Characterisation of transposable elements of cassava (Manihot esculenta Crantz)

Transposable elements (TEs) are fundamental components of probably all eukaryotic genomes. They contribute to the size, structure, variation and diversity of the genome and can have tremendous effects on gene function. Sequencing projects have revealed the diversity of TEs in many organisms and shown that they constitute a very high percentage o f the genome, especially in plants with large genome sizes. PCR-based techniques using degenerate primers designed from conserved enzyme domains are being used to make quick and extensive surveys of TEs in many plants, thus making the study of TEs diversity, abundance and applications possible in species where full genome sequence data are not yet available. Using PCR degenerate primers, cassava reverse transcriptase fragments characteristic of Tyl/copia-Wke retrotransposons was isolated, cloned and sequenced. Sequences analysis of cassava Tyl/copia clones (Mecops) lead to the identification of highly diverse and heterogeneous members of this group of elements with the cassava elements forming clusters with elements from distantly related plants. Southern hybridisation supports the presence of multiple copies of Mecops in the genome of all cassava cultivars tested. The use of degenerate PCR primers anchored on both the integrase and reverse transcriptase has proved a robust and reliable tool for the isolation and characterisation of the polyprotein fragment diagnostic of Ty3/gypsy-\ik& retrotransposon in cassava. Phylogenetic analysis o f cassava and other plant Ty3/gypsy polyproteins revealed a level of heterogeneity in cassava elements that has not been reported in many plant Ty3/gypsy group retrotransposons. Three lineages including the Athila and Calypso/Cyclops of Ty3/gypsy group retrotransposons were revealed. The implication o f env activity bearing retrotransposons in evolution is discussed. Cassava transposase fragments characteristic of En/Spm-Vke transposons were isolated, cloned and sequenced using PCR primer from conserved transposase domains. Sequence analysis showed that the cassava En/Spm-Vks transposases, Meens, are not heterogeneous, with all of the cassava elements forming clusters within a clade. On the other hand, preliminary investigation into the level of diversity of Mutator like elements (MULEs) in the cassava genome revealed at least five different wwdrT-related classes of clones. The copy number has been estimated to be 10 per haploid genome of cassava. Southern hybridisation suggested highly methylated retrotransposons and En/Spm-Mke, sequences of cassava as shown by the minor differences between the hybridisation pattern of the DNA digested by the isoschizomers, Hpa II and Msp I. On the other hand, similar experiment with cassava MULE sequences revealed significant differences between the hybridisation patterns o f these two enzymes suggesting low level of methylation. Interestingly, mudrA-related elements’ RNA transcripts of cassava were detected but none of Mecop, Megyp and Meens transcripts was detected. These lend strong support to the widely acclaimed association between decrease in DNA methylation and increased activity of transposable elements.