BAGY2 Retrotransposon Analyses in Barley CalliCultures and Regenerated Plantlets

Sibel Yilmaz • Sevgi Marakli • Nermin Gozukirmizi

Received: 28 February 2013 / Accepted: 25 October 2013

� Springer Science+Business Media New York 2014

Abstract The stability of aging barley calli and regenerated plantlets from those

calli was investigated by the BAGY2 retrotransposon-specific IRAP technique.

Mature embryos of barley (Hordeum vulgare cv. Golden Promise) were cultured in

Murashige and Skoog medium supplemented with 4 mg/L dicamba and maintained

on the same medium for 45 and 90 days. Two IRAP-based primers were used, and

the levels of variation of DNA isolated from 45- and 90-day-old calli and regen-

erated plantlets were found to be increased 0–21%, depending on the mature

embryo material and the age of the callus. It has been observed that culture con-

ditions cause genetic variations and evident BAGY2 retrotransposon alterations.

Internal domains of BAGY2 were also analyzed by qPCR, and copy numbers were

found to be increased. These findings are expected to contribute to understanding of

how retrotransposons affect features like tissue culture (especially callus tissue)

formation and genetic engineering studies.

Keywords Hordeum vulgare � Somaclonal variation � BAGY2 �Polymorphism � Copy number variations

Introduction

Somaclonal variation is defined as a genetic and phenotypic variation among clonally

propagated plants of a single donor clone (Larkin and Scowcoft 1981). During tissue

S. Yilmaz (&) � S. Marakli � N. Gozukirmizi

Department of Molecular Biology and Genetics, Istanbul University, Vezneciler, 34134 Istanbul,

Turkey

e-mail: sibelyilmaz22@hotmail.com

S. Marakli

e-mail: sevgi.marakli@istanbul.edu.tr

N. Gozukirmizi

e-mail: nermin@istanbul.edu.tr

123

Biochem Genet

DOI 10.1007/s10528-014-9643-z

culture some genetic and epigenetic changes (such as cytogenetic abnormalities, sequence

changes, DNA methylation variations, and transposon movements) can occur in calli,

regenerated plants, and their progeny (Kaeppler et al. 2000; Evrensel et al. 2011).

Retrotransposons are ubiquitous components of DNA and play a major role for genome

evolution (Kumar and Bennetzen 1999). Long terminal repeat (LTR) retrotransposons

constitute a subclass of transposons. They transpose in a replicative manner using an RNA

intermediate mechanism (Grzebelus 2006). The highest percentage of retrotransposons

encodes proteins needed for their life cycle. They have a coding region containing gag and

pol (protease, reverse transcriptase, RNaseH, integrase) genes flanked by long terminal

repeats (Sabot and Schulman 2006). They resemble retroviruses because of their physical

structure. Some retrotransposons also have an envelope (ENV) domain needed for

retrovirus infection. Due to their copy-and-paste mechanism, genomes diversify through

the insertion of new retrotransposon copies. Their abundance in the genome is generally

correlated with genome size (Schulman and Kalendar 2005). Cereal genomes especially

contain retrotransposons up to 95%. In barley, BARE1 is the most active retrotransposon:

BARE1[BAGY2 = Sukkula[ Nikita[Sabrina (Leigh et al. 2003). BAGY2 is a

gypsy-like retrotransposon, containing the ENV domain, and has 47% similarity with

BAGY1 (Shirasu et al. 2000; Leigh et al. 2003).

The mobility of a retrotransposon can be easily detected by retrotransposon-based

markers. One such marker technique is inter-retrotransposon amplified polymorphism

(IRAP). It relies on the amplification of the genomic regions flanked by two LTR

retrotransposons or solo LTRs (Kalendar et al. 1999). In this method, the polymorphism

is detected by the presence or absence of the PCR product, and lack of amplification

indicates the absence of the retrotransposon at the particular locus. IRAP has been used

to investigate genetic relationships between varieties and related species (Guo et al.

2006), gene mapping (Manninen et al. 2000), characterization of somaclonal variation

(Campbell et al. 2011; Evrensel et al. 2011; Bayram et al. 2012), and somatic genome

variations (Muhammad and Othman 2005). It also has been adapted to human

endogenous virus screening studies (Guliyev et al. 2013).

In the current study, we investigated BAGY2 retrotransposon insertion patterns of

45- and 90-day-old calli in barley and their regenerated shoots. In addition, both

sequence analyses were conducted and copy number alterations of BAGY2 internal

domains (GAG, PR, RT, RH, INT, and ENV) were investigated. This is the first

report on BAGY2 movements and copy number variations of the internal domains in

cultured barley materials.

Materials and Methods

Plant Material and DNA Isolation

Barley (Hordeum vulgare cv. Golden Promise) seeds were surface-sterilized with

commercial bleach for 20 min and rinsed three times with sterile dH2O for 10 min.

After sterilization, mature embryos were removed from seeds and dipped into absolute

ethanol for 30 s. Ethanol was removed and embryos were rinsed three times with sterile

dH2O for 1 min and dried on sterile filter papers. Each embryo was given a number and

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123

cultured on MS medium (Murashige and Skoog 1962) of 3% sucrose and 0.9% agar (pH

5.7), supplemented with 4 mg/L dicamba, and was maintained on the same medium for

45 days. At the end of 45 days of cultivation time, each callus was cut into three pieces

and each piece was numbered with the starting embryo’s number. One of the callus

pieces was used for genomic DNA isolation, the second for shoot regeneration in MS

medium supplemented with 0.5 mg/L zeatin, and the third was subcultured under the

same callus culture conditions for another 45 days. At the end of the second 45 days,

each callus was cut into two pieces and numbered. One of the pieces was used for shoot

regeneration and the other for DNA isolation. At the end of the tissue culture, we

obtained four experimental plant materials (45- and 90-day-old calli and their

regenerated shoots) from one embryo; these were considered a single group. IRAP was

performed with three different groups. Genomic DNA was isolated from those three

groups and three control groups using Tri Reagent (Sigma T9424) according to the

manufacturer’s instructions. The control groups consisted of noncultured mature

embryos that were kept in water for 16 h (first control), the leaf of a seedling germinated

between filter papers (second control), and the leaf of a seedling germinated in MS0

medium (third control).

IRAP

IRAP was performed with forward and reverse primers designed for LTR sequences

of BAGY2 retrotransposon (Table 1, Primers 1 and 2). Amplification was carried out

in a 20 lL reaction volume containing 3.5 lL nuclease-free dH2O, 0.5 lL dNTP

mixture (10 mM), 2 lL of each primer (10 nmol/lL), 2 lL template genomic DNA

(10 ng/lL), and 10 lL 29 Sapphire enzyme mix. PCR conditions were an initial

denaturation step at 94�C for 3 min; followed by 30 cycles at 94�C for 20 s, 52�C

for 20 s, and 72�C for 2 min; and a final extension step at 72�C for 10 min.

Evaluation of PCR Products

A 10 lL aliquot of IRAP PCR product was separated on 8% nondenaturing

polyacrylamide (29:1 Ac:Bis) gel at 200 V for 4 h in 19 TBE buffer. A molecular

weight marker (Fermentas, SM0321) was also loaded to determine the size of

amplicons. Gel was stained in 39 GelRed solution (Biotium, 41003) for 30 min.

Well-resolved bands were evaluated as present (1) or absent (0). The binary matrix

(1/0) was used to calculate the similarity by Jaccard’s coefficient (Jaccard 1908)

among samples (controls, 45- and 90-day-old calli, and their regenerated shoots).

Sequence Analysis of BAGY2 Domains

BAGY2 internal domains for GAG, protease (PR), reverse transcriptase (RT),

RNaseH (RH), integrase (INT), and envelope (ENV) were amplified using specific

primers for each domain. The reference sequence was obtained from NCBI

(accession no. AF254799.1). Primers were designed by IDT PrimerQuest (Table 1,

Primers 3–14). BAGY2 internal domains were amplified from genomic DNA of

embryos by Phusion High-Fidelity DNA Polymerase (Thermo, F-530S). PCR was

Biochem Genet

123

performed in a total volume of 20 lL, containing 11.3 lL nuclease-free dH2O, 4 lL

59 buffer (with 7.5 mM MgCl2), 0.5 lL dNTP mixture (10 mM), 1 lL of each

primer (10 nmol/lL), 2 lL template genomic DNA (10 ng/lL), and 0.2 lL Phusion

DNA polymerase. PCR conditions were initial denaturation at 98�C for 30 s;

followed by 30 cycles of denaturation at 98�C for 10 s, annealing at 53�C for 20 s,

and extension at 72�C for 20 s; and completion with an additional extension at 72�C

for 5 min. PCR products were resolved on 1% agarose gel in 19 TAE buffer at

70 V for 40 min. Expected bands were purified from agarose gel with a DNA

extraction kit (Roche, 11696505001). Extracted fragments were ligated into

pJET1.2/blunt cloning vector (Fermentas, K1231) and transformed to JM-107

Escherichia coli cell line according to the manufacturer’s instructions. Then, colony

PCR was performed to detect recombinant colonies. Recombinant colonies for each

domain were cultured in LB broth (containing 100 lg/mL ampicillin) at 37�C for

Table 1 Primer sequences used in this study

No Name Direction Sequence (50?30) Expected

band size

Purpose

1a BAGY2/

E0521

Forward TCGAAAGGTCTATGATTGATCCC Variable IRAP

2a BAGY2/

E0520

Reverse CATGAAAGCATGATGCAAAATGG

3 GAG-F Forward GGATCATCACATCCAGACTTGCATC 925 bp Sequence

analysis4 GAG-R Reverse TAACATGCCCGTCTATGTCTTCGG

5b PR-F Forward GTGATATGGGTGCTAGTGTTAGTGC 261 bp Sequence

analysis6 PR-R Reverse TGACAGTGTTGAGAAAGGGTCTGC

7 RT-F Forward GGAAGCAGGAATCATTTATCCTGTTGCTCA 702 bp Sequence

analysis8 RT-R Reverse AGAAACCAGCATGACCTAGGAAAC

9 RH-F Forward TGCGGCGCTAGTGATTATGCTGTT 322 bp Sequence

analysis10 RH-R Reverse GGTTATCAGCACCCTTTCGGTCAA

11 INT-F Forward AAGCCTACAGAGGACACCATGCTA 960 bp Sequence

analysis12 INT-R Reverse CACCGGAACGATATACTTCCTCGACA

13a ENV-F Forward CCAAGGTCTATGGGACTTGGAACC 385 bp Sequence

analysis14a ENV-R Reverse CAAGGGGATTGCCCATACCAATGC

15 GAG2F Forward TCGGCTTTCTCATGAAGATCGTACC 100 bp qPCR

analysis16 GAG2R Reverse GATTCTTTCCAATAAATTCCACTTGTGGTC

17 PR-2R Reverse GCGTAATAGTGACATCAATGGGTTC 105 bp qPCR

analysis

18 RT-2F Forward CCGTTTACGGTTCTTCCTTTGACG 102 bp qPCR

analysis19 RT-2R Reverse AGTGGCACTTCTCCCAGTTCAGGA

20 RH-2F Forward GAAACTATGCCACTACGGAGAAGG 109 bp qPCR

analysis21 RH-2R Reverse GCAGCATGATCAGAGTGAATAATGACC

22 INT-2F Forward TGGCGGTTCACACTTCATTCATGG 108 bp qPCR

analysis23 INT-2R Reverse TCCACTTGACCACTAGACTGAGGA

a Primer sequence obtained from Vicient et al. (2001)b Primer used in sequence and qPCR analyses

Biochem Genet

123

18 h. Plasmids were isolated using a Qiagen Plasmid Mini Kit (Qiagen, 12125). The

plasmids were digested by BglII restriction enzyme (Fermentas, FD0083) and

sequenced. The sequences were compared with both nucleotide (Blastn) and amino

acid (tBlastn) sequences of BAGY2 domains at the NCBI web page by Blast. Amino

acid sequences of domains were obtained from GyDB (Gypsy DataBase).

Copy Number Analysis of BAGY2 Domains by qPCR

We employed qPCR to determine the copy number alterations of BAGY2 internal

domains during tissue culture. To form a standard curve, a plasmid series with copy

number from 105 to 109/lL was used. An additional qPCR with actin primers was

performed for confirmation of the equivalence of all DNA sample concentrations. The

qPCR was performed using Bio Rad CFX96. We designed qPCR primers using IDT

PrimerQuest (Table 1, Primers 5 and 15–23). The qPCR was performed in a total

volume of 10 lL containing 2.2 lL nuclease-free dH2O, 5 lL 29 EvaGreen Enzyme

Mix (Bio-Rad, 730001462), 0.4 lL of each primer (10 nmol/lL), and 2 lL template

genomic DNA (10 ng/lL). The qPCR conditions were an initial denaturation at 98�C

for 3 min; followed by 40 cycles of denaturation at 98�C for 5 s, annealing and

extension at 55�C for 5 s; and a melt curve step (from 65�C, gradually increasing

0.5�C/s, to 95�C, with acquisition data every 1 s).

To determine the statistical significance of copy number alterations, we classified

the tissue culture samples into seven groups (45-day-old calli, 90-day-old calli,

45-day-old shoots, 90-day-old shoots, and controls 1, 2, and 3) according to their

ages and types. Starting-quantity values of qPCR were evaluated using a one-way

ANOVA test to find out whether the copy number of each domain had a statistically

significant (p \ 0.05) alteration in different samples. Multiple comparison tests for

equal (Tukey) or unequal (Games Howell) variances were used to determine which

groups have significant copy number alterations.

Results and Discussion

IRAP Results

In the current study, BAGY2 retrotransposon movements were investigated in 45-

and 90-day-old calli and their regenerants. All four plant materials originating from

the same embryo were grouped. IRAP-PCR was performed with three groups to

determine whether there are any different polymorphisms between samples

originating from different embryos. Furthermore, we used three control groups

(four individuals of each) to compare polymorphism rates between calli and shoots.

After IRAP analysis of the first control group, we observed 41 bands under

1,000 bp, and all of these bands were monomorphic in the group (Fig. 1). This

result may indicate that there are not any natural polymorphisms between individual

embryos regarding BAGY2 transposition. Despite their mutagenic feature, it might

be said that retrotransposons have an excellent mechanism for conservation of their

transposition between individuals of the same species. The band patterns of the

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second and third control groups were also monomorphic, but they differed from the

first control group in that we observed two novel bands and one missing band

(Fig. 1). The polymorphism rate was calculated at 7% between control groups by

Jaccard’s coefficient.

IRAP band patterns of each callus and regenerated shoot were different from both

control groups and other calli and shoots (Fig. 2). We calculated the polymorphism

rate by comparing each sample with other samples and controls and found it to be

0–21% (Table 2). These polymorphism rates between control groups and tissue

culture samples (calli and shoots) clearly show that culturing conditions and time

have an effect on BAGY2 movements. Further, different polymorphism percentages

between all samples indicated that tissue culture might not have the same effect on

the samples even if they originated from the same embryo. For example, the calli in

Fig. 2 cultured for 45 (lane 2) and 90 (lane 4) days have 12% polymorphism,

although they originated from the same embryo.

Leigh et al. (2003) reported that BARE1 is the most active and BAGY2 the second

most active retrotransposon in barley. In our previous studies, we also observed

polymorphisms of BARE1 and Nikita retrotransposons during tissue culture of

barley (Evrensel et al. 2011; Bayram et al. 2012; Temel and Gozukirmizi 2013), and

the polymorphism rates for BARE1 were calculated as 14–25% (Evrensel et al.

2011). In this study, the polymorphism percentages for BAGY2 were lower than for

Fig. 1 IRAP results for control groups. Lanes 1–4 (first control group) mature embryo, lanes 5–8 (secondcontrol group) leaf of seedlings germinated between filter papers, lanes 9–12 (third control group) leaf ofseedlings germinated on MS medium, NC PCR negative control. Arrow a novel bands in second and thirdcontrols, arrow b unique band in first control, arrow c novel bands in second and third controls

Biochem Genet

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BARE1. These results are consistent with the barley retrotransposon activation

order.

Previously, some retrotransposons were shown to be activated by biotic and

abiotic stress (Grandbastien 1998; Hamad-Mecbur et al. 2014). Tissue culture

conditions can also be accepted as a stress factor. Copy number alterations of

tobacco Tto1 (Hirochika 1993) and rice Tos17 (Hirochika et al. 1996) elements were

shown during the tissue culture process. Somaclonal variations induced by

retrotransposons have been studied for the last few years (Evrensel et al. 2011;

Bayram et al. 2012). Campbell et al. (2011) used the IRAP technique to detect

somaclonal variation in barley tissue cultures; they stated that retrotransposon-based

marker systems, such as IRAP, are valuable tools for the characterization of

mutations that arise during tissue culture. Tissue culture conditions, however, did

not show the same effect in every individual, leading to variable polymorphism

rates. Therefore, determination of general aspects of retrotransposon-induced

somaclonal variations and the prediction of average polymorphism rates are

challenging. Hence, these results show that differences in both tissue culture

conditions and culturing time alter retrotransposon movements.

Fig. 2 IRAP results for callus and shoot tissue culture materials. Lane 1 first control. In lanes 2–5 (group1), lanes 6–9 (group 2), and lanes 10–13 (group 3), even-numbered lanes are barley callus and odd-numbered lanes are regenerated shoot. Tissues in lanes 2, 3, 6, 7, 10, and 11 were cultured 45 days.Tissues in lanes 4, 5, 8, 9, 12, and 13 were cultured 90 days. Arrowheads indicate polymorphic bands

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Sequence Analysis of BAGY2 Domains

In addition to the IRAP analyses, we also amplified the internal domains of BAGY2

from both control groups and tissue culture samples. We designed specific primers

for the GAG, PR, RT, RH, and INT domains of the BAGY2 sequence (NCBI

accession no. AF254799.1). Primer sequences for the ENV domain were obtained

from Vicient et al. (2001). All the internal domains were amplified from both

control and tissue culture samples (Fig. 3). Further, we cloned and sequenced all the

domains from the first control sample (mature embryo). The comparisons with Blast

sequences (Table 3) show high degrees of nucleotide (87–95%) and amino acid

(86–94%) sequence similarity.

Copy Number Analysis of BAGY2 Domains by qPCR

In order to determine copy number alterations of the BAGY2 internal domains we

performed qPCR with specific primers (Table 1, Primers 5 and 15–23). The qPCR

results of actin primers showed that all reactions have nearly the same starting

quantities, and the ANOVA test (p [ 0.05) indicated that all DNA samples have

equal concentrations. The starting-quantity values of the BAGY2 internal domains

were variable, however, some samples had statistically significant copy number

increments and others did not (Table 4). Especially in calli samples, copy numbers

of all domains were found to be dramatically increased. This might be caused by the

unstable nature of the cell genome in calli culture. On the other hand, in shoots, the

copy numbers of domains were found to be lower than in calli, indicating that cells

Table 2 Polymorphism percentage of calli and regenerated shoots

Group Group 1 Group 2 Group 3

Culture 45 days 90 days 45 days 90 days 45 days 90 days

Tissue BC RS BC RS BC RS BC RS BC RS BC RS

First control 5 5 14 5 14 10 12 2 7 7 7 10

1 45 BC – 0 12 13 10 7 7 5 5 7 5 7

RS – 12 15 8 8 0 0 3 0 0 3

90 BC – 18 8 11 17 13 15 15 15 15

RS – 21 15 15 20 17 15 9 12

2 45 BC – 6 19 20 14 9 6 15

RS – 12 17 14 12 9 12

90 BC – 0 5 0 0 16

RS – 3 0 0 5

3 45 BC – 3 3 13

RS – 0 8

90 BC – 8

BC barley callus, RS regenerated shoot from the callus

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123

with a more stable genome might have a higher chance for regeneration. In the

literature, there are no studies of copy number analyses of BAGY2 internal domains

in tissue culture, but BAGY2 has been demonstrated to be one of the active

retrotransposons in barley (Shirasu et al. 2000; Leigh et al. 2003). It is expressed in

embryo, leaf, root, flower, and callus (Vicient et al. 2001; Marakli et al. 2012). In

this study we showed that BAGY2 could be active in both expression and insertion

processes.

Besides the copy number increase of all domains in tissue culture samples, we

also observed that those increments varied among domains. This may be explained

by the existence of a BAGY2 partial internal sequence in the genome. Previously,

Wicker et al. (2007) demonstrated that BAGY2 is one of the most abundant

retrotransposons in the barley genome, constituting 5.15% of the genome. Many

copies of BAGY2 could have lost some or all of the internal domains during

evolutionary processes by homolog recombination (Shirasu et al. 2000; Wicker

et al. 2009). It has also been reported that the BAGY2 transcript is alternatively

spliced (Vicient et al. 2001), and this might result in a BAGY2 copy with a partial

internal sequence. These results together might prove that time and tissue culture

conditions increase BAGY2 copy numbers and thus cause genome enlargement.

In conclusion, IRAP analyses of all groups resulted in polymorphisms of 0–21%.

It is clear that some retrotransposition events have occurred during calli

Fig. 3 PCR results for BAGY2 domains. Amplified bands for each domain from the first control group:Lane 1 GAG, 2 PR, 3 RT, 4 RH, 5 INT, 6 ENV. Band sizes as in Table 3

Table 3 Sequence analysis of BAGY2 internal domains

Domain Size Similarity

Amino acid Nucleotide Amplified band Nucleotide (Blastn) Amino acid (tBlastn)

GAG 318 954 925 95 90

PR 92 279 261 87 86

RT 243 730 702 93 92

RH 118 354 322 93 94

INT 329 978 960 96 92

ENV – – 385 92 –

Biochem Genet

123

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Biochem Genet

123

development and shoot regeneration. In addition, qPCR analyses proved that copy

numbers of BAGY2 internal domains increase during tissue culture. Our results are

the first data on BAGY2 movement alterations in cultured material and qPCR

analyses of internal domains.

Acknowledgments This study was supported by the Research Fund of Istanbul University (Project Nos.

17704 and 5501).

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