y. Oil Set. 71, 111-120 (1984)
111
Printed in Great Britain © The Company of Biologists Limited 1984
G AND/OR C-BANDS IN PLANT CHROMOSOMES?
I. SCHUBERT, R. RIEGER
Zentralinslitut fur Genetik und Kulturpflanzenforschung der Akademie der
Wissenschaften der DDR, DDR-4325 Gatersleben, DDR
AND P. DOBEL
Institut fur Biochemie der Pflanzen der Akademie der Wissenschaften der DDR, DDR4010 Halle/Saale, DDR
SUMMARY
Similarities and differences become evident from comparisons of centromeric and noncentromeric banding patterns in plant and animal chromosomes.
Similar to C and G-banding in animals (at least most of the reptiles, birds and mammals),
centromeric and nucleolus-organizing region bands as well as interstitially and/or terminally located
non-centromeric bands may occur in plants, depending on the kind and strength of pretreatment
procedures. The last group of bands may sometimes be subdivided into broad regularly occurring
'marker' bands and thinner bands of more variable appearance.
Non-centromeric bands in plants often correspond to blocks of constitutive heterochromatin that
are rich in simple sequence DNA and sometimes show polymorphism; they thus resemble C-bands.
However, most of these bands contain late-replicating DNA. Also they are sometimes rich in A-T
base-pairs, closely adjacent to each other and positionally identical to Feulgen + and Q + bands, thus
being comparable to mammalian G-bands.
Although banding that is reverse to the non-centromeric bands after Giemsa staining is still
uncertain in plants, reverse banding patterns can be obtained with Feulgen or with pairs of A-T
versus G-C-specific fluorochromes.
It is therefore concluded that not all of the plant Giemsa banding patterns correspond to Cbanding of mammalian chromosomes. Before the degree of homology between different Giemsa
banding patterns in plants and G and/or C-bands in mammals is finally elucidated, the use of the
neutral term 'Giemsa band', specified by position (e.g. centromeric, proximal, interstitial, terminal), is suggested to avoid confusion.
INTRODUCTION
By means of various treatment procedures before Giemsa staining a variety of
banding patterns has been demonstrated in the chromosomes of many species, thus
allowing their lengthwise differentiation. The two fundamental patterns are the socalled C and G-banding patterns originally established for mammalian chromosomes
by Pardue & Gall (1970), Arrighi & Hsu (1971) and Sumner, Evans & Buckland
(1971), respectively. (The techniques were later improved, modified, and adapted to
a variety of species by many investigators.) On the basis of different reactions to
various banding techniques, Comings (1978) subdivided the chromatin of mammals
into three types: (1) constitutive heterochromatin resulting in C-bands; (2) interstitial
(A-T-rich and late-replicating) heterochromatin* resulting in G-bands (positionally
•This type of heterochromatin is in accordance with Nagl's (1976) 'functional' heterochromatin
but was called 'ontogenetic' (non-transcribed) euchromatin by Goldman et al. (1984).
112
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correlated with Q-bands, see below); and (3) euchromatin resulting in R-bands
(reverse to the G-banding pattern). While G-banding results in closely adjacent bands
along the chromosomes, which in man and Chinese hamster correspond to the
chromomere pattern in pachytene chromosomes (Luciani, Morrazzani & Stahl, 1975;
Okada & Comings, 1974), C-banding reveals blocks of constitutive heterochromatin
located mainly around the centromeres of mammalian chromosomes (ISCN, 1978).
For more than a decade, a large number of studies have been done to differentiate
plant chromosomes, by the application of techniques identical or similar to those used
for animal chromosomes. Most authors called the banding patterns obtained in plant
chromosomes C-banding.
The main reasons for the widespread use of this term for chromosome banding in
plants are: (1) the morphological similarity of these banding patterns, in many cases
at least, to C-banding patterns of animal chromosomes (large dark bands, but in most
cases not at the centromeric position, and large chromosome regions without bands);
(2) the similarity of the procedures (often Ba(OH)2 pretreatment) resulting in this
type of banding patterns; (3) the argument of Greilhuber (1977) that the more dense
compaction of plant chromosomes during mitosis, as compared to mammalian
chromosomes, prevents microscopic resolution of G-bands in plant chromosomes.
While the first two arguments are not without exceptions (see below), the third one
became less convincing since Anderson, Stack & Mitchell (1982) found, under comparable conditions, that the ratios of DNA to volume and DNA to length are similar
in both plant and mammalian chromosomes (see also Bennett, Heslop-Harrison,
Smith & Ward, 1983). Also, Takayama(1976) reported that the G-banding procedure
results, parallel to the contraction of chromatin in G+-band regions, in an extension
of chromatin in G~-band regions.
In this paper we report on the similarities and differences of the Giemsa banding
patterns observed in mammalian and plant chromosomes that allow us to reach
preliminary conclusions and recommendations with respect to the nomenclature of
the various types of chromosome bands in plants.
SIMILARITIES
OF CENTROMERIC
AND
NO N-C ENTROM ERIC
BANDS IN PLANTS AND C- AND G-BANDS IN MAMMALS
GIEMSA
(1) As in mammals, two main types of Giemsa banding patterns may occur in the
same plant. One, similar to C-banding in mammals, involves heterochromatin adjacent to centromeres and secondary constrictions only. It has been observed in Vicia
faba (Dobel, Schubert & Rieger, 1978), Allium cepa, Rhoeo discolor, Ornithogalum
vtrens, Tradescantia edwardsiana (Stack, 1974), Avena tuiestii (Yen & Filion,
1977), Tulipagesneriana (Filion &Blakey, 1979) andNigella damascena (KlaSterska
& Natarajan, 1975). In N. damascena, the three terminal or subterminal Giemsa
bands, which occur in addition to the centromeric bands, indicate nucleolusorganizing region (NOR) positions as demonstrated by AgNO3 staining (Hizume,
Tanaka & Shigematsu, 1982; Schubert, unpublished).
A second pattern, consisting of interstitial and/or terminal bands, appears in
G andfor C-bands in plant chromosomes
113
V. faba and A. cepa following a procedure published by Schwarzacher, Ambros &
Schweizer (1980) or after using a urea/trypsin technique (Dobel et al. 1978;
Schubert, Ohle & Hanelt, 1983). \r\Avena wiestii and T. gesneriana, the same type
of pattern occurred after shortening of HCl-hydrolysis time before Ba(OH)2 treatment (Yen & Filion, 1977; Filion & Blakey, 1979).
(2) C and G-banding do not exclude each other; intermediate forms may in fact be
observed. For mammalian chromosomes, Daniel & Lam-Po-Tang (1973) demonstrated the stepwise transition of one banding pattern into the other. Comings,
Avelino, Okada & Wyandt (1973) wrote: 'relatively mild conditions allow staining of
both C- and G-bands. To obtain only C-bands the chromosomes must be treated more
harshly to disrupt or destroy the G-bands'. Similar observations have been made in
V. faba. The harsher Ba(OH)2 pretreatment resulted in a C-like pattern. Shortening
of the Ba(OH)2 treatment time allowed the stepwise appearance of single interstitial
bands, additional to the centromeric bands. On the other hand, the procedure usually
giving rise to interstitial bands sometimes also results in centromeric bands. inAvena
xviestii and T. gesneriana, a transition between both types of Giemsa bands has also
been observed (Yen & Filion, 1977; Filion & Blakey, 1979) after slight changes of the
pretreatment conditions.
(3) A urea pretreatment technique, originally developed for G-banding in human
cells by Berger (1971) and Shiraishi & Yoshida (1972), resulted in interstitial bands
when applied to V. faba (Dobel, Rieger & Michaelis, 1973); and in at least two
chromosomes, these bands were close to each other thus resembling mammalian Gbanding patterns (Dobel et al. 1978).
A pattern with many closely adjacent Giemsa bands was also reported for Leopoldia
comosa by Bentzer & Landstrom (1975), iorPinus resinosa by Drewry (1982) and for
Zea mays by T. C. Hsu & Shi Liming (personal communication).
(4) Comparable to mammalian G-bands, some of the interstitial Giemsa bands of V.
faba may (depending on the degree of chromosome contraction) appear as a large block
(in rather contracted chromosomes of late colchicine metaphases) or subdivided into
two bands in more extended chromosomes of early metaphase cells (Dobel et al. 1978).
(5) Like mammalian G-bands (Grzeschik, Kim & Johannsmann, 1975; Dutrillaux,
Couturier, Richer & Viegas-Pequignot, 1976; Holmquist, Grey, Porter & Jordan,
1982) most interstitial Giemsa bands of V. faba contain late-replicating DNA as
demonstrated after incorporation of [3H]thymidine or bromodeoxyuridine during late
5-phase (Dobel et al. 1978). The only exception is the NOR-associated
heterochromatin. It shows up as a dark band after pretreatment, resulting in either
interstitial or centromeric bands, respectively, and as a single dark band after Nbanding* (see Funaki, Matsui & Sasaki, 1975; D'Amato, Bianchi, Capineri & Marchi,
1979; Schubert, 1984), which was found to be the earliest replicating region of the V.
faba genome (Schubert & Rieger, 1979).
• In conformity with the nomenclature for mammalian chromosome banding, the term used for
description of a banding pattern should follow the phenomenon specified rather than the procedure
used. Thus, N-banding should be reserved for the exclusive staining by Giemsa of NORs or NORassociated heterochromatin.
114
/. Schubert, R. Rieger and P. Dobel
(6) In human and mouse chromosomes, dark bands (F + bands) appear after prolonged hydrolysis with HCl before Feulgen staining. These are positionally coincident
with G + bands (Rodman, 1974; Rodman & Tahiliani, 1973). In V.faba, prolonged
hydrolysis with HCl and subsequent Feulgen staining revealed dark bands (Tempelaar, deBoth& Versteegh, 1982; Schubert, unpublished), which-notwithstanding
the different staining intensities of some of these bands — correspond positionally to
the interstitial Giemsa bands (except for the secondary constriction, which never
shows up as a dark band after Feulgen staining). Also in other plants, Anemone
blanda, Fritillaria lanceolata (Marks, 1983), Buglossoides purpurocaerulea
(D'Amato, Capineri & Marchi, 19816)), rather striking homologies between interstitial Giemsa bands and F + bands have been reported.
(7) In mammals, the position of G-bands is similar to that of the so-called Q-bands
obtained after treatment with quinacrine dihydrochloride or quinacrine mustard,
respectively (Evans, Buckton & Sumner, 1971; ISCN, 1978); these regions are
thought to be rich in A-T base-pairs (Holmquist et al. 1982). In V. faba all the
brightly fluorescing chromosome regions after staining with Hoechst 33258 — a
fluorochrome with specific affinity for A-T base-pairs (Latt & Wohlleb, 1975;
Schweizer, 1981) - corresponded positionally to the sites of interstitial Giemsa bands
(Dobel & Schubert, 1981). A similarfluorescencepattern was obtained by Caspersson
et al. (1969) using quinacrine mustard. After treatment of Vicia chromosomes with
quinacrine dihydrochloride, either enhancement or depression of fluorescence
occurred in regions characterized by interstitial Giemsa bands (Dobel et al. 1978;
Dobel & Schubert, 1981). In Vicia melanops (D'Amato, Bianchi, Capineri & Marchi,
1980) and Allium carinatum (Vosa & Marchi, 1972) terminal Giemsa bands coincide
with Q+-bands, in A. cepa (Vosa, 1976) with Hoechst 33258+ and Q~ bands, and in
some other cases, e.g. Tulbaghia leucantha (Vosa & Marchi, 1972), Alliumfistulosum
(Kamizyo & Tanaka, 1978), Buglossoides purpurocaerulea (D'Amato et al. 19816),
Scilla siberica (Deumling & Greilhuber, 1982), also with Q~-bands.
DIFFERENCES
BETWEEN
GIEMSA
BANDING
PATTERNS
IN
PLANTS
AND
MAMMALS
(1) Non-centromeric Giemsa bands (those located neither at nor adjacent to the
centromere) in plants occur, in many cases, in the form of blocks, leaving large parts
of the chromosome free of bands; multiple closely adjacent bands, like mammalian
G-bands, are less frequently observed.
(2) In some plants, the interstitial bands may be subdivided into a group of regularly
occurring large bands (called 'marker bands' by Dobel et al. 1973, 1978) and a group
of thinner bands, which either occur after special modifications of the pretreatment
procedures, as chromosomal polymorphisms in inbred lines (e.g. see Lelley, Josifek
& Kaltsikes, 1978), or even in different metaphases of the same slide. Such additional
interstitial bands occur in V.faba (Greilhuber, 1975; Dobel et al. 1978), P. resinosa
(Drewry, 1982), Secale cereale (Lelley et al. 1978; Schlegel & Kynast, personal
communication), in some Fritillaria species (La Cour, 1978) and (probably) in
G and/or C-bands in plant chromosomes
115
A. cepa (Schubert et al. 1983). In the last case, additional interstitial chromosome
regions other than those normally showing up as Giemsa bands are late-replicating
(Cortes, Gonzalez-Gil & Lopez-Saez, 1980). In some plant inbred lines, e.g. Zea
mays (Hadlaczky & Kalman, 1975) and Hordeum vulgare (Linde-Laursen, 1978),
pronounced Giemsa bands, which show polymorphism - similar to C-bands but
unlike G-bands in mammals (see Schweizer, 1980) - have also been observed.
(3) Contrary to the situation in mammals, no complete R(reverse)-banding has so
far been described for plants after Giemsa staining. Schweizer (1973) showed
that occasionally reverse Giemsa banding patterns may occur in chromosomes of
F. lanceolata and F. recurva.
However F~-banding reverse to Giemsa banding was described by Marks (1983)
for F. lanceolata after short hydrolysis with HC1 before Feulgen staining; prolongation of HC1 hydrolysis led to F + bands at sites positionally identical to those of Giemsa
bands. Indications of a similar phenomenon in Ornithogalum montanum were reported by D'Amato, Bianchi, Capineri & Marchi (1981a). In V.faba, reverse banding
(F*~-bands) obtained by modified HC1 hydrolysis conditions before Feulgen staining
was restricted to the metacentric satellite chromosome (Takehisa, 1970; D'Amato et
al. 1981a).
In spite of the absence of unambiguous reverse Giemsa banding patterns, reverse
banding patterns comparable to the situation observed in mammals (ISCN, 1978),
have also been obtained after application of different fluorochromes in some plant
species. A fluorescence pattern the reverse of that of the A-T-specific fluorochrome
4'-6-diamino-2-phenylindole (DAPI) has been detected in the metacentric satellite
chromosome of V.faba after counterstaining with the G-C-specific combination of
methylgreen plus fluorochrome chromomycin A3 (Schweizer, 1976). The same is true
for the whole karyotype of V. faba when the Hoechst 33258 fluorescence pattern
(Dobel & Schubert, 1981) is compared to the counterstaining pattern obtained after
chromomycin + distamycin + DAPI (Schweizer, personal communication). Any of
the positive fluorescent regions after DAPI or Hoechst staining corresponds to a
negatively fluorescing region after counterstaining. Only the G-C-rich NOR, the
single chromosomal site exhibiting negative fluorescence after Hoechst 33258 or
DAPI, shows intensive fuorescence after counterstaining. Pairs of reverse
fluorescence patterns have also been found in A. cepa (Vosa, 1976), S. siberica, 0.
caudatum, P. mugo and Anemone blanda (Schweizer, 1976, 1980).
(4) Eiberg (1974) reported on Cd (centromeric dot) bands in human chromosomes.
In spite of the morphological similarity between some centromeric Giemsa bands in
plants and Cd-bands, they do not correspond to each other, since Cd-banding never
includes nucleolus-organizing secondary constrictions and is obtained after quite
another pretreatment procedure than used for obtaining most of the centromeric
Giemsa banding patterns described in plants.
(5) Unlike G-bands, but similar to C-bands in mammals, interstitial and terminal
Giemsa bands in plants frequently contain a large amount of simple sequence DNA.
This is true for some species of Secale (Bedbrook et al. 1980) and Scilla (Deumling
& Greilhuber, 1982), as well as for Luzulapurpurea (Ray & Venketeswaran, 1979),
116
/. Schubert, R. Rieger and P. Dobel
barley, wheat (Dennis, Gerlach & Peacock, 1980) and maize (Peacock, Dennis,
Rhoades & Pryor, 1981). In V.faba many highly repetitive DNA sequences, even
when cloned in the plasmid pBR322, gave random distribution of silver grains along
the whole chromosome complement (Kaina, Baumlein, Wobus & Schubert, 1979;
Rowland, 1980). However, Bassi, Cremonini & Cionini (1982) found preferential in
situ hybridization of a repetitive palindromic sequence in chromosome regions known
to be sites of intercalary Giemsa bands.
CONCLUSIONS
From the observations summarized above the following conclusions may be drawn.
(1) As in animals, there are different types of heterochromatin in plants (e.g. see
Vosa, 1970; Stack, Clarke, Cary & Muffly, 1974; Schweizer & Nagl, 1976; La Cour,
1978; Sato, Kuroki & Ohto, 1979; Sato, 1980; Dobel & Schubert, 1981).
(2) Giemsa banding patterns of plant and mammalian chromosomes show a number
of similarities as well as differences.
(3) Centromeric Giemsa bands of plants correspond to C-bands of mammals, rather
than to Cd-bands of human chromosomes, since Cd-banding does not include the
nucleolus-organizing secondary constrictions. The formal similarity to the Cd-bands
is probably due to the fact that centromeric heterochromatin in plants is often
quantitatively less represented than in many animal chromosomes.
(4) Whether the existence of two main types of Giemsa banding patterns in plants
— a mainly centromeric one obtained after harsh pretreatment procedures, and interstitial/terminal patterns obtained after application of milder conditions — are the rule
or the exception deserves further study. However, the fact that such patterns were
found in at least four unrelated genera makes it rather probable that thorough future
investigations will reveal additional positive examples. The same may be true for
reverse banding patterns.
(5) Provided the above-mentioned similarities between interstitial/terminal Giemsa
bands in plant chromosomes and mammalian G-bands on the one hand, and
centromeric Giemsa bands in plants and animal C-bands on the other are genuine and
the differences not fundamental in nature, then one should very carefully consider
which banding pattern in plant chromosomes is to be called G or C-banding, respectively (see Drewry, 1982).
(6) If, however, differences (e.g. in the content of simple sequence DNA and the
degree of polymorphism) between Giemsa banding patterns in plants and G or Cbanding in mammals are due to fundamental differences between the various
heterochromatin fractions in mammals and plants, respectively (as suggested by
Nagl, 1979), the use of the term C-banding should be avoided in connection with
Giemsa bands in plant chromosomes.
(7) The different Giemsa banding patterns obtained in various plants are evidently
not always identical to C-banding in mammals. Therefore, one should be aware of the
fact that the term 'C-banding' used (often uncritically) for plant chromosomes does
not necessarily describe the same phenomenon referred to as C-banding in mammals.
C and/or C-bands in plant chromosomes
117
(8) Before a final solution with respect to the homology of the different types of
Giemsa banding patterns in mammals and plants is at hand, the use of the neutral term
'Giemsa banding', specified by a description of the band positions, e.g. centromeric,
proximal, intercalary/terminal (and possibly the main pretreatment procedure used)
is suggested for plants. This will avoid confusion.
We thank Dr G. E. Marks (John Innes Institute, Norwich, England), Dr G. P. Holmquist
(Baylor College of Medicine, Houston, Texas, U.S.A.) and Professor Dr D. Schweizer (Institute
of Botany, University of Vienna, Austria) for their critical review of the manuscript and helpful
suggestions.
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{Received 9 April 1984-Accepted 14 May 1984)