Recently, genetic investigations and the development of markers for subspecies of plants that cannot be classified visually or phenotypically have been active. Even for similar subspecies, the differences in physiological activity and components are clear, so these studies are receiving increasing attention [
26,
27]. In the field of marker development, a comprehensive investigation of new or combined species-specific variable markers in the chloroplast region of plants has begun. However, genetic information of plants is essential for differentiation and research is almost impossible if there is no reference genome. In this study, we aimed to genetically differentiate
S. indica L. and
S. indica var.
tsusimensis (H. Hara) Ohwi, which are difficult to classify by phenotype. In some cases, most of the specimens identified as
S. indica L. were actually
S. indica var.
tsusimensis (H. Hara) Ohwi [
2]. In the NCBI GenBank database, about 17 species in the genus
Scutellaria L. have reported the complete chloroplast genomes. However, among the five species of
Scutellaria L. selected for this study, only
S. baicalensis Georgi has been reported as a complete chloroplast genome. A specific primer developed using the reference genome was tested but classification failed because it did not show specific amplification. For this reason, we performed whole-genome de novo assembly to obtain the genome of the subspecies
S. indica var.
tsusimensis (H. Hara) Ohwi. Most of the genes did not have hits and 279 genes matched the genus
Scutellaria. Primers were prepared by screening these 279 genes and nine annotated chloroplast barcoded DNA loci. Since the primers prepared with each sequence registered in GenBank did not show specific amplification, we aimed to design primers that were specific to each species by using the gene matched with the genus
Scutellaria via BLAST. As a result, four specifically amplifying primers were designed and three species could be classified. The SP primer was specific to
S. pekinensis var.
transitra (Makino) H. Hara (
S. pekinensis var.
transitra (Makino) H. Hara voucher KUS:2006-1082 trnH-PsbA intergenic spacer, partial sequence; and PsbA (psbA) gene, partial CDS). It was prepared using the base sequence of the chloroplast (KX060016.1). Primers (ST1 and ST2 primers) specific to
S. indica var.
tsusimensis (H. Hara) Ohwi were prepared using the annotated chloroplast barcode DNA AtpI_0 and rbCL_3 nucleotide sequences. The sequences of these two barcode DNAs were registered in the NCBI GenBank database and given accession numbers MZ714561 and MZ714562, respectively. For
S. baicalensis Georgi, the complete chloroplast genome sequence (
S. baicalensis Georgi isolate BOP028266 chloroplast, complete genome (MF521633.1)) was deposited in GenBank, so it was possible to obtain the specific SB primer. The SL primer was produced using the nucleotide sequence of the
S. indica L. TCP transcription factor (CYC2B) gene, partial CDS (KM526800.1), but amplified DNA in two species,
S. indica L. and
S. indica var.
tsusimensis (H. Hara) Ohwi. However, when this primer was used with the ST1 or ST2 primer,
S. indica L. could be discerned. The SD1 primer amplifies DNA in the
Scutellaria insignis chloroplast, the complete genome (KT750009.1) was produced using the sequence and classification was possible because the primer amplified DNA only in
S. barbata D. Don. However, it was concluded that this result alone was insufficient evidence, and a differentiation method was developed using both the SD2 and SD1 primers, which amplified DNA in all five species. The SD2 primer is specific to
Scutellaria indica var.
tsusimensis voucher SWUS (Kim 2008-008 psbK-psbI intergenic spacer, partial sequence). It was prepared using the chloroplast (KX059898.1) base sequence. This study focused on classifying five species of the genus
Scutellaria L. native to Korea as a precedent study for our future research. Prior to this study, five species whose usefulness was confirmed in the screening process for new drug development were selected. For in-depth studies, it was necessary to investigate and standardize the active compounds for each of the five species. Except for
S. barbata D. Don and
S. baicalensis Georgi, the morphologies between the three species were very similar. Due to the similar shape of the three species, it was difficult to conduct research because it was difficult to supply and sample accurate raw materials. Therefore, research on classification and identification by species was urgently needed. Species-specific primer development was attempted by comparing each gene, but the three species lacked reference genomes and did not overlap, so SNP or barcode genes could not be targeted and failed to generate classification markers using the reported reference genome. Species-specific primers were prepared using the annotated cpDNA portion of the WGS results,
S. indica L. and
S. indica var.
tsusimensis (H. Hara) Ohwi could be classified, and a classification method for five species was made with the additionally prepared primers. However, genetic studies on
S. indica var.
tsusimensis (H. Hara) Ohwi,
S. indica L. and
S. pekinensis var.
transtra (Makino) H. Hara are still lacking, and further studies are needed. In addition, it cannot be regarded as a perfect genetic classification because species-specific primers focusing on the SNP part were not prepared by comparing cpDNA or barcode genes with each of the five overlapping species. Nevertheless, it is very meaningful to be able to classify five species of
Scutellaria L. genus and we will make up for the shortcomings through future genetic research.