Introduction
Retrotransposons, also called class I transposable elements, are mobile genetic elements that undergo replicative transposition through reverse transcription of RNA intermediates (Kumar and Bennetzen, Reference Kumar and Bennetzen1999). Retrotransposons are the major components of cereal genomes that are largely located in repetitive DNA (Barakat et al., Reference Barakat, Carels and Bernardi1997). Polymorphic retrotransposon insertions can be used as a molecular marker system for genetic studies in plant species (Ellis et al., Reference Ellis, Poyser, Knox, Vershinin and Ambrose1998; Queen et al., Reference Queen, Gribbon, James, Jack and Flavell2004). One of the most popular transposon-based marker methods is the sequence-specific amplified polymorphism (SSAP) technique (Waugh et al., Reference Waugh, McLean, Flavell, Pearce, Kumar, Thomas and Powell1997) that was designed to analyse the insertion polymorphism of high copy number long terminal repeat (LTR) retrotransposons in plant genomes. SSAP techniques can be powerful experimental genomic tools that can be applied to molecular mapping, marker-assisted selection, diversity analysis and evolutionary studies.
Materials and methods
The recombinant inbred line (RIL) mapping population Opata85 × Synthetic W7984 was used to map Jeli LTR retrotransposon (gypsy-like family) insertions in common wheat (Triticum aestivum L.) genome. Total DNA was extracted from individual plants by using hexadecyltrimethylammonium bromide protocol (Torres et al., Reference Torres, Weeden and Martin1993) with minor modification. SSAP analysis was performed, as described by Konovalov et al. (Reference Konovalov, Goncharov, Goryunova, Shaturova, Proshlyakova and Kudryavtsev2010). Six primer combinations were used to amplify the DNA sequences between the Jeli LTRs and TaqI restriction sites. The LTR primer sequence 5′-CCCTAGGAACATAGCTTCATCA-3′ was based on the Jeli sequence TREP3458 obtained from the International Triticeae Mapping Initiative Triticeae Repeat Sequence Database (Wicker et al., Reference Wicker, Matthews and Keller2002). To reduce the number of amplification products, two to three selective bases were added to the 3′ ends of the LTR primers (AC, AG, CTG, GAC, GGA and TC). The PCR reaction products were separated by electrophoresis in a polyacrylamide sequencing gel using a 38 × 50 cm Bio-Rad SequiGen GT cell and visualised using silver staining. The polymorphic SSAP markers were mapped within a framework of known restriction fragment length polymorphism markers (http://www.graingenes.org/). The marker presence/absence data were analyzed using MAPMAKER 3.0 (Lander et al., Reference Lander, Green, Abrahamson, Barlow, Daly, Lincoln and Newburg1987). A logarithm of odds score threshold of 5.0 was used to detect genetic linkage. Markers that were not assigned into linkage groups were discarded.
Results and discussion
Polymorphic Jeli insertions represented by SSAP bands were scored in a RIL mapping population Opata85 × Synthetic W7984 (Fig. 1). The total number of SSAP bands for the six primer combinations was 316 with a mean of 52.7 bands/primer combination. The number of polymorphic bands was 65, ranging from 5 (selective bases CTG) to 18 (selective bases AG) with a mean of 10.8. The polymorphism level (proportion of polymorphic bands in the total number of bands) for Jeli observed in our study was 0.18, which was close to the polymorphism level of the markers based on the retrotransposons Tar1 and TAGERMINA, but higher than that of the markers based on Thv19 and BARE-1/Wis-2-1A elements (Queen et al., Reference Queen, Gribbon, James, Jack and Flavell2004). Seventeen of the 65 markers could not be assigned into linkage groups, and therefore, were discarded; thus, 48 markers were placed on the chromosome maps. The number of SSAP markers mapped on each chromosome varied from zero (chromosomes 1D, 4D, 5D and 7D) to six (chromosome 2A) markers/chromosome.
Jeli-based SSAP marker (LTR primer with selective bases AG) segregation in an Opata85 × Synthetic W-7984 mapping population. M, 10-bp DNA ladder; O, Opata85; S, Synthetic W7984.
The mapping experiments demonstrated approximately the same general distribution of Jeli markers between the three wheat genomes as the nulli-tetrasomic analysis had revealed earlier (Konovalov et al., Reference Konovalov, Goncharov, Goryunova, Shaturova, Proshlyakova and Kudryavtsev2010): 30 insertion sites in the A genome (63%); 14 in the B genome (14%) and 4 in the D genome (4%). The A-genome preference of the Jeli polymorphic insertions was revealed by our marker system. Such an unequal distribution of the retrotransposon-based markers could be explained by a burst of Jeli amplification in the diploid A-genome donor (Triticum urartu Thum. ex Gandil.) with lower retrotransposition activity in the B and D genome donors (Konovalov et al., Reference Konovalov, Goncharov, Goryunova, Shaturova, Proshlyakova and Kudryavtsev2010) that led to a higher copy number of Jeli itself in the A genome; another alternative involves possible differences in the LTR sequences of the Jeli lineages from the A, B and D genomes that could result in preferable primer annealing at the Jeli copies from the polyploid wheat genome A.
Tight clustering of Jeli markers was observed in some cases (chromosomes 1B, 2A, 4A, 5A and 6A). Some clustering of BARE-1/Wis-2-1A markers on the linkage map has been shown (Queen et al., Reference Queen, Gribbon, James, Jack and Flavell2004). SSAP marker clustering may be because of cases wherein one polymorphic change is scored as two markers (e.g. if an single-nucleotide polymorphism at a restriction site changes the SSAP band length) or by clustering of the retrotransposons themselves, but a more detailed investigation is needed to confirm this hypothesis.
The SSAP system based on the Jeli retrotransposon provides multiple genetic markers for common wheat. Our marker system preferably revealed A-genome Jeli insertions, and therefore can be used for targeted analysis of the A genome in evolutionary studies, genetic mapping, polymorphism screening and marker-assisted selection. The number of Jeli insertion sites that can be revealed by different primer/restriction enzyme combinations can be estimated as being at least several hundreds. It may also be a promising A-genome identification tag when used as a probe in fluorescent in situ hybridization analyses.
