Chromomere mapping in maize pachytenes

Vol. 56, no. 1: 53-56, 2003
CARYOLOGIA
Chromomere mapping in maize pachytenes
EVELINE TEIXEIRA CAIXETA and CARLOS ROBERTO DE CARVALHO*
Universidade Federal de Viçosa , Departamento de Biologia Geral, 36571-000, Viçosa, Minas Gerais, Brazil.
Abstract - The study of chromomere maps has been of great usefulness for chromosome identification and in the elucidation of different aspects of the higher
organisms genetics. The number, size, distribution and pattern of chromomeres
observed in the maps are species-specific during a given stage of cellular development. The consistence of this type of data, makes it suitable for using as cytological markers. In plants, the importance of chromomere analysis is even higher, due to the difficulties in obtaining other types of cytological markers. Considering the importance of chromomere structure, the objective of this work was
to develop a method for the elaboration of a maize chromomere map. The
slide preparation method allowed us to obtain isolated pachytenic chromosomes, with good cytogenetic quality and adequate resolution for the distinction
of longitudinally arranged chromomeric structures. The chromomere map for
bivalent 8 was prepared using this method. A total of 35 chromomeres were
identified in the map, 10 in the short arm and 25 in the long arm, including
knobs and telomeres.
Key words: Chromomere map, cytological marker, knob, maize, pachytene.
INTRODUCTION
Chromosome analysis during pachytene
reveals several evident longitudinal differentiations, which are useful in cytogenetic studies.
Among these types of differentiation are shorter
bead-like regions of high density, termed chromomeres (FINCHAM 1994). The number, size, distribution and pattern of chromomeric structures
are species-specific during a given stage of cellular development. The consistence of this type of
data makes it suitable for using as cytological
markers (McCLINTOCK 1978; SWANSON et al.
1982).
Chromomere maps have been prepared and
analyzed in some animals (FANG and JAGIELLO
1981; JHANWAR and CHANGATI 1981), plants
(DUNDAS et al. 1983; WU 1989; SHEN and WU
1992) and in humans (FANG and JAGIELLO 1991;
* Corresponding author: fax +55 31 3899 2549; e-mail:
[email protected]
VERMA and BABU 1995). The analysis of chromomeric patterns described in these maps
increases the detail of ideograms, facilitating the
recognition and identification of specific chromosomes (SWANSON and WEBSTER 1977; JHANWA R and C H A G A N T I 1981). The variation
observed in the chromomeric pattern of different
tissues in an individual is relevant to understand
the genetic organization of eukaryotic chromosomes (LIMA-DE-FARIA et al. 1973). Such cytogenetic markers are important in the localization of break points involved in chromosome
rearrangements (SHEN and WU 1992; SYBENGA
1992) and facilitate the elaboration of gene maps
(SHEN et al. 1987; SHEN and WU 1992). The
combination of chromomere pattern analysis and
in situ hybridization has been considered to be a
powerful tool in gene mapping studies (SHEN
and WU 1992). Another contribution of chromomere maps is the analysis of chiasmas.
Mapped chromomeric structures allow the determination of the number and localization of chi-
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CAIXETA
asmas, greatly facilitating the evaluation of many
strategic questions related to crossing-over
(FANG and JAGIELLO 1988). The usefulness of
chromomere maps has also been demonstrated
in studies of chromatin structure and function,
with emphasis in the parallelism of the interchromomeric region with GC-rich DNA sites
(FANG and JAGIELLO 1991).
Considering the importance of the chromomere as a cytological marker, especially in plants,
where few additional cytological markers are
available, a method for the preparation of maize
pachytenic chromosomes and subsequent chromomere mapping was developed.
MATERIALS AND METHODS
Male inflorescences of maize inbred line L-869 of
the Universidade Federal de Viçosa, Brazil, were
selected in order to obtain only anthers in pachytene
stage. Approximately 15 anthers, previously fixed
and CARVALHO
in fresh cold methanol-acetic acid (3:1), were placed
in a scooped plate containing distilled water and
transferred to an adapted 0.5 ml microfuge tube,
which had its bottom removed and replaced with a
100 mm nylon screen. The material was washed,
three times, in distilled water for 15 minutes and
the excess water was removed with filter paper. The
tube containing the anthers was immersed in an
undiluted enzymatic solution of Flaxzyme (NOVO),
enough to cover the anthers, and placed in an incubator at 35oC, for 50 minutes. After digestion, the
anthers were washed again. The adapted microfuge
tube was then placed into a 1.5 ml microfuge tube,
filled with enough distilled water to reach the nylon
screen. The anthers were mechanically ground,
releasing the pollen mother cells (PMCs) into the 1.5
ml microfuge tube. The cell suspension formed in
the 1.5 ml tube was centrifuged at 150 g for 5 minutes. The supernatant was removed, leaving 0.5 ml
of the cell suspension, and 30 µl of the previously
mentioned enzymatic solution was added. The cells
were carefully resuspended. After digestion (at 35ºC,
for 1 hour), the tube was filled with 1.0 ml of distilled water and centrifuged at 150 g for 5 minutes.
Fig. 1 – Chromomere map for maize bivalent 8. (a) Three 8 bivalents. “C” refers to the centromere, and “k” to a knob. The chromomeres are indicated by the black dots. (b) Diagram of the chromomeres identified in the bivalents from Fig. 1a. Bar 5 µm.
CHROMOMERE MAPPING IN MAIZE
This washing procedure was repeated four times.
The supernatant was removed and, to the cellular
sediment, a fresh fixative solution (methanol-acetic
acid, 3:1) was dripped. After 10 minutes of incubation in a freezer, the fixative solution was changed
twice. The supernatant was removed by centifugation, leaving approximately 0.3 ml of material. Three
to four drops of cell suspension were dropped in a
cold slide, from a height of approximately 20 cm.
Slides were air-dried, placed onto a hot plate at 50oC
and stained with 5% Giemsa in phosphate buffer,
pH 6.8, for 7 minutes, followed by two washes in
distilled water and air drying.
Images of pachytenes and individual bivalents
were captured with a video camera and digitalized by
an image analysis system attached to the microscope
with immersion objective (100x).
RESULTS AND DISCUSSION
The slide preparation method allowed us to
obtain isolated pachytene chromosomes, with
good cytogenetic quality and adequate resolution to distinguish longitudinally arranged chromomeric structures. As an example, a chromomere map for bivalent 8 was prepared using this
method. Fig. 1a shows three bivalents 8 obtained
from cells in medium to late pachytene. The
chromomeric distribution was correspondently
accompanied by black dots. Detected chromomeres were linearly represented (Fig. 1b). In the
three bivalents shown, a correspondence of the
linear distribution of the chromomeric pattern
was observed.
The map (Fig. 1b) clearly shows the presence, in the short arm, of 10 chromomeres,
including the telomere. In the long arm, comprising the region from the centromere to the
large knob, it is possible to observe: five proximal chromomeres; one gap, followed by three
chromomeres separated from two more evident
ones; another gap; and 11 uniformly distributed
chromomeres. From the large knob to the distal
end of the chromosome, a region of poorly
defined chromomeres is observed, with the
identification of just the small knob, one adjacent chromomere and the telomere. A total of
35 chromomeres were identified in the map, 10
in the short arm and 25 in the long arm, including knobs and telomeres. The map was prepared
based only on the quantitative analysis of the
chromomeres, in order to obtain their number
and position.
55
SHEN and WU (1989) reported that, in maize,
a detailed correlation between physical and
genetic maps is difficult due to the absence of
intrachromosomal markers. In order to obtain
such markers, these authors prepared a chromomere map for chromosome 6 of this species,
based on electron microscopy data. Mapping of
the remaining bivalents was reported by W U
(1989) and SHEN and WU (1992). The pattern of
bivalent 8, in the work of WU (1989), shows 9
chromomeres in the short arm and 27 in the
long arm, without any mention of the presence
of knobs. In the present work, 10 chromomeres
were observed in the short arm, and 25 in the
long arm, including 2 knobs (Fig. 1). The difference between the number of chromomeres
reported here and in the literature can be
explained by: a) differences in the lineages, i.e.,
distinct lineages could show a variation in the
chromomeric pattern; b) different chromomeric patterns due to differences between the resolutions of light and electron microscopes; and
c) variation in the level of compactation of the
bivalents analyzed.
Small variations in the number of chromomeres were also observed in the same bivalent
from different pachytenes, which can be
explained by the different level of compaction of
the chromosomes. This type of variation has
already been mentioned by L UCIANI et al.
(1984), who affirmed that the number of chromomeres observed along human meiotic chromosomes varies as a function of the degree of
compaction of the pachytene from which they
originated.
The method developed here constitutes a
powerful tool for the elaboration of chromomere maps. The analysis of such maps can help
in the identification and characterization of
whole or segments of chromosomes, as well as in
studies of origins and evolution, relationships
among lineages and varieties, genomic organization and chromatin structure and function.
Therefore, the study of maize chromomeric
structures may contribute to the elucidation of
phenomena related to classical and molecular
genetics of this plant, and of higher organisms in
general.
Acknowledgements – The study was supported by the Fundação de Amparo a Pesquisa do
Estado de Minas Gerais (FAPEMIG) and Conselho Nacional de Pesquisa (CNPq).
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CAIXETA
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Received May 28, 2002; accepted August 27, 2002