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

Abstract

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.