Retrotransposon Cassandra copies estimating in plums using real-time PCR approach

*Corresponding author: Jana Žiarovská, Department of Genetics and Plant Breeding, Slovak University of Agriculture, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic. E-mail: jana.ziarovska@uniag.sk Received: 04 May 2015; Revised: 03 November 2015; Accepted: 04 November 2015; Published Online: 04 November 2015 Emirates Journal of Food and Agriculture. 2015. 27(12): 883-888 doi: 10.9755/ejfa.2015-04-165 http://www.ejfa.me/ Žiarovská, et al.: Cassandra copies in plums 884 Emir. J. Food Agric ● Vol 27 ● Issue 12 ● 2015 performed not only by high sensitive hybridization with specific DNA probes (Walsh et al. 1992) or by competitive PCR (Handt et al. 1996), but more recently, by using real-time quantitative PCR (Walker et al. 2003). Real-time PCR detects amplification products directly as they are produced. Subsequently, the Ct value is setting as the cycle at which fluorescence significantly exceeds the background (Higuchi et al. 1993). Ct value describes the state when product accumulation correlated with the amount of starting template. A lower Ct value describes a higher starting quantity of the template. Threshold is desirable to be achieved withing the exponential phase, where no limitation in the PCR exist and Ct values are reproducible (Higuchi et al. 1993). Optimizing this has the result of precision in DNA quantitation. Quantitation of the template in analysed accessions is performed by a standard curve approach or by using of internal controls approach (Gentle et al. 2001, Raeymaekers 2000, Song et al. 2002). Generating the standard curve results in linear plot of a set of standards initial copy number logs versus Ct value. Subsequently, Ct-s of analysed accessions is determined, and constructed standard curve is what determines their starting copy number (Higuchi et al. 1993, Song et al. 2002). Internal control approach works with an internal endogenous housekeeping gene that i sused for normalization of the amount of template (Livak and Schmittgen 2001, Schmidt and Parrott 2001). Oantitation in this approach is based on the same efficiencies for the template and internal housekeeping genes (Li et al. 2004). In this study, a quantitative real-time PCR protocol was established for Cassandra retroelement analysis in the genome of Prunus domestica, L. Based on this protocol the Cassandra copies number was calculated using an absolute quantitation method. Finally, different plant accessions were compared for their Cassandra copies. MATERIAL AND METHODS Genomic DNA extraction for real-time PCR Twenty-three plum (P. domestica, L.) genotypes were used in analysis. A detailed characteristic is listed in Table 1. Approximately 100 mg of leaf tissue was ground to a fine powder in liquid nitrogen. Total genomic DNA was isolated using the isolation kit Invisorb Spin Plant Mini Kit (Invitek), according user ́s manual. The obtained DNA was quantified by fluorometer (QubitTM). Oligonucleotid primer design and real time PCR assays Using data deposited in NCBI database about Cassandra retroelements in the plum genome (AY860314) were retrieved and used as template for designing real-time PCR oligos. 18S rRNA sequence for setting a standart curve was also obtained from NCBI database (FJ899098). Real-time PCR oligonucleotide primers were manually designed for both of them (Table 2) using internet-based interface of Primer 3 software. Real-time PCR assays Real-time PCR runs were performed in 96-well optical plates in triplicate, each containing 12.5 PCR master mix (SYBR Green BioRad), 0.2 pmol of forward and reverse primer 2 μl template DNA (diluted to 20 ng/μl), for 35 cycles using a BioRad CFX 96. Cycle conditions were as follows; 5 min at 95 °C followed by 35 cycles of 10 s at 95 °C, 10 sec at 58 °C and 30 sec at 72 °C. A standard curve was drawn by plotting the natural log of the threshold cycle against the natural log of the number of molecules. The equation drawn from the graph was used to calculate the precise number of specific 18S rRNA molecules. Verifying of amplification specifity by direct sequencing of Cassandra internal domain After the control of the PCR specifity by the melting point analysis of obtaining amplicons (that was set at 84.5 °C), cleaning step was performed for PCR products before the sequencing procedure. This was performed using Agencourt AMPure XP (Beckman Coulter) following the manufacturer ́s instructions. Sequencing PCRs were optimized for the molar ratio of ~ 40:1 (primer to template) using the Dye Terminator Cycle Sequencing Genome LabTM Kit (Beckman Coulter). Cycling was as follows: 96 °C 20 sec; 50 °C 20 sec and final 60 °C 4 minutes. Purification of sequencing PCR was performed by Agencourt® CleanSEQ® (Beckman Coulter) following the manufacturer ́s instructions. Sequencing of amplicons was performed in CEQTM Genetic Analyses System using the LFR-1 protocol. All the direct sequencing reactions were repeated twice. Data analysis using absolute qantification In order to compare samples, it was necessary to use appropriate form of calibration. The common method to employ a reference gene – 18S rRNA was used. The quantity of Cassandra retrotransposon copies was interpolated from a range of standarts of known quantity. A 10-fold dilution series was created from a sample with a known concetration of 18S rRNA gene, resulting in the set of standards containing up to the 112 994 copies of the gene. The copy numbers were calculated based on the following formula, in which n means the number of the base pairs, m is the mass of the DNA, NA is the Avogadro ́s number (6,02 × 1023 bp/mol) and M is the average molecular weight of a base pair. n = (m × NA)/M; m = n × 1,096 × 10-21 g/bp. The following equation to determine the quantity of an unknown sample was used N = 10 ((Ct – b)/m), where Ct is a threshold, b is the regression Žiarovská, et al.: Cassandra copies in plums Emir. J. Food Agric ● Vol 27 ● Issue 12 ● 2015 885 constant and m is the regression coefficient. Obtained quantity values where converted into the number of copies of both Cassandra retrotransposon and 18S rRNA by using the plum genome size of 323 Mbp (Loureiro et al. 2007) and following formula for calculating the number of copies from quantifying DNA concentration: m = (n) (1,096e-21 g/bp), where n is a genome size in bp, m is a mass and e-21 is equal to the x 10-21. RESULTS AND DISCUSSION Comparing the known sequences of Cassandra has shown the conserved regions among species. They were defined in the both of LTRs and in the part of the internal domain in the primer binding site sequence. This sequence is considered to be conserved in Cassandra (Kalendar et al 2010), more, the authors based on the PBS sequence a new approach for Cassandra insertional polymorphism analyses the iPBS method. Another study (Kalendar et al. 2008) has proved all of the known TRIM elements as containing a highly conserved region for 5S RNA that corresponds to the LTRs. Using the first, or the second above mentioned approach, molecular DNA based analyses of Cassandra provide a wide range of germplasm evaluation. Here, a real-time PCR approach is reported for a estimation of Cassandra retrotransposon copies in the genome of Prunus domestica, L., as both, genomic DNA and reverse transcribed cDNA can be used as templates for real-time PCR. Total genomic DNA of plum was used for estimation of TRIM retrotransposon copies when comparing it to the standart curve based on the 18S rRNA gene. Specifity of Cassandra amplification was inspected both, by melt analysis (data not shown) and by direct sequencing, too. Obtained sequences were BLASTed against the Cassandra sequence of Prunus domestica internal domain region previously registered in the NCBI database (AY860314). They were returned as identical (data not shown). First, the real-time PCR protocol for the amplification of 18S sRNA was optimized for futher analysis. 18S sRNA provided a good constitutive control as was shown by constitutive level of expression. After optimization of Table 1: List of analysed Prunus domestica, L. accessions No. Genotype Origin/Locality Pedigree 1 P. domestica, L. Slovakia – Pečovská Nová Ves Unknown 2 P. domestica, L. Slovakia – Torysa Unknown 3 P. domestica, L. Slovakia – Podolínec Unknown 4 P. domestica, L. Slovakia – Lipany Unknown 5 Gabrovská Bulgaria Kjustendilska×montfortska 6 Chrudimská Czech Republic Accidental seedlings 7 Švestka domácí Czech Republic Accidental seedlings 8 Čačanská lepotice Former Yugoslavia Wangenheimova×požegača 9 Anna spätch Hungary Accidental seedlings 10 Čačanská ranná Former Yugoslavia Wangenheimova×požegača 11 Čačanská rodná Former Yugoslavia Stanley×požegača 12 Elena Germany Vlaška×stanley 13 Hamanova švestka Czech Republic Not specified 14 Hanita Germany President×auerbacher 15 Katinka Germany Ortenauer×gerstetter 16 Lutzelsachsenská Germany Accidental seedlings 17 Presenta Germany Ortenauer×president 18 Stanley USA Agenská × grand duke 19 Tegera Germany Ortenauer×gerstetter 20 Valjevka Former Yugoslavia Agenská × stanley 21 Vlaška Italy Not specified 22 Wangenheimova Germany Not specified 23 Zimmerova Germany Accidental seedlings Table 2: Real‐time quantitative PCR primers Primer name Primer Sequence (5 ́‐ 3 ́) Concentration Amplicon size PRUN18S‐A‐F Forward acgagagagcgcgtccccat 400 nM 450 bp PRUN18S‐A‐R Reverse ggggtcgcgttgaaagccga 400 nM PRUN‐ID‐F Forward ccgtgagggcagagaggggg 200 nM 364 bp PRUN‐ID‐R Reverse gctcccaaaaggcctcgtgct 200 nM Žiarovská, et al.: Cassandra copies in plums 886 Emir. J. Food Agric ● Vol 27 ● Issue 12 ● 2015 amplification conditions it was used as internal control for the subsequent experiments. Dissociation curves of 18S sRNA amplified products calculated by plotting the negative derivative of fluorescence emitted by the PCR sample during the melting procedure showed a single peak with Tm of 87.6°C. This was identical for all analysed samples, indicating the specifity of PCR product (Fig. 1). Moreover, agarose gel electro


INTRODUCTION
Retrotransposons are an inevitable part of the throughout all life domens and large plant genomes consist of >50% of these elements.One of them, terminal repeat retrotransposon in miniature posses no reading frames for retrotransposon specific proteins.They consist from following: LTRs, reverse transcriptase priming site and an intervening segment.TRIMs are expected to be activated by transcomplementation (Kalendar et al. 2008).They are used widely in genome diversity studies for the purpose of the assessment of agricultural biodiversity on interspecific and intraspecific level (Žiarovská et al., 2012;Trebichalský et al., 2013;Balážová et al., 2014).
Cassandra as one of the TRIM elements, posses conserved 5S rRNA sequences and in its LTR is a promoter for RNA polymerase III.Very similar as another members from this group, Cassandra is highlu copied throughout the higher plants (Kalendar et al. 2008).They are a dynamic component of the genome, affecting both gene expression and overall genome structure.Until very recently, the nonautonomous retrotransposons were considered the junk of the junk (Schulmann 2007).Retroelement parasites such as Cassandra have no coding capacity but appear to exploit not only proteins of other retrotransposons but also those of the cell.Furthermore, the plant cell must regulate the replicative capacity of the retrotransposon to prevent unlimited, disruptive integration of new daughter copies (Schulmann 2007).Cassandra elements have not autonomous transposition and as reported by many studies, they are dependend on the help of mobility-related proteins that are encoded by other retrotransposons (Witte et al. 2001).
Traditionally used methods for the determination of gene copies is Southern blot hybridization.In this approach, two possible counting strategies are reported.In the first method, single restriction site in the cassette is chosen to digest the gDNA, and this is used for Southern blot hybridization with specific probes.If a sinle band is obtained in Southern blot, the copy number should be one (Bubner and Baldwin 2004).In the second method, the same amount of gDNA from different assessions is digested using two restriction enzymes that cutt flanking regions to obtain a fragment of an expected size, that is subsequently hybridized with a specific probe (Yuan et al. 2008).
performed not only by high sensitive hybridization with specific DNA probes (Walsh et al. 1992) or by competitive PCR (Handt et al. 1996), but more recently, by using real-time quantitative PCR (Walker et al. 2003).Real-time PCR detects amplification products directly as they are produced.Subsequently, the Ct value is setting as the cycle at which fluorescence significantly exceeds the background (Higuchi et al. 1993).Ct value describes the state when product accumulation correlated with the amount of starting template.A lower Ct value describes a higher starting quantity of the template.Threshold is desirable to be achieved withing the exponential phase, where no limitation in the PCR exist and Ct values are reproducible (Higuchi et al. 1993).Optimizing this has the result of precision in DNA quantitation.Quantitation of the template in analysed accessions is performed by a standard curve approach or by using of internal controls approach (Gentle et al. 2001, Raeymaekers 2000, Song et al. 2002).Generating the standard curve results in linear plot of a set of standards initial copy number logs versus Ct value.Subsequently, Ct-s of analysed accessions is determined, and constructed standard curve is what determines their starting copy number (Higuchi et al. 1993, Song et al. 2002).Internal control approach works with an internal endogenous housekeeping gene that i sused for normalization of the amount of template (Livak andSchmittgen 2001, Schmidt andParrott 2001).Oantitation in this approach is based on the same efficiencies for the template and internal housekeeping genes (Li et al. 2004).
In this study, a quantitative real-time PCR protocol was established for Cassandra retroelement analysis in the genome of Prunus domestica, L. Based on this protocol the Cassandra copies number was calculated using an absolute quantitation method.Finally, different plant accessions were compared for their Cassandra copies.

Genomic DNA extraction for real-time PCR
Twenty-three plum (P.domestica, L.) genotypes were used in analysis.A detailed characteristic is listed in Table 1.Approximately 100 mg of leaf tissue was ground to a fine powder in liquid nitrogen.Total genomic DNA was isolated using the isolation kit Invisorb Spin Plant Mini Kit (Invitek), according user´s manual.The obtained DNA was quantified by fluorometer (Qubit™).

Oligonucleotid primer design and real time PCR assays
Using data deposited in NCBI database about Cassandra retroelements in the plum genome (AY860314) were retrieved and used as template for designing real-time PCR oligos.18S rRNA sequence for setting a standart curve was also obtained from NCBI database (FJ899098).Real-time PCR oligonucleotide primers were manually designed for both of them (Table 2) using internet-based interface of Primer 3 software.

Real-time PCR assays
Real-time PCR runs were performed in 96-well optical plates in triplicate, each containing 12.5 PCR master mix (SYBR Green BioRad), 0.2 pmol of forward and reverse primer 2 µl template DNA (diluted to 20 ng/µl), for 35 cycles using a BioRad CFX 96.Cycle conditions were as follows; 5 min at 95 °C followed by 35 cycles of 10 s at 95 °C, 10 sec at 58 °C and 30 sec at 72 °C.A standard curve was drawn by plotting the natural log of the threshold cycle against the natural log of the number of molecules.The equation drawn from the graph was used to calculate the precise number of specific 18S rRNA molecules.

Verifying of amplification specifity by direct sequencing of Cassandra internal domain
After the control of the PCR specifity by the melting point analysis of obtaining amplicons (that was set at 84.5 °C), cleaning step was performed for PCR products before the sequencing procedure.This was performed using Agencourt AMPure XP (Beckman Coulter) following the manufacturer´s instructions.Sequencing PCRs were optimized for the molar ratio of ~ 40:1 (primer to template) using the Dye Terminator Cycle Sequencing Genome Lab™ Kit (Beckman Coulter).Cycling was as follows: 96 °C 20 sec; 50 °C -20 sec and final 60 °C -4 minutes.Purification of sequencing PCR was performed by Agencourt® CleanSEQ® (Beckman Coulter) following the manufacturer´s instructions.Sequencing of amplicons was performed in CEQ TM -Genetic Analyses System using the LFR-1 protocol.All the direct sequencing reactions were repeated twice.

Data analysis using absolute qantification
In order to compare samples, it was necessary to use appropriate form of calibration.The common method to employ a reference gene -18S rRNA was used.The quantity of Cassandra retrotransposon copies was interpolated from a range of standarts of known quantity.A 10-fold dilution series was created from a sample with a known concetration of 18S rRNA gene, resulting in the set of standards containing up to the 112 994 copies of the gene.The copy numbers were calculated based on the following formula, in which n means the number of the base pairs, m is the mass of the DNA, N A is the Avogadro´s number (6,02 × 10 23 bp/mol) and M is the average molecular weight of a base pair.n = (m × N A )/M; m = n × 1,096 × 10 -21 g / bp.The following equation to determine the quantity of an unknown sample was used N = 10 ((Ct -b)/m), where Ct is a threshold, b is the regression constant and m is the regression coefficient.Obtained quantity values where converted into the number of copies of both Cassandra retrotransposon and 18S rRNA by using the plum genome size of 323 Mbp (Loureiro et al. 2007) and following formula for calculating the number of copies from quantifying DNA concentration: m = (n) (1,096e -21 g/bp), where n is a genome size in bp, m is a mass and e -21 is equal to the x 10 -21 .

RESULTS AND DISCUSSION
Comparing the known sequences of Cassandra has shown the conserved regions among species.They were defined in the both of LTRs and in the part of the internal domain -in the primer binding site sequence.This sequence is considered to be conserved in Cassandra (Kalendar et al 2010), more, the authors based on the PBS sequence a new approach for Cassandra insertional polymorphism analyses -the iPBS method.Another study (Kalendar et al. 2008) has proved all of the known TRIM elements as containing a highly conserved region for 5S RNA that corresponds to the LTRs.Using the first, or the second above mentioned approach, molecular DNA based analyses of Cassandra provide a wide range of germplasm evaluation.
Here, a real-time PCR approach is reported for a estimation of Cassandra retrotransposon copies in the genome of Prunus domestica, L., as both, genomic DNA and reverse transcribed cDNA can be used as templates for real-time PCR.Total genomic DNA of plum was used for estimation of TRIM retrotransposon copies when comparing it to the standart curve based on the 18S rRNA gene.Specifity of Cassandra amplification was inspected both, by melt analysis (data not shown) and by direct sequencing, too.Obtained sequences were BLASTed against the Cassandra sequence of Prunus domestica internal domain region previously registered in the NCBI database (AY860314).They were returned as identical (data not shown).
First, the real-time PCR protocol for the amplification of 18S sRNA was optimized for futher analysis.18S sRNA provided a good constitutive control as was shown by constitutive level of expression.After optimization of amplification conditions it was used as internal control for the subsequent experiments.Dissociation curves of 18S sRNA amplified products calculated by plotting the negative derivative of fluorescence emitted by the PCR sample during the melting procedure showed a single peak with Tm of 87.6°C.This was identical for all analysed samples, indicating the specifity of PCR product (Fig. 1).Moreover, agarose gel electrophoresis of these products confirmed the amplification of a single fragment and no primer-dimer formation during the PCR (Fig. 2).
Control reactions did not generate any products with significant fluorescence.Cassandra retrotransposons are reported to be abundant and insertionally polymorphic (Cloix et al. 2000, Kalendar et al. 2008).Kalendar et al. (2008) found 6,697 ± 588 copies of Cassandra element.
In this study a probe using approach was chosen when the probe was constructed in a manner that it includes only a 5S domain of Cassandra.
Analysis of the rice genome obtained 436 Cassandra 5S RNA sequences.A similar amount of 5S genes, concretelly 384, were identified in rice (Cloix et al. 2000).Kalendar et al. (2008) also determine thousands of Cassandra in the ferns.These copies number corresponds to our findings in the hexaploid plum genome.
As the principle of absolute quantification is to determine the most exact copies of the target present in the template, by relating the fluorescence signal to a standart curve, therefore requiring that the absolute quantities of the standart be known (Mackay 2004).This approach was used in a slightly modified version, where the quantity of Cassandra retroelement copies (Table 3) was interpolated from a range of 18S rRNA standarts of known quantity in the hexaploid plum´s genome.Beside the absolute quantification, the relative fold of amplification increase of Cassandra sequences was determined (Fig. 3) to comparing the calculated number copies with changes showed within the relative fold of Cassandra copies.
Similar method where the absolute quantification approach of standart curve construction was used for determining gene copies in transgenic mice (Yun et al. 2006).The authors reported that the quantification of gene targets such this provides a more effective form of data obtaining than relative quantification, but the universally accepted methodologies for achieving absulote quantification still not exist (Yun et al. 2006).
As the real-time PCR is sensitive and specific enough to distinguish a 1-or 2-fold difference in target template, the counting of transgene copies in wheat was compared with results from Southern blot analysis (Li et al. 2004).Concordant results when comparing both of them were obtained for the nuclear DNA content for the most of analysed accessions -70% of samples in the range of >1000-30 pg (Alonso et al. 2003).30 % of the analysed samples in the study of the same authors resulted negative for the blot analysis and positive when analyzed them by the real-time PCR.Authors have summarized this as strongly indicating that the real-time PCR is an efficient and more sensitive method than the blot hybridization.The concordance between real-time PCR and Southern blot results was approximately 90% (Li et al. 2004).
Cassandra elements as a fairly abundant family in plants genomes are reported in the case of rice (Kalendar et al. 2008).On the applied level, Cassandra elements have the potential to be a very useful molecular markers and the analysis of their functionality will aid exploitation in mapping and genome diversity studies.The portion of nonautonomous retroelements such as short nuclear interspersed elements and TRIMs in genomes of eukaryotics may be the result of a unique evolutionary relationship with their autonomous elements partners and the host genome, so the specific information about them are still required and the estimation of their number of copies is an inevitable part of them.Analyses of the rice genome provided by Pryer et al. 2001 resulted in that 15% of Cassandra long terminal repeats and 21% of complete elements are the parts of the genes, but only 1% of them is in coding sequences.When compared, retrotransposon Tos17 possess a similar distribution in the rice genome but about half the genic insertions are into exons.Unlike Cassandra, Tos17 is generally silent and rare, being found in one to five copies (Pryer et al. 2001).
Among the species in the Hordeum genus, BARE1 is present on an average in 14,000 copies, with 16,000 copies in barley (Vicient et al. 1999); the number varies across the genus.Based on these copy numbers and on the genome sizes, full-length BARE1 comprises 0.8% to 5.7% of the genome in genus, and this measure also varies across the genus Kankanpää et al. 1996).Vukich et al. (2009) reported, that sunflowers repeated sequences ot the three Gypsy-like sequences and one Copia -likesequence have a copy number per haploid genome ranging from 4,000 to 16,000.Woloszynska et al. (2006) used real-time PCR in the verification of the differences in the copy numbers and steady-state transcript levels of mitochondrial genes coding for proteins or RNAs of four Phaseolus vulgaris lines.
Real-time PCR has provided the method of choice for not time consuming, relatively cheap, and effective estimation of copy number (Ingham et al. 2001, Mason et al. 2002, Bubner and Baldwin 2004).Various approaches have been tested to improve the effectivity of real-time PCR for this application using both the standard curve method and the ΔCt method involving an internal reference gene (Yang et al. 2005).Some details of several methods and their statistical models that were developed for these methods were discussed by Yuan et al. 2008.Thus, the results presented in literature and obtained by this paper findings shown the strength of the real-time PCR approach in the genome elements copy number estimation.

CONCLUSION
In conclusion, it has been demonstrated the utility of realtime PCR for the quantification of genome transposable elements represented by the retrotransposon Cassandra.This methodology offers several advantages with respect to the hybridization methods, including: higher sensitivity, unnecessary post-PCR processing, the possibility to perform qualitative analysis of different aspects from different genomes, and the automation feasibility.1.
and writing the manuscript, and prepared the figures.Danka Bošeľová performed nucleic acids extractions and helped with experimental procedures.Milan Bežo collaborated in data analysis and helped in drafting the manuscript.All authors were involved in revising the manuscript in progress and critically approving the final manuscript.

Fig 3 .
Fig 3. Visualization of SYBR detected real-time PCR based changes in Cassandra product amplification.Number of plum variety identification as in Table1.

.
Melting analyses of 18S rRNA PCR product of Anna Späch plum variety.Electrophoreotic visualization of the specifity of 18S rRNA PCR product amplification.Number of plum variety identification as in Table1.