An Unusual Family of Retrotransposons from White Clover

Athena Mascarenhas, and Advisor Howard Laten Program in Bioinformatics Loyola University Chicago, Chicago, Illinois USA 60660
INTRODUCTION
Transposable elements are mobile DNA elements that constitute the majority of the DNA in many higher plants and animals and play major roles in gene expression and genome evolution. Retrotransposons are a type of element that use a copy and paste mechanism involving reverse transcriptase to replicate and amplify their populations within genomes. Long terminal repeat (LTR) retrotransposons, a major component of many plant genomes, have LTRs that flank an internal region that codes for structural proteins (GAG) and enzymes (POL). The pol gene encodes protease, integrase, reverse transcriptase and RNAse H. Loss of any of the above proteins and enzymes can hinder the completion of the lifecycle (see Fig 3). However, we have identified a defective LTR retrotransposon in the genome of white clover (Trifolium repens) that has amplified to high copy-numbers without its own integrase (int) but retains functional copies of all other genes. These types of retrotransposons that lack some or all of its protein coding region, but are able to complete their lifecycles are said to be nonautonomous. This is achieved by the complementation in trans by proteins expressed by an autonomous partner that can restore its ability to transpose. The int-less or int TreLTRRT2 retrotransposon is a member of the Ty3/gypsy superfamily and presumably arose from members with an int gene. Fig. 1 depicts the canonical structure of a Ty3/gypsy element and Fig. 2 illustrates the retrotransposon life cycle.
Figure 6. Ethidium bromidestained PCR products electrophoresis on 1% agarose. Lane 1: T. repens 1; lane 2: T. repens 2; lane 3: T. pallescens; lane 4: T. occidentale.

Figure 4. Phylogenetic tree of one section of the genus Trifolium. The recent interspecific hybrid ancestry of T. repens is highlighted

METHODS
Because only small fragments of the T. repens genome have been released to public databases and no DNAs from the other species have been released, the first step in our study was to use the existing TreLTRRT2 sequence information (Fig. 5) to design a PCR experiment to amplify other copies of this element from all three Trifolium species. DNAs were PCR amplified (Fig 5) and these amplicons were characterized by gel electrophoresis, gel purified, and cloned. The DNAs from 30 clones were then Sanger sequenced.

Figure 1.Organization of genes within Ty3-gypsy LTR retrotransposon. Gag: structural protein. PR: Protease essential for gag protein cleavage. RT: Reverse transcriptase. RH: RNAse H. INT: Integrase necessary for integration.

Figure 7. tBlastn results using the 2-kb band of T. occidentale as query. This sequence is reported as Gmr30 in Soybase.

Figure 2. Lifecycle of LTR Retrotransposon. IN: integrase. PR: protease. RT: Reverse transcriptase. VLP: Virus-Like Particles. Black triangles are LTRs.

There appears to be low levels of the int+ form in the former two species. The int+ forms may provide the integrase that supports the amplification of the int forms. It seems clear that T. repens did not receive its population of TreLTRRT2 elements from T. occidentale. Our sequence results indicated that we recovered several different copies of both the int+ and int forms of TreLTRRT2 from T. occidentale. Using parallel experiments, we were also able to recover different copies of the int+ and int forms from T. pallescens and T. nigrescens (see Fig. 4). We are in the process of trying to recover and sequence what we expect to be the int+ forms from T. repens (see Fig. 6). It will be interesting to pin-point the origin of the int elements. Using computational tools, we were also able to find a int copy in Gmr30 as well (Fig 8).

While the int gene is missing from the high copy-number clover retrotransposon, TreLTRRT2, the RT-RH region is virtually identical to that of the Gmr30 retrotransposon from soybean (Glycine max) (Fig. 3). Two full-length copies of TreLTRRT2 have previously been sequenced from BAC clones. Based on the alignments of the two full-length TreLTRRT2, these insertions have occurred in the past 30,000 years, making this a very young family with recent activity.

Figure 5. Annotated partial sequence of TreLTRRT2 consensus showing locations of primers (dark green).

Figure 8. Using Gmr30 int+ as a reference, we mapped the int copies of Gmr30 and T. repens. Figure 9. Alignment scores of int+ and int copies of Gmr30 and int copy of T. repens.

RESULTS AND DISCUSSION

Amplification generated a 200-bp amplicon as expected, but a weak 2-kb amplicon was generated from T. repens and T. pallescens (Fig. 6, Laten, unpublished). However, in T. occidentale, the 200-bp product was weak and the 2-kb product was strong. DNA sequencing confirmed that the 200-bp bands were >99% identical to the expected amplicon from Fig. 5. Single bp substitutions were observed among clones of the same species and between species. DNA sequences derived from the T. occidentale 2-kb band clones generated a DNA sequence whose conceptual translation matched the integrase from the soybean Gmr30 element (Fig. 7). We recovered an amplicon consistent with the expected size based on the soybean Gmr30 int sequence from T. occidentale. The resulting DNA sequences were highly similar to that of the Gmr30 int, and virtually identical at the amino acid level (Fig. 7). The distribution of int+ and int forms of TreLTRRT2 in T. repens is similar to T. pallescens, while their distribution is inverted in T. occidentale.

The sequence identity between TreLTRRT2 and Gmr30 is remarkable considering these species are separated by tens of millions of years of evolution. It is possible that Gmr30 was horizontally transferred to Trifolium more recently.

REFERENCES
Laten et al. 2010. Repbase Rep. 10: 2176-2177. Du et al. 2010. Plant J. 63: 584598. Tam et al.(2005). Theor Appl Genet 110: 820-831. Sabot, F. & Schulman, A. (2006) Heredity 97: 381-388. Williams et al. (2012) BMC Plant Biol 12: 55. Schulman , A. (2012) Plant Transposable Elements no: 71-88.

Figure 3. Alignment of the carboxyl end of pol from TreLTRRT2 and Gmr30 from a tBlastn search.

ACKNOWLEDGEMENTS
Co-investigator: Piotr Weglarz Supported by Loyola Bioinformatics Program and LUROP. Thanks also to Stasa Pintur, Kailey Becker, Mariam Bahia, Christina Jreisat, Samer Martini and Haley Luebke for providing the DNA.

White clover is a recent allotetraploid that is thought to have formed ~15,000 years ago through interspecific hybridization between two diploids, T. pallescens and T. occidentale, both of which still grow wild in several locales (Fig. 4).