Genomic introgression and expression profiling of the KTI null allele in soybean through elite-by-elite backcrossing

Soybean stands out with a high protein (35-45 %) and oil content (18-22 %) is one of the important plant-based protein sources. However, the presence of Kunitz Trypsin Inhibitor (KTI), an anti-nutritional factor, reduces the bioavailability of soybean proteins. While thermal inactivation of KTI is possible, it compromises protein solubility and leaves residual activity. Therefore, eliminating KTI through genetic approaches is crucial to improving soybean's nutritional profile. This study aimed to develop soybean genotypes devoid of KTI through marker-assisted selection (MAS) to enhance their nutritional value and global marketability. Hybridization was conducted between recipient parents (AMS-MB-5-18 and AMS-MB-5-19) and donor parents (NRC-101 and NRC-127) to produce four cross combinations. Marker-assisted screening was used to identify plants carrying the null KTI allele across successive backcross generations (BC₁F₁ to BC₁F₄). A total of 342 SSR markers were used to assess molecular polymorphism and determine recurrent parent genome content (RPGC) recovery. Absence for KTI peptide was confirmed with native polyacrylamide gel electrophoresis (PAGE). In addition, expression of the KTI3 gene was evaluated during seed developmental stages (R5 and R6) using quantitative PCR. Hybridization efforts yielded 60 F1 seeds with a 12 % success rate. Backcrossing resulted in the production and screening of 259 BC₁F₂ plants, 45 BC₁F₃ plants, and 8 BC₁F₄ plants carrying the null KTI allele. RPGC recovery increased across generations, with BC₁F₄ plants achieving 84.15 % in Cross A (AMS MB 5-18 X NRC 101) and 83.72 % in Cross B (AMS MB 5-18 X NRC 127). Native PAGE confirmed the absence of KTI peptide in 8 BC₁F₄ plants. Expression profiling of KTI3 gene via qRT-PCR revealed that expression was significantly higher during the R5 stage compared to R6, with fold changes ranging from 1.39 to 267.64 in R5 and 8.19 to 130.77 in R6. Null parent lines exhibited minimal KTI3 expression, consistent with their genetic background. These findings confirmed the successful introgression of the null KTI allele and its desired effect on trait in advanced backcross populations.