CYTOGENETIC AND CYTOGENOMIC ANALYSES OF THREE MAJOR MEDICINAL PLANTS OF GENUS SENNA, SOLANUM NIGRUM COMPLEX, AND ARALIACEAE FAMILY USING RECENTLY DEVELOPED FISH TECHNIQUE = 주요 약용 식물 결명자속, 까마중속 및 두릅나무과 식물에 대한 새로운 FISH 기법을 활용한 세포유전학적 및 세포유전체학적 분석

다국어 초록 (Multilingual Abstract)

GENERAL ABSTRACT
Cytogenetic and Cytogenomic Analyses of Three Major Medicinal Plants of
Genus Senna, Solanum nigrum Complex, and Araliaceae Family
Using Recently Developed FISH Technique
by Hong Thi Nguyen
Medicinal plants have long been foundational to traditional medicine and pharmacology, with their therapeutic effects deeply rooted in complex genomic structures and evolutionary histories. Advances in plant genomics and cytogenetics have equipped researchers with powerful tools to explore these genetic foundations, enhancing our understanding of genome organization, chromosomal dynamics, and evolutionary pathways contributing to medicinal efficacy. Cytogenetic and cytogenomic markers, such as DNA tandem repeats, unique sequences or chloroplast DNA, are essential for mapping plant genomes, tracing phylogenetic relationships, and understanding speciation. This study leverages these markers through advanced molecular techniques and bioinformatics to investigate three medicinally valuable plant taxa: genus Senna, Solanum nigrum complex, and Araliaceae family. Each chapter of this research highlights a unique aspect of these plants' genomic structures, offering insights into their chromosomal evolution and the genetic basis underlying their medicinal properties.
In the first chapter, we investigated the medicinally significant Senna genus within the Fabaceae family, focusing on the role of tandem repeats (TRs) in genome organization and karyotype evolution across 18 species. Using fluorescence in situ hybridization (FISH) with Senna tora-specific oligo probes, the study revealed diverse TR distribution patterns influenced by polyploidy and dysploidy, showing significant variations in chromosome numbers across species. Among the TRs analyzed, StoTR01_86 was generally conserved, while StoTR04_55 was exclusive to S. obtusifolia, and StoTR03_178 displayed different localization patterns depending on ploidy levels—appearing in pericentromeric regions in dysploid species, subtelomeric regions in diploids, and both regions in polyploids. TRs were especially abundant in species with dysploid karyotypes, concentrating in (peri)centromeric regions known as hotspots for chromosomal rearrangement. These findings reveal the complex evolutionary dynamics within the Senna genus, enhancing our understanding of karyotypic diversity and genome evolution in medicinal plants.
Chapter 2 explored to the chloroplast genomes of seven Senna species, utilizing next-generation sequencing to address gaps in their phylogenetic relationships. The chloroplast genomes exhibited a quadripartite structure and conserved gene content, with eight identified hotspots serving as potential species-specific markers. Phylogenetic analysis based on these genomes clarified the evolutionary relationships within Senna, offering a foundation for studies in population genetics and ecological adaptation. This chapter underscores the utility of chloroplast DNA in advancing phylogenetic research and understanding lineage evolution within a single medicinal genus.
Chapter 3 examined the Solanum nigrum complex within the Solanaceae family, known for its agricultural and medicinal significance, using an advanced oligo-based FISH barcoding technique to facilitate high-resolution chromosomal identification across S. americanum, S. villosum, and S. nigrum. As the largest genus in Solanaceae, Solanum includes major crops like potatoes, tomatoes, and eggplants, with the wild relatives in the S. nigrum complex providing valuable insights into polyploid evolution and genetic diversity. The study introduces oligo-FISH barcoding, which employs 60,000 oligos targeting single-copy DNA regions to generate unique fluorescence "barcodes" for each chromosome, revealing extensive karyotypic conservation and identifying chromosomal rearrangements across species. These findings support the hypothesis that S. americanum may be a progenitor of both S. villosum and S. nigrum, contributing chromosomal sets to these polyploids, emphasizing the genetic diversity and adaptability gained through polyploidization. This research offers a crucial cytogenomic framework for understanding polyploid evolution in Solanum and highlights the potential of oligo-FISH barcoding for enhancing taxonomic, phylogenetic, and breeding studies across Solanaceae, especially in improving crop traits like disease resistance and stress tolerance.
Chapter 4 investigated the Araliaceae family, widely valued for its medicinal properties, focusing on tandem repeats to explore chromosomal evolution and speciation. Using a read clustering algorithm and whole genome sequencing of ten available Araliaceae species, four family-specific TRs (ArTR_01, ArTR_02, ArTR_03, and ArTR_04) were identified. FISH analysis revealed that ArTR_04, a 160 bp repeat, was prevalent in the centromeric regions across most Araliaceae species but notably absent in Panax ginseng, suggesting structural divergence within the family and a possible role in chromosome stability. This study highlights the significance of centromeric TRs as cytogenomic markers, contributing to a comprehensive cytogenomic framework for Araliaceae, which offers valuable tools for chromosome identification, structural variation tracking, and breeding research. These insights are foundational for advancing genomic research, taxonomy, and breeding strategies, ultimately supporting the conservation and enhancement of economically valuable Araliaceae species.
Collectively, these four chapters provide a comprehensive approach to understanding the genetic architecture and evolutionary mechanisms of medicinally significant plants. Through the integration of cytogenetic and cytogenomic techniques - PLOP-FISH, oligo-based chromosome barcoding, chloroplast genome sequencing, and bioinformatics—this research contributes to plant cytogenomics. These data are a useful foundation for future studies on genetic diversity, genome evolution, and species differentiation, with applications in conservation, pharmacology, and plant breeding.

목차 (Table of Contents)

• ACKNOWLEDGEMENTS I
• LIST OF FIGURES IX
• LIST OF TABLES XII
• LIST OF ABBREVIATIONS XIII
• GENERAL INTRODUCTION 4
• LITERATURE REVIEW 8
• A. Cytogenetic markers 8
• 1. Ribosomal DNA and telomeric repeats 8
• 2. Tandem repeats 9
• 3. Genome size estimation using flow cytometry 9
• B. Cytogenomic markers 10
• 1. PLOP-FISH and advanced chromosomal mapping 11
• 2. Barcode oligo-FISH for comparative chromosome mapping 11
• 3. Chloroplast genome sequences 11
• C. The medicinal plants: Senna, Solanum nigrum complex, and Araliaceae species 12
• 1. Senna species 13
• 2. Solanum nigrum complex 13
• 3. Araliaceae species 14
• REFERENCES 15
• CHAPTER 1: CHROMOSOMAL DYNAMICS IN SENNA: COMPARATIVE PLOP–
• FISH ANALYSIS OF TANDEM REPEATS 20
• ABSTRACT 21
• I. INTRODUCTION 22
• II. MATERIALS AND METHODS 24
• A. Plant materials 24
• B. Chromosome spread preparation 25
• C. Repeat mining, probes preparation, and fluorescence in situ hybridization
• (FISH) 25
• III. RESULTS 29
• A. Chromosome counts 29
• B. Distribution of nine S. tora-TR probes 29
• IV. DISCUSSION 56
• V. CONCLUSION 62
• REFERENCES 63
• CHAPTER 2: THE COMPLETE CHLOROPLAST GENOME SEQUENCES OF
• SEVEN SENNA SPECIES: INSIGHTS INTO GENOME EVOLUTION AND
• PHYLOGENETIC RELATIONSHIPS WITHIN GENUS SENNA 69
• ABSTRACT 70
• I. INTRODUCTION 71
• II. MATERIALS AND METHODS 74
• A. Plant material and DNA extraction 74
• B. Chloroplast genome sequencing, assembly, mapping, annotation, and
• structure 74
• III. RESULTS 78
• A. Chloroplast genome structure and features 78
• B. Repeats structure analysis 82
• C. Codon usage bias 83
• E. Comparative chloroplast genomic analysis within seven Senna species 87
• F. Divergent hotspots in the Senna cp genomes 88
• G. Phylogenetic analysis 89
• IV. DISCUSSION 91
• A. Chloroplast genome structure and features 91
• B. Repeats structure analysis 91
• C. Codon usage bias 92
• D. Inverted repeat contraction and expansion 92
• E. Comparative chloroplast genomic analysis 93
• F. Divergent hotspots in the Senna cp genomes 93
• G. Phylogenetic analysis 94
• V. CONCLUSION 95
• REFERENCES 96
• CHAPTER 3: POLYPLOID EVOLUTION AND CHROMOSOMAL DYNAMICS IN
• THE SOLANUM NIGRUM COMPLEX: INSIGHTS FROM OLIGO-FISH
• CHROMOSOME BARCODING 101
• ABSTRACT 102
• I. INTRODUCTION 104
• II. MATERIALS AND METHODS 107
• A. Plant materials 107
• B. Chromosome barcode design 107
• C. Chromosome preparation 108
• D. Fluorescence in situ hybridization (FISH) 108
• III. RESULTS 110
• A. Development of oligo-FISH barcode probes for chromosome identification
• using single-copy sequences 110
• B. Karyotyping of S. americanum 112
• C. Karyotyping of S. villosum using S. americanum chromosome barcode 114
• D. Karyotyping of S. nigrum using S. americanum chromosome barcode 117
• IV. DISCUSSION 123
• V. CONCLUSION 125
• REFERENCES 126
• VIII
• CHAPTER 4: COMPARATIVE CYTOGENOMICS OF ARALIACEAE: GENOME
• SIZE EXPANSION, CHROMOSOME EVOLUTION, AND CONSERVATION OF
• FAMILY-SPECIFIC TANDEM REPEATS 129
• ABSTRACT 130
• I. INTRODUCTION 131
• II. MATERIALS AND METHODS 133
• A. Plant materials 133
• B. Repeat mining and quantification 134
• C. Probe design 137
• D. Chromosome counting and FISH karyotype analysis 139
• E. Flow cytometry and genome size estimation 139
• III. RESULTS 141
• A. Chromosomal distribution of rDNAs and telomeric repeat 141
• B. Genome size estimation 142
• C. Holoploid and monoploid genome sizes 146
• D. Total and monoploid karyotype lengths 147
• E. FISH revealed a predominant (peri)centromeric distribution of Araliaceae
• family-specific tandem repeats 147
• IV. DISCUSSION 151
• A. Genome size and chromosomal information in the evolutionary context 151
• C. Genome dynamics in Araliaceae 152
• D. Family-specific TRs in Araliaceae species 153
• E. Direction in Araliaceae evolutionary cytogenomics research 154
• V. CONCLUSION 156
• REFERENCES 157
• 국문 초록 161