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Genetic Composition of Korean Ginseng Germplasm by Collection Area and Resource Type

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Genetic Composition of Korean Ginseng Germplasm by Collection Area and Resource Type agronomy Article Genetic Composition of Korean Ginseng Germplasm by Collection Area and Resource Type Kyung Jun Lee , Raveendar Sebastin, Seong-Hoon Kim, Eunae Yoo, Sookyeong Lee, Gyu-Taek Cho, Manjung Kang and Do Yoon Hyun * National Agrobiodiversity Center, National Institute of Agricultural Sciences (NAS), RDA, Jeonju 54874, Jeollabuk-do, Korea; [email protected] (K.J.L.); [email protected] (R.S.); [email protected] (S.-H.K.); [email protected] (E.Y.); [email protected] (S.L.); [email protected] (G.-T.C.); [email protected] (M.K.) * Correspondence: [email protected]; Tel.: +82-63-238-4912 Received: 24 September 2020; Accepted: 22 October 2020; Published: 26 October 2020 Abstract: To improve crops, it is important to secure plant genetic source material and evaluate the genetic diversity. Ginseng (Panax ginseng C.A. Meyer) has long been used as a medicinal herb in Korea and China. Since ginseng originated from wild ginseng with low genetic diversity, it is also expected to have low genetic diversity. In this study, the genetic diversity of 451 ginseng accessions conserved in the National Agrobiodiversity Center (NAC) at Korea was analyzed using 33 SSR markers. Another objective was to establish a strategy for NAC to manage ginseng germplasm based on these results. The 451 accessions were collected from 22 cities in six provinces in South Korea. Among the 451 ginseng accessions, 390 (86.5%) and 61 (13.5%) were landraces and breeding lines, respectively. In the STRUCTURE results for the accessions, there was no relationship between assigned genotypes and collection areas, but there was a population genetic structure. In addition, genetic differentiation within populations of each analysis was low, indicating that the ginseng accessions conserved at NAC are extensively dispersed throughout the collection areas. The results of this study suggest that NAC should increase the genetic diversity of ginseng accessions for breeding programs, and alternatives are needed for securing ginseng genetic resources. Keywords: ginseng; genetic composition; genetic diversity; SSR 1. Introduction Plant breeding in agriculture has decreased the diversity of many crops, which has caused bottlenecks in crop domestication, dispersal, and modernization. Researchers, in particular, have identified significant negative effects of plant breeding on diversity following the modernization bottleneck [1]. The loss of crop variation caused by the modernization of agriculture has been described as genetic erosion [2]. Since loss of genetic variation could decrease the potential of species to persist in the face of abiotic and biotic environmental changes, genetic erosion could pose a severe threat to long-term global food security [3]. Crop improvement largely depends on immediate conservation of genetic resources for their effective and sustainable utilization. For this, it is necessary to conserve and breed the vast genetic variation found in populations of the wild progenitors and landraces of cultivated plants [4,5]. Plant genetic resources are the most important components of agro-biodiversity, which encompasses primitive forms of cultivated plant species and landraces, modern and obsolete cultivars, breeding lines and genetic stocks, weedy types, and related wild species [6]. Since the International Board for Plant Genetic Resources was established in 1974, over 1750 gene banks have been activated worldwide, about 130 of which hold more than 10,000 accessions each [7,8]. In Korea, Agronomy 2020, 10, 1643; doi:10.3390/agronomy10111643 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1643 2 of 13 the National Agrobiodiversity Center (NAC) was established in 2008, and the center now has conserved 258,984 accessions of 3126 species [9]. The Panax genus of the Araliaceae family comprises 17 species, of which P. ginseng, P. notoginseng, and P. quinquefolium are widely cultivated for medicinal value [10,11]. The Korean ginseng, P. ginseng C.A. Meyer, is an important medical plant, which is well-known for its remarkable pharmacological effects [10–14]. Generally, the origination area and time of P. ginseng are known to be in Sangdang, China and the first century B.C. during the Han dynasty, era (Chinese origin theory of ginseng). However, it is fairly obvious that this theory does not answer appropriately to the fundamental questions of the origin of ginseng; (1) why ginseng suddenly appeared in that time, Han China, (2) how explain clearly the formation of ginseng character. Since P. ginseng naturally exists in only three regions: Korea (33.7◦–43.1◦), Manchuria (43◦–47◦), and the Littoral province of Siberia, it is sometimes referred that the originating place is not Shangdang of Shansi area of China, but Manchuria and Korea [15]. Many previous researchers have studied the medical and pharmacological effects and genetic diversity of P. ginseng [16–23]. Based on the growing environment and the method of cultivation, commercial trade ginseng is classified into three grades: cultivated, mountain cultivated, and mountain wild [24]. In general, the cultivated ginseng is known to have originated from wild ginseng, a rare, endangered plant [25]. The genetic consequence of rare or endangered species is increased genetic drift and inbreeding, and these increased phenomena contribute to a reduction in genetic diversity [26,27]. Geographic differences in the distribution of genetic diversity are extremely common. Populations can vary in all aspects of diversity such as number of alleles, identities of alleles, and the effect on the characteristics in the population. The breeding system of the species is very important in determining the differences between populations from different geographic locations [28]. In a previous study, the researchers reported on narrow genetic diversity among 1109 ginseng accessions conserved in the NAC, but there was a lack of sufficient information on the genetic diversity of ginseng accessions such as the collection site [29]. In this study, the genetic diversity of 451 Korean ginseng accessions with information on the collection area was analyzed; in particular, each accession was genetically differentiated and sorted by the collection area. Based on these results, this study includes proposals for how to efficiently manage the ginseng germplasm in NAC. 2. Materials and Methods 2.1. Plant Materials A total of 451 ginseng accessions were obtained from the NAC at the Rural Development Administration in South Korea (Table S1). 2.2. DNA Extraction Genomic DNA was extracted from the leaves of ginseng accessions using Qiagen DNA extraction kit (Qiagen, Hilden, Germany). DNA quality and quantity were measured using 1% (w/v) agarose gel and a spectrophotometry (Epoch, BioTek, Winooski, VT, USA). Extracted DNA was diluted to 30 ng/uL and stored at 20 C until further PCR amplification. − ◦ 2.3. SSR Genotyping For SSR (simple sequence repeat) analysis, a total of 33 SSRs were selected previous studies [30,31] (Table S2). They were fluorescently labelled (6-FAM, HEX, and NED) and used to detect amplification products. PCR reactions were carried out in a 25 uL reaction mixture that contained 30 ng template DNA, 1.5 mM MgCl2, 0.2 mM of each dNTPs, 0.5 um of each primer, 1 U Taq polymerase (Inclone, Yongin, Korea). The amplification was performed under the following cycling conditions: initial denaturation at 94 ◦C for 5 min, followed by 35 cycles of denaturation at 95 ◦C for 30 s, annealing at 55 ◦C for 30 s, extension at 72 ◦C for 1 min, and a final extension step at 72 ◦C for 10 min. Each amplicon was resolved Agronomy 2020, 10, 1643 3 of 13 on an ABI3500 DNA sequence (Thermo Fisher Scientific Inc., Wilmington, DE, USA) and scored using Gene Mapper Software (Version 4.0, Thermo Fisher, Wilmington, DE, USA). 2.4. Genetic Diversity Analysis The number of alleles (Na), the number of genotypes (Ng), Shannon-Wiener index (H), Nei’s genetic diversity (I), Eveness, polymorphic information content (PIC) were calculated using R software [32]. Analysis of molecular variance (AMOVA) within and between gene pools was performed using GenAlEx software v. 6.5 [33]. 2.5. Population Structure Analysis Population structure was analyzed using STRUCTURE v.2.3.4 [34]. In STRUCTURE analysis, Bayesian clustering was performed; three independent runs were tested with K from 1 to 15, each run with a burn-in period of 50,000 iterations and 500,000 Monte Carlo Markov iterations, assuming an admixture model. The output was subsequently visualized with STRUCTURE HARVESTER v.0.9.94 [35]. 3. Results 3.1. Distribution of Collection Areas in Korean Ginseng Accessions The 451 ginseng accessions in this study were collected from 22 cities in six provinces in South Korea (Figure1 and Table S3). The collection areas were four cities in Gangwon (GW), five in Gyeonggi (GG), two in Gyeongsangbuk (GB), two in Jeollabuk-do (JB), five in Chungcheongnam (CN), and four in Chungcheongbuk (CB). Among 451 ginseng accessions, 203 (45.0%) were collected from CN, 124 (27.5%) from GW, 70 (15.5%) from GG, 24 (5.3%) from JB, 20 (4.4%) from CB, and 10 (2.2%) from GB. Of 22 cities, the largest ginseng accessions were collected from Geumsan (19.3%), followed by Hoengseong (14.4%), with less than 10% collected from other cities. Among the 451 accessions, 390 (86.5%) and 61 (13.5%) were landraces and breeding lines, respectively. All 61 breeding lines were collected from Geumsan in CN. 3.2. Profiling of SSR Markers in Korean Ginseng Accessions A total of 226 alleles were detected among the 451 ginseng accessions (Table1 and Table S4). Polymorphic information content (PIC) of 33 SSR markers ranged from 0.025 (GM104) to 0.770 (GM116), with an average of 0.469. In the 390 landraces and 61 breeding lines, there were 223 and 148 alleles, respectively. The number of observed alleles (Na) ranged from 2 to 24, with an average of 6.85, of which there were 6.76 and 4.49 ginseng landraces and breeding lines, respectively.
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