Composition and structure of the centromeric region of rice chromosome 8

Abstract

Understanding the organization of eukaryotic centromeres has both fundamental and applied importance because of their roles in chromosome segregation, karyotypic stability, and artificial chromosome-based cloning and expression vectors. Using clone-by-clone sequencing methodology, we obtained the complete genomic sequence of the centromeric region of rice (Oryza sativa) chromosome 8. Analysis of 1.97 Mb of contiguous nucleotide sequence revealed three large clusters of CentO satellite repeats (68.5 kb of 155-bp repeats) and >220 transposable element (TE)-related sequences; together, these account for approximately 60% of this centromeric region. The 155-bp repeats were tandemly arrayed head to tail within the clusters, which had different orientations and were interrupted by TE-related sequences. The individual 155-bp CentO satellite repeats showed frequent transitions and transversions at eight nucleotide positions. The 40 TE elements with highly conserved sequences were mostly gypsy-type retrotransposons. Furthermore, 48 genes, showing high BLAST homology to known proteins or to rice full-length cDNAs, were predicted within the region; some were close to the CentO clusters. We then performed a genome-wide survey of the sequences and organization of CentO and RIRE7 families. Our study provides the complete sequence of a centromeric region from either plants or animals and likely will provide insight into the evolutionary and functional analysis of plant centromeres.

Figure 1.

Genetic, Physical, and Sequence Maps of the Centromeric Region of Rice Chromosome 8. Genetic map, PAC/BAC contigs, and sequence map are shown from the top to bottom. The green boxes above and below the sequence map represent transposable elements that showed homology to known sequences within GenBank (overall alignment coverage of >50% and sequence identity of >80%). Sequences of a transposable element–related element interrupted internally by DNA insertions are grouped with horizontal black lines and (if necessary) an additional arched arrow. The positions of the seven RIRE7-related elements that formed two groups are indicated by arrowheads. Yellow boxes represent the positions of CentO clusters on the central part of the sequenced region that are enlarged at the bottom; the number and orientation of the 155-bp satellite repeats are given. Ac/Ds, Activator/Dissociation; cM, centimorgan.

Figure 2.

Distribution of G+C Fractions, Repetitive Sequences, and DNA Motifs along the Centromeric Region. The horizontal axis represents the 1.97-Mb contiguous sequence from the short to long arm. The vertical axis shows the G+C content and frequency of various repeat sequences obtained by analyzing the sequence every 10 kb using a 1-kb sliding window and the occurrence of the DNA motif A(/T)AT(/C)ATT within every 1 kb. (A) G+C content. (B) All repeat sequences. (C) DNA transposons. (D) Retrotransposons. (E) Miniature inverted repeat transposable elements (MITEs). (F) A(/T)AT(/C)ATT motifs.

Figure 3.

Annotation Map of the 1.97-Mb Centromeric Region of Chromosome 8. White boxes represent the predicted genes. Green and gray boxes indicate the TEs. The names of TEs or TE-like sequences identical to those in GenBank as described in the text are shown above the corresponding sequences (also see Supplemental Tables 1 and 2 online for details). Arrows represent LTR sequences and their orientations. Arched arrows indicate that the TE sequence is interrupted by DNA insertions. Red bars indicate elements containing sequences similar to the rice centromere-specific element RCE1 (accession number AB013613). Yellow boxes represent CentO clusters; the red arrows above show the orientations of the 155-bp satellite repeats. The positions of five genetic markers without recombination are indicated by their names in orange.

Figure 4.

Sequence Variations among Each CentO Satellite Repeat. (A) The most frequent bases (consensus shown above the graph) detected among the 393 units of 155-bp satellite repeats that showed >99.99% sequence accuracy. (B) The base frequency including the transversion sequence. The eight alternative bases comprising six transversions (AT and GC) and two transitions (TC) are indicated by arrows and are found within the regions with gray shading in (A).

Figure 5.

Sequence Comparison of a RIRE7-Related Element Found in the Centromeric Region of Chromosome 8 with GenBank Data. The sequence alignment between each element on the basis of BLASTN analysis is shown with closed boxes. Sequence identities for the aligned segments ranged from 80 to 100%. Sequences identical to CRR sequences (accession numbers AF078903 for RCS1, AF058903 for RCH1, AF058904 for RCH2, and AF058905 for RCH3) within the RIRE7-related elements are represented with open boxes. (A) The maize retrotransposon CRM (accession number AY129008). (B) The RIRE7-related element from nucleotide 155,040 to 162,737 of this study. (C) The rice retrotransposon RIRE7 (accession numbers AB033234 for LTR regions and AB033235 for the internal gag-pol polyprotein region).