Recent rapid changes in climate have resulted in rising temperature and salinity stresses, causing challenge for stable ginseng production (Kim et al. 2008; Jin et al. 2009). Particularly, salinity stress has decreased the annual ginseng yield up to 20% (Kim et al. 2015). The optimal soil salinity concentration for ginseng production is as low as 0.5 ds/m, and thus, it is a salinity-sensitive crop (Kim et al. 2014). Its growth is inhibited at high salinity concentrations and the plant withers in severe cases (Cho and Kim 2004). Thus, National Institute of Horticultural and Herbal Science (NIHHS) prepared an in vitro test system to develop a salinity-resistant ginseng cultivar and has selected salinity-resistant lines from the collected genetic resources. ‘G07006’ was selected for salt tolerance based on the changes of test markers, such as physiological responses and chlorophyll fluorescence, following immersion of petioles in 5 ds/m mixed salinity solution (21 mM KNO₃, 6.1 mM KH₂PO₄, 6.2 mM MgSO₄·7H₂O) at the NIHHS in 2014–2016 (Jo et al. 2015).

In order to develop this line into a new variety, it must be genetically stabilized as a pure line through repeated selections and segregations. To maintain the pure line during development, the genetic homogeneity of seeds must be tested, which requires a system that can distinguish the new variety from existing ones (Bang et al. 2015).

DNA barcoding is a standard method used to distinguish species based on single nucleotide polymorphisms (SNPs) in the same gene loci in the genomes of different organisms (Jo et al. 2014). No standardized gene locus is currently available to distinguish plant species; therefore, coding regions of chloroplast DNA, including matK, rbcL, rpoB, and rpoC1, and non-coding regions, including atpF-atpH, psbK-psbI, and psbA-trnH spacers, have been suggested (Kress et al. 2005). Chloroplast is a plant organelle containing its own circular DNA molecules that vary in size from 120 kb to 160 kb depending on the plant species. Chloroplast DNA (CpDNA) is largely composed of LSC, SSC, and they encode 4 rRNAs, 30 tRNAs, and 80 proteins on average, which are involved in photosynthesis or the expression of chromosomal genes (Palmer 1985; Jansen et al. 2005; Wang et al. 2008).

CpDNA, compared to genomic DNA, shows greater evolutionary conservation, facilitating phylogenetic analysis and species identification based on SNPs. In addition, CpDNA is inherited maternally, enabling evolutionary taxonomy analysis of plants, and is shorter than genomic DNA, thereby decreasing the cost of DNA sequencing and analysis by NGS (Nock et al. 2011; Straub et al. 2012; McPherson et al. 2013). The first full CpDNA sequence was reported in tobacco leaves in 1986 (Yamada et al. 1986). Following this report, numerous studies have been conducted on chloroplasts, and the full CpDNA sequences of approximately 300 species have been deposited in the NCBI database to date. Recently, Yang et al. (2015) analyzed the CpDNA sequence of nine varieties of ginseng and developed SNP- and indel-based DNA markers that can differentiate between the Chunpoong and Sunhyang varieties. Thus, this study was conducted to develop DNA markers capable of determining distinctness and homogeneity among the salt-resistant varieties of ginseng. For this, the full CpDNA sequence of ‘G07006’, a salt-resistant line, was analyzed using NGS and compared with the recently released CpDNA sequences of nine ginseng varieties (Chunpoong, Yunpoong, Gopoong, Gumpoong, Sunpoong, Sunun, Sunone, Cheongsun and Sunhyang).

Plant material

The P. ginseng breeding line ‘G07006’, was selected for salt tolerance through salinity screening of mature leaves at the NIHHS of the Rural Development Administration (RDA) in 2014-2016. Fourth years of its growing season, in July, the leaves were harvested, powdered using liquid nitrogen and stored in a deep freezer (-80°C) until further processing.

DNA extraction, Cp genome assembly and annotation

Total genomic DNA was extracted from fresh leaves of 4 year old plants of P. ginseng breeding line (G07006) by using Dneasy Plant Mini Kit (Qiagen, Hilden, Germany). The quantity and quality of DNA samples were measured using a NanoDrop ND-1000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA). An Illumina paired-end (PE) library with 300-bp insert size was constructed using genomic DNA and sequenced using an Illumina HiSeq 2000 instrument (Illumina, San Diego, CA) according to the instructions of the manufacturer. High quality PE reads of 3.95 Gb were assembled using CLC genome assembler (v. beta 4.6, CLC Inc., Aarhus, Denmark), after trimming of reads (Phred scores of 20 or less). Afterward, representing chloroplast genome sequences were joined by comparison with reference sequences (P. ginseng cv. Yunpoong, NC_006290) (Kim and Lee 2004) and validated by PE read mapping. The chloroplast genome was annotated using DOGMA (http://dogma.ccbb.utexas.edu/) and manual curation based on BLAST searches.

In this study, to identify SNP mutations in the CP genome of the decoded G07006 system based on the NCBI DB, CP genome sequences for nine cultivars of P. ginseng were down loaded for comparative analysis (KM067386, KM067387, KM067388, KM067390, KM067391, KM067392, KM067393, KM088019, KM088020). Base sequences were edited using the BioEdit program, saved as no gap, and arranged as Cluster W (ver. 1.83) to identify the location and type of common SNP.

Characterization of the chloroplast genome

NGS was used to obtain 3.95 Gb data from the decoded DNA base nucleotides of the G07006 system. Results showed that 492,591 contigs were within the cpDNA region and the coverage was identified to be 939× (Table 1). The total size of the completely mapped CP genome of the G07006 was 156,356 bp. This CP genome encodes 114 unigenes (80 protein-coding genes, four rRNA genes, and 30 tRNA genes), in which 18 are duplicated in the IR regions (Fig. 1). Comparative analyses of nine cultivars of P. ginseng with the CP genome showed that CP genome size of the G07006 was identical to that of the Gumpoong cultivar.

Table 1 . Data regarding cpDNA of ‘G07006’ generated by the Illumina HiSeq sequencer.

Species	Organism	Raw data	Aligned reads to	Cp genome	Cp genome length
	source	bases (bp)	the cp genome (#)	coverage(x)	(bp)
P. ginseng	GO7006	3,955,213,989	492,591	939	156,356

Figure 1.

Circular gene map of the breeding line of Panax ginseng (G07006) chloroplast genome. Genes shown inside the circle are transcribed clockwise, and those outside the circle are transcribed counterclockwise

SNP detection of a breeding line in Korean ginseng

Comparative analyses of previously identified CP genome sequences of nine cultivars of P. ginseng and that of the G07006 showed five SNPs (Fig. 2). Sunhyang cultivar had a thymine (T) nucleotide at 7,159 bp in the rps16-trnUUG region, and other accessions had guanine (G) in that region. Further, Sunhyang had a T nucleotide in the ndhF-rpl32 region, in contrast with that of other accessions, which had G. Therefore, base substitutions in rps16-trnUUG and ndhF-rpl32 could be used as cultivar-specific markers to identify the Sunhyang. In addition, the rpoC2 gene samples showed SNPs (located in the total CP genome of 21,344 bp): Gumpoong, Chungsun, and G07006 had T and other accessions had cytosine (C). The Chunpoong CP genome at 22,287 bp nucleotides (rpoC1 gene) had T and the other accessions had G. Chunpoong had a unique root shape, and thus, was an appropriate strain for red ginseng production. Therefore, the specific SNP to Chunpoong at the rpoC1 region could be used as a molecular marker to select suitable cultivar for red ginseng process production and the management of product quality. Lastly, at the ccsA gene region, SNP types in accessions were divided into two groups. Gumpoong and Chungsun had G, and the others had adenine (A). Since three of five SNPs were cultivar-specific to Chunpoong and Sunhyang, they could be easily distinguished from other accessions. However, when arranging SNPs according to their gene locations, the G07006 genotype was ‘GTGGA’, which was distinct from other accessions (Table 2). G07006 is a salt-resistant line selected by a simplified test for salt resistance from 2014 to 2016. For such a cultivar to be cultivated, it is important to maintain a genetically uniform pure line. Therefore, the five selected SNPs from the completed CP genome sequence of the G07006 will provide valuable information to distinguish salt-resistant breeding line from other cultivars and maintain and preserve the pure line.

Figure 2.

Sequence alignment representing DNA variation detected in rps16-trnUUG, rpoC2, rpoC1, ndhF-rpl32 and ccsA gene regions of cpDNA of ten Korean ginseng accessions. The boxes indicate SNP positions among Korean ginseng accessions

Table 2 . SNP combinations among Korean ginseng accessions by comparison of the cp genome.

Type	Locus	SNP Position (bp)	P. ginseng cultivarsa & breeding lineb
SNP			CP	YP	GO	GP	SP	SU	SO	SH	CS	GO7006
	rps16-trnUUG	7159	G	G	G	G	G	G	G	T	G	G
	rpoC2	21344	C	C	C	T	C	C	C	C	T	T
	rpoC1	22287	T	G	G	G	G	G	G	G	G	G
	ndhF-rpl32	115594	G	G	G	G	G	G	G	T	G	G
	ccsA	117376	A	A	A	G	A	A	A	A	G	A
SNP combination			GCTGA	GCGGA	GCGGA	GTGGG	GCGGA	GCGGA	GCGGA	TCGTA	GTGGG	GTGGA

Underlined italic letters indicate a cultivar-specific SNP and allele combination.

^aCP, ‘Chunpoong’; YP, ‘Yunpoong’; GO, ‘Gopoong’; GP, ‘Gumpoong’; SP, ‘Sunpoong’; SU, ‘Sunun’; SO, ‘Sunone’; SH, ‘Sunhyang’ CS, ‘Cheongseon’

^bG07006; salt-resistant breeding line

This work was carried out with the support of the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ010104012016),” Rural Development Administration, Republic of Korea.