Ac/Ds-induced chromosomal rearrangements in rice genomes

Abstract

A closely-linked pair of Ac/Ds elements induces chromosomal rearrangements in Arabidopsis and maize. This report summarizes the Ac/Ds systems that generate an exceptionally high frequency of chromosomal rearrangements in rice genomes. From a line containing a single Ds element inserted at the OsRLG5 locus, plants containing a closely-linked pair of inversely-oriented Ds elements were obtained at 1% frequency among the population regenerated from tissue culture. Subsequent regeneration of the lines containing cis-paired Ds elements via tissue culture led to a high frequency (35.6%) of plants containing chromosomal rearrangements at the OsRLG5 locus. Thirty-four rearrangement events were characterized, revealing diverse chromosomal aberrations including deletions, inversions and duplications. Many rearrangements could be explained by sister chromatid transposition (SCT) and homologous recombination (HR), events previously demonstrated in Arabidopsis and maize. In addition, novel events were detected and presumably generated via a new alternative transposition mechanism. This mechanism, termed single chromatid transposition (SLCT), resulted in juxtaposed inversions and deletions on the same chromosome. This study demonstrated that the Ac/Ds system coupled with tissue culture-mediated plant regeneration could induce higher frequencies and a greater diversity of chromosomal rearrangements than previously reported. Understanding transposon-induced chromosomal rearrangements can provide new insights into the relationship between transposable elements and genome evolution, as well as a means to perform chromosomal engineering for crop improvement. Rice is a staple cereal crop worldwide. Complete genome sequencing and rich genetic resources are great advantages for the study of the genomic complexity induced by transposable elements.(1) (-) (2) The combination of tissue culture with genetic lines carrying a pair of closely located Ac/Ds elements greatly increases the frequency and diversity of rearrangements in rice genomes. The methodology and its efficiency and significance are briefly summarized.

Figure 1. Structure of the Ds T-DNA vector and the polymorphic display of Ds elements in regenerated plants. (A) A BAR selection marker and GUS reporter gene were contained within the Ds T-DNA vector. The short vertical arrow indicated by ‘E’ is an EcoRI restriction site inside the GUS coding region. The horizontal lines below the map indicate the sizes of the Ds vector (5.9 kb) and GUS coding region (1.8 kb). The numbers “5” and “3” above the Ds T-DNA vector indicate the 5′ and 3′ Ds termini, respectively. (B) Southern blot hybridization was performed to identify Ds transpositions in plants regenerated from OsRLG5::Ds seeds. EcoRI-digested genomic DNA was hybridized with a 1.8 kb DNA fragment from the GUS coding region. The 4.7 kb arrow indicates the location of the original Ds element. Transposed Ds elements of regenerated plants were collectively named t.Ds.

Figure 2. Models for single chromatid transposition (SLCT) with a distal target site. The inversion/deletion process derived from single chromatid transposition is depicted in three steps (A–C). (A) Ac transposase (circles) cuts at the 5′ end of Ds-y1 and the 3′ end of Ds-y2. (B) Ligation of the host sequences flanking the excised Ds termini leads to generation of a footprint (‘x’) and inversion of the inter-transposon segment c–d. (C) Reinsertion of the 5′ and 3′ termini of Ds-y1 and Ds-y2 into distal target site a–b (black arrow). Reinsertion in either of two possible orientations results in products shown in parts (C1) and (C2). Part (C1) shows the inversion of fragment a, and inversion of both Ds-y1 and Ds-y2. Part (C2) shows the deletion of fragment a, deletion of Ds-y2, and inversion of Ds-y1.

Figure 3. Phenotypic expression of plants homozygous for deletions. (A) The 280 kb genomic block of rice chromosome 1 contains 36 receptor-like kinase genes (RLKs), as shown in the upper diagram. RLK 19 and RLG 5 represent the same gene. Below the map of the RLK cluster, deleted regions and the names of deletion lines are shown as dotted lines and numbers, respectively. Vertical lines indicate the deletion endpoints in each deletion lines. All deletions begin at RLG5 and extend to either the distal or proximal region. (B) Plants homozygous for deletions 25 and E106 (25DD and E106DD) show a necrotic phenotype. Leaves of 2-week-old plants are shown.