
Korean ginseng (
The chloroplast, one of many types of plastids, plays vital roles in photosynthesis (Pfannschmidt
Various studies on the ginseng genome have been carried out, and a draft genome comprising approximately 3 Gbp of assembled sequence was recently published (Jayakodi
DNA molecular markers are useful genetic tools that could be used for species authentication (Kim
In the current study, we uncovered plastome diversity in ginseng and established a standard haplotype grouping (haplotyping) system based on the different genotypes of ginseng plastomes. We identified 18 polymorphic sites, including 11 SNPs and 7 InDels, from a comparative analysis of the plastomes of 44 cultivated and wild ginseng accessions from Northeast Asian countries. We developed 10 KASP markers based on the SNP variations, and applied them to diverse genetic resources to identify different haplotypes and their cultivation history. The results of this study provide valuable information about the genetic diversity of different ginseng populations. Moreover, our digital haplotyping system using the 10 newly developed KASP markers could be used as a fundamental tool for ginseng breeding.
A total of 203 ginseng genetic resources were used in this study, including cultivated and wild accessions from Nor-theast Asian countries. Origin and provider information of the samples were described in Supplementary Table 1. Among these resources, 156 individuals were collected from various regions in Korea, 30 from China, 8 from in Japan, and 9 from Russia. Among these samples, 146 ginsengs were the same samples used in our previous study (Jang
Among the DNA samples, DNA from 27 ginseng indi-viduals was sent to Phyzen (Seongnam, Korea) for library construction and 0.5-1x low-coverage whole genome sequencing. Libraries were prepared using TruSeq Nano DNA kit (Illumina Inc., San Diego, USA) in accordance with the manufacturer’s instructions. Sequencing was per-formed using the multiplexing method on the NextSeq platform (Illumina Inc., San Diego, USA), generating 150 bp paired-end reads. The reads from each sample were sorted based on specific index sequences and used to assemble the plastome sequences with the dnaLCW method (Kim
Comparative analysis was performed using 44 plastome sequences, including previously reported sequences (Kim
The newly designed KASP primer pairs were validated using 182 ginseng genetic resources excluding the sequenced samples. PCR amplification was carried out in a 10 mL volume containing 10 ng genomic DNA, 5 mL of KASP Master mix solution, and 0.14 mL of KASP Assay mix solution (LGC genomics, Teddington, UK). The reaction mixtures and two 10 mL samples of sterile distilled water (as negative controls) were dispensed into a 96-well plate (Semagn
To examine the distribution for InDel variations in the ginseng population, gel-based genotyping analysis was performed by selecting 4 InDel variations (ID2, ID3, ID6, and ID7). The markers developed in a previous study (Kim
We obtained 27 plastome sequences from ginseng genetic resources collected from Korea, China, Japan, and Russia using low-coverage NGS data. Our analysis was focused on the plastomes of 44 accessions identified in the current and previous studies (Kim
A comparative analysis of the 44 ginseng plastome sequences from cultivated and wild ginseng accessions identified 18 polymorphic sites, including 11 SNPs and 7 InDels. These polymorphic sites were distributed throu-ghout the entire sequence, including 10 in the LSC region, 5 in the SSC region, and 3 in IR regions (Fig. 1 and Table 1). In addition to the six SNPs identified in our previous study (Kim
Table 1 . Summary of the variations identified in the ginseng plastomes.
Variation type | Plastome type | 1-1 | 1-2 | 1-3 | 1-4 | 1-5 | 2 | 3-1 | 3-2 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | ||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Region | Positionz) | VariationNo.u) | 21Acc.t) | YSS | JF2 | HY1 | JYH | SH | R2 | 3Acc.s) | 3Acc.r) | JF3 | KF1 | 2Acc.q) | M1 | 3Acc.p) | 3Acc.o) | |||
SNP | LSC | 7,159v) | S1 | G | G | G | G | G | T | G | G | G | G | G | G | G | G | G | ||
21,344y),v) | S2 | C | C | C | C | C | C | T | T | C | C | C | C | C | C | C | ||||
22,287x),v) | S3 | G | G | G | G | G | G | G | G | T | G | G | G | G | G | G | ||||
23,946w),v) | S4 | A | A | A | A | A | A | A | A | A | C | A | A | A | A | A | ||||
40,011y),w),v) | S5 | C | C | C | C | C | C | C | C | C | C | T | C | C | C | C | ||||
44,895w),v) | S6 | G | G | G | G | G | G | G | G | G | G | G | T | G | G | G | ||||
IR | 90,858/151,519y),w),v) | S7 | A | A | A | A | A | A | A | A | A | A | A | A | G | A | A | |||
SSC | 115,466 | S8 | G | G | G | G | G | T | G | G | G | G | G | G | G | G | G | |||
117,376x),v) | S9 | A | A | A | A | A | A | A | G | A | A | A | A | A | A | A | ||||
118,525y),w),v) | S10 | T | T | T | T | T | T | T | T | T | T | T | T | T | A | T | ||||
127,069x),v) | S11 | A | A | A | A | A | A | A | A | A | A | A | A | A | A | T | ||||
InDel | LSC | 5,473 | ID1 | (C)8 | (C)9 | (C)8 | (C)8 | (C)8 | (C)8 | (C)9 | (C)9 | (C)8 | (C)8 | (C)8 | (C)9 | (C)8 | (C)8 | (C)8 | ||
7,189 | ID2 | 13×1 | 13×1 | 13×1 | 13×1 | 13×1 | 13×2 | 13×1 | 13×1 | 13×1 | 13×1 | 13×1 | 13×1 | 13×1 | 13×1 | 13×1 | ||||
32,850 | ID3 | 59×1 | 59×1 | 59×1 | 59×1 | 59×1 | 59×2 | 59×1 | 59×1 | 59×1 | 59×1 | 59×1 | 59×1 | 59×1 | 59×1 | 59×1 | ||||
38,191w) | ID4 | (C)10 | (C)10 | (C)9 | (C)10 | (C)10 | (C)10 | (C)10 | (C)10 | (C)10 | (C)10 | (C)10 | (C)10 | (C)10 | (C)10 | (C)10 | ||||
IR | 105,431/136,936 | ID5 | (G)11 | (G)11 | (G)11 | (G)10 | (G)11 | (G)10 | (G)11 | (G)11 | (G)11 | (G)11 | (G)11 | (G)11 | (G)11 | (G)11 | (G)11 | |||
111,304/130,897 | ID6 | 57×4 | 57×4 | 57×4 | 57×4 | 57×3 | 57×4 | 57×4 | 57×4 | 57×3 | 57×4 | 57×4 | 57×4 | 57×4 | 57×4 | 57×3 | ||||
SSC | 115,833 | ID7 | 7×2 | 7×2 | 7×2 | 7×2 | 7×2 | 7×2 | 7×2 | 7×2 | 7×3 | 7×2 | 7×2 | 7×2 | 7×2 | 7×2 | 7×2 |
The nucleotides in gray boxes indicate variations. Each germplasm is represented by the abbreviation defined in
z)Variation positions are based on the plastome sequence of
y)Non-synonymous SNP.
x)Synonymous SNP.
w)Newly identified variation.
v)SNP position developed as KASP marker.
u)Variation number shown in Fig. 1.
t)ChS2, CF1, CF2, CF3, CF5, DMY, EMY, G8, G13, G17, GLS, GO, JK, JF1, JF4, JF5, R1, SP, SU, SW, and YP.
s)CS, GP, and G2.
r)CP, KF2, and KF3.
q)G15 and KF4.
p)CF4, DJ, and G16.
o)HS, JL, and KF5.
Acc.: Accessions, CDS: Coding sequence, IR: Inverted repeat, KASP: Kompetitive allele-specific PCR, LSC: Large single copy, SSC: Small single copy.
Based on the SNP and InDel compositions, the assem-bled 44 ginseng plastomes were classified into 15 types (Table 1). When only 11 SNPs were considered, the 44 plastomes were grouped into 10 major types. The most common type (Type 1) was identified in 25 plastomes and was further subdivided based on the presence of InDel variations (Type 1-1–1-5). The nine other types of plas-tomes contain one or two additional SNPs derived from the type 1 plastome. Each of the seven SNPs is present in the respective plastome type, where mutations occurred at independent positions. The four other SNPs coexist in two types of plastomes in pairs (S1&S8 and S2&S9). In contrast to SNPs, InDels are present in shuffled patterns in the different plastome types. The assembled plastome sequences contain one to five variations.
Two plastome sequences of
We tried to develop allele-specific markers based on the 11 SNPs and their flanking sequences in
We genotyped all 203 ginseng genetic resources based on plastome sequence information, 10 KASP markers, and 4 InDel markers (Supplementary Table 4-6). Excluding the five indivi-duals showing heterozygous genotypes, the 198 remaining genetic resources were classified into 12 groups based on digitalized 10 SNP genotypes (Table 2). Among the 198 genetic resources, 119 (60.1%) accessions belonged to the most common haplotype (named haplotype A), which might be the original plastome type. The most abundant SNP (S2) was identified in 43 (21.8%) accessions (haplotype B). Haplotype B was sub-classified based on one additional SNP (S9) that was identified in 10 (5.1%) of 43 accessions (haplotype B’). An unexpected combination of two SNPs (S3 and S4) that was not observed in the comparative analysis of the 44 plastome sequences was also identified in three accessions (haplotype IJ). The cultivated and wild ginseng populations displayed similar proportions of each haplotype group.
Table 2 . Number and proportion of individuals for each plastome haplotype.
Haplotype | Variation No.z) | Country of origin | Germplasm type | Total (%) | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
S2 | S9 | S6 | S10 | S7 | S11 | S1 | S5 | S3 | S4 | K | C | J | R | Cultivatedginseng | Wildginseng | ||||
A | C | A | G | T | A | A | G | C | G | A | 91 | 23 | 4 | 1 | 98 | 21 | 119 (60.1) | ||
B | T | · | · | · | · | · | · | · | · | · | 23 | 2 | - | 8 | 24 | 9 | 33 (16.7) | ||
B’ | T | G | · | · | · | · | · | · | · | · | 10 | - | - | - | 6 | 4 | 10 (5.1) | ||
C | · | · | T | · | · | · | · | · | · | · | 5 | 1 | - | - | 5 | 1 | 6 (3.0) | ||
D | · | · | · | A | · | · | · | · | · | · | 5 | 1 | - | - | 4 | 2 | 6 (3.0) | ||
E | · | · | · | · | G | · | · | · | · | · | 2 | - | 2 | - | 4 | - | 4 (2.0) | ||
F | · | · | · | · | · | T | · | · | · | · | 2 | 1 | - | - | 3 | - | 3 (1.5) | ||
G | · | · | · | · | · | · | T | · | · | · | 2 | - | - | - | 2 | - | 2 (1.0) | ||
H | · | · | · | · | · | · | · | T | · | · | 1 | - | - | - | 1 | - | 1 (0.5) | ||
I | · | · | · | · | · | · | · | · | T | · | 7 | - | 1 | - | 7 | 1 | 8 (4.0) | ||
J | · | · | · | · | · | · | · | · | · | C | 2 | - | 1 | - | 2 | 1 | 3 (1.5) | ||
IJ | · | · | · | · | · | · | · | · | T | C | 2 | 1 | - | - | 3 | - | 3 (1.5) |
Grayboxes represent the SNP positions where mutations occurred.
z)Variation number shown in Fig. 1.
C: China, J: Japan, K: Korea, R: Russia.
The results of genotyping based on InDel variations showed more dynamic patterns among the genetic resources (Fig. 3 and Supplementary Table 6). Three InDels (ID2, ID3, and ID7) showed two types of copy number variation (CNV) for each TR as they were found in the assembled plastome sequences, whereas ID6 showed new types of CNV not previously identified in the plastome sequences. Various CNV patterns were detected in the same haplo-types, which divided each main haplotype into different subgroups. Haplotype A, representing the original plastome form, was subdivided into four subgroups based on the CNV patterns of the four TRs (Fig. 4).
Plastomes are conservatively inherited and show few variations within a species. Due to this characteristic, variations in the plastome hold valuable information for utilization in DNA barcoding (Dong
Ginseng breeding has been performed via pure line selection in which individual plants are selected from arable fields of mixed local landrace populations (Zhang
Molecular DNA markers developed from plastome sequences do not usually exhibit heterozygous genotypes because they are generally uniparentally inherited. However, markers S3 and S7 exhibited bi-alleles resembling hetero-zygous genotypes in some individual ginseng plants (Fig. 2 and Supplementary Table 5). To explore this issue, we compared the 50 bp flanking sequences of the SNP targets with the nuclear genome sequences (Jayakodi
Plastomes provide useful information about the breeding history of self-pollination plants such as ginseng because most plastomes follow uniparental inheritance patterns (Jakobsson
Cultivated and wild accessions were intermingled in each haplotype, indicating that there are no differences in genetic diversity between the two populations. A similar result was obtained in a study that analyzed the differences in genetic diversity between two populations using simple sequence repeat markers developed from the nuclear genome (Jang
Since ginseng is mainly a self-fertilizing plant, its geo-graphical distribution could be inferred using plastome haplotypes (Vettori
In this study, we characterized 44 complete plastomes of cultivated and wild ginsengs from four Northeast Asian countries. Eighteen polymorphic variations were identified, and 10 KASP markers were developed to evaluate the genetic diversity. By applying these markers to various genetic resources, we validated the usefulness of the mar-kers and established a standard haplotyping system for ginseng. Also, we explored the breeding history of ginseng based on the accumulation patterns of plastome variations and the genetic relationship between cultivated ginseng and wild collections. These results expand our understanding of the genetic diversity and cultivation history of ginseng. Furthermore, the KASP markers and standard plastome haplotyping system developed in this study provide useful genetic tools for the efficient breeding of ginseng.
All plastid sequence data from this article can be found in the GenBank data libraries under accession numbers des-cribed in Supplementary Table 2.
Supplementary data to this article can be found online at https://doi.org/10.9787/PBB.2022.10.3.174. Supplementary Table 1. Origin and provider information of 203
This work was carried out with the support of 2019 grant from the Korean Society of Ginseng and “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ015903)” and 2022 the RDA Fellowship Program of National Institute of Horticultural and Herbal Science, Rural Development Administration, Republic of Korea.
The authors declare that there are no conflicts of interest.
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