Title Page
Contents
ABSTRACT 13
GENERAL INTRODUCTION 17
CHAPTER 1. Taxonomic status and phylogeny of Veratrum section Veratrum (Melanthiaceae) in Korea and Japan based on chloroplast and nuclear sequence data 22
I. INTRODUCTION 23
II. MATERIALS AND METHODS 29
A. Plant materials 29
B. Selecting DNA regions 29
C. DNA extraction, PCR amplification, and Sequencing 30
D. Alignment and phylogenetic analysis 31
III. RESULTS 33
A. Nuclear DNA analysis with ITS 33
B. Chloroplast DNA analysis with matK, psbA-trnH, rpL16, and trnS-trnG 34
C. Combined nuclear and chloroplast DNA analysis 38
D. Distributions of cpDNA haplotypes in Veratrum section Veratrum 39
IV. DISCUSSION 41
A. Taxonomic status Veratrum stamineum in Japan 41
B. Species delimitation and phylogenetic relationships of Veratrum album complex in Korea and Japan 42
CHAPTER 2. Development and characterization of microsatellite markers for Veratrum album complex in Korea by Illumina MiSeq 71
I. INTRODUCTION 72
II. MATERIALS AND METHODS 75
A. Sampling and DNA extraction 75
B. NGS and primer design 75
C. Polymer chain reaction (PCR) and genotyping 77
D. Data analysis 78
III. RESULTS AND DISCUSSION 79
IV. CONCLUSIONS 82
CHAPTER 3. Physiological activities of Veratrum oxysepalum extracts in Korea 98
I. INTRODUCTION 99
II. MATERIALS AND METHODS 103
A. Plant materials and preparation of extracts of Veratrum oxysepalum 103
B. Investigation of physiological activities of Veratrum oxysepalum 104
C. Data analysis 112
III. RESULTS AND DISCUSSION 113
A. Anti-oxidant activity of Veratrum oxysepalum 113
B. Anti-atopic activity of Veratrum oxysepalum 114
C. Anti-inflammatory activity of Veratrum oxysepalum 116
D. Immunostimulating activity of Veratrum oxysepalum 118
IV. CONCLUSION 122
REFERENCES 135
Appendix 152
Appendix 1. Sequence variations in DNA regions studied of section Veratrum within ingroup 152
Appendix 2. Allele frequencies of seven microsatellite markers in five populations of Veratrum album complex in Korea 156
국문초록 158
Table 1-1. Subclassification and species of Veratrum section Veratrum according to... 49
Table 1-2. Distribution of taxa of section Veratrum occur in Korea, Japan, and neighboring regions. 51
Table 1-3. Morphological characters of the section Veratrum in Korea and Japan from original description by Loesener (1926, 1927)... 52
Table 1-4. Listed species in section Veratrum in Korea and Japan treated in The Plant List... 53
Table 1-5. Collection and voucher information for taxa used in this study, and GenBank accession numbers assigned to each DNA region. 54
Table 1-6. ITS, matK, psbA-trnH, trnS-trnG, and rpL16 primers in PCR and sequencing. 56
Table 1-7. Summary statistics of parsimony analysis for the genomic regions of all samples. 57
Table 1-8. Haplotypes of section Veratrum within ingroup from four cpDNA regions (matK,... 58
Table 2-1. Eleven tested microsatellite markers which developed in previous studies. 83
Table 2-2. Characteristics of seven microsatellite primer pairs developed in Veratrum album complex. Colored characters indicate... 84
Table 2-3. Results of next generation sequencing and microsatellite search from JJ (Jeju)... 85
Table 2-4. Characteristics of the seven microsatellite markers analyzed in five populations... 86
Table 2-5. Genetic diversity of five populations of Veratrum album complex in Korea on seven microsatellite loci. N: number of individuals... 87
Table 3-1. Extraction yield of Korean Veratrum oxysepalum from 10 populations. The... 124
Table 3-2. IC50 values (µg/ml) obtained in the anti-oxidant assay of DPPH scavenging of...(이미지참조) 125
Fig. 1-1. Taxa of section Veratrum photographed in native habitats within Korea and Japan. A: V.... 60
Fig. 1-2. Sampling sites for taxa of section Veratrum in Korea and Japan. Abbreviations... 61
Fig. 1-3. Strict consensus of 24 equally most parsimonious trees (TL=89, CI=0.978,... 62
Fig. 1-4. Maximum likelihood tree resulting from analysis of nuclear ITS DNA sequence... 63
Fig. 1-5. Strict consensus of 541 equally most parsimonious trees (TL=242, CI=0.938,... 64
Fig. 1-6. Maximum likelihood tree resulting from combined analysis of matK, psbA-trnH,... 66
Fig. 1-7. Strict consensus of 1,623 equally most parsimonious trees (TLT=290, CI=0.962,... 67
Fig. 1-8. Maximum likelihood tree resulting from combined analysis of nuclear ITS and... 68
Fig. 1-9. Median-joining haplotype network constructed for matK, psbA-trnH, rpL16, and trnS-trnG cpDNA sequence data for section Veratrum. 69
Fig. 2-1. Presence of indel in amplified regions using microsatellite marker developed by previous study. 88
Fig. 2-2. Five populations of Veratrum album complex sampled in Korea (AS=Mt. An;... 89
Fig. 2-3. Amplification scheme for PCR fragments using three primer system (Schuelke... 90
Fig. 2-4. The percentages of SSRs found from JJ and SB samples using Illumina MiSeq... 91
Fig. 2-5. The interpretation of polymorphic allele peaks of KVera131V locus derived from... 92
Fig. 2-6. Allele frequency distributions of seven loci in five populations of Veratrum album complex in Korea. Abbreviations were... 94
Fig. 2-7. Expected heterozygosity (HE) and observed heterozygosity (HO) across the seven...(이미지참조) 96
Fig. 2-8. Allele patterns across the five populations of V. album complex revealed by the... 97
Fig. 3-1. Chemical structure of (A) veratridine and (B) cevadine. 126
Fig. 3-2. Collection sites of Veratrum oxysepalum in Korea. 127
Fig. 3-3. DPPH radical scavenging activity of VOE. (A) aerial part (B) underground part. Abbreviations were followed Fig. 3-2. 128
Fig. 3-4. Effects of VOE on HaCaT cell viability. (A) aerial part (B) underground part. HaCaT cells were treated with 50, 100, 200... 129
Fig. 3-5. Effects of VOE on MDC/TARC production in TNF-α/IFN-γ-stimulated HaCat cells. (A) aerial part (B) underground part.... 130
Fig. 3-6. Effects of VOE on NO production and cytotoxicity in LPS-stimulated RAW 264.7 cells. (A) aerial part (B) underground part.... 131
Fig. 3-7. Effects of VOE on NO production and cell viability in RAW 264.7 cells. (A) aerial part (B) underground part. RAW 264.7... 132
Fig. 3-8. Effects of VOE on iNOS and COX-2 protein expression in LPS-stimulated RAW... 133
Fig. 3-9. Effects of VOE on LPS-stimulated TNF-α, IL-4, IL-10 production in RAW 264.7... 134