Abstract
The internal transcribed spacer (ITS) is one of the most extensively utilized in the taxonomy of the genus Morus due to its generally concerted evolution. Although non-concerted evolution of nuclear ribosomal DNA (nrDNA) has been reported in some species, genome-wide nrDNA characteristics in the genus Morus remain poorly understood. In this study, 158 single-nucleotide polymorphisms (SNPs) and 15 insertions and deletions (InDels) were identified within the nrDNA regions of 542 mulberry accessions representing sixteen Morus species. These wide occurrences of heterogeneous SNPs and InDels revealed the intra-individual polymorphism within the nrDNA region of Morus, indicating the incomplete concerted evolution of nrDNA. Notably, 66 out of 158 SNPs and 13 out of 15 InDels were localized within the ITS regions (ITS1-5.8S-ITS2), indicating a high degree of polymorphism in the ITS, which was further validated through classical cloning and Sanger sequencing methodologies. The 13/16 bp InDel located in the ITS1 region was utilized to develop a rapid and reliable cleaved amplified polymorphic sequence (CAPS) marker-based method for distinguishing M. alba and M. notabilis from other Morus species, eliminating the need for a clone-based sequencing step or comparative phenotypic analysis. Phylogenetic analysis based on nrDNA SNPs from 542 mulberry accessions revealed six distinct clades, corresponding to the six Morus species. These findings offer novel new insights into the taxonomy, conservation, and breeding improvement of Morus species.