tivar and 10% for location, indicating that neither variable was causing large expression changes, when considering %CV. Glycinin G2 also showed low overall variation, however, the disparity between variation from location and cultivar was large, 15 and 2% respectively. In contrast, one of the two lower abundance forms, Glycinin G3, has the largest percent variation. Location resulted in variation exceeding 60%, which was approximately twice the variation observed among cultivars.
The levels of Glycinin G1 and G2 were affected by the environment and to a lesser extent by the cultivar (Table 1). Kansas and Pennsylvania had significantly higher levels of Glycinin G1 compared to the other locations across four cultivars (Figure 3). Conversely, Ontario had significantly lower levels of Glycinin G2 compared to the other locations across four cultivars. The level of Glycinin G3 was impacted by both the environment and the cultivar, but the environmental factor had a greater impact on the expression of this allergen as indicated by both the F statistics and P value (Table 1). Ontario and Pennsylvania had significantly higher levels of Glycinin G3 compared to the other locations across four cultivars.
The levels of Glycinin G4 were affected by the environment (Table 1); with cultivars grown in Georgia and Ontario having the lowest levels (Figure 3). The difference in cultivar also affected the expression of Glycinin G4 (Table 1). Cultivar 92B12 and 93B15 had the lowest levels of Glycinin G4 across all six locations. The expression level of Gly m Bd 28k and β-conglycinin α subunit were affected mainly by the environment it was grown in (Table 1). Iowa and Kansas had the highest levels of Gly m Bd 28k compared to the other sites across four commercial varieties (Figure 3). In contrast, cultivars grown in Georgia were consistently lower than those grown in Iowa and Kansas (Figure 3). Nevertheless, a lower level of β-conglycinin α subunit was observed in all the cultivars grown in Ontario (Figure 3).
The total allergen content was similar between Ontario, Georgia, Nebraska, and Iowa (Figure 4). Likewise, the allergen content was similar between Pennsylvania, and Kansas. Cultivar 92B12 grown in Ontario had the lowest level of total allergens whereas; cultivars 92M72 and 93B15 grown in Pennsylvania had the highest levels of total allergens (Figure 4).
The following can be seen from Figure 5: (1) Across the 24 samples (four cultivars at six sites), Glycinin G2, Glycinin G4, and Beta conglycinin α were positively correlated (a small acute angle), and KTI 3-1 and KTI 3-2 were positively correlated (an acute angle). Glycinin G3 was negatively correlated with Glycinin G2, KTI 1, KTI 3, Beta conglycinin α, and Glycinin G4 (obtuse angles). (2) Samples tended to be clustered together by sites. All four cultivars from the GA site were at the lower left quadrant of the biplot. All four cultivars from the ON site were at the left part of the biplot. All four cultivars from the PA site were at the upper part of the biplot. All four cultivars from the KS site were at the right part of the biplot. All four cultivars from IA and NE sites were at the lower part of the biplot and showed similar pattern by cultivar. From PCA analysis it can be concluded that variation among sites (i.e., environment) was the dominant variation for allergens.
In this study, eight allergens were quantified in 24 samples with each sample measured four times. In order to perform this ambitious study we addressed the individual seed variability by taking large samplings to average this component. This was performed using standard procedure for random sampling of material from test plots. It should also be noted that subsequent quantification was performed on single representative AQUA™ peptides as explained in the Section “Materials and Methods.”
Once protein was extracted, processing from protein dissolution to calculations for absolute quantity took approximately 6 days, with an average percent coefficient of variance for the experiment at 10.0%. In order to achieve this r