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Genomic affinities between maize and Zea perennis using classical andmolecular cytogenetic methods (GISH Y FISH)
G. Gonzalez1,3*, C. Comas2, V. Confalonieri2, C. A. Naranjo1 & L. Poggio1,2
1 Instituto Fitote cnico de Santa Catalina (FCAF, UNLP) Y CIGen (CONICET-UNLP-CIC) C.C. 4, 1836
Llavallol, Buenos Aires, Argentina; 2 Departamento de Ecologı a, Gene tica y Evolucio n (FCEN, UBA), Buenos
Aires, Argentina; 3 Liniers no. 1169 Dpto. B. Te mperley, C.P. 1834, Buenos Aires, Argentina; Tel: +54-011-
4298-7669; Fax: +54-011-4282-0233; E-mail: [email protected]
*Correspondence
Received 23 February 2006. Received in revised form and accepted for publication by Adrian Sumner 11 April 2006
Key words: amphiploidy, GISH Y FISH, hybrids, in-situ hybridization, meiosis, Zea
Abstract
In this study we have analysed and compared the genomic composition, meiotic behaviour, and meiotic affinities
of Zea perennis and Zea mays ssp. mays. To do so we studied the parental taxa and the interspecific hybrid Zea
perennis  Zea mays ssp. mays, using classical cytogenetic methods, as well as GISH and FISH. GISH enabled
us to recognize the genomic source of each chromosome involved in the meiotic configurations of this hybrid,
and established the genomic affinities between their parental species. The results obtained here reinforce the
hypothesis of the amphiploid origin of Zea perennis and, together with previous research, indicate that the
chromosomes with divergent repetitive sequences in maize and Zea luxurians could be the remnants of a relict
parental genome not shared with Zea perennis.
Introduction
The genus Zea (Poaceae/Tribe Maydeae) comprises
two sections: Luxuriantes and Zea (Doebley 1990).
The section Luxuriantes (Doebley & Iltis), includes
the perennials Zea diploperennis Iltis (Doebley &
Guzman) and Zea perennis (Reeves & Mangelsdorf),
and the annuals Zea luxurians (Durieu & Ascherson)
(Bird) and Zea nicaragu ensis (Iltis & Benz 2000).The section Zea includes the annual Zea mays L.,
with its four subspecies Zea mays ssp. mays (maize),
Zea mays ssp. mexicana, Zea mays ssp. parviglumis
and Zea mays ssp. huehuetenanguensis.
The analysis of the meiotic behaviour of the chro-
mosomes in Zea hybrids has been frequently used to
evaluate their genomic homologies (Naranjo et al.
1990, 1994, Poggio et al. 1990, 1999b). Studies of
interspecific hybrids with 2n = 30 chromosomes were
especially important, as they provided strong cyto-
genetic evidence that x = 5 is the basic number of
these species, thus confirming the cryptic polyploid
nature of the genus (Naranjo et al. 1990, Poggio
et al. 1990, Poggio & Naranjo 1995). Based on
analyses of meiotic behaviour in parental species and
hybrids, we developed genomic formulae that denote
the genomic composition of each species (Poggioet al. 2005). It was thereby established unequivocally
for the first time that the extant n = 10 genomes of
Zea mays ssp. mays and its wild relatives are of
tetraploid origin, and that the higher ploidy level of
Zea perennis (n = 20) corresponds to an octoploid
origin (Naranjo et al. 1990, Poggio & Naranjo 1995).
The polyploid hypothesis was further confirmed by
evidence from genetic linkage maps (Moore et al.
Chromosome Research (2006) 14:629–635
DOI : 10.1007/s10577-006-1072-3
# Springer 2006
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1995, Gaut & Doebley 1997, White & Doebley 1998,
Gaut 2001). More recent cytogenetic and molecular
data have begun to expose evolutionarily significant
mechanisms of genetic diploidization in some of
these taxa (Lal et al. 2003, Lal & Hannah 2005,
Morgante et al. 2005, Buckler et al. 2006).
In-situ hybridization using total genome DNA
(GISH) as a probe is also a powerful tool to detect
genomic affinities between related species, especially
in hybrid plants and allopolyploids (Heslop-Harrison
et al. 1988, Schwarzacher et al. 1989, Bennett 1995,
Raina & Rani 2001). Used jointly with procedures
for blocking (competitive in-situ hybridization with
unlabelled DNA), GISH can enable one to discrim-
inate between closely related genomes in plants
(Anamthawat-Jonsson et al. 1990). Application of
this methodology revealed the genomic relatedness
between maize and other teosinte species with 2n = 20chromosomes, and also between maize and Tripsacum
(Poggio et al. 1999a,b, 2000, 2005, Gonzalez 2004,
Gonzalez et al. 2004). These studies provided new
evidence about their evolutionary relationships, and
also uncovered cryptic genomic divergences between
closely related taxa.
The present work deals with the analysis of the
meiotic behaviour of the Zea perennis  Zea mays
ssp. mays hybrid. Using GISH and FISH we have
established the genomic affinities between the paren-
tal species and, by analysing meiotic configurations
in the hybrid, have identified the genomic source of each chromosome.
Materials and methods
Plant material
The materials used in this study included Zea mays
ssp. mays (race Amarillo Chico) and Zea perennis
from Ciudad Guzman, Jalisco, Mexico. The seeds
of Zea perennis that we used were provided by Dr Prywed, Chapingo University, Mexico, and were culti-
vated at the Instituto Fitotecnico de Santa Catalina
(FCAF-UNLP), Llavallol, province of Buenos Aires
(35.0-S; 58.0-W).
Cytological analysis
Young panicles from Zea mays ssp. mays, Zea peren-
nis and their F1 hybrids were fixed in 3:1 (absolute
alcohol:acetic acid) solution and squashed in 2%
acetic haematoxylin. The pairing configurations were
determined at diakinesis-metaphase I. Only plates
showing well-spread cells were scored.
Fluorescence in-situ hybridization
Genomic DNA probes were isolated from adult leaves
of Zea mays ssp. mays and Zea perennis with the
Wizard Genomic DNA Purification Kit (Promega).
The pTa 71 plasmid, containing the 18S-5.8S-25S
ribosomal sequences fromTriticum aestivum (Gerlach
& Bedbrook 1979), was used as a probe. These probes
were labelled with Dig High Prime or Biotin Nick
Translation Kit (Boehringer Mannheim, Germany),
according to the manufacturer’s procedures.
Root tips were pretreated in 0.02 M 8-hydroxy-quinoline (Merck) for 3 h at room temperature, and
fixed in 3:1 ethanol:acetic acid for 24 Y 48 h. Fixed
root tips and young panicles were washed in 0.01 M
citric acid Y sodium citrate buffer (pH 4.6) to remove
fixative. They were then transferred to an enzyme solu-
tion containing 2% cellulase Onozuka R10 (Merck)
and 20% liquid pectinase (Sigma), and squashed in
a drop of 45% acetic acid. Slides were selected by
phase-contrast light microscopy. After removal of
coverslips by freezing, the slides were air-dried. The
in-situ hybridization technique was carried out as
described by Gonzalez et al. (2004). Slides wereexamined with a Carl Zeiss Axiophot epifluorescence
microscope. Photographs were taken using Kodak
Gold 400 colour print film.
Results
Meiotic behaviour
Zea mays ssp. mays (2n = 20, genomic formulaAmAmBmBm) shows regular meiosis, forming 10
bivalents (II) in metaphase I (Figure 1A). Zea
perennis (2n = 40, ApApA¶pA¶pBp1Bp1Bp2Bp2) is an
amphioctoploid showing a IV (tetravalent) range
from 2 to 6, the most frequent configuration being
5 IV + 10 II (Table 1, Figure 1B). The hybrid
Zea perennis  Zea mays ssp. mays (2n = 30,
ApA¶pAmBmBp1Bp2) forms five trivalents (III) + five
bivalents (II) + five univalents (I) as the most
630 G. Gonza lez et al.
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Figure 2. A,B: Mitotic metaphase of Zea mays ssp. mays. A: GISH using labelled Zea perennis DNA as probe, detected with yellow-green
FITC; B: DAPI counterstaining; arrows indicate four chromosomes with weaker fluorescence, and arrowheads show knobs without
fluorescence signals. C: GISH of mitotic metaphase of Zea perennis using labelled Zea mays ssp. mays DNA as probe, detected with yellow-
green FITC. D,E: FISH using the pTa71 probe detected with Cy3; D: two signals on metaphase chromosomes of Zea mays ssp. mays (DAPI
counterstaining); E: four signals on metaphase chromosomes of Zea perennis (propidium iodide counterstaining). F Y L: Meiotic chromosomes
of F1 hybrid Zea perennis  Zea mays ssp. mays. F,K,L: FISH using the pTa71 probe, detected with Cy3 (DAPI counterstaining); F,K: three
signals are observed on a trivalent, L: two signals on a bivalent and one on a univalent. G,I: GISH using unlabelled Zea perennis DNA for
blocking, and labelled Zea mays ssp. mays DNA as probe, detected with yellow-green FITC; G: univalent showing hybridization signal and
the bivalent without signal; I: Ffrying pan_-shaped trivalent showing a stronger hybridization signal on the chromosome Bhandle^ of the
Bfrying pan^; H,J: DAPI counterstaining. Bar = 10 mm.
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frequent configuration, in 55% out of 69 studied cells
(Table 1); the trivalents have the Ffrying pan_ shape
and the bivalents are homomorphic (Figures 1C,D
and 2F Y L).
In-situ hybridization experiments
Initial in-situ hybridization experiments targeted
mitotic chromatin of Zea mays ssp. mays and Zea
perennis. When total DNA of Zea perennis was
hybridized as a probe onto Zea mays ssp. mays chro-
mosomes, the fluorescence signal was absent from at
least two pairs of metacentric chromosomes per cell,
and from all heterochromatic (DAPI-positive) knobs
of maize (Figure 2A,B). A dispersed signal was
observed in the rest of the chromosomes. Different
results were observed in GISH experiments, wherelabelled maize DNA was hybridized to maize
chromosomes competitively with unlabelled total
DNA from Zea perennis. In this case there was
strong differential fluorescence on all DAPI-positive
knobs in maize. On the other hand, total labelled
DNA of Zea mays ssp. mays hybridized to Zea
perennis chromosomes yielded a hybridization signal
uniformly dispersed across the whole complement
(Figure 2C). Furthermore, GISH was carried out on
meiotic chromatin of the hybrid Zea perennis  Zea
mays ssp. mays (2n = 30). In this experiment the
chromosomes were blocked with unlabelled Zea perennis genomic DNA and probed with labelled
total genomic DNA from Zea mays ssp. mays. This
resulted in a fluorescence signal on all the univalents,
but on none of the bivalents (Figure 2G,H), further
indicating their homomorphic composition. Triva-
lents, where observed, showed a strong fluorescence
signal on the Fhandle_ of the Ffrying pan_ config-
urations (Figure 2I,J).
FISH experiments were also carried out using the
pTa71 probe (45S rDNA from Triticum aestivum),
which labels the nucleolar organizer regions. Whenthe pTa71 probe was hybridized to Zea mays ssp.
mays and Zea perennis mitotic cells, two and four
signals were detected respectively (Figure 2D,E).
When the rDNA probe was hybridized to meiotic cells
of the Zea perennis  Zea mays ssp. mays hybrid,
three fluorescence signals were observed on a single
trivalent (80% of 50 cells analysed) (Figure 2F,K), or
two signals on a bivalent plus one on a univalent
(20% out of 50 cells analysed) (Figure 2L).
Discussion
The association of homologous or homoeologous
chromosomes during meiosis reveals the relative
affinities between the parental genomes of the hybrids
and polyploid species. Moreover, these meiotic
configurations detect chromosomal rearrangements
that may act as reproductive isolation mechanisms.
When we did this type of analysis on Zea species,
and on artificial hybrids between species with equal
and different ploidy levels, we could deduce their
polyploid nature and the genomic formulae of all
species (Poggio et al. 2005). Accordingly, two dif-
ferent genomes were postulated to occur in these
cryptic polyploids, each with x = 5 chromosomes,
which were arbitrarily named FA_ and FB_. The
hypothetical formula proposed for 2n = 20 species
was AxAxBxBx, and for Zea perennis (2n = 40)ApApA¶pA¶pBp1Bp1Bp2Bp2 (Naranjo et al. 1994).
This paper reports the meiotic analysis of the
hybrid Zea perennis  Zea mays ssp. mays, whose
putative genomic formulae is ApA¶p AmBp1 Bp2Bm.
This hybrid formed 5 III + 5 II + 5 I, as the most
frequent configuration at metaphase I. It would not
be possible to recognize reliably the parental source
of the chromosomes involved in each meiotic config-
uration (i.e. III, II, I) using classical plant chromo-
some staining methods. We therefore used genomic
in-situ hybridization to deduce the chromosomal
composition of the meiotic configurations on agenome-of-origin basis in this hybrid, and thus
determine the actual meiotic affinities of the respec-
tive genomic components of these polyploids.
In the GISH experiment carried out on meiotic
cells of the hybrid we used a hybridization mixture
composed of labelled DNA from Zea mays ssp. mays
and unlabelled DNA from Zea perennis. We consis-
tently observed fluorescence signals on only one of
the chromosomes forming the Ffrying pan_-shaped
trivalents. This result indicates that the two unla-
belled chromosomes, due to the blocking procedure,belong to the Zea perennis parent, while the remain-
ing labelled chromosome belongs to Zea mays ssp.
mays. The bivalents never showed hybridization
signals, demonstrating that they were always derived
from Zea perennis, and univalents were always
labelled, showing that they belong to the Zea mays
ssp. mays parent. Therefore we state the following:
(a) trivalents are formed by autosyndetic pairing
(pairing of chromosomes coming from the same par-
Genomic affinities between maize and Zea perennis 633
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ental gametes) of genomes ApA¶p from Zea perennis
and by allosyndetic pairing (pairing of chromosomes
coming from different parental gametes) of genomes
Am from maize; (b) bivalents result from autosyn-
detic pairing of genomes Bp1 and Bp2 from Zea
perennis; (c) univalents correspond to genome Bmof
Zea mays ssp. mays. Similar results were obtained by
Poggio et al. (2000) when analysing the hybrid Zea
luxurians  Zea perennis. We therefore conclude
that the formation of bivalents and univalents is not
random, and that the FA_ genome of 2n = 20 species
is more homologous to the FA_ genomes of Zea perennis
than to its own FB_ genome, strongly suggesting a
hybrid origin for the genus, with a common progenitor
for both taxa.
The FISH experiment with the ribosomal 45S
showed that Zea perennis has four hybridization sig-
nals, whereas Zea mays ssp. mays has only two.Consequently, the hybrid showed three signals,
which usually appeared on all three chromosomes
involved in a trivalent. This observation would
indicate that ribosomal genes have remained linked
with homologous sequences in both species, which
would correspond to the A genome.
When total DNA of Zea perennis was hybridized
to Zea mays ssp. mays chromosomes, Takahashi
et al. (1999) observed a uniform hybridization signal
across the whole complement. However, when we
hybridized total DNA of Zea perennis onto Zea mays
ssp. mays chromosomes under high-stringency con-ditions, fluorescence was absent from two pairs of
chromosomes, and from all the maize heterochro-
matic knobs. Southern blot analysis previously
confirmed that knob sequences present in maize, and
the annual grass teosintes, Zea diploperennis and
Tripsacum, are absent in the Zea perennis genome
(Dennis & Peacock 1984, Ananiev et al. 1998,
Poggio et al. 2000). Therefore, the lack of hybrid-
ization of maize knobs can be taken as a negative
control for the hybridization experiment, giving
support to the fact that at least two pairs of Zeamays ssp. mays chromosomes have low homology in
repetitive sequences with Zea perennis. It is impor-
tant to note that four chromosomes of Zea luxurians
also have weak fluorescence signals when hybridized
with the Zea perennis probe (Poggio et al. 1999b).
These results reinforce the hypothesis of the amphi-
ploid origin of Zea perennis, and would indicate that
the chromosomes with divergent repetitive sequences
both in maize and Zea luxurians could be remnants
of a relict parental genome not shared with Zea
perennis.
Acknowledgements
This research was supported by grants from the
Agencia Nacional de Promocion Cientıfica y Tecno-
logica (ANDPCT-FONCYT) and Universidad de
Buenos Aires. We thank Mr Diego Fink (CONICET)
for image technical assistance. L.P., C.A.N. and V.C.
belong to the Researcher Carrier of the Argentine
Research Council (CONICET). G.G. has a scholar-
ship at the Universidad Nacional de la Plata.
References
Anamthawat-Jonsson K, Schwarzacher T, Leitch AR, Bennett MD,
Heslop-Harrison JS (1990) Discrimination between closely
related Triticeae species using genomic DNA as a probe. Theor
Appl Genet 79: 721 Y 728.
Ananiev EV, Phillips RL, Rines HW (1998) A knob associated
tandem repeat in maize capable of forming fold-back DNA
segments: are chromosome knobs megatransposons? Proc Natl
Acad Sci 95: 10785 Y 10790.
Bennett MD (1995) The development and use of genomic in situ
hybridization (GISH) as a new tool in plant biosystematics. In
Brandham PE, Bennett MD, eds., Kew Chromosome Conference
IV , Royal Botanic Gardens, Kew, pp. 167 Y 183.
Buckler ES, Gaut BS, McMullen MD (2006) Molecular and
functional diversity of maize. Current Opin Plant Biol 9: 1 Y 5.
Dennis E, Peacock W (1984) Knob heterochromatin homology in
maize and its relatives. J Mol Evol 20: 341 Y 350.
Doebley JF (1990) Molecular systematics of Zea (Gramineae).
Maydica 35: 143 Y 150.
Gaut BS (2001) Patterns of chromosomal duplication in maize and
their implication for comparative maps of the grasses. Genome
Res 11: 55 Y 66.
Gaut BS, Doebley JF (1997) DNA sequence evidence for the
segmental allotetraploid origin of maize. Proc Natl Acad Sci 94:
6809 Y 6814.
Gerlach WL, Bedbrook JR (1979) Cloning and characterization of
ribosomal RNA genes from wheat and barley. Nucleic Acids Res
7: 1869 Y 1885.
Gonzalez G (2004) Afinidades genomicas y mapeo cromosomico
en maız y especies relacionadas, a traves de estudios de cito-
genetica clasica y de hibridacion in situ. Tesis doctoral. Facultad
de Ciencias Exactas y Naturales, Universidad de Buenos Aires.
Gonzalez G, Confalonieri V, Comas C, Naranjo CA, Poggio L
(2004) GISH reveals cryptic genetic differences between maize
and its putative wild progenitor Zea mays ssp. parviglumis.
Genome 47: 497 Y 452.
Heslop-Harrison JS, Schwarzacher T, Leitch AR, Anamthawat-
Jonsson K, Bennett MD (1988) A method of identifying DNA
634 G. Gonza lez et al.
8/4/2019 trabajo Per x maíz
http://slidepdf.com/reader/full/trabajo-per-x-maiz 7/7
sequences in chromosomes of plants. European patent amplifi-
cation number 8828130.8.
Iltis HH, Benz BF (2000) Zea nicaraguensis (Poaceae), a New
Teosinte from Pacific Coastal Nicaragua. Novon 10: 382 Y 390.
Lal SK, Hannah LC (2005) Helitrons contribute to the lack of gene
colinearity observed in modern maize inbreds. Proc Natl Acad
Sci USA 102: 9993 Y 9994.
Lal SK, Giroux MJ, Brendel V, Vallejos CE, Hannan LC (2003) Themaizegenomecontainsa helitron insertion. Plant Cell 15: 381 Y 391.
Moore G, Devos KM, Wang Z, Gale MD (1995) Cereal genome
evolution. Grasses, line upand form a circle. Current Biol 5: 737 Y 739.
Morgante M, Brunner S, Pea G, Fengler K, Zuccolo A, Rafalski A
(2005) Gene duplication and exon shuffling by helitron-like
transposons generate interspecies diversity in maize. Nat Genet
37: 997 Y 1002.
Naranjo CA, Molina M, Poggio L (1990) Evidencias de un numero
basico x = 5 en el genero Zea y su importancia en estudios del
origendelmaız. Acad Nac Cs Ex Fı s Nat, Buenos Aires 5: 43 Y 53.
Naranjo CA, Poggio L, Molina M, Bernatene E (1994) Increase in
multivalent frequency in F1 hybrids of Zea diploperennis  Zea
perennis by colchicine treatment. Hereditas 120: 241 Y 244.
Poggio L, Naranjo CA (1995) Origen del maız: evidencias cito-geneticas. Reunio n Latinoam Zona Andina Inv Maı z, pp.
969 Y 980.
Poggio L, Molina M, Naranjo CA (1990) Cytogenetic studies in
the genus Zea. 2: Colchicine induced multivalents. Theor Appl
Genet 79: 461 Y 464.
Poggio L, Confalonieri V, Comas C, Cuadrado A, Jouve N,
Naranjo CA (1999a) Genomic in situ hybridization (GISH) of
Tripsacum dactyloides and Zea mays ssp. mays with B-
chromosomes. Genome 42: 687 Y 691.
Poggio L, Confalonieri V, Comas C, Gonzalez G, Naranjo CA
(1999b) Genomic affinities among Zea luxurians, Zea perennis
and Zea diploperennis: meiotic behaviour in the F1 and genomic
in situ hybridization (GISH). Genome 42: 993 Y 1000.Poggio L, Confalonieri V, Comas C, Gonzalez G, Naranjo CA
(2000) Evolutionary relationships in the genus Zea: analysis of
repetitive sequences used as cytological FISH marker. Genet
Mol Biol 23: 1021 Y 1027.
Poggio L, Gonzalez G, Confalonieri V, Comas C, Naranjo CA
(2005) The genome organization and diversification of maize
and its allied species revisited: evidences from classical and
FISH Y GISH cytogenetics analysis. Cytogenet Gen Res 109:
259 Y 267.
Raina SN, Rani V (2001) GISH technology in plant genome
research. Meth Cell Sci 23: 83 Y 104.
Schwarzacher T, Leitch AR, Bennett MD, Heslop-Harrison JS
(1989) In situ localization of parental genomes in a wide hybrid.
Ann Bot 64: 315 Y 324.Takahashi C, Marshall JA, Bennett MD, Leitch IJ (1999) Genomic
relationships in maize and its wild relatives. Genome 42: 1201 Y
1207.
White S, Doebley J (1998) Of genes and genomes and the origin of
maize. Trends Genet 14: 327 Y 332.
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