DOI: 10.5061/dryad.r4xgxd2k9
title: Phenotypic characterization of southeastern United States open-pollinated maize landraces

description: Maize is the most important crop in the United States, but food production
 and niche uses such as distilling often rely on varieties that are not
 bred explicitly for these purposes. Farmers targeting niche food grain
 markets have expressed interest in historical open-pollinated varieties of
 maize, but few such populations are widely available, and even fewer are
 well-characterized. We planted field trials of a subset of 76
 open-pollinated heirloom varieties available from catalogs and the USDA
 germplasm collection along with four F1 hybrid cultivars. We measured 24
 traits across three years and three locations to characterize the selected
 varieties and measure their phenotypic relationships. We identified a
 subset of 19 traits useful for phenotypic analysis. Our results
 demonstrate that many historical accessions contain substantial genetic
 variation that should permit improvement from within-population selection.
 Variety name and origin are often not useful indicators of phenotypic
 relationships or potential crop value. Cluster analysis identifies nine
 morphologically distinct groups within the varieties tested, which are not
 fully in agreement with currently accepted landrace groupings, suggesting
 a need for genetic analysis of larger samples of USA open-pollinated
 populations to better define the natural classification of maize
 indigenous to the United States.
description: In 2017, a single plot of each variety was planted at Clayton,
 NC.  Each plot consisted of four 7.6-m rows of the same variety, seeded at
 a rate of 25 seeds per plot (100 seeds total per population). Seed was
 produced from each plot by bulking pollen within the plot each day and
 pollinating as many ears as possible by hand. Seed was bulked from each
 population and used as source seed for subsequent experiments. Check plots
 of T175 × T177 and T177 × T179 F<sub>1</sub> hybrids were
 planted in three replicates distributed throughout the experiment
 field.  In 2018 and 2019 an 8×10 alpha-lattice design
 with three full replications was planted in each of three locations:
 Clayton, NC; Pendleton, SC; and Columbia, MO. Experimental units were
 single 7.6-m rows sown with 25 seeds of each variety.  Experimental fields
 were irrigated as needed with overhead irrigation at Columbia and Clayton
 and drip irrigation at Pendleton.  At Clayton, fields received nitrogen at
 53.2 kg ha<sup>-1</sup> pre-planting, and then 190.5 kg
 ha<sup>-1</sup> in a split application during the growing
 season.  At Columbia, fields received 134.5 kg ha<sup>-1</sup>
 N pre-planting, then 11.2 kg ha<sup>-1</sup> in a single
 application during the growing season. At Pendleton, fields received 44.8
 kg ha<sup>-1</sup> N pre-planting.   We
 collected data on a subset of the morphological traits previously
 recommended for maize racial classification by Sánchez G. et al. (1993)
 from each of the four rows of the seed increase plots in 2017 and from
 each experimental plot in 2018 and 2019 (Table S1). Days to anthesis
 (DTA), days to silking (DTS), anthesis-silk interval, mean numbers of ears
 and tillers per plant, and lodging percentage were measured on a
 plot-basis. DTA and DTS were the first dates on which half or more of the
 plants in a plot were shedding pollen or silking, respectively.
 Anthesis-silking interval (ASI) was calculated as the difference between
 DTS and DTA. Mean numbers of ears and tillers per plant were measured as
 the total count of ears or tillers per plot, divided by number of plants
 per plot. The end plants in each plot were not included in counts of ears
 or tillers or in the total count of plants per plot. Lodging percentage
 was the proportion of plants within a plot that exhibited root lodging
 (stalks leaning from base of the plant by more than 30° from upright) or
 stalk lodging (stalks broken below the primary ear or dropped primary
 ear). Other traits were measured on one randomly
 selected individual plant per row in 2017 and on two plants per row in
 2018 and 2019. Plant and ear heights were measured approximately two weeks
 after pollination. Plant height was measured from base to node of the leaf
 subtending the tassel; ear height was measured from base to the primary
 ear node. Ear position, the ear height relative to plant height, was
 calculated as (Ear height/Plant height)*100. Leaf length was measured
 along the midrib from ligule to tip on the leaf above the primary ear.
 Leaf width was measured at the widest point on the same leaf. Tassels were
 detached from the plant and measured in the field.  The number of primary
 branches per tassel were counted.  Tassel length was measured from lowest
 branch node to tip of main spike.             In year
 2017, one sib-pollinated ear was selected from each row to measure ear and
 kernel traits. In years 2018 and 2019, two plants per plot were randomly
 chosen for self- or sib-pollination, and at maturity, two self-pollinated
 ears from each plot were collected, along with two open-pollinated ears
 from other plants in the plot (not including the end plants) for ear and
 trait measurements.  Sib- or self-pollinated ears were used to measure
 husk and shank traits, and the range of kernel colors among all
 sibbed/selfed ears per population were noted (Table 1).  Open-pollinated
 ears generally had more complete pollination and remained healthier during
 maturation, and thus were used to measure other ear and kernel traits in
 2018 and 2019.             Husk color was recorded as a
 binary trait for presence or absence of anthocyanin in the outer husk
 leaves.  Husk number was the number of husk leaves covering each ear. 
 Shank length was measured with a Fowler Xtra-Valu caliper (54-101-300-1;
 Newton, MA). Ear diameter was measured by caliper at the middle of the ear
 with seeds attached to the cob.  Kernel row number was counted at the
 middle of the ear.  Each ear was assigned a percentage ear rot score based
 on the proportion of the ear exhibiting signs of rot diseases incited by
 <em>Fusarium</em>, <em>Aspergillus</em>, or
 <em>Diplodia</em> fungi.  Ears were dried in a forced-air
 drier for two weeks and kernels were removed from ears to permit cob and
 kernel measurements. Cob length was measured with a caliper from the base
 of the ear to the tip after removal of staminate tips (where present). Cob
 color was noted on a 5-point scale from 1 (white cob, no pigment) through
 5 (dark red; Figure S1).  Cob diameter was measured at the middle of the
 cob with a caliper. Kernel width was estimated as the mean of four
 individual kernel values measured with calipers per ear. A sample of 100
 kernels was randomly selected from each ear to measure 100-kernel weight.
    
description: R, Rstudio. ASReml-R used for some analyses, this is commercial
 software requiring a license. Echidna is a free software that could be
 used as an alternative to ASReml-R
description: # Characterization of Southeastern United States Open-Pollinated Maize
 Landraces --- Maize (Zea mays L. subsp. mays) is the most important crop
 in the United States, but maize food and distillation products often rely
 on varieties that are not bred explicitly for these purposes. Farmers
 targeting niche food grain markets have expressed interest in historical
 open-pollinated varieties of maize, but few such populations are widely
 available, and even fewer are well-characterized. We planted field trials
 of 76 open-pollinated heirloom varieties available from seed catalogs and
 the USDA germplasm collection along with four F1 hybrid cultivars. We
 measured 24 traits across three years and three locations to characterize
 the selected varieties and measure their phenotypic relationships. We
 identified a subset of 19 traits with heritability &gt; 0.5 and pairwise
 correlation coefficients not exceeding 0.9 for phenotypic analysis. Our
 results demonstrate that many historical accessions contain substantial
 genetic variation that should permit improvement from within-population
 selection. Variety name and origin are often not useful indicators of
 phenotypic relationships or potential crop value, although Hickory King
 varieties have maintained their identity even following global dispersion.
 Cluster analysis identifies nine morphologically distinct groups within
 the varieties tested, which are largely in agreement with previously
 proposed landrace groupings. Our results suggest a complex intermixing of
 Southern Dents with other maize types, rather than a clear distinction
 between Southern Dents and Derived Southern Dents, however. Genetic
 analysis of larger samples of USA open-pollinated populations will help
 better define the natural classification of maize indigenous to the United
 States. ## Description of the data and file structure Exp 32 was our local
 codename for this experiment, therefore it is used as a prefix in many of
 the file names.\ Exp32_data.csv is the primary plot-level data file. It
 contains 2922 records with 32 columns. Columns (and measurement units)
 are: Year Location: evaluation location, NC, MO, or SC Rep: field complete
 block Block: field incomplete block Plot: randomized plot number Sample:
 not used Population: Name of variety or population DTA: days from planting
 to 50% anthesis (days) DTS: days from planting to 50% silking (days) ASI:
 days between silking and anthesis (days) Tillers: Number of tillers per
 plant (mean count) Ears: Average number of ears per plant (mean count)
 Lodging: percent of lodged plants (proportion) Plant Height: height to
 uppermost node (cm) Ear Height: height to primary ear node (cm) Leaf
 Length: length of leaf above ear (cm) Leaf Width: width of leaf above ear
 (cm) Tassel Branches: number of primary branches per tassel (count) Tassel
 MSL: Main stem length (MSL) was measured from lowest branch node to
 highest branch node (cm) Tassel Length: distance from lowest branch node
 to tip of main spike (cm) Tassel Spike: Tassel spike length was calculated
 as the difference between tassel length and tassel MSL (cm) Tassel
 Internode: Tassel internode length was calculated as the count of branches
 divided by tassel MSL (cm) Husk Color: binary score for presence of
 anthocyanins in husk leaves (0/1 score) Husk Num: number of husk leaves
 (count) Shank Length: length of lateral branch from main stem node to base
 of ear (cm) Cob Length: length of cob from base to tip, excluding
 masculinized tips (cm) Kernel Row Number: number of kernel rows per ear
 (count) Ear Diam: diameter of ear, including seeds, at middle (cm) Cob
 Color: color intensity score (1 - 5 score, white to dark red) Cob Diam:
 diameter of shelled cob at middle (cm) Weight100k: weight of 100 randomly
 selected kernels (g) Kernel Width 1: first sample measure of kernel width
 (mm) Kernel Width 2: 2nd sample measure of kernel width (mm) Kernel Width
 3: 3rd sample measure of kernel width (mm) Kernel Width 4: 4th sample
 measure of kernel width (mm) Ear Rot: percent of kernels with visible
 symptoms of fungal infection (proportion) Missing values are recorded as
 "." or as NA The following data summary files are generated by
 running the R scripts included on this initial data set:
 SE_OPV_standardized_within_population_genetic_variances.csv
 SE_OPV_9ClusterTraitMeans.csv SE_OPV_data_cleaned.csv
 SE_OPV_Heritability_Pop_Means.csv SE_OPV_Observation_counts_per_trait.csv
 SE_OPV_Population_Cluster_Assign.csv SE_OPV_Population_Means.csv
 SE_OPV_PopulationMeans_ClusterAssign.csv SE_OPV_Trait_Correlations.csv
 SE_OPV_Table_2.csv #Estimates of h2, ratio of within-genetic to
 among-population variances (Vw_g_Vp_ratio_mn), ratio of pop-by-env to
 among pop variances (Vpe_Vp_ratio_mn) for each trait The following html
 files are knitted versions of the R markdown files, showing code,
 comments, and output together:
 Step1_SE_OPV_raw_data_QC_cleaning_summary.html
 Step2_SE_OPV_Pop_mean_heritability.html
 Step3_SE_OPV_Population_variances.html Step4_SE_OPV_cluster_and_PCA.html
 Step5_Final_Tables.html The following Word and csv files contain
 Supplemental Figures and Tables:
 Southeastern_OPV_Maize_Supplemental_Figures.docx
 Table_S1_Trait_Definitions.docx Table_S2_SE_OPV_Trait_Correlations.csv
 Table_S3_SE_OPV_PopulationMeans_ClusterAssign.csv
 Table_S3_SE_OPV_standardized_within_population_genetic_variances.csv
 Table_S5_SE_OPV_9ClusterTraitMeans.csv ## Sharing/Access information NA ##
 Code/Software The following R codes in R markdown format were used to
 analyze the data: Step1_SE_OPV_raw_data_QC_cleaning_summary.Rmd
 Step2_SE_OPV_Pop_mean_heritability.Rmd
 Step3_SE_OPV_Population_variances.Rmd Step4_SE_OPV_cluster_and_PCA.Rmd
 Step5_Final_Tables.Rmd