Identification of potential transcriptionally active Copia LTR retrotransposons in Eucalyptus

Background Long Terminal Repeat retrotransposons (LTR-RTs) represent the most abundant genomic component in all plant genomes thus far investigated. They are transposable elements that replicate through a “copy/paste” mechanism that relies on reverse transcription and integration of a RNA intermediate. Plant LTR-RTs can be divided in two major superfamilies: Copia and Gypsy[1]. LTR-RTs have impact on genome size variation, as well as in the expression of adjacent genes in their host genomes, providing a “genomic plasticity” [2]. Their transcription was believed to be extremely repressed in plants. However, despite their potential mutagenic and deleterious effects, LTR-RTs were proven to be transcriptionally active in several plant species [3]. Eucalyptus is one of the most commercially important forest genus in the world, due to their superior growth, broad adaptability and multipurpose wood properties. Most molecular studies in Eucalyptus are focused on cellulose production and wood development, and there are few works on genome composition, structure and evolution. Pinus and Populus, the tree genera with most available genomic resources, have several works analyzing their repertoire of LTR-RTs [i. e 4, 5], but only one study characterized LTR-RTs in Eucalyptus[6], with no detailed manual checking or phylogenetic analysis. Here, we used FOREST database as a starting point to identify transcriptionally active Copia LTR-RTs in Eucalyptus, that were further analyzed regarding their in silico expression, evolutionary diversity, and distribution in public genomic databases. Methods A previous survey with 88 CopiaLTR-RTs from diverse plants defined six major common evolutionary Copialineages [7]. The 22 Arabidopsis thaliana families analyzed in that study were used as queries to the identify EucalyptusEST sequences related to Copiaelements in FORESTS database [8], by tBLASTx (e-value >1e-50). Sequences were then analyzed in RepBase [9] to confirm their similarity to Copia LTR-RTs. EucalyptusESTs with >200bp of copia-like retrotransposon fragments were used to identify complete copies in Eucalyptusgrandis genome v 1.0 in a BLASTn search (identity >80%; in a region >250bp). We picked up 10000bp surrounding the aligned region, that were analyzed using LTR-Finder [10] and LTR_STRUC [11]. Full-length LTR-RTs were then used as queries in GenBank to retrieve related EucalyptusEST sequences (>200bp; >80% identity). Phylogenetic analyses using the reverse transcriptase of these elements (alignment in MUSCLE, Maximimum Likelihood method, bootstrap 1000 replicates) were done using MEGA 5.01 [12].