ABSTRACT: Background:Baitouweng is a traditional Chinese medicine with a long history of different applications. Although referred to as a single medicine, Baitouweng is actually comprised of many closely related species. It is therefore critically important to identify the different species that are utilized in these medicinal applications. Knowledge about their phylogenetic relationships can be derived from their chloroplast genomes and may provide additional insights into development of molecular markers. Methods:Genomic DNA was extracted from six species of Pulsatilla and then sequenced on an Illumina HiSeq 4000. Sequences were assembled into contigs by SOAPdenovo 2.04, aligned to the reference genome using BLAST, and then manually corrected. Genome annotation was performed by the online DOGMA tool. General characteristics of the cp genomes of the six species were analyzed and compared with closely related species. Additionally, phylogenetic trees were constructed, based on single nucleotide polymorphisms (SNPs) and 51 shared protein-coding gene sequences in the cp genome among all 31 species via maximum likelihood. Results:The size of cp genomes of P. chinensis (Bge.) Regel, P. chinensis (Bge.) Regel var. kissii (Mandl) S. H. Li et Y. H. Huang, P. cernua (Thunb.) Bercht. et Opiz f. plumbea J. X. Ji et Y. T. zhao, P. dahurica (Fisch.) Spreng, P. turczaninovii Kryl. et Serg, and P. cernua (Thunb.) Bercht. et Opiz. were 163,851 bp, 163,756 bp, 162,481 bp, 162,450 bp, 162,795 bp, and 162,924 bp, respectively. Each species included two inverted repeat regions, a small single-copy region, and a large single-copy region. A total of 134 genes were annotated, including 90 protein-coding genes, 36 tRNAs, and eight rRNAs across all species. In simple sequence repeat analysis, only P. dahurica was found to contain hexanucleotide repeats. A total of 26, 39, 32, 37, 32 and 43 large repeat sequences were identified in the genic regions of the six Pulsatilla species. Nucleotide diversity analysis revealed that the rpl36 gene and ccsA-ndhD region have the highest Pi value. In addition, two phylogenetic trees of the cp genomes were constructed, which laced all Pulsatilla species into one branch within Ranunculaceae. Conclusions:We identified and analyzed the cp genome features of six species of P. Miller, with implications for species identification and phylogenetic analysis.
