Identification of Kunitz trypsin inhibitor mutations using SNAP markers in soybean mutant lines

The Kunitz trypsin inhibitor (KTi) in soybean has several polymorphic types that are controlled by multiple alleles, which behave in a co-dominant fashion. Of these, Tia and Tib, which differ by nine amino acids, are the predominant types. In order to develop a single nucleotide amplified polymorphism (SNAP) marker for the classification of the predominant KTi types, Tia and Tib, and evaluate KTi activities by differing KTi type total 451 soybean mutant lines (M₁₂-M₁₆ generation) were incorporated in this study. Among 451 soybean mutants, 144 and 13 mutant lines showed decreased and increased trypsin inhibitor activity when compared with the original cultivars, respectively. To identify the KTi type, we designed a SNAP marker. Among 451 mutant lines from 12 soybean cultivars and landraces, 8 mutant lines derived from cvs. Baekwoon, Paldal and Suwon115 showed a change in KTi type when compared with the original cultivars using the SNAP marker. Five mutant lines in Suwon115 changed from Tib to Tia, while two mutant lines derived from cv. Baekwoon and one mutant line derived from cv. Paldal were changed from Tia to Tib. These changes of KTi types were confirmed by sequencing of the KTi genes and non-denaturing polyacrylamide gel electrophoresis of the KTi proteins. To identify the effect of KTi activity based on the change in KTi type, we measured the KTi activity using the three cultivars and eight mutant lines that showed changes in KTi type. Two mutant lines (BW-1 and 7-2) derived from cv. Baekwoon and one mutant line (PD-5-10) from cv. Paldal that changed from Tia to Tib showed lower activity than the original cultivar. In cv. Suwon115, five mutant lines that changed from Tib to Tia showed higher activity than the original cultivar. These results indicate that the designed SNAP marker was capable of identifying the KTi type and that Tia activity was higher than Tib activity in soybean.