Extensive nrDNA Polymorphism in Morus L. and Its Application

The internal transcribed spacer (ITS) is one of the most extensively utilized in the taxonomy of the genus <i>Morus</i> 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 <i>Morus</i> 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 <i>Morus</i> species. These wide occurrences of heterogeneous SNPs and InDels revealed the intra-individual polymorphism within the nrDNA region of <i>Morus</i>, 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 <i>M</i>. <i>alba</i> and <i>M. notabilis</i> from other <i>Morus</i> 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 <i>Morus</i> species. These findings offer novel new insights into the taxonomy, conservation, and breeding improvement of <i>Morus</i> species.