Abstract
<i>Lycium barbarum</i> L. (<i>L. barbarum</i>), an important medicinal and food crop from the Solanaceae family, is known for excellent tolerance to saline–alkaline and drought stresses. Therefore, it has significant research and application value. The nuclear and chloroplast genomes of <i>L. barbarum</i> have been previously reported; however, its mitochondrial genome is still unexplored. In this study, mitochondrial DNA sequencing and assembly in <i>L. barbarum</i> were studied via second- and third-generation sequencing technologies using the anthers collected from the main cultivars “Ningqi No.1” (fertile line) and its male-sterile natural mutant “Ningqi No.5” from Ningxia, China. The results revealed that “Ningqi No.1” and “Ningqi No.5” have the same mitochondrial circular genome structure with a total length of 413,881 bp. In total, 61 genes were annotated, including 35 protein-coding genes, four rRNA genes, 22 tRNA genes, and 2,242 open reading frames with unknown functions. The repeat sequences in mtDNA were analyzed, in which short repeats (30–59 bp) exhibited the highest number. Overall, 23 protein-coding genes in “Ningqi No.1” and “Ningqi No.5” exhibited RNA-editing phenomenon, with different RNA-editing site number and distribution position. Phylogenetic analysis demonstrated that <i>L. barbarum</i> has the closest relationship with <i>Hyoscyamus niger</i>. These results provided a basis for a comprehensive understanding of the mitochondrial genome of <i>L. barbarum</i> and the relevant data for omics studies on <i>L. barbarum</i>.