivar, $${\varepsilon _{ij}}$$ is the random experimental error effect, and $${w_{ijk}}$$ is the residual effect that represents the effect of individual plant measurements within a plot. The null hypothesis of no cultivar effects was tested with a Wald F-test, and cultivar best linear unbiased estimates (BLUEs) were estimated. Mixed model analyses were fit with ASReml-R version 4.2 (Butler et al., 2023).

Cultivar BLUEs were used to estimate pairwise correlations among traits. Trait pairs with correlation greater than r = 0.95 were pruned, dropping the trait with more missing data. Data were missing for various reasons, including poor germination in a few plots and plants not producing ears. The resulting matrix of cultivar-by-trait means had 10% missing data. Multivariate analyses required a complete data matrix, so missing cultivar-trait means were imputed. First, Black Beauty was missing ear and plant height data (because of decomposition at time of height measurements), but this cultivar was evaluated in a common trial grown in the same location in summer 2024 with the other temperate maize cultivars used in this experiment, so its missing ear height value was imputed based on relative differences to other cultivars observed. Oaxaca 321 was missing data for silking date, anthesis–silk interval, and ear height because of its photoperiod sensitivity, which resulted in no ear development. Because it was very tall and very late (based on plant height and anthesis observations), we imputed these missing values as equal to the maximum mean values among all entries in the experiment. All remaining missing values were imputed with the mice package in R (van Buuren & Groothuis-Oudshoorn, 2011).

The complete matrix of cultivar-by-trait means was subjected to principal components (PCs) analysis. Trait loadings on the first two PCs and cultivar scores on the first four PCs were visualized with the ggplot2 package in R (Wickham, 2016).

2.4 DNA sequencing

Five plants per cultivar were sampled and equal amounts of tissue for each plant were bulked for DNA extractions. Several cultivars were represented by two independent samples of five plants each. Inbreds B73 and CML311 were also sampled multiple times to serve as quality control checks for sequencing. Oaxaca 17 seeds did not germinate well enough to include in the DNA sequencing study. A cultivar named Strubbes Green from Sand Hill Preservation Center was also included in the sequencing study. Multiplexed reduced representation sequencing was used to sequence all cultivars following the library preparation methods of Manching et al. (2017) using an Illumina NovaSeq 6000. Sequences were aligned to the B73 version 5 reference genome (Hufford et al., 2021). Single-nucleotide polymorphisms (SNPs) were called using bcftools mpileup and call functions (Danecek et al., 2021). SNPs with more than 23% missing data were filtered out, leaving 39,708 SNPs remaining. A distance matrix was computed in TASSEL version 5 (Bradbury et al., 2007). Cluster analysis and multidimensional scaling were performed on the resulting distance matrix using functions in base R (R Core Team, 2019). A dendrogram was visualized using the factoextra package in R (Kassambara & Mundt, 2024).

2.5 Kernel histology

Two mature kernels each of Oaxacan Green Dent, Minnesota 13, and Black Beauty were analyzed after being immersed in water for 48 h and embedded in Tissue Freezing Medium (Leica Biosystems). A cryostat (Leica CM1860) was used to produce 20-µm thick sections of kernel crowns (Kumar et al., 2024). Sections were mounted by applying paraffin oil as a mounting medium on the surface of the glass slide. Sections were examined under an Axiovert 200 M microscope (Carl Zeiss Microscopy) with a 10x objective. Images were captured with an AxioCamMR3 camera coupled to the microscope with Axiovision Release 4.8 (Carl Zeiss Microscopy) software.

3 RESULTS AND DISCUSSION

The null hypothesis of no cultivar differences was rejected for all traits as the p-values for cultivar effects ranged from 0.03 (ear diameter) to 0.0006 (cob length) to less than 10⁻¹⁵ for all other traits. Cultivar mean value distributions for most traits were quite distinct for Corn Belt and Oaxacan cultivars (Figure 2; Tables 2 and 3). Corn Belt cultivars flowered much earlier with smaller anthesis–silk intervals, were much shorter and had lower relative ear heights, and had more intensely colored cobs and shorter tassel branched part lengths than Oaxacan cultivars (Figure 2). Oaxacan Green Dent cultivars generally were near the median of the Corn Belt cultivar distributions for these traits, although they were intermediate between the two groups for ear position and tassel branched part length (Figure 2). Oaxacan cultivars had uniformly white or very lightly pink-colored cobs (mean scores near 1 on a 5-point color intensity scale), whereas Corn Belt Dent cultivars had red cobs, although the Corn Belt popcorn cultivar Black Beauty had white cobs (Figure 2; Table 2). Oaxacan Green Dent cultivars had red cobs like the Corn Belt Dent cultivars, although slightly less intensely colored (Table 2). For flowering time traits, anthesis–silk interval, ear and plant height, relative ear position, tassel branched part length, and cob color, the mean values for Oaxacan Green Dents were significantly different from Oaxacan cultivars but not from US Corn Belt cultivars (Table 3). Kernel row number is the only trait for which Oaxacan Green Dents were significantly different from US Corn Belt cultivars but not Oaxacan cultivars (Table 3).

FIGURE 2

Boxplots of distributions of cultivar mean values by group category for anthesis–silk interval, days to anthesis, cob color, ear position, plant height, and length of branched part of tassel. For all traits in this figure, the Oaxacan Green Dent group mean is significantly (p ≤ 0.01) different from Oaxacan group mean but not from US Corn Belt group mean.

TABLE 2. Maize cultivar mean values for highly heritable traits measured during 2021 in Clayton, NC.

Cultivar	Seed source	DTA (day)	DTS (day)	ASI (day)	Ear height (cm)	Plant height (cm)	Ear position	Num tassel branches	Tassel branch length (mm)	Cob length (mm)	Cob diameter (mm)	Ear diameter (mm)	Kernel row number	Kernel width (mm)	Wt 100 kernels (g)	Cob color (1–5 scale)
US Corn Belt cultivars
Black Beauty	PI 452061	60	62	2				9.3	56	123	17.3	39.9	12.3	6.0	11.3	1.0
ES Green Dent	PI 452039	64	69	5	56	132	0.42	18.3	71	121	23.8	40.3	11.0	8.9	28.2	3.3
Minnesota 13	Ames 23519	61	64	3	64	146	0.44	15.6	91	126	25.3	41.9	13.3	8.4	26.8	3.3
Reid's Yellow Dent	PI 213698	71	74	3	112	210	0.53	18.8	128	165	26.3	47.0	15.4	8.6	32.7	3.8
Reid's Yellow Dent	PI 213709	67	70	3	90	195	0.46	14.5	90	156	25.0	44.9	15.6	8.2	29.5	3.1
Oaxacan Green Dent cultivars
Oaxacan Green	Restoration seeds	67	69	3	92	184	0.50	20.8	121	139	24.4	40.6	10.8	10.1	32.3	3.2
Oaxacan Green	Sand hills	66	69	3	87	172	0.51	17.7	95	139	25.3	40.2	12.0	9.7	28.9	2.8
Oaxacan Green	Seed savers exchange	67	70	3	82	173	0.47	17.8	103	155	24.7	41.3	11.1	10.2	31.0	3.3
Oaxacan cultivars
Oaxaca 1	PI 645928	101	108	9	215	333	0.66	24.3	168	159	23.8	36.9	13.0	8.6	30.9	1.3
Oaxaca 17	PI 484842	103	118	15	241	341	0.71	19.0	168	106	17.3		11.0	7.5		1.3
Oaxaca 24	PI 484136	93	96	3	189	276	0.68	23.4	165	180	24.9	42.6	14.3	8.5	30.9	1.0
Oaxaca 26	PI 515390	80	94	15	82	170	0.48	12.5	105
Oaxaca 40	PI 484137	83	93	10	145	270	0.58	18.5	139	123	19.7	38.9	10.0	9.7	39	1.0
Oaxaca 43	PI 628469	87	101	14	172	277	0.62	18.0	185	110	19.1	31.6	9.6	8.3	30.3	1.0
Oaxaca 55	PI 628470	70	73	4	111	210	0.53	19.8	135	117	24.9	43.0	11.8	9.4	32.3	1.1
Oaxaca 62	PI 645933	68	81	13	107	226	0.47	12.2	119	115	21.4	33.1	10.0	9.3	29.4	1.0
Oaxaca 210	PI 628479	77	93	17	153	256	0.60	16.4	153	159	23.1	39.5	11.3	8.6	34.8	1.0
Oaxaca 321	PI 645952	111						18.8	162	64	20.4	22.2	13.0	7.5		1.0
Oaxaca 425	PI 645957	90	107	17	174	288	0.60	24.5	175	126	24.9	38.5	15.3	8.9		1.0
Oaxaca 406	PI 645955	95	118	26	163	292	0.57	23.9	196
Oaxaca 446	PI 645958	83	98	16	140	254	0.56	16.4	142	160	23.5	37.4	11.5	9.5	39.4	1.5
Benz 933	PI 705721	108	117	9	263	385	0.68	20.5	189	191	21.2	32.4	11.1	8.6	25.6	1.0
Benz 935	PI 705722	95	99	5				20.5	157		21.1			7.3
Avg LSD		4	8	8	30	38	0.07	5.6	29	53	3.3	12.4	2.4	1.0	7.7	0.8

• Note: Avg LSD, average least significant difference (p < 0.05) between two cultivar means.
• Abbreviations: ASI, anthesis–silk interval; DTA, days to anthesis; DTS, days to silk.

TABLE 3. Maize cultivar group means, group differences, and p-values of group differences for highly heritable traits measured during 2021 in Raleigh, NC.

Group	DTA (day)	DTS (day)	ASI (day)	Ear height (cm)	Plant height (cm)	Ear position	Num tassel branches	Tassel branch length (mm)	Cob length (mm)	Cob diameter (mm)	Ear diameter (mm)	Kernel row number	Kernel width (mm)	Wt 100 kernels (g)	Cob color (1–5 scale)
Group means
Oaxacan cultivars	89.7	99.7	12.1	166	275	0.59	19.2	157	134	22.0	36.0	11.8	8.6	32.5	1.1
Oaxacan Green Dent	66.8	69.4	2.7	87	176	0.49	18.8	106	144	24.8	40.7	11.3	10.0	30.7	3.1
US Corn Belt cultivars	64.9	67.9	3.1	80	171	0.46	15.3	87	138	23.5	42.8	13.5	8.0	25.7	2.9
Group mean differences
Oaxaca–Oaxacan Green Oaxaca–Corn Belt Oaxacan Green–Corn Belt	22.9	30.3	9.4	79	99	0.10	0.5	51	−10	−2.8	−4.7	0.5	−1.4	1.8	−2.0
	24.8	31.8	9.0	85	105	0.13	3.9	70	−4	−1.6	−6.8	−1.7	0.6	6.8	−1.8
	1.9	1.5	−0.4	7	5	0.03	3.5	19	6	1.3	−2.1	−2.2	2.0	5.0	0.2
p-values of group mean comparisons
Oaxaca vs. Oaxacan Green	0.000	0.000	0.010	0.000	0.000	0.001	0.425	0.000	0.337	0.030	0.207	0.317	0.001	0.313	0.000
Oaxaca vs. Corn Belt	0.000	0.000	0.004	0.000	0.000	0.000	0.028	0.000	0.418	0.100	0.078	0.031	0.061	0.015	0.000
Oaxacan Green vs. Corn Belt	0.188	0.358	0.461	0.336	0.400	0.213	0.116	0.101	0.412	0.230	0.372	0.038	0.000	0.106	0.297

• Abbreviations: ASI, anthesis–silk interval; DTA, days to anthesis; DTS, days to silk.

Oaxaca 55 and Oaxaca 62 were distinct from most of the other Oaxacan cultivars; they flowered earlier and were shorter than the other Oaxacan cultivars (Table 2). They can be distinguished from the Corn Belt Dent cultivars by their shorter and white cobs, lower kernel row numbers, and wider kernels (Table 2). Oaxaca 55 was previously classified as Zapalote Chico, which is renowned for its earlier flowering time, shorter stature, and smaller ears compared to other Mexican cultivars (Wellhausen et al., 1952). Although the plant type of Zapalote Chico is closer than other Oaxacan cultivars to the Corn Belt cultivars in our long daylength evaluation environment, the ear and kernel shapes and colors of Zapalote Chico (Wellhausen et al., 1952) are distinct from Oaxacan Green Dent (Figure S2). Oaxaca 62 is classified as Bolita, a racial group with a diminutive stature, early flowering, and has 8–10 kernel rows with a rounded semi-flint to flint grain type from which the name is derived (“little ball”; Figure S3). Bolita varies for grain color, including white, yellow, blue–black, red–purple, and tonal variations of these colors (Comisión Nacional para el Conocimiento y Uso de la Biodiversidad, 2025).

Correlation analysis revealed that several trait pairs were highly correlated, with correlation coefficients greater than 0.95: DTA with DTS and ear height; and ear height with plant height and ear position (Table S2). Therefore, days to silk and ear height were removed from the matrix of cultivar trait means before conducting multivariate analysis. PCs analysis of the resulting data matrix revealed that the first PC accounted for 43% of the variation with larger values associated with earlier maturity, smaller plant size and ear position, higher cob color intensity, and wider ears and cobs (Figure S4; Table S3). The second PC (23% of variation) was mostly negatively influenced by anthesis–silk interval and positively influenced by cob diameter and length (Figure S4; Table S3). Plotting the cultivar scores on the first two phenotypic PCs revealed that the first PC perfectly discriminates the true Oaxacan cultivars from US cultivars, with Oaxacan Green Dent falling on the positive side of the first PC with the other US cultivars (Figure 3). The second PC largely separated cultivars within these groups, with five Oaxacan landrace accessions and Black Beauty representing the most negative scores on this axis due to its small cob size, but Black Beauty was clearly distinct from the Oaxacan cultivars on PC1, having the largest positive score on PC1 (Figure 3). The Oaxacan Green Dent cultivars grouped more closely to the Corn Belt Dent cultivars (in particular to a Reid's Yellow Dent) than to the Oaxacan cultivars (Figure 3). Oaxaca 55 was not well separated from the Oaxacan Green Dent cultivars; however, as described above, this similarity was driven by its short plant stature and early maturity rather than by ear, cob, or kernel traits. PC3 is mostly influenced by the difference between kernel row number and kernel width (Table S3). Plotting cultivars on the basis of their PC1 and PC3 morphology scores reveals that Oaxacan Green Dents are most similar to ES Green Dent on these dimensions (Figure S5). Together, these results suggest that Oaxacan Green Dent is most closely related to Corn Belt Dents, perhaps with some influence of introgression from a source of kernel color genes.

FIGURE 3

Biplot of cultivar scores on the first two principal components of phenotypic data. PC, principal component.

A genetic similarity matrix among all cultivars was created from about 40,000 SNPs obtained from DNA sequencing samples of each cultivar. We included multiple samples of inbred lines B73 (temperate Corn Belt origin) and CML311 (subtropical origin) in the sequencing study as quality control checks. A dendrogram based on cluster analysis of the SNP data revealed that the two B73 samples were almost identical and the five CML311 samples were also nearly identical to each other and very distinct from the B73 samples (Figure 4). In addition, we included two samples of five plants each of Black Beauty, ES Green Dent, Strubbe's Green, Oaxacan Green Dent from Seed Savers Exchange, and Oaxaca collections 55, 62, 321, and Benz 933. In all cases, the two samples from the same cultivar clustered more closely than to any other cultivar (Figure 4). The genetic similarities of samples within cultivars were not as close as the similarities between replicates of inbred lines, as expected for open-pollinated cultivars that contain significant genetic diversity. These results indicate good reliability of these data for measuring genetic relationships.

FIGURE 4

Dendrogram of cultivars from cluster analysis of genetic distances based on 39,708 single-nucleotide polymorphisms (SNPs) from DNA sequencing. Sample names followed by “_1″ represent a second independent sample of individuals from the same cultivar.

The cluster analysis of SNP data revealed four major groups of samples: (1) a green dent cluster containing Strubbe's Green, ES Green Dent, and all three Oaxacan Green Dent cultivars; (2) a Corn Belt cluster containing the three Corn Belt Dent cultivars, inbred B73 (derived from Corn Belt Dents), and a more loosely related Black Beauty Corn Belt popcorn cultivar; (3) CML311 subtropical inbred line; and (4) all Oaxacan plant introduction collections (Figure 4). The multidimensional scaling analysis reveals greater genetic distance between Oaxacan Green Dent and the Oaxacan cultivars than between Oaxacan Green Dent and the other Corn Belt cultivars (Figure 5). Black Beauty is intermediate between Corn Belt Dents and the Oaxacan Green Dents, suggesting that it may have been a source of kernel color genes introgressed into a Corn Belt Dent cultivar to form ES Green Dent and the Oaxacan Green Dent cultivars. The genetic data unambiguously demonstrate that Oaxacan Green Dent is more closely related to Corn Belt Dents than to any sampled Oaxacan cultivar.

FIGURE 5

Position of cultivars along two dimensions of a multidimensional scaling analysis of genetic distances based on 39,708 single-nucleotide polymorphisms (SNPs). Sample names followed by “_1″ represent a second independent sample of individuals from the same cultivar.

Our phenotypic, genetic, and historical records provide no evidence that Oaxacan Green Dent is a cultivar from Mexico. Mexico has a robust cadre of maize genetic resources scientists who, in association with international organizations, have conducted numerous evaluations of maize germplasm collections made throughout Mexico from the 1940s until 2011. These evaluations have shown no evidence of any maize similar to Oaxacan Green Dent (Anderson, 1946; Aragón-Cuevas et al., 2006; Bellon et al., 2003; Chávez-Servia et al., 2011; Jaspeado et al., 2013; Ortega Corona et al., 2011; Ortega Paczka, 2003; Perales & Golicher, 2014; Rendón-Aguilar et al., 2015; Ron Parra et al., 2006; Sánchez G. & Goodman, 1992; Wellhausen et al., 1952). All maize originated in the area that is now Mexico, so in the broadest sense, even the most elite temperate commercial maize hybrids “originated” in Mexico, but that origin was on the scale of 1000 or more years ago (Matsuoka et al., 2002; Smith, 2017; Swarts et al., 2017; Yang et al., 2023). The oldest use of the cultivar name Oaxacan Green Dent, of which we are aware, is in the 1993 seed catalog by the seed saver organization Seeds of Change.

Claiming Oaxacan Green Dent as a Mexican cultivar is inaccurate and misleading. The cultural and culinary history of Mexican maize includes 700 distinct maize dishes closely associated with specific cultivar groups (razas or races; Fernández Suárez et al., 2013) but not foods made from green-colored grains (Echevarría & Arroyo, 2000; Sahagun Castellanos, 2015). The Mexican national database of collected maize cultivars includes 22,931 collections and has descriptors for 19 distinct grain colors but not for green kernels (Comisión Nacional para el Conocimiento y Uso de la Biodiversidad, 2025). A search of this database for collections with “verde” (“green”) in the cultivar common name identifies 43 collections, all from Guerrero (not Oaxaca), and all of which are indicated as having white or yellow grains or have “crema” (cream colored) in the cultivar name if grain color is not indicated. In the region of these collections, “maíz verde” refers to maize with the stay-green trait of delayed vegetative senescence (Juan Manuel Hernández Casillas and Noel Gómez Montiel, personal communication, 2025). A comprehensive and detailed analysis of 1818 indigenous populations of maize collected in Oaxaca between 1943 and 2005 describes the range of variation in grain color but does not report any cultivars with green-colored grain (Aragón-Cuevas et al., 2006). Household surveys of more than 240 farm families in the Central Valleys of Oaxaca in 1998 detailed 13 different color and texture combinations of maize grain but did not report the existence of green grains (Smale et al., 1999). Green-colored tortillas are rare but exist; however, their green color results from masa produced from white or yellow maize mixed with culinary green leaves of Chipilín (Crotalaria longirostrata Hook. and Arn.), Moringa (Moringa oleifera Lam.), Hoja Santa (Piper auritum Kunth), or chili peppers (Capsicum L. species), or from masa mixed with a puree of nopal (Opuntia Mill. species; Guevara-Arauza et al., 2012; Amanda Gálvez Mariscal, personal communication, 2025).

The most likely source of what is being marketed as Oaxacan Green Dent is ES Green Dent, developed by Ernest Strubbe of Minnesota. The original plant introduction inventory data for ES Green Dent (PI 452039) described its origin (United States Department of Agriculture Agriculture Research Service, 1980):

452039. A-1788. ES Green Dent. United States. Alberta, Minnesota. E. H. Strubbe. Black popcorn x yellow dent field corn. Collected April 18, 1977. Selected for 40 years. Cobs to 12 cm, red.

In an oral history (Whealy & Adelmann, 1986), Ernest Strubbe was quoted as follows on the origin of ES Green Dent:

In 1929 I crossed some yellow field corn with some black popcorn Black Beauty which Shumway dropped a few years ago. What I got was a dark colored black/bronze dented ear. At first the color was real spotty with bronze and brownish/purples. I worked with it for quite awhile and finally got the color really even and uniform. I was pretty proud of that….[ES Green Dent] came from the original bronze/black corn that I got from crossing the black popcorn and the yellow field corn. I kept a little patch of it by itself just to see what developed. One fall I happened to be looking at it in the sun to see how many different colors I could get to reflect. There were some blues, some purples, some brownish-bronze and the like. And one time here was this bright green reflection. You had to hold it just right. That fired my imagination. I wondered if it would ever be possible to produce a green color. I could see the possibility of producing most of these others, but green? I hunted and hunted through that stuff and every time I would find a kernel that had the faintest suggestion of a green reflection I picked it out and grew that in isolation…. For the first ten years or so it seemed quite hopeless, just plain frustrating. Because only occasionally would I find a kernel that was really green. But all at once it looked a little more promising, so I kept going. After about 40 years, lo and behold one fall when I was picking I found a couple of ears that were really green….But there's still a lot of work to be done on it yet. Even with the ear here in the case, if I were to plant that I would throw out all of the lighter green kernels. And a few of the kernels might have just a little too much of a purple cast to them. Throw them out because they are just going to pollute whatever you're after….One year Gurney Seed and Nursery wrote to me and wanted to grow 50 bushels!

Another possible origin of Oaxacan Green Dent is John Deere Corn, a US Midwestern novelty maize cultivar with green aleurone. John Deere Corn was used in genetic studies of maize aleurone color, but “the John Deere line may be identical to an open-pollinated cultivar known as Oaxacan Green Dent” (Stinard & Sachs, 2002). The most likely explanation is that ES Green Dent is the original source of both Oaxacan Green Dent and John Deere Corn. Besides the Oaxacan Green Dent/John Deere cultivar, we are aware of only one other maize cultivar that produces green colored kernels: Glass Gem, which segregates a wide range of colors on small popcorn-like kernels and was developed by Carl Barnes from crosses among numerous open-pollinated cultivars (Curry, 2022). Stinard and Sachs (2002) reported that they received their original seed of John Deere corn from Carl Barnes. Thus, it seems likely that Strubbe's Green Dent was a donor of gene combinations conferring green kernel color to Glass Gem.

Both John Deere Corn and Oaxacan Green Dent were sources of unique alleles of anthocyanin inhibitor genes (Inr1-JD and Inr2-JD; Stinard & Sachs, 2002). Alleles at these two genes reduce expression of anthocyanins in aleurone tissue by epistatically interacting with alleles at the R locus. The level of anthocyanin inhibition varied among R alleles; Inr1-JD and Inr2-JD in combination with some R alleles resulted in a range of purple color intensities, including very light coloration (Stinard & Sachs, 2002). Anthocyanins confer blue to red colors (Wallace & Giusti, 2015); we are unaware of anthocyanins conferring green colors. Oaxacan Green Dent and Strubbe's Green Dent both have an unusual combination of yellow vitreous endosperm and blue anthocyanins in the aleurone, whereas most cultivars with blue kernels have white endosperm. Thus, it is possible that the rare green kernel phenotype of Strubbe's Green Dent is due to a translucent layer of blue anthocyanins (with inhibited expression due to the InR1-JD or InR12-JD genes) over yellow endosperm.

To determine if Oaxacan Green Dent produced green colors specifically in aleurone tissue, we sectioned kernel crown tissue from Oaxacan Green Dent, Minnesota 13, and Black Beauty. All three cultivars have colorless pericarp tissue (Figure 6). Minnesota 13 kernels have no anthocyanin in the aleurone but have yellow carotenoids in both the aleurone and vitreous endosperm. (Figure 6a). In contrast, Black Beauty kernels lack carotenoids or yellow color in all tissues but have an intensely dark blue/purple pigmented aleurone. (Figure 6c). The hue of aleurone anthocyanins varies within a single kernel of Black Beauty around the circumference of the kernel crown, ranging from magenta to dark purple (Figure S6). Oaxacan Green Dent combines and modifies the coloration patterns of Black Beauty and Minnesota 13, with a yellow endosperm and blue-green aleurone (Figure 6b). The green color of Oaxacan Green Dent kernels occurs specifically in the aleurone layer, also with slight variation around the kernel crown within a single ear (Figure S6). It remains unclear, however, whether this green color is due to a combination of a light accumulation of blue anthocyanins and yellow carotenoids together in the aleurone or if this cultivar makes a novel green-colored compound.

FIGURE 6

Visible light micrographs of transverse sections of maize kernels of (a) Minnesota 13, (b) Oaxacan Green Dent, and (c) Black Beauty. Top images show the entire kernel section (black bar = 2 mm); bottom images are close-up views of a section at edge of kernel (represented by a black box in top image; black bar = 40 µm), showing endosperm (e), aleurone (a), and pericarp (p) tissues.

In summary, our results demonstrate that Oaxacan Green Dent is from Minnesota, not from Oaxaca. Many Mexican maize cultivars have been selected over centuries for their flavors, aromas, textures, and culinary properties, among them tortilla making (Fernández Suárez et al., 2013; Hernández Xolocotzi, 1985; Ortega Paczka, 2003; Sahagun Castellanos, 2015). Appropriating the name of Oaxaca in the marketing of a Corn Belt Dent cultivar implies to the consumer excellent culinary quality (and, in some cases is directly stated, Table S1) and may diminish the culinary reputation of Oaxacan maize by linking it to corn primarily used for animal feed. We recommend that seed dealers discontinue calling this cultivar Oaxacan Green and, instead, use its original and correct name, ES Green Dent or Strubbe's Green.

AUTHOR CONTRIBUTIONS

James B. Holland: Conceptualization; formal analysis; investigation; writing—original draft; writing—review and editing. Martha C. Willcox: Conceptualization; writing—original draft; writing—review and editing. Luis Fernando Samayoa: Investigation; writing—review and editing. Matthew Smith Woore: Conceptualization; investigation; writing—review and editing. Miriam Nancy Salazar Vidal: Investigation; writing—review and editing. William F. Tracy: Funding acquisition; supervision; writing—review and editing.

ACKNOWLEDGMENTS

We thank Jason Brewer and Francois de Wet for managing the field experiment and collecting trait data. Jay Bost provided us with evidence of the early use of Oaxacan Green Dent cultivar name from 1993. Juan Manuel Hernández Casillas, Noel Gómez Montiel, Jesús Sánchez González, and Flavio Aragón Cuevas generously helped us by checking the records of maize collections in Mexico to confirm the absence of maize with green-colored kernels in the collections of indigenous maize. Amanda Gálvez Mariscal provided valuable information on tortillas in Mexico. MCW thanks Ricardo Salazar for bringing this issue to her attention and Glenn Roberts for reiterating the same question, “Is this really from Oaxaca?”

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Open Research

DATA AVAILABILITY STATEMENT

Sequencing data are publicly available as BioProject PRJNA1183640 at https://www.ncbi.nlm.nih.gov/bioproject/. Trait data and analysis scripts are deposited at Dryad (https://doi.org/10.5061/dryad.r7sqv9sp6) for permanent storage.