ially available varieties of soybeans were each grown at six different locations (Georgia, Iowa, Kansas, Nebraska, Ontario, and Pennsylvania, Figure 1). Soybean allergens were measured similarly to Houston et al. (2010) and absolute quantities of soybean allergens reported. Principal Component Analysis (PCA) was conducted to explore the patterns of multivariate data and a biplot (Gabriel, 1971) was utilized to provide results graphically. Genotypic variation and environmental variation were quantified relative to the overall mean and relative to residual variation using Analysis of Variance (ANOVA)-based F-test. The data suggest that over a broad geographical region, the environment plays a larger role than genotype in determining allergen/anti-nutrient protein levels.
Unless stated otherwise, all reagents were purchased through Research Products International, Mount Prospect, IL, USA.
Four commercially available non-GM soybean varieties (92B12, 92M10, 92M72, and 93B15 were utilized for this study. A similarity matrix using all the markers available that are in common between the varieties was conducted and it was found that the lines have anywhere from 62 to 71% similarity. The study design included six sites located in the commercial soybean-growing regions of North America: Quitman, GA, USA; Richland, IA, USA; Larned, KS, USA; York, NE, USA; Branchton, ON, Canada; and Hereford, PA, USA. The field phase of this study was conducted during the 2007 growing season. The mean temperature during the day for each site was as follows: GA (90.5°F); IA (81.5°F); KS (86.5°F); NE (80.3°F); ON (77.5°F); PA (86.8°F). The mean temperature during the night for each site was as follows: GA (68.2°F); IA (60.5°F); KS (59.2°F); NE (58.7°F); ON (53.5°F); PA (58.2°F). Average rainfall for each site is as follows: GA (4.98″); IA (4.79″); KS (2.23″); NE (3.45″); ON (1.99″); PA (2.87″). A randomized complete block design containing three blocks, with each block containing the 92B12, 92M10, 92M72, and 93B15 commercial soybean varieties, was utilized at each site. Each soybean variety was planted in a two-row plot, which was bordered on either side by one row of non-GM commercial soybean variety of similar relative maturity. Sites were surrounded by at least 10 feet of bare ground buffer area. All plants grew well at all locations and harvesting was conducted at typical maturity/dryness levels. Typical would refer to 95% of pods on the plant are mature and seed weight adjusted to 13% seed water content. Seed samples for each cultivar (300 g from each block) were harvested and pooled. There was no morphology/physiology differences among the seeds collected from the different locations. The pools were ground and a portion removed for each cultivar. Samples were shipped at ambient temperatures to the University of Missouri for analysis. Samples were stored at room temperature in the dark until extracted.
Protein was isolated in quadruplicate from 0.25 g of soybean meal using the extraction method described in (Stevenson et al., 2009). Briefly, soybean meal was suspended in 2.5 mL of Tris-buffered phenol (pH 8.0; Fisher Scientific, Inc., Pittsburgh, PA, USA) and 2.5 mL extraction buffer [0.9 M sucrose, 0.1 M Tris-HCl pH 8.0, 10 mM EDTA, 0.4% (v/v) 2-mercaptoethanol]. The mixture was homogenized using a T25 Basic S1 Disperser (IKA Works, Inc., Wilmington, NC, USA) on speed 4 with the S25N-10 tool. The emulsion was inverted at 4°C for 2 h and then centrifuged at 5000 × g and 4°C for 20 min. Phenol was removed and a single back extraction was performed in the same manner. Phenol fractions were combined and protein precipitated using five volumes of 95% (v/v) methanol with 0.1 M ammonium acetate and incubation at −20°C overnight. Precipitated protein was pelleted by centrifugation at 5000 × g and 4°C for 5 min, and washed by re-suspending in 10 mL fresh methanol-ammonium acetate solution three times, 80% (v/v) acetone two times, and 70% (v/v) ethanol once, with 20 min incubations at −20°C before pelleting. Protein was stored in 80% (v/v) acetone at −20°