Digital PCR enables direct root biomass quantification and species profiling in soil samples.

Roots support plant growth and resilience and are a major route for carbon sequestration. Thus, the study of roots in agricultural and natural systems is essential to develop strategies to mitigate and adjust to climate change. Methods to quantify root biomass in mono- and mixed crop systems are therefore in high demand. A promising approach is to exploit the correlation between root biomass and nuclear DNA. The use of qPCR for the quantitative analysis of root samples has been reported. Here, we show how digital PCR can be used to quantify root DNA from soil samples harboring single species or species mixtures. This molecular method has several advantages over more time-consuming methods, including enhanced sensitivity and absolute quantification of target DNA, increased accuracy and reliability, and the ability to quantify roots directly from soil in different species mixtures. We developed a DNA-based digital droplet PCR (ddPCR) method for root species profiling and biomass quantification directly from soil samples under semi-field conditions. Our findings suggest that implementing this ddPCR method can substantially simplify and improve root quantification of specific species, even in crop mixtures. This method offers a more time- and labor-efficient alternative to traditional techniques (e.g. root separation or C13 labeling). The complement of primer-probe sets presented here can be continuously expanded to include additional plant species, thus broadening the scope of this DNA-based ddPCR method.