Hong-Il Choi

Development of Reproducible EST-derived SSR Markers and Assessment of Genetic Diversity in Panax ginseng Cultivars and Related Species

Little is known about the genetics or genomics of Panax ginseng. In this study, we developed 70 expressed sequence tag-derived polymorphic simple sequence repeat markers by trials of 140 primer pairs. All of the 70 markers showed reproducible polymorphism among four Panax speciesand 19 of them were polymorphic in six P. ginseng cultivars. These markers segregated 1:2:1 manner of Mendelian inheritance in an F2 population of a cross between two P. ginseng cultivars, ‘Yunpoong’ and ‘Chunpoong’, indicating that these are reproducible and inheritable mappable markers. A phylogenetic analysis using the genotype data showed three distinctive groups: a P. ginseng-P. japonicus clade, P. notoginseng and P. quinquefolius, with similarity coefficients of 0.70. P. japonicus was intermingled with P. ginseng cultivars, indicating that both species have similar genetic backgrounds. P. ginseng cultivars were subdivided into three minor groups: an independent cultivar ‘Chunpoong’, a subgroup with three accessions including two cultivars, ‘Gumpoong’ and ‘Yunpoong’ and one landrace ‘Hwangsook’ and another subgroup with two accessions including one cultivar, ‘Gopoong’ and one landrace ‘Jakyung’. Each primer pair produced 1 to 4 bands, indicating that the ginseng genome has a highly replicated paleopolyploid genome structure.

EST-SSR Marker Sets for Practical Authentication of All Nine Registered Ginseng Cultivars in Korea

Panax ginseng has been cultivated for centuries, and nine commercial cultivars have been registered in Korea. However, these nine elite cultivars are grown in less than 10% of ginseng fields, and there is no clear authentication system for each cultivar even though their values are higher than those of local landraces. Here, we have developed 19 microsatellite markers using expressed gene sequences and established an authentication system for all nine cultivars. Five cultivars, ‘Chunpoong’, ‘Sunpoong’, ‘Gumpoong’, ‘Sunun’, and ‘Sunone’, can each be identified by one cultivar-unique allele, gm47n-a, gm47n-c, gm104-a, gm184-a (or gm129-a), and gm175-c, respectively. ‘Yunpoong’ can be identified by the co-appearance of gm47n-b and gm129-c. ‘Sunhyang’ can be distinguished from the other eight cultivars by the co-appearance of gm47n-b, gm129-b, and gm175-a. The two other cultivars, ‘Gopoong’ and ‘Cheongsun’, can be identified by their specific combinations of five marker alleles. This marker set was successfully utilized to identify the cultivars among 70 ginseng individuals and to select true F1 hybrid plants between two cultivars. We further analyzed the homogeneity of each cultivar and phylogenetic relationships among cultivars using these markers. This marker system will be useful to the seed industry and for breeding of ginseng.

Major repeat components covering one-third of the ginseng (Panax ginseng C.A. Meyer) genome and evidence for allotetraploidy

Ginseng (Panax ginseng) is a famous medicinal herb, but the composition and structure of its genome are largely unknown. Here we characterized the major repeat components and inspected their distribution in the ginseng genome. By analyzing three repeat-rich bacterial artificial chromosome (BAC) sequences from ginseng, we identified complex insertion patterns of 34 long terminal repeat retrotransposons (LTR-RTs) and 11 LTR-RT derivatives accounting for more than 80% of the BAC sequences. The LTR-RTs were classified into three Ty3/gypsy (PgDel, PgTat and PgAthila) and two Ty1/Copia (PgTork and PgOryco) families. Mapping of 30-Gbp Illumina whole-genome shotgun reads to the BAC sequences revealed that these five LTR-RT families occupy at least 34% of the ginseng genome. The Ty3/Gypsy families were predominant, comprising 74 and 33% of the BAC sequences and the genome, respectively. In particular, the PgDel family accounted for 29% of the genome and presumably played major roles in enlargement of the size of the ginseng genome. Fluorescence in situ hybridization (FISH) revealed that the PgDel1 elements are distributed throughout the chromosomes along dispersed heterochromatic regions except for ribosomal DNA blocks. The intensity of the PgDel2 FISH signals was biased toward 24 out of 48 chromosomes. Unique gene probes showed two pairs of signals with different locations, one pair in subtelomeric regions on PgDel2-rich chromosomes and the other in interstitial regions on PgDel2-poor chromosomes, demonstrating allotetraploidy in ginseng. Our findings promote understanding of the evolution of the ginseng genome and of that of related species in the Araliaceae.

Genome-Wide Comparative Analysis of 20 Miniature Inverted-Repeat Transposable Element Families in Brassica rapa and B. oleracea

Miniature inverted-repeat transposable elements (MITEs) are ubiquitous, non-autonomous class II transposable elements. Here, we conducted genome-wide comparative analysis of 20 MITE families in B. rapa, B. oleracea, and Arabidopsis thaliana. A total of 5894 and 6026 MITE members belonging to the 20 families were found in the whole genome pseudo-chromosome sequences of B. rapa and B. oleracea, respectively. Meanwhile, only four of the 20 families, comprising 573 members, were identified in the Arabidopsis genome, indicating that most of the families were activated in the Brassica genus after divergence from Arabidopsis. Copy numbers varied from 4 to 1459 for each MITE family, and there was up to 6-fold variation between B. rapa and B. oleracea. In particular, analysis of intact members showed that whereas eleven families were present in similar copy numbers in B. rapa and B. oleracea, nine families showed copy number variation ranging from 2- to 16-fold. Four of those families (BraSto-3, BraTo-3, 4, 5) were more abundant in B. rapa, and the other five (BraSto-1, BraSto-4, BraTo-1, 7 and BraHAT-1) were more abundant in B. oleracea. Overall, 54% and 51% of the MITEs resided in or within 2 kb of a gene in the B. rapa and B. oleracea genomes, respectively. Notably, 92 MITEs were found within the CDS of annotated genes, suggesting that MITEs might play roles in diversification of genes in the recently triplicated Brassica genome. MITE insertion polymorphism (MIP) analysis of 289 MITE members showed that 52% and 23% were polymorphic at the inter- and intra-species levels, respectively, indicating that there has been recent MITE activity in the Brassica genome. These recently activated MITE families with abundant MIP will provide useful resources for molecular breeding and identification of novel functional genes arising from MITE insertion.

Evidence of genome duplication revealed by sequence analysis of multi-loci expressed sequence tag-simple sequence repeat bands in Panax ginseng Meyer.

Background Panax ginseng, the most famous medicinal herb, has a highly duplicated genome structure. However, the genome duplication of P. ginseng has not been characterized at the sequence level. Multiple band patterns have been consistently observed during the development of DNA markers using unique sequences in P. ginseng. Methods We compared the sequences of multiple bands derived from unique expressed sequence tag-simple sequence repeat (EST-SSR) markers to investigate the sequence level genome duplication. Results Reamplification and sequencing of the individual bands revealed that, for each marker, two bands around the expected size were genuine amplicons derived from two paralogous loci. In each case, one of the two bands was polymorphic, showing different allelic forms among nine ginseng cultivars, whereas the other band was usually monomorphic. Sequences derived from the two loci showed a high similarity, including the same primer-binding site, but each locus could be distinguished based on SSR number variations and additional single nucleotide polymorphisms (SNPs) or InDels. A locus-specific marker designed from the SNP site between the paralogous loci produced a single band that also showed clear polymorphism among ginseng cultivars. Conclusion Our data imply that the recent genome duplication has resulted in two highly similar paralogous regions in the ginseng genome. The two paralogous sequences could be differentiated by large SSR number variations and one or two additional SNPs or InDels in every 100 bp of genic region, which can serve as a reliable identifier for each locus.

A refined Panax ginseng karyotype based on an ultra-high copy 167-bp tandem repeat and ribosomal DNAs

Background Panax ginseng Meyer (Asian ginseng) has a large nuclear genome size of > 3.5 Gbp in haploid genome equivalent of 24 chromosomes. Tandem repeats (TRs) occupy significant portions of the genome in many plants and are often found in specific genomic loci, making them a valuable molecular cytogenetic tool in discriminating chromosomes. In an effort to understand the P. ginseng genome structure, we characterized an ultrahigh copy 167-bp TR (Pg167TR) and explored its chromosomal distribution as well as its utility for chromosome identification. Methods Polymerase chain reaction amplicons of Pg167TR were labeled, along with 5S and 45S rDNA amplicons, using a direct nick-translation method. Direct fluorescence in situ hybridization (FISH) was used to analyze the chromosomal distribution of Pg167TR. Results Recently, we reported a method of karyotyping the 24 chromosome pairs of P. ginseng using rDNA and DAPI (4′,6-diamidino-2-phenylindole) bands. Here, a unique distribution of Pg167TR in all 24 P. ginseng chromosomes was observed, allowing easy identification of individual homologous chromosomes. Additionally, direct labeling of 5S and 45S rDNA probes allowed the identification of two additional 5S rDNA loci not previously reported, enabling the refinement of the P. ginseng karyotype. Conclusion Identification of individual P. ginseng chromosomes was achieved using Pg167TR-FISH. Chromosome identification is important in understanding the P. ginseng genome structure, and our method will be useful for future integration of genetic linkage maps and genome scaffold anchoring. Additionally, it is a good tool for comparative studies with related species in efforts to understand the evolution of P. ginseng.

Rapid amplification of four retrotransposon families promoted speciation and genome size expansion in the genus Panax

Genome duplication and repeat multiplication contribute to genome evolution in plants. Our previous work identified a recent allotetraploidization event and five high-copy LTR retrotransposon (LTR-RT) families PgDel, PgTat, PgAthila, PgTork, and PgOryco in Panax ginseng. Here, using whole-genome sequences, we quantified major repeats in five Panax species and investigated their role in genome evolution. The diploids P. japonicus, P. vietnamensis, and P. notoginseng and the tetraploids P. ginseng and P. quinquefolius were analyzed alongside their relative Aralia elata. These species possess 0.8–4.9 Gb haploid genomes. The PgDel, PgTat, PgAthila, and PgTork LTR-RT superfamilies accounted for 39–52% of the Panax species genomes and 17% of the A. elata genome. PgDel included six subfamily members, each with a distinct genome distribution. In particular, the PgDel1 subfamily occupied 23–35% of the Panax genomes and accounted for much of their genome size variation. PgDel1 occupied 22.6% (0.8 Gb of 3.6 Gb) and 34.5% (1.7 Gb of 4.9 Gb) of the P. ginseng and P. quinquefolius genomes, respectively. Our findings indicate that the P. quinquefolius genome may have expanded due to rapid PgDel1 amplification over the last million years as a result of environmental adaptation following migration from Asia to North America.

Fruit Quality and Chemical Contents of Hybrid Boysenberry ( Rubus ursinus ) Lines Developed by Hybridization and Gamma Irradiation

The Rubus fruit is an economically important berry crop that contains various functional compounds. The objective of this study was to analyze fruit qualities (i.e., pH, soluble solids content, titratable acidity, and mineral content) as well as fatty acid and phenolic compounds (i.e., ellagic acid and anthocyanins) among hybrid boysenberry lines developed by hybridization and gamma irradiation. There were no significant differences in the hybrid boysenberry fruit pH and titratable acidity (%) among the tested genotypes. However, the soluble solids content was higher in the BSA119 and BSA144 mutants than in the original genotype (BS_Hybrid). Meanwhile, linoleic acid was the most abundant fatty acid in the analyzed hybrid boysenberry fruits. The fatty acid composition did not differ significantly among the genotypes. The ellagic acid content of all genotypes ranged from 8.72 mg/100 g to 46.10 mg/100 g, with the highest concentration observed for the BSB127 genotype. Additionally, cyanidin-3-O-sophoroside (M-H, 611 m/z) and cyanidin-3-O-glucoside (M-H, 449 m/z) were the two major anthocyanins detected in the boysenberry and mutant genotypes, while cyanidin-3-O-glucoside was the predominant blackberry anthocyanin. The total anthocyanin concentrations of four mutant genotypes (i.e., BSA036, BSA078, BSA101, and BSB127) were significantly higher than that of the original genotype (382.0 mg/100 g). The highest total anthocyanin concentration was observed for the BSA078 genotype (467.9 mg/100 g). These results may be useful for identifying the optimal genotypes for breeding new cultivars with enhanced qualities and potential health benefits.

The complete chloroplast genome sequence of Panax vietnamensis Ha et Grushv (Araliaceae)

Abstract The complete chloroplast genome sequence of Panax vietnamensis, a medicinal herb belonging to Araliaceae family, was generated by de novo assembly using whole genome next-generation sequences. The chloroplast genome was a circular form of 155 992 bp long and showed typical chloroplast genome structure consisting of a large single-copy region of 86 177 bp, a small single copy region of 17 935 bp and a pair of inverted repeats of 25 940 bp. The chloroplast genome had 79 protein-coding genes, 29 tRNA genes and 4 rRNA genes. The phylogenetic analysis with the reported chloroplast genomes revealed that four Panax species were grouped in the same clade and P. vietnamensis is more closely related to P. notoginseng than P. ginseng and P. quinquefolius.

Identification of Mutagenized Plant Populations

The recent availability of large-scale genomic data has allowed researchers to begin deciphering plant gene function. Plant mutagenesis is a powerful tool for the identification and characterization of the function of specific genes linked to phenotypes. TILLING (Targeting Induced Local Lesions IN Genomes) is a general reverse-genetics tool that combines traditional mutagenesis with high-throughput methods of mutation discovery among mutagenized populations. The aim of TILLING is to identify mutagenized genotypes that affect specific phenotypes. Securing genetic diversity and selecting efficient progeny are the most important factors in plant breeding. During the evolutionary process, the gene pool has skewed towards a direction that is favorable to humans and some essential alleles may have been lost during the selection processes. Genome editing using an engineered nuclease is a target-directed, controlled, and predictable approach that can provide a genetically diverse gene pool and shorten cultivar development time. Depending on engineered nucleases and plant species types, the mutagenesis rate and outcomes show significant differences, indicating that nuclease types and characteristics, efficient target mutagenesis, construction and delivery of the nuclease, selection and verification of the mutants, and off-target mutagenesis need to be considered. Genomic sequence variations generated by chemical and/or physical mutagenesis can be strongly related to changes in phenotype. Haplotype analysis allows plant breeding knowledge to be expanded and can help improve understanding of diversity at the genomic sequence level. Experiments using SNP haplotypes can provide insights into plant evolution, and can contribute to phenotypic analysis, and expectation and characterization of mutagenized plant phenotypes.