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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 Â 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



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 Â 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 Â 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.





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 Â 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.



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 Â 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 Â 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 Â 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



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 Â 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.

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