FAD2A Allele Frequency
The causal mutation in FAD2A is present on the Affymetrix Axiom Arachis2 48K SNP array (Clevenger et al. 2018) as AX_147234396. Data on 200 Virginia-type lines from the NCSU program run on the array was collected to investigate the allele frequencies of the FAD2A mutation (Hancock 2018). DNA was isolated from young leaf tissue of an additional 136 lines not run on the array with the Qiagen DNeasy Plant Mini kit per the manufacturer’s instructions. All of the additional lines were either used as a parent or entered into advanced yield trials by the NCSU program from 2016-2019. Following quantification and normalization, 5ng of DNA was combined with 2.1μL molecular biology grade water, 2.5μL PACE 2.0 Genotyping Master Mix (3CR Bioscience Part # 003-0002), and 0.069μL FAD2A genotyping assay (Table 2). Thermocycling conditions followed the manufacturer’s instructions with 42 cycles during Step 3 and afterwards were read on a BMG Labtech GmbH PHERAstar plate reader.
Plant Materialand Field Design
The eight lines listed in Table 1 were planted for two years (2019 and 2020) in two-row 31-foot plots at the Peanut Belt Research Station in Lewiston-Woodville, NC. The same seed source was used in both years. Of the eight lines, only Bailey is NO while the remaining seven are HO. Peanuts were dug at three dig dates in order to test for the effects of maturity: an optimum dig date (145 days after planting (DAP)), early (131 DAP, i.e. two weeks early) and late (159 DAP, i.e. two weeks late). Dig dates necessitated a strip-plot design in order to accommodate machinery, plots that needed to be dug on the same date needed to be in the same strip. After digging, a small sample bag was harvested and shelled from each plot. There were two replications for 96 total experimental units or plots.
Oleic acid content was determined by running 96 seeds from each plot (9,216 total seeds) on a Brimrose Luminar 3076 Seedmeister. This is the same machine used by the North Carolina Foundation Seed Producers (NCFSP) to verify seed lots as high oleic. This machine transmits light to a seed, measures how the light is reflected by the seed, and uses those measures in a model determined by gas chromatography to predict oleic acid content. After phenotyping and in an orderly fashion, each seed was placed in an individual well of a 24-square well microplate (Spex Sample Prep, Product # 2230) for tissue collection. This allowed the phenotype of each individual seed to be paired with its genotypic data.
Design of Manual Seed Chipper
To facilitate the tissue collection and individual genotyping of all 9,216 phenotyped seeds, a manual seed chipper was designed and constructed in the NCSU Biological and Agriculture Engineering Research Shop. A detailed overview of the seed chipper and its operation is presented in Supplementary File 1. The seed chipper consisted of a 2-ton Dayton Arbor Press (Model 467L16) with 24, 3.0mm reusable rapid punch biopsy kits (World Precision Instruments # WP3030) mounted in a 6-by-4 layout within a stainless steel bracket. The bracket included an ejector plate that, when lowered, depressed the plunger on all 24 biopsy kits simultaneously, thus ejecting all 24 seed chips simultaneously. The entire apparatus was mounted at the base of the arbor press ram and moved in concert with the ram. A stainless steel base was designed that could be rapidly alternated to accommodate the two different microplates needed, as described below. A spring-loaded lever arm was also added to maintain a consistent sampling height for the biopsy kits.
The 24-square well microplate fit perfectly in the groove of the seed chipper’s base, centering each biopsy kit over its respective seed. Therefore, samples were always taken from the middle of the seed, leaving the embryo at either end of the seed intact. With depression of the lever arm, all 24 biopsy kits moved down simultaneously into the 24-well plate and sampled the respective seed. After pushing the lever arm back up, seeds remained in their wells while samples were within the biopsy kit. The 24-well plate was removed and a flat metal sheet placed over the grooved base. A 96-round well microplate (Spex Sample Prep, Product # 2210) was placed onto the metal sheet and moved under the biopsy kits. By design, the 96-well plate could fit under the biopsy kits in four different positions designated by ordinal directions (Supplemental File 1). Depression of the ejector plate ejected the 24 seed chips into the 96-well plate. This process was repeated three more times until each 96-well plate was full. Once four 96-well plates were full, a 4mm stainless steel grinding ball (Spex Sample Prep, Product # 2150) was added to each well using a grinding ball dispenser (Spex Sample Prep, Product # 2100), sealed with a cap mat (Spex Sample Prep, Product # 2211), and ground for 30 seconds at 1,350rpm in a Spex Sample Prep 1600 MiniG automated tissue homogenizer. Following grinding, plates were briefly spun at 250G to collect ground material. To determine the effect of tissue collection on seed viability, 96 chipped and 96 unchipped seed of Bailey II were planted under routine greenhouse conditions and germination rates recorded. Seeds were considered germinated if cotyledons were visible one week after planting.
Crude DNA Isolation & Genotyping at FAD2B
The DNA isolation protocol was loosely based on an existing protocol used in cotton (Zheng et al. 2015). To each well, 100μL of 100mM NaOH, 2% Tween 20 was added and plates were vortexed for one minute. Plates were then incubated at 65oC for ten minutes in a forced air oven (VWR Cat No. 89511-412). Following incubation, 100μL of 100mM Tris-HCl, 2mM EDTA was added to each well and shaken vigorously for ten seconds. After a brief spin to collect all material in a well, 400μL molecular biology grade water (VWR Cat No. 02-0201-1000) was added to each well. Then 30μL of supernatant was transferred to a new plate and mixed with 30μL of molecular biology grade water. All centrifuge steps were performed in a Beckman Coulter Avanti J-15 benchtop centrifuge with a JS-4.750 swing-bucket rotor. Without quantification or normalization, 1μL of DNA from each seed was genotyped as described above for FAD2A except the FAD2B genotyping assay (Table 2) was used instead.
Development of the SNP Caller Web Application
The SNP caller application was developed in Python using Plotly Dash and deployed freely through Heroku (snp-caller.herokuapp.com). The application accepts input files from microplate readers that have been formatted as shown in the application’s ‘Example Data Sets,’ including the header. The user-defined parameters are determined based on the plate layout (384-well [default]) and cluster number (four [default]) with customizable options for switching back-and-forth between markers within the plate layout/design and adding marker names. In addition, there are manual options for changing marker calls, including the default genotype calls for ‘Y’ (homozygous for HEX dye), ‘H’ (heterozygous), ‘X’ (homozygous for FAM dye) and ‘Fail’ (genotype not present), along with ‘No Call’ (indistinguishable genotype). When a user adds data from the plate reader, the application produces a data table and figure based on the default plate layout and cluster options. Two data columns are immediately added to the input data file, the plate markers associated to each well and the initial SNP calls based on the default layout and cluster numbers. Clusters are determined using the ‘KMeans’ module contained within the SciKit Learn package in Python and then SNP calls are assigned based on the coordinate arrangement of the cluster centroids in the figure map. To change the plate layout, select the radio button corresponding to the layout examples presented in the application. In addition, if negative controls i.e. ‘blank’ wells and/or heterozygotes do not exist, the number of clusters can be adjusted down to reflect the anticipated genotype groups displayed in the figure. After adjusting the plate layout and cluster number, the data table updates the plate marker and SNP call data columns to reflect these changes. Once all parameters and marker options are modified to the user’s needs, the figures and data table can be downloaded to a user’s computer. For additional information on the SNP caller functionality, see user’s manual located in the application. Accompanying example data sets are located in the application corresponding to the actions in the user’s manual. The SNP caller should be compatible with other allele-specific assays such as KASPTM.
Statistical analysis for the strip-split plot design was performed using the PROC GLM procedure in SAS version 9.4 (Cary, NC, USA). Within each year, the eight peanut lines were arranged among each of the dig dates (strip-plots) and then grouped in two blocks, representing replications. For each factor, the strip- and split-plots were randomly assigned within the blocks and strip-plots, respectively. The analysis of the strip-split plot factors was conducted using appropriate F-tests based on the expected mean squares. Observations for the analysis were limited to only those seed with a homozygous FAD2B genotype (i.e. seeds which ‘failed’ to produce a FAD2B genotype or were heterozygous were excluded). All means separation tests were conducted using Fisher’s protected least significant difference (LSD) at a significance of α = 0.05, and were presented for dig date, line, and their interaction.