Journal of Experimental Botany, Vol. 47, No. 297, pp. 583-588, April 1996
Journal of
Experimental
Botany
Chromosome identification and nuclear architecture in
triticale x tritordeum F1 hybrids
J. Lima-Brito1'2, H. Guedes-Pinto1, G.E. Harrison2 and J.S. Heslop-Harrison2'3
1
Department of Genetics and Biotechnology, University of Tras-os-Montes and Alto Douro, 5000 Vila Real,
Portugal
2
Karyobiology Group, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
Received 16 October 1995; Accepted 20 December 1995
Abstract
Introduction
In situ hybridization with cloned, repetitive DNA probes
and total genomic DNA enables the parental origin of
all chromosomes to be established in metaphases of
triticale x tritordeum F, hybrids (2n = 6x = 42). Nuclei
contain seven chromosomes of Hordeum chilense
origin, seven from Secale cereale and 28 of wheat
origin. When used as a probe, total genomic rye DNA
labelled the rye chromosomes strongly and uniformly
along their lengths, with brighter regions coincident
with the terminal heterochromatin. The probe labelled
the wheat-origin chromosomes weakly and was almost
undetectable on the H. chilense-orig'm chromosomes.
In contrast, under the same conditions, H. chilense
DNA hybridized strongly to the H. chilense- and, with
intermediate strength, to the S. cerea/e-origin chromosomes, excluding the subtelomeric heterochromatin:
it hybridized only weakly to the wheat chromosomes,
in some experiments revealing characteristic bands on
wheat chromosomes. Cloned repetitive DNA probes
from rye and H. chilense were used as probes to
identify the linkage groups of all of their own-species
chromosomes. Analysis of hybridization patterns of
various probes to prophase and interphase nuclei indicated that there are many non-random features in the
localization of both repetitive DNA and whole chromosomes, although general patterns of nuclear organization have yet to emerge. Both the particular lines used
and the techniques developed here are likely to be
valuable for production and characterization of plant
breeding material.
Many species in agriculture are of hybrid origin: for
example, bread wheat (Triticum aestivum L.) is an allohexaploid with three genomes, A, B and D, of different
origins. The possibility of increasing the genetic base of
crop plants by use of synthetic hybrids has not been
ignored, and the amphiploid between wheat and rye,
triticale (x Triticosecale Wittmack) is now grown extensively, particularly on dry and sandy soils in Poland and
Canada. Triticale proved to be tolerant to many diseases
(Skovmand et al., 1984), and high levels of aluminium
(Pinto-Camide et al., 1991). Agronomically, it is of
interest for both grain and forage production (Carnide
and Guedes-Pinto, 1991). Such hybrids can also be used
in breeding programmes by further crossing of the hybrids
and wheat, with the aim of transferring whole chromosome segments or groups of genes into a new wheat line
which carries useful genes from the alien species.
Hybrids between Hordeum (barley species), particularly
H. chilense, and wheat (Martin and Sanchez-Monge
Laguna, 1982), known as tritordeum (x Tritordeum
Ascherson et Graebner), are not yet widely grown, but
are currently under trial in the south of Spain and
elsewhere. They are being assessed for use as animal feed,
and tritordeum flour has the potential for use in bread
or biscuit making instead of wheat flour (Alvarez et al.,
1992). Although the yield of the amphiploid is substantially depressed over wheat (by 60-80%), tritordeum is
of considerable agricultural interest because of its high
protein content, earliness, drought and temperature tolerance (Cubero et al., 1986), and disease resistance
(Rubiales et al., 1993). These characters are combined
with desirable characteristics from its parents including
high chromosome stability, high fertility, full grains with
Key words: In situ hybridization, triticale, cytogenetics,
plant breeding, Hordeum chilense.
!
To whom correspondence should be addressed. Fax: +44 1603 451704. E-mail: [email protected]
© Oxford University Press 1996
584
Lima-Brito et al.
good seed morphology, and desirable ear form (Martin,
1988).
In the present experiments, the chromosomes of hybrids
between tritordeum and triticale have been analysed. Such
trigeneric hybrids are of potential value because they are
a source for the production of substitution or recombinant
lines which may produce lines with improved agronomic
performance. Although no such lines are widely grown as
yet, a few cultivated hexaploid triticales (genome designation normally AABBRR), include chromosomes originating from all three wheat genomes and fewer than 14 ryeorigin chromosomes (e.g. the variety Bacum lacks one rye
chromosome and has a D genome chromosome substituted; Neves et al., unpublished results). Use of multiple
hybrids enables the production of substitution lines which
may correct certain deficiencies in varieties. Furthermore,
they may give rise to lines with recombinant chromosomes,
which again may be useful in agriculture: the widely grown
wheats with a 1B-1R translocation chromosome are
an important example of the value of recombinants
(Lukaszewski, 1990; Carver and Rayburn, 1994).
Wide hybrids provide a useful scientific model for
examining nuclear organization (Heslop-Harrison and
Bennett, 1990). Because the various genomes contain
markedly different genes and repetitive DNA sequences,
their behaviour as independent entities can be studied in
detail (Schwarzacher et al., 1992a). The organization of
the nucleus may have implications for the control of gene
expression (including that of the nucleolar organizing
chromosomes) and meiotic recombination or stability.
In the present work, in situ hybridization was used to
examine the chromosome complements in trigeneric
hybrids. Interphase and prophase nuclei were then examined to see if any features of nuclear architecture and
non-random positioning of the genomes were evident.
Materials and methods
Plant material
Root tips were obtained from F, hybrids (Lima-Brito and
Guedes-Pinto, 1993), genomic constitution AABBRH ch (2n =
6.Y = 4 2 ) between 6.v triticale advanced line UTAD17/85
(AABBRR) (selected in the Department of Genetics and
Biotechnology, University of Tras-os-Montes and Alto Douro)
and 6,v tritordeum advanced line HT67 (AABBH ch H ch ) kindly
given by A. Martin, Spain.
Chromosome
preparation
For root tip preparations, seeds were germinated on moist filter
paper for 48 h at 25 °C and then kept at 4 °C for 24 h followed
by 28-29 h at 25 °C to synchronize cell divisions. The excised
root-tips were then transferred to ice water for 24 h at 0 °C to
accumulate metaphases before fixation in ethanol: acetic acid
(3:1). Spread preparation essentially followed the methods
described by Schwarzacher et al. (1994). Fixed root-tips were
partially digested with cellulase and pectinase before squashing
in 45% acetic acid. Cover slips were removed after freezing with
dry ice and the slides air-dried. Preparations were used
immediately.
Probe preparation and in situ hybridization
Total genomic DNA from Hordeum chilense and Secale cereale
cv. Petkus was mechanically sheared to 10-12 kb fragments
and labelled with digoxigenin-11-dUTP (Boehringer) by nick
translation for use as a probe for in situ hybridization. Total
genomic DNA from T. aestivum cv. Chinese Spring was
fragmented to pieces about 250 bp long by autoclaving and
used as blocking DNA (Heslop-Harrison et al., 1990). The
ribosomal rDNA sequence pTa71 contains a 9 kb EcoRI
fragment of rDNA isolated from wheat T. aestivum L. em.
Thell. (Gerlach and Bedbrook, 1979) recloned into pUC19 and
was provided by R.B. Flavell and M. O'Dell (JI Centre,
Norwich); it contains coding sequences for the 18S, 5.8S and
25S rRNA genes and the intergenic spacer sequences. The
rDNA sequence was labelled with TRITC (tetramethyl rhodamine isothiocyanate)-coupled dUTP (Amersham, Fluorored) by
nick translation. pHcKB6, a tandemly repeated DNA fragment
isolated from Hordeum chilense (Anamthawat-Jonsson and
Heslop-Harrison, 1993), was labelled with biotin-11-dUTP
(Sigma) by PCR. pSc200 is a clone isolated from Secale cereale
(Vershinin et al., 1995) and was labelled with biotin-11-dUTP
(Sigma) by PCR.
The in situ hybridization and probe detection protocols
followed Schwarzacher et al. (1994) with minor modifications.
The probe concentrations (ng/slide) used in the hybridization
mixture were: 75 ng of total genomic DNA from Hordeum
chilense or from rye, 100 ng of pHcKB6 and pSc200, 125 ng of
the rDNA clone and blocking DNA at 35 times the amount of
genomic probe DNA. The hybridization mixture also contained
50% formamide, 1 x SSC, 20% (w/v) dextran sulphate, 3.75%
salmon sperm DNA, and 0.25% (w/v) SDS (sodium dodecyl
sulphate). The hybridization mixture was denatured at 70 °C
for 10 min, chilled on ice for 5 min and 40 /u.1 was applied to
the preparation. After covering with a plastic coverslip, slides
were denatured at 70 °C for 5 min and hybridization was carried
out overnight at 37 °C in a humid chamber (Schwarzacher
et al., 1994), followed by stringent washes in 20% (v/v)
formamide in 0.1 x SSC at 42 °C for 10 min. Final stringency
was typically 80-85%.
For detection of sites of probe hybridization, sheep antidigoxigenin conjugated to fluorescein (F1TC; Boehringer) was
used for digoxigenin-labelled probes and streptavidin-CY3
(Sigma) for the biotin-labelled probes. Chromosomes were
counterstained with DAPI (4 /*gml~'; 4'-6' diamidino-2phenylindole). Slides were mounted in citifluor-glycerol and
analysed on an epi-fluorescence Leitz Aristoplan microscope
with appropriate filters. Photographs were taken on Fuji 400
colour print film and negatives digitized to photo CD. Images
were printed after overlaying and contrast optimization (applied
to the whole image only) using Adobe Photoshop.
Where required, preparations were reprobed and treated as
described by Heslop-Harrison et al. (1992). Briefly, the slides
were washed for 3 x 30 min in 4 x SSC in 0.1 Tween 20 and
then for 2 x 5 min in 2 x SSC to remove the mountant. Slides
were then dehydrated through an alcohol series and air-dried.
For reprobing, the preparations on the slides were covered with
hybridization mixture containing the labelled probe, hybridized,
and detected as described above.
Results
Chromosome discrimination at metaphase
Figure 1 shows a metaphase of the F! hybrid tritordeum
x triticale with the chromosome constitution AABBRH ch
Triticale x tritordeum hybrids
{2n = 6x=42). The probe pHcKB6 gives a characteristic
hybridization pattern on all seven H. chilense origin
chromosomes (Fig. IB). pHcKB6 shows some minor
hybridization signals on wheat-origin chromosomes, but
the weak red fluorescence seen on the rye chromosomes
is largely cross-excitation of the rye genomic DNA probe
(known because of theoretical considerations and the
identical band pattern seen in yellow under blue light
excitation). The chromosomes originating from rye are
strongly and relatively specifically probed with rye genomic DNA probe, yellow (Fig. 1C).
After reprobing and superposition of the image
(Fig. 1C, red), the major sites of 18S-25S rDNA were
visible on chromosomes IB, 6B (both subterminal on
wheat chromosomes), 1R (rye) and 5Hch and 6Hch (H.
chilense).
Figure 5 shows the karyotype and banding pattern of
H. chilense chromosomes probed with pHcKB6 based on
measurements of eight probed cells. Each arm and each
chromosome shows a characteristic pattern.
After hybridization with H. chilense genomic DNA,
the seven small H. chilense-ohgin chromosomes give
uniform bright hybridization (Fig. 2) but the seven large
rye-origin chromosomes also fluoresce green. The image
also shows hybridization sites of the repetitive probe
pSc200 giving a characteristic hybridization pattern in
the telomeric and subtelomeric regions of rye-origin
chromosomes.
Nuclear organization
When prophase nuclei were labelled with two probes to
discriminate the wheat, rye and H. chilense-origin chromosomes, the chromosomes originating from the three genera
were not found in intermixed domains (Fig. 3). In typical
prophase nuclei, of which two are shown, the chromosomes from H. chilense and from rye were each clustered
in one to three domains. The clustering was clear in both
early and mid-prophases. No clear separation of the 42
chromosomes into groups was evident at metaphase
(Fig. 1), although no statistical tests were applied nor
were three dimensional reconstructions made.
At interphase, non-intermixed domains of H. chilense
(Fig. 4B), rye (Fig. 4C) and wheat chromosomes were
visible and entirely consistent with the non-intermixed
appearance of the prophase nuclei, although the axes of
the chromosomes could not be distinguished (Fig. 4).
585
here. Surprisingly, there was a relatively high level of
cross hybridization between the H. chilense and rye chromosomes, such that translocations would not be easily
detectable when H. chilense DNA was used as a probe.
This indicates that at least some of the highly repeated,
dispersed sequences in H. chilense are also present in rye,
although in reduced copy number. Probing with rye DNA
specifically detected rye-origin chromosome and any
translocation would be detected clearly. Fluorescent in
situ hybridization of the repetitive DNA sequence
pHcKB6, isolated from H. chilense, to triticale x
tritordeum Fl hybrids revealed that multiple sites of
hybridization were present on all seven H. chilense-origin
chromosomes although paracentromeric regions often
had less hybridization signal, and a banded karyotype of
H. chilense could be made (Fig. 5). Only two major sites
of rDNA hybridization were detected on H. chilenseorigin chromosomes (5Hch and 6Hch) confirming the
results of Cabrera and Martin (1991) who found four
active NORs in the H. chilense chromosome complement
by using silver-banding.
Cabrera et al. (1995), using in situ hybridization of the
repetitive DNA sequence pAsl isolated from T. tauchii
by Rayburn and Gill (1986) to H. chilense found multiple
sites of hybridization on the seven Hch genome chromosomes that made possible the identification of all seven
pairs of H. chilense chromosomes. The probe pHcKB6
revealed a similar pattern of hybridization sites although
more sites were seen in most chromosomes. While the
difference might be explained by the large amount of
polymorphism present in H. chilense (Linde-Laursen
et al., 1989), there could also be differences in target
sequences or in hybridization stringency. The sequence of
pAsl is not known.
Anamthawat-Jonsson and Heslop-Harrison (1993)
used Southern hybridization to examine the presence of
pHcKB6, isolated from H. chilense, in wheat, rye and
Aegilops speltoides. Little hybridization was detected to
rye and Ae. speltoides, although some hybridization to
limiting mobility DNA and a 340 bp Dral fragment from
wheat was detected. Presumably, this fragment arises
from the wheat D genome and is homologous to the
external Dral fragment of pAs 1 in the restriction map of
Rayburn and Gill (1986, their Fig.l), but the higher
stringency of hybridization increases the specificity of the
pHcKB6 probe.
Nuclear architecture
Discussion
As expected from results with interspecific hybrids and
derivatives, genomic in situ hybridization from both
rye (Heslop-Harrison et al., 1990) and H. chilense
(Schwarzacher et al., 19926) was able to identify chromosomes of the two species in the complex hybrids analysed
Extensive reconstruction work has shown that there is
good correlation between results seen in spread preparations and reconstructions (Leitch et al., 1991).
Nevertheless, extreme care must be taken in the interpretation of structure in three-dimensional nuclei from twodimensional nuclei in spread chromosome preparations.
586 Lima-Brito et al.
Fig. 1. Root tip metaphase of the F, hybrid triticale x tritordeum after staining and in situ hybridization. (A) The 42 chromosomes stained with
DAPI; (B) The probe pHcKB6, isolated from H. chilense, gives characteristic banding patterns along the length of the H. chilense-origjn
chromosomes; numbers indicate their homoeologous groups. (C) Genomic rye DNA probe labels the seven chromosomes of rye origin in green.
Under the conditions used, major heterochromatic bands appear brighter green and enable identification of individual chromosomes. Major rDNA
sites (arrows) are seen by red fluorescence.
Fig. 2. Root tip metaphase of the F, triticale x tritordeum hybrid. After in situ hybridization with H. chilense genomic DNA probe, seven large and
seven small chromosomes show green fluorescence signal. After overlaying the image showing hybridization sites of the repetitive probe pSc200 a
characteristic hybridization pattern in the telomeric and subtelomeric regions of rye-origin chromosomes is revealed.
Fig. 3. Root tip prophases of the triticale x tritordeum F, hybrid digitized and processed to make overlay after staining with DAPI (blue) and
simultaneous in situ hybridization with total genomic rye DNA (green) and pHcKB6 (red).
Fig. 4. Root tip interphase of the triticale x tritordeum F, hybrid stained with DAPI (A; blue) and probed with pHcK.B6 (B; red) and total genomic
rye DNA (C; green).
Triticale x tritordeum hybrids 587
References
X
A.R.
1.20
1.36
1.29
1.10
X
2.00
1.15
1.67
Fig. 5. Karyotype of H. chilense probed with pHcKB6. Chromosome
numbers indicate homoeologous groups. A.R. = arm ratio.
The results here indicate (Figs 3, 4) that chromosomes
from two different genomes tend not to intermix at
interphase, but individual chromosomes and groups of
chromosomes from each genome remain in discrete
domains. This contrasts with the situation in diploid Fj
hybrids between Triticeae species where complete separation of the genomes, with one either surrounding or lying
next to the other, seems to be frequent (Leitch et al.,
1991; Heslop-Harrison and Bennett, 1990). In most
Triticeae hybrids, dominance of activity of particular
NOR chromosomes is seen (Lacadena et al., 1988), and
this pattern reflects the position of the active nucleolar
chromosomes in the nucleus. It will be interesting to
examine such relationships of gene expression to position
in more detail in complex hybrids such as those examined here.
Conclusions
Multiple hybridization of combinations of repetitive DNA
clones and genomic DNA is a valuable method to identify
chromosomes in hybrids involving multiple genera. Both
in the specific case using the probes and derivatives of
the lines described here and, in general, the ability to
identify individual chromosomes in hybrids using these
methods is likely to be useful for linking agriculturally
desirable traits to chromosomes and for following known
chromosomes or recombinant chromosome segments
through plant breeding and hybridization programmes.
Acknowledgements
This work was supported by Junta Nacional de Investigacao
Cientifica e Tecnologica (JNICT) grant BD 2661/93-IE and we
are grateful to the British Council for support through the
Treaty of Windsor programme.
Alvarez J, Ballesteros J, Silero JA, Martin LM. 1992.
Tritordeum: a new crop of potential importance in food
industry. Hereditas 116, 193-7.
Anamthawat-Jonsson K, Heslop-Harrison JS. 1993. Isolation
and characterization of genome-specific sequences in Triticeae
species. Molecular and General Genetics 240, 151-8.
Cabrera A, Friebe B, Jiang J, Gill BS. 1995. Characterization
of Hordeum chilense chromosomes by C-banding and in situ
hybridization using highly repeated DNA probes. Genome
38, 435-42.
Cabrera A, Martin A. 1991. Cytology of the amphiploid
Hordeum chilense (4x) x Aegilops squarrosa (4.v). Theoretical
and Applied Genetics 81, 758-60.
Carnide V, Guedes-Pinto H. 1991. Forage aptitude of primary
8x triticale compared with rye and wheat progenitors.
Proceedings of the 2nd International Triticale Symposium,
Mexico. D.F: CIMMYT, 536-41.
Carver BF, Rayburn AL. 1994. Comparison of related wheat
stocks possessing IB or 1RS.1BL chromosomes: agronomic
performance. Crop Science 34, 1505-10.
Cubero JI, Martin A, Millan T, Gomez-Cabrera A, de Haro A.
1986. Tritordeum: a new alloploid of potential importance as
a protein source crop. Crop Science 26, 1186-90.
Gerlach WL, Bedbrook JR. 1979. Cloning and characterization
of ribosomal RNA genes from wheat and barley. Nucleic
Acids Research 7, 1869-85.
Heslop-Harrison JS, Bennett MD. 1990. Nuclear architecture in
plants. Trends in Genetics 6, 401-5.
Heslop-Harrison JS, Harrison G, Leitch IJ. 1992. Reprobing of
DNA:DNA in situ hybridization preparations. Trends in
Genetics 8, 372-3.
Heslop-Harrison JS, Leitch AR, Schwarzacher T, AnamthawatJonsson K. 1990. Detection and characterization of 1B/1R
translocations in hexaploid wheat. Heredity 65, 385-92.
Lacadena JR, Cermeno MC, Orellana J, Santos JL. 1988.
Nucleolar competition in Triticeae. Kew Conference HI,
151-2.
Leitch AR, Schwarzacher T, Mosgoller W, Bennett MD, HeslopHarrison JS. 1991. Parental genomes are separated throughout the cell cycle in a plant hybrid. Chromosoma 101, 206-13.
Lima-Brito J, Guedes-Pinto H. 1993. Tritordeum x triticale
crossability studies in diallelic crosses. In: Abstract Book of
XXIX
Jornadas Luso-Espanholas de Genetica. Faro:
Portugal, 126.
Linde-Laursen IB, von Bothmer R, Jacobsen N. 1989. Giemsa
C-banded karyotypes of South American Hordeum (Poaceae).
Hereditas 110, 289-305.
Lukaszewski AJ. 1990. Frequency of 1RS.1AL and 1RS.1BL
translocations in United States wheats. Crop Science 30,
1151-3.
Martin A. 1988. Tritordeum: the first ten years. Rachis 7, 12-15.
Martin A, Sanchez-Monge Laguna E. 1982. Cytology and
morphology of the amphiploid Hordeum chilense x Triticum
turgidum conv. durum. Euphytica 31, 261-7.
Pinto-Carnide O, Guedes-Pinto H, Vaz E. 1991. Aluminium
tolerance behaviour of F 2 6JC triticales and their progenitors.
Proceedings of the 2nd International Triticale Symposium,
Mexico. D.F: CIMMYT, 268-73.
Rayburn AL, Gill BS. 1986. Isolation of a D-genome-specific
repeated DNA sequence from Aegilops squarrosa. Plant
Molecular Biology Reporter 4, 102-9.
Rubiales O, Niks RE, Dekens RG, Martin A. 1993. Histology
of the infection of tritordeum and its parents by cereal brown
rusts. Plant Pathology 42, 93-9.
588 Lima-Brito et al.
Schwarzacher T, Heslop-Harrison JS, Anamthawat-Jonsson K,
Finch RA, Bennett MD. 1992a. Parental genome separation
in reconstructions of somatic and premeiotic metaphases of
Hordeum vulgare x H. bulbosum. Journal of Cell Science
101, 13-24.
Schwarzacher T, Anamthawat-Jonsson K, Harrison GE, Islam
AKMR, Jia JZ, King IP, Leitch AR, Miller TE, Reader SM,
Rogers WJ, Shi M, Heslop-Harrison JS. 19926. Genomic in
situ hybridization to identify alien chromosomes and chromosome segments in wheat. Theoretical and Applied Genetics
84, 778-86.
Schwarzacher T, Leitch AR, Heslop-Harrison JS. 1994.
DNA:DNA in situ hybridization - methods for light microscopy. In: Harris N, Oparka KJ, eds. Plant cell biology: a
practical approach. Oxford: IRL/Oxford University Press,
127-55.
Skovmand BP, Fox PN, VillaReal LR. 1984. Triticale in
commercial agricultural: progress and promise. Advances in
Agronomy 37, 1-45.
Vershinin A, Schwarzacher T, Heslop-Harrison JS. 1995. The
large-scale genomic organization of repetitive DNA families
at the telomeres of rye chromosomes. The Plant Cell
7, 1823-33.