Kalendar R, Schulman AH 2006. IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nature Protocols, 1(5): 2478-2484.
Kalendar R, Antonius K, Smykal P, Schulman AH 2010. iPBS: A universal method for DNA fingerprinting and retrotransposon isolation. Theoretical and Applied Genetics, 121(8): 1419-1430.
Kalendar R 2011. The use of retrotransposon-based molecular markers to analyze genetic diversity. Field and Vegetable Crops Research, 48(2): 261-274. DOI:10.5937/ratpov1102261K
Schulman A, Flavell A, Paux E, Noel Ellis TH 2012. The application of LTR retrotransposons as molecular markers in plants. Methods in Molecular Biology, 859: 115-153. DOI:10.1007/978-1-61779-603-6_7
Kalendar R, Schulman AH 2014. Transposon based tagging: IRAP, REMAP, and iPBS. Molecular Plant Taxonomy: Methods and Protocols, Methods in Molecular Biology, Besse Pascale (Ed.), Humana Press, 1115: 233-255.
Kalendar R, Aizharkyn KS, Khapilina ON, Amenov AA, Tagimanova DS 2017. Plant diversity and transcriptional variability assessed by retrotransposon-based molecular markers. Russian Journal of Genetics: Applied Research, 21(1): 128-134. DOI:10.18699/VJ17.231
© 2006-2018 All Rights Reserved, Ruslan Kalendar, University of Helsinki.
Retrotransposons can be used for markers because their integration creates new joints between genomic DNA and their conserved ends. To detect polymorphisms for retrotransposon insertion, marker systems generally rely on PCR amplification between these ends and some component of flanking genomic DNA. We have developed two methods, REMAP (REtrotransposon-Microsatellite Amplified Polymorphism) and IRAP (Inter-Retrotransposon Amplified Polymorphism), which require neither restriction enzyme digestion nor ligation to generate the marker bands. The IRAP products are generated from two nearby retrotransposons using outward-facing primers. In REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands. Here, we describe protocols for the IRAP and REMAP techniques, including methods for PCR amplification with a single primer or with two primers and agarose gel electrophoresis of the product using optimal electrophoresis buffers and conditions. This protocol can be completed in one to two days.
Long Terminal Repeat (LTR) retrotransposons, or Type I transposable elements, replicate by a process of reverse transcription resembling that of the lentiviruses (such as the HIV) 1. The retrotransposons themselves encode the proteins needed for their replication and integration back into the genome 2. Their “copy and paste” life cycle means that they are excised in order to insert a copy elsewhere in the genome. Hence, genomes diversify by the insertion of new copies, but old copies persist. Their abundance in the genome is generally highly correlated with genome size. Large plant genomes contain hundreds of thousands of these elements, together forming the vast majority of the total DNA 3. Human and other mammalian genomes also contain an abundance of retrotransposons. The majority of these, however, are not LTR retrotransposons but LINEs and SINEs, which replicate by a somewhat different copy-and-paste mechanism 4, 5. The L1 family of LINE elements and the Alu family of SINE elements comprise together roughly 30% of human genomic DNA and nearly two million copies 6. The features of integration activity, persistence, dispersion, conserved structure and sequence motifs and high copy number together suggest that retrotransposons are well suited genomic features on which to build molecular marker systems.
The REMAP method is similar to IRAP, but one of the two primers matches an SSR motif with one or more non-SSR anchor nucleotides present at the 3’ end of the primer. Microsatellites of the form (NN)n, (NNN)n or (NNNN)n are found throughout plant and animal genomes. In cereals, they furthermore appear to be associated with retrotransposons 21. Due to phenomena including polymerase slippage, microsatellites have high mutation rates and therefore may show much variation at individual loci within a species. Differences in the number of SSR units in a microsatellite are generally detected using primers designed to unique sequences flanking microsatellites. Alternatively, the stretches of the genome present between two microsatellites may be amplified by ISSR 22, in a way akin to IRAP. In REMAP, anchor nucleotides are used at the 3’ end of the SSR primer both to avoid slippage of the primer within the SSR, which would produce a “stutter” pattern in the fingerprint, and to avoid detection of variation in repeat numbers within the SSR. REMAP uses primer types that are shared by IRAP and ISSR. Although it would appear that the SSR primers in REMAP should also yield ISSR products and the LTR primers also IRAP products, in practice this is rarely the case. This is probably due to a combination of factors including both genome structure and competition within the PCR reactions.
The retrotransposon methods described above provide consistent data 40. Although S-SAP is somewhat more general than IRAP or REMAP, requiring only a restriction site near the outer flank of a retroelement, its requirement for two additional enzymatic steps introduces the possibility of artifacts from DNA impurities, methylation, and incomplete digestion or ligation. Furthermore, S-SAP generally requires selective nucleotides on the 3’ ends of the retrotransposon primers in order to reduce the number of amplification products and increase their yield and resolvability. As for IRAP and REMAP, the resulting subsets of amplifiable products are not additive 8. Although RBIP confers the power of codominance, developing flanking primers to nested retrotransposon insertions, which can constitute many of the insertions in cereals, is difficult and a method is therefore required for efficient mining of unique flanks. The strength of all these methods is that the degree of phylogenetic resolution obtained depends on the history of activity of the particular retrotransposon family being used. Hence, it is possible to analyse both ancient evolutionary events such as speciation as well as the relationships and similarities of recently derived breeding lines. The IRAP and REMAP can be generalised, furthermore, to other transposable element systems such as to MITEs, and to other organisms. For example, the SINE element Alu of humans has been used in a method called Alu-PCR in a way similar to IRAP and REMAP 41.
PCR reaction buffers: several PCR reaction buffers are suitable for PCR: ThermoPol® Buffer, 1X: 20 mM Tris-HCl (pH 8.8, 25°C), 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4; Buffer 2, 1X: 10 mM Tris-HCl (pH 8.8, 25°C), 2 mM MgCl2, 50 mM KCl, 0.1% Triton X-100; Buffer 3, 1X: 50 mM Tris-HCl (pH 9.0, 25°C), 2 mM MgCl2, 15 mM (NH4)2SO4, 0.1% Triton X-100. ▲Critical The PCR reaction and its efficiency depend on which buffer and enzyme combination is used. Most enzymes are supplied with their own recommended buffer; these buffers are often suitable for other thermostablepolymerases as well. The concentration of MgCl2 (MgSO4) can be varied from 1.5 to 3 mM without influencing the fingerprinting results. A higher MgCl2 concentration can increase the PCR efficiency and allow reduction in the number of PCR cycles from 32 to 30 and also help in PCR reactions containing somewhat impure DNA. Additional components such as (listed at their final concentration in the reaction buffer) 5% acetamide (Sigma A6082), 0.5 M betaine (N,N,N-trimethylglycine, Sigma B-2629), 3% DMSO (Sigma D9170), 5% glycerol (Sigma G8778), 5% PEG 8000 and 5-20 mM TMA (Tetramethylammonium chloride, Sigma T3411) can increase the PCR efficiency for multiple templates and PCR products 42.
1 Design a PCR primer to match an LTR sequence near to either its 5’ or 3’ end, and orient the primer so that the amplification direction is towards the nearest end of the LTR. Generally it is best to base the design on a sequence alignment for representative LTRs from a particular family of elements and to place the primer within the most conserved region for that family. For long LTRs, it is often useful to test primers at several locations within the LTR and in both orientations, particularly if there is evidence for nested insertions in the genome. Primers can be placed directly at the end of the LTR facing outward, provided that they do not form dimers or loops. For primers placed at the edge of the LTR, one or more additional selective bases can be added at 3’end in order to reduce the number of amplification targets. This can be tried in a second round of primer design, if the initial primer yields amplification products containing too many weak individual species for analysis by gel electrophoresis. If the LTRs are short (<300 bp), the primers may also be designed to match internal regions, but this will concomitantly increase the size of the amplified products. Microsatellite primers for REMAP or ISSR should be designed according to two principles: first, primer length should be between 19 - 22 bases; second, the last base at 3’-end of the primer is designed as selective base, which is absent in repeat unit itself. Examples of LTR conservation and consequent primer design for LTRs and microsatellites are shown in
▲CRITICAL STEP We have designed LTR primers using the “FastPCR” software (R. Kalendar). Database searches can sometimes be used to find un-annotated, native LTR sequences matching characterised retrotransposons from other species. However, care should be taken in defining the ends of the LTRs. Generally, mapping of the reverse transcriptase primer binding sites PBS and PPT is needed in order to define the LTR ends with confidence.
: Example of IRAP and REMAP primer design: conserved 3’ end of Sukkula LARD LTRs and matching primer
* * * **** * * * ****** ***** ** ** ** * *** ** * *** *** ********** * * ***
(CT)n microsatellites: 5’(CT)11G, 5’(CT)11T, or 5’(CT)11A
(CA)n microsatellites: 5’(CA)11G, 5’(CA)11T, or 5’(CA)11A
(TG)n microsatellites: 5’(TG)11G, 5’(TG)11C, or 5’(TG)11A
(AG)n microsatellites: 5’(AG)11G, 5’(AG)11C, or 5’(AG)11T
(AC)n microsatellites: 5’(AC)11G, 5’(AC)11C, or 5’(AC)11T
(CTC)n microsatellite: 5’(CTC)7G, 5’(CTC)7T, or 5’(CTC)7A
(GTG)n microsatellite: 5’(GTG)7C, 5’(GTG)7T, or 5’(GTG)7A
(CAC)n microsatellite: 5’(CAC)7G, 5’(CAC)7T, or 5’(CAC)7A
(ACC)n microsatellite: 5’(ACC)7G, 5’(ACC)7T, or 5’(ACC)7C
(TCG)n microsatellite: 5’(TCG)7G, 5’(TCG)7C, or 5’(TCG)7A
Polymerase chain reaction (PCR) ● timing ~ 2-2.5 h
2 Perform PCR in a 25 µl reaction mixture containing 25 ng DNA, 1x ThermoPol® buffer (20 mM Tris-HCl (pH 8.8, 25°C), 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4), 0.5 µM primer(s), 200 µM dNTP, 1U Taq polymerase, and recommended to add 0.005 unit of Pfu DNA Polymerase). The standard PCR reaction (120 min) should consist of: a 3 min denaturation step at 95°C; 28-31 cycles of 15s at 95°C, 30s at 60°C and 90s at 72°C; a 5 min a final extension at 72°C.
▲Critical STEP The amount of template DNA plays an important role in the quality of the resulting fingerprint. Most commonly, 1ng DNA per 1µl of reaction volume is ideal. Much higher DNA concentrations will produce smears between the bands, which is a sign of over-amplification.
▲Critical STEP DNA and primers should be stored in a 1xTE solution, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
▲Critical STEP The final primer concentration(s) in the reaction can vary from 200 to 400 nM. Although higher primer concentrations increase PCR efficiency and the rapidity of DNA amplification, they also produce over-amplified products, such as shown in Figure 2b.
▲Critical STEP DNA quality is very important for obtaining high-quality results. Standard DNA extraction methods are sufficient to yield DNA of high quality from most of samples. DNA should be free of polysaccharides, pigments, and secondary metabolites. Some plant materials contain polysaccharides, pigments, oils or polyphenols, which can reduce the efficiency of PCR. Furthermore, contaminated DNAs will decline in PCR performance during prolonged (a month or more) periods of storage, due to chemical modification. Such DNAs (for example, from Brassica spp.) should be extracted with methods involving sodium hydroxide, followed by two ethanol DNA precipitations. High-quality DNA can be stored at 4°C for many years without showing any PCR inhibition or decrease in amplification efficiency for the longer bands.
▲Critical STEP PCR thermal conditions can be varied without large effects on the resulting band pattern. The denaturation step in PCR can be carried out at 94°C for 30s or 95°C for 20s or 98°C for 5s. The length of the annealing step can vary from 10 to 30 second at 60°C. The annealing temperature varies with the melting temperature of the primer; it should be between 55 and 68°C (60°C is optimal for almost all primers and their combinations in IRAP and REMAP).
●PAUSE POINT PCR reactions can be stored at 4°C overnight with loading buffer or without.
Casting the agarose gel ● TIMING ~2-3 h
3 Prepare 200 ml of 1.5% (w/v) agarose containing 1xTHE buffer in a 500 ml bottle. This volume is required for one gel with the dimensions 0.4 cm x 20 cm x 20 cm. Dissolve and melt the agarose in a microwave oven. The bottle should be closed, but the plastic cap must not be tightened! The agarose gel must be completely melted in the microwave and then allowed to slowly cool until its temperature drops to about 60-65°C. At that point, if desired, add the ethidium bromide solution at a rate of 100 ul per 100 ml, to bring the final concentration to 0.5 ug per ml (alternatively the gel can be stained at the end of the run).
▲CAUTION Take care not to boil over the agarose. Add ethidium bromide only after removing the agarose from the microwave oven to minimise risks from boil-over.
▲Critical STEP The agarose gel must melt and dissolve properly. Small undissolved inclusions will severely hamper the quality of the results. Do not allow the gel to cool unevenly before casting, for example by leaving it stand on the benchtop or in cool water. The best way to cool the agarose is by shaking it at 37°C for 15 minutes.
4) Pour the agarose into the gel tray (20 x 20 cm). Allow the agarose to solidify at room temperature for one hour minimum.
▲Critical STEP For optimal resolution, cast horizontal gels 3 – 4 mm thick. The volume of gel solution needed can be estimated by measuring the surface area of the casting chamber and then multiplying by gel thickness.
Sample preparation and loading ● timing ~ 15 min
6 Add an equal volume of 2x loading buffer to the completed PCR reactions in tube or plate and mix well. Collect the mixture by a short centrifugation (by turning a benchtop microcentrifuge on and immediately off again). Load the gels with a sample volume of 10 µl.
▲Critical STEP The DNA concentration plays an important role in gel resolution. Overloaded lanes will result in poor resolution.
Gel electrophoresis ● timing ~3-5 h
7 Select running conditions appropriate to the configuration of your electrophoresis box. For a standard 20 x 20 cm gel, carry out electrophoresis at a constant 80-100 V for 3-8 h (a total of 600 volt-hours). Electrophoresis may cause the gels to after several hours; their temperature should not be allowed to exceed 50°C, above which electrophoretic resolution will be impaired. Still better results are obtained with a slower run. We routinely use 100 V for 6 h (600 volt-hours).As the end of the run approaches, it is helpful to check the run with a UV-transluminator.
▲Critical STEP For samples with many or large (> 1000 bp) bands, perform the gel electrophoresis at a constant voltage of 50 V overnight (14 h) as shown in Figure 3.
DNA visualization ● timing ~15 min
8) DNA can be visualized directly by casting ethidium bromide into gel as described above, or by incubating in an ethidium bromide solution of equivalent strength following electrophoresis.
Scan the gels on an Typhoon FLA 9500 imaging system (GE Healthcare Life Sciences)
Primer design (step 1), ~1-2 h
Polymerase chain reaction (PCR; step 2), ~ 2-2.5 h
Casting the agarose gel (steps 3-5), ~2-3 h
Sample preparation and loading (step 6), ~ 15 min
Gel electrophoresis (step 7) ~3-7 h
DNA visualization (step 8) ~15 min
Occasionally, not all primers (derived from either retrotransposons or microsatellites) will work in the PCR. The genome may contain too few retrotransposon or microsatellite target sites, or they may be too dispersed for the generation of PCR products. Alternatively sequence divergence in old retrotransposons or polymorphisms between heterologous primers and native elements may lead to poor amplification. Some primers generate smears under all PCR conditions. Many sources can contribute to this problem, ranging from primer structure to variability in the target site and competition from other target sites. Generally, it is more efficient to design another primer than to try to identify the source of the problem. Furthermore, primers which produce a single, very strong band are not suitable for fingerprinting.
Development of a new marker system for an organism in which retrotransposons have not been previously described generally takes one to six months. The availability of heterologous and conserved primers as well as experience in primer design, sequence analysis, and testing speeds up the development cycle. Routine analysis of samples with optimised primers and reactions may be carried out thereafter. Retrotransposons have several advantages as molecular markers. Their abundance and dispersion can yield many marker bands, the pattern possessing a high degree of polymorphism due to transpositional activity. The LTR termini are highly conserved even between families, yet longer primers can be tailored to specific families. Unlike DNA transposons, the new copies are inserted but not removed. Even intra-element recombination resulting in the conversion of a full-length element to a solo LTR does not affect its performance in IRAP or REMAP. Retrotransposon families may vary in their insertional activity, allowing the matching of the family used for marker generation to the phylogenetic depth required. The primers for different retrotransposons and SSRs can be combined in many ways to increase the number of polymorphic bands to be scored. Furthermore, the length and conservation of primers to the LTRs facilitate cloning of interesting marker bands and the development of new retrotransposons for markers. The IRAP and REMAP fingerprinting patterns can be used in a variety of applications, including measurement of genetic diversity and population structure
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Figure 1 Retrotransposon-based marker methods. The figure displays the genomic features and the positions of the priming sites for the major methods described in the text. (a) IRAP. Amplification is carried out between the LTRs of two retrotransposons. Genomic DNA (gDNA) is shown as a solid blue line, primers as arrows above and below the genome segment, the retrotransposon as comprised of LTRs and a core domain (core). Other features may be present in the genome, such as microsatellites (SSRs) or restriction sites (R), but IRAP method does not take them into account. (b) REMAP. Amplification is carried out between primers matching an LTR and a microsatellite domain (SSRs). (c) SSAP. Amplification is carried out between primers matching an LTR and a restriction site adapter ligated to genomic DNA digested with a restriction enzyme. (d) RBIP. Full sites (containing a retrotransposon) are generally scored by an amplification reaction with an LTR primer and a primer in flanking, single copy DNA. Empty sites are scored by amplification between the left and right flanks of the presumptive integration site.
Figure 2 IRAP gel fingerprints. The figure illustrates the results achieved following agarose gel electrophoresis with correct (a) and incorrect (b) conditions. (a) Standard amplification, almost all DNA samples at the same concentration (b) PCR over-amplification, resulting from a too high primer concentration, too many cycles, too much template or loaded sample, or a combination thereof.