Approach for qPCR primer designing and optimization for KTI3 gene expression profiling in elite backcross population of soybean

Abstract

Soybean (Glycine max L.) is a globally important legume crop valued for its high protein content and wide applications in food and feed industries. However, its nutritional potential is limited by the presence of anti-nutritional factors, particularly protease inhibitors that reduce protein digestibility. The Kunitz Trypsin Inhibitor (KTI3) gene plays a crucial role in determining the anti-nutritional profile of soybean, influencing both seed quality and end-use suitability. Accurate quantification of KTI3 transcript levels is essential for functional studies and breeding programs aimed at developing low-KTI cultivars. However, the reliability of quantitative real-time PCR (qPCR) largely depends on primer specificity, efficiency, and optimization. In this study, we designed and optimized KTI3-specific primer to achieve high amplification efficiency, single distinct melt curve peaks, and absence of non-specific products. The primer pair was initially evaluated in silico for GC content, secondary structure formation, and target specificity using NCBI Primer-BLAST. The primer pair was subjected to gradient PCR to determine the optimal annealing temperature, followed by validation through amplification efficiency testing using serial dilutions of cDNA. Gene expression analysis was performed in selected soybean lines at reproductive stages R5 and R6 using Actin as a reference gene and the comparative Ct (ΔΔCt) method. Most lines exhibited higher KTI3 expression at R5, indicating predominant expression during early seed filling. The highest R5 expression was recorded in A41(8)-226 (3.81), A41-16 (3.74), A33-4 (3.70), B37(35)232 (2.88), and B94-87 (2.94), while the null parents NRC-101 and NRC-127 showed negligible expression (0.00–0.01) at both stages. Expression data revealed consistent and significant downregulation of KTI3 at R6 in all lines, with the largest fold change between stages observed in A41-16 (2.97), A33-4 (2.82), B37(35)232 (2.38), and A41(8)-226 (2.01). This study establishes a validated primer optimization workflow for KTI3 qPCR analysis and provides detailed expression profiles, offering a robust tool for soybean quality improvement programs targeting reduced trypsin inhibitor content.