Validation of Molecular Markers for Low Kunitz Trypsin Inhibitor Content in European Soybean (Glycine max L. Merr.) Germplasm

Abstract

Trypsin inhibitors (TI) in raw soybean grain, mainly represented by the Kunitz trypsin inhibitor protein (KTI), prevent the normal activity of the digestive enzymes trypsin and chymotrypsin in humans and monogastric livestock. The inactivation of TI is achieved through costly and time-consuming heat treatment. Thermal processing also impairs the solubility and availability of the soybean grain protein. Therefore, the genetic elimination of KTI has been proposed as a suitable alternative to heat treatment. The aim of this study was to screen the collection of European soybean cultivars with six genetic markers (one SSR marker and five SNP markers) previously proposed as tightly linked to the KTI3 gene encoding the major Kunitz trypsin inhibitor seed protein of soybean and validate their usability for marker-assisted selection (MAS). The six markers were validated on a subset of 38 cultivars with wide variability in KTI content and in the F₂ and F_3:5 progenies of two crosses between the known high- and low-KTI cultivars. Three genetic markers (SSR Satt228 and two SNP markers, Gm08_45317135_T/G and Gm08_45541906_A/C) were significantly associated with KTI content in a subset of 38 cultivars. Low-KTI alleles were detected in both low- and high-KTI genotypes and vice versa, high-KTI alleles were found in both high- and low-KTI genotypes, indicating a tight but not perfect association of these markers with the KTI3 gene. The genetic marker SSR Satt228 showed a significant association with KTI content in the F₂ progeny, while the SNP markers Gm08_45317135_T/G and Gm08_45541906_A/C allowed significant discrimination between progeny with high- vs. low-KTI progenies in the F_3:5 generation. These three markers could be applied in MAS for low-KTI content but not without the additional phenotyping step to extract the desired low-KTI genotypes.

Keywords: Kuntz trypsin inhibitor; antinutrient; marker-assisted selection; soybean.

Figure 1

Allele frequencies at six marker loci associated with KTI content in the 165 soybean cultivars. Blue and orange colors indicate low- and high-KTI alleles, respectively.

Figure 2

Frequency distribution of KTI content (µg g⁻¹) in seeds of 38 soybean cultivars for low- and high-KTI alleles at six marker loci. Blue and orange bars indicate low- and high-KTI alleles, respectively. P > |t|—the significance of the t-test.

Figure 3

Distribution of KTI content (µg g⁻¹) for three genotypic classes (250/250, 250/217, and 217/217) at SSR locus Satt228 in F₂ generation of the crosses ‘Ascasubi’ × ‘DH_5170’ and ‘Bahia’ × ‘ES_Mentor’. Differences among genotype means followed by the same letter (indicated next to box plots) are not significantly different at p < 0.05 according to the Fisher LSD test. Means of low-KTI and high-KTI parents are indicated with green and red circles, respectively. MP indicates midparent value.

Figure 4

Distribution of KTI content (µg g⁻¹) for homozygous genotypes at SNP loci Gm08_45317135_T/G and Gm08_45541906_A/C in F_3:5 of the crosses ‘Ascasubi’ × ‘DH_5170’ (N = 12) and ‘Bahia’ × ‘ES_Mentor’ (N = 32). Pr > |t| indicates the significance of the t-test.