Cytogenetics and Plant Breeding

Cytogenet Genome Res 109:335343 (2005)

DOI: 10.1159/000082417
Production of alien chromosome additions and
their utility in plant genetics
S.-B. Chang and H. deJ ong
Laboratory of Genetics, Wageningen University, Wageningen (The Netherlands)
Received 20 October 2003; manuscript accepted 25 February 2004.
Request reprints from Hans de Jong, Laboratory of Genetics
Wageningen University, Arboretumlaan 4
NL6703 BD Wageningen (The Netherlands)
telephone: +31 317 482618; fax: +31 317 483146; e-mail: hans.dejong@wur.nl
ABC
Fax +41 61 306 12 34
E-mail karger@karger.ch
www.karger.com
2005 S. Karger AG, Basel
03010171/05/10930335$22.00/0
Accessible online at:
www.karger.com/cgr
Abstract. Breeding programs aiming at transferring desira-
ble genes from one species to another through interspecific
hybridization and backcrossings often produce monosomic and
disomic additions as intermediate crossing products. Such
aneuploids contain alien chromosomes added to the comple-
ments of the recipient parent and can be used for further intro-
gression programs, but lack of homoeologous recombination
and inevitable segregation of the alien chromosome at meiosis
make them often less ideal for producing stable introgression
lines. Monosomic and disomic additions can have specific
morphological characteristics, but more often they need addi-
tional confirmation of molecular marker analyses and assess-
ment by fluorescence in situ hybridization with genomic and
chromosome-specific DNA as probes. Their specific genetic
and cytogenetic properties make them powerful tools for funda-
mental research elucidating regulation of homoeologous re-
combination, distribution of chromosome-specific markers
and repetitive DNA sequences, and regulation of heterologous
gene expression. In this overview we present the major charac-
teristics of such interspecific aneuploids highlighting their ad-
vantages and drawbacks for breeding and fundamental re-
search.
Copyright 2005 S. Karger AG, Basel
Plant geneticists and breeders have gained great interest in
extending genetic variation of crop plants using exotic germ-
plasm from related species (Kalloo and Chowdhury, 1992; Kik,
2002). In a long term crossing program, known as introgressive
hybridization, economically or otherwise important genes were
incorporated into the recipient parent by sexual or somatic
hybridization between the crop and a related species or genus,
followed by consecutive backcrossings with the recipient parent
while selecting for the favourable trait(s). In the offspring fami-
lies aneuploid individuals could thus be isolated containing
only a single alien chromosome added to the full cell comple-
ment of the recipient parent. Monosomic additions were first
described by Leighty and Taylor (1924), but use and
potential were better demonstrated in the comprehensive study
of OMara (1940). Khush (1973), Gupta (1995) and Sybenga
(1992) gave a full overview of alien additions (the commonly
used term monosomic addition lines is incorrect because lines
assume homozygosity for a particular gene or chromosome
variant, and so will not segregate in the next generation) in rela-
tion to other aneuploids in plant genetics. Although the number
of papers on alien additions are almost countless, reports on full
sets with all alien chromosomes appeared only for monosomic
additions with chromosomes of Beta webbiana, B. patellaris
and B. procumbens to beet, Solanum lycopersicoides to tomato,
tomato to potato, Oryza officinalis to rice, a number of Triti-
ceae species, including rye and barley, to Triticum aestivum,
maize to oat, and onion to Allium fistulosum. Table 1 presents
an overview of complete sets, together with their parental spe-
cies, marker selections, cytogenetics and references. In this
paper we will discuss the most common techniques and prob-
lems encountered in the production of monosomic additions,
their chromosomal and molecular characterization and the sig-
nificance of these aneuploids for chromosome research and
plant breeding.
D
o
w
n
l
o
a
d
e
d

b
y
:

U
n
i
v
e
r
s
i
t
y

L
i
b
r
a
r
y

U
t
r
e
c
h
t





























1
3
1
.
2
1
1
.
2
0
8
.
1
9

-

6
/
6
/
2
0
1
4

9
:
1
4
:
3
6

P
M
336 Cytogenet Genome Res 109:335343 (2005)
Table 1. Overview of the complete sets of additions, their parental species, way of selection and references. Karyotype analysis includes both chromosome
counts and morphology.
Donor species Recipient species No. of full alien
addition sets
Marker selection Cytogenetics Reference
Aegilops speltoides Triticum aestivum (wheat) 7 Plant phenotype
RFLP
C-banding
FISH with repeat probes
Friebe et al., 2000b
Allium cepa (onion) Allium fistulosum 8 isozymes karyotype analysis Shigyo et al., 1996
Beta webbiana Beta vulgaris (beet) 9 plant phenotype and isozymes karyotype analysis Reamon Ramos and Wricke, 1992
Beta patellaris Beta vulgaris (beet) 9 repetitive DNA fingerprinting karyotype analysis Mesbah et al., 1997a
Beta procumbens Beta vulgaris (beet) 9 plant phenotype karyotype analysis van Geyt et al., 1988
Lycopersicon esculentum
(tomato)
Solanum tuberosum
(potato)
12 RFLP GISH Ali et al., 2001
Oryza officinalis Oryza sativa (rice) 12 plant phenotype karyotype analysis Jena and Khush, 1989
Solanum lycopersicoides Lycopersicon esculentum 12 plant phenotype karyotype analysis Chetelat et al., 1998
Wheat Various species 20 morphology karyotype analysis Shepherd et al., 1988
Zea mays (maize) Avena sativa (oat) 10 SSR markers karyotype analysis Kynast et al., 2001
Production of additions
Three major barriers can be distinguished in introgressive
hybridization: i) incompatibility or incongruence between the
parental species, and hence the difficulties in producing viable
interspecific or intergeneric hybrids; ii) sterility of the F1 and
sometimes also of the BC1, thus hampering transfer of alien
chromosomes through backcrossing and iii) rare or negligible
meiotic recombination between the alien chromosomes and
one of its homoeologous counterparts, preventing incorpora-
tion of alien chromatin into the recipient genome. Methods
have been developed to overcome these natural crossing bar-
riers. One of them makes use of chromosome doubling to pro-
duce allopolyploids from which fertile allotriploids can be
derived. A second method makes use of pollen mixes, contain-
ing pollen for syngamy and mentor pollen from the maternal
species to facilitate fertilization by foreign pollen (Brown and
Adiwilaga, 1991). Alternatively, desirable traits can be trans-
ferred indirectly using a bridge cross with a related (wild) spe-
cies or cultivar that is compatible with either parental species.
Although time consuming, such interim crosses have success-
fully been applied to various crop species including alfalfa
(McCoy and Echt, 1993), beet (Savitsky, 1960), lettuce (Eenink
et al., 1982) and onion (Khrustaleva and Kik, 2000).
The development of cell culture technology for protoplast
fusion and regeneration, for the production of somatic hybrids
from fused protoplasts of related species that are incompatible
in a sexual cross was a further step to bridge the gap between
non-crossable species. Melchers et al. (1978) was the first to
make somatic hybrids, later followed by many others (for
review in tomato genetics, see Wolters et al., 1994). A highly
advanced example of somatic hybridization technology was
developed by Ramulu et al. (1996) who isolated micronuclei
containing one or few chromosomes from the donor species
and fused them with complete euploid protoplasts from the
recipient crop. This microprotoplast fusion technique directly
produced asymmetric somatic hybrids with the cell comple-
ment of a monosomic addition, thus skipping the problems of
interspecific incongruity and BC1 sterility. Although potential-
ly very promising, this advanced strategy never received large-
scale practical application.
Hybrid embryo abortion after fertilization is an important
problem generally occurring in (somatic) hybrids from wide
crosses and reflects disharmony between the genomes of the
parental species that results in embryo mortality, endosperm
break down, seed inviability and hybrid sterility. Different in
vitro embryo rescue techniques were developed to overcome
this problem of hybrid embryo degeneration and have been
applied to a large variety of crops and ornamental species (for
review see Sharma et al., 1996; van Tuyl et al., 1997). Once the
backcross progenies were recovered, aneuploid individuals
with alien chromosomes, including monosomic additions
could be isolated. As maintenance of monosomic additions was
difficult due to sterility, inferior viability and low chromosome
transmission through the germline, additions of potato, onion
and beet background were often kept in vitro or retained by
vegetative propagation. Transmission of the alien chromo-
somes and viability of the additions can differ considerably so
that certain monosomic additions with specific chromosomes
were often difficult to obtain. Production of disomics was only
possible when the alien chromosome was also transmitted
through the male germ line, or they can be obtained by meiotic
non-disjunction of the alien chromosome in the monosomic
addition parent. Use of derived ditelosomic additions, in which
single individual alien chromosomes were replaced by their tel-
ocentrics, often improved chromosomal transmission consider-
ably, as was shown for rice (Yasui and Iwata, 1998).
The final step in introgressive hybridization programs in-
volves meiotic recombination between the alien chromosome
and one of its homoeologous counterparts in order to stably
incorporate its chromatin into the recipient parental genome.
Generally, additions derived from wide interspecific or inter-
generic hybrids display little or no homoeologous recombina-
tion at all, and hence leave the alien chromosomes as univalents
at metaphase I. In the corresponding substitution lines, associa-
tion of homoeologous chromosomes may be far higher due to
the absence of homologous partners (Ji and Chetelat, 2003).
Transfer of desired chromosome regions to wheat is generally
most successful in the Ph1 background of wheat allowing high
levels of homoeologous recombination (Ceoloni, 1984). Other
means of alien chromosome introgression can be achieved by
breakage and fusion of chromosome fragments, either spon-
D
o
w
n
l
o
a
d
e
d

b
y
:

U
n
i
v
e
r
s
i
t
y

L
i
b
r
a
r
y

U
t
r
e
c
h
t





























1
3
1
.
2
1
1
.
2
0
8
.
1
9

-

6
/
6
/
2
0
1
4

9
:
1
4
:
3
6

P
M
Cytogenet Genome Res 109:335343 (2005) 337
Fig. 1. Genomic painting (genomic in situ
hybridization) shows the number and behaviour
of alien chromosomes in backcross individuals.
Here we show the monosomic addition carrying
chromosome 3 of tomato added to tetraploid
potato (2n = 4x + 1 = 49) hybridized with total
genomic tomato DNA as probe and 50 unla-
beled potato DNA as competitor. (a) Mitotic me-
taphase complement. (b) Pachytene complement.
Bars equal 10 m.
taneously by radiation-induction (Sears, 1956; Friebe et al.,
1993; Ahmad et al., 2000; Okagaki et al., 2002) or by a gameto-
cidal factor in Aegilops cylindrical (Friebe et al., 2000a), which
have especially been applied in the cereal species.
Characterization of the alien chromosome(s)
Monosomic additions can be selected on the basis of specific
alien traits, like disease resistance, aberrant plant phenotype,
species-specific molecular markers and karyotype analysis to
demonstrate the presence of an extra chromosome. Morpholog-
ical traits can be qualitative, like the characteristic liguleless
leaves of the maize chromosome 3 monosomic addition in oat
(Muehlbauer et al., 2000) and the monogenic dominant resis-
tance genes, or inherit quantitatively, such as plant size and
spike morphology. The morphological traits in the monosomic
addition set of Beta vulgaris carrying an extra chromosome
from B. procumbens or B. patellaris appear only partly chromo-
some-specific, and hence are not adequate for the identification
of all alien chromosomes without additional markers (Mesbah
et al., 1997a). Monosomic additions derived from hybrids
between genetically related parental species can have pheno-
types often resembling its corresponding primary trisomics
(Chetelat et al., 1998).
Alien chromosomes in the additions may sometimes be
morphologically distinguishable, as was shown in a karyotype
analysis of four nematode resistant monosomic additions of
beet containing different alien chromosomes from Beta pro-
cumbens or B. patellaris (de Jong et al., 1985). Chromosome
morphology was for a long time described in terms of centro-
mere position, arm lengths, C- and N-banding profiles, and
heterochromatin pattern in cell complements at pachytene
(Multani et al., 1994). Flow cytometry can be helpful when
chromosome size and GC/AT ratio are sufficient to identify the
alien chromosome in the flow karyogram (Shigyo et al., 2003)
and can even be applied to isolate large numbers of alien chro-
mosomes, as was demonstrated for a monosomic addition with
maize chromosome 9 to oat (Li et al., 2001).
The by far most important tool to visualise alien chromo-
somes is genomic in situ hybridization (GISH) or genome
painting, a fluorescence in situ hybridization protocol using
total genomic donor DNA as probe and non-labelled DNA
from the recipient parent as competitor (Schwarzacher et al.,
1989). An example of GISH of a monosomic addition of toma-
to chromosome 3 in potato is shown in Fig. 1a and b. Reports
on establishing the number of alien chromosomes in intergen-
eric backcross families are numerous (Raina and Rani, 2001),
like tomato to potato (Jacobsen et al., 1995), maize to oat
(Riera-Lizarazu et al., 1996), Beta corolliflora in beet (Gao et
al., 2001), Solanum brevidens to potato (Dong et al., 2001), and
S-genome chromosomes in wheat (Belyayev et al., 2001). The
technique is also considered effective in studying individual
chromosomes in plants (Schubert et al., 2001). GISH has also
the advantage of demonstrating interspecific and intergeneric
translocations (Mukai et al., 1993), and shows recombinant
chromosomes resulting from homoeologous recombinations
(Barthes and Ricroch, 2001; King et al., 2002).
For the identification of the alien chromosome in the addi-
tion stocks, additional analysis is required using chromosome-
specific wild morphological traits (Fernandez and Jouve,
1988; Jena and Khush, 1989; Morgan, 1991; Reamon Ramos
and Wricke, 1992; Littlejohn and Pienaar, 1995; Shigyo et al.,
1996; Mesbah et al., 1997a; Gao and Jung, 2002), isozyme
markers (Quiros et al., 1987; Peffley and Currah, 1988; van
Geyt et al., 1988; Reamon Ramos and Wricke, 1992; Delos et
al., 1998), molecular markers including RFLPs (see Fig. 2, and
in Friebe et al., 2000b; Garriga-Calder et al., 1997, 1998; Jia et
al., 2002), RAPDs (Jorgensen et al., 1996; Kaneko et al., 2000),
AFLPs (van Heusden et al., 2000), microsatellites (Hernandez
et al., 2002; Malysheva et al., 2003) and repetitive sequence
DNA fingerprints (Riera-Lizarazu et al., 1996; Mesbah et al.,
1997a; Gao et al., 2001).
The integrity and identification of the alien chromosome in
a monosomic addition carrying a Solanum brevidens chromo-
some in potato background were obtained by sequential ge-
nomic painting and FISH with BACs diagnostic for each chro-
mosome (Dong et al., 2001). The advantage of this 2-step pro-
cedure is that both genomic origin and genetic identity of the
alien chromosome can be established in a single experiment.
D
o
w
n
l
o
a
d
e
d

b
y
:

U
n
i
v
e
r
s
i
t
y

L
i
b
r
a
r
y

U
t
r
e
c
h
t





























1
3
1
.
2
1
1
.
2
0
8
.
1
9

-

6
/
6
/
2
0
1
4

9
:
1
4
:
3
6

P
M
338 Cytogenet Genome Res 109:335343 (2005)
Fig. 2. RFLP molecular markers for the characterization and selection of
monosomic additions with a specific alien chromosome. Southern hybridiza-
tion of DraI/EcoRI digested genomic DNA with the tomato chromosome 3
specific marker tg251. Lanes: M = /HindIII marker, T = tomato, P = potato
and 111, a series of offspring plants from a monosomic addition 3 back-
cross.
Properties of additions
The potential of alien additions for breeding programs
largely depends on the genetic distance of the parental species
and hence, on the possibility of the alien chromosome to
recombine with one of its homoeologous counterparts by cross-
ing over. When parents can be combined in a sexual cross, such
as wheat and rye, maize and Pennisetum, Festuca and Lolium,
crossovers between homoeologous chromosomes are not rare,
which may reveal recombinant chromosomes, even in the first
backcross generations. In the case of distant parental species
when somatic hybridization and embryo rescue are required
for the production of monosomic additions, the alien chromo-
some generally fails to synapse and/or recombine at meiotic
prophase I, remains lagged behind in the equatorial plane at
anaphase I and may get lost at later stages (cf. Khush, 1973;
Sybenga, 1992). Its univalence may result in centromere break-
age and thus producing monotelosomic and isotelosomic addi-
tions in the progeny (Lange et al., 1993; Yasui and Iwata,
1998). In the derived disomic additions, the two alien chromo-
somes will pair and form bivalents and will segregate at ana-
phase I, but in a few cases they may fail to pair or form chiasma-
ta, resulting in the formation of univalents and hence, in unbal-
anced gametes (Khush, 1973).
The extent of crossover recombination between the ho-
moeologues in the monosomic additions depends primarily on
the genetic relation between the parental species, but can also
vary between the different alien chromosomes in the mono-
somic additions and in different genetic backgrounds. When
homoeologous recombination in the interspecific hybrids is
rare, it will be even more seldom or entirely absent in the
derived backcross generations due to high preferential pairing.
Additional causes for suppression of crossover and recombina-
tion are the heterochromatic pericentromeric chromosome re-
gions (Chetelat et al., 2000) and heterozygosity for (small) chro-
mosomal rearrangements like duplications, inversions and
translocations or pericentromeric regions (Tanksley et al.,
1992; Chetelat et al., 2000; Ji and Chetelat, 2003).
Geneticists have undertaken several strategies to improve
homoeologous recombination. Wheat genotypes lacking the
Ph1 locus that controls suppression of homoeologous recombi-
nation are often used in breeding programs for transferring
desirable genes from rye, and other related cereal species to
wheat (Ceoloni, 1984). More recent studies on the mode of Ph1
have shown that the locus does not directly control homoeolo-
gous recombination suggesting the need for an entirely new
approach to the introgression of alien genetic variation into
wheat (Miller et al., 1998). Very little is known about other
genetic systems controlling synapsis and recombination be-
tween homoeologous chromosomes in plant polyploids. In Lo-
lium amphidiploids Jenkins and Jimenez (1995) found that
diploidizing genes carried by A-chromosomes and supernu-
merary B-chromosomes controlled bivalent formation. This
system has so far not been applied to introgressive hybridiza-
tion programs.
Diploid plants with one chromosome replaced by its ho-
moeologue can be easily obtained in backcross offspring fami-
lies of interspecific hybrids and monosomic additions. The
heteromorphic (homoeologous) bivalents in such monosomic
substitutions generally demonstrate higher levels of crossover
recombination between the alien chromosome and its homoeo-
logous counterpart than in the corresponding monosomic addi-
tion, and are therefore more appropriate for producing recom-
binant chromosomes (Ji and Chetelat, 2003). King et al. (2002)
produced a large series of substitution lines from Festuca pra-
tensis Lolium perenne hybrids and used GISH to establish the
sites of homoeologous crossover events in the recombinant
chromosomes. The range of substitution lines each with differ-
ent recombinant chromosomes provided excellent material for
physical mapping the introgressed F. pratensis chromosome
segments and comparing genetic and physical maps for the
molecular markers on these chromosomes.
Gamma-ray irradiation of monosomic oat-maize additions
was used to produce radiation hybrid lines containing centric
maize chromosome fragments or oat-maize translocation chro-
mosomes. These lines, which lack one or more parts of the orig-
inal maize chromosome, allow mapping of molecular markers
on a sub-chromosome level of the maize genome (Riera-Lizara-
zu et al., 2000; Okagaki et al., 2002).
Transmission rates are generally far higher through the
female than the male line, and vary greatly between and among
the addition sets, as was found for Oryza australiensis to rice
(Multani et al., 1994), Solanum lycopersicoides to tomato (Ji
and Chetelat, 2003) and onion to A. fistulosum (Shigyo et al.,
2003). In the monosomic additions of Solanum lycopersicoides
D
o
w
n
l
o
a
d
e
d

b
y
:

U
n
i
v
e
r
s
i
t
y

L
i
b
r
a
r
y

U
t
r
e
c
h
t





























1
3
1
.
2
1
1
.
2
0
8
.
1
9

-

6
/
6
/
2
0
1
4

9
:
1
4
:
3
6

P
M
Cytogenet Genome Res 109:335343 (2005) 339
chromosomes to tomato, transmission for chromosome 10 was
24%, whereas for chromosome 6 no transmission at all could be
recorded (Chetelat et al., 1998). In the extensive study of Ali et
al. (2001) transmission values amounted to 032% for chromo-
some 9, whereas that of chromosome #6 varied from 14 to 88%
between the different families. Transmission of the alien chro-
mosomes in the eight Allium monosomic additions varied from
9 to 49% and was always far less than 10% through the female
line (Shigyo et al., 2003). Transmission rate can vary consider-
ably for the different alien chromosomes and is generally higher
for the larger chromosomes (Garriga-Calder et al., 1998). A
recent study encompassing three consecutive generations in the
Brassica rapa monosomic additions in Raphanus sativus, re-
vealed average transmission rates ranging from 26 to 44% (Ka-
neko et al., 2003).
Applications and scientif ic impact of additions
Introgressive hybridization
We have seen that alien additions are primarily produced to
add specific desirable genes to a crop or model species, via sta-
ble disomic additions or substitutions with (almost) complete
transmission rates, or by introgression of the favourable chro-
mosomal segment by homoeologous recombinations. Although
molecular and cell biological strategies based on site-specific
transformations are more effective in cases of known genes or
DNA sequences, introgressive hybridizations through alien
chromosomal additions remain an important strategy in cases
of complex polyfactorial or quantitative inheritable traits. Ka-
ryotype analyses of different nematode resistant, monosomic
additions of beet (Beta vulgaris) carrying a chromosome from
B. procumbens or B. patellaris revealed that different chromo-
somes from the donor species contain genes for resistance, each
with specific properties on their resistance level (de Jong et al.,
1985; Mesbah et al., 1997b).
Dhaliwal et al. (2002) characterized the transfer of rust
resistance from Aegilops ovata into bread wheat (Triticum aes-
tivum L.). Of special interest is the production and character-
ization of aneuploids carrying genes for apomixis, and so pro-
duce seeds clonally. As meiosis is skipped, at least for the reduc-
tional part, offspring is almost identical to the mother, so
monosomic additions and other aneuploids can be retained for
breeding purposes. Kindiger et al. (1996) demonstrated the
power of this technique to assign gene(s) for apomixis on the
alien chromosomes in Tripsacum maize hybrids, while Mor-
gan et al. (1998) established mapping of the apomixis trait on
alien chromosomes in Pennisetum hybrids. Also Gao and Jung
(2002) pointed out that identification of monosomic additions
from Beta vulgaris B. corollinae hybrids with apomictic and
disease resistance characters offers the possibility of transfer-
ring those genes to sugar beet.
Due to self-sterility or chromosomal instability in the
monosomic additions that were produced for introgressing
genes into crops, Taketa and Takeda (2001) obtained a com-
plete set of wheat-wild barley (Hordeum vulgare ssp. sponta-
neum) chromosome additions through disomic or double mon-
osomic additions.
Gene/marker localization
The correlation between specific alleles and molecular
markers or repeat fingerprints and the presence of an alien
chromosome not only helps to identify the alien chromosome
in the monosomic and disomic additions, but also allows
assigning loci or linkage groups on chromosomes. As to the
former, identification and characterization of the alien chro-
mosomes are often based on RFLP, AFLP or other molecular
marker assays in combination with chromosome banding or
genomic painting to check chromosome integrity (Jacobsen et
al., 1995; Suen et al., 1997; Fox et al., 2001; Zhang et al., 2002).
As to the latter, many genes of cultivated rye and barley have
been assigned to chromosomes using the addition stocks, or
marker variants that are unique for a given addition, but pre-
viously not mapped due to lack of polymorphism in the corre-
sponding donor species, could now be assigned to the alien
chromosomes (van Heusden et al., 2000; Okagaki et al., 2001;
Chang et al., our own unpublished results).
Construction of chromosome-specific libraries
Another important application of additions is the produc-
tion of DNA libraries from specific chromosomes using ge-
nomic DNA of the alien addition. Using this strategy, Ananiev
et al. (1997) constructed a cosmid library of maize chromo-
some-specific sequences using disomic additions with maize
chromosome 9 in oat, that were screened along with their par-
ents as control with a mixture of labelled highly repetitive
maize sequences. In a total of 5000 clones from the disomic
addition 29 of them were shown to contain maize DNA, of
which eight produced a chromosome-specific pattern that
could be used for chromosome identification. In a so called
representational difference analysis, Chen et al. (1998) used
repeated genomic subtraction of DNA of the monosomic addi-
tion carrying chromosome 3 in oat to reveal a subtraction libra-
ry in which maize DNA sequences were enriched from 1.8 to
72% of the total DNA. Most of the DNA sequences contained
multiple or repeated DNA sequences that do not cross-hybrid-
ise to oat sequences.
Heterologous gene expression
Alien additions also provide a unique system to investigate
heterologous expression of genes of alien chromosomes in the
genetic background of a wide relative. The aberrant morpho-
logical trait in a monosomic addition carrying maize chromo-
some 3 in oat was used to study the ectopic expression of the
maize liguleless 3 homoeobox gene that in this monosomic
addition results in few characteristic morphological abnormali-
ties of leaf and panicle and outgrowth of axillary buds (Muehl-
bauer et al., 2000). This type of research is especially important
for ectopic expression of disease resistance genes, their depen-
dence of genes on other chromosomes of the donor species, and
the effect of different genetic backgrounds of the recipient spe-
cies, but little molecular experiments have so far been carried
out (Hu et al., 1996).
D
o
w
n
l
o
a
d
e
d

b
y
:

U
n
i
v
e
r
s
i
t
y

L
i
b
r
a
r
y

U
t
r
e
c
h
t





























1
3
1
.
2
1
1
.
2
0
8
.
1
9

-

6
/
6
/
2
0
1
4

9
:
1
4
:
3
6

P
M
340 Cytogenet Genome Res 109:335343 (2005)
Organization of the alien chromosomes
Aspects of chromosome arrangements into the nucleus as
the relative position of homologues can be studied in disomic
additions using GISH (an example is provided by Corredor et
al. in this issue). Alkhimova et al. (1999) studied rye chromo-
some variability in a set of monosomic addition and substitu-
tion lines of rye chromosomes in Chinese Spring wheat back-
ground, along with their parental species, using FISH with the
pSc200 and pSc250 tandem repeats as probes. The in situ
hybridizations showed chromosome-specific localizations al-
lowing identification of most rye chromosome arms. Polymor-
phisms for the repeats in the monosomic additions were dem-
onstrated in all chromosomes except 4R and 6R. The substitu-
tion lines only showed a structural change in chromosome 2R
compared to the parent, suggesting that substitution lines pos-
sess far better chromosome stability than the additions.
More evidence for chromosomal instability came from a
study of the telomere ends of a somatic tomato (+) potato
hybrid, and its derived backcrossings. Using the Arabidopsis
type TTTAGGG telomere and the tomato-specific subtelomere
tandem repeat TGR1 as probes for Southern analysis of BglII/
EcoRV digested genomic DNA and FISH analyses of extended
DNA fibres, dramatic loss of both telomere repeats were
reported for the somatic hybrid and backcrossings. FISH obser-
vations of the repeats on mitotic chromosomes demonstrated
further confirmed loss of TGR1 sites (De Jong et al., 2000). A
comparable example of terminal deletions was reported for
Nicotiana sylvestris monosomic additions in N. plumbaginifol-
ia background (Chen et al., 2001).
In plant chromosomes a greater part of the tandem repeats
(apart from the 2 rDNAs) and most of the dispersed repeats
occur on all chromosomes. Elucidating their precise distribu-
tion and genome wide molecular organization is therefore
extremely laborious and time consuming. FISH of such repeats
on the alien chromosomes in monosomic and disomic addi-
tions is a more powerful alternative way to assess the position
and molecular size of the repetitive sequences on the chromo-
somes and extended DNA fibres. De Jong et al. (2000) used
this approach to reveal the size of the telomeric repeat
(TTTAGGG) and the tomato-specific subtelomeric repeat
TGR1 in the short arm of chromosome 6 of tomato using FISH
on mitotic and meiotic cell spreads and extended DNA fibres
of a monosomic addition with that tomato chromosome in
potato. The molecular length of these tandem arrays were
shown to correspond exactly with one of the telomere domains
as previously demonstrated for the tomato parent (Zhong et al.,
1998). Mesbah et al. (2000) used a series of monosomic addi-
tions to establish the physical localization and organization of
the Procumbentes-specific repetitive DNA sequence, PB6-4,
on the chromosomes of Beta procumbens (2n = 18), using FISH
to mitotic chromosomes and extended DNA fibres. As this
repeat hybridizes to all B. procumbens chromosomes and not to
any of the B. vulgaris, PB6-4 could be well studied in the
monosomic additions displaying characteristic fluorescent sig-
nals on the alien chromosomes. FISH to extended DNA fibres
revealed different classes of fluorescent tracks for the alien
chromosomes. Measurements of the fluorescent tracts allowed
classification into discrete groups, varying from one to three
groups per B. procumbens chromosome.
Studies on chromosome pairing of alien chromosome
pairs using GISH
Disomic additions are particularly suitable for studying
chromosome pairing at meiosis. Several studies of additions of
rye and barley chromosomes in wheat using FISH and confocal
microscopy with total genomic rye DNA on cell spreads and
intact pollen mother cells from anther sections deal with pre-
meiotic associations of (alien) homologues in cereals, and their
alignment and pairing at meiotic prophase, and focus on the
effect of ph1 on these processes (Schwarzacher, 1997; Mikhai-
lova et al., 1998; Martinez-Perez et al., 1999; Maestra et al.,
2002).
Use in genomics and physical mapping
Flow sorting can be effective in isolating large samples of
alien chromosomes from metaphase suspensions if the flow
karyograms from sorted monosomic addition complements
demonstrate distinct peaks not present in those of the parental
species. Li et al. (2001) applied this method for flow sorting the
maize chromosome 9 using its monosomic addition in oat. The
same band could not be isolated from the maize flow karyo-
gram itself. The efficiency of sorting was 6 10
3
chromosomes
from a chromosome suspension obtained from 30 root tips, and
at a purity of more than 90%. Kubala kova et al. (2003) used a
monosomic addition set with rye chromosomes in wheat for
isolating the rye chromosomes 2R7R that could not be dis-
criminated in the flow karyotype of Secale cerale Imperial
itself.
An alternative method for isolating chromosome-specific
DNA sequences is microdissection of chromosomes that are
morphologically recognisable in mitotic or meiotic cell comple-
ments. When chromosomes are morphologically too similar for
distinction in the karyotype, they can often be distinguished in
metaphase I complements in aneuploids, either as trivalents in
trisomics, or as univalents in monosomics and monosomic
additions (Potz et al., 1996; Tian et al., 2000; Dong et al., 2002;
Yuzhu et al., 2002).
Ananiev et al. (1997) used a disomic addition library with
the maize chromosomes 2, 3, 4, 7, or 9 in hexaploid oat to con-
struct chromosome-specific cosmid libraries. The multi-probes
of the maize-specific repetitive sequences selected the maize
chromosome-specific cosmid clones. Instead of constructing
each maize chromosome (or precisely maize repetitive se-
quences)-specific libraries, we propose here an efficient and
accurate method to enrich chromosome-specific BAC libraries.
Several laboratories have constructed BAC genome libraries of
a certain number of species. Use monosomic additions-
genomic DNA as a probe to select the chromosome-specific
BAC clones subtracted from the host genome. This approach
makes the libraries enriched not only for alien repetitive
sequences but also unique elements. In most plants, repetitive
D
o
w
n
l
o
a
d
e
d

b
y
:

U
n
i
v
e
r
s
i
t
y

L
i
b
r
a
r
y

U
t
r
e
c
h
t





























1
3
1
.
2
1
1
.
2
0
8
.
1
9

-

6
/
6
/
2
0
1
4

9
:
1
4
:
3
6

P
M
Cytogenet Genome Res 109:335343 (2005) 341
sequences are abundant and distributed along the chromo-
somes complex. Getting chromosome-specific BAC clones can
thus be achieved by the additional blockage of the Cot10100
fraction, which is a pool of high, middle and low copy repetitive
sequences.
Conclusions and f uture perspectives
Additions can basically be subdivided into two classes, each
with their own properties and applications: i) Class 1 in which
homoeologous recombination between the alien chromosome
and one of its homoeologous counterparts occurs at reasonable
frequency. These additions produce various recombinants and
so are particularly suitable for introgressive hybridization, for
comparing physical and genetic maps, and for studying the
effect of individual chromosomes on crossover (chiasma) dis-
tribution and frequencies. More knowledge is required espe-
cially about the genes controlling homoeologous recombina-
tion, chiasma distribution and unreduced gamete formation,
and for genetic programs aiming at producing monosomic sub-
stitutions in order to force homoeologous chromosomes to pair
and synapse. ii) In class 2 homoeologous recombination does
not occur in a normal genetic background, i.e., not disturbed in
genes controlling meiotic recombination, homoeologous re-
combination or unreduced forms of meiosis (like first and sec-
ond division restitution). This group of interspecific aneuploids
are less attractive for breeders as long as introgressive hybridi-
zation cannot be achieved by homoeologous recombination,
but can provide significant information for various cytogenetic
and genomic studies including chromosome disposition, mo-
lecular organization of repetitive and single copy sequences,
heterologous gene expression and starting material for flow-
sorted or microdissected alien chromosome samples for chro-
mosome-specific DNA sequences.
Ref erences
Ahmad F, Comeau A, Chen Q, Collin J, St Pierre CA:
Radiation induced wheat-rye chromosomal trans-
locations in triticale: optimizing the dose using flu-
orescence in situ hybridization. Cytologia 65:16
(2000).
Ali SNH, Ramanna MS, Jacobsen E, Visser RGF:
Establishment of a complete series of a monosomic
tomato chromosome addition lines in the culti-
vated potato using RFLP and GISH analyses. The-
or Appl Genet 103:687695 (2001).
Alkhimova AG, Heslop Harrison JS, Shchapova AI,
Vershinin AV: Rye chromosome variability in
wheat-rye addition and substitution lines. Chro-
mosome Res 7:205212 (1999).
Ananiev EV, Riera Lizarazu O, Rines HW, Phillips RL:
Oat-maize chromosome addition lines: a new sys-
tem for mapping the maize genome. Proc Natl
Acad Sci USA 94:35243529 (1997).
Barthes L, Ricroch A: Interspecific chromosomal rear-
rangements in monosomic addition lines of Al-
lium. Genome 44:929935 (2001).
Belyayev A, Raskina O, Nevo E, Hernandez P: Detec-
tion of alien chromosomes from S-genome species
in the addition/substitution lines of bread wheat
and visualization of A-, B- and D-genomes by
GISH, in Proceedings of the Fourth International
Triticeae Symposium, Cordoba, Spain, September
2001. Hereditas Lund 135:23, 119, 122 (2001).
Brown CR, Adiwilaga KD: Use of rescue pollination to
make a complex interspecific cross in potato. Am
Potato J 68:813820 (1991).
Ceoloni C: Transfer of a mildew resistance gene from
Triticum longissimum to common wheat by in-
duced homoeologous recombination. Genetica
Agraria 38:326327 (1984).
Chen CC, Kao YY, Lee FM, Lin RF: Somatic hybriza-
tion between Nicotiana sylvestris Speg. and Comes
and N. plumbaginifolia Viv, in Nagata, Bajaj (eds):
Somatic Hybridization in Crop Improvement II
Biotechnology in Agriculture and Forestry 49:292
303 (2001).
Chen ZJ, Phillips RL, Rines HW: Maize DNA enrich-
ment by representational difference analysis in a
maize chromosome addition line of oat. Theor
Appl Genet 97:337344 (1998).
Chetelat RT, Rick CM, Cisneros P, Alpert KB, DeVer-
na JW: Identification, transmission, and cytologi-
cal behavior of Solanum lycopersicoides Dun.
monosomic alien addition lines in tomato (Lyco-
persicon esculentum Mill.). Genome 41:4050
(1998).
Chetelat RT, Meglic V, Cisneros P: A genetic map of
tomato based on BC1 Lycopersicon esculentum
Solanum lycopersicoides reveals overall synteny
but suppressed recombination between these hom-
eologous genomes. Genetics 154:857867 (2000).
Corredor E, Dez M, Shepherd K, Naranjo T: The posi-
tioning of rye homologous chromosomes added to
wheat through the cell cycle in somatic cells un-
treated with colchicine. Cytogenet Genome Res
109:112119 (2005).
De Jong JH, Speckmann GJ, de Bock TSM, van Voorst
A: Monosomic additions with resistance to beet
cyst nematode obtained from hybrids of Beta vul-
garis and wild Beta species of the section Patel-
lares. II. Comparative analysis of the alien chromo-
somes. Z Pflanzenzuechtg 95:8494 (1985).
De Jong JH, Zhong X-B, Fransz P, Wennekes-Van
Eden J, Jacobsen E, Zabel P: High resolution FISH
reveals the molecular and chromosomal organisa-
tion of repetitive sequences of individual tomato
chromosomes, in: Chromosomes Today Vol 13, pp
267275 (Birkhuser Verlag, Basel 2000).
Delos RBG, Khush GS, Brar DS: Chromosomal loca-
tion of eight isozyme loci in rice using primary tri-
somics and monosomic alien addition lines. J
Hered 89:164168 (1998).
Dhaliwal HS, Harjit S, William M: Transfer of rust
resistance from Aegilops ovata into bread wheat
Triticum aestivum L. and molecular characterisa-
tion of resistant derivatives. Euphytica 126:153
159 (2002).
Dong F, McGrath JM, Helgeson JP, Jiang J: The genet-
ic identity of alien chromosomes in potato breed-
ing lines revealed by sequential GISH and FISH
analyses using chromosome-specific cytogenetic
DNA markers. Genome 44:729734 (2001).
Dong YZ, Liu ZL, Liu B, Bu XL, He MY, Huang BQ,
Hao S: Microdissection of individual chromo-
somes of Thinopyrum intermedium and isolation
of molecular markers that are useful in detecting
Th. intermedium chromatin introgressed into
wheat. Cer Res Commun 30:253260 (2002).
Eenink AH, Groenwold R, Dieleman FL: Resistance of
lettuce (Lactuca) to the leaf aphid Nasonovia ribis
nigri. 1. Transfer of resistance from Lactuca virosa
to Lactuca sativa by interspecific crosses and selec-
tion of resistant breeding lines. Euphytica 31:291
300 (1982).
Fernandez JA, Jouve N: The addition of Hordeum chi-
lense chromosomes to Triticum turgidum conv. du-
rum. Biochemical, karyological and morphological
characterization. Euphytica 37:247259 (1988).
Fox SL, Jellen EN, Kianian SF, Rines HW, Phillips RL:
Assignment of RFLP linkage groups to chromo-
somes using monosomic F-1 analysis in hexaploid
oat. Theor Appl Genet 102:320326 (2001).
Friebe B, Jiang J, Gill BS, Dyck PL: Radiation-induced
nonhomoeologous wheat Agropyron interme-
dium chromosomal translocations conferring resis-
tance to leaf rust. Theor Appl Genet 86:141149
(1993).
Friebe B, Kynast RG, Gill BS: Gametocidal factor-
induced structural rearrangements in rye chromo-
somes added to common wheat. Chromosome Res
8:501511 (2000a).
Friebe B, Qi LL, Nasuda S, Zhang P, Tuleen NA, Gill
BS: Development of a complete set of Triticum aes-
tivum Aegilops speltoides chromosome addition
lines. Theor Appl Genet 101:5158 (2000b).
Gao D, Jung C: Monosomic addition lines of Beta cor-
olliflora in sugar beet: Plant morphology and leaf
spot resistance. Plant Breeding 121:8186 (2002).
Gao D, Guo D, Jung C: Monosomic addition lines of
Beta corolliflora Zoss in sugar beet: cytological and
molecular-marker analysis. Theor Appl Genet
103:240247 (2001).
Garriga-Calder F, Huigen DJ, Filotico F, Jacobsen E,
Ramanna MS: Identification of alien chromo-
somes through GISH and RFLP analysis and the
potential for establishing potato lines with mono-
somic additions of tomato chromosomes. Genome
40:666673 (1997).
Garriga-Caldere F, Huigen DJ, Angrisano A, Jacobsen
E, Ramanna MS: Transmission of alien tomato
chromosomes from BC1 to BC2 progenies derived
from backcrossing potato (+) tomato fusion hy-
brids to potato: The selection of single additions for
seven different tomato chromosomes. Theor Appl
Genet 96:155163 (1998).
D
o
w
n
l
o
a
d
e
d

b
y
:

U
n
i
v
e
r
s
i
t
y

L
i
b
r
a
r
y

U
t
r
e
c
h
t





























1
3
1
.
2
1
1
.
2
0
8
.
1
9

-

6
/
6
/
2
0
1
4

9
:
1
4
:
3
6

P
M
342 Cytogenet Genome Res 109:335343 (2005)
Hernandez P, Dorado G, Cabrera A, Laurie DA, Snape
JW, Martin A: Rapid verification of wheat Hor-
deum introgressions by direct staining of SCAR,
STS, and SSR amplicons. Genome 45:198203
(2002).
Hu C, Hole DJ, Albrechtsen RS, Hu CJ: Barley chro-
mosome location and expression of dwarf bunt
resistance in wheat addition lines. Plant Disease
80:12731276 (1996).
Jacobsen E, De Jong JH, Kamstra SA, van den Berg
PMM, Ramanna MS: Genomic in situ hybridiza-
tion (GISH) and RFLP analysis for the identifica-
tion of alien chromosomes in the backcross proge-
ny of potato (+) tomato fusion hybrids. Heredity
74:250257 (1995).
Jena KK, Khush GS: Monosomic alien addition lines
of rice: production, morphology, cytology, and
breeding behaviour. Genome 32:449455 (1989).
Jenkins G, Jimenez G: Genetic control of synapsis and
recombination in Lolium amphidiploids. Chro-
mosoma 104:164168 (1995).
Ji Y, Chetelat RT: Homoeologous pairing and recombi-
nation in Solanum lycopersicoides monosomic ad-
dition and substitution lines of tomato. Theor Appl
Genet 106:979989 (2003).
Jia J, Zhou R, Li P, Zhao M, Dong Y, Jia JZ, Zhou RH,
Li P, Zhao ML, Dong YS: Identifying the alien
chromosomes in wheat Leymus multicaulis de-
rivatives using GISH and RFLP techniques. Eu-
phytica 127:201207 (2002).
Jorgensen RB, Chen BY, Cheng BF, Heneen WK,
Simonsen V: Random amplified polymorphic
DNA markers of the Brassica alboglabra chromo-
some of a B. campestris alboglabra addition line.
Chromosome Res 4:111114 (1996).
Kalloo G, Chowdhury JB (eds): Distant hybridization
of crop plant. Monographs on Theoretical Applied
Genetics vol 16, pp 271 (Springer, Berlin 1992).
Kaneko Y, Bang SW, Matsuzawa Y: Early-bolting trait
and RAPD markers in the specific monosomic
addition line of radish carrying the e-chromosome
of Brassica oleracea. Plant Breeding 119:137140
(2000).
Kaneko Y, Yano H, Bang SW, Matsuzawa Y: Genetic
stability and maintenance of Raphanus sativus
lines with an added Brassica rapa chromosome.
Plant Breeding 122:239243 (2003).
Khrustaleva LI, Kik C: Introgression of Allium fistulo-
sum into A. cepa mediated by A. roylei. Theor Appl
Genet 100:1726 (2000).
Khush GS: Cytogenetics of Aneuploids (Academic
Press, New York 1973).
Kik C: Exploitation of wild relatives for the breeding of
cultivated Allium species, in Rabinowitch HD,
Currah L (eds): Allium Crop Science Recent
Advances, pp 81100 (CABI International, Wal-
lingford 2002).
Kindiger B, Bai D, Sokolov V: Assignment of a gene(s)
conferring apomixis in Tripsacum to a chromo-
some arm: cytological and molecular evidence. Ge-
nome 39:11331141 (1996).
King J, Armstead IP, Donnison IS, Thomas HM, Jones
RN, Kearsey MJ, Roberts LA, Thomas A, Morgan
WG, King IP: Physical and genetic mapping in the
grasses Lolium perenne and Festuca pratensis. Ge-
netics 161:315324 (2002).
Kubala kova M, Vala rik M, Bartos J, Vra na J, Cihal-
kova J, Molna r-La ng M, Doleel J: Analysis and
sorting of rye (Secale cerale L.) chromosomes using
flow cytometry. Genome 46:893905 (2003).
Kynast RG, Riera-Lizarazu O, Vales MI, Okagaki RJ,
Maquieira SB, Chen G, Ananiev EV, Odland WE,
Russell CD, Stec AO, Livingston SM, Zaia HA,
Rines HW, Phillips RL: A complete set of maize
individual chromosome additions to the oat ge-
nome. Plant Physiol 125:12161227 (2001).
Lange W, de Bock TSM, Speckmann GJ, De Jong JH:
Disomic and ditelosomic alien chromosome addi-
tions in beet (Beta vulgaris), carrying an extra chro-
mosome of B. procumbens or telosome of B. patel-
laris. Genome 36:261267 (1993)
Leighty CE, Taylor JW: Hairy neck wheat segregates
from wheat-rye hybrids. J Agr Res 28:567576
(1924).
Li LJ, Arumuganathan K, Rines HW, Phillips RL,
Riera Lizarazu O, Sandhu D, Zhou Y, Gill KS:
Flow cytometric sorting of maize chromosome 9
from an oat-maize chromosome addition line. The-
or Appl Genet 102:658663 (2001).
Littlejohn GM, Pienaar RV: Thinopyrum distichum
addition lines: production, morphological and cy-
tological characterisation of 11 disomic addition
lines and stable addition-substitution line. Theor
Appl Genet 90:3342 (1995).
Maestra B, de Jong JH, Shepherd K, Naranjo T: Chro-
mosome arrangement and behaviour of two rye
homologous telosomes at the onset of meiosis in
disomic wheat-5RL addition lines with and with-
out the Ph1 locus. Chromosome Res 10:655667
(2002).
Malysheva L, Sjakste T, Matzk F, Roder M, Ganal M:
Molecular cytogenetic analysis of wheat-barley hy-
brids using genomic in situ hybridization and bar-
ley microsatellite markers. Genome 46:314322
(2003).
Martinez-Perez E, Shaw P, Reader S, Aragon-Alcaide
L, Miller T, Moore G: Homologous chromosome
pairing in wheat. J Cell Sci 112:17611769 (1999).
McCoy TJ, Echt CS: Potential of trispecies bridge
crosses and random amplified polymorphic DNA
markers for introgression of Medicago daghestani-
ca and Medicago pironae germplasm into alfalfa
(Medicago sativa). Genome 36:594601 (1993).
Melchers G, Sacrista n MD, Holder AA: Somatic hybrid
plants of potato and tomato regenerated from
fused protoplasts. Carlsberg Res Commun 43:203
218 (1978).
Mesbah M, De Bock TSM, Sandbrink JM, Klein Lank-
horst RM, Lange W: Molecular and morphological
characterization of monosomic additions in Beta
vulgaris, carrying extra chromosomes of B. pro-
cumbens or B. patellaris. Mol Breed 3:147157
(1997a).
Mesbah M, Scholten OE, Bock TSMd, Lange W, De
Bock TSM: Chromosome localisation of genes for
resistance to Heterodera schachtii, Cercospora beti-
cola and Polymyxa betae using sets of Beta pro-
cumbens and B. patellaris derived monosomic ad-
ditions in B. vulgaris. Euphytica 97:117127
(1997b).
Mesbah M, Wennekes-Van Eden J, De Jong JH, De
Bock TSM, Lange W: FISH to mitotic chromo-
somes and extended DNA fibres of Beta procum-
bens in a series of monosomic additions to beet
(B. vulgaris). Chromosome Res 8:285293 (2000).
Mikhailova EI, Naranjo T, Shepherd K, Wennekes-
Van Eden J, Heyting C, De Jong JH: The effect of
the wheat Ph1 locus on chromatin organisation and
meiotic chromosome pairing analysed by genome
painting. Chromosoma 107:339350 (1998).
Miller TE, Reader SM, Shaw PJ, Moore G, Slinkard
AE: Towards an understanding of the biological
action of the Ph1 locus in wheat, in: Proceedings of
the Ninth International Wheat Genetics Sympo-
sium, Saskatoon, Saskatchewan, Canada, 27 Au-
gust 1998 (University Extension Press, Extension
Division, University of Saskatchewan, Saskatoon
1998).
Morgan RN, Ozias-Akins P, Hanna WW: Seed set in an
apomictic BC3 pearl millet. Int J Plant Sci 159:89
97 (1998).
Morgan WG: The morphology and cytology of mono-
somic addition lines combining single Festuca dry-
meja chromosomes and Lolium multiflorum. Eu-
phytica 55:5763 (1991)
Muehlbauer GJ, Riera Lizarazu O, Kynast RG, Martin
D, Phillips RL, Rines HW: A maize chromosome 3
addition line of oat exhibits expression of the
maize homeobox gene liguleless-3 and alteration of
cell fates. Genome 43:10551064 (2000).
Mukai Y, Friebe B, Hatchett JH, Yamamoto M, Gill
BS: Molecular cytogenetic analysis of radiation
induced wheat-rye terminal and intercalary chro-
mosomal translocations and the detection of rye
chromatin specifying resistance to Hessian fly.
Chromosoma 102:8895 (1993).
Multani DS, Jena KK, Brar DS, Reyes BGdl, Angeles
ER, Khush GS, De los Reyes BG: Development of
monosomic alien addition lines and introgression
of genes from Oryza australiensis Domin. to culti-
vated rice, O. sativa L. Theor Applied Genet 88:
102109 (1994).
Okagaki RJ, Kynast RG, Livingston SM, Russell CD,
Rines HW, Phillips RL: Mapping maize sequences
to chromosomes using oat-maize chromosome ad-
dition materials. Plant Physiol 125:12281235
(2001).
Okagaki R, Kynast R, Odland WE, Stec AO, Russell
CD, Zaia HA, Rines HW, Phillips RL: A radiation
hybrid system for the genetic and physical map-
ping of the corn genome. Maize Genet Cooperation
Newsletter 76:88 (2002).
OMara JG: Cytogenetic studies on Triticinae. I. A
method for determining the effect of individual
Secale chromosomes on Triticum. Genetics 25:
401408 (1940).
Peffley EB, Currah L: The chromosomal locations of
enzyme-coding genes Adh-1 and Pgm-1 in Allium
fistulosum L. Theor Appl Genet 75:945949
(1988).
Potz H, Schubert V, Houben A, Schubert I, Weber WE:
Aneuploids as a key for new molecular cloning
strategies: Development of DNA markers by mi-
crodissection using Triticum aestivum Aegilops
markgrafii chromosome addition line B. Euphytica
89:4147 (1996).
Quiros CF, Ochoa O, Kianian SF, Douches D: Analysis
of the Brassica oleracea genome by the generation
of B. campestris oleracea chromosome addition
lines: characterization by isozymes and rDNA
genes. Theor Appl Genet 74:758766 (1987).
Raina SN, Rani V: GISH technology in plant genome
research. Meth Cell Sci 23:83104 (2001).
Ramulu KS, Dijkhuis P, Rutgers S, Blaas J, Krens FA,
Dons JJM, Colijn-Hooymans CM, Verhoeven HA:
Microprotoplast mediated transfer of single chro-
mosomes between sexually-incompatible plants.
Genome 39:921933 (1996).
Reamon Ramos SM, Wricke G: A full set of mono-
somic addition lines in Beta vulgaris from Beta
webbiana: morphology and isozyme markers. The-
or Appl Genet 84:411418 (1992).
Riera-Lizarazu O, Rines HW, Phillips RL: Cytological
and molecular characterization of oat maize par-
tial hybrids. Theor Appl Genet 93:123135
(1996).
Riera-Lizarazu O, Vales MI, Ananiev EV, Rines HW,
Phillips RL: Production and characterization of
maize chromosome 9 radiation hybrids derived
from an oat-maize addition line. Genetics 156:
327339 (2000).
Savitsky H: Meiosis in an F1 hybrid between a Turkish
wild beet (Beta vulgaris, ssp. maritima) and Beta
procumbens. J Am Assoc Sugar Beet Technologists
ASSBT 11:4967 (1960).
Schubert I, Fransz PF, Fuchs J, de Jong JH: Chromo-
some painting in plants. Meth Cell Sci 23:5769
(2001).
Schwarzacher T: Three stages of meiotic homologous
chromosome pairing in wheat: cognition, align-
ment and synapsis. Sex Plant Reprod 10:324331
(1997).
D
o
w
n
l
o
a
d
e
d

b
y
:

U
n
i
v
e
r
s
i
t
y

L
i
b
r
a
r
y

U
t
r
e
c
h
t





























1
3
1
.
2
1
1
.
2
0
8
.
1
9

-

6
/
6
/
2
0
1
4

9
:
1
4
:
3
6

P
M
Cytogenet Genome Res 109:335343 (2005) 343
Schwarzacher T, Leitch AR, Bennett MD, Heslop-Har-
rison JS: In situ hybridization of parental genomes
in a wide hybrid. Ann Bot 64:315324 (1989).
Sears ER: The transfer of leaf rust resistance from Aegi-
lops umbellulata to wheat. Brookhaven Sym Bio
9:122 (1956).
Sharma DR, Kaur R, Kumar K, Laibach F: Embryo
rescue in plants a review. Euphytica 89:325337
(1996).
Shepherd KW, Islam AKMR, Miller TE, Koebner
RMD: Fourth compendium of wheat-alien chro-
mosome lines, in Miller TE, Koebner RMD (eds):
Procedings of the 7th International Wheat Genet-
ics Symposium, pp 13731398 (Bath Press, Bath
1988).
Shigyo M, Tashiro Y, Isshiki S, Miyazaki S: Establish-
ment of a series of alien monosomic addition lines
of Japanese bunching onion (Allium fistulosum L)
with extra chromosomes from shallot (A. cepa L
Aggregatum group). Genes Genet Syst 71:363371
(1996).
Shigyo M, Wako T, Kojima A, Yamauchi N, Tshiro Y:
Transmission of alien chromosomes from selfed
progenies of a complete set of Allium monosomic
additions: The development of a reliable method
for the maintenance of a monosomic addition set.
Genome 46:10981103 (2003).
Suen DF, Wang CK, Lin RF, Kao YY, Lee FM, Chen
CC: Assignment of DNA markers to Nicotiana syl-
vestris chromosomes using monosomic alien addi-
tion lines. Theor Appl Genet 94:331337 (1997).
Sybenga J: Cytogenetics in plant breeding. Monographs
on Theoretical and Applied Genetics, pp 469
(Springer, Berlin 1992).
Taketa S, Takeda K: Production and characterization
of a complete set of wheat-wild barley (Hordeum
vulgare ssp. spontaneum) chromosome addition
lines. Breed Sci 51:199206 (2001).
Tanksley SD, Ganal MW, Prince JP, de Vicente MC,
Bonierbale MW, Broun P, Fulton TM: High densi-
ty molecular linkage maps of the tomato and pota-
to genomes. Genetics 132:11411160 (1992).
Tian C, Lu Y, Deng J, Li B, Zhang X, Liu G, Tian C, Lu
YF, Deng JX, Li B, Zhang XY, Liu GT: Microdis-
section of additional chromosome in common
wheat Th. intermedium TAI-27 and screening of
its special probe. Science in China Series C Life
Sciences 43:105112 (2000).
van Geyt JPC, Oleo M, Lange W, de Bock TSM: Mono-
somic additions in beet (Beta vulgaris) carrying
extra chromosomes of Beta procumbens. 1. Identi-
fication of the alien chromosomes with the help of
isozyme markers. Theor Appl Genet 76:577586
(1988).
van Heusden AW, Shigyo M, Tashiro Y, Vrielink-van
Ginkel R, Kik C: AFLP linkage group assignment
to the chromosomes of Allium cepa L via monos-
omic addition lines. Theor Appl Genet 100:480
486 (2000).
van Tuyl JM, van Kronenburg BCM, Meijer B: Inter-
specific lily hybrids A promise for the future, in
Lilien-Kipnis H, Halevy AH, Borochov A (eds):
Proceedings of the International Symposium on
Flower Bulbs, Acta Hort ISHS 430:465476
(1997).
Wolters AM, Jacobsen E, O CM, Bonnema G, Ramulu
KS, De JH, Schoenmakers H, Wijbrandi J, Koorn-
neef M: Somatic hybridization as a tool for tomato
breeding. Euphytica 79:265277 (1994).
Yasui H, Iwata N: Development of monotelosomic and
monoacrosomic alien addition lines in rice (Oryza
sativa L.) carrying a single chromosome of O. punc-
tata Kotschy. Breed Sci 48:181186 (1998).
Yuzhu D, Zhenlan L, Bao L, Xiuling B, Mengyuan H,
Baiqu H, Shui H: Microdissection of individual
chromosomes of Thinopyrum intermedium and
isolation of molecular markers that are useful in
detecting Th. intermedium chromatin introgressed
into wheat. Cer Res Comm 30:253260 (2002).
Zhang JY, Li XM, Wang RRC, Cortes A, Rosas V,
Mujeeb Kazi A: Molecular cytogenetic character-
ization of E
b
-genome chromosomes in Thinopyrum
bessarabicum disomic addition lines of bread
wheat. Int J Plant Sci 163:167174 (2002).
Zhong X-B, Fransz PF, Wennekes van Eden J, Raman-
na MS, Kammen Av, Zabel P: FISH studies reveal
the molecular and chromosomal organization of
individual telomere domains in tomato. Plant J
13:507517 (1998).
D
o
w
n
l
o
a
d
e
d

b
y
:

U
n
i
v
e
r
s
i
t
y

L
i
b
r
a
r
y

U
t
r
e
c
h
t





























1
3
1
.
2
1
1
.
2
0
8
.
1
9

-

6
/
6
/
2
0
1
4

9
:
1
4
:
3
6

P
M