Chromosomal distribution of pTa-535, pTa-86, pTa-713, 35S rDNA repetitive sequences in interspecific hexaploid hybrids of common wheat (Triticum aestivum L.) and spelt (Triticum spelta L.)
Chromosomal distribution of pTa-535, pTa-86, pTa-713, 35S rDNA repetitive sequences in interspecific hexaploid hybrids of common wheat (Triticum aestivum L.) and spelt (Triticum spelta L.)
Klaudia Goriewa-Duba 1 2
Adrian Duba 2
Michaø Kwiatek 0 2 3
Halina Wiśniewska 2 3
Urszula Wachowska 2
Marian Wiwart 1 2
0 Institute of Plant Genetics, Polish Academy of Sciences , Poznań, Wielkopolskie Voivodeship , Poland
1 Department of Plant Breeding and Seed Production, University of Warmia and Mazury in Olsztyn, Olsztyn, Warmian-Masurian Voivodeship, Poland, 2 Department of Entomology , Phytopathology and Molecular Diagnostics , University of Warmia and Mazury in Olsztyn , Olsztyn, Warmian-Masurian Voivodeship , Poland
2 Editor: Guangyuan He, Huazhong University of Science and Technology , CHINA
3 Department of Genetics and Plant Breeding, Poznań University of Life Sciences , Poznań, Wielkopolskie Voivodeship , Poland
Fluorescent in situ hybridization (FISH) relies on fluorescent-labeled probes to detect specific DNA sequences in the genome, and it is widely used in cytogenetic analyses. The aim of this study was to determine the karyotype of T. aestivum and T. spelta hybrids and their parental components (three common wheat cultivars and five spelt breeding lines), to identify chromosomal aberrations in the evaluated wheat lines, and to analyze the distribution of polymorphisms of repetitive sequences in the examined hybrids. The FISH procedure was carried out with four DNA clones, pTa-86, pTa-535, pTa-713 and 35S rDNA used as probes. The observed polymorphisms between the investigated lines of common wheat, spelt and their hybrids was relatively low. However, differences were observed in the distribution of repetitive sequences on chromosomes 4A, 6A, 1B and 6B in selected hybrid genomes. The polymorphisms observed in common wheat and spelt hybrids carry valuable information for wheat breeders. The results of our study are also a valuable source of knowledge about genome organization and diversification in common wheat, spelt and their hybrids. The relevant information is essential for common wheat breeders, and it can contribute to breeding programs aimed at biodiversity preservation.
Data Availability Statement: Detailed information
about wheat accessions used in this study, primers
sequences and PCR conditions for wheat repetitive
sequences amplification are available from
figshare. The auxiliary table containing information
about pTa-535, pTa-86, pTa-713 and 35S rDNA
repetitive sequences distribution on wheat
chromosomes is also available from figshare. The
links to access these data sets are as follows:
The preservation of biodiversity in living organisms, including major crop species, is one of
the key challenges in the 21st century. The aim of modern breeding programs is to produce
high-yielding cultivars. Over the years, the above has considerably narrowed down genetic
Funding: The authors received no specific funding
for this work.
pools of many crop species, including common wheat (Triticum aestivum L). In contemporary
wheat cultivars, reduced genetic variation increases susceptibility to environmental stressors.
Wild emmer wheat, Triticum dicoccoides (KoÈrn. ex Asch. & Graebn.) Schweinf. [
], is the
donor of A and B genomes to contemporary tetraploid wheats. It is believed that the A genome
originates from diploid wheat Triticum urartu Thum. ex Gandil, and the B genomeÐfrom the
species Aegilops speltoides Tausch. [
]. Mutations in the above genomes and variations
resulting from crosses with related taxa have led to the emergence of new species such as durum
wheat (Triticum durum Desf.), Polish wheat (Triticum polonicum L.) and Khorasan wheat
(Tritcum turanicum Jakubz.). Hexaploid wheats carry the third genome, D, which was
introduced when wheat the AABB amphiploid was crossed with Aegilops tauschii Coss. (= Triticum
tauschi Coss.) with the DD genome [
]. Common wheat and spelt (Triticum spelta L.) belong
to the above group [
]. Genetic drift and natural and artificial selection have led to the
emergence of local varieties that were very well adapted to specific environmental conditions.
Today, the above varieties are of low economic significance, and they have been replaced by
high-yielding cultivars that account for more than 90% of global wheat production . These
cultivars have been developed by crossing a relatively small number of local varieties and
cultivars. In consequence, they are characterized by low genetic variation and a relatively high
degree of relatedness [
In the past two decades, consumers have shown a growing interest in high-quality food
products. Due to a steady decrease in the genetic variation of common wheat and lower
nutritional value of wheat grain proteins in comparison with other cereals, breeders increasingly
often rely on species closely related to T. aestivum to produce cultivars with improved
nutritional value [
]. The growing popularity of spelt can be attributed to its relatively low
agronomic requirements and high resistance to abiotic and biotic stress. Spelt is characterized by
hulled grains and genetic polymorphism, which effectively prevent the spread of pathogenic
]. Wiwart et al. [
] demonstrated that spelt is more resistant to Fusarium
culmorum infections than common wheat. Today, spelt is produced mainly in organic farming
]. Growing levels of consumer awareness contribute to the popularity of spelt.
According to Waga et al. [
] and Escarnot et al. [
], spelt is a valuable source of genes
responsible for high nutritional value and a high protein content of grain. As a result, spelt
grain is characterized by high concentrations of essential amino acids, including tyrosine,
leucine and isoleucine, as well as valuable nutrients such as zinc, magnesium and iron ions.
According to Waga et al. [
], spelt and common wheat hybrids are characterized by similar
nutritional value. There is evidence to indicate that spelt delivers health benefits and can be
used in the production of hypoallergenic foods [
]. However, the non-allergenic properties
of spelt were not confirmed by Waga et al. , Ruibal-Mendieta et al. [
] or Pahr et al. [
Spelt grain proteins have higher nutritional value than common wheat proteins, and the
protein content of spelt can exceed 16.5% of dry weight [
]. The quality and nutritional value of
T. spelta grain is superior to that T. aestivum. Common wheat grain is characterized by free
threshability, considerable resistance to lodging and high yield potential, which prompts
breeders to develop hybrids of these two closely related species. Research into common wheat
and spelt hybrids revealed that the expression of heterosis in F1 individuals can reach 40%. The
grain of common wheat and spelt hybrids was also characterized by higher nutritional value
and processing suitability than the grain of parental forms [
]. Studies investigating hybrids'
resistance to selected pathogens have demonstrated that progeny resistant to brown rust can
be produced if one of the parental components is resistant to this pathogen [
wheat and spelt hybrids are characterized by new agriculturally useful traits, in particular high
nutritional value and processing suitability of grain [
2 / 16
T. aestivum and T. spelta hybrids have never been subjected to cytogenetic analyses. The
aim of this study was to: (1) describe the karyotype of T. aestivum and T. spelta hybrids and
their parental components (three common wheat cultivars and five spelt breeding lines), (2) to
identify chromosomal aberrations in the analyzed genomes, and (3) to analyze the distribution
of polymorphism of repetitive sequences in the examined hybrids.
The experimental material comprised the grain of F7 hybrids from single crosses between T.
spelta x T. aestivum and T. aestivum x T. spelta and their parental forms: spring spelt breeding
lines (denoted S10, S11, S12, S13 and S14), selected at the Department of Plant Breeding and
Seed Production of the University of Warmia and Mazury in Olsztyn, Poland, and three
cultivars of common wheat: Torka, Kontesa and Zebra (Table 1, S1 Table). The field experiment
was performed at the Agricultural Experiment Station in Baøcyny, Poland (53Ê36'N, 19Ê51'E).
Spelt and wheat were grown and harvested in accordance with good agricultural practice
standards. Parental forms and their hybrids were subjected to cytogenetic analyses to determine
their karyotypes, to localize and identify chromosomal aberrations, and to develop a physical
map of repetitive sequences.
The leaves of three-week-old seedlings of spelt, common wheat and wheat hybrids were
collected. DNA was isolated with a ready-to-use Genomic Micro AX Plant Gravity Kit (A&A
Biotechnology, Poland). The quantity and quality of DNA was determined with a
spectrophotometer (nanoMaestro Gen, Poland) at 260 nm and 280 nm wavelength. Extracted DNA was
additionally purified before further analysis with the use of the Anti-Inhibitor Kit (A&A
Biotechnology, Poland). The 1BL/1RS translocation in investigated parental components and
common wheat-spelt hybrids was identified by PCR according to the method described by
Iqbal and Rayburn [
] with specific primers JO71F1
5’-TAAGCCGTAAAGCATGGTGCAC3’ and J07IR1 5’-CTTCAACGAAAT GTT TTC CTC TTC-3’. Total reaction volume
was 20 μl.
Preparation of chromosome spreads
Seed germination and the accumulation of chromosomes during metaphase division in
embryonic wheat roots were determined according to the method described by Kwiatek et al.
]. Mitotic chromosome preparations were obtained from root tips digested in an enzyme
KONTESA x S14
ZEBRA x S10
ZEBRA x S11
ZEBRA x S12
ZEBRA x S13
ZEBRA x S14
S10 x TORKA
S11 x TORKA
S12 x TORKA
S13 x TORKA
S14 x TORKA
S10 x KONTESA
S11 x KONTESA
S12 x KONTESA
S13 x KONTESA
S14 x KONTESA
mixture: 20% (v/v) pectinase (Sigma), 1% (w/v) cellulase (Calbiochem) and 1% (w/v) Onozuka
R-10 cellulase (Serva) diluted in 0.01M sodium citric buffer (pH 4.8). Root tips were macerated
for at least 150 minutes at 37ÊC. After maceration, the enzyme mixture was removed, and
roots were rinsed with sodium citric buffer at room temperature. Root tips were placed on a
slide in a drop of ice-cold 60% acetic acid and dispersed with a metal needle. The dispersed
material was covered with a coverslip and pressed down. Slide quality was verified by
DNA probes three repetitive sequences, pTa-86 (GenBank accession number KC290896.1),
pTa-535 (KC290894.1) and pTa-713 (KC290900.1), were amplified from the clones listed in
the BAC library of wheat developed by Komuro et al. [
]. Additional repetitive sequence, 35S
rDNA (KC290907) was also used in this study [
]. Specific primers were designed in the
Primer3 program [
] to amplify selected sequences (S2 Table). Primer properties were
verified with OligoCalc [
]. PCR conditions were as follows: 95ÊC for 5 minutes, 35 cycles of
95ÊC for 30 seconds, annealing temperature appropriate for each primer pair (pTa-86: 58.5ÊC,
pTa-535: 58ÊC, pTa-713: 59ÊC, 35S rDNA: 59ÊC) for 30 seconds, 72ÊC for 90 seconds and
72ÊC for 5 minutes. All sequences were labeled with the nick-translation kit (Sigma) according
to the manufacturer's instructions. Probe pTa-86 was labeled with digoxigenin-11-dUTP
(Roche), probe pTa-535 ±with tetramethyl-5-dUTP-rhodamine (Roche) and probe pTa-713 ±
with Atto 647 (Jena BioScience). 35S rDNA probe was labeled with Atto 647 (Jena BioScience).
Fluorescent in situ hybridization
The FISH procedure was carried out according to the protocol described by Kwiatek et al.
]. Chromosomes were treated with RNase (100 μg per milliliter) and incubated in a moist
chamber for 60 minutes at a temperature of 37ÊC. After incubation, the samples were twice
rinsed in 2x SSC for 5 minutes. They were deproteinized in formaldehyde (4%) diluted with 1x
PBS for 15 minutes at room temperature. Deproteinized samples were twice rinsed in 2x SSC
for 5 minutes and dehydrated in a graded series of alcohols: 70%, 90% and 100%.
Chromosomal DNA in the presence of hybridization mixture (50% formamide, 10% dextran sulfate,
20x SSC, 0,1% SDS, 30 μg of SHS, 70 ng of probes and water) was denatured at 75ÊC for 10
minutes in under cover slip and stabilized on ice. Drops of the hybridization mixture were
applied to glass slides, the specimens were incubated overnight in a moist chamber at 37ÊC.
The next day specimens were rinsed in 2x SSC, 0,1x SSC, 0,1x SSC and 2x SSC solutions in a
water bath at a temperature of 42ÊC, followed by 2x SSC at room temperature. Before the
application of antibody solutions, the specimens were additionally rinsed in 4x SSC+0.2%
Tween 20 at room temperature. 20 μg per milliliter of anti-digoxigenin-fluorescein antibody
(Roche) was applied, then the specimens were incubated in a moist chamber for 60 minutes to
increase signal intensity and were rinsed in 4x SSC+0.2% Tween 20 heated to 37ÊC and in 2x
SSC at room temperature. The specimens were dehydrated in a graded series of alcohols: 70%,
90% and 100%, and were mounted with DAPI and then with Vectashield. Image processing
was carried out using Olympus Cell-F (version 3.1; Olympus Soft Imaging Solutions GmbH:
MuÈnster, Germany) imaging software and Photoshop CS3 software (version 10.0.1; Adobe
Systems, USA). After documentation of the FISH sites, the slides were washed as described in
Probe elution section and dried and used for second FISH experiment. In the first FISH trial,
three probes were applied: pTa-86, pTa-535 and pTa-713. After elution, probes pTa-86 and
pTa-535 were also applied (in order to identify the particular chromosomes) and third probe
pTa-713 was added. The identification of particular chromosomes were made by comparing
4 / 16
the signal patterns of tested probes hybridized to hexaploid wheat according to Komuro et al.
] and Kwiatek et al. [
After documentation of the FISH sites, the analyzed slides were washed (2×60 min in 4×SSC
Tween, 2×5min in 2×SSC, at room temperature) and after alcohol washes were dried and used
for second FISH experiment. They were dehydrated in a graded series of alcohols: 70%, 90%
Parental forms (T. spelta and T. aestivum) and their simple-cross hybrids were characterized
by similar genome composition (BBAADD, 2n = 6x = 42). Unlike in common wheat
accessions, the chromosomes in spelt lines bred by the authors were difficult to separate due to high
cytoplasm density (Fig 1).
The FISH method enabled the identification of chromosome structure and possible
aberrations. Structural aberrations such as translocations, inversions and deletions were not observed
in parental wheat cultivars (T. spelta breeding lines S10-S14 and T. aestivum cultivars Torka,
Kontesa and Zebra). In our experiment, attempts were also made to identify the 1BL/1RS
translocation, however, it was not detected in any of the progenitors or their hybrids.
PCRbased identification of 1BL/1RS translocation also showed the absence of 1BL/1RS
Physical mapping of repetitive sequences
The FISH method was also used to analyze the distribution of repetitive sequences in wheat
chromosomes. A-, B- and D-genome chromosomes were identified by comparing the labeling
patterns developed by Komuro et al. [
]. In our study, the distribution analysis of pTa-535,
pTa-86 and pTa-713 and 35S rDNA repetitive sequences in the chromosomes of common
wheat and spelt lines and their hybrids (Fig 2) revealed considerable similarity with selected
polymorphic sites. The long arm of a chromosome is termed the L arm and the short arm is
indicated by S letter. Both indicators are located next to particular chromosome number.
Fig 1. A cluster of chromosomes in spelt line (S14) (A) and separated chromosomes of common wheat cultivar
5 / 16
Fig 2. A physical map of repetitive sequences pTa-535 (red signals), pTa-86 (green) and pTa-713 (yellow) in
analyzed lines of common wheat and spelt and their hybrids. Arrowheads indicate centromere positions. In the
absence of an arrowhead, a chromosome is considered metacentric.
The pTa-535 sequence produced the highest number of specific signals in A-genome
chromosomes (Figs 2 and 3). The labeling patterns of the pTa-535 repetitive sequence were present in
each A-genome chromosome. However, the intensity of the pTa-535 probe signals was rather
low in all investigated wheat accessions. Hybridization patterns differed across chromosome
types, but were similar in all tested wheat cultivars and their hybrids. Hybridization signals in
subtelomeric regions of both arms of chromosomes 1A, 4AL and 5AL and in telomeric regions
of 1AS, 6AS and both arms of chromosomes 2A and 7A were detected in hexaploid
progenitors. A centromeric hybridization pattern was detected in two chromosomes (2A, 3A).
Chromosomes carried 1 to 4 hybridization sites. Some hybrids were characterized by a lower
number of repetitive sequences, which decreased signal intensity (Fig 3). Only several pTa-86
sites were identified in A-genome chromosomes. Signals were observed in the long arm of
chromosome 4A and in the short arm of chromosome 5A, both in telomeric regions (Figs 2
and 3). The hybridization pattern of probe pTa-713 was detected in three A-genome
chromosomes: 5AS, 6AL (telomeric region) and 7A (centromeric region) in most investigated
accessions. Only the pTa-713 hybridization pattern was polymorphicÐin chromosomes 4A and
6A. In chromosome 4A of T. spelta accession S10, the subtelomeric pTa-713 signal was not
detected in the long arm of the chromosome (Fig 2). A comparison of hybrids revealed the
polymorphic site of subtelomeric pTa-713 labeling in chromosome of 4A in Torka x S10 and
S10 x Kontesa crosses (Figs 2 and 3). Probe pTa-713 did not produce a signal in the Torka x
S10 accession in chromosome 6A (Figs 2 and 3).
Repetitive sequences pTa-535 were not present in the majority of B-genome chromosomes,
and they generated weak and variable signals in the tested lines (Figs 2 and 4). pTa-535 labeling
was present only in chromosomes 3B and 7B. Nonetheless, pTa-535 signal distribution
6 / 16
Fig 3. Karyograms of Torka x S10, Torka x S11, S10 x Kontesa hybrids and Torka, Kontesa, Zebra, S10-S14 parental components showing
A-genome chromosomes after FISH with pTa-535 (red), pTa-86 (green) and pTa-713 (yellow) probes. Abbreviations: accession number
1Torka x S10, 2- Torka x S11, 20- S10 x Kontesa, 25- Torka, 26- Kontesa, 27- Zebra, 28- S10, 29- S11, 30- S12, 31- S13, 32- S14.
facilitated chromosome identification. In all accessions, the most informative pTa-535 patterns
were detected in chromosome 7B in the subtelomeric region of the short arm.
The pTa-86 hybridization pattern in B-genome chromosomes was strong and detectable in
each chromosome of the analyzed accessions. Minor changes in signal intensity were observed
between the examined common wheat cultivars and spelt lines and their hybrids (Fig 4).
pTa86 signals were detected mainly in telomeric and subtelomeric regions. Polymorphic sites of
pTa-86 probe were detected in the short arms of chromosomes 1B and 6B in the telomeric
regions. In Torka x S10 and S10 x Kontesa hybrids and S10 and S14 parental components, the
pTa-86 labeling in 1B was replaced with pTa-713 probe signal (Fig 4). Another polymorphic
hybridization pattern of pTa-86 was reported in 6B chromosome in telomeric region of short
arm of chromosome. In accessions Torka x S11 (Fig 4), Torka x S12, Kontesa x S11, S10 x
Kontesa (Fig 4), S11 x Torka and S12 x Torka, S11, S12, Torka signal was not observed.
The signal intensity of the pTa-713 probe differ in B-genome chromosomes. An intense
signal generated by pTa-713 was detected in the centromeric region of chromosomes 1B and 4B
7 / 16
Fig 4. Karyograms of Torka x S10, Torka x S11, S10 x Kontesa hybrids and Torka, Kontesa, Zebra, S10-S14 parental components showing
B-genome chromosomes after FISH with pTa-535 (red), pTa-86 (green) and pTa-713 (yellow) probes. Abbreviations: accession number
1Torka x S10, 2- Torka x S11, 20- S10 x Kontesa, 25- Torka, 26- Kontesa, 27- Zebra, 28- S10, 29- S11, 30- S12, 31- S13, 32- S14.
and pericentromeric region of chromosome 6B. The pTa-713 signals in subtelomeric regions
of chromosomes 5B was weaker but still strong. Weak, polymorphic pTa-713 signal was
present in two parental components: S10 and S14 and two hybrids: Torka x S10 and S10 x Kontesa
In the FISH procedure, the pTa-535 probe produced strong and intense signals in D-genome
chromosomes which were most informative in the group of the tested probes (Figs 2 and 5).
Only pTa-535 tandem sequences were observed in D-genome chromosomes. pTa-535 labeling
was present at various positions across entire chromosomes. Exceptionally high staining
8 / 16
Fig 5. Karyograms of Torka x S10, Torka x S11, S10 x Kontesa hybrids and Torka, Kontesa, Zebra, S10-S14 parental
components showing D-genome chromosomes after FISH with pTa-535 (red), pTa-86 (green) and pTa-713 (yellow) probes.
Abbreviations: accession number 1- Torka x S10, 2- Torka x S11, 20- S10 x Kontesa, 25- Torka, 26- Kontesa, 27- Zebra, 28- S10,
29S11, 30- S12, 31- S13, 32- S14.
intensity was found in chromosome 4D (long arm, subtelomeric region) and in both arms of
chromosome 7D (telomeric regions) in the analyzed accessions. The repetitive sequences
pTa86 were only present in chromosome 2D. The pTa-713 signal was detected only in the
pericentromeric and centromeric regions of chromosomes 6D and 7D, respectively (Fig 5).
35S rDNA mapping
The metaphases of wheat cultivars and their descendants had 3 or 4 pairs of chromosomes
with 35S rDNA signals in a total of 42 chromosomes. Of these, 3 pairs of chromosomes (1B,
6B and 5D) always had the 35S rDNA hybridization pattern (Fig 6). An additional 35S rDNA
signal was observed in chromosome 1A of T. aestivum cultivar Torka progenitors in the
telomeric region of the short arm (Fig 6). Signal intensity was arranged in the following order:
9 / 16
Fig 6. Karyograms of Torka x S10, Torka x S11, S10 x Kontesa hybrids and Torka, Kontesa, Zebra, S10-S14 parental components showing
1A, 1B, 6B and 5D chromosomes after FISH with pTa-535 (red), pTa-86 (green) and 35S rDNA (yellow) probes. Abbreviations: accession
number 1- Torka x S10, 2- Torka x S11, 20- S10 x Kontesa, 25- Torka, 26- Kontesa, 27- Zebra, 28- S10, 29- S11, 30- S12, 31- S13, 32- S14.
The analyzed T. aestivum x T. spelta and T. spelta x T. aestivum single-cross hybrids and their
parental forms are allohexaploids (2n = 6x = 42). The structural similarity of chromosomes
and their conjugation in hybrids produces BBAADD genomes [
]. Cytogenetic analyses
involving the appropriate molecular techniques are increasingly used in breeding programs.
Fluorescence in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent
probes to identify chromosomes according to their sequence [
]. None of the structural
aberrations (translocations, inversions and deletions) were present in parental wheat cultivars and
A specific PCR reaction was initially performed to identify the 1BL/1RS translocation in the
analyzed parental lines. Despite the fact that the 1BL/1RS translocation is widely used in
breeding programs (in rye, the 1RS chromosome arm carries genes encoding resistance to rust (Yr9,
Sr31, Lr26) and powdery mildew (Pm8)) [
], it was not detected in any of the parental spelt
lines or common wheat cultivars. Negative PCR results for the 1BL/1RS translocation in all
analyzed wheat lines was the confirmation of its absence in FISH results. The observed absence
of the 1BL/1RS translocation was partially consistent with the findings of Kowalczyk et al. [
who analyzed translocations in the short arm of rye (Secale cereale L.) chromosome 1RS onto
common wheat. In the cited study, the 1BL/1RS translocation was absent in T. aestivum cv.
Torka Our study made the first ever attempt to detect the 1BL/1RS translocation in common
wheat cultivars (Kontesa and Zebra), self-bred lines of spelt and their crosses. This
translocation is globally widespread in common wheat due to the presence of genes encoding resistance
to selected diseases and pests. The 1BL/1RS translocation has been retained in many breeding
] because it contributes to an increase in yield potential [
] and wheat growth
dynamics under drought conditions [32±34]. It should also be noted that the 1BL/1RS
translocation increases dough stickiness and decreases dough strength, traits that are not admissible
10 / 16
in the grain of bread-making quality . The absence of the 1BL/1RS translocation is a
desirable trait which increases the bread-making quality and nutritional value of common wheat
and spelt hybrids.
Karyotypes of investigated parental components of common wheat and spelt and their
hybrids confirmed the BBAADD genome composition [
]. Repetitive sequences considerably
influence genome evolution, and they can be used in analyses of genome diversity and
phylogenetic reconstruction. Repetitive sequences also provide information about chromosomal
]. In higher plants, including members of the genus Triticum, physical
maps are usually developed based on repetitive DNA sequences which are easier to identify
and describe than low-copy genes [
]. Physical mapping of four repetitive sequences
pTa-535, pTa-86 and pTa-713 and 35S rDNA in the chromosomes of common wheat cultivars,
spelt lines and their hybrids revealed some polymorphic sites within analyzed parental
components and their hybrids. Additionally, differences in repetitive sequences distribution have
been detected between genomes of investigated wheats and standard cultivar for wheat
cytogenetic research- T. aestivum cv. Chinese Spring . Probes used in this study were enabled
to distinguish wheat A-,B- and D-chromosomes (S3 Table). Moreover, these FISH markers
captured the differences between investigated hybrids and their parental components. Few
polymorphic sites in chromosomes were observed. Moreover, the use of pTa-535, pTa-86,
pTa-713 and 35S rDNA probes allowed to trace the intensity of the signals. The presence of
the cytoplasmic residues on chromosomes resulted in non-specific green background in
certain wheat accessions (for example spelt accessions: 32 (Fig 3) or 30 (Fig 5)). However, it did
not affect the signals reading. The observed signal strength and the number of pTa-535
repetitive sequences in A-genome chromosomes in hybrids and their progenitors were indicative of
low polymorphism. A-genome chromosomes were characterized by decreased number of
pTa-535 repetitive elements in comparison with D-genome chromosomes. In accessions with
significant lower number of pTa-535, the signal was weak and (in some accessions) detected
only in the red channel in the imaging program (Fig 3). In the A-genome chromosomes,
pTa86 labeling pattern was crucial for the identification of chromosomes 4A. A comparison of
hybrids revealed the absence of subtelomeric pTa-713 labeling in chromosome 4A in Torka x
S10 and S10 x Kontesa crosses (Figs 2 and 3). In both accessions, T. spelta breeding line S10
was one of the progenitors. It can be assumed that in these hybrids, one chromosome 4A was
inherited from the S10 progenitor of T. spelta. The pTa-713 signals in the centromeric region
of chromosomes 7A contributed to more precise identification of the studied hybrids,
especially during the identification of chromosomes 7A and 7D. A comparison with the study of
Komuro et al. [
] who analyzed the distribution of repetitive sequence signals in
chromosomes of T. aestivum cv. Chinese Spring revealed some differences in repetitive elements
distribution. In the long arms of chromosomes 3A and 4A of the analyzed lines, the pTa-535 signals
were not observed in telomeric and subtelomeric regions, respectively. Probe pTa-713 was not
detected in chromosomes 1A and 6A. However, it should be noted that the signals obtained
from probe pTa-713 in chromosome 1A of T. aestivum cv. Chinese Spring was rather not
], which suggests that in our study, the absence of the signals could be attributed to
a small number of repetitive sequences in the chromosome.
In B-genome chromosomes, hybridization patterns of the analyzed probes were more
diversified than in A-genome chromosomes (Fig 4). Differences in the intensity of the signals
were observed especially in chromosomes 1B, 3B, 5B. The pTa-86 labeling pattern of
chromosomes 4B was discriminative and crucial for its identification. The intensity and distribution of
the pTa-86 signals in 4B were constant in all accessions. Kwiatek et al. [
] pointed out that 4B
chromosomes had lower diversity than rest of the B-genome chromosomes. Polymorphic sites
were observed in 1B and 6B chromosomes (Fig 4). Our findings are consistent with the
11 / 16
previous study [
]- the telomeric region of the short arm of chromosome 6B is polymorphic,
probably due to evolutionary changes. The pTa-713 probe was highly helpful in identifying
Our study also revealed other differences. In the analyzed accessions, the hybridization
patterns of the tested probes differed from those reported by Komuro et al. [
] in the Chinese
Spring cultivar. The pTa-86 signals were more intense in the long arm of chromosome 1B, and
additional signals generated by pTa-713 (Torka x S10, S10 x Kontesa, S10 and S14) or pTa-86
(Torka x S11, Torka x S12, Kontesa x S11, S11 x Torka and S12 x Torka, S12, Torka) were
detected in the short arm of chromosome 1B (Fig 4). The presence of additional pTa-713 and
pTa-86 signals in hybrids where spelt was the paternal component suggests that polymorphism
is determined by this paternal form. In Kontesa x S11 hybrids, an additional pTa-86 signals
were not detected on the short arm of chromosome 1B of the Kontesa maternal component,
but it was identified on the S11 paternal component. In S11 x Kontesa hybrids, where spelt line
S11 was the maternal component and common wheat cultivar Kontesa was the paternal
component, an additional pTa-86 labeling pattern was not observed. The above could imply that
when an additional pTa-86 signals are detected in both parental lines, such as cultivar Torka
and spelt lines S11 and S12, the signals will also be present in hybrids (Torka x S11, Torka x
S12, S11 x Torka and S12 x Torka). The identity of maternal and parental components was not
important in the above lines.
Intraspecific polymorphisms between common wheat cv. Chinese Spring and other
cultivars of common wheat and spelt could have resulted from minor changes in the genome.
Komuro et al. [
] compared repetitive sequence signals in cultivar Chinese Spring and also
detected intraspecific polymorphisms in the analyzed accessions: an absence of a clear
pTa713 signals, a stronger pTa-535 signals in the telomeric region of the long arm of chromosome
3B, a single pTa-713 signal in chromosome 5B, different localization of the pTa-713
hybridization pattern (in the short rather than the long arm of the chromosome) and an absence of
pTa86 labeling in the telomeric region of 7BL. The observed hybridization patterns of the tested
probes in parental components and their hybrids indicate that B-genome chromosomes are
more diversified than A-, and D-genome chromosomes. Significant variations in B-genome
chromosomes of wheat were also reported by Salina et al. [
] and Levy and Feldman .
The D-genome of wheat appears to be less susceptible to evolutionary changes, and it is
characterized by low chromosome diversity [
]. In this study, polymorphic sites were not
identified in these chromosomes (Fig 5). The distribution of pTa-535, pTa-86 and pTa-713
signals was stable and repeatable. The intensity of hybridization patterns was similar across the
According to Komuro et al. [
] and Badaeva et al. [
], pTa-535 is a useful tool for
identifying A- and D-genome chromosomes in common wheat. The labeling patterns in common
wheat and spelt were highly similar, and they can be used to identify these chromosomes in
common wheat x spelt hybrids and spelt x common wheat hybrids. The results of the analysis
of pTa-535, pTa-86, pTa-713 and 35S rDNA hybridization patterns suggest the presence of a
close relationship between common wheat and spelt. According to many authors, T. spelta was
the first hexaploid wheat species whose random mutations gave rise to other wheat species,
including T. aestivum [
]. The degree of polymorphism between different cultivars of these
hexaploid wheats and their hybrids is rather low and confirms their close affinity. The
identified variations are not associated with large-scale chromosome rearrangements [
wheat is an allohexaploid species that originated from a small number of interspecific and
intergeneric hybridizations. Processes such as a genetic bottleneck and the founder effect are
responsible for its low phenotypic and genotypic variation [
]. However, due to the absence
of selection processes in the past, spelt is characterized by considerable genetic variability, and
12 / 16
it could be a potential donor of desirable genes [
]. The above could explain why selected
common wheat and spelt hybrids are characterized by polymorphic distribution of repetitive
sequences in chromosomes. Numerous authors have suggested that the B-genome of wheat is
the most diversified genome with the highest number of polymorphic markers in
allohexaploid wheat [48±51]. Gill [
] found that B-genome chromosomes are characterized by more
C-banding than A- and D-genome chromosomes. As anticipated, the distribution pattern of
repetitive sequences in B-genome chromosomes exhibited the highest number of polymorphic
sites between the analyzed lines relative to the labeling patterns of wheat cultivar Chinese
]. The D-genome may be less diversified because allohexaploid wheat originated
around 8500±9000 BC, and it is a source of repetitive sequence polymorphisms. The
polymorphisms observed in common wheat and spelt hybrids carry important information for wheat
breeders. The results of our study are also a valuable source of knowledge about genome
organization and diversification in common wheat, spelt and their hybrids. The relevant
information is essential for common wheat breeders, and it can contribute to breeding programs
aimed at biodiversity preservation.
S1 Table. Description of the accessions used in this study.
S2 Table. Primers sequences and PCR conditions for wheat repetitive sequences amplification.
S3 Table. The pTa-535, pTa-86, pTa-713 and 35S rDNA repetitive sequences distribution on chromosomes.
Conceptualization: Klaudia Goriewa-Duba.
Data curation: Klaudia Goriewa-Duba, Adrian Duba, Urszula Wachowska.
Formal analysis: Klaudia Goriewa-Duba, Adrian Duba.
Funding acquisition: Marian Wiwart.
Investigation: Klaudia Goriewa-Duba, Adrian Duba.
Methodology: Michaø Kwiatek, Halina Wiśniewska.
Project administration: Klaudia Goriewa-Duba, Marian Wiwart.
Resources: Michaø Kwiatek, Halina Wiśniewska.
Software: Klaudia Goriewa-Duba, Adrian Duba, Michaø Kwiatek, Halina Wiśniewska.
Supervision: Marian Wiwart.
Visualization: Klaudia Goriewa-Duba.
Writing ± original draft: Klaudia Goriewa-Duba.
Writing ± review & editing: Klaudia Goriewa-Duba.
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