ASSESSMENT OF GENETIC DIVERSITY IN YEAST AND BARLEY BY RETROTRANSPOSON-BASED MOLECULAR MARKERS

urvival of a species and its adaptability to the environment depends on genetic diversity which is so important for representing the heritable variation between and within populations of organisms (Rao and Hodgkin, 2002). The presence of different alleles in the gene pool is reflected by genetic diversity among individuals and hence different genotypes within populations (Templeton, 1991 & 1993). Genetic diversity has been the major focus of evolutionary biology since the beginning of the 20 th century.

Assessment of genetic diversity has become more advanced, whether through the utilization of variation in enzymes (allozymes) or through PCR-based marker systems, allowing direct examination of DNA sequence variation (Mondini et al., 2009;Poczai et al., 2012).Molecular markers have a great influence in the characterization and evaluation of genetic diversity within and between species and populations (Russel et al., 1997).DNA markers as RAPD, ISSRs, SSRs and AFLP and RFLP-PCR are considered the most suitable means for estimating genetic diversity at the DNA level because of their abundant polymorphisms and the fact that they are independently of environmental conditions (Zietkiewicz et al., 1994;Esselman et al., 1999;Holderegger et al., 2006).
Retrotransposons are a subclass of mobile genetic elements and are abundant components of the DNA in many eukaryotic organisms (Kumar and Bennetzen, 1999;Fedoroff, 2000;Zou et al., 2009).They replicate through an mRNA intermediate (Boeke and Sandmever, 1991;Havecker et al., 2004).One of the powerful molecular tools; Retrotransposonbased molecular markers, although these markers are not readily available due to the difficulty in obtaining species-specific retrotransposon primers (Du et al., 2013).Three retrotransposon-based techniques; Inter retrotransposon amplified polymorphism (IRAP), Retrotransposon Microsatellite Amplified Polymorphism (REMAP) and RAPD-retrotransposon amplified polymorphism (RRAP) were used to study the genetic diversity in different organisms; in rice (Branco et al., 2007), in fungus (Santana et al., 2012) and in tomato (Mansour et al., 2010).IRAP technique was carried out between two retrotransposons-Long Terminal Repeats (LTRs) to measure the distance between them using PCR (Kalendar and Schulman, S 2006), while PCR-amplification for the REMAP was carried out between primers matching an LTR sequence and a microsatellites domain (SSRs) to measure the distance between LTR and the nearest microsatellites locus (Kalendar and Schulman, 2006).On the other hand, RRAP technique uses a combination of primers for RAPD and LTRretrotransposon to amplify the distance between LTR and the binding site of the RAPD primer (Aalami et al., 2012).
The aim of this study was to evaluate the efficiency of RAPD, ISSRs and IRAP-PCR as molecular markers and the new developed RRAP & REMAP-PCR techniques in assessment of genomic diversity using two eukaryotes; yeast and barley as model genomes.
PCR reaction was performed in a gradient thermal cycler (Eppendorf, Germany).The reaction mixture of 10 μl con-sisted of 50 ng of genomic DNA, 0.5 U of Taq DNA polymerase, 1 μl of 10 X PCR amplification buffer, 0.2 mM dNTP, 10 p moles each of the primers and 1.5 mM MgCl 2 .Amplification for the RAPD and ISSR-PCR was done by initial denaturation at 94C for 4 minutes, followed by 30 cycles of denaturation at 94C for 45 second, annealing temperature of the primers was 37-44C for 45 second, extension at 72C for 2 minutes and the final extension was conducted at 72C for 10 minutes.For the IRAP, RRAP and REMAP-PCR, amplification was done by initial denaturation at 94C for 2 minutes, followed by 30 cycles of denaturation at 94C for 45 second, annealing temperature of primers was 50-61C for 1 minute, extension at 72C for 2 minutes and the final extension was conducted at 72C for 10 minutes.Amplification production was analyzed by submarine gel electrophoresis using 1.2% agarose and ethidium bromide at 8V/cm and the reaction product was visualized under Gel doc/UV transilluminator.

Primer design and gel analysis
Freeware Clustal X 2.1 was used to align LTR sequences to get the conserved regions which were used to design primers.The aligned sequences were formatted by CLC-Free-Workbench 3.2.3 to be more readable to the human eye.PerlPrimer 1.1.21freeware was used to design the IRAP primers for yeast genome.To confirm that the designed primers are appropriate to match only LTR positions of the yeast retrotransposons, the PrimerSelect program in the Lasergene7 software package was applied.Gel images were analyzed using the freeware (GelAnalyzer 3) to determine molecular sizes of the amplified fragments scoring for their polymorphic type either monomorphic or polymorphic.

RESULTS AND DISCUSSION
Retrotransposons consist of a conserved domain encoding products required for transposition, bounded by direct repeats (long terminal repeats, LTRs).The domain order in the polyprotein encoded between the LTRs defines retrotransposons as ''copia -like'' or ''gypsy -like'', after the type elements of Drosophila melanogaster, and defines two ancient lineages shared by plants, animals and yeasts (Xiong and Eickbush, 1990).
The LTRs are unique to each retrotransposon family, and this specificity has been exploited in genetic analyses by using primers within the LTRs to amplify flanking regions.In barley there are six retrotransposon families: BARE -1, Sukkula, Sabrina, Nikita, BAGY-1 and BAGY-2 (Leigh et al., 2003).Whereas there are five retrotransposon families in yeast: Ty1, Ty2, Ty3, Ty4 and Ty5 (Kim et al., 1998)

Tagging new genomic regions using IRAP retrotransposons molecular marker
IRAP analysis can be carried out with a single primer matching either 5´or 3´ end of the LTR but oriented away from the LTR itself, or with two LTR primers from the same or a different family of retrotransposons (Kalendar and Schulman, 2006).Two newly designed primers; ScM1 and ScM2, which carried out through PearlPrimer freeware parameters depending on the conserved regions of ten Ty1-retrotransposon sequences retrieved from NCBI database, were used with the five strains of yeast and showed moderate polymorphism percentages of 46.15% with ScM1 and 63.16% with ScM2 as a single primer, whereas the combination between primers gave 81.82% polymorphism (Table 1).The total number of bands ranged from 11 to 19 bands with molecular sizes from 207 to 2586 bp.
Whereas the combination between the primers gave less percentages of polymorphisms; 24% and 54.55% with 5'LTR+Sukkula and 5'LTR+Wltr2105, respectively.The total number of bands using IRAP method ranged from 8 to 25 with molecular sizes ranged from 134 to 2632 bp.(Table 1).

Efficiency of RAPD and R-RAP-PCR for genetic diversity detection
The ten RAPD primers showed high percentages of polymorphisms almost 100% either with yeast strains or barley cultivars.Total number of bands ranged from 8 to 31 with molecular sizes from 109 to 2718 bp in yeast, while it ranged from 6 to 26 with molecular sizes from 81 to 2399 bp in barley (Table 2).In yeast, seven RAPD primers were selected with the highest percentages of polymorphisms to be combined with the two newly designed IRAP primers, so they gave different percentages of polymorphisms between 31.25% with ScM1+OPB08 and 100% with ScM1+OPA20.The total number of bands ranged from 15 to 30 with molecular sizes from 125 to 2941 bp as shown in Table (3).At the same time in barley six RAPD primers were chosen to be combined with the three IRAP primers and they also gave different percentages of polymorphisms between 12.5% with 5'LTR+OPO14 and 72.73% with 5'LTR+OPO10.The total number of bands ranged from 11 to 27 with molecular sizes from 64 to 1963 bp as shown in Table (4).

Efficiency of ISSRs and REMAP for genetic diversity detection
Nine ISSRs primers were examined and gave high percentages of polymorphisms which reached 100% with yeast strains, and about 75% with barley cultivars (Table 5).Total number of bands; ranged from 10 to 24 with molecu-lar sizes from 159 to 2221 bp in yeast, while it ranged from 6 to 22 with molecular sizes from 127 to 2014 bp in barley.In yeast, six ISSR primers with the highest percentages of polymorphisms were selected to be combined with the two newly designed IRAP primers to make REMAP, they showed different percentages of polymorphisms between 33.33% with ScM2+17898B primers and 88.46% with ScM2+HB14 primers.The total number of bands ranged from 16 to 33 with molecular sizes from 113 to 3876 bp as shown in Table (6).Likewise in barley, six ISSRs primers were chosen to be combined with the three IRAP primers to obtain REMAP and gave different percentages of polymorphisms between the smallest 11.11% with 5'LTR+HB13 primers to the highest 61.90% with Wltr2105+ HB13 primers.The total number of bands ranged from 9 to 226 with molecular from size 97 to 2341 bp as shown in Table (7).

Comparative assessment of genetic diversity in yeast strains and barley cultivars using RAPD, ISSRs, IRAP, REMAP and RRAP molecular markers
Data of both the five yeast strains and the six barley cultivars were arrayed together to compare between the five used techniques; RAPD, ISSRs, IRAP, RRAP and REMAP in the assessment of genetic diversity.Depending on the marker technique used, the resulted banding patterns varied dramatically.However, each of them targeted the genome differently and thus results in a different banding pattern.For instance, RAPD primers targeted ran-dom homologous genomic regions (Williams et al., 1990) while ISSRs primers amplify the highly repetitive inter-simple sequence repeats of the microsatellite region in the genome (Zietkiewicz et al., 1994).In contrast, IRAP primers amplify specific genomic retrotransposons inside the middle repetitive region (Kalendar et al., 1999).The comparison among the data obtained from the five techniques was made using three parameters: polymorphism percentages, number of bands for each technique and developing specific bands for each genotype (Table 8) as following:  Polymorphism percentages Table (8) and Fig. (1) show the comparison between the five techniques based on their ability to detect polymorphism among the studied genotypes.RAPD technique exhibited the highest mean of polymorphism percentages per primer comparing with the other four techniques in both yeast strains and barley cultivars.ISSRs primers represented polymorphism percentages more than IRAP primers in yeast, but they exhibited vice versa in barley.Both REMAP and RRAP showed the lowest polymorphism percentage in barley, while were higher than IRAP in yeast.
In a comparative study among RAPD, ISSRs, IRAP and REMAP markers to assess the genetic diversity and relationship among the Citrus and its relative genotypes, Biswas et al. (2010) reported that RAPD technique generated the highest number of polymorphic bands and the average number of polymorphic bands per primer.Bublyk et al. (2013) investigated in formativeness and effectiveness of different marker types including ISSRs, IRAP and REMAP, and found that ISSRs showed the highest polymorphism among the other techniques.In a study on ten Ficus sycomorus L. genotypes, Saleh (2013) reported that RAPD markers displayed polymorphism percentages lower than those of IRAP markers (71.59% and 84.83%, respectively).

 Number of bands for each technique
Retrotransposon-based techniques showed higher number of bands than those of non-retrotransposon basedtechniques which may confirm the genetic diversity.As illustrated in Table (8

 Developing specific bands for each genotype
In yeast, a total of 178 unique bands were observed; each of them ap-peared only in a specific yeast strain (unique band).RAPD-PCR showed the highest number of strain-specific bands (71).RRAP, ISSRs and REMAP showed about 36, 33 and 29 unique bands, respectively.IRAP revealed only nine unique bands which may be due to the small number of the used IRAP primers.Barley cultivars displayed a total of 74 cultivarspecific bands.More than the half of barley unique bands was obtained by RAPD-PCR technique.IRAP-PCR technique displayed ten unique bands, the other three techniques showed equal number of 8 unique bands (Table 8).It is evident that RAPD technique excelled all others used here in this respect.
However some investigators advocate the notion that, many features of retrotransposons make them appealing as the basis of molecular marker systems.They are ubiquitous, abundant, dispersed components of eukaryotic genomes.Their activity simultaneously leads to genome diversification and provides means of their detection.Retrotransposons are long and produce a large genetic change at the point of insertion, thereby providing conserved sequences that can be used to detect their own integration.This event is not linked to removal of the transposable element from another locus, as it is for DNA transposons (Schulman, 2007).An ideal molecular marker technique should have the following criteria: 1) be polymorphic and evenly distributed throughout the genome, 2) provides adequate resolution of genetic differences, 3) generates multiple, independent and reliable markers, 4) be sim-ple, quick and inexpensive, 5) needs small amount of DNA sample, 6) requires no prior information about the genome for an organism and 7) has linkage to distinct phenotype (Agrawal et al., 2008).As yet, no molecular marker has been known to have all of these aspects together, but scientists should select a suitable marker according to the aim of the research.Retrotransposon transcriptional activation will lead to an increase in copy number and genome size if the newly transposed copies survive selection.They claimed that, all retrotransposon-based methods create high polymorphism between and within species and can meet most of the above mentioned criteria (Kalendar et al., 2011).and REMAP use agarose gels and inexpensive reagents but generate several different polymorphic markers to be used in studies of genetic variability and high reproducibility by using specific primers (Kalendar et al., 1999;Leigh et al., 2003;Le et al., 2008;Santana et al., 2012).Saleh (2013) studied the accuracy, repeatability and reproducibility of the RAPD and IRAP techniques for determining the genetic variability and found that the IRAP marker was more efficient than the RAPD assay.Usually, the REMAP pattern was considerably more variable than the corresponding ISSRs pattern; and often, but not always, depending on LTR sequence.

SUMMARY
Five Molecular genetics techniques (RAPD, ISSRs, IRAP, REMAP and RRAP) were used to study the genetic diversity in five yeast strains and six barley cultivars.New two retrotransposonbased primers (ScM1 and ScM2) were designed.RAPD technique represented the highest polymorphism percentages per primer compared with the other four techniques.ISSR primers represented polymorphism percentages more than IRAP primers in yeast, but lower in barley.Both REMAP and RRAP combinations showed similar results either in yeast or barley.Retrotransposon-based techniques (IRAP, RAP and REMAP) showed more number of bands more than those nonretrotransposon (RAPD and ISSRs) based techniques which make them a useful approach as molecular markers.
. In this work, PAPD and ISSRs-based PCR together with IRAP, R-RAP and REMAP retrotransposons were used to study the genetic diversity of the five strains of yeast (Saccharomyces cerevisiae) and the six cultivars of barley (Hordeum vulgare L.).
) and Fig. (2), yeast REMAP-PCR technique produced the highest band numbers (24) whereas RAPD and RRAP-PCR technique showed equal number of bands (20).In barley, RRAP-PCR technique revealed the highest band numbers (20), while the lowest number of bands (12) was scored for ISSRs-PCR technique.Bublyk et al. (2013) studied the genetic diversity of Iris pumila L. depending on different marker types including RAPD, ISSRs, IRAP and REMAP.They found that ISSRs were the best markers among the other techniques in amplifying different loci on the genome of Iris pumila L.
Retrotransposon-based technique can be used to obtain molecular markers, due to many features and advantages, where IRAP and REMAP techniques have more advantages, over other techniques already used; versatility, as they allow for the combination of various primers that anneal to conserved regions of retrotransposons (IRAP) or microsatellites (REMAP), easy handling because the techniques involve straight forward PCR, lower cost and less labour than the AFLP and the microsatellite techniques, IRAP

Fig. ( 1
Fig. (1): Histogram of polymorphism percentages using the five techniques in yeast and barley.

Fig
Fig. (2): Histogram of comparison of bands number in yeast and barley using the five techniques.

Table ( 1
): IRAP-PCR banding patterns for the five strains of yeast and the six cultivars of barley using IRAP primers and their combinations.

Table ( 5
): Results of the Nine ISSR based PCR primers with the five strains of yeast and the six cultivars of barley.

Table ( 6
): REMAP-PCR banding patterns for the five strains of yeast (Saccharomyces cerevisiae L.) using primers combinations.

Table ( 8
): Comparative assessment of different molecular markers in the yeast strains and barley cultivars.