Tools and methodologies for cytogenetic studies of plant chromosomes
A brief overview is presented in advances in cytogenetic methodology and development of aneuploid stocks since the 1920s.The methodologies range from first reports of chromosome numbers of major organisms, the development of chromosome karyotypes, then aneuploid stocks in the major crop plants. Mole...
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irk-123456789-80952010-05-05T12:01:12Z Tools and methodologies for cytogenetic studies of plant chromosomes Fedak, G. Nam-Soo Kim A brief overview is presented in advances in cytogenetic methodology and development of aneuploid stocks since the 1920s.The methodologies range from first reports of chromosome numbers of major organisms, the development of chromosome karyotypes, then aneuploid stocks in the major crop plants. Molecular inputs included chromosome banding techniques, molecular marker maps and in situ hybridization methodologies. All of the new techniques greatly increased the degree of resolution obtained from cytogenetic studies. Представлен краткий обзор развития методов цитогенетики и анеуплоидых stocks с 20-х годов. Методология варьирует от первых сообщений о хромосомных наборах большинства организмов, развития хромосомных кариотипов, а затем анеуплоидых stocks для большинства возделываемых растений. Вклад молекулярной биологии включает технологии исчерчености хромосом, карты молекулярных маркеров и методы гибридизации in situ. Все эти новые методики значительно увеличили точность цитогенетических исследований. 2008 Article Tools and methodologies for cytogenetic studies of plant chromosomes / G. Fedak, Nam-Soo Kim // Цитология и генетика. — 2008. — Т. 42, № 3. — С. 64-80. — Бібліогр.: с. 73-80. — англ. 0564-3783 http://dspace.nbuv.gov.ua/handle/123456789/8095 en Інститут клітинної біології та генетичної інженерії НАН України |
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A brief overview is presented in advances in cytogenetic methodology and development of aneuploid stocks since the 1920s.The methodologies range from first reports of chromosome numbers of major organisms, the development of chromosome karyotypes, then aneuploid stocks in the major crop plants. Molecular inputs included chromosome banding techniques, molecular marker maps and in situ hybridization methodologies. All of the new techniques greatly increased the degree of resolution obtained from cytogenetic studies. |
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Fedak, G. Nam-Soo Kim |
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Fedak, G. Nam-Soo Kim Tools and methodologies for cytogenetic studies of plant chromosomes |
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Fedak, G. Nam-Soo Kim |
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Fedak, G. |
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Tools and methodologies for cytogenetic studies of plant chromosomes |
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Tools and methodologies for cytogenetic studies of plant chromosomes |
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Tools and methodologies for cytogenetic studies of plant chromosomes |
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Tools and methodologies for cytogenetic studies of plant chromosomes |
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Tools and methodologies for cytogenetic studies of plant chromosomes |
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tools and methodologies for cytogenetic studies of plant chromosomes |
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Інститут клітинної біології та генетичної інженерії НАН України |
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2008 |
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Tools and methodologies for cytogenetic studies of plant chromosomes / G. Fedak, Nam-Soo Kim // Цитология и генетика. — 2008. — Т. 42, № 3. — С. 64-80. — Бібліогр.: с. 73-80. — англ. |
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2025-07-02T10:49:04Z |
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64 ISSN 0564–3783. Цитология и генетика. 2008. № 3
A brief overview is presented in advances in cytogenetic
methodology and development of aneuploid stocks since the
1920s.The methodologies range from first reports of chromo"
some numbers of major organisms, the development of chro"
mosome karyotypes, then aneuploid stocks in the major crop
plants. Molecular inputs included chromosome banding tech"
niques, molecular marker maps and in situ hybridization
methodologies. All of the new techniques greatly increased the
degree of resolution obtained from cytogenetic studies.
Historical perspectives
Prior to the 1920s, cytological studies were car�
ried out on biological tissues that were embedded in
paraffin, sectioned, and stained (Wilson, 1925;
Darlington, 1937). The methods that were in vogue
were not sufficiently refined to allow for the detec�
tion of such gross morphological features as cen�
tromeres, secondary constrictions, and satellites of
chromosomes. They merely facilitated the determi�
nation of chromosome numbers and permitted
detection of approximate size differences among the
chromosomes in somatic and meiotic cells of many
eukaryotic species. For example, in the genus
Triticum, Sakamura (1918) showed that the somatic
cells of the species monococcum, turgidum, and aes"
tivum possessed 14, 28, and 42 similar�sized chro�
mosomes, respectively.
During the 1920s and 1930s innovations were
introduced which facilitated cytological and karyo�
typic analyses. In 1921, Belling described a tech�
nique for studying meiosis in plant species that
involved the squashing of anthers. This method
permitted the separation of PMCs and facilitated
the spreading of their chromosomes. In 1929,
Kagawa, working with Triticum and Aegilops
species, demonstrated that treatment with chloral
hydrate before fixing and staining the cells short�
ened the chromosomes. This made it easier to sep�
arate them and study their gross morphological
features: centromeres, secondary constrictions,
and satellites. Pretreatment with other agents such
as alpha bromonaphthalene (Schmuck and Kostoff,
1935), colchicine (O’Mara, 1939), paradichloro�
benzene (Meyers, 1945) and cold water (Hill and
Myers, 1945) also permitted identification of these
chromosomal substructures. By the early 1940s the
squashing technique, concomitant with appropri�
ate modifications and pre�treatment, completely
replaced the method of microtome sectioning of
tissues in chromosome studies using somatic and
meiotic tissues of most species (Hillary, 1938;
Aase, 1935; O’Mara, 1939).
The squash technique, with appropriate modi�
fications, was also used successfully in chromo�
some studies of insects, amphibians, and other
animals (White, 1954), excluding mammals (Hsu,
1979). Mammalian cytology had to await the
innovations of hypotonic solutions (Hsu, 1952), in
vitro culturing of tissues and cells (Hsu and
Pomerat, 1953), and their colchicine pretreatment
(Hsu and Pomerat, 1953) for obtaining good
spreads of somatic chromosome complements.© GEORGE FEDAK, NAM�SOO KIM, 2008
GEORGE FEDAK1, NAM�SOO KIM2
1Eastern Cereals and Oilseeds Research Centre
Agriculture and Agri�Food Canada
960 Carling Avenue
Ottawa, Notario, K1A 0C6
Canada
2 Department of Molecular Biosciences
Kangwon National University
Chunchon, Korea 200–701
TOOLS AND METHODOLOGIES
FOR CYTOGENETIC STUDIES
OF PLANT CHROMOSOMES
65
Tools and methodologies for cytogenetic studies of plant chromosomes
ІSSN 0564–3783. Цитология и генетика. 2008. № 3
Using the innovative procedures the chromosome
number in man was first correctly determined to be
2n = 46 by both Tjio and Levan, and Ford and
Hamerton in 1956. In the ensuing years this tech�
nique, combined with the use of phytohemagglu�
tinin (Nowell, 1960) to stimulate cell divisions,
was applied to karyotyping euploids, aneuploids
and individuals with chromosomal abnormalities
in numerous mammalian species (see Hsu and
Benirschke 1967–1977). The various techniques
that have been used to this day for karyotyping
hundreds of species using gross chromosomal
morphological features are detailed in La Cour
(1947), Darlington and La Cour (1960), Sharma
and Sharma (1965), and Haskell and Wills (1968).
Standard somatic karyotypes in plant species
In plant species, such as maize (Rhoades and
McClintock, 1935), tomato (Rick and Barton,
1954; Rick et al., 1964) and rice (Chu, 1967;
Kurata and Omura, 1978) the individual chromo�
somes (and therefore trisomics) could not be iden�
tified in somatic cells using standard straining pro�
cedures because the chromosomes were either too
small and/or similar in morphology. In most of the
plant species, however, at least a few of the chro�
mosomes and trisomics could be identified in stan�
dard somatic karyotypes. For example, in barley
(Tsuchiya, 1960) and Petunia axiillaris (Reddi and
Padmaja, 1982), three of the chromosome pairs
and trisomics could be identified using standard
procedures. In beets (Romagosa et al., 1986) eight
of nine chromosomes and trisomics could be iden�
tified in the standard fashion and in A. strigosa,
Rajhathy (1975) was able to distinguish all chro�
mosomes and identify all trisomics from standard
somatic karyotypes.
Although numerous karyotypic studies have
been carried out since 1939 in several species of
the genus Triticum, only the species T. monococ"
cum (AA), T. turgidum (AABB), and T. aestivum
(AABBDD) will be reviewed here. Studies by
Camara (1943), Coucoli and Skorda (1966),
Giorgi and Bozzini (1969b) and Kerby and
Kuspira (1988) have shown that there are 14 simi�
lar chromosomes in the diploid complement of T.
monococcum; one ST pair, two M pairs, and four
SM pairs. Depending on the accession line stud�
ied, either one or two chromosome pairs were
found to possess satellites (Camara, 1943; Riley et
al., 1958; Coucoli and Skorda, 1966). The kary�
otype of T. turgidum consists of two SAT pairs, two
ST pairs, seven SM pairs, and three M pairs of
chromosomes (Giorgi and Bozzini, 1969a; Kerby
and Kuspira, 1988). Since the A genome has the
chromosome constitution given above, the kary�
otype of the B genome in T. turgidum must consist
of two SAT pairs, one ST pair, three SM pairs, and
one M pair of homologues. Two of these chromo�
some pairs possess large satellites (Pathak, 1940;
Riley et al., 1958) which belong to the B genome
(Okamoto, 1957). The satellites in the A genome
in T. turgidum are suppressed (Riley et al., 1958).
At most, four to six of the chromosomes in the
somatic complement of this species can be distin�
guished using standard procedures.
Depending on the genotype studied, either one
(Sears, 1954), two (Morrison, 1953), three (Pathak,
1940, Camara, 1943) or four (Kagawa, 1929,
Schulz�Schaeffer and Haun, 1961) satellited chro�
mosomes are observed in T. aestivum. These be�
long to the B and D genome (Sears, 1954; Schulz�
Schaeffer and Haun, 1961). Camara (1943), Mor�
rison (1953), Sears (1954, 1958), Schulz�Schaeffer
and Haun (1961), and Gill (1987) have shown that
the chromosomes in the somatic complement of
bread wheat are similar in size. For example, Gill
(1987) reported that they range in length from 8.4
um for chromosome ID to 13.8 um for chromo�
some 3D. The latter observations also show that (i)
except for the chromosome 4A pair, all other pairs
in homoeologous groups 1, 4 and 5 are highly het�
erobrachial (ST), (ii) except for the chomosome
7B pair, all other pairs in homoeologous groups 6
and 7 are M, and (iii) chromosome pairs in
homoeologous groups 2 and 3 as well as chromo�
some pairs 4A and 7B are SM. Even in the best
somatic metaphase spreads, only a limited number
of chromosomes and chromosome pairs can be
distinguished by standard methods.
Chromosome identification during meiosis
Standard staining procedures render nucleoli to
be clearly visible at pachytene stage and permit the
detection of chromosomes that carry NORs.
Moreover, in corn (Rhoades and McClintock,
1935), tomato (Rick and Barton, 1954), and rice
(Khush et al., 1984) it is possible to identify each
univalent, bivalent, and multivalent association on
the basis of its length and chromomere pattern
66
George Fedak, Nam3Soo Kim
during pachytene. Thus a chromosome in tripli�
cate in these species is easily identified by exami�
nation of the trivalent configuration at pachytene
using standard staining techniques.
Banding techniques. In almost all species the
usefulness of standard staining procedures, howev�
er, has been limited. Although they have facilitated
the ascertainment of chromosome numbers and
gross morphological features of chromosomes, they
have not permitted an accurate and unequivocal
identification of all the chromosomes, and there�
fore the aneuploids, of a species. An exhaustive
analysis of the karyotype requires the use of stain�
ing procedures that can reveal each chromosome as
a specific, unique, and constant pattern of alternat�
ing dark and light banding regions, topologically
equivalent to the bands in the polytene chromo�
somes in salivary gland cells of D. melanogaster.
Darlington and La Cour (1940) demonstrated
that with cold treatment of somatic cells of
Trillium erectum some regions of chromosomes
revealed unique patterns by appearing thinner and
less intensely stained than the rest of the chromo�
somes. The utilization of fluorescent and other
dyes together with various modifications in pre�
treatment of cytological material in the late 1960s
heralded a new era of cytogenetics. New and reli�
able staining procedures were introduced, each of
which was capable of revealing a unique banding
pattern of the chromosomes of a given species. By
1972 the application of one or another of five
major banding techniques (Q, G, R, C, and N) for
the purpose of karyotypic analysis came into
vogue. These have led to a more precise cytogenet�
ic and phylogenetic analysis of various eukaryotes.
QHbanding
Caspersson and his colleagues in 1968 were first
to demonstrate that fluorescent dyes such as
quinacrine and quinacrine mustard bind preferen�
tially to certain regions of normal mitotic chromo�
somes of Cricetulus griseus, V. faba and T. erectum.
As a result, unique patterns of brightly fluorescent
regions alternating with non�fluorescent (dark)
regions were produced in each chromosome.
Weisblum and de Haseth (1972) and Burkholder
(1988) have shown that fluorescent dyes interact
with AT base pairs and those regions of DNA that
are sufficiently AT�rich (70–100 %) fluoresce and
appear as bright bands (Q bands). Q�banding per�
mits an identification of all the chromosomes and
their homologues in most species. For example, in
man all 23 pairs of homologous chromosomes can
be distinguished on the basis of their Q�banding pat�
terns (Caspersson et al., 1971). In Scilla sibirica all
eight chromosome pairs can be identified (Casper�
son et al., 1969). Q�banding does not require any
pretreatment and is the simplest of all the banding
methods. Compared to other banding techniques, it
has several disadvantages; the fluorescent bands are
not permanent; the technique requires the use of
ultraviolet light, and does not stain the ends of chro�
mosomes. As a consequence Q�banding has been
used to a limited extent, and since the late 1970s
(Pinkel et al., 1988) has been largely replaced by
other banding methods. In plants, Q�banding stud�
ies have been limited to a few in Trillium, Scilla,
Allium, Crepis, Lilium, Secale, and Vicia (Caspersson
et al., 1969; Vosa and Marchi, 1972a; Kongsuwan
and Smyth, 1977; Schweizer, 1980; Rowland, 1981).
GHbanding
In 1971, Drets and Shaw, Patil et al., Seabright,
and Sumner et al. independently developed a pro�
tocol for animal species whereby each chromo�
some segment and chromosome revealed a unique
pattern of bands. Each of the protocols, by using a
variety of treatments before fixing chromosomes
and staining with Giemsa, yielded a banding pat�
tern in normal mitotic chromosomes that was sim�
ilar to the one revealed by the Q�banding tech�
nique. The dark regions are the topological
equivalents to Q�bands and are called G�bands
whereas the light regions are equivalent to the
nonfluorescent dark ones revealed with the use of
fluorescent dyes (Drets and Shaw, 1971; Dutrillaux
and Lejeune, 1975). Application of G�banding
methods to prophase and prometaphase chromo�
somes in animals revealed a larger number of
bands than at metaphase which permited more
precise karyotyping and cytogenetic analysis
(Yunis, 1981; Iannuzzi, 1990).
The basis for G�banding is currently unknown.
One plausible explanation is that of Comings (1978)
who postulated that prophase and metaphase chro�
mosomes contain a basic chromomeric structure
that can be enhanced. This enhancement occurs by
inducing some rearrangement of the fibers away
from the light bands toward the G�bands, possibly
some extraction of light�band DNA with denatured
ISSN 0564–3783. Цитология и генетика. 2008. № 3
67
Tools and methodologies for cytogenetic studies of plant chromosomes
nonhistone proteins, followed by the marked
enhancement of this pattern through the ability of
thiazin dyes in Giemsa to side stack on available
DNA. Sumner (1982) and Burkholder (1988) have
proposed alternate mechanisms.
Although the technique has been attempted in
many plant species, G�bands have been generated
in the chromosomes of only a few species: Tulipa
gesneriana (Filion and Blakey, 1979), Pinus
resinosa (Drewry, 1982), and Vicia hajsatana
(Wang and Kao, 1988). The failure to produce G�
bands in the chromosomes of most plant species,
including those in the Triticeae, has been attrib�
uted to the increased condensation of the plant
chromosomes (Greilhuber, 1977; Drewry, 1982).
Anderson, et al. (1982), however, failed to show
consistent differences in the degree of compaction,
based on measurements of lengths and volumes of
chromosomes from several plant and animal
species. Wang and Kao (1988) demonstrated that
improper pretreatment of plant chromosomes
alters the organization of their chemical con�
stituents and renders them unresponsive to the G"
banding procedure.
Reverse (R)Hbanding
This banding technique was developed by
Dutrillaux and Lejeune in 1971. Mild denaturation
by heat and subsequent staining of chromosomes
with Giemsa or a fluorochrome dye revealed a
banding pattern that is the reverse of the patterns
produced by the G�and Q�banding methods
(Bobrow et al., 1972; Comings, 1973; Dutrillaux et
al., 1973). Specifically, if the chromosomes are
stained with Giemsa, the dark bands (R bands)
produced with this technique are equivalent to the
light bands produced by the G�banding technique
and vice�versa (Dutrillaux and Lejeune, 1971,
1975). If a flurochrome dye such as acridine orange
or olivomycin is used, fluorescent R�banding is the
reverse of Q�banding in that the R�bands fluoresce
bright green and the non�R�bands show a faint red
color (Schweizer 1976; Lin et al. 1980; Schmid and
Guttenback 1988). R�banding is particularly useful
in the detection of structural rearrangements
involving ends of chromosomes in that it stains
telomeres as T�bands (Dutrillaux et al. 1973).
R�bands have been detected in only a few plant
species e.g., S. sibirica, V. fava, Allium spp., none
of which belong to the Tririceae tribe (Schweizer,
1980; DeumIing and Greilhuber, 1982; Loidl,
1983). Moreover, since the R�bands in these
species are few in number and faint in expression,
they have not been used for karyotyping and cyto�
genetic studies.
R�bands can be produced by GC�specific fluo�
rochromes (Schweizer, 1976; Van de Sande et al.,
1977; Holmquist et al., 1982), although the mech�
anism of R�banding is unknown (Burkholder,
1988). The mechanism proposed by Comings
(1978) may also explain R�banding if the DNA
and proteins in the G�and R�bands are selectively
denatured under different conditions of pH, salt
concentrations, and temperature.
CHbanding
Pardue and Gall (1970) and Jones (1970) inde�
pendently demonstrated a procedure which with
stringent treatment of chromosomes prior to fixa�
tion and staining with Giemsa, stained only the
regions of constitutive heterochromatin in chro�
mosomes of Mus musculus. The regions, now
referred to as C�bands, were observed to be proxi�
mal to the centromeres of all the chromosomes in
this species and have since been demonstrated in
chromosomes of the guinea pig (Yasmineh and
Yunis, 1975), Drosophila spp. (Gall and Atherton,
1974; Brutlag et at., 1977), Rattus rattus (Yosida
and Sagai, 1975) and many other animal species.
Constitutive heterochromatin usually appears as
satellite�DNA when nuclear chromosomal DNA is
fragmented and centrifuged (Kit 1961). It consists
of short, highly repeated base pair sequences in
tandem (Southern 1970; Corneo et al. 1970; Gall
and Atherton, 1974; Brutlag et al., 1977) in one or
more regions of all or more chromosomes in most
species. Arrighi and Hsu (1971) showed that C�
bands are located next to the centromeres of each
chromosome, next to the secondary constrictions
of chromosomes 1,9, and 16 as well as the satellites
of acrocentric chromosomes in man. With few
exceptions, constitutive heterochromatin in ani�
mal species reveals a consistent pattern of distribu�
tion. Therefore, C�banding in animal species does
not correspond to a banding pattern in a strict
sense. It’s application in these species is limited
because it does not allow precise recognition of
individual chromosomes.
Several lines of evidence indicate that the pro�
duction of C�banding is due to the extraction of
ІSSN 0564–3783. Цитология и генетика. 2008. № 3
68
George Fedak, Nam3Soo Kim
non�C�band DNA and denaturation of proteins in
these regions. The DNA in constitutive hete�
rochromatin is resistant to extraction, remains
within the chromosomes and is therefore stainable
by Giemsa (Pathak and Arrighi, 1973; Dille et al.,
1987; Burkholder, 1988).
Since the initial studies in V. faba by Vosa and
Marchi in 1972, chromosomes of many species of
Aegilops, Agropyron, Elymus, Hordeum, Secale, and
Triticum (Gill and Kimber, 1974a and b; Linde�
Laursen, 1975; Vosa, 1976; Gerlach and Peacock,
1980; Singh and Tsuchiya, 1981b; Seal, 1982; Teoh
and Hutchinson, 1983; Endo, 1986; Morris and
Gill, 1987) and other plant species (Linde�Laursen
et al., 1980; Loidl, 1983) have revealed C�bands.
These studies show that there is a fundamental dif�
ference in the distribution of constitutive hete�
rochromatin within chromosomes of animals and
plants. C�bands in the chromosomes of plants can
be located at various sites including the regions they
characteristically occupy in the chromosomes of
animals. Moreover, in most of the plant species
e.g., Allium carinatum (Loidl, I983), Hordem spp.
(Linde�Laursen et al., 1980) and Agropyron elonga"
tum (Endo et al., 1984a), some of the chromosomes
do not reveal C�bands next to their centromeres.
Thus, in many plant species a unique C�banding
pattern occurs in each arm of each chromosome in
the genome. This allows individual chromosomes
in the somatic cells to be identified on the basis of
their patterns (loc. cit.).
Gill and Kimber (1974a) published the first
report on C�banding patterns of chromosomes of
T. aestivum. Despite the efforts of many investiga�
tors in the interim, Endo (1986), using an
improved C�banding technique, was able to
unequivocally identify all 21 chromosomes in the
genome of cvs. Chinese Spring and Norin 61 of T.
aestivum. These banding patterns are currently
accepted as standard patterns for the chromo�
somes of common wheat (Gill, 1987; Gill et al.,
1988). Ferrer et al. (1984) applied the C�banding
protocol to the study of chromosomes in meio�
cytes and clearly identified nine of the 21 chromo�
some pairs.
Except for the work of Simeone et al. (1988),
the efforts of other investigators (Zurabishibili et
al., 1978; Seal, 1982; Lukaszewski and Gustafson,
1983) to identify the A and B genomes of T.
turgidum on the basis of their C"banding patterns
have been inconsistent. Simeone et al. (1988)
reported C�banding patterns for chromosomes of
T. turgidum that were equivalent to those of their
homologues in the A and B genomes of T. aestivum
and therefore permitted their unequivocal identifi�
cation. Shang et al. (1989), used the HKG (HCI�
KOH�Giemsa) method to produce banding pat�
terns in chromosomes of T. turgidum that were in
part similar to their C�banding patterns, thus
allowing identification of some of the chromo�
somes.
C�banding of the chromosomes of T. monococ"
cum has been reported by both Gill and Kimber
(1974a) and Kuz’menko et al. (1987). The banding
patterns of the chromosomes in these two investi�
gations were partially dissimilar. Moreover, the C�
banding patterns of some of the chromosomes in
these publications were different from those for the
A�genome chromosomes in T. aestivum, thus pre�
cluding their identification. Using the HKG
method, Shang et al. (1988 and 1989) reported
banding patterns for chromosomes of T. monococ"
cum, partially, resembled their C�banding patterns.
Although the banding patterns revealed by the
HKG method rendered some of the chromosomes
distinguishable from the others, they did not allow
for their unequivocal identification.
C�banding has been used in the identification of
aneuploids (Linde�Laursen, 1978b, 1982; Zeller et
al., 1987), translocations and other structural
rearrangements (Gill and Kimber, 1977; Lukas�
zewski and Gustafson, 1983; Lapitan et al., 1984)
and the precise physical mapping of genes (Kota
and Dvorak, 1986; Jampates and Dvorak, 1986).
C�banding analysis of durum"timopheevi and
durum"speltoides hybrids by Chen and Gill (1983)
has supported Dvorak’s suggestion (1983) that
chromosomes 4A and 4B should be reassigned to
the B and A genomes, respectively. Moreover, C�
banding has clarified and further substantiated
phylogenetic conclusions based on chromosome
pairing in interspecific and intergeneric hybrids
(Gill and Kimber, 1974a; Hutchinson and Miller,
1982; Chen and Gill, 1983; Morris and Gill, 1987).
NHbanding
In 1973 Matsui and Sasaki developed a tech�
nique they called N�banding, which selectively
stained NORs in the chromosomes of mammalian
species. Funaki et al. (1975) improved this proce�
ISSN 0564–3783. Цитология и генетика. 2008. № 3
69
Tools and methodologies for cytogenetic studies of plant chromosomes
dure and demonstrated that N�bands were con�
fined to the NORs of the chromosomes of 27
eukaryotic species that they studied. Faust and
Vogel (1974) and Pimpinelli et al. (1976) observed
that the bands obtained with this procedure are not
NOR�specific in D. melanogaster and the mam�
malian species studied. Nevertheless, these non�
NOR bands were and continue to be referred to as
N�bands. Using the method of Funaki et al.
(1975), with slight modifications, Gerlach (1977)
and Jewel1 (1979), working with Triticum and
Aegilops species respectively, clearly demonstrated
that N�bands do not necessarily correspond to
NORs. Moreover, at least some of the chromo�
somes in the species analyzed had unique N�band�
ing patterns, permitting their identification.
Gerlach (1977) identified nine of the 21 chromo�
somes of common wheat on the basis of their N�
banding patterns. Subsequently, Endo and Gill
(1984a) identified 16 of the 21 chromosomes of
common wheat, including five in the A genome,
using an improved N�banding protocol. Jewell
(1979) identified all 14 chromosomes of Aegilops
variabilis on the basis of their N�banding patterns.
The technique has also been used to identify chro�
mosomes in barley (Singh and Tsuchiya, 1982b),
rye (Jewell, 1981; Schlegel and Gill, 1984), lentils
(Mehra et al., 1986) and Elymus spp. (Morris and
Gill, 1987). N�banding has also been used to iden�
tify various types of aneuploids (Singh and
Tsuchiya, 198Zb; Zeller et al., 1987), alien addi�
tion and substitution lines (Islam, 1980), and
translocations and deletions (Jewell, 1978). It
should be noted that N�banding has been attempt�
ed in T. monococcum by B.S. Gill (personal com�
munication) and in our laboratory without suc�
cess. Why this should be, since some of the A
genome chromosomes in T. aestivum contain N�
bands, is unknown.
Gerlach (1977), and subsequently others,
noted that many of the N�bands occupy the same
positions as C�bands, implying that the N�banding
technique, like the C�banding one, identifies con�
stitutive heterochromatin and that at least two
classes of heterochromatin occur in wheat, rye and
other species. This was confirmed by Schlegel and
Gill (1984) and Endo and Gill (1984a). Some het�
erochromatic regions in each chromosome stain
positively using both C� and N�banding proce�
dures. These regions are referred to as C+N�
bands. Other such regions stain positively only
with C�banding techniques. These heterochro�
matic segments are called C+N�bands. Schlegel
and Gill (1984) have shown that only N�bands
(C+N+ bands) possess multiple copies of the
(GAA) n (GAG) n sequence DNA. The base pair
sequences in C+N–bands have not been identified.
Gill (1987) and Gill et al. (1988) have proposed
banding nomenclatures for the chromosomes of T.
aestivum cv. Chinese Spring.
Identification of NORs and Nucleoli. Nucleolus
organizer regions (NORs) are the sites of rRNA
genes in the chromosomes of animal (Ritossa and
Spiegelman, 1965; Wallace and Birnstiel, 1966;
Henderson et al., 1972, 1974) and plant species
(Phillips et al., 1971; Flavell and O’Dell, 1975;
Hutchinson and Miller, 1982). Methods have been
developed for the selective staining of these chro�
mosomal regions both in animals (Goodpasture
and Bloom, 1975; Howell et al., 1975; Verma and
Babu, 1984) and plants (Hizume et al., 1980;
Lacadena et al., 1984; Mehra et al., 1985; Cunado
et al., 1986). The Ag�As (ammoniacal silver)
method selectively stains those sites on chromo�
somes which correspond exactly to regions that
can be detected by in situ hybridization with rDNA
probes (Howell et al., 1975; Miller et al., 1976a
and b). It seems that this procedure stains only the
NORs that are functionally active during the pre�
ceding interphase (Howell, 1977; Schmiady et
a1. 1979). There is evidence to suggest acidic or
nonhistone proteins associated with the rDNA
regions are responsible for the selective staining of
NORs (Howell et a1., 1975; Wang and Juurlink,
1979; Howell, 1985).
In situ hybridization and its application to wheat
cytogenetics. Gall and Pardue (1996) and John et
al. independently reported a procedure that facili�
tated the cytological detection of hybrid nucleic
acid regions. This technique involves the annealing
of radioactively labelled nucleic acid sequences to
cytological (chromosomal) preparations in situ (on
slides) and subsequent detection of the hybrid
regions by autoradiography. Specific DNA
sequences in the chromosomes of animal and plant
species have been localized with this technique.
These include some highly repetitive short base�
pair sequences in the chromosomes of animals
(Pardue and Gall, 1970; Brutlag et al., 1977) and
plants (Gerlach and Peacock, 1980; Appels and
ІSSN 0564–3783. Цитология и генетика. 2008. № 3
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George Fedak, Nam3Soo Kim
McIntyre, 1985; Ganal et al., 1988; Lapitan et al.,
1989), moderately repeated sequences such as
rRNA genes in the chromosomes of animals
(Wimber and Steffensen, 1970, 1973; Henderson
et al., 1972) and plants (Wimber et al., 1974;
Gerlach and Bedbrook, 1979; Mascia et al., 1981;
Clark et al., 1989), and some single copy genes in
animals (Harper and Saunders, 1981; Henderson,
1982; Olsen el al., 1989) and plants (Ambros et al.,
1986; Huang et al., 1988).
Gerlach and Peacock (1980) isolated a highly
repetitive DNA sequence from T. aestivum cv.
Chinese Spring and hybridized it to cytological
preparations of T. aestivum, T. dicoccoides, T.
monococcum and Ae. squarrosa. A number of heavi�
ly labelled chromosomes were observed in the
preparations of T. aestivum and T. dicoccoides, but
not in those of T. monococcum and Ae. squarrosa.
On the basis of these results the authors concluded
that most of the heavily labelled chromosomes
belong to the B genome. C�banding studies by Endo
(1986) and Gill (1987) and N�banding reports by
Endo and Gill (1984b) support this conclusion.
Peacock et al. (1981) demonstrated that the DNA
sequence is composed of repeated (GAA) n and
(GAG)n units. Rayburn and Gill (1985) showed
that the major C"and N�bands correspond to sites
which contain this satellite sequence. In situ
hybridization studies with a highly repetitive D�
genome specific DNA sequence isolated from Ae.
squarrosa were used by Rayburn and Gill (1986) to
identify D�genome chromosomes in hexaploid
wheat.
At least four pairs of chromosomes (lA, 1B, 5D,
and 6B) of T. aestivum contain NORs (Crosby,
1957; Darvey and Driscoll, 1972). If NORs are the
sites of ribosomal RNA (rRNA) genes, then all
these chromosomes should be expected to possess
clusters of rRNA genes. Flavell and Smith (1974a,
1974b) and Flavell and O’Dell (1976, 1979)
showed that in hexaploid wheat a large proportion
of the rRNA genes are on chromosomes 1B and
6B, with only a small proportion of the genes
residing on chromosomes 1A and 5D. Gerlach and
Bedbrook (1979) cloned the 18S+26S rRNA genes
of T. aestivum into a bacterial plasmid and showed
that the probe derived from this clone hybridized
to regions on chromosomes 1B and 6B. Miller et
al. (1980) showed that the same rDNA probe
hybridized to minor NORs on chromosomes 1A
and 5D in bread wheat. A similar approach with a
different rDNA probe enabled Appels et al. (1980)
to confirm the location of rRNA genes on chro�
mosomes lB, 5D, and 6B only. They speculated
that too low a level of rRNA genes on chromosome
1A may have precluded their detection by in situ
hybridization experiments. In situ hybridization
experiments in T. turgidum and T. timopheevi with
rDNA probes confirmed the assignment of rDNA
loci to chromosomes 1B and 6B, (Appels and
Dvorak, 1982; Dvorak and Appels, 1982). Miller et
al. (1983) showed that, in T. urartu, a labelled
rDNA probe hybridized in situ to a region on chro�
mosome 5A that corresponds to the NOR. Some
genotypes of T. urartu, and other diploid wheat,
have been shown to have two chromosome pairs
with nucleolus organizers (Gerlach et al., 1980).
The second NOR must, by deduction, be located
on chromosome 1A. Frankel et al. (1988), using a
synthetic tetraploid AABB and a 3H�labelled
rDNA probe, clearly demonstrated that the NORs
of two pairs of A�genome chromosomes were
labelled after in situ hybridization. Apart from one
being more heavily labelled than the other, the two
chromosome pairs (1A and 5A) could not be dis�
tinguished cytologically.
Information on the location of 5S rRNA genes
in wheat species is scanty. Appels et al. (1980)
localized the 5S rRNA gene cluster to chromo�
some 1B of T. aestivum at a site distinct from and
distal to the NOR region. Kota and Dvorak (1986)
mapped these genes to a single site on the p arm of
chromosome 5B, using a line with a spontaneous
deletion. Lassner and Dvorak (1985) and Kota and
Dvorak (1986) suggested that chromosomes 5A
and 5D may also carry the genes for 5S rRNA.
Scoles et al. (1987), using cloned 5S rDNA
sequences obtained unequivocal evidence for the
presence of 5S rRNA genes on chromosomes 1B,
1D, and 5B in T. aestivum. Studies by Dvorak et al.
(1989) have shown that chromosomes 1A and 5A
of T. monococcum var. aegilopoides carry 5S rRNA
genes. Moreover, they indicate that the 5S rDNA
on chromosome 1B is linked to the Nor�B1 locus.
Production of molecular probes. Molecular
probes, are derived from cloned recombinant DNA
molecules e.g., plasmids such as pBR 322 with
cDNA genes. These molecules are generated using
restriction and other DNA modifying enzymes and
then cloned in a proper host (Mertz and Davis
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Tools and methodologies for cytogenetic studies of plant chromosomes
1972; Watson et al., 1983). Protocols for generat�
ing recombinant DNA molecules, cloning them,
excising the relevant DNA fragment e.g., 5S
rDNA genes from these molecules and subsequent
use in in situ hybridization and other experiments
are detailed in Watson et al. (1983) and Winnacker
(1987). The procedures used in the cereals are
given by Gerlach and Bedbrook (1979) and
Lawrence and Appels (1986).
Restriction fragment length polymorphism (RFLP)
for chromosome identification. RFLP markers are
currently being extensively used in genetic mapping
(Wyman and White 1980, Helentjaris 1987, White
and Lalouel, 1988) and determining genome
homologies among crop species (Bonierbale et al.,
1988; Sharp et al., 1989). They have also been used
in the identification of the critical chromosomes in
aneuploid plants in tomato (Young et al., 1987) and
bread wheat (Gale et al., 1988).
Sharp et al. (1989) isolated specific RFLP
probes for each arm of the seven chromosomes of
the homoeologous genomes of Triticum. These
14 homoeologous probes permit the identification
of chromosomes in each genome of the diploid
and polyploid wheats.
Aneuploid stocks
Several types of aneuploid stocks have been
produced in wheat that have been used for decades
for studies of physical mapping. These stocks are at
the series of 21 mulli tetras produced by Ernie
Sears (Sears, 1966) that have been used for assign�
ing genes to individual chromosomes (Sears, 1954;
1966) and the 24 ditelo stocks for mapping genes
to individual chromosome arms (Sears and Sears,
1978). These stocks have also been used for a num�
ber of studies; from assigning some of the original
molecular markers to individual chromosomes in
the construction of the first molecular marker
maps (Anderson et al., 1992) and to assigning
ESTs to physical positions on chromosome arms
(Han et al., 2005). The trisomic series in barley and
the ditelo series in barley (Tsuchiya, 1960, Fedak et
al., 1971, 1972) and trisomic series in T. monococ"
cum (Friebe et al., 1990) and rye (Kamanoi and
Jenkins, 1962) have also been used for physical
marker mapping. Another aid in wheat cytogenet�
ics was the publication of a standard karyotype and
nomenclature system for hexaploid wheat (Gill et
al., 1995). The addition lines of Betzes barley in
Chinese Spring wheat (Islam, 1980) have also been
used in initial studies of assigning molecular mark�
ers to chromosomes in the initial phases of devel�
oping molecular maps in barley.
The deletion stocks in wheat are another useful
cytogenetic tool. They were produced by the
gametocidal action of a particular genotype of Ae.
speltoides (Endo and Gill, 1996) that caused a
series of terminal deletions on all wheat chromo�
somes followed by restoration of the telomere
function. The deletion stocks consist of 101 lines
with 119 chromosome segment deletions for sub�
arm mapping. These stocks provide complete cov�
erage of the wheat genome, subdividing it into
159 chromosome bins. These stocks were recently
used to physically map 16,000 wheat ESTs (Qi et
al., 2004). Such a map is having numerous appli�
cations such as a source of SNP discovery (Somers
et al., 2003) microsatellite discovery (Thiel et al.,
2003) comparative mapping, hastening the speed
of gene discovery in wheat plus providing a frame�
work for constructing BAC�contigs of wheat. The
stocks have also been useful on a smaller scale, as
for example the physical mapping of ESTs upregu�
lated in wheat following fungal attack (Han et al.,
2005). Another tool for cytogenetic analysis of
genomes, cloning and tagging of genes is the BAC
libraries that have been constructed in diploid
(Lyavetsky et al., 1999) and hexaploid wheats.
Chromosome Addition Lines
Another set of cytogenetic stocks that permit
analysis of single donor chromosomes are the
addition lines of maize in oat (Riera�Lizarazu et
al., 2000) which were produced by means of oat by
maize pollination (Riera�Lizarazu et al., 1999).
Yet another cytogenetic tool is that of chromosome
sorting. Chromosome sorting is a technique to
provide aliquots enriched in particular wheat chro�
mosomes (Kubalakova et al., 2005) that can be
used for molecular cytogenetic studies.
Complete series of addition lines from numer�
ous alien species have been produced in a wheat
background. Chromosomes from more than
20 alien species have been added to wheat; in some
cases a complete set of addition lines has been pro�
duced. From 18 of these combinations, chromo�
some substitution lines have also been produced.
In many cases, translocations have been induced
between the chromosomes of the crop plant and
ІSSN 0564–3783. Цитология и генетика. 2008. № 3
72
George Fedak, Nam3Soo Kim
the alien species. A compendium of such cytoge�
netic stocks appears as an appendix in the pro�
ceedings of international wheat genetics symposia
(e.g. Proceedings of the Seventh International
Wheat Genetics Symposium, Cambridge E, July
13–19,1998. pp1374–1387). Various molecular
methods have been devised to monitor the transfer
of traits from alien species into crop plant chromo�
somes (Fedak,1999). Numerous translocations
have been induced between alien and wheat chro�
mosomes from the alien addition lines as a means
of transferring disease resistance to wheat (Friebe
et al., 1996).
Molecular Marker Maps
Another class of fairly recent genetic tools are
the molecular linkage maps. These are not physi�
cal chromosome tools but are very useful in chro�
mosome and genome identification and monitor�
ing alien introgressions. RAPD markers were
basically random base sequences that were exten�
sively used to tag genes controlling agronomic
traits (Williams et al., 1990). The next class of
markers were RFLPs. Extensive maps consisting of
1000 RFLP markers were generated in wheat
(Gale et al., 1995). These markers were character�
ized as too expensive and cumbersome for applica�
tion to marker assisted selection but played a sig�
nificant role in uncovering relationships between
diverse grass genomes (Gale and Devos, 1998). For
example overall colinearity between wheat, barley,
maize and rice genomes was established (Feuillet
and Keller, 1999) which permitted the sharing of
markers across the maps. Because the rice genome
was fully sequenced (Goff et al., 2002), this per�
mitted the extensive use of rice sequences to per�
form fine mapping in wheat genetic studies (Liu et
al., 2006). RFLP technology, particularly the use
of cDNA probes has been and still is a valuable
method of monitoring alien chromatin introgres�
sion into the wheat genome, mainly because of
sequence conservation and systemic relationship
among the Triticeae. In more recent fine mapping
experiments it is being discovered that the
collinearity between chromosomes of species such
as wheat and rice is not as close as initially
assumed (La Rota and Sorrels, 2004).
Other molecular maps were developed with
PCR�based markers including RAPDs (William et
al., 1990) AFLPs (Vos et al., 1995) and microsatel�
lites (SSRs) (Roder et al., 1998). High density
microsatellite maps coupled with high throughput
capillary electrophoresis are the essential compo�
nents of a marker assisted breeding program. The
high density consensus map of Somers et al.,
2004 is adequate for QTL detection.
High density maps are essential for map based
cloning by increasing the probability of discovering
markers flanking the gene in question and by
reducing the number of BAC clones containing
the gene.
FISH and GISH analysis
Another class of methodologies that facilitate
the identification of specific genomes, individual
chromosomes or chromosomal segments is the use
of fluorescent signals. The first procedure to use flu�
orescent labels to distinguish plant chromosomes
was the process of genomic in situ hybridization
(GISH) (Shwarzacher et al., 1989). This technique
was then used to identify parental genomes and
genome organization, plus alien genome/chromo�
some introgression (Jiang and Gill, 1994). The
identification of the three genomes of hexaploid
wheat has been achieved, but proved to be difficult
to repeat consistently (Mukai et al., 1993; Sanchez�
Moran et al., 1999). A modification of the multi�
colour GISH technique (Han et al., 2003, 2004)
permitted the unequivocal identification of all three
wheat genomes plus the presence of alien chromo�
somes and translocations. Other cytogenetic tools
such as the Afa family, a repetitive sequence, cloned
from Ae. squarossa (Rayburn and Gill, 1986);
PSc119.2�a rye subtelomeric heterochromatic
sequence (Bedbrook et al., 1980); and PTa7l, an
rDNA clone of the 18 S�8.5S�26S ribosomal
sequence (Gerald and Bedroock, 1979) can be used
singly, in combination or in combination with FISH
to identify individual chromosomes of wheat or bar�
ley. For example, using a combination of FISH, Afa
and PSc119.2. Kubalakova et al. (2005) were able to
identify all chromosomes of durum wheat.
Additional tools that complement the use of
FISH technology for cytogenetic analysis of indi�
vidual chromosomes are 5S and 25S genes plus
BAC clones that can be differentially stained to
identify individual chromosomes (Zhang et al.,
2004). The fiber FISH technique, using appropri�
ate probes which is applied to stretched somatic
chromatin can provide better resolution of gene
ISSN 0564–3783. Цитология и генетика. 2008. № 3
73
Tools and methodologies for cytogenetic studies of plant chromosomes
and repeated sequence arrangements on wheat and
bareley chromosomes (Valanik et al., 2004).
It is obvious from this review that in recent
years classic cytogenetic analysis has been replaced
by molecular methods. In recent years, however,
there is a renewed interest in minichromosomes.
These are structures that consist of a centromere
and minimal proximal chromatin into which genes
of interest will potentially be integrated. The
minichromosomes will not pair with those of the
regular complement, will behave normally at
meiosis and provide stable transmission of the
introgressed trait. This technology is most
advanced in mamalian cells. In plant cells it is
being developed in maize from B chromosomes
(Yu et al., 2007).
It is interesting to note that Gus Wiebe, a
Research Coordinator for USDA had developed
lines of 16 chromosome barley in the 1960s. His
theory was that removing the telomeres from the
additional chromosomes would limit their pairing
with normal chromosomes and would thus stably
carry additional traits for the barley genome.
Another recent development is the use of rice
sequence information for fine mapping in wheat,
barley and maize. In many cases the colinearity of
sequences between rice and the other species is not
as precise as originally predicted. More recently it
has been suggested that the genome of
Brochypodium could be used as a model system for
the study of grass genomes (Bossolini et al., 2007).
George Fedak, Nam"Soo Kim
TOOLS AND METHODOLOGIES FOR
CYTOGENETIC STUDIES OF PLANT
CHROMOSOMES
Представлен краткий обзор развития методов цито�
генетики и анеуплоидов с 20�х годов. Методология ва�
рьирует от первых сообщений о хромосомных наборах
большинства организмов, развития хромосомных ка�
риотипов, а затем анеуплоидов для большинства воз�
делываемых растений. Вклад молекулярной биологии
включает технологии исчерченности хромосом, карты
молекулярных маркеров и методы гибридизации in
situ. Все эти новые методики значительно увеличили
точность цитогенетических исследований.
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