차세대 염기서열 분석법을 이용한 더덕의 SSR마커 개발

다국어 초록 (Multilingual Abstract)

Codonopsis lanceolata is an herbaceous perennial plant. It is predominantly cultivated in East Asia, and is used as a medicinal plant as well as a vegetable. However, there is a lack of research about genetic studies of C. lanceolata. Therefore, we sequenced genomic DNA using next generation sequencing (NGS) and searched the simple sequence repeats (SSRs) for marker development in C. lanceolata. A total of 250,455 SSRs were identified and dinucleotides and trinucleotides accounted for the majority of all the SSRs. A total of 26,334 primer sets were designed from dinucleotide to octanucleotide motifs. We investigated 2,626 SSRs (trinucleotide to pentanucleotide motifs) and found 573 SSRs showed in silico polymorphism. One-hundred seventy-seven SSR primer sets were selected to confirm that the 573 SSRs showed polymorphism in silico. As a result, 160 primer sets amplified expected amplicons and all of them showed polymorphism. From the results of genotyping using 39 SSR markers, the number of alleles ranged from 2 to 13, and the mean value of PIC was 0.54. In addition, 33 SSR markers were applied to 26 C. lanceolata collections (95 individual plants), and genetic diversity and population structure analysis were performed. Genetic relationship analysis showed that C. lanceolata collected from South Korea were not grouped by collection sites and showed genetic diversity. The results of population structure analysis showed that the members of most of the C. lanceolata collections were not grouped into one population and were considered an admixture. Consequently, we developed SSR markers from C. lanceolata that were polymorphic enough to analyze genetic diversity even though the markers did not distinguish the C. lanceolata by collection sites.

목차 (Table of Contents)

• Ⅰ. Introduction 14
• Ⅱ. Material and Methods 21
• 1. SSR marker development 21
• 1.1. Plant material 21
• 1.2. Next generation sequencing 23
• 1.2.1. DNA extraction 23
• 1.2.2. Genomic DNA sequencing 24
• 1.2.3. Sequence assembly 25
• 1.3. SSR marker development 26
• 1.3.1. SSR finding and primer design 26
• 1.3.2. In silico polymorphic SSR searching 27
• 1.3.3. SSR primer screening 28
• 1.3.4. Validation of the SSR markers 29
• 2. Genetic diversity and population structure analysis 30
• 2.1. Plant material 30
• 2.2. DNA extraction 33
• 2.2. PCR amplification and capillary electrophoresis 34
• 2.4. Genotyping and data analysis 35
• Ⅲ. Results 37
• 1. Next generation sequencing 37
• 1.1. Sequencing and assembly 37
• 1.2. Identification and characterization of SSR 41
• 2. SSR marker development 43
• 2.1. SSR finding and primer design 43
• 2.2. In silico polymorphic SSR marker searching 47
• 2.3. SSR primer screening 48
• 2.4. Validation of SSR markers 48
• 3. Genetic diversity and population structure analysis 54
• 3.1. Genetic diversity analysis 54
• 3.2. Phylogenetic relationship of Codonopsis lanceolata 57
• 3.3. Population structure analysis 61
• Ⅳ. Discussion 68
• Ⅴ. References 72
• Abstract in Korean 81