Design and application of species-specific primers to Quercus cerris roots' identification in urban forests

Accurate species identification, the first crucial step for effective root studies, is a time-demanding, experience-based and error-prone process. Molecular methods are therefore needed to ensure this process, especially in urban settings where root sampling is challenging. Here, we developed a novel molecular method for root identification in complex environments. Specifically, we focused on detecting Quercus cerris-a species common in European cities and non-urban areas and used in afforestation-from bulk root samples, including those collected non-invasively. To achieve this, we conducted the first comprehensive analysis of candidate DNA regions to discriminate among Quercus species. Among the candidate sequences tested, ITS and ITS2 showed the highest discriminatory power compared to commonly used barcodes such as matK, psbA-trnH, rbcL, rpoC1, trnL-trnF. Based on this results, we designed specific primers to target ITS and ITS2 and we developed a PCR-based protocol capable of reliability and specificity detecting Q. cerris within mixed Quercus root samples. This method was then successfully applied to root bulk samples collected via excavation and non-invasive soil coring in the urban area of Campobasso (central Italy), with results validated through traditional identification techniques. The outcome is a novel, rapid, low-cost, and non-invasive molecular approach for monitoring Q. cerris roots. More broadly, this tool enable in situ root identification and mapping which support the study of root functioning and dynamics in ecosystems and is particularly valuable in challenging urban environments.