Utilization of low-coverage whole genome sequences for diversity analysis in plant genomes

학위논문 (박사)-- 서울대학교 대학원 농업생명과학대학 식물생산과학부, 2017. 8. 양태진.Recently, a rapid progress was achieved for whole-genome shotgun sequencing (WGS) based on support of next generation sequencing (NGS) technology. Although NGS greatly enhanced WGS, obtaining the complete reference genome sequence is a big challenge and is usually geared towards model plants or major crops based on massive WGS data and a variety of supporting data. Meanwhile, genomes of most resource plants are still unrevealed. In this study, a multi-directional approach was taken to understand the genome of resource plants, which have no previous genomic data, with only low coverage WGS data. This research was conducted to obtain a variety of fundamental information and nucleotide diversity on the inter- or intra-species level: assembly of complete chloroplast (cp) genome and nuclear ribosomal DNA (nrDNA) sequences, development of a tool to detect polymorphic single sequence repeat (pSSR) markers, and quantification of major repeats among relative species in various plant genomes. In the first chapter, complete cp genome and nrDNA sequences for two genotypes of Euonymus hamiltonianus, a medicinal and ornamental plant, were obtained. Furthermore, a pipeline to identify WGS reads harboring simple sequence repeat (SSR) motifs and develop pSSR by systematic comparison of two WGS reads was developed. The pipeline is composed of several steps which include end joining of paired reads, isolation of WGS reads harboring SSR motifs, identification of SSR reads derived from unique non-repetitive regions, identification of pSSR via comparison of counterpart WGS reads derived from another individual plant, design of pSSR primer sets and validation. Phylogenetic analysis using assembled and complete 157,360 bp of cp genome and 5,824 bp of 45S nrDNA was conducted. A total of 161 pSSR contigs showing polymorphism were identified between the two different E. hamilotnianus genotypes. Among them, 20 primer pairs were designed and seven were validated as real pSSR markers. In the second chapter, the pipeline for pSSR was applied to Peucedanum japonicum which is an indigenous medicinal and edible plant in Korea. A total of 452 pSSR candidates were identified between two P. japonicum. Among them, ten primer pairs were designed, nine of which were validated as real pSSR markers for seven P. japonicum genotypes. In the third chapter, the genome proportion of the major repeats was measured using small amount of WGS data for five Panax species and a related species Aralia elata. The diploids P. japonicus, P. vietnamensis, and P. notoginseng and the tetraploids P. ginseng and P. quinquefolius possess 2.0 to 4.9 Gb genome sizes for the haploid genome equivalent. About 39-52% of the genome is comprised by four long terminal repeat retrotransposon (LTR-RT) family members, PgDel, PgTat, PgAthila, and PgTork. In particular, PgDel1 LTR-RT occupied 23-35% of the Panax genomes and directly impacted their genome size variation. The genome size difference between P. quinquefolius (4.9 Gb) and P. ginseng (3.6 Gb) is explained by a burst of 0.9 Gb of PgDel during environmental adaptation after migration from Asia to North America one million years ago. The genome proportion of PgDel2 LTR-RT is 2.5% in tetraploids and approximately 5% in diploids. Fluorescence in situ hybridization analysis of PgDel1 and PgDel2 supported the in silico estimation across three Panax species, P. notoginseng, P. ginseng and P. quinquenfolius. Our data revealed the role of four major repeats, which occupy almost 50% of the genome proportion, for evolution of the Panax genus. These results suggest that the study of only small amount of WGS could lead to a better understanding of the genome of plants including non-model plants and minor crops.GENERAL INTRODUCTION 1 REFERENCES 3 CHAPTER I. Chloroplast genomes, nuclear ribosomal genes and polymorphic SSR markers derived from two whole genome sequences of Euonymus hamiltonianus genotypes 5 ABSTRACT 6 INTRODUCTION 7 MATERIAL AND METHODS. 10 Plant materials, genomic DNA extraction and NGS sequencing. 10 Sequence assembly and phylogenetic analysis of cp genome and 45S nrDNA 10 Sequence preparation (quality control and paired end joining), identification SSR motif and clustering. 11 Discovery of polymorphic SSR between two WGS data. 11 PCR validation of designed SSR markers 12 RESULTS 13 Complete cp genome and 45s nuclear ribosomal DNA assembly. 13 Establishment of pipeline for detection of polymorphic SSR using low coverage of WGS (dpsLCW) 19 Verification of dpsLCW protocol with E. hamiltonianus 22 Validation of predicted pSSR for E. hamiltonaunis. 25 DISCUSSION. 30 Complete cp genomes and 45S nrDNA sequences of E. hamiltonianus 30 False pSSRs derived from dpsLCW in E. hamiltonianus. 30 The advantages of dpsLCW. 33 REFERENCES. 36 CHAPTER II. High-throughput development of polymorphic simple sequence repeat markers using two whole genome sequence data in Peucedanum japonicum 41 ABSTRACT 42 INTRODUCTION 43 MATERIAL AND METHODS.. 46 Plant materials, genomic DNA extraction and NGS sequencing. 46 Sequence preparation (quality control and paired end joining), identification of SSR motif and clustering.47 Discovery of pSSRs between two WGS reads 47 PCR validation of SSR markers. 48 RESULTS 49 Identification of pSSR using two WGS in P. japonicum 49 Validation of pSSR and application for P. japonicum germplasm. 51 DISCUSSION. 57 REFERENCES. 59 CHAPTER III. Rapid amplification of four retrotransposon families promoted speciation and genome size expansion in the genus Panax 64 ABSTRACT 65 INTRODUCTION 67 MATERIAL AND METHODS 69 Plant materials, genomic DNA isolation, and Illumina sequencing. 69 Major repeat sequences of Panax ginseng 70 Quantification of major repeats using WGS 70 Fluorescence in situ hybridization (FISH) analysis. 71 RESULTS 73 Whole genome sequence (WGS)-based quantification of major repeats in P. ginseng. 73 Genomic quantification of major repeats in five Panax species. 81 Dynamics of the PgDel1 subfamily members in Panax species. 85 Cytogenomic mapping of PgDel1 and PgDel2 in three Panax species. 87 Contribution of major repeats to genome size variation. 89 DISCUSSION. 91 REFFRENCE. 99 GENERAL INTRODUCTION 106 REFERENCES 108 ABSTRACT IN KOREAN 109Docto