DOI: 10.5061/dryad.r7sqv9sp6
title: Data and analysis files from: Oaxacan green dent maize is not from Oaxaca

description: “Oaxacan Green Dent” is a maize (Zea mays L.) cultivar marketed as an
 introduced Mexican heritage cultivar adapted to the higher latitudes of
 the USA. 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 USA 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.
description: <em>Germplasm</em> 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 (Preston et al.,
 2011; Curry, 2022). 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
 USA Corn Belt cultivars and 15 Oaxacan cultivars from the National Plant
 Germplasm System maize collection (Volk et al., 2023). The USA cultivars
 included two populations of Reids Yellow Dent and one population of
 Minnesota 13, which represent important germplasm sources that contributed
 to modern commercial corn hybrid pedigrees (Troyer, 1999; Smith et al.,
 2022). 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).
 <em>Field Experiment</em> 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<sup>−1</sup> pre-planting and then at 190.5 kg
 ha<sup>−1</sup> 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, 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 at 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. <em>Trait data
 analysis</em> Traits with one measurement per
 plot were analyzed with the following linear mixed model:
 Y_ij=μ+R_i+C_j+ε_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 ε_ij is the residual effect.
 Traits with more than one measurement per plot were analyzed with the
 following linear mixed model:
 Y_ijk=μ+R_i+C_j+ε_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, ε_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., 2017). 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 <em>mice</em> package in R (van
 Buuren and Groothuis-Oudshoorn, 2011). The complete
 matrix of cultivar-by-trait means was subjected to principal components
 analysis. Trait loadings on the first two principal components and
 cultivar scores on the first four principal components were visualized
 with the <em>ggplot2</em> package in R (Wickham,
 2016). <em>DNA sequencing</em>
 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 B73
 version 5 reference genome (Hufford et al., 2021). 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 <em>factoextra</em> package in
 R (Kassambara, 2024).
description: # Data and analysis files from: Oaxacan green dent maize is not from
 Oaxaca
 [https://doi.org/10.5061/dryad.r7sqv9sp6](https://doi.org/10.5061/dryad.r7sqv9sp6) ## Description of the data and file structure Plants sampled from Corn Belt, Oaxacan and 'Oaxacan Green Dent' populations were grown and measured in the field in North Carolina 2021 and also sequenced for comparison. ### Files and variables #### File: OGD\_plot\_phenotypes.csv **Description:** Flowering time data on Oaxacan Green field experiment, Clayton, NC 2021. NA values refer to missing data (not available). ##### Variables * REP: Replication block * PLOT: Plot number * MATERIAL: Population/cultivar/accession name * SEED_ID: Local seed source * ENTRY: Population and seed source * PLANTING_DATE: Planting date * DELAY: Number of days planting delayed * DTA: days from planting to anthesis * DTS: days from planting to silk * ASI: anthesis-silk interval (days) * STAND: Number of plants in plot if 50% or less of target stand #### File: OGD\_ind\_plant\_phenotypes.csv **Description:** Plant, ear, and tassel trait data on individual plants in Oaxacan Green field experiment, Clayton, NC 2021. NA values refer to missing data (not available). ##### Variables * REP: replication block number * PLOT: Plot number * MATERIAL: Population/cultivar/accession name * SEED_ID: Local seed source * ENTRY: Population and seed source * PLANT: Individual plant within population number * Ear_height: height to primary ear-bearing node (cm) * Num_tassel_branches: Number of tassel branches * Tass_branched_length: Tassel branched part length (mm) * Plant_height: Height to topmost node (cm) * Ear_position:  Ear_height/Plant_height * KColor: Kernel color category (W = white, Y = yellow, PG and Pale G = pale green, DG = dark green, W/P and W/Purple = segregating white and purple, PG/Y = segregating pale green and yellow, Multi = segregating for multiple colors, Red/Y/Blue = segregating red, yellow, and blue, WCP/YP = segregating white cap purple and yellow purple, WC/Y = white cap and yellow, PG/Brown = segregating pale green and brown * Cob_length: Cob length (mm) * Ear_diameter: Ear diameter including intact kernels (mm) * Kernel_row_number: Number of kernel rows * Cob_color: Intensity of cob color (5-point scale: 1 = white, 5 = dark red) * Cob_diameter: Cob diameter (mm) * KWidth1: Kernel width, 1st sample (mm) * KWidth2: Kernel width, 2nd sample (mm) * KWidth3: Kernel width, 3rd sample (mm) * KWidth4: Kernel width, 4th sample (mm) * Wt_100_kernels: Weight of 100 kernels (g) * Kernel_Width: mean of KWidth1, KWidth2, KWidth3, KWidth4 (mm) #### File: CL24\_Heirloom\_Data\_Overlap\_2021.xlsx **Description:** Data from 2024 Clayton, NC heirloom evaluation experiment for populations also included in 2021 Clayton, NC experiment ##### Variables * studyYear: Year of experiment * germplasmName:  Population/cultivar/accession name * replicate: Replication block * plotNumber: plot number * Ear Height [cm]|cycle 1|COMP:0000014: Ear height, 1st plant * Ear Height [cm]|cycle 2|COMP:0000015: Ear height, 2nd plant * Ear Height [cm]|cycle 3|COMP:0000016: Ear height, 3rd plant * Plant Height[cm]|cycle 1|COMP:0000026: Plant height, 1st plant * Plant Height[cm]|cycle 2|COMP:0000027: Plant height, 2nd plant * Plant Height[cm]|cycle 3|COMP:0000028: Plant height, 3rd plant #### File: Oaxacan\_Green\_Experiment\_DNA\_Samples.csv **Description:** Metadata for DNA sequencing samples ##### Variables * Code for Fernando: Sample code used in sequencing files * Material:  Population/cultivar/accession name * Accession: Plant Introduction collection number * Source: Seed source * N_planted: Number of seeds germinated for leaf tissue sampling * N_sampled: Number of seedlings sampled for DNA extraction * Comment: comment #### File: OGD\_dist\_mat.txt **Description:** Genetic distance matrix among sequenced samples based on SNPs ##### Variables * Row and column names are the population name corresponding to the genetic distance in the row or column #### File: OGD\_mindp2\_maxdp50\_sort.vcf **Description:** Variant call format file for sequenced samples ## Code/software R version 4.4.1 used for analysis. R packages used: tidyverse asreml mice cluster factoextra dendextend ggrepel Oaxacan Green Dent 2021 Field Experiment.Rmd is an R markdown file used to merge the three different field experiment data files, fit linear mixed models, estimate population BLUEs, impute missing values, estimate trait correlations, perform principal components analysis, and display phenotypic relationships among the populations. Oaxacan Green Dent 2021 Field Experiment.html is a knitted R markdown file of analysis of field experiment. OGD_Genetic_Relationships.Rmd is an R markdown file used to do cluster and MDS analysis and display genetic relationships among populations based on SNP genetic distance matrix. The SNP genetic distance estimates were made using TASSEL software, reading from the vcf, numericalizing the SNP calls, filtering for no more than 23% missing data per SNP, and then estimating genetic distances. OGD_Genetic_Relationships.html is a knitted R markdown file of analysis of SNP data