| Summary: | <i>Fortunella venosa</i> (Rutaceae) is an endangered species endemic to China and its taxonomic status has been controversial. The genus <i>Fortunella</i> contains a variety of important economic plants with high value in food, medicine, and ornamental. However, the placement of Genus <i>Fortunella</i> into Genus <i>Citrus</i> has led to controversy on its taxonomy and Systematics. In this present research, the Chloroplast genome of <i>F. venosa</i> was sequenced using the second-generation sequencing, and its structure and phylogenetic relationship analyzed. The results showed that the Chloroplast genome size of <i>F. venosa</i> was 160,265 bp, with a typical angiosperm four-part ring structure containing a large single copy region (LSC) (87,597 bp), a small single copy region (SSC) (18,732 bp), and a pair of inverted repeat regions (IRa\IRb) (26,968 bp each). There are 134 predicted genes in Chloroplast genome, including 89 protein-coding genes, 8 rRNAs, and 37 tRNAs. The GC-content of the whole Chloroplast genome was 43%, with the IR regions having a higher GC content than the LSC and the SSC regions. There were no rearrangements present in the Chloroplast genome; however, the IR regions showed obvious contraction and expansion. A total of 108 simple sequence repeats (SSRs) were present in the entire chloroplast genome and the nucleotide polymorphism was high in LSC and SSC. In addition, there is a preference for codon usage with the non-coding regions being more conserved than the coding regions. Phylogenetic analysis showed that species of <i>Fortunella</i> are nested in the genus of <i>Citrus</i> and the independent species status of <i>F. venosa</i> is supported robustly, which is significantly different from <i>F. japonica</i>. These findings will help in the development of DNA barcodes that can be useful in the study of the systematics and evolution of the genus <i>Fortunella</i> and the family Rutaceae.
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