The accurate identification of walnut (Juglans regia L.) varieties is crucial for nurseries to detect any mistakes that might occur during vegetative propagation, enabling them to certify their plant material. It is also essential for farmers to resolve any uncertainties about the plant material used in orchard plantations. The availability of a practical, accurate, and reliable tool for walnut genotyping will be of obvious interest. Traditional genotyping methods, such as microsatellite (Simple Sequence Repeats – SSR) analysis, are often too time-consuming and expensive for routine use. This study introduces High-Resolution Melting (HRM) analysis as an innovative, cost-effective, and rapid technique for effective walnut variety genotyping.
HRM analysis utilizes real-time PCR to amplify variable genomic regions, followed by melting curve analysis to distinguish between different varieties. This method streamlines the genotyping process, making it both accessible and practical for routine applications. PCR conditions were optimized to improve the accuracy and efficiency of HRM analysis, and its effectiveness was demonstrated by comparing the results with those obtained through SSR analysis.
Leaves collected from four J. regia varieties (Tulare, Lara, Howard, and Chandler), grown in different orchards in the Alentejo region of Portugal, were used as plant material to establish the methodology. Polymorphic regions corresponding to thirteen SSR loci were examined, and three proved to be effective in differentiating the varieties (WGA202, WGA321, and WGA376).
Validation via conventional microsatellite analysis substantiated the reliability of the HRM-based method. The implementation of HRM analysis can mitigate the risk of varietal mixing, thereby ensuring superior quality control and compliance with market standards. Furthermore, future studies could extend this technique to the identification of walnut varieties at the fruit level, facilitating the correct identification of market-bound fruits and ensuring higher quality for consumers.
