Genome-wide Characterization of MITEs and their Utility for Breeding in Brassica Species

Abstract

학위논문 (박사)-- 서울대학교 대학원 : 식물생산과학부(작물생명과학전공), 2014. 2. Tae-Jin Yang.Miniature inverted-repeat transposable elements (MITEs) are non-autonomous class II transposable elements. Tens of thousands MITE members were widely distributed in most of the eukaryotic organisms. However, characterization and utilization of MITEs on Brassica genome has been poorly done. The main theme of this research provides the genome-wide characterization and comparative analysis of 20 MITE families. At the first chapter, I conducted the genome-scale intensive analysis for 20 MITE families and identified 5894 and 6026 MITE members from the available 283 Mb and 385 Mb whole genome pseudo-chromosome sequences of Brassica rapa and B. oleracea, respectively. Meanwhile, only four of 20 families, including 573 members, were identified in Arabidopsis genome indicating most of them were activated in the Brassica genus after divergence with Arabidopsis. Though MITE family has conserved between the B. rapa and B. oleracea genome, members were differentially amplified indicates the recent activity of MITE in Brassica genome. Six MITE families showed different copy numbers, up to 16 fold variation, between B. rapa and B. oleracea suggest the species specific amplification and will be an important source to study the genetic relationship between B. rapa and B. oleracea. MITE insertion characterization on various genomic regions revealed that 54% and 51% of the MITEs were present in the vicinity of less than 2 kb to the gene, hence a large portion of the MITEs were associated with geneic regions. In addition, I propose an effective utilization of MITE elements as insertion polymorphic markers, for breeding and evolution of duplicated genes. Insertion polymorphisms analysis of 289 targets against three different Brassica species (B. rapa, B. oleracea and B. napus) showed high level of polymorphism, 52% and 23% inter- and intra-species level, respectively. Also, lots of MITE elements resided inside of genes suggest that MITE might responsible for genetic differentiation and diversification of Brassica genome. Consequently, the newly identified MITEs will provide a foundation for the further research on roles of MITEs in gene and genome evolution in Brassica species. In second chapter, I deeply characterized a high copy Stowaway family MITE, named as BraMi-1in three Brassica crops and showed its putative role in the evolution of the highly duplicated Brassica genome based on comparative genomics analysis. MIP analysis revealed that the BraMi-1 elements were dispersed into whole Brassica genome by gradual amplification. I also propose effective utilization of the elements as DNA markers for breeding and evolution of duplicated genes. Third chapter describes about BrassicaTED, contains the characterization of the 20 MITE families, 5 TRIM families and 16 SINE families with detailed annotation of its members (around 18556) on the B. rapa and B. oleracea genome. BrassicaTED offers convenient utilization of incorporated MITE information which will be a very valuable repository for scientist and breeders in order to make an efficient research on Brassica species. BrassicaTED has unique user-friendly visualization tool, K BLAST and microarray expression data comparison tool in B. rapa. Forth chapter describes Next generation sequencing (NGS)-based MITE display, a new highly efficient method for rapid and high throughput identification of MITE insertion polymorphism among different Brassica accessions. Using this approach high amount of polymorphic marker can be developed (47%) even without reference genome sequences in B. rapa Hence, This study shows that the MITE are potential target for development of marker with high polymorphism. My research provides the genome-wide identification and characterization of MITEs in B. rapa and B. oleracea genome. This findings would helpful to understand the MITE dynamics and development of variety of markers for breeding purpose in Brassica genome. BrassicaTED, database for the MITEs was established to take full advantage of the compiled MITE information for further study. Moreover, NGS-MITE display will be an important tool for potential marker system for genomics and breeding studies in Brassica genome, gives more insight about the utilization of MITEs in other crops. Furthermore this study laid foundation to understand the MITE transposition activity, amplification and evolution in Brassica genomes.MITEs were actively amplified at gene-rich regions in Brassica genome 51 BraTo-9 could play a role in the evolution of duplicated genes in B. rapa 52 MITEs as valuable sources of DNA marker 53 REFERENCES 55 APPENDICES 62 CHAPTER II. Characterization of a new high copy Stowaway family MITE, BRAMI-1 in Brassica genome ABSTRACT 80 INTRODUCTION 82 RESULTS 84 Characterization of BRAMI-1 in Brassica 84 Phylogenetic analysis of the BRAMI-1 elements 89 Role of the BRAMI-1 elements for gene evolution in the B. rapa genome 91 Transcriptional changes of B. rapa genes containing BRAMI-1 insertions 101 Survey of MITE insertion polymorphisms (MIPs) and estimation of activation dates 103 DISCUSSION 108 Structure, distribution and evolution of BRAMI-1 in the B. rapa genome 108 Rapid amplification of BRAMI-1 elements in the Brassica genus 108 The putative role of BRAMI-1 in B. rapa genome evolution 109 BRAMI-1 elements are active up to the present in Brassica genera 111 MATERIALS AND METHODS 112 Identification and characterization of BRAMI-1 112 Estimation of BRAMI-1 copy number 112 Expression analysis of B. rapa genes with BRAM1-1 insertions 113 MITE Insertion Polymorphism 114 REFERENCES 116 CHAPTER III. BrassicaTED - a public database for miniature Transposable Elements in Brassica species ABSTRACT 123 INTRODUCTION 125 Architecture and Contents of the BrassicaTED 128 Browse 129 Search 131 Tools 138 UTILITY AND DISCUSSION 141 REFERENCES 147 CHAPTER IV. NGS-Transposon Display, A new method for rapid marker development using Miniature Transposable Elements (mTEs) through NGS approach ABSTRACT 153 INTRODUCTION 154 RESULTS and DISCUSSION 157 Transposon display approach has limitations for developing high quality markers 157 Next generation sequencing -based TD (NGS-TD) 157 MATERIALS AND METHODS 166 REFERENCES 173 ABSTRACT IN KOREAN 176Docto