The FaRE1 LTR-retrotransposon Based SSAP Markers Reveal Genetic Polymorphism of Strawberry (Fragaria x ananassa) Cultivars

The genetic diversity of strawberry F. x ananassa varieties with respect to FaRE1 LTR retrotransposon integration sites was studied for the first time. The SSAP method with a single combination of primers revealed 35 polymorphic sites of FaRE1 LTR retrotransposon integration in the strawberry genome. Most of the studied varieties (36 out of 46) had unique combinations of polymorphic FaRE1 insertions but 5 pairs of varieties did not differ in their SSAP markers. Assessment of genetic similarity of strawberry varieties using SSAP markers did not reveal distinct genetic clusters, which may be explained by a relatively short history of selection of this cultivar and by crossing within the same genetic pool.

The strawberry (Fragaria x ananassa Duch.) is the most common small-fruit crop that is cultivated under various agroclimatic conditions.F. x ananassa is an octoploid (2n=8x=56) originally produced by crossing of the octoploid species F. chiloensis and F. virginiana, and has a genome AAA'A'BBB'B' comprising four different subgenomes: A, A', B, and B' (Bringhurst & Gill, 1990).The crossing of F. chiloensis and F. virginiana took place about 300 years ago, which makes F. x ananassa a "young" crop (Hancock, 1999); however, active selection has resulted in hundreds of existing varieties (Faedi et al., 2002).
There is vast literature devoted to the studies of diversity of strawberry and other Fragaria species using different molecular marker systems.The use of protein and DNA-based markers in the studies of strawberries was described in detail by Hokanson and Maas (Hokanson & Maas, 2001).Molecular genetic research of Fragaria species was carried out using such marker systems as SSR (Simple Sequence Repeats) (Njuguna, 2010), RAPD (Randomly Amplified Polymorphic DNA) (Garcia et al., 2002), RFLP (Restriction Fragment Length Polymorphism) (Vilanova et al., 2008), and AFLP (Amplified Fragment Length Polymorphism) (Lerceteau-Köhler et al., 2003;Tyrka et al., 2002).The strawberry genome was mapped using ISSR (Inter Simple Sequence Repeats) markers (Hussein et al., 2008).One more system of genetic markers used in the study of strawberries was CAPS (Cleaved Amplified Polymorphic Sequences) (Kunihisa et al., 2005).
Another efficient genetic marker system, based on polymorphism of retrotransposon integration sites, holds some advantages over the systems mentioned above.Retrotransposons are mobile genetic elements which undergo reverse transcription (RNA→DNA) during replication, after which the new copies are integrated into the genome.Retrotransposons represent a significant component of the plant genome and may constitute over 50% of the nuclear DNA (Wicker et al., 2009;Pearce et al., 1996;SanMiguel & Bennetzen, 1998;Meyers et al., 2001).Owing to the ubiquity of these mobile elements in various plant species, their integration activity, the conservative structure of their sequences, and a large number of copies in the euchromatic chromosome regions, they can be used to study genetic diversity in plants and create genetic markers (Feschotte et al., 2002;Kalendar et al., 2011).Molecular markers based on polymorphism of retrotransposon integration sites are widely used in assessment of retrotransposon activity, phylogenetic studies, gene mapping, studies of somaclonal variation, and estimation of genetic diversity of cereals (Waugh et al., 1997;Queen et al., 2004;Konovalov et al., 2010), legumes (Ellis et al., 1998;Pearce et al., 2000;Vershinin et al., 2003;Sanz et al., 2007), potato (Lightbourn et al., 2007), vine (Labra et al., 2004), sunflower (Tang et al., 2004), flax (Smykal et al., 2011), and many other plants (Feschotte et al., 2002;Kalendar et al., 2011).
We have used the SSAP approach to analyze the varieties of strawberry (Fragaria x ananassa).This method is based on amplification of the DNA sequence between the conservative region of the retrotransposon (the LTR-Long Terminal Repeat sequence in our case) and the nearest restriction site for a suitable site specific restriction enzyme (Taq α I in our case).The amplification primers are designed from the transposon conservative sequence and from a special adapter ligated to the end of the DNA cut by the restriction enzyme.The adapter is designed in such a way as to prevent adapter-to-adapter PCR amplification.
It should always be borne in mind that the polymorphism revealed by SSAP analysis is not solely determined by transposon insertion at specific sites of the genome, but may also result from SNP polymorphism at the nearest restriction site.We have performed SSAP analysis using FaRE1 retrotransposon from the Ty1-copia group.The transposon is 5104 bp long and includes an LTR sequence 490 bp long.Its copy number is approximately 96 per genome, which corresponds to about 0.33% of the genome (He et al., 2010).

Method
The plant material of strawberry varieties (Fragaria x ananassa) was obtained from the Russian State Agrarian University.In all, we studied 46 varieties grown in Russia.
DNA was extracted from the leaves following the technique of Torres et al. (Torres et al., 1993) with minor modifications.
The DNA sequence between the LTR region of FaRE1 retrotransposon and the Taq α I restriction site was amplified.
The primers to the LTR region of FaRE1 retrotransposon were designed based on a sequence obtained from GenBank (accession number FJ871121) (He et al., 2010), with addition of a single selective nucleotide to reduce the number of amplified DNA fragments.The following LTR primers were used: Amplification was performed in two stages.At the first stage only the primer to the LTR region of FaRE1 retrotransposon was used; the PCR mix contained 5 µl of ligation mix, 1 U of Taq polymerase, Taq polymerase buffer, 0.5 mM MgCl 2 , 20 µM dNTP, and 5 pmol of LTR primer.The first amplification program was 95 °C for 5 min, then 30 cycles (95 °C for 30 s, 62 °C for 1 min, 72 °C for 2 min), then 72 °C for 10 min.
At the second stage of amplification, the adapter primer 5'-GTTTACTCGATTCTCAACCCGA-3' (Konovalov et al., 2010) and one of the primers fitted to the LTR region of FaRE1 retrotransposon were used.The PCR mix contained 15 µl of the first-stage PCR product, 1 U of Taq polymerase, Taq polymerase buffer, 1.5 mM MgCl 2 , www.ccsen 200 µM dN 95 °C for 5 The PCR Gresshoff, samples of The genet Dice's form using Split

Results
In order to with FaRE polyacryla electropho  Elizabeth, Bereginya and Vicoda did not differ in the identified FaRE1 insertions.This may be accounted for by these varieties being closely related.Strawberry is a "young" crop; since varieties with the best production characteristics are commonly used as parents in breeding practice, some distinguished varieties are present in the pedigree of most cultivars.Besides, the varieties chosen for crossing are usually characterized by low genetic diversity, so that the progeny is likely to have a low level of polymorphism.The close relations among all the varieties are illustrated by the dendrogram (Figure 3) which contains no distinct genetic clusters.In our work, using a single primer to FaRE1 retrotransposon, 78% of the studied varieties could be reliably identified by their SSAP markers.Our results demonstrate the possibility of using molecular markers of this kind to identify varieties in the planting stock.
FaRE1 is not the only retrotransposon found in strawberries.Other LTR retrotransposons of F. x ananassa (Ma et al., 2008) have been characterized; the transposon families of F. vesca have been identified, of which LTR retrotransposons proved to be the most abundant (Pontaroli et al., 2009;Davis et al., 2010).The sequenced genome of F. vesca has revealed 576 transposons (Shulaev et al., 2011), among which, according to the cited authors, LTR retrotransposons are the most abundant and comprise about 16% of the nuclear genome.The extensive data available on the strawberry transposon sequences allow the researcher to overcome one of the few drawbacks of retrotransposon-based markers, namely the need of knowing the LTR retrotransposon sequences to design the primers.The high similarity of sequences of some transposons in strawberry indicates their recent transcriptional activity (Shulaev et al., 2011); the SSAP markers based on different insertion sites of such retrotransposons may be highly polymorphic, varying even between closely related varieties.In view of the above, the SSAP technique adapted to strawberry in our work can be applied to other LTR retrotransposons identified in Fragaria.An advantage of the SSAP technique in this case is the possibility of using the same ligation mix and adaptor primer but different LTR primers, which provides a much easier way to obtain markers based on other retrotransposons.In addition, this method is theoretically capable of identifying varieties by their DNA with 100% accuracy.
Figure 1 Lanes 1, 2 Comparison of the SSAP markers obtained for strawberry showed most of its varieties (36 out of 46) to possess unique combinations of polymorphic insertions of FaRE1 retrotransposon.However, five pairs of varieties, namely Alena and Kubata, Holiday and Tsarskosel'skaya, Slavutich and Junija Smaids, Ruslan and Queen