ISSN 1021�4437, Russian Journal of Plant Physiology, 2014, Vol. 61, No. 6, pp. 828–833. © Pleiades Publishing, Ltd., 2014.

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1INTRODUCTION

Sukkula elements are long terminal repeat (LTR)retrotransposons, and the LTR pairs together with theintervening domains constitute complete, but non�autonomous elements belonging to a novel group ofretroelements. The members of this group are similarto TRIMs in their lack of a protein�coding domain.However, they are unique in their possession of a largeinternal domain that is highly conserved in primarysequence and secondary structure despite the lack ofcoding capacity. This group was named as large ret�rotransposon derivative (LARD) elements [1]. Theinsertion contains Sukkula, which means “shuttle” inFinnish. Sukkula internal domains were first identifiedelsewhere in the barley genome [2]. Thus, it is likelythat the Sukkula sequences in the contig represent soloLTRs of a novel barley retrotransposon that were gen�erated by recombination between two LTRs of theoriginal element.

Some marker techniques were developed to deter�mine retrotransposon movements in the genome [3].One of these techniques, Inter�RetrotransposonAmplified Polymorphism (IRAP) depends on theamplification of genomic distance between two LTR

1 This text was submitted by the authors in English.

retrotransposons [4]. However, there are other tech�niques, such as Inter�Primer Binding Site Amplifica�tion (iPBS) [5]. In this technique, polymorphism isdetected by the presence or absence of the PCR prod�uct. The lack of amplification indicates the absence ofthe retrotransposon at the particular locus [6, 7].

Generally, many retrotransposons are inactivatedby various mechanisms (such as DNA methylation)during plant development [8]. However, they may beactivated by various biotic or abiotic stress conditionslike wounding, pathogen attack, various drug orchemical applications [9–11]. Bonchev et al. [12]showed ethyl methanesulfonate (EMS)�induced tran�scriptional activation of BARE1 and WIS 2�1A ret�rotransposons. In addition, in our previous study [13],we have shown that epirubicin, one of the anthracy�cline antibiotics, that is commonly used in cancer che�motherapy, induced BARE1 variation.

Plant tissue culture applications have been knownas one of the stressful conditions. Somaclonal varia�tions are common mutations in tissue culture andresult in genetic and phenotypic variations amongclonally propagated plants of a single donor [14]. Theycan occur by different kinds of genetic and epigeneticmechanisms, such as chromosome breakage and DNAmethylation alterations [15, 16]. Up to date, manystudies on somaclonal variation induced by tissue cul�ture have been published. Cytogenetic abnormalities,sequence changes, DNA methylation variations, and

RESEARCH PAPERS

Sukkula Retrotransposon Insertion Polymorphisms in Barley1

G. Kartal�Alacam, S. Yilmaz, S. Marakli, and N. Gozukirmizi Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Vezneciler, 34134 Istanbul, Turkey;

fax: +90 212 455 58 11; e�mail: nermin@istanbul.edu.trReceived October 30, 2014

Abstract—Tissue culture could be considered as one of the stress factors that affect the activation of ret�rotransposons. In this study, mature barley embryos (Hordeum vulgare L., cv. Tokak 157/37) were cultured forcallus formation. Sukkula (a non�autonomous retrotransposon) polymorphism was investigated in calli withdifferent culturing time (40� and 80�day�old), which was derived from the same embryo in MS medium sup�plemented with 3 mg/L of Dicamba. Polymorphism was detected using Inter�Retrotransposon AmplifiedPolymorphism (IRAP) and Inter�Primer Binding Site Amplification (iPBS) techniques with two differentSukkula�specific primers. Noncultured five mature embryos were used as a control. While up to 14% poly�morphism rate was observed in the case of noncultured embryos, 61 and 0% polymorphism rates wereobserved with IRAP analyses for 40� and 80�day�old calli, respectively. iPBS analysis revealed the polymor�phism ratio up to 29% in noncultured material, and up to 58 and 70% in 40� and 80�day�old callus materials,respectively. In addition, there were 50 and 74% polymorphism rates between 40� and 80�day�old calli byIRAP and iPBS analyses, respectively. The results obtained showed that tissue culture conditions and callusage affected Sukkula retrotransposon movements, and all individuals did not present the same effect.

Keywords: Hordeum vulgare, Sukkula, IRAP, iPBS, retrotransposon

DOI: 10.1134/S1021443714060107

Abbreviations: IRAP—Inter�Retrotransposon Amplified Poly�morphism; iPBS—Inter�Primer Binding Site Amplification;LTR—Long Terminal Repeats.

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SUKKULA RETROTRANSPOSON INSERTION POLYMORPHISMS IN BARLEY 829

transposon movements were found in calli, regener�ated plants, and their progeny [17–19]. Although itsinsertion (therefore the efficiency of its transposition)remains unclear, Sukkula has been shown to be theone of the most moveable elements in barley. In thisstudy, we investigated Sukkula retrotransposon move�ments in callus materials of different age (40� and80�day�old), which were obtained from the sameembryo and the variations in the genome comparedwith noncultured mature embryos.

MATERIALS AND METHODS

Callus culture. Barley (Hordeum vulgare L., cv.Tokak 157/37) seed sterilization was performedaccording to Bayram et al. [20]. Sterilized and dis�sected embryos were blotted with sterile filter paperand cultured on MS medium supplemented with3 mg/L of Dicamba [21].

Each embryo was given with a number and incu�bated in plant growth chamber at 25 ± 2°C in darknessfor 40 days. At the end of the incubation time, eachcallus was cut into two pieces, and the embryo numberwas given to each piece. One piece was used forgenomic DNA isolation, and the second one was sub�cultured at the same conditions to obtain 80�day�oldcallus. 40� and 80�day�old calli originated from thesame embryos were accepted as one group, and IRAPand iPBS were carried out with three groups originatedfrom three different embryos. Furthermore, five non�cultured mature embryos were used as a control todetermine whether there is any naturally polymor�phism related to Sukkula transposition between indi�viduals.

Genomic DNA isolation. Genomic DNA was iso�lated from noncultured embryos and 40� and 80�day�old calli according to Tchorbadjieva and Pantchev [22].The quality of DNA was controlled on 1% agarose gel,and the quantity of DNA was measured with a spec�trophotometer.

Inter�Retrotransposon Amplified Polymorphism(IRAP)�PCR. IRAP was performed with theSukkula specific primer (3'�GGAACGTCG�GCATCGGGCTG�5'). Primer sequence wasobtained from Leigh et al. [23]. IRAP�PCR was car�ried out in 20 μL of the reaction mixture containing6 μL of nuclease�free dH2O, 10 μL of SapphireAmpFast PCR Master Mix (A801�1, 2X), 2 μL of primer(10 μM), 2 μL of 10 ng/μL template genomic DNA.The amplification conditions were as follows: one ini�tial denaturation step at 94°C for 2 min followed by30 cycles of 94°C for 20 s, 60°C for 20 s, and 72°C for1.5 min. The reaction was completed by a final exten�sion step at 72°C for 7 min.

Inter�Primer Binding Site Amplification(iPBS)�PCR. iPBS was performed with the Sukkulaspecific primer (5'�GCTCTGATACCA�3'). Primersequence was obtained from Kalendar et al. [5].iPBS�PCR was performed in 20 μL of the reaction

mixture containing 5 μL of nuclease�free dH2O, 10 μLof SapphireAmp Fast PCR Master Mix (A801�1,Takara, 2X), 3 μL of primer (10 μM), 2 μL of 10 ng/μLtemplate genomic DNA. The amplification condi�tions were as follows: one initial denaturation step at95°C for 3 min followed by 30 cycles of 95°C for 15 s,50°C for 60 s, and 68°C for 60 s. The reaction wascompleted by a final extension step at 72°C for 5 min.

Evaluation of PCR products. PCR products wereloaded to 6% nondenaturing PAAG (29 : 1, acryla�mide : bis), and gel was run at 200 V for 4 h in 1X TBEbuffer. A molecular weight marker (GeneRuler™ 1�kbDNA Ladder, SM0312, Fermentas) was also loaded todetermine the size of amplicons. Gel was stained andphotographed on a UV transilluminator. Well�resolvedbands were scored with a binary value: (1) for presenceand (0) for absence. The binary matrix (1/0) was used tocalculate the similarity between embryos and 40� and80�day�old calli. Jaccard’s similarity index was calcu�lated using the formula: NAB/(NAB + NB + NA); whereNAB is the number of bands shared by two samples,NA represents amplified fragments in sample A, andNB represents amplified fragments in sample B [24].

RESULTS AND DISCUSSION

In this study Sukkula retrotransposon�mediatedpolymorphism was investigated by IRAP and iPBSmethods in noncultured mature embryos, 40� and80�day�old barley calli. Polymorphism ratios were cal�culated with Jaccard’s coefficient.

IRAP Results

IRAP results showed some polymorphic bandsamong control, 40� and 80�day�old calli. In Fig. 1,some unique and missing bands are shown with arrowsmarked by different letters (a–h). Arrow a shows aband just observed in the 1st and 2nd control samples,while arrow b indicates a missing band for the 3rd, 6th,7th, 8th, and 11th samples. The arrow c is a bandappeared in the 1st, 2nd, 5th, and 8th samples,whereas arrow d⎯in the 1st, 2nd, 3rd, 5th, 6th, and7th samples. Arrow e denotes a band in the 6th, 7th,9th, 10th, and 11th samples. Arrow f also denotes aband obtained from the 6th and 7th samples. Arrows gand h show unique and missing bands for the 8th sam�ple, respectively.

In accordance with this data, polymorphism rateswere calculated in the range of 4–14% in control sam�ples (Fig. 1 and Table 1, lines 1–5). Previously, in somesimilar studies of retrotransposon movements in callusculture, it was demonstrated that there was no poly�morphism between individual embryos arising fromBARE1 and BAGY2 retrotransposons [25, 26]. A rea�son why the control embryos had polymorphism inour experiments might be derived from barley cultivarthat was used in this study. We used Tokak 157/37 cul�tivar, and it might have natural polymorphism between

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KARTAL�ALACAM et al.

individuals. Another reason could be the usage of dif�ferent retrotransposons. In the previous studies,BARE1 and BAGY2 retrotransposons were used, whileSukkula was employed in this study. Therefore, ourresults might indicate that Tokak 157/37 cultivar ofH. vulgare had natural polymorphism caused by Suk�kula transpositions [27, 28].

Polymorphism rates of 40�day�old calli were calcu�lated as 24, 37, and 61% when each callus was com�pared with other 40�day�old calli (Fig. 1, Table 1, lines

6–8). These polymorphism ratios of 40�day�old calliwere found higher than the natural polymorphism thatwe obtained between noncultured embryos. This resultmight prove that tissue culturing affected Sukkulamovements in the barley genome. In addition, IRAPresults for 40�day�old calli compared with noncul�tured embryos showed that polymorphism rates wereranged from 22 to 47%.

IRAP results for 80�day�old calli revealed thatthere was no polymorphism among individuals (Fig. 1and Table 1, lines 9–11). These results might showthat all 80�day�old calli were homomorphic. However,this homomorphism most likely was caused by someretrotransposon events occurring between the 40thand 80th days of tissue culture. These retrotransposonevents might cause homomorphic results between callias determined by IRAP, although these calli are stillpolymorphic. When 80�day�old calli were comparedwith noncultured embryos, polymorphism ratios werecalculated as 19 to 25%. We also observed polymor�phism between 40� and 80�day�old calli even theyoriginated from the same embryo. Polymorphismratios in 40� and 80�day�old calli of the first groupwere 34% (between 6 and 9 in Table 1 and Fig. 1), thesecond group⎯50% (between 7 and 10 in Table 1 andFig. 1), and the third group⎯41% (between 8 and 11in Table 1 and Fig. 1).

iPBS results

iPBS results also showed some polymorphic bandsamong control, 40� and 80�day�old calli. In Fig. 2,some unique and missing bands are shown with arrowsmarked with different letter (a–i). Arrow a shows amissing band in the 6th, 7th, 9th, and 10th samples,

bp

10000

1000

800

M 1 2 3 4 5 6 7 8 9 10 11

ab

d

he

f

g

Fig. 1. IRAP results. (1–5) Noncultured mature embryos; (6–8) 40�day�old calli; (9–11) 80�day�old calli formed from the same embryo. Polymor�phic bands are shown with arrows (a–h).

Table 1. Polymorphism percentage of calli by IRAP markertechnique

Nos. 1 2 3 4 5 6 7 8 9 10

2 8 –

3 14 13 –

4 14 13 10 –

5 5 4 10 9 –

6 31 31 22 29 18 –

7 35 47 41 37 38 24 –

8 35 35 26 41 35 37 61 –

9 24 25 19 23 22 34 50 41 –

10 24 25 19 23 22 34 50 41 0 –

11 24 25 19 23 22 34 50 41 0 0

(1–5) Noncultured mature embryos, (6–8) 40�day�old calliformed from embryo, (9–11) 80�day�old calli formed from thesame embryo

b

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SUKKULA RETROTRANSPOSON INSERTION POLYMORPHISMS IN BARLEY 831

while arrow b⎯in the 7th, 8th, and 11th samples.Arrow c also indicates a missing band in the 3rd, 6th,7th, 8th, and 11th samples. Moreover, arrow d denotesa band appeared in the 8th, 9th, and 10th samples.Arrow e is a missing band only in the 7th and 11th sam�ples. Arrow f just appeared in the 6th, 7th, and 10thsamples. On the other hand, arrow g shows a missingband for the 2nd, 4th, 7th, 8th, and 10th samples.Arrow h was observed only in the 7th, 8th, and 11thsamples. Finally, arrow i appeared only in the 4th and5th samples.

We calculated polymorphism rates up to 29% incontrol samples using iPBS data (Fig. 2 and Table 2,lines 1–5). These unequal polymorphism rates ofcontrol samples between IRAP and iPBS techniques(4–14% and up to 29%, respectively) may cause acontradiction. However, more certain detection of ret�rotransposon polymorphisms can be achieved by usingeither at least two marker systems (IRAP and iPBS) orone marker system with different primer combina�tions. For this reason, we employed both iPBS andIRAP marker systems to obtain more certain results,although they resulted in different polymorphismrates. In addition, in a previous study different poly�morphism rates were found in barley calli with 25 dif�ferent combinations of BARE1 primers [27]. Thatstudy proved that different polymorphism rates couldbe obtained from a marker system by using differentprimer combinations even they are specific for thesame retrotransposon. Therefore, we can say that dif�ferent polymorphism rates obtained from two markersystems are an expected result. Nonetheless, ourresults obtained from IRAP and iPBS techniquescould prove that Tokak 157/37 cultivar had a naturalpolymorphism between individuals for Sukkula inser�tion patterns. Therefore, we took into account this

natural polymorphism while evaluating our tissue cul�ture samples using IRAP and iPBS techiques.

Polymorphism rates were calculated as 44, 55, and58% among 40�day�old calli by using iPBS analysis(Fig. 2 and Table 2, lines 6–8). Polymorphism ratios of40�day�old calli in iPBS analysis were also foundhigher than the natural polymorphism that we obtainedbetween noncultured embryos, like in the case of IRAPanalysis. When iPBS results for 40�day�old calli werecompared with noncultured embryos, polymorphismrates were found variable from 23 to 60%. Further�

M 1 2 3 4 5 6 7 8 9 10 11bp

200015001000

750

500

250

abc

c

g

i

f

d

h

Fig. 2. iPBS results. (1–5) Noncultured mature embryos; (6–8) 40�day�old calli; (9–11) 80�day�old calli formed from the same embryo. Polymor�phic bands are shown with arrows (a–i).

Table 2. Polymorphism percentage of calli by iPBS markertechnique

Nos. 1 2 3 4 5 6 7 8 9 10

2 17 –

3 24 29 –

4 28 24 20 –

5 23 28 13 7 –

6 23 28 25 25 24 –

7 50 53 56 58 60 44 –

8 45 42 41 44 47 55 58 –

9 32 37 35 39 33 33 67 47 –

10 35 32 39 33 37 28 55 42 18 –

11 65 70 64 74 68 68 72 74 61 70

(1–5) Noncultured mature embryos, (6–8) 40�day�old calliformed from embryo, (9–11) 80�day�old calli formed from thesame embryo

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KARTAL�ALACAM et al.

more, different polymorphism ratios observedbetween 40�day�old calli might show that tissue cul�ture affected every individual independently.

In contrast to IRAP technique, iPBS analysisresulted in 18, 61, and 70% polymorphism rates among80�day�old calli (Fig. 2 and Table 2, lines 9–11). Thesetwo opposite results obtained by IRAP and iPBS anal�yses might prove that 80�day�old calli are still poly�morphic when compared with each other. Thus, wecan conclude that one marker technique is not suffi�cient to detect this polymorphism. Furthermore, acomparison between noncultured embryos and80�day�old calli resulted in the 32 to 74% polymor�phism ratios. Similar to the IRAP results, we alsoobserved polymorphisms by iPBS between 40�and80�day�old calli even when they originated from thesame embryo. Polymorphism ratios in 40� and 80�day�old calli of the first group were 33% (between 6 and 9in Table 2 and Fig. 2), the second group – 55%(between 7 and 10 in Table 2 and Fig. 2), and the thirdgroup – 74% (between 8 and 11 in Table 2 and Fig. 2).

Sukkula is known as the second most active ret�rotransposon in barley genome similar to BAGY2 afterBARE1: BARE1 > BAGY2 = SUKKULA > NIKITA,according to data obtained from three different barleyvarieties [23]. Up to date, Sukkula has been studied forthe determination of genetic variability [29, 30]. In ourstudy, we proposed that Sukkula retrotransposon isalso responsible for intraspecies variations and poly�morphism rates also related to the age of the callusmaterials (Tables 1, 2). These results demonstratedthat the culture period was one of the most importantfactors for genome variations in barley, like it wasreported previously [15, 16, 25].

So in this study, tissue culture�induced polymor�phism of Sukkula retrotransposon was investigated inmature embryos, 40� and 80�day�old calli by IRAPand iPBS marker techniques. Our results showed thatsome retrotransposition events occurred both in con�trol mature embryos and during callus development.Despite the importance of retrotransposons togenome dynamics and gene activity, our understand�ing of their biology is still in a primitive state. In termsof Sukkula, a non�autonomous retrotransposon, isthere a specific autonomous partner for the movementof this retrotransposon and what are the evolutionaryimpacts of Sukkula for genome expansion in barley?Our results could be important for the understandingof answer to these questions.

ACKNOWLEDGMENTS

This study was supported by the Research Found ofIstanbul University (project nos. 5501 and 8530).

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