Most ginseng cultivars bear red berry and only one cultivar ‘Gumpoong’ (GU) bears golden berry. GU is an elite cultivar bred by pedigree selection from a golden berry landrace (a mixed population) ‘Hwangsook’ (HS). We developed three unique polymorphic markers from complete chloroplast genome sequences of GU and HS. A population of GU showed uniform band amplicon against three chloroplast markers whereas HS population displayed mixed genotypes for both GU and HS. Using the characteristics of mixed genotypes in HS population, we developed a convenient method to differentiate GU and HS population by application of pooled DNA template for PCR analysis (pooling method). The pooling method revealed that the GU pool was identical with GU genotype while the HS pool showed both GU and HS genotype. The pooling method is a cost and time effective method for accurate authentication of both golden berry ginseng cultivars. The method is useful to protect GU products from its tentative counterfeits from seeds to mature plant stages as well as processed root products.
The most elite cultivars were bred by identification of individual plant from farmer’s field and inbreeding as pedigree in Korea (Kwon
Most of the ginseng landraces have reddish color berry (red berry) except HS, which has golden color berry (golden berry). Therefore, HS is easily distinguished from other
Chloroplast genome has been used to investigate inter-and intra-species genetic relationship of plants (Kim
From our previous study (Kim
PCR amplification was proceeded in total 25 μL of reaction volume containing the following: 20 ng of DNA template, 10 μM of primer set, 5 mM of dNTP, and one unit
To differentiate GU and HS efficiently on population level, we applied DNA pooling method by creating a DNA pool of GU and HS population for PCR analysis, as the concept of bulked analysis (Michelmore
Golden berry is distinct characteristics compare to red berry in ginseng. Most popular cultivating ginseng cultivars are red berry bearing cultivars (Fig. 1A) but GU is the only cultivar bearing golden berry (Fig. 1C). Our previous study found 12 intra-species polymorphisms (6 SNPs and 6 InDels) in complete chloroplast genomes among 11
From the five polymorphic sites, three were chosen for marker designing and analysis. Two markers, pgycf1 identifying copy number variation of 57 bp tandem repeat (TR) in
All three markers successfully amplified their specific PCR amplicons for each individual in GU and HS population. Each individual plant could be genotyped as GU type or HS type for each of three markers (Fig. 4). All 32 individual plants in GU populations from both KGC and RDA populations showed the same genotype as that of control GU plant when applied all three markers. Meanwhile, 32 individual plants in HS population did not show identical genotypes but mixed genotypes for both GU and HS for all three markers (Table 2, Fig. 4).
The frequency of genotypes was different in two HS populations. For pgycf1 marker, two plants in KGC HS population (plant nos. 4 and 8) and all 18 plants in RDA HS population (plant nos. 15–32) showed GU genotype and the remaining 12 plants showed HS genotype (Fig. 4). For pgcpd02 and pgcp_ccsA marker, one plant in KGC HS (plant no. 4) and 11 plants in RDA HS (plant nos. 15–19, 21–24, and 28) were identified as GU genotype and the remaining 20 plants were identified as HS genotype (Fig. 4).
Although our three DNA markers could discriminate genotype of each GU and HS individual clearly, it was obvious that large number of individuals in a population should be inspected to determine homogeneity of GU and HS population. Analysis of large number of plants is easy technically but it is high costs and time-consuming task. Thus, we proposed a pooling method to authenticate each population without PCR analysis of large numbers of plants. The genomic DNAs from GU and HS population plants were pooled, respectively, and the PCR amplification was performed using the pooled DNA as template with the three markers, pgcpd02, pgcp_ccsA, and pgycf1. GU pool (GU-P) displayed same and uniformed band pattern of amplicon as control GU sample, whereas HS pool (HS-P) displayed mixed band patterns of two types of GU and HS band (Fig. 5).
In history of
As sequencing technology and bioinformatics tools have advanced, the studies on complete assembly of chloroplast genome and development of barcoding markers to differentiate the plant species or cultivars have also been increased. We identified 12 polymorphic markers from 11 ginseng cultivars (Kim
The landraces are locally adapted plant population over centuries and have more genetic diversities than cultivars. Originally, the cultivar GU was bred by pureline selection of one superior individual of landrace HS and followed pedigree selection. Thus, some individuals in HS population may contain same genotype as GU such as shown by three markers in this study (Fig. 4). All of GU plants showed same genotype for three markers, indicating the GU has uniform genotype that make GU as registered cultivar. With such characteristic shared by landrace HS and GU, the uncertainty would arise when we use small number of plant samples and markers for authentication of GU and HS. We observed also that pgycf1 showed GU genotype for all of RDA HS population (Fig. 4. Plant nos. 21–32). Furthermore, 12 HS individual plants such as no. 4, 15–19, 21–24, 28, and 32 show GU genotypes for all three markers (Fig. 4) that would lead to wrong decision to distinguish of HS from GU. We suggest to use a couple of markers together against more than thirty samples per population to authenticate the golden berry bearing population. It is also expected that the differentiation accuracy would be improved with increasing number of plant samples and DNA markers.
Based on our results, we realized that the discrimination power is low when we use small number of plant samples. Applying markers onto large numbers of plants or population would require more cost and efforts. To utilize the molecular marker into efficient and high-throughput manner, we designed and used a pooling method. Each of three markers used in this study was capable of differentiating GU from HS population. Even though each marker could differentiate GU or HS pools, we strongly suggest to use all three markers for evaluating GU or HS population.
The general profile of plant authentication analysis relies more on morphological trait differences or molecular marker system among inbred cultivars. However, under the condition like differentiating cultivar and landrace, new concepts and analysis methods are necessary. In this study, we have evaluated molecular markers to efficiently differentiate cultivar GU from landrace HS by utilizing pooling method. This is the first study that focused on authentication of an elite ginseng cultivar from its original landrace mixed population at the population level. We believe that this method can be applied in discriminating other
The pooling method used in this study is cheap, practical and exact to discriminate the mixed population. The authentication could be more precise by increasing individuals in each population without extra cost, which is a big advantage for practical application. The pooling method can also be applied to tissue pools derived from a population. Furthermore, the method can be applied to large number of seeds to evaluate the authentic seeds of GU cultivar and seed purity, collection of small piece of leaves derived from large numbers of plants in same field, as well as collection of piece of roots in a root bulk in the ginseng market or processing line of ginseng product industry.
This study was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project Nos. PJ01103001)”, Rural Development Administration, and a grant (16172MFDS229) from Ministry of Food and Drug Safety in 2016, Republic of Korea. We thank Dr. Sang-Choon Lee for critical comments.
Polymorphic sites and marker information in chloroplast genomes of GU and HS.
|Type||Position||Nucleotide position GU / HS||Polymorphisms GU / HS||Primer ID||Product size (bp) GU/HS||Restriction enzyme|
|InDel||111,305 / 111,304||57×4 / 57×3||pgycf1z)||729/672|
|InDel||5,473||(C)9 / (C)8|
|SNP||21,345 / 21,344||T / C||pgcpd02z)||177/200|
|SNP||117,427 / 117,369||G / A||pgcp_ccsAy)||199/223|
|SNP||127,069||A / T|
Polymorphic sites and marker information were from Kim
y)Developed in this study.
The genotyping data of individual plant in GU and HS population using three markers.
|Primer ID||Gumpoong (GU)||Hwangsook (HS)|
|No. of GU type||No. of HS type||No. of GU type||No. of HS type|