Identification of high-copy number long terminal repeat retrotransposons and their expansion in Phalaenopsis orchids

Background: Transposable elements (TEs) are fragments of DNA that can insert into new chromosomal locations. They represent a great proportion of eukaryotic genomes. The identification and characterization of TEs facilitates understanding the transpositional activity of TEs with their effects on the orchid genome structure. Results: We combined the draft whole-genome sequences of Phalaenopsis equestris with BAC end sequences, Roche 454, and Illumina/Solexa, and identified long terminal repeat (LTR) retrotransposons in these genome sequences by using LTRfinder and classified by using Gepard software. Among the 10 families Gypsy-like retrotransposons, three families Gypsy1, Gypsy2, and Gypsy3, contained the most copies among these predicted elements. In addition, six high-copy retrotransposons were identified according to their reads in the sequenced raw data. The 12-kb Orchid-rt1 contains 18,000 copies representing 220 Mbp of the P. equestris genome. Southern blot and slot blot assays showed that these four retrotransposons Gypsy1, Gypsy2, Gypsy3, and Orchid-rt1 contained high copies in the large-genome-size/large-chromosome species P. violacea and P. bellina. Both Orchid-rt1 and Gypsy1 displayed various ratios of copy number for the LTR sequences versus coding sequences among four Phalaenopsis species, including P. violacea and P. bellina and small-genome-size/small-chromosome P. equestris and P. ahprodite subsp. formosana, which suggests that Orchid-rt1 and Gypsy1 have been through various mutations and homologous recombination events. FISH results showed amplification of Orchid-rt1 in the euchromatin regions among the four Phalaenopsis species. The expression levels of Peq018599 encoding copper transporter 1 is highly upregulated with the insertion of Orchid-rt1, while it is down regulated for Peq009948 and Peq014239 encoding for a 26S proteasome non-ATP regulatory subunit 4 homolog and auxin-responsive factor AUX/IAA-related. In addition, insertion of Orchid-rt1 in these three genes are all in their intron regions. Conclusion: Orchid-rt1 and Gypsy1–3 have amplified within Phalaenopsis orchids concomitant with the expanded genome sizes, and Orchid-rt1 and Gypsy1 may have gone through various mutations and homologous recombination events. Insertion of Orchid-rt1 is in the introns and affects gene expression levels.