[보고서]인삼 유전체 완성도 향상 및 유전다양성 연구

양태진

인삼 유전체 완성도 향상 및 유전다양성 연구
Genome sequencing and genomic diversity analysis of ginseng and its relatives 원문보기

보고서 정보
주관연구기관	서울대학교 Seoul National University
연구책임자	양태진
참여연구자	이상춘 , 박지영 , 구현조 , 김남훈 , 박현승 , 이준기 , 이원경 , 장예은 , Nguyen Van Binh , Jayakodi Murukarthick , 김현희 , Nomar E. Waminal , 윤성민
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2018-02
과제시작연도	2017
주관부처	농촌진흥청 Rural Development Administration(RDA)
등록번호	TRKO201800043241
과제고유번호	1395049030
사업명	차세대바이오그린21
DB 구축일자	2018-12-15
키워드	고려인삼.전장유전체 해독.비교유전체 연구.전사체 분석.형광동소 보합법.Ginseng.Whole genome sequencing.Comparative genomics.Transcriptome analysis.FISH.
DOI	https://doi.org/10.23000/TRKO201800043241

초록

- 인삼유전체 draft version 1.0 완성 및 annotation
- 인삼의 이질배수체 구조 확인 및 인삼의 진화모델 구축
- 비교유전체 연구를 통한 인삼 종내 유전다양성 연구
- 전사체 및 대사체 통합 분석을 통한 인삼 내재해성 유전자군 발굴과 조직별 발편패턴 분석
- 인삼 부정근 이용한 형질전환체 제작 및 대사산물 분석
- 반복서열을 이용한 인삼 핵형분석 및 근연종의 분자세포유전학적 비교유전체 분석

(출처 : 보고서 요약서 3P)

Abstract ▼

Purpose & Contents
< Purpose >
- Improving the completion of the ginseng genome and its comparative genomic studies
- Transcriptome and metabolome research on ginseng
- Constructing the physical map of the ginseng and validating the assembled genome sequence
< Contents >
- The production of long read sequencing data and a hybrid assembly with Illumina short reads for a more complete ginseng genome
- GBS analysis of Yunpoong x Chunpoong F2 population used for constructing a high-resolution genetic map and drawing a linkage map by scaffold mapping
- Research on genetic diversity through comparative analysis among ginseng intraspecies and related species
- Identification of related genes and expression profiling through transcriptome analysis
- Chromosome anchoring of scaffolds using molecular cytogenetic mechanisms and using this to assemble and validate the sequence, identify gaps, and analyze the heterochromatin structure

Results
- Completion of ginseng genome draft version 1.0 and annotation
- Verification of the allopolyploid structure and construction of an evolutionary model for ginseng
- Research the genetic diversity within ginseng species through comparative genomics analysis
- Detection of abiotic stress related gene family through transcriptome and metabolome research and analysis of expression patterns in different tissues
- Creation of transformants and metabolite analysis with ginseng adventitious root
- Karyotyping ginseng using repetitive sequences and comparative analysis with related species

Expected Contribution
- Completion of a reference genome for ginseng to construct a genome database and give domestic ginseng research groups priority rights to use the data
- A scientific approach on the mysterious effects of ginseng and provide a research base for biotechnology through molecular breeding research on ginseng and by uncovering the ginseng genome
- Discovering useful genes related to the medicinal components and securing intellectual property rights

(출처 : Summary 5P)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
국 문 요 약 문 ... 4
Summary ... 5
목차 ... 6
제 1 장 연구 개발 과제의 개요 ... 7
제1절 연구 개발 목적 ... 7
제2절 연구 개발의 필요성 ... 8
제3절 연구 개발 범위 ... 8
제 2 장 국내외 기술개발 현황 ... 10
제 3 장 연구 수행 내용 및 결과 ... 12
제 4 장 목표달성도 및 관련분야 기여도 ... 140
제1절 : 목표대비 달성도 ... 140
제2절 : 정량적 성과(논문게재, 특허출원, 기타)를 기술 ... 143
제 5 장 연구 결과의 활용 계획 ... 144
제 6 장 연구 과정에서 수집한 해외 과학 기술 정보 ... 145
제 7 장 연구 개발 결과의 보안 등급 ... 146
제 8 장 국가과학기술지식정보서비스에 등록한 연구시설·장비 현황 ... 146
제 9 장 연구개발과제 수행에 따른 연구실 등의 안전조치 이행실적 ... 146
제 10 장 연구개발과제의 대표적 연구실적 ... 147
제 11 장 기타사항 ... 148
제 12 장 참고문헌 ... 149
끝페이지 ... 154

표/그림 (161)

표 Assembly Error Correction (AEC) 프로그램의 개념도. Mate-paired read 들과 정확히 매치되는 부분만 데이터를 뽑아내어 오류를 수정한다
표 두 개의 제한효소를 이용한 인삼 GBS library 제작과 시퀀싱 개념도
표 Hi-C 기술을 이용한 라이브러리 제작 과정
표 Overview of the architecture of the Ginseng Genome Database
표 베트남삼 NGS 데이터의 pre-quality check 결과
표 베트남삼 NGS 시퀀싱 정보
표 기존 버전들과의 assembly 향상 비교
표 유전체서열 검증을 위한 13개의 BAC 클론과 스캐폴드서열의 비교
표 인삼 draft genome 서열상의 TE 분포
표 인삼 유전자 annotation pipeline
표 인삼 유전체 초안서열에서 예측된 유전자 정보
표 Improvement of P . ginseng gene models by PacBio transcript sequences
표 Top 10 INTERPRO domains in the IPGA gene set version 1.1
표 Gene Ontology (GO) annotation. Top GO terms were shown in this figure. GO terms were categorized into biological process, molecular function and cellular component
표 유전체해독이 된 쌍자엽 4종 식물(당근, 토마토, 애기장대, 포도)들과의 gene family 분석
표 5종의 paralog 및 ortholog쌍들의 Ks 분포. (a), 애기장대(At), 당근(Dc), 인삼(Pg)의 paralog쌍들의 Ks 분포. (b), 인삼과 애기장대, 당근, 포도(Vv), 토마토(Sl)의 ortholog 쌍의 Ks 분포
표 Phylogenetic tree and divergence time of 19 Araliaceae species. The tree was generated based on cp genomes by Bayesian Inference analysis using BEAST (version 1.8.1). Divergence time was calculated based on 49 million years ago (MYA) when Araliaceae species were diverged from a common ancestor with Apiaceae species (represented by Daucus carota). The number above each branch refers to the Bayesian posterior probability
표 Phylogenetic tree and divergence time of 11 Araliaceae species. (a, b) The tree was generated based on cp protein-coding and 45S nrDNA sequences by Bayesian Inference analysis using BEAST (version 1.8.1). Divergence time was calculated based on 49 million years ago (MYA) when Araliaceae species were diverged from a common ancestor with Apiaceae species (represented by Daucus carota). The number above each branch refers to the Bayesian posterior probability
표 Evolutionary history of P. ginseng. The pale blue triangles signify species number in the Araliaceae (1500) and Apiaceae (3700)
표 Panax 종들의 현재 분포 및 두 번의 대륙이동 모식도
표 인삼 paralog 스캐폴드간 dotplot 분석
표 두 번의 전장유전체배가에 의한 인삼 paralog 블락들의 상동성 분석
표 λDNA, 인삼, 당귀, 구절초 DNA에 다양한 제한효소를 처리한 결과
표 샘플별 생성된 read의 수
표 F2 coverage와 read depth에 따른 SNP와 scaffold 개수
표 인삼 유전지도의 scaffold anchoring 결과
표 인삼과 당근의 synteny 분석
표 당근염색체 기반한 인삼의 superscaffold 구성과 인삼유전체 구조 분석. (a) 당근 염색체 기반 18개의 가상 superscaffold 구성. (b) 18개의 superscaffold 내의 원형 맵과 SNP(A), 반복인자 (B), 유전자 분포(C)
표 Hi-C를 이용한 Proximity-Guided Assembly 방법
표 Hi-C 정보를 이용한 인삼 Clustering Heatmap
표 인삼 genome database 구축
표 인삼 draft 서열에서의 유전자 annotation 예
표 인삼 20종의 시퀀싱 현황
표 인삼 천풍 유전체 서열과 인삼 20종의 다형성 분석 결과
표 함양 특이적인 SNP 및 INDEL 변이 지역
표 함양산삼과 다른 품종간의 nonsynonymous mutation 유전자 지역
표 Chloroplast genome map of 13 P. ginseng cultivars. Colored boxes are conserved chloroplast genes classified based on product function
표 인삼 14 종의 엽록체 서열과 45S nrDNA 서열 완성 결과
표 Validation of copy number variation (CNV) of TRs in rps16 ~ trnUUG region. (A) Schematic diagram of CNV of TR units among 11 P. ginseng cultivars and P. quinquefolius. Arrowheads and polygons indicate TR units of 13 bp and 33 bp, respectively. P. ginseng cv. SH and P. ginseng other cvs. indicate cultivar Sunhyang and the remaining 11 accessions listed in Table 1 and P. quinquefolius. (B) PCR analysis of CNV regions using 097f2*r primer set in 11 P. ginseng cultivars and P. quinquefolius. Abbreviated cultivar names are denoted on the gel. PQ and M denote P. quinquefolius and 100-bp DNA ladder, respectively
표 Summary of nucleotide polymorphisms in cp genomes and 45S nrDNA sequences of 11 P. ginseng accessions
표 Primers to detect polymorphism among P . ginseng accessions Primer ID
표 Primers to detect polymorphism among P. ginseng accessions Primer ID. Validation of SNP and InDel polymorphic sites. (A, B) PCR analysis of InDel regions in ycf1 (A) using primer set pgycf1 and trnUUC-trnGGU (B) using primer set pgcp137 (Table II-1-5). (C) SNP analysis using dCAPS primers, pgcpd02, designed for the SNP site in the rpoC2 gene (Table II-1-5). ScaI digestion of the amplicon produced a digested fragment except in GU and CS. (D) SNP analysis with dCAPS primers, pgcpd01, designed for the SNP site in rpoC1 gene (Table 3). XbaI digestion of the amplicon produced a digested fragment except in ChP and PQ. Abbreviated cultivar names are denoted on the gels. PQ and M denote P. quinquefolius and 100-bp DNA ladder, respectively
표 Summary of nucleotide polymorphisms in cp genomes of 14 P. ginseng accessions
표 베트남삼 WGS데이터와 인삼 유전체의 매핑 결과
표 베트남삼과 인삼의 비교를 통한 다형성 분석 결과
표 Complete chloroplast genomes of seven Panax species. Colored boxes show conserved chloroplast genes, classified based on product function. Genes shown inside the circle are transcribed clockwise, and those outside the circle are transcribed counterclockwise. Genes belonging to different functional groups are color-coded. Dashed area in the inner circle indicates the GC content of the chloroplast genome
표 Chloroplast genome sequences used in this study
표 The numbers of nucleotide substitutions in the seven Panax chloroplast genomes
표 Single nucleotide polymorphic sites in 79 CDS genes of 7 Panax species. The inner track showing 79 chloroplast CDS genes, track A represents total SNPs in all 7 Panax species. The track B – G represent SNPs in P. trifolius, P. stipuleanatus, P. vietnamensis, P. japonicas, P. notoginseng, and P. quinquefolius compared to P. ginseng, respectively. Red, green, blue and black lines on each track indicate the four kinds of SNPs (T, A, C and G nucleotides), respectively. Yellow lines indicate InDel regions
표 ML phylogenetic tree of P. stipuleanatus and P. trifolius with 17 related species in the Araliaceae family based on entire chloroplast genome sequences. Numbers in the nodes are the bootstrap support values from 1000 replicates. Black triangles indicate tetraploid Panax species. The chloroplast sequence of carrot (Daucus carota) was used as an outgroup
표 Schematic representation of gene transfer between chloroplast and mitochondrial genome of Panax species. Each gray line within the circle shows the regions of chloroplast genome that have been inserted into different locations of mitochondrial genome. Colored boxes show conserved chloroplast genes, classified based on product function. Genes shown inside the circle are transcribed clockwise, and those outside the circle are transcribed counterclockwise
표 Validation of 18 dCAPS markers derived from CDS SNP regions of seven Panax chloroplast genomes. The 18 denoted dCAPS markers, Pgdm1 – 3, Pqdm4 – 6, Pndm7 – 9, Pjdm10 and 11, Pvdm12 and 13, Psdm14 – 16, Ptdm17 and 18 are unique specific for P. ginseng, P. quinquefolius, P. notoginseng, P. japonicus, P. vietnamensis, P. stipuleanatus, and P. trifolius, respectively. Abbreviated species names shown on amplicons: Pg, P. ginseng; Pq, P. quinquefolius; Pn, P. notoginseng; Pj, P. japonicus; Pv, P. vietnamensis; Ps, P. stipuleanatus; Pt, P. trifolius; M, 100-bp DNA ladder
표 Summary of GP calculation for major repeats in WGS data sets with various genome coverage of P. ginseng cv. Chunpoong
표 GP of 13 repeats in the 11 cultivars of Panax ginseng. (CP: cv. Chunpoong, CS: cv. Cheongsun, GO: cv. Gopoong, GU: cv. Gumpoong, HS: cv. Hwangsook, JK: cv. Jakyung, SH: cv. Sunhyang, SP: cv. Sunpoong, SU: cv. Sunun, SO: cv. Sunone and YP: cv. Yunpoong)
표 Genomic proportion of the major repeats in Panax species and a related species. Genomic proportion (GP) of 13 repeats in seven Panax species and the related species A. elata
표 Fluorescence in situ hybridization (FISH) analysis of PgDel1 and PgDel2 in P. ginseng and P. notoginseng chromosomes. The FISH signals of PgDel1 on somatic metaphase chromosomes in (a) P. notoginseng (purple), (b) P. ginseng and (c) P. quinquefolius. The FISH signals of PgDel2 on somatic metaphase chromosomes in (d) P. notoginseng, (e) P. ginseng (blue) and (f) P. quinquefolius (blue). Bar = 10 um
표 Summary of P . ginseng root transcriptome data acquired in this study
표 Unigene set and functional annotation of P . ginseng root transcriptome
표 GO annotation of root transcripts in P. ginseng. A total of 41,244 transcripts in the Nr unigene set were assigned to at least one GO term in three categories, i.e., biological process, molecular function, and cellular component
표 KEGG pathway assignments of P. ginseng root transcripts. Among Nr unigene sequences, 5,720 transcripts were assigned to KEGG pathways by KASS analysis. The percentage of the 5,720 transcripts assigned to the indicated pathways is shown
표 Expression profiles of differentially expressed transcripts among root samples. A total of 364 transcripts were identified to be differentially expressed among four root samples using the edgeR Bioconductor package based on individual FPKM values of three biological replicates for each root sample. Heatmap shows the hierarchical clustering of average FPKM values obtained from individual FPKM values of three replicates
표 Expression profiles of specifically expressed transcripts in each of four root samples. A total of 39 transcripts were found to be specifically expressed based on the criteria of FPKM value > 3 in one sample and < 1 FPKM in the other samples. Heatmap shows the hierarchical clustering of average FPKM values obtained from individual FPKM values of three replicates. A indicates one-year-old whole roots, and B, C, and D represent main bodies, lateral roots, and rhizomes of six-year-old root samples, respectively. Transcript IDs are shown to the right of the heatmap
표 Expression profiles of transcripts encoding enzymes involved in ginsenoside biosynthesis in P. ginseng. Enzymes functioning in each step of the MVA and MEP pathways are indicated by bold letters. The expression patterns of transcripts encoding these enzymes are shown in the heatmap, which was constructed using average FPKM values obtained from individual FPKM values of three replicates. a indicates one-year-old whole roots and b, c, and d represent main bodies, lateral roots, and rhizomes of six-year-old root samples, respectively. The number of transcripts identified is shown in parentheses to the left of heatmap
표 Expression profiles of 184 UGT genes identified in the Nr unigene set. Heatmap shows the hierarchical clustering of average FPKM values obtained from individual FPKM values from three replicates. A indicates one-year-old whole roots, and B, C, and D represent main bodies, lateral roots, and rhizomes of six-year-old roots, respectively
표 Venn diagram showing P. ginseng abiotic stress-responsive genes identified in this study. Differentially expressed (DE) genes were identified by comparison of gene expression under cold (4℃for 24hr), salt (100mM for 24hr), drought (air-drying for 24hr), and heat (30℃ for 1 week and 3 weeks) with control plants, using EdgeR with parameters of FDR adjusted P-vaule cutoff 0,01 and more than 2 fold changes
표 Classification of DE genes. The DE genes identified were subjected to annotation and GO analysis and functionally classified. DE genes related to transcriptional regulation were also separately grouped and sub-classified
표 22 P. ginseng samples used for Pacbio Iso-Seq analysis
표 Results of Pacbio Iso-Seq analysis of 22 P. ginseng samples
표 1. PCA loading plots of five Panax ginseng cultivars analyzed by UPLC-ESI/MS in negative mode
표 Relative abundance of eight ginsenosides in adventitious roots from five ginseng cultivars analyzed by UPLC-ESI/MS in negative mode
표 Transcriptome analysis of adventitious roots of CS (청선), SH (선향), SU (선운)
표 Proposed ginsenoside biosynthesis pathway in Panax ginseng
표 FPKM value of genes involved in ginsenoside synthesis pathway in adventitious roots from three ginseng cultivars (CS: 청선, SH: 선향, SU: 선운)
표 Expression level of the genes involved in ginsenoside biosynthesis pathway and relative abundance of intermediates in three ginseng cultivars (CS, SH, and SU). Expression levels of the genes were calculated by FPKM values and represented in log2. Differentially expressed unigenes among three ginseng cultivars (p<0.05) were indicated in blue. Relative abundance of intermediate compounds were analyzed by GC-MS and indicated by different letters in bar-graph
표 Expression levels of the genes involved in ginsenosides biosynthesis pathway and relative abundance of intermediates in the biosynthetic pathway in MeJA-treated adventitious roots from three ginseng cultivars (CS, SU, and SH). Expression level of each gene was analyzed by realtime PCR. Fold changes of genes expression level at 12 and 24 hr compared to that at 0 hr were represented with yellow-red color code using R. Relative abundance of intermediates was represented in bar-graph and those significant differences (p<0.05) were indicated by letters
표 Ginsenoside content analyzed by LC-UV-DAD in three ginseng cultivars adventitious roots following MeJA treatment for 7 days
표 Representative chromatograms of MJ-treated CS adventitious roots analyzed by UPLC-QTOF/MS
표 PCA and PCA loading plots of MJ-treated CS adventitious roots analyzed by UPLC-QTOF/MS
표 Identified metabolites in MeJA-treated or non-treated CS adventitious roots
표 Relative abundance of 20 marker compounds identified in MJ-treated or non-treated CS adventitious roots
표 Proposed ginsenosides biosynthesis pathway in Panax ginseng adventitious roots and characterized UGTs
표 Expression levels of genes and contents of metabolites involved in ginsenosides biosynthesis pathway in Panax ginseng adventitious roots at 0, 12, 24 and 48hr after MeJA treatment. The gene expression levels were calculated by FPKM value and the values were represented in heatmap with white/red color gradient
표 Expression pattern of candidate UGTs in MJ-treated CS adventitious roots. Expression values were calculated by FPKM value and represented by Heatmap with green-red color in Log2 scale. Different letters indicates the unigenes that those expression was significantly increased after MeJA treatment (p<0.05)
표 Growth pattern of P . ginseng adventitious roots for 56 day
표 Consumption pattern of major nutrients in liquid media culturing P. ginseg adventitious roots
표 Biomass composition of P . ginseng adventitious roots at 28 and 42 day
표 Ginseng root-specific metabolic network model
표 Representative 1H-NMR spectrum of adventitious root sample
표 Representative 1H-13CHSQC NMR spectrum of adventitious root sample. 1:Glucose, 2:Sucrose, 3:Fructose, 4:Glutamine, 5:myo-Inositol, 6:Pyroglutamate, 7:4-Aminobutyrate (GABA), 8:Aspargine, 9:Asparatate, 10:Alanine, 11:Choline, 12:Valine, 13:Threonine
표 Structures of quantified primary metabolites in adventitious roots
표 Representative HPLC-RI chromatogram of culture media
표 Representative BPI chromatograms of adventitious root and culture medium
표 Structures of identified ginsenosides from adventitious roots and media. G: ginsenoside, m: malonyl, Glc: glucose, Ara(p): arabinopyranose, Ara(f): arabinofuranose, Rha: rhamnose, GlcA: glucuronic acid
표 (A) Score plot and (B) loading plot of adventitious roots using NMR data
표 Score/loading plots of adventitious roots (A, B) and media (C, D) using LC-MS data
표 Quantification of identified sugars in media (a) and adventitious roots (b)
표 Quantification of identified other metabolites in adventitious roots
표 Heatmap representing ion intensities of metabolites from media (a), adventitious roots (b). G: ginsenoside, m: malonyl
표 Identification and classification of transcription factor (TF) genes in P. ginseng genome. A total of 3,588 TF genes were identified and classified into 66 families, of which top 10 abundant TF families were shown here, together with those from other 6 plant genomes
표 Identification and classification of transcriptional regulator (TR) genes in P. ginseng genome. A total of 851 TR genes were identified and classified into 25 families, of which top 10 abundant TR families were shown here, together with those from other 6 plant genomes
표 Identification and classification of protein kinase (PK) genes in P. ginseng genome. A total of 2,209 PK genes were identified and classified into 91 families, of which top 10 abundant PK families were shown here, together with those from other 6 plant genomes
표 Over-represented TF genes in P. ginseng genome. Seventeen over-represented (>=3.0 times) TF genes in P. ginseng genome were identified, as compared with carrot genome. Eua, Ast, and All indicate the over-represented P. ginseng genes when compared with average gene numbers of all Euasterid, all Asterid, and all 19 plant genomes, respectively
표 Over-represented TR genes in P. ginseng genome. Eight over-represented (>=3.0 times) TR genes in P. ginseng genome were identified, as compared with carrot genome. Ast indicates the over-represented P. ginseng genes when compared with average gene numbers of all Asterid plant genomes
표 Over-represented PK genes in P . ginseng genome. Twelve over-represented (>=3.0 times) PK genes in P. ginseng genome were identified, as compared with carrot genome. Eua, Ast, and All indicate the over-represented P. ginseng genes when compared with average gene numbers of all Euasterid, all Asterid, and all 19 plant genomes, respectively
표 Phylogenetic relationship of HSF genes. Phylogenetic tree was generated by the Neighbor-joining (NJ) method with 1,000 bootstraps using MEGA 6.0, after alignment of deduced amino acid sequences by MUSCLE. Classes A, B, and C were indicated by different line color. HSF genes that were not grouped into the three classes were indicated as not assigned. The bootstrap support values and taxon names are omitted for a legible illustration
표 Heatmap of 51 HSF genes in P. ginseng genome. Expression profiles in various tissues at various developmental stages (A) and under abiotic stresses (B and C) were determined using normalized FPKM values obtained from RNA-Seq data. Expression values (FPKM) were scaled per row (i.e. per gene) to visualize expression peaks of genes among samples. Heatmap was generated using heatmap.2 function provided by the R-package gplots. D1, adventitious roots; D2, seeds (stratified and stored at 4℃); D3, seeds (imbibed at 4℃ for 3 weeks); D4, seedlings of 30 days old; D5, leaves of 1-year old plants; D6, roots of 1-year old plants; D7, leaves of 5-year old plants; D8, stems of 5~6-year old plants; D9, main roots of 6-year old plants; D10, rhizomes of 6-year old plants; D11, lateral roots of 6-year old plants; D12, dormant roots of 7-year old plants; D13, flowers; D14, immature berries; D15, mature berries; S1, control plants (1-year old whole plants); S2, plants treated with 100 mM NaCl for 24 hr; S3, plants treated with cold (4℃) for 24 hr; S4, plants treated with drought (air-drying) for 24 hr; H1, leaves of control plants (1-year old, cv. Chunpoong); H2, leaves of plants (1-year old, cv. Chunpoong) treated with heat (30℃) for 1 week; H3, leaves of plants (1-year old, cv. Chunpoong) treated with heat (30℃) for 3 weeks; H4, leaves of control plants (1-year old, cv. Yunpoong); H2, leaves of plants (1-year old, cv. Yunpoong) treated with heat (30℃) for 1 week; H3, leaves of plants (1-year old, cv. Yunpoong) treated with heat (30℃) for 3 weeks
표 Phylogenetic tree of AP2/ERF family genes. The deduced amino acid sequences of total 1,108 genes from 6 plant genomes were aligned using MUSCLE and maximum likelihood tree was constructed by MEGA 6.0. Group I to IV belong to CBF/DREB subfamily and Group V to VL-L belong to ERF subfamily. The red bar at IX group of ERF subfamily indicates location of Apiales specific ERF gene cluster. Subfamilies and subgroups were indicated by different line colors. The bootstrap support values and taxon names are omitted for a legible illustration
표 Phylogenetic relationship of CSD protein genes. Phylogenetic tree was generated by the Maximum Likelihood (ML) method with 1,000 bootstraps using MEGA 7.0, after alignment of deduced amino acid sequences by MUSCLE. The bootstrap support values and taxon names are omitted for a legible illustration
표 Heatmap of CSD protein genes in P. ginseng genome. Expression profiles in various tissues at various developmental stages (A) and under abiotic stresses (B and C) were determined using normalized FPKM values obtained from RNA-Seq data. Expression values (FPKM) were scaled per row (i.e. per gene) to visualize expression peaks of genes among samples. Heatmap was generated using heatmap.2 function provided by the R-package gplots. D1, adventitious roots; D2, seeds (stratified and stored at 4℃); D3, seeds (imbibed at 4℃ for 3 weeks); D4, seedlings of 30 days old; D5, leaves of 1-year old plants; D6, roots of 1-year old plants; D7, leaves of 5-year old plants; D8, stems of 5~6-year old plants; D9, main roots of 6-year old plants; D10, rhizomes of 6-year old plants; D11, lateral roots of 6-year old plants; D12, dormant roots of 7-year old plants; D13, flowers; D14, immature fruits; D15, mature fruits; S1, control plants (1-year old whole plants); S2, plants treated with 100 mM NaCl for 24 hr; S3, plants treated with cold (4℃) for 24 hr; S4, plants treated with drought (air-drying) for 24 hr; H1, leaves of control plants (1-year old, cv. ChP); H2, leaves of plants (1-year old, cv. ChP) treated with heat (30℃) for 1 week; H3, leaves of plants (1-year old, cv. ChP) treated with heat (30℃) for 3 weeks; H4, leaves of control plants (1-year old, cv. YuP); H2, leaves of plants (1-year old, cv. YuP) treated with heat (30℃) for 1 week; H3, leaves of plants (1-year old, cv. YuP) treated with heat (30℃) for 3 weeks
표 Phylogenetic relationship of HRT genes. Phylogenetic tree was generated by the Maximum Likelihood method (ML) method with 1,000 bootstraps using MEGA 7.0, after alignment of deduced amino acid sequences by MUSCLE. The bootstrap support values and taxon names are omitted for a legible illustration
표 Heatmap of HRT genes in P. ginseng genome. EExpression profiles in various tissues at various developmental stages (A) and under abiotic stresses (B and C) were determined using normalized FPKM values obtained from RNA-Seq data. Expression values (FPKM) were scaled per row (i.e. per gene) to visualize expression peaks of genes among samples. Heatmap was generated using heatmap.2 function provided by the R-package gplots. D1, adventitious roots; D2, seeds (stratified and stored at 4℃); D3, seeds (imbibed at 4℃ for 3 weeks); D4, seedlings of 30 days old; D5, leaves of 1-year old plants; D6, roots of 1-year old plants; D7, leaves of 5-year old plants; D8, stems of 5~6-year old plants; D9, main roots of 6-year old plants; D10, rhizomes of 6-year old plants; D11, lateral roots of 6-year old plants; D12, dormant roots of 7-year old plants; D13, flowers; D14, immature fruits; D15, mature fruits; S1, control plants (1-year old whole plants); S2, plants treated with 100 mM NaCl for 24 hr;S3, plants treated with cold (4℃) for 24 hr; S4, plants treated with drought (air-drying) for 24 hr; H1, leaves of control plants (1-year old, cv. ChP); H2, leaves of plants (1-year old, cv. ChP) treated with heat (30℃) for 1 week; H3, leaves of plants (1-year old, cv. ChP) treated with heat (30℃) for 3 weeks; H4, leaves of control plants (1-year old, cv. YuP); H2, leaves of plants (1-year old, cv. YuP) treated with heat (30℃) for 1 week; H3, leaves of plants (1-year old, cv. YuP) treated with heat (30℃) for 3 weeks
표 Identification of lncRNAs in P . ginseng genomes
표 Identification of miRNAs in P . ginseng genomes
표 Multiple alignment of the deduced protein sequences of cloned four genes and Arabidopsis homologous genes. The conserved AP2/EREBP domain is indicated by the red boxes. Shaded boxes indicate conserved residues among compared proteins. The alignment was made using ClustalW and GeneDoc software
표 Expression profiles of cloned genes under MeJA treatment. Expression of cloned four genes, PgDREB-Ya1Z1, PgERF-Yh1Z12, PgERF-Yh1Z11, and PgERF-Yi1Z21, was analyzed by searching RNA-seq databases of MeJA (200 uM)-treated P. ginseng cv. Cheungsun (CS) and cv. Sunhyang (SH) for the indicated times on X-axis. FPKM values were determined by mapping of RNA-seq reads on those sequences and then relative expression was determined after normalization with FPKM value of PgActin gene. Vertical bars indicate the average relative expression value+standard error from three independent RNA-seq analyses. Significant difference was determined by comparison with control (0 h) using Student t-test at *p < 0.05 and **p <0.01. Fold-change value compared to control (0 h) is shown above bar
표 Expression profiles of cloned genes at various tissues and developmental stages. Expression of cloned four genes, PgDREB-Ya1Z1, PgERF-Yh1Z12, PgERF-Yh1Z11, and PgERF-Yi1Z21, was analyzed by searching RNA-seq databases of 15 P. ginseng samples representing various tissues and developmental stages. FPKM values were determined by mapping of RNA-seq reads on those sequences and then relative expression was determined after normalization with FPKM value of PgActin gene. A, seeds (stratified); B, seeds (imbibed at 4℃ for 3 weeks); C, adventitious roots; D, seedlings (30 days old); E, roots (1 year old); F, leaves (1 year old); G, main roots (6~7 years old); H, rhizomes (6~7 years old); I, lateral roots (6~7 years old); J, roots (dormant, 7 years old); K, shoot (without leaves); L, leaves of 6~5 year old plants); M, flowers; N, immature fruits (green); O, mature fruits (red)
표 Classification and expression of FAD genes. (a) Phylogenetic analysis of FAD genes. (b) Mapping coverage for coding (CDS) genes using 10x coverage WGS reads from tetraploid P. ginseng cv. YuP (white bars) and diploid P. vietnamensis (red bars).Orthologous FADs in diploid P. notoginseng (PN) denoted as * under the bar graphs. (c) TMM normalized expression of each gene under control (CTR), drought, salt and cold stress conditions. The FAD subclass is represented at the top to show expansion of specific sub classes and its role in abiotic stress responses
표 Callus growth rates of the two ginseng landraces and two cultivars
표 Duncan test for callus growth rates of the two ginseng landraces and two cultivars
표 Hairy roots of two ginseng landraces (A 괴산, D 강화) and two cultivars (B 금풍, C 연풍) cultured in solid media (½ Murashige and Skoog medium including cefotaxime and kanamycin) for 2 months
표 Efficiency of Agrobacterium rhizogenes transformation of two ginseng landraces (괴산, 강화) and two cultivars (금풍, 연풍)
표 Hairy roots of 강화 in solid and subsequent liquid media
표 Confirmation of transformation in 강화 hairy root genomic DNA or RNA. RolB and RolC genes are co-transformed into genome during transformation. WT: wild type (non-transformed), HR1: hairy root 1, HR2: hairy root 2, POS: positive control (GUS gene)
표 GUS histochemical staining of hairy roots from GUS-transformed landrace, Kanghwa. Hairy root was cultured in liquid media for a month. Blue color indicates the activity of transformed GUS that disassembles X-gluc to the primary reaction product, 5-bromo-4-chloro-3-indolyl, which is colorless and soluble but further oxidized and dimerized in the visualization reaction to form an insoluble and intensely blue final reaction product (indigo)
표 GC/MS로 확인한 클로닝된 몇몇 OSC의 기능. 클로닝된 OSC를 효모에서 발현시켰으 며 GC/MS로 확인한 total chromatogram과 주요 peak의 mass spectra를 보여준다. Peak 1: dammarenediol, Peak 2: α-amyrin, Peak 3: β-amyrin, Peak 4: cycloartenol, Peak 5: lupeol, Peak ‘#’: lanosterol, Peak ‘&’:taraxasterol-like, Peak ‘*’:cycloartenol-related (based on mass spectra)
표 다양한 oxidosqualene cyclase에 의한 여러 가지 물질의 생합성 경로
표 GC/MS로 확인한 CAS2 돌연변이체의 기능. 클로닝된 OSC를 효모에서 발현시켰으며 GC/MS로 확인한 total chromatogram과 주요 peak의 mass spectra를 보여준다. 각 peak에 대한 물질은 Fig. III-7-1과 같다
표 GC/MS로 확인한 DDS2 돌연변이체의 기능. 클로닝된 OSC를 효모에서 발현시켰으며 GC/MS로 확인한 total chromatogram과 주요 peak의 mass spectra를 보여준다. 각 peak에 대한 물질은 Fig. III-7-1과 같다
표 Probe labeling을 위한 stock solution 및 labelling 반응
표 Direct-labelled multi-color FISH (mcFISH) using five different fluorochromes for simultaneous cytogenetic mapping of repetitive DNA elements in P. ginseng. A) PgDel1 (Cy5-white) showing disperse genomic hybridization, B) PgDel2 (DEAC-blue) showing subgenomic pericentromeric hybridization to 24 chromosomes, C) PgTRb (FAM-green) distribution to 24 chromosomes, D) Total PgTR signals (Texas Red- red) showing hybridization to all chromosomes, E) Telomeric repeat of TTAGGG (Cy3-orange) showing telomeric hybridization, and F) merged signals of all probes
표 Genomic distribution of PgDel2 (Choi et al, 2014, green arrows) and PgDel5 show distinct subgenome distribution albeit presence of some colocalization in some chromosomes
표 Top 30 contigs from dnaLCW analysis with description form customized ginseng repeat database
표 List of CACTA elemnets characterized in this analysis
표 Characterization of a ginseng CACTA element (PgCACTA) showing the average depth of mapped whole genome sequence reads to reference PgCACTA element. Conservation of element regions such as tnpD, TnpA, and Pg167TR regions are shown from blast of the element to the current ginseng genome assembly. Low GC content, an indicator of CACTA insertion sites, are shown at both ends of the PgCACTA element. Conservation of the terminal inverted repeats are shown at the bottom row
표 Diagrammatic representation of representative PgCACTA elements. Black graphs at the upper portion of each element represent mapping depths of 1x coverage WGS reads to each element at log 100 scale. Transposase coding sequences are shown, corresponding to the highly conserved regions of the elements. Pg167TR units at the 3
표 Phylogenetic analysis of conserved tnpD transposase regions present in the ginseng genome. A) Phylogenetic tree of 11 PgCACTA elements with conserved and non-truncated tnpD regions showing two major groups. Stars indicate elements used to quantity PgCACTA element in the ginseng genome. B) Phylogenetic tree of all conversed tnpD sequences in the ginseng genome supporting the grouping in panel A. Marked elements represent the 11 elements in panel A, while the shaded marks indicate elements used for quantification. C) Quantification of PgCACTA elements in the genus Panax and those outside the genus but related species
표 List of regulatory elemnet motifs found in PgTR sequences
표 Sequence characterization of Pg167TR revealed two major groups with high curvature propensity. A) Multiple alignment of Pg167TR from representative PgCACTA elements and top blast hits from scaffold sequences. Elements ranged from 140~168bp. Red and blue background denote high and low GC content, respectively. B) Curvature propensity plot of three Pg167TR units. Curvature peaks at the left panel correspond to high AT content, Pg167TR unit curvature simulation is presented at the left panel. This shows the contribution of satellite DNAs in DNA condensation. C) K-mer based phylogenetic tree of sequences from panel A showing two major groups (1 and 2), with three subgroups of group 2. Analysis of sequences revealed sequence polymorphism at nt 110~112 showing AGT and TCG major types
표 Reference mapping of whole genome sequence reads to Pg167TRa and Pg167TRb showed differential abundance of each type within Panax and outside Panax. A and B) Genome representation (GR) of each Pg167TR type in kb within and without the genus Panax repectively. It is important to note that diploid species have considerably lower Pg167TR than tetraploid species. In particular, P. ginseng show a burst in Pg167TR amplification compared with its closely related tetraploid species, P. quinquefolius. C and D) Ratio of Pg167TRa and Pg167TRb within and without the genus Panax. It is important to note that Pg167TRa type is more abundant (up to >80 of all Pg167TR in the genus Panax, while the opposite is true in related species outside the genus. This implies evolutionary contribution of Pg167TR in shaping the species in Panax and related species
표 PCR-based PgTRa and PgTRb probe desgin. Differential forward PCR primers were designed from the TCG/AGT regions of both PgTRs, reverse primers were the common for both sequences
표 A) PCR amplification of PgTRa and PgTRb using subgroup-specific primers. B) High-molecular weight PgTRa amplicons were digested with TaqI restriction enzyme
표 BLAST estimation and sequence similarity index of 18 variant oligonucleotides. Polymorphic regions of PgTR units after multiple sequence alignment were extracted and subject to BLAST against Panax ginseng v.1 assembly to estimate their respective abundance at e-value of 1.0. PgTRa and PgTRb has similar hits, but PgTRa (var_a) has the hightest hit
표 Distribution of oligoprobes PgTRa (Var_a, A) and PgTRb (B). Overlapped image of panels A and B is shown in panel C. PgTRa (Var_a) hybridized to all chromosomes with different pattern from that of PCR-based combined PgTRa/PgTRb. 20 chromosomes hybridized with PgTRb (arrows, four are ambiguous, compared to that in Fig. 7). 12 chromosomes show PgTRa-specific signals (blue arrowheads). Ten chromosomes showed very intense PgTRa signals but weak colocalized PgTRb (pink arrows). Eight chromosomes showed intense signals for both PgTRTs (white arrows). The distal PgTR locus in chromosome 3 showed PgTRb-specificity (yellow arrows). Bars=10 μm
표 A) Karyogram showing PgTRb distribution. Asterisks show the 12 chromosomes bearing PgTRb loci. B) Karyotype idiogram of P. ginseng somatic metaphase complement based on chromosome length and tandem repeat and retrotransposon cytogenetic markers. Blue, green, red, and yellow bars indicate DAPI, 5S rDNA, 45S rDNA, and PgTR bands. PgTR bands with red borders indicate PgTRb. Purple and outlined chromosomes represent PgTRb and PgDel2-rich (Choi et al, 2014) chromosomes, respectively. Bars= 10 μm
표 Chromosome-specific micro- and satellite repeats in P. ginseng
표 Chromosomal distribution of novel chromosome-specific tandem repeats in P. ginseng. PgCL79 has homologous site at nt 66~95 to the IGS region of P. ginseng 45S rDNA
표 Characterization of a novel chromosome-specific satDNA in P. ginseng, PgCL79. A) Repeat analysis revealed a 201-bp satDNA with homologous site at nt 66~95 to the IGS region of P. ginseng 45S rDNA. FISH data revealed strong signal at chromosome 8 (red arrows) while also revealing signals at the NOR site (chr. 14, yellow arrows). B) Quantification of PgCL79 abundance by WGS read mapping revealed relative abundance in a diploid relative, P. japonicus, suggesting that the P. ginseng 45S rDNA is highly homologous with that of P. japonicus
표 A) Design of PCR primers from unique regions (red and blue bars above scaffold) from adjacent scaffolds. To analyze the assembly of Pg_scaffold0266 and Pg_scaffold2259, unique regions with a total length of 26.4 and 44.4 kb were designed, respectively. The abundance of repeats in Pg_scaffold0266 caused a looser distribution of target size compared with Pg_scaffold2259. Upper and lower syntenic regions show two recent paralogous sites that share more ancient paralogous sites. With this design, we expect a range of 4~8 signals in root metaphase chromosomes, and 2~4 signals in pachytene chromosomes, that could vary in intensity or fragmentation. B) PCR amplicons derived from the designed primers. All targeted products were amplified. In some cases, four signals were amplified, reflecting tetraploidy and length polymorphism of the genic region. C, D) FISH of unique sequences from Pg_scaffold0266 (green) and Pg_scaffold2259 (red) on Panax ginseng pachytene chromosomes. These two pachytene images show the co-localization of both scaffold signals (yellow arrows) indicating the area where Pg_scaffold0266 and Pg_scaffold2259 localize. Independent Pg_scaffold0266 (green arrow) and Pg_scaffold2259 (red arrow) signals indicate corresponding paralogous sites. Inset diagram depicts the actual distribution of the FISH signals. Bar=10 μm
표 Left panel: Detection of OsCENH3 in one O. sativa nucleus. Middle: Characterization of PgCENH3 amino acid sequence. Red bar denotes the region from which antibody was designed. Right panel: Initial immunocytochemistry test of PgCENH3 antiserum before affinity purification for specificity of antibody
표 Western blot to check specific affinity of our designed antibody to PgCENH3. Once purified, it can then be used for immunodetection using cytogenetic techniques
표 Identification a low-abundance 168-bp satellite repeat (left panel) with a 71-bp (66..136) region homologous to Pg167TR (right panel). Green and pink arrows indicate PCR primers and oligoprobe for FISH analysis, designed in regions
표 Preliminary FISH result using PgCL82_op_4 as FISH probe showed faint centromeric signals to several chromosomes (white arrows to selected chromosomes with centromeric signals). Bar = 10 μm
표 GISH analysis of three diploid ginseng related species, P. notoginseng, P. japonicus, and P. vietnamensis to P. ginseng chromosomes. The three genomes hybridized to mostly similar chromosomal regions with varying intensity, while about 12 chromosomes showed no or weak signals (white arrows). This reveals the homology of the 45S rDNA between P. japonicus and P. ginseng (circles), corroborating data from 그림 2. Bars = 10 ìm
표 FISH analysis of rDNA and Pg167TR in P. quinquefolius (2n = 48. A) 5S rDNA signal B) 45S rDNA signals C) Pg167TR signals D) merged signals. Intense grey "knobs" are inherent DAPI bands. E) FISH karyogram utilizing rDNA and Pg167TR repeats. Bars = 10 μm
표 FISH analysis of rDNA and Pg167TR in P. notoginseng 2n = 48. A) 5S rDNA signal B) 45S rDNA signals C) Pg167TR signals D) merged signals. E) FISH karyogram utilizing rDNA and Pg167TR repeats. Bars = 10 μm
표 FISH analysis of rDNA and telomere in Aralia elata (2n = 24). A) 5S rDNA signal B) 45S rDNA signals C) TTTAGGGn telomere signals D) merged signals, and E) FISH karyogram. Bars = 10 μm
표 FISH analysis of rDNA and telomere in Eleutherococcus sessiliflorus (2n = 48). A) 45S rDNA signal B) 5S rDNA signals C) TTTAGGGn telomere signals D) merged signals, and E) FISH karyogram. Bars = 10 μm
표 FISH analysis of rDNA and telomere in Kalopanax septemlobus (2n = 48). A) 45S rDNA signal B) 5S rDNA signals C) TTTAGGGn telomere signals D) merged signals, and E) FISH karyogram. Bars = 10 μm
표 FISH analysis of rDNA and telomere in Dendropanax morbifera (2n = 48). A) 45S rDNA signal B) 5S rDNA signals C) TTTAGGGn telomere signals D) merged signals, and E) FISH karyogram. Bars = 10 μm
표 Major repeats found in the Araliaceae species analayzed
표 Raw FISH signals of Aral_CR160 to A) A. elata B) D. morbifera, and C) E. sessiliflorus, showing centromeric hybridization to all species but with sequence divergence. Bars = 10 μm

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인삼 유전체 완성도 향상 및 유전다양성 연구
Genome sequencing and genomic diversity analysis of ginseng and its relatives 원문보기