Principle

Knowledge of the Long Terminal Repeat (LTR) sequence is essential for developing retrotransposon-based markers such as S-SAP and REMAP. However, LTRs are extremely heterogeneous and poorly conserved even among the same Ty1-copia family of retrotransposons. Furthermore, knowledge of the Coffea genome is too limited to allow identification of LTRs by screening a database, as available sequences are few and far between. Therefore, to isolate at least part of the LTR, we exploited the 3’ end of the adjacent RNase H gene, which contains a very conserved ADIFTK motif. By PCR-amplification of the tract between this RNAse H motif and a restriction site in the adjacent LTR sequence, it is possible to isolate and characterize sequences containing the necessary part of the LTR, without the need to construct a DNA library.

Subsequently, primers are designed in the LTR sequence to be used as multilocus markers.

Isolation of TY1-Copia RNase H sequences

(adapted from Pearce et al., 1999)

Production of “RNase H/Mse I” PCR products

An amount of 2.5 µg of total genomic DNA, extracted from seeds of Coffea arabica var. Jamaica Blue Mountain, was partially digested with Mse I in 100 µl 1x RL buffer at 37°C. Aliquots were taken at 2, 5, 10, 20 and 40 minutes, followed by 20’ at 80°C to stop the reaction. 5 µl of each aliquot were run in a 1,5% agarose gel to determine fragment size and the sample with an average length of 1 Kb was selected.
To 15 µl of the restriction solution, 1 µl of 50 mM adapters (plus strand, 5’-GACGATGGATCCTGAG-3’; minus strand, 5’-TACTCAGGATCCAT-3’), 0,1 units of T4 DNA ligase, 1 µl of 0,1 M ATP and 1x RL buffer to a final volume of 20 µl were added, the tubes were incubated overnight at room temperature, and the resulting DNA was diluted a hundredfold. A pre-selective PCR reaction was performed in a 50 µl volume as follows:

• 10 µl of restricted/ligated DNA fragments (approx. 40 ng)
• 1x Taq Gold PCR buffer (Perkin Elmer)
• 0,025 units/µl Taq Gold DNA polymerase (Perkin Elmer)
• 0,2 mM dNTPs
• 2 mM MgCl2
• 300 mM of biotinylated “RNase H-1” primer (5’-MGNACNAARCAYATHGA-3’)
• 200 mM of adapter primer “Mse + 0” (5’-GATGGATCCTGAGTAA-3’)

on an MWG Primus thermal cycler with the following program:
94°C x 5’ – 25 x (94°C x 30” – 45°C x 30” – 72°C x 1’) – 72°C x 10’

PCR products were size-fractionated by electrophoresis in a 1,5% agarose gel and products ranging from 0.5 to 1 Kb were recovered using a QIAGen QIAQuick™ Spin kit, then eluted in a 40 µl volume. 30 µl of streptavidin-coated magnespheres (Promega) were added to the eluate, washed three times with 0,2 x SSC to remove non-biotinylated products, then resuspended in 50 µl of 1x PCR buffer. 2 µl of the magnespheres suspension were PCR-amplified in a 50 µl volume with the following conditions:

• 300 nM “RNase H-2” primer (5’- GCNGAYATNYTNACNAA-3’)
• 100 nM adapter primer “Mse + 0”

All other PCR conditions and program were left unchanged.

Cloning

PCR products were purified again with QIAQuick™ Spin and cloned in a pGEM®-T vector (Promega), following manufacturer’s protocol and performing the A-tailing procedure to maximize ligation efficiency.

Sequence analysis

Insert size for all clones obtained was verified by electrophoresis of the PCR products (with M13 primers) on a 2% agarose gel. 50 inserts, with a size larger than 120 bp were selected; PCR products from the selected clones were cleansed from dNTPs and primers with QIAQuick™ Spin, their concentration determined by observation in a 2% agarose gel, and sequenced on an ABI PRISM® 3100 genetic analyser, using the M13 forward primer. 44 sequences were obtained and analysed individually to remove vector- and adapter-derived or ambiguous tracts, then a tBLASTx search was performed against the NCBI database. 36 clones had significant similarity to retrotransposable elements; furthermore, 20 displayed a clearly identifiable polypurine tract (PPT) at the end of the coding sequence, a further evidence for the retrotransposon origin of these sequences. One of the discarded sequences was recognized for a cloning artifact and it consisted, in fact, of two distinct RNase H fragments joined in head-to-head orientation, bringing the number of retrotransposon sequences to 38.

Primer design

Primers were designed using the BioOligo program from BioGene, with the following characteristics:

• complementary to the LTR sequence and as close as practical to the PPT tract, in order to minimize the expected size of polymorphic PCR products;
• pointing towards the 5’ end of the retrotransposon;
• labelled with a 6-FAM at the 5’ end for detection in the ABI PRISM ® 373 genetic analyser;
• optimal annealing temperature of around 53°C.