Accelerating moss identification through the development of specific DNA barcodes based on the whole chloroplast genome

Mosses represent the most species-diverse clade of bryophytes and are among the earliest land plants. These diminutive organisms possess considerable ecological significance and diverse applications. However, their study, development, and utilization are impeded by the complex identification process and scarcity of researchers specializing in moss taxonomy. The advancement of DNA barcoding technology presents an opportunity for precise moss identification. While several chloroplast DNA barcodes have been proposed for mosses, these molecular markers primarily originate from angiosperm research and may not be optimal for moss species. This study aims to identify suitable DNA barcodes for mosses at the chloroplast genome level. Utilizing 61 complete chloroplast genome datasets of mosses, this research presented the first construction of a reliable phylogenetic tree at the family level of mosses using whole chloroplast genomes, enabling accurate identification of most samples. Based on nucleotide polymorphism in the complete chloroplast genome, 12 highly variable regions were selected as candidate DNA barcodes for mosses. Experimental validation of newly designed primer universality demonstrated high universality (>90%) for the primers developed in this study. The resolution verification experiment, employing DNA barcodes from 103 samples representing 21 families and 48 genera, confirmed the efficacy of atpB-rbcL, psaI-accD, ycf2, ycf1, matK, rpoB-trnC, and clpP as reliable DNA barcodes for mosses. The study also revealed inconsistencies in the chloroplast genome structures of mosses submitted to public databases, which hinder subsequent research. Consequently, we recommend that researchers upload data with a designated reference genome (such as Bryum argenteum) in future submissions.