Next-Generation Sequencing Based Transposon Display to Detect High-Throughput Insertion Polymorphism Markers in Brassica

Miniature transposable elements (mTEs) such as miniature inverted-repeat transposable element (MITE), terminal repeat retrotransposon in miniature, and short interspersed element are exquisite sources for marker development. mTEs are short, non-autonomous and stably inherited. The high-copy members are widely distributed into the gene rich euchromatic regions. Here, we conducted a modified transposon display (TD) for a high-copy MITE family, BraSto-2 (Bs2). The Bs2-specific primers derived from conserved sequences of Bs2 members as well as MseI adapter primers were used for polymerase chain reaction (PCR) in two Brassica rapa accessions, ‘Chiifu’ and ‘Kenshin’. The pooled PCR products were sequenced by Illumina sequencing platform instead of high-resolution gel electrophoresis. Subsequent in silico-based insertion polymorphism (IP) analysis (next-generation sequencing [NGS]-based Bs2 transposon display) was conducted, which generated more than 99 putative polymorphic insertion sites between ‘Chiifu’ and ‘Kenshin’. Among 90 successful PCR amplification, 34 showed Bs2 IP (IP-Bs2) between ‘Chiifu’ and ‘Kenshin’ accessions, 27 and seven ‘Chiifu’- and ‘Kenshin’-unique insertions, respectively. When the 90 IP-Bs2 primer sets were applied to 10 Brassica accessions, including four additional B. rapa and B. oleracea accessions, 69 (76%) showed insertion olymorphism among accessions. The IP-Bs2 were evenly distributed through all the chromosomes and provide rich polymorphism among various B. rapa and B. oleracea accessions demonstrating the usefulness of these markers for various genetic diversity and molecular breeding studies in Brassica. In addition, NGS-based TD will be applicable to various high copy transposable elements family for high throughput and rapid polymorphic marker development which will be helpful for efficient plant genomics and breeding purposes.