CompCytogen 10(4): 543–554 (2016)                                                                               COMPARATIVE                   A peer-reviewed open-access journal
                                       Towards a FISH-based karyotype of Rosa L. (Rosaceae)                                                                         543
doi: 10.3897/CompCytogen.v10i4.9536                               RESEARCH ARTICLE                              Cytogenetics
http://compcytogen.pensoft.net                                                                                         International Journal of Plant & Animal Cytogenetics,
                                                                                                                              Karyosystematics, and Molecular Systematics
Towards a FISH-based karyotype of Rosa L. (Rosaceae)
   Ilya V. Kirov1,2,3, Katrijn Van Laere3, Nadine Van Roy4, Ludmila I. Khrustaleva1,2
1 Center of Molecular Biotechnology, Russian State Agrarian University - Moscow Timiryazev Agricultural
Academy, Timiryazevskay str. 49, 127550, Moscow, Russia 2 Department of Genetics and Biotechnology, Rus-
sian State Agrarian University - Moscow Timiryazev Agricultural Academy, Timiryazevskay str. 3, 127550,
Moscow, Russia 3 Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Applied Gene-
tics and Breeding, Caritasstraat 39, 9090, Melle, Belgium 4 Center of Medical Genetics, Faculty of Medicine
and Health Sciences, Ghent University, De Pintelaan 185, 9000, Ghent, Belgium
Corresponding author: Ilya V. Kirov (kirovez@gmail.com)
Academic editor: A. Joachimiak | Received 29 June 2016 | Accepted 8 September 2016 | Published 4 November 2016
                                        http://zoobank.org/4B2D9EE5-1366-4BB0-B49B-86C166FD82FD
Citation: Kirov IV, Van Laere K, Van Roy N, Khrustaleva LI (2016) Towards a FISH-based karyotype of Rosa L.
(Rosaceae). Comparative Cytogenetics 10(4): 543–554. doi: 10.3897/CompCytogen.v10i4.9536
Abstract
The genus Rosa Linnaeus, 1753 has important economic value in ornamental sector and many breeding
activities are going on supported by molecular studies. However, the cytogenetic studies of rose species are
scarce and mainly focused on chromosome counting and chromosome morphology-based karyotyping.
Due to the small size of the chromosomes and a high frequency of polyploidy in the genus, karyotyping
is very challenging for rose species and requires FISH-based cytogenetic markers to be applied. Therefore,
in this work the aim is to establish a FISH-based karyotype for Rosa wichurana (Crépin, 1888), a rose spe-
cies with several benefits for advanced molecular cytogenetic studies of genus Rosa (Kirov et al. 2015a). It
is shown that FISH signals from 5S, 45S and an Arabidopsis-type telomeric repeat are distributed on five
(1, 2, 4, 5 and 7) of seven chromosome pairs. In addition, it is demonstrated that the interstitial telom-
eric repeat sequences (ITR) are located in the centromeric regions of four chromosome pairs. Using low
hybridization stringency for ITR visualization, we showed that the number of ITR signals increases four
times (1–4 signals). This study is the first to propose a FISH-based R. wichurana karyotype for the reliable
identification of chromosomes. The possible origin of R. wichurana ITR loci is discussed.
Keywords
Cytogenetic markers, fluorescence in situ hybridization, interstitial telomeric repeat (ITR), 5S rDNA, 45S
rDNA, Rosa wichurana
Copyright Ilya V. Kirov et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

544             Ilya V. Kirov et al. / Comparative Cytogenetics 10(4): 543–554 (2016)
Introduction
Rosa Linnaeus, 1753 is an economically important ornamental genus belonging to
the Rosaceae. Of the approximately 200 described Rosa species (Wissemann and Ritz
2005), only 8 to 15 species contributed to the original germplasm of the modern
rose cultivars. Rosa is one of the most widely cultivated ornamental plants worldwide,
but few basic molecular cytogenetic studies in Rosa have been performed, including
chromosome counts and karyotyping (Wylie 1954, Price et al. 1981, Liu and Li 1985,
Subramanian 1987, Ma et al. 1997, Fernandez-Romero et al. 2001, Akasaka et al.
2002, 2003, Jian et al. 2013a, 2013b). Performing molecular cytogenetics in roses is
a big challenge due to their very small genome size (the diploid genome size is 0.83 to
1.30 pg/2C, Roberts et al. 2009), small chromosomes (Kirov et al. 2014a), low mitotic
index in roots and shoots, and weak root development (Ma et al. 1996). Moreover,
most wild roses are polyploids (Vamosi and Dickinson 2006), ranging from diploid
(2n = 2x = 14) to decaploid (2n = 10x = 70) (Roberts et al. 2009, Jian et al. 2010).
     Rosa wichurana (Crépin, 1888) is a valuable model species for molecular cytoge-
netic studies in Rosa genus (Kirov et al. 2015b). It is a diploid species (2n = 2x = 14)
with suitable apical and root meristems that can be used for chromosome prepara-
tions. Rosa wichurana is involved in the origin of modern rose cultivars and is one of
the parental species used for the construction of several rose genetic maps (Crespel et
al. 2002, Dugo et al. 2005, Shupert et al. 2007, Spiller et al. 2011, Moghaddam et
al. 2012). To increase the efficiency of FISH experiments, we recently developed the
“SteamDrop” protocol for the preparation of high quality chromosome slides (Kirov
et al. 2014b). Using this “SteamDrop” protocol and Tyramide-FISH it was possible to
physically map several single-copy genes on the mitotic and meiotic chromosomes of R.
wichurana (Kirov et al. 2014a, Kirov et al. 2015a) and to anchor three linkage groups
of the genetic map (Moghaddam et al. 2012) to three R. wichurana chromosomes.
     Because the chromosomes are difficult to distinguish, further progress in cytogenetic
mapping depends on the development of cytogenetic markers useful for chromosome iden-
tification. The conservative tandemly organized repetitive sequences 5S and 45S rRNA
genes are valuable sources of cytogenetic markers, and have been used for chromosome iden-
tification in many plant species including Rosa species (Ma et al. 1997, Fernandez-Romero
et al. 2001, Akasaka et al. 2002, 2003, Lim et al. 2005, Jian et al. 2012, Kirov et al. 2014a).
Other conservative repeats, such as the Arabidopsis-type telomeric repeat (Fuchs et al. 1995,
He et al. 2013) might be used for chromosome identification. Typically, telomeric repeats
(TRs) occupy the end (telomere) of the chromosomes (Fuchs et al. 1995). However, the lo-
cation of TRs on plant chromosomes is not restricted to the telomere ends and telomere-like
sequences have been found in centromeric, subtelomeric and interstitial regions in several
genera (Fuchs et al. 1995, Uchida et al. 2002, Tek and Jiang 2004, Mlinarec et al. 2009,
Mandakova et al. 2010, Gong et al. 2012, He et al. 2013, Sousa et al. 2014). The unique
position of these interstitial telomeric repeats (ITRs) on some chromosomes and their high
copy number make them valuable cytogenetic markers. The position of ITR on chromo-
somes can also reflect ancient chromosomal rearrangement as telomeric sequences and

                       Towards a FISH-based karyotype of Rosa L. (Rosaceae)             545
their remnants are involved in chromosomal rearrangements via illegitimate recombination
between centromeric/telomeric repeats (Murat et al. 2010) and can be associated with fragile
sites of chromosomes (Grabowska-Joachimiak et al. 2015). In addition, the chromosomal
location of ITR can be used to detect descending dysploidy (Sousa and Renner 2015).
     Development of an effective cytogenetic marker system is an important step in
answering many biological questions (Jiang and Gill 2006). FISH-based markers have
shown their effectiveness and ease-to-use. The modern methods of probe labeling and
the application of directly labeled oligonucleotides make FISH-based chromosome iden-
tification a robust and fast procedure (Kato et al. 2004, Fu et al. 2015, Tang et al. 2014,
Cuadrado et al. 2009). Up-to-date FISH based karyotyping was established for many
plant species including wheat, maize, rice, soybean, common bean and others (Cheng et
al. 2001, Kato et al. 2004, Findley et al. 2010, Iwata-Otsubo et al. 2015). Cytogenetic
markers are widely used to trace individual chromosomes in hybrids accelerating transfer-
ring of desirable traits from wild relatives (Szinay et al. 2010). FISH-based karyotyping is
used to shed light on speciation and allopolyploid formation (Badaeva et al. 2016). And
a relatively new application came with the development of a FISH-based chromosome
sorting procedure, allowing individual chromosome identification, sorting and further
sequencing (Giorgi et al. 2013). These and other applications clearly demonstrate the im-
portance of having a system of cytogenetic markers enabling chromosome identification.
     This study aims to explore the opportunities of ITRs, 5S and 45S rDNA as cy-
togenetic markers allowing to distinguish individual chromosomes of Rosa. FISH with
5S rDNA, 45S rDNA and the Arabidopsis-type telomeric repeat was performed. These
FISH results were combined with chromosome morphology measurements (Kirov et
al. 2014a), in order to identify all seven mitotic chromosomes of R. wichurana. In
addition, we also attempted to identify pachytene bivalents by FISH using the 45S
rDNA and Arabidopsis-type telomeric repeat probes.
Materials and methods
Plant material
Rosa wichurana plants were grown in the field. For chromosome slide preparations, cut-
tings were made. Rooted cuttings were transferred to terracotta stone pots and grown
in the greenhouse (moderate climatic conditions, East Flanders, Belgium). To prepare
mitotic chromosome slides, young meristems were harvested. For meiotic (pachytene)
chromosome slides, flowers buds with a hypanthium size of 3 mm were harvested.
Probe labeling
Plasmids containing 5S rRNA genes of rye (pSCT7, Lawrence and Appels 1986) and 45S
rRNA genes of wheat (pTA71, Gerlach and Bedbrook 1979) were labeled by Digoxigenin-

546              Ilya V. Kirov et al. / Comparative Cytogenetics 10(4): 543–554 (2016)
and Biotin- Nick Translation Mix (Roche, Germany), respectively, according to the manu-
facturer’s protocol. The Arabidopsis-type telomere repeat (CCCTAAA)3, labeled by TAMRA
at the 5’ end (Syntol, Russia) was used.
Chromosome preparation and fluorescence in situ hybridisation
Pachytene and mitotic chromosomes were prepared according to the “SteamDrop”
protocol (Kirov et al. 2014b).
     For FISH we used the protocol described in Heslop-Harrison et al. (1991) with
some modifications. Briefly, slides were incubated overnight at 37°C. Chromosomes
were pretreated with 4% paraformaldehyde in 2xSSC (pH 8.3–8.5) for 6 min and
dehydrated in ethanol (70%, 90% and 100%). Hybridization mixture consisted of
50% (v/v) deionized formamide, 10% (w/v) dextran sulphate, 2xSSC, 0.25% sodium
dodecyl sulphate, 2.00 ±1.00 ng/µl probe DNA. The mixture was denatured at 75°C
for 5 min, placed on ice for 5 min and 60 µl was applied on each slide. Slides were
denaturated at 75°C for 5 min and incubated in a humid chamber for 15–16 hours
at 37°C (the common hybridization condition) or at 23–25°C (the low stringency
hybridization condition). For stringency washing 0.1xSSC solution was used at 48°C
(2 times 7 minutes). Biotin and digoxigenin labeled probes were detected by Strepta-
vidin-Cy3 (Sigma-Aldrich, USA), diluted 1:200 in TNB buffer, and anti-digoxigenin-
FITC (Roche, Germany), diluted 1:200 in TNB buffer, respectively.
     For sequential FISH experiments, the slides were washed in the series of ethanol
(70%, 90% and 100%) after the first round of FISH and then the above-mentioned
FISH procedure was applied.
Microscopy and image analysis
Images were acquired using a Zeiss AxioImager M2 fluorescence microscope (400×
and 1000× magnification) equipped with an AxioCam MRm camera and Zen software
(Zeiss, Belgium). Final image adjustments were performed using Photoshop (Adobe
Inc., USA). Measurements of chromosome lengths and karyotyping was done in Mi-
croMeasure version 3.2 (Reeves and Tear 2000) for at least 10 well-spread metaphases.
Results
FISH using Arabidopsis-type telomere repeat, 5S rDNA and 45S rDNA allows
unambiguous identification of 3 Rosa wichurana mitotic chromosomes
FISH using the common hybridisation temperature of 37°C with 45S rDNA revealed
a signal on chromosome 7, while the Arabidopsis type telomere-based probe hybridized
on chromosome 5 (Fig. 1A).

                         Towards a FISH-based karyotype of Rosa L. (Rosaceae)                  547
Figure 1. FISH on the chromosomes of R. wichurana. A FISH with Arabidopsis-type telomere probe
(red) and 45S (green) under hybridization at 37°C B FISH with Arabidopsis-type telomere probe under
the low hybridization stringency condition (23-25°C). Arrows indicate the major ITRs on chromosome
5 and arrowheads show the ITRs which are visible under the low hybridization stringency condition
C The same metaphase as in 1B rehybridized with 5S rDNA under the common hybridization stringency
(37°C). Arrows indicate the 5S rDNA signals. Sacale bar: 5 µm.
     To further evaluate the value of the telomeric repeat (TR) as a cytogenetic marker,
FISH was carried out at room temperature (the low hybridization temperature). We
observed the Arabidopsis-type TR signals on all chromosome ends (Fig. 1B). Besides the
telomeric signals, a bright fluorescent signal in the centromeric region on chromosome
5 and weak signals in the centromeric region on three other chromosomes 1, 2 and 7
were observed. Remarkably, the weak centromeric signals on chromosomes 1, 2 and 7
were not observed when performing a hybridization at 37°C (Fig. 1A). No ITRs were
present on chromosomes 3, 4 and 6. FISH with 5S rDNA using the common hybridiza-
tion temperature of 37°C showed fluorescent signals on the long arm of chromosomes
4 and 7 (Fig. 1C) but the signal frequency across the metaphases was low (20–40%).
     Sequential FISH at the low hybridization temperature with the Arabidopsis-type
telomere-based probe and 5S rDNA showed co-localization of these signals on chro-
mosome 7. We also performed double-color FISH with the Arabidopsis-type telomere
repeat-based probe and the 45S rDNA probe under the low temperature of hybridiza-
tion (Fig. 2) which confirmed the identification of four (1, 2, 5 and 7) out of seven
chromosomes.
     A summary of the karyotypic features and distribution of FISH probes is given
in Fig. 3. Taken together, three chromosomes (4, 5 and 7) of R. wichurana could be
unambiguoulsy identified by 5S rDNA, 45S rDNA and the Arabidopsis-type TR using
common FISH hybridisation conditions (Fig. 3).
     All the other chromosomes can only be distinguished at this time based on their
morphological parameters. Differentiation between chromosome 1 and 2 is possible
by their centromeric indices which are 46.00 ±1.2% and 40.30 ±1.3%, respectively
(Kirov et al. 2014a) and by the presence of an ITR when using FISH at low tempera-
ture hybridization conditions. Chromosomes 3 and 6 have centromeric indices on the
level of 44.3 ±1.0% and 41.8 ±1.1%, respectively (Kirov et al. 2014a). However, these
chromosomes still remain very difficult to distinguish from each other.

548               Ilya V. Kirov et al. / Comparative Cytogenetics 10(4): 543–554 (2016)
Figure 2. Double-color FISH under the low hybridization conditions using the Arabidopsis-type telomere
repeat-based (red) and 45S rDNA (green) probes to R. wichurana mitotic chromosomes. Scales bar: 10 µm.
ITRs are located on the centromere of chromosome 5
FISH experiments with 5S rDNA, 45S rDNA, and the Arabidopsis-type TR on rose
pachytene chromosomes provide a much higher resolution of the mapped sequences.
5S rDNA-FISH on pachytene chromosomes did not reveal any reliable signals, while
FISH with the 45S rDNA probe resulted in a clear signal at the subtelomeric region
of the NOR-bearing chromosome (Fig. 4). FISH with the Arabidopsis-type TR probe
resulted in signals on all ends of pachytene chromosomes and one bright signal on the
centromeric region of chromosome 5 (Fig. 4). Since centromeres of rose pachytene
bivalents are clearly visible after DAPI staining as being the weakest part of the chro-
mosomes (Kirov et al. 2015a), comparison between the DAPI stained chromosomes
(Fig. 4B’) and the ITR signal positions (Fig. 4A’) revealed that the ITRs are located
exactly on the centromere of chromosome 5.

                         Towards a FISH-based karyotype of Rosa L. (Rosaceae)                      549
Figure 3. Distribution of the repetitive sequences on the mitotic R. wichurana chromosomes.
1
  – ITR1: signals that are visible under hybridization at 37°C as well as at low temperature (23–25°C).
2
  – ITR2: signals that are visible only under hybridization at low temperature (23–25°C).
Figure 4. High resolution physical mapping of ITR on R. wichurana pachytene chromosomes. FISH
with the Arabidopsis-type telomere repeat probe (red) and 45S (green). Merged (A) and the DAPI gray
scale (B) pictures are shown. FISH was performed under the low hybridization stringency condition.
Dotted lines show the regions that were digitally enlarged (A’ and B’). Scales bar: 5 µm.

550             Ilya V. Kirov et al. / Comparative Cytogenetics 10(4): 543–554 (2016)
Discussion
Rosa mitotic and meiotic chromosomes are difficult to distinguish by common karyo-
type analysis (Kirov et al. 2014, Kirov et al. 2015a). The development of cytogenetic
markers is necessary for individual chromosome identification and further cytogenetic
studies in Rosa. In our study, we positively evaluated the use of the conservative tan-
dem repeats, Arabidopsis-type telomere, 45S and 5S probes, as FISH-based cytoge-
netic chromosome markers for R. wichurana. However, the 5S rDNA probe cannot
be considered as a good cytogenetic marker for R. wichurana chromosomes due to the
low reliability of the FISH-signals. Application of FISH with the 5S rDNA probe to
chromosome slides prepared by an alternative method (spread protocol of Pijnacker
and Ferwerda (1984)) and using FAM labeled 5S oligos or a R. wichurana 5S clone as
probes, did not improve FISH results (data not shown). Thus the reason for weak 5S
rDNA FISH signals on R. wichurana chromosomes remains unclear. FISH with the
Arabidopsis-type TR under low hybridization conditions (hybridization at 23-25°C in-
stead of 37°C) provided us an additional tool for identification of Rosa chromosomes.
     In this study, FISH with the 45S rDNA and the Arabidopsis-type telomere probe,
reliably identified 2 (chromosome 5 and 7) of the 7 pachytene bivalents of R. wichura-
na. These markers will accelerate the ongoing physical mapping of pachytene chromo-
somes of R. wichurana as their identification by morphological parameters or specific
heterochromatin patterns is impossible (Kirov et al. 2015a).
     ITRs can be used to trace ancient chromosomes rearrangements such as chromosome
fusions, Robertsonian translocations and duplications resulting in dysploidy (Mandakova
et al. 2010, Sousa et al. 2014). However, Rosa species have a basic chromosome number n
= 7, suggesting that no descending dysploidy, which usually results in basic chromosome
number changes, has occurred. Therefore, it seems unlikely that the observed ITRs are the
indications of such chromosome fusions or translocations. ITRs might also be the traces
of intrachromosomal rearragements implicating telomeres (e.g., inversions and duplica-
tions) (Murat et al. 2010). In our study, the Arabidopsis telomere-like motif was found in
centromeric repeats of Rosa wichurana, as is also observed in several other genera (Tek and
Jiang 2004, He et al. 2013, Emadzade et al. 2014). The FISH signal from ITRs on chro-
mosome 5 is significantly stronger than those observed in the telomeres of R. wichurana
chromosomes. Thus, we hypothesize that the occurrence of ITRs in the centromeric re-
gions of R. wichurana chromosomes is the result of insertion of Arabidopsis telomere-like
sequence into centromeric sequence followed by massive amplification of centromeric
tandem repeat(s) containing an Arabidopsis telomere-like motif. To check this hypothesis
identification of centromeric repeats of R. wichurana should be done (Tek and Jiang
2004). The events leading to insertion of ITR sequences into centromere are unknown.
     Interestingly, FISH under the low hybridization temperature – and thus low strin-
gency – revealed more chromosomes possessing the telomeric repeat compared to FISH
performed under the common hybridization temperature. This result suggest that these
chromosomes (1, 2 and 7) may contain truncated or diverged telomere motifs. As a
consequence for our experiments, the telomeric probe may be much more informative
as cytogenetic marker when hybridized at a lower temperature than at 37°C (Fuchs et al.

                        Towards a FISH-based karyotype of Rosa L. (Rosaceae)                    551
1995, Tek and Jiang 2004, Sousa et al. 2014, Sousa and Renner 2015). However, the
application of ITR markers under the low-hybridization stringency and simultaneous
mapping of other probes (e.g. genes) can be challenging as non-specific hybridization
signals may occur due to low stringency. In this case sequential FISH can be applied.
     High-resolution FISH on pachytene chromosomes with the telomere probe re-
sulted in a signal in the centromere of chromosome 5, indicating that the telomere-like
motifs may be the components of the R. wichurana functional centromere as it has
been shown for potato (Tek and Jiang 2004).
     This is the first report describing valuable cytogenetic markers for four mitotic chro-
mosomes and two pachytene bivalents of R. wichurana. Moreover, by combining our
FISH results with the chromosome morphology measurements (Kirov et al. 2014a),
all 7 mitotic chromosomes of R. wichurana could be identified. Because R. wichurana
has many advantages as a model species for cytogenetic studies of the Rosa genus, the
development of a complete set of cytogenetic markers should facilitate the physical
mapping of its genome. Designing new DNA probes based on NGS data covering all
chromosomes of R. wichurana is a scope for our future research. These markers will be
indispensable for high-resolution physical mapping experiments (Kirov et al. 2015a)
that are currently ongoing for this species.
Acknowledgements
The authors would like to thank Oleg S. Alexandrov for providing the telomere probe.
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