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

Abstract

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.