Novel protein domain-specific primers strategy for qPCR/ endpoint PCR to identify resistance gene analogous

Abstract

Abstract Background: In the quest to identify new resistance genes analogous to those found in other plant species, a novel primer designing strategy is introduced for the first time. Unlike traditional methods that rely on prior information about degeneracy positions, this new approach involves designing primers based on specific domain positions within the candidate resistance gene and eliminates the need for prior knowledge of degeneracy. By using this new approach, it becomes possible to uncover resistance genes and understand their functional interactions with pathogens. Additionally, this approach sheds light on the redundancy and diversity of resistance genes. Notably, this primer designing strategy exhibits remarkable sensitivity, allowing the detection of elusive low-abundance target sequences that were previously challenging to identify using degeneracy-based designs. Results: The qPCR primers, designed using the novel approach of protein domain-specific regions, underwent standardization and validation in endpoint PCR. Subsequent melt curve analysis in qPCR revealed that out of the ten primers tested, six NB-ARC family protein domain-specific qPCR primers (NB-ARC_2, NB-ARC_3, NB-ARC_4, NB-ARC_8, NB-ARC_12, and NB-ARC_17) exhibited a single peak melt curve, indicating precise amplification of the conserved NB-ARC domain of the R-protein. This confirms their specificity and reliability for target detection, enabling the identification of new resistance gene analogues. Conclusion: Our innovative protein domain-specific qPCR primer design approach allows for precise and accurate PCR amplification, overcoming the limitations of traditional degenerate primers. It enables targeted amplification of specific domain regions within resistance proteins, uncovering both conserved domains and novel resistance genes or gene analogs. The use of these primers also captures the redundancy of resistance genes, offering improved accuracy and reliability in target gene identification. This breakthrough represents a significant advancement in molecular biology research and opens new possibilities for identifying resistance gene analogs. To the best of our knowledge, this is the first report of identifying resistance gene analogs using “protein domain-specific” region based qPCR primer design approach.