Comparative Analysis of Chloroplast DNA Sequences of Codonopsis Lanceolata and Platycodon Grandiflorus and Application in Development of Molecular Markers
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Appl Biol Chem (2017) 60(1):23–31 Online ISSN 2468-0842 DOI 10.1007/s13765-016-0248-6 Print ISSN 2468-0834 ARTICLE Comparative analysis of chloroplast DNA sequences of Codonopsis lanceolata and Platycodon grandiflorus and application in development of molecular markers Sun-Goo Hwang1 . Ju Hee Kim1 . Jun-Cheol Moon2 . Jin-Hyuk Kim1 . Cheol Seong Jang1 Received: 11 October 2016 / Accepted: 2 December 2016 / Published online: 23 December 2016 Ó The Korean Society for Applied Biological Chemistry 2016 Abstract Codonopsis lanceolata and Platycodon grandi- Introduction florus (order Asterales) originate from East Asia. Despite the high commercial availability of C. lanceolata and P. Asterales is an order of dicotyledonous flowering plant grandiflorus, limited genetic research has been performed species and includes 11 families evolved from one com- on these plants. We applied a targeting enrichment method mon ancestor. Many herbaceous plant species of Asterales to detect genetic diversity in C. lanceolata and P. grandi- are used as spices and traditional medicines. In previous florus and recovered their chloroplast genomes from total phylogenetic research, Asterales have been included in the DNA sequence data. Chloroplast DNAs (cpDNAs) were Campanulid clade, which is similar to the Euasterids II 61,154 bp (C. lanceolata) and 81,214 bp (P. grandiflorus) group of the Angiosperm Phylogeny Group III as one type in length. Sixteen simple sequence repeats and 15 long of taxonomic classification (Bremer et al. 2002). The repeat sequences were determined, which are useful as molecular evidence suggests that Asterales originated c. potential markers in both plant species. We surveyed the 100 MaB.P. in the Cretaceous (Bremer and Gustafsson phylogenetic relationships with increased resolution in 14 1997). plant species, including other 8 species from the order In Korea, Codonopsis lanceolata, order Asterales, is Asterales and 4 from the order Apiales. In addition, we known as Deodeok. Its roots are used in traditional food demonstrated the availability of recovered chloroplast dishes. Platycodon grandiflorus, order Asterales, is a genomes through cpDNA marker development to deter- herbaceous flowering perennial plant, known as Doraji in mine the authenticity of food fraud at the DNA level of Korea, and its roots are a recognized ingredient in salads plant species. and traditional cuisine. Both plants originate in East Asia and are used as natural and traditional medicine due to their Keywords Chloroplast genome Á Codonopsis lanceolata Á own functional compounds. In addition, Panax ginseng, Marker Á Platycodon grandiflorus order Asterales, has been utilized in China as a traditional medicine for the treatment of ailments related to the central nervous system, and endocrine and adrenocortical systems, and to control blood pressure and diabetes, (Nah et al. 2007;Kim2012). The complete chloroplast genome of Korean ginseng (Panax schinseng Nees) is 156,318 bp and North American ginseng (Panax quinquefolius)is & Cheol Seong Jang 156,359 bp (Kim and Lee 2004; Han et al. 2015). Also, [email protected] Zhao et al. (2015) reported the comparative analysis of 1 P. ginseng Plant Genomics Lab, Department of Applied Plant Sciences, chloroplast DNAs (cpDNAs) for four strains Kangwon National University, Chuncheon 24341, Republic including Damaya, Ermaya, Gaolishen, and Yeshanshen, in of Korea which the complete chloroplast genome size (156,354 bp) 2 Agriculture and Life Sciences Research Institute, Kangwon of P. ginseng Damaya was equal to the total length of each National University, Chuncheon 24341, Republic of Korea 123 24 Appl Biol Chem (2017) 60(1):23–31 cpDNA of P. ginseng Ermaya and Gaolishen, unlike that of relationships and structural diversity of cpDNA among P. ginseng Yeshanshen. The different minor allele sites in various plant species (Zhao et al. 2015). the large single copy and inverted repeat regions of the Here, we performed the DNA-seq from C. lanceolata chloroplast genome were also identified, suggesting an and P. grandiflorus and applied a target enrichment method inferred evolutionary relationship among these four P. for convenient cpDNA recovery from total DNA sequence ginseng strains. However, high-resolution studies of evo- data. Phylogenic analysis was performed using 12 chloro- lutionary relationships using numerous sequences have not plast genome sequences of Asterales, including C. lance- been reported for C. lanceolata and P. grandiflorus olata and P. grandiflorus, and 4 sequences of Apiales with chloroplast genomes. P. ginseng. To develop molecular markers for discrimi- Recently, the consumption of functional foods for nating three species, we selected two gene families, in improving health and wellness has increased. However, which their segregating sites were 191 for rpo and 20 for commercial fraud related to food products has become a ndh, and then designed gene-specific primers for quanti- concern due to the differences in the cost of food materials, tative real-time polymerase chain reaction (qRT-PCR). e.g., the price of ginseng is generally higher than that of Finally, the specific primers were tested in total genomic Deodeok or Doraji; therefore, the latter cheaper options are DNA isolated from three plant species, C. lanceolata, P. being used in products and being marketed as original grandiflorus, and P. ginseng. ginseng. Therefore, consumer concerns related to unorigi- nal ginseng food products, which are mixed with rather cheap foods such as Deoduck and Doraji, have increased. Materials and methods However, it is difficult to discriminate the ingredients of ginseng products, especially ginseng powders, visually. DNA sequencing of C. lanceolata and P. grandiflorus Molecular markers can be used to determine the authen- ticity of food at the DNA level of plant species. For Seeds of C. lanceolata and P. grandiflorus were obtained example, molecular markers derived from rpoB and rpoC2 from the Gyeonggi-do Agricultural Research and Exten- successfully detected the cpDNA of rice grain flour in sion Services, and seedlings were grown in a growth different mixed-flour samples using quantitative real-time chamber at 25 °C for 4 weeks. Total DNA was isolated PCR (qRT-PCR) (Hwang et al. 2015). Furthermore, Moon from the leaves of 2-week-old seedlings of C. lanceolata et al. (2016) determined unique species among five plants, and P. grandiflorus using the CTAB method (Saghai-Ma- including rice, barley, adlay, wheat, and maize, using a roof et al. 1984), and the extracted DNA was used to cpDNA marker developed from rpoC2, which was used for conduct the DNA-seq by an Illumina HiSeq2000 following detecting the cpDNA for each plant species in seven the manufacturer’s instructions. commercial mixed-flour products. Molecular markers have been developed on the basis of sequence differences; Recovery of the chloroplast genome from the total however, the genetic information of C. lanceolata and P. DNA sequence data grandiflorus mostly remains unknown, even though the chloroplast genome sequence of P. ginseng has been To recover chloroplast genome sequences from DNA-seq completed (Zhao et al. 2015). data, we used the modified target enrichment method Photosynthesis, which occurs in chloroplasts, is an (Mandel et al. 2014). In summary, for quality control of important process for energy production of green plants. raw data, the FASTQ files data obtained from DNA-seq The chloroplasts contain a genome ranging in size from were filtered using the PRINSEQ tool with a minimum 120 to 170 kb (Clegg et al. 1994). The entire nucleotide read length of 40, minimum quality score of 15, minimum sequence of cpDNA has been widely used for phylogenetic length of 30-end poly-N tail of 10, and ambiguous base N studies and molecular marker development due to its percentage of 20 (http://prinseq.sourceforge.net/index. highly conserved genetic structure. Bremer et al. (2002) html). The filtered reads were annotated based on the reported that the three coding and three non-coding cpDNA complete chloroplast genome of ten plant species from the markers provided improved evidence for resolution among order Asterales obtained from NCBI (http://www.ncbi.nlm. the Asterids order at higher taxonomic levels. Recently, the nih.gov/) using BLAST search, and then the annotated rapid development of the next generation sequencing reads identified as potential cpDNA sequences of C. technique provided a comprehensive methodology for lanceolata and P. grandiflorus were collected using PERL monitoring genetic diversity in plant species. For example, script. The collected cpDNA reads were used for de novo the complete chloroplast genome sequences of P. ginseng assembly using VelvetOptimiser (http://www.vicbioinfor obtained from DNA sequencing (DNA-seq) provided matics.com/software.velvetoptimiser.shtml). The assem- genome-wide genetic information, such as the phylogenetic bled contigs were rearranged to produce one chromosome 123 Appl Biol Chem (2017) 60(1):23–31 25 genome sequence using the ABACAS tool (Assefa et al. (Fig. 2). Total DNA was extracted from 2-week-old seedling 2009). The Campanula takesimana chloroplast genome as leaves of C. lanceolata, P. grandiflorus, and P. ginseng using a reference sequence was used for contig assembly due to the CTAB method, respectively (Saghai-Maroof et al. 1984), the higher genetic similarity of cpDNA with C. lanceolata and then used for qRT-PCR. qRT-PCR was performed using and P. grandiflorus in Asterales. The gene information of a Real-Time System with 20 lL reaction