The reverse transcriptase (RT) domain of the copia retrotransposons was amplified from genomic DNA of an aquatic perennial grass, Zizania latifolia (Griseb.)-a wild relative of rice. In total, 71 different sequences were obtained, which, according to alignment and phylogenetic analysis, can be classified into seven distinct families, with five each containing multiple sequences and two each containing a single sequence. The nucleotide sequence similarity values varied widely among the sequences, indicative of their high degree of heterogeneity. Of the 71 sequences, 25 are intact whereas the others have a frameshift, a stop codon, or both. Southern blot analysis revealed differential occurrence of the Z. latifolia copia retrotransposons among representative species of the grass family, and upon which three groups (1, 11 and 111) can be divided. An interesting observation is that whereas the occurrence patterns of groups I and 11 among the grass species is compatible with their organismal phylogeny, which of group III shows apparent incongruity. Because cytosine methylation has been proposed as a major mechanism controlling transpositional behavior, amplification, and hence distribution, of mobile elements, the extent and pattern of this epigenetic modification were analyzed for the Zizania copia retrotransposons. Southern blotting with a pair of isoschizomers, Hpall and Mspl, with differential sensitivity to cytosine methylation revealed an atypical methylation pattern of the Z. latifolia copia retrotransposons. It was found that only two of the seven families (families 1 and 4) showed typical methylation patterns as found in other plants, i.e., CG is more heavily methylated than CNG, while the rest five families showed an atypical pattern, i.e., CNG is more methylated than CG, which was further verified by using the Z. latifolia copia-mixture as a probe. In addition, the homologues of those Z. latifolia copia elements that exist in both cultivated rice and the common wild rice are largely undermethylated and highly conserved, though some of which showed evidence for methylation alteration in several rice-Zizania introgression lines. Thus, the atypical patterns of cytosine methylation of the copia retrotransposons in Z. latifolia might have played a role in their differentiation into distinct groups, and differential occurrence of their homologs in other species of the grass family. (C) 2008 Elsevier B.V. All rights reserved.