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Integrated Karyotyping of Woodland Strawberry (Fragaria vesca) with

Oligopaint FISH Probes

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DOI: 10.1159/000485283

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 Original Article
Cytogenet Genome Res
DOI: 10.1159/000485283
Integrated Karyotyping of Woodland
Strawberry (Fragaria vesca) with
Oligopaint FISH Probes
Manman Qu Kunpeng Li Yanli Han Lei Chen Zongyun Li Yonghua Han
Jiangsu Key Laboratory of Phylogenomics and Comparative Genomics, School of Life Sciences, Jiangsu Normal
University, Xuzhou, China
Keywords
FISH · Fragaria vesca · Karyotype · Oligonucleotide probes ·
rDNA
Abstract
Chromosome identification is critical for many aspects of
cytogenetic research. However, for Fragaria vesca, definite
identification of individual chromosomes is almost impos-
sible because of their small size and high similarity. Here, we
demonstrate that bulked oligonucleotide (oligo) probes can
be used as chromosome-specific DNA markers for chromo-
some identification in F. vesca. Oligos specific to entire pseu-
dochromosomes in the draft genome of F. vesca were identi-
fied and synthesized as libraries. In all, we synthesized 6 oligo
libraries corresponding to 6 pseudochromosomes of F. ves-
ca. These libraries were amplified and labeled as probes for
fluorescence in situ hybridization (FISH). Two rounds of mul-
ticolor FISH analysis were sequentially conducted on the
same metaphase cells with each round including 3 probe
libraries, which permitted simultaneous identification of all
chromosomes of F. vesca. Moreover, 45S and 5S rDNA were
mapped to chromosomes 1, 2, and 7, respectively. A karyo-
type of metaphase chromosomes was constructed, repre-
senting the first FISH-based molecular cytogenetic karyo-
© 2017 S. Karger AG, Basel
E-Mail karger@karger.com
www.karger.com/cgr
Accepted: September 15, 2017
by M. Schmid
Published online: December 21, 2017
type of F. vesca. Our study can serve as a basis for future
comparative cytogenetic research through cross-species
chromosome painting using bulked oligo probes and will
facilitate the application of breeding technologies that rely
on the identification of chromosomes in the genus Fragaria.
© 2017 S. Karger AG, Basel
The strawberry genus Fragaria includes about 25 spe-
cies, the majority being diploid with 2n = 2x = 14; the
polyploids range from tetraploid, hexaploid, and octa-
ploid to decaploid [Liu and Davis, 2011]. Many Fragaria
species are valued for their edible fruits. The economi-
cally most important of these is the cultivated strawberry
(Fragaria × ananassa) that is grown in over 60 countries.
The knowledge of phylogenetic relationships and the ge-
netic affinities among Fragaria species can help to choose
parents from various species for introgression of wild
genes into cultivars. However, our current understanding
of the relationships between the diploid Fragaria species
and the origin of the polyploid species is limited although
various analyses have been carried out [Rousseau-Gueu-
tin et al., 2009; Njuguna et al., 2013; DiMeglio et al., 2014;
Hirakawa et al., 2014; Liston et al., 2014; Sargent et al.,
2016].
130.241.16.16 - 12/21/2017 10:12:27 AM
Zongyun Li or Yonghua Han
Jiangsu Key Laboratory of Phylogenomics and Comparative Genomics
School of Life Sciences, Jiangsu Normal University
Göteborgs Universitet
Xuzhou 221116 (China)
Downloaded by:
E-Mail zongyunli @ jsnu.edu.cn or hanyonghua @ jsnu.edu.cn
 Chromosomal karyotype information is an important
key to clarify phylogenetic relationships and evolutionary
origins among related species [Stebbins, 1971]. Higher
similarity in chromosome morphology is considered to
be indicative of closer phylogenetic relatedness. Karyo-
type analysis relies on accurate and consistent chromo-
some identification. However, for the genus Fragaria,
definite identification of the individual chromosomes is
almost impossible because of their small size and high
similarity. Therefore, although some papers have de-
scribed the karyotypes of several diploid and polyploidy
species based on morphometric measurements of mitotic
metaphase chromosomes [Iwatsubo and Naruhashi,
1989, 1991; Nathewet et al., 2009, 2010], most species are
still not analyzed.
The advent of fluorescence in situ hybridization (FISH)
has led to the development of an array of methods for
chromosome identification based on FISH signals which
can serve as excellent cytological markers [Jiang and Gill,
2006]. The most common FISH probes used for chromo-
some identification are repetitive DNA elements or large
genomic DNA clones [see Han et al., 2015]. However,
such probes are unavailable for Fragaria species except
for the conservative repetitive 5S and 45S rRNA genes
which have been used for chromosome identification in
many plant species. In the genus Fragaria, the application
of rDNA-FISH has enabled the construction of the mo-
lecular karyotypes of F. vesca and F. nilgerrensis with 3
and 2 pairs of marker chromosomes, respectively [Lim,
2004; Shulaev et al., 2011; Rho et al., 2012]. However, it
remains difficult to accurately distinguish the remaining
chromosomes, even to match the homologs.
Recently, massively synthesized oligonucleotides (oli-
gos) have been successfully labeled as FISH probes in
mammalian and Drosophila species [Boyle et al., 2011;
Yamada et al., 2011; Beliveau et al., 2012]. We first devel-
oped an oligo-based chromosome painting technique in
plants [Han et al., 2015] and successfully labeled specific
chromosomes using bulked oligo libraries as probes in
cucumber, melon, and watermelon [Han et al., 2015; Li et
al., 2016a, b]. To date, the draft genome of F. vesca “Ha-
waii 4” was reported and anchored to the genetic linkage
map in 7 pseudochromosomes [Shulaev et al., 2011], pro-
viding an approach for developing chromosome-specific
probes for chromosome identification in F. vesca.
In the present study, oligos specific to entire pseudo-
chromosomes in the draft genome of F. vesca “Hawaii 4”
were identified and synthesized as libraries. In all, we syn-
thesized 6 oligo libraries corresponding to 6 pseudochro-
mosomes of F. vesca. These libraries were amplified and
2 Cytogenet Genome Res
DOI: 10.1159/000485283
labeled as FISH probes. Using different mixes of oligo li-
braries in 2 successive hybridizations allowed the simul-
taneous identification of all chromosomes of F. vesca in a
single cell, and a molecular cytogenetic karyotype was
constructed. The set of oligo probes developed here will
enable studying karyotype evolution among Fragaria
species in a more detailed way through cross-species
chromosome painting, and will also facilitate the applica-
tion of breeding technologies that rely upon identifica-
tion of individual chromosomes in this genus.
Materials and Methods
Plant Materials and Chromosome Preparation
F. vesca subsp. vesca (PI 551890) and F. cascadensis (PI 551527)
were obtained from the National Clonal Germplasm Repository
(NCGR), Corvallis, OR, USA. The other F. vesca accession, F.
nilgerrensis Schlecht, F. pentaphylla Lozinsk, F. viridis Duch, F.
corymbosa Lozinsk, and F. chiloensis Duch were obtained from the
Shenyang Agricultural University. Young root tips of runners were
excised when they reached 1–2 cm in length, and were then treat-
ed with 2 mM 8-hydroxyquinoline liquid at room temperature for
4 h. The materials were rinsed in distilled water before being fixed
in Carnoy solution (acetic acid:ethanol 1: 3, v/v). Then they were
stored at −20 ° C in 70% ethanol. The fixed root tips were washed
in distilled water prior to enzyme treatment (2% cellulose and 1%
pectolyase) at 37 ° C for 4 h. The digested root tips were macerated
on glass slides in 50% acetic acid with fine-pointed forceps and
then “flame-dried.”
rDNA Probes and Oligo Probes
The plasmids containing 5S and 45S rDNA were kindly pro-
vided by K. Arumuganthan, University of Nebraska. These probes
were labeled via nick translation with digoxigenin- and biotin-
dUTP, respectively. The oligo libraries were synthesized by
MYcroarray (Ann Arbor, MI, USA). Each synthesized oligo con-
tained 48 bp of genomic sequence, a 5′ F primer, which included
the T7 RNA polymerase promoter sequence, and a 3′ R primer.
Amplification and labeling of the oligo libraries was as described
by Han et al. [2015].
Fluorescence in situ Hybridization
FISH was performed according to published protocols [Jiang et
al., 1995; Cheng et al., 2001]. High-quality chromosome prepara-
tions were used for repeated probing. After the first round of prob-
ing and image capture, coverslips were taken off carefully, and the
slides were washed 3 times in 1× PBS (5 min each). The slides were
then dehydrated in an ethanol series (70, 90, and 100%, 5 min each),
denatured again in 70% formamide at 80 ° C for 2 min, dehydrated
in a second ethanol series, and reprobed with different probes. Bi-
otin-labeled probes were detected by Alexa Fluor 488 streptavidin;
digoxigenin-labeled probes were detected by rhodamine anti-di-
goxigenin. When 3 probes were used in a FISH experiment, the first
and the second probe were labeled by biotin and digoxigenin, re-
spectively; the third probe was labeled by biotin and digoxigenin
(1: 1), which resulted in a yellow detection color. Chromosomes
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Göteborgs Universitet
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were counterstained with DAPI in a VectaShield antifade solution
(Vector Laboratories). Images were captured digitally using a CCD
camera (QIMAGING, RETIGA-SRV, FAST 1394) attached to an
Olympus BX63 epifluorescence microscope. Gray-scale images
were captured for each color channel and then merged. Final image
adjustments were done with Adobe Photoshop (Adobe Systems).
Measurements
For each F. vesca accession, 3 of the best images were selected
for measuring the long and short arm lengths. The images of
DAPI-stained chromosomes were converted into black-white im-
ages to clearly show the centromere position. The “measurement”
tool of Adobe Photoshop was used for all size estimations. Relative
chromosome length and arm ratio were calculated based on mea-
suring 3 metaphase cells. Chromosomes were classified according
to Levan et al. [1964].
Results
The draft genome of F. vesca has 7 pseudochromo-
somes. To identify the cytological chromosome corre-
sponding to each pseudochromosome of F. vesca, 6 oligo
libraries numbered library 1 to 6 corresponding to pseu-
dochromosomes 1 to 6, respectively, were synthesized.
Each library contained all oligos specific to an entire
pseudochromosome at a density of 2–3 oligos per kilo-
base based on the draft genome assembly of woodland
strawberry (F. vesca) [Shulaev et al., 2011]. Then, these
libraries were amplified and labeled as “chromosome
painting” probes for FISH.
First, we simultaneously hybridized 2 oligo libraries
(labeled by biotin and digoxigenin, respectively) to so-
matic metaphase cells of F. vesca subsp. vesca (Fig. 1a–c).
Although there are some background signals on other
chromosomes, all libraries generated bright and specific
FISH signals only on a pair of homologous chromosomes,
indicating that these chromosome-specific oligo libraries
can serve as reliable molecular cytological markers to un-
ambiguously identify chromosomes of F. vesca. Based on
the above FISH results, libraries 1 (digoxigenin-labeled),
2 (biotin-labeled), and 3 (labeled by biotin and digoxi-
genin) were pooled and used to simultaneously hybridize
to metaphase cells. As expected, the 3 libraries produced
bright FISH signals on 3 pairs of chromosomes numbered
chromosomes 1 to 3, respectively (Fig.  1d). Then, the
slides were washed and reprobed with libraries 4 (labeled
by biotin and digoxigenin), 5 (digoxigenin-labeled), and
6 (biotin-labeled) simultaneously. The chromosome pairs
4 to 6 were identified based on the color of signals (Fig. 1e).
The pair of chromosomes without signal labeling should
be chromosome 7 (Fig. 1f). This allowed the simultaneous
Integrated Karyotyping of F. vesca with
Oligopaint FISH Probes
a b c
d e f
g
h
Fig. 1. Karyotyping of Fragaria vesca subsp. vesca using oligo li-
braries. a–c Simultaneous hybridization of 2 oligo libraries on mi-
totic metaphase cells. a Libraries 1 (green) and 2 (red). b Libraries
3 (green) and 4 (red). c Libraries 5 (red) and 6 (green). d Simulta-
neous hybridization of libraries 1 (red), 2 (green), and 3 (yellow).
e The same cell as in d reprobed with libraries 4 (yellow), 5 (red),
and 6 (green). f The same cell as in d reprobed with 5S (red) and
45S (green) rDNA probes. g The DAPI-stained chromosomes in
d were converted into black-white images to clearly show the cen-
tromere position, cut out, and arranged. h Representative idio-
grams. The chromosome nomenclature follows the pseudochro-
mosomes in the draft genome assembly of F. vesca [Shulaev et al.,
2011]. The relative length and the arm ratio of each chromosome
are drawn based on the data in Table 1. Scale bars, 5 μm.
identification of all chromosomes in a cell, and the 7 pseu-
dochromosomes were assigned to specific cytological
chromosomes. Finally, to determine the location of 5S
and 45S rDNA, the slides were washed and reprobed with
probes for 5S rDNA (digoxigenin-labeled) and 45S rDNA
(biotin-labeled) simultaneously. As shown in Figure 1f,
chromosomes 1 and 2 bear 45S rDNA sites. One chromo-
some 7 has both 5S and 45S rDNA sites. However, the
other homolog of chromosome pair 7 has no 45S but only
a 5S site (Fig.  1f). The DAPI-staining in Figure 1d was
converted into a black-white image to clearly show the
position of the centromeres, and the chromosomes were
cut out and arranged in Figure 1g. The karyotype data and
an idiogram of F. vesca subsp. vesca are given in Table 1
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 c

a b d

a b g

c e f h

Fig. 2. Karyotyping of Fragaria vesca from Shenyang Agricultural
University using oligo libraries. a Simultaneous hybridization of
libraries 1 (red), 2 (green), and 3 (yellow). b The same cell as in a
reprobed with libraries 4 (yellow), 5 (red), and 6 (green). c The
DAPI-stained chromosomes in a were converted into black-white
images to clearly show the centromere position, cut out, and ar-
ranged. The results of the karyotype analysis are given in Table 1.
Scale bar, 5 μm.
Fig. 3. FISH of oligo probes and 5S and 45S rDNA in Fragaria
vesca subsp. vesca (a–d) and F. vesca from Shenyang Agricultural
University (e–h). a, e Simultaneous hybridization of libraries 1
(green) and 2 (red). b, f The same cells as in a and e were reprobed
with 5S rDNA (red) and 45S rDNA (green). c, g The chromosomes
with the 5S rDNA (red) and 45S rDNA (green) signals in b and f,
respectively. DAPI-stained chromosomes were converted into
black-white images to clearly show satellites and secondary con-
strictions. d The chromosomes with 5S (red) and 45S rDNA
(green) signals from Figure 1f. h The chromosomes with 5S (red)
and 45S rDNA (green) signals from another metaphase cell of F.
vesca from Shenyang Agricultural University. Scale bars, 5 μm.

Table 1. Relative lengths and arm ratios of mitotic metaphase chro-
mosomes of 2 Fragaria vesca accessions
Accession Chromosome Relative
number lengtha, %
F. vesca subsp. 1 12.88 ± 1.10
vesca 2 14.89 ± 0.06
3 16.12 ± 0.66
4 14.62 ± 0.33
5 13.55 ± 0.59
6 16.99 ± 0.74
7 10.95 ± 0.86
F. vesca from 1 12.09 ± 1.08
Shenyang 2 14.12 ± 0.52
3 15.79 ± 0.08
4 13.78 ± 0.95
5 15.27 ± 1.36
6 17.95 ± 1.01
7 11.00 ± 0.91
a
Relative length = 100 × chromosome length/total complement length.
b
Arm ratio = length of the long arm/length of the short arm.
c
m, metacentric; sm, submetacentric.
and Figure 1h, with chromosome nomenclature follow-
ing the pseudochromosomes in the draft genome of F.
vesca “Hawaii 4” [Shulaev et al., 2011].
Arm ratiob Chromosome
morphologyc We also analyzed the karyotype of the F. vesca acces-
sion obtained from Shenyang Agricultural University
1.99 ± 0.13 sm
1.32 ± 0.13 m
with this set of oligo probes. This accession exhibited sim-
1.23 ± 0.06 m ilar karyotype results to F. vesca subsp. vesca, although the
1.29 ± 0.21 m descending orders of chromosome lengths were different
1.58 ± 0.21 m for both F. vesca accessions (Fig. 2; Table 1). Both acces-
1.17 ± 0.15 m sions had 5 pairs of metacentric chromosomes (chromo-
2.39 ± 0.19 sm
somes 2 to 6) and 2 pairs of submetacentric chromosomes
1.77 ± 0.17 sm (chromosomes 1 and 7). The longest chromosome was
1.48 ± 0.20 m
1.19 ± 0.04 m
chromosome 6, and the shortest one was chromosome 7.
1.42 ± 0.11 m However, the 2 accessions differed from each other in the
1.65 ± 0.15 m number of 45S rDNA sites. In F. vesca subsp. vesca, only
1.05 ± 0.02 m one chromosome 7 had 45S rDNA signals at the satellite
2.07 ± 0.01 sm and secondary constriction (Fig. 3c, d, indicated by ar-
rows). The other chromosome 7 displayed no 45S rDNA
signals and did not have the satellite and secondary con-
striction. In contrast, in the F. vesca accession from
Shenyang Agricultural University, both chromosomes 7
had 45S rDNA sites (Fig. 3f–h). Furthermore, differences
in signal size and intensity were observed among the three
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DOI: 10.1159/000485283
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a b c
d e f So far, several karyotypes of F. vesca have been report-
Fig. 4. FISH of libraries 1 (green) and 2 (red) in 6 representative
Fragaria species. a F. nilgerrensis. b F. pentaphylla. c F. viridis. d F.
corymbosa. e F. chiloensis. f F. cascadensis. Scale bars, 5 μm.
45S rDNA loci as well as between homologs at each 45S
locus in the 2 F. vesca accessions (Fig. 3b–d, f–h). The 45S
rDNA signals on chromosome pair 7 were always much
larger and more intensive than those on chromosome
pairs 1 and 2. In the F. vesca subsp. vesca accession, the
45S rDNA signal on one chromosome 1 was obviously
larger and stronger than that on the other one (Fig. 3b–d).
The signal patterns were generally consistent among the
examined cells, suggesting that this variability was not
due to experimental artifacts. Shulaev et al. [2011] also
observed a polymorphism in signal intensity and/or pres-
ence versus absence between homologous 25S rDNA sites
in many diploid Fragaria genotypes.
To evaluate the potential of cross-species detection of
homeologous chromosomes using the bulked oligo
probes, we performed FISH using library 1 and library 2
as probes on metaphase chromosomes from 15 addition-
al Fragaria species including 8 diploid, 3 tetraploid, 3 oc-
toploid, and 1 decaploid species. The 2 probes generated
FISH signals on specific chromosomes in all examined
species. The hybridization results from 6 representative
species are shown in Figure 4. Libraries 1 and 2 hybrid-
ized to 2 chromosomes in the 3 diploid species, including
F. nilgerrensis (Fig. 4a), F. pentaphylla (Fig. 4b), and F.
viridis (Fig. 4c), to 4 chromosomes in tetraploid F. corym-
bosa (Fig. 4d), to 8 chromosomes in octoploid F. chiloen-
sis (Fig. 4e), and to 10 chromosomes in decaploid F. cas-
cadensis (Fig. 4f). Obvious variability in signal intensity
was observed among homologous chromosomes in octo-
ploid and decaploid species (Fig. 4e, f).
Integrated Karyotyping of F. vesca with
Oligopaint FISH Probes
Discussion
The development of reliable and easy-to-use tech-
niques for chromosome identification is critical for cyto-
logical analyses, as well as subsequent studies in phy-
logenetics, taxonomy, and the evolution of polyploidy.
Unfortunately, chromosome identification is a major
challenge in many plant species with small chromosomes.
The somatic metaphase chromosomes of F. vesca are
small in size and are morphologically similar. Thus, un-
ambiguous identification of individual F. vesca chromo-
somes based on their morphology is almost impossible.
ed based on chromosome length of mitotic metaphase
cells, and chromosomes were arranged in descending or-
der, with the longest one being chromosome 1 (or A) and
the shortest one chromosome 7 (or G) [Iwatsubo and Na-
ruhashi, 1989; Lim, 2004; Shulaev et al., 2011; Rho et al.,
2012]. However, significant discrepancies exist among
the previously published karyotypes. For example, Iwat-
subo and Naruhashi [1989] reported that the karyotype
of F. vesca species was composed of 4 pairs of metacentric,
2 pairs of submetacentric, and 1 pair of subtelocentric
chromosomes. But Rho et al. [2012] reported 5–6 pairs of
metacentrics, 1–2 pairs of submetacentrics, and no sub-
telocentrics in 2 F. vesca accessions. Moreover, the chro-
mosomes carrying rDNA sites also differ in different ac-
cessions of F. vesca, although all above studies reported
that F. vesca species had 3 pairs of 45S and 1 pair of 5S
rDNA sites [Lim, 2004; Shulaev et al., 2011; Rho et al.,
2012]. The use of different F. vesca accessions and mea-
surement variability in the different studies can poten-
tially result in these discrepancies in assigning chromo-
some numbers since the chromosomes of F. vesca are
small and compact. More importantly, the chromosome
numbering systems in these previous F. vesca karyotypes
do not correlate with one another, and none of them is
associated with the numbering system of 7 pseudochro-
mosomes in the draft genome of F. vesca [Shulaev et al.,
2011]. Shulaev et al. [2011] only identified tentative cor-
respondences between 2 cytologically marked chromo-
somes and genomically defined pseudochromosomes by
finding sequence homology between 25S and 5S rDNA
probes and scaffolds mapped to the pseudochromosomes
of the “Hawaii 4.” They proposed that the chromosome
pair G (the shortest one), double marked by 25S and 5S
rDNA sites, appears to correspond to pseudochromo-
some VII, which is consistent with the results of our pres-
ent study. However, it is inconsistent with our results
about chromosome pair F which may correspond to
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pseudochromosome VI. In this study, we integrated
chromosome nomenclature between the 7 pseudochro-
mosomes and cytological chromosomes; the cytological
chromosomes were numbered from 1 to 7 corresponding
to pseudochromosomes I to VII, respectively. We found
that both F. vesca accessions had one 5S locus on chromo-
some 7 and three 45S rDNA loci on chromosomes 1, 2,
and 7. No 45S rDNA locus was detected on chromosome
6 (chromosome pair F in Shulaev et al. [2011]). We think
that the scaffold containing 25S sequences on pseudo-
chromosome VI may have been placed incorrectly when
the draft genome of F. vesca “Hawaii 4” was assembled.
In the present study, to avoid the limitations associ-
ated with the previously published karyotypes, we devel-
oped a set of chromosome-specific oligo libraries. This set
of oligo libraries allowed the simultaneous identification
of all 14 mitotic metaphase chromosomes of F. vesca in a
single cell, and a molecular cytogenetic karyotype of F.
vesca was constructed. Several distinct advantages exist in
our molecular cytogenetic karyotype compared with pre-
viously published karyotypes. First, every mitotic chro-
mosome can be readily and unambiguously identified.
Moreover, the 7 pseudochromosomes in the draft ge-
nome of F. vesca were assigned to their corresponding
cytological chromosomes. The 5S and 45S rDNA loci
were also mapped to specific chromosomes. Second, in
the current molecular cytogenetic karyotype, the F. vesca
chromosomes are identified and numbered in accor-
dance with the pseudochromosomes in the draft genome
of F. vesca instead of previous traditional systems that use
only chromosomal size parameters to assign a chromo-
some number. Thus, chromosome identification and no-
menclature based on this set of oligo probes will also be
consistent among different Fragaria species, assuming
that there are no large rearrangements involving different
chromosomes in these species. We found that 2 oligo li-
braries from F. vesca produced clear signals on specific
chromosomes in all examined 15 Fragaria species. Thus,
the homoeologous chromosomes can be identified, and
the karyotypes can be developed in Fragaria species
through cross-species chromosome painting with this set
of oligo probes from F. vesca. This will enable the study
of karyotype evolution among Fragaria species in a more
detailed way, allowing further investigations concerning
synteny and chromosomal rearrangements among ge-
nomes of Fragaria species. Therefore, it will be possible
to clarify the phylogenetic relationships of all Fragaria
species and the origin of polyploid strawberry species by
karyotype analysis. Furthermore, chromosome-specific
oligo probes can be used to characterize Fragaria addi-
tion, substitution, and introgression lines cytologically,
and trace individual chromosomes in hybrids, which can
hardly be characterized using conventional cytogenetic
techniques. Thus, the set of cytogenetic markers devel-
oped here will facilitate the application of breeding tech-
nologies in the Fragaria genus that rely upon identifica-
tion of individual chromosomes.
Acknowledgements
The authors are grateful to Kassandra Semrau from MYcro-
array (http://mycroarray.com) for designing the oligo probes. This
research was supported by the Graduate Innovation Project of
Jiangsu Normal University (2016YYB093) and the Priority Aca-
demic Program Development of Jiangsu Higher Education Insti-
tutions (PAPD).
Disclosure Statement
The authors have no conflicts of interest to declare.

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