“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Challenges to genome integrity: conflict and resolution
N. JONES# AND I. PASAKINSKIENE*
#
University of Wales Aberystwyth, Institute of Biological Sciences,
Aberystwyth SY23 3DD, U.K.,
*Lithuanian Institute of Agriculture, Dotnuva-Akademija, 5051 Kedainiai, Lithuania
#
Correspondence: phone + 44 (0)1970 622230, rnj@aber.ac.uk
In hybrids and introgression lines unfamiliar genomes are brought together into an alien
nuclear environment, and into a cytoplasm derived from one parent. Genomes are composed
of a number of components which may differentiate them structurally (repetitive DNA
elements), and functionally (genes, alleles and regulatory sequences), and this presents an
operational conflict. The challenge is exacerbated by the way in which the genome is
embedded in chromatin, and by histone modification and chromatin remodelling which
influences the gene environment. Resolution of conflict manifests itself in a spectrum of
responses, some of which are cytologically visible and some more cryptic. One extreme
involves the outright rejection of one partner genome, as is made use of in the production of
doubled haploids. In allopolyploids which have more than two genomes the resolution may be
more complex, even resulting in diploidisation and somatic recombination, and building
‘novel diploids’. Less dramatic are the changes in ‘genome balance’, where one genome of a
hybrid exceeds more than its share of chromatin. In some cases very large differences in
nuclear DNA amount between two species can be resolved in mysterious ways to facilitate
homoeologous pairing and bivalent formation at meiosis, from a starting point of homology
search chaos. Sequence analysis is now revealing cryptic and instantaneous changes in
sequence variation, and in the elimination / silencing / activation of genes, in allopolyploids.
The environment can also present stress on genomes, and modulate the copy number of
retrotransposons and other repetitive elements, within a single generation, and generate intraspecific variation in DNA amounts between populations. The genome is dynamic in ways
which we are only just beginning to appreciate. What opportunities this dynamism offers
breeding is very much an open question.
1
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
2
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
The exploitation of chromosome recombination between Lolium and
Festuca to assemble, locate, and identify, gene- combinations that may
underpin future sustainable grassland production*
M. W. HUMPHREYS
Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth,
SY23 3EB, Wales, UK
The anticipated complexity of multifunctional grasslands with environment-friendly and
sustainable management practices, demands better understanding of traits, their interactions,
and their genetic control. Intergeneric hybrids between closely related Lolium and Festuca
species are being used to broaden the gene pool and provide the plant breeder with options to
combine complementary traits aimed at high quality but more robust grass varieties for the
future. New techniques in introgression-mapping provide opportunities for precision breeding
whereby desirable gene combinations transferred from one species into another are selected
preferentially, with the exclusion of deleterious alien genes. The close homology between
genomes of Lolium and Festuca species allows high levels of chromosome pairing and
recombination. Using genomic in situ hybridisation (GISH) on Lolium/Festuca hybrids and
their derivatives, recombination between Lolium and Festuca chromosomes may be observed
at nearly every location (although the recombination frequency will vary with the
chromosome site). The system provides unlimited access to any combination of Lolium and
Festuca DNA sequence. Moreover, genes transferred between homoeologous chromosome
sites are expected to function normally at their new locations. Further chromosome
recombination between homoeologous Lolium and Festuca sequences enables Festuca
introgressions to be "dissected", and recombination series created. The reduced size of alien
chromosome segments aids the exclusion of deleterious gene combinations and thereby
reduces linkage drag.
Additional approaches to aid introgression mapping have employed androgenesis
techniques and have led to novel genotypes rarely observed as outcomes of breeding
programmes.
Molecular markers such as AFLPs, SSRs, SNPs, or RFLPs are being targeted to genes
of interest to allow their selection through different generations. Relatively-simple PCR-based
marker systems as breeders' toolkits are being designed from molecular markers that flank the
targeted genes and are being applied directly to aid genotype selection in large-scale plant
breeding programmes.
Breeders' toolkits also provide opportunities to isolate genes that determine important
agronomic traits. Other approaches at gene isolation involve knowledge of synteny and gene
sequences within model species amongst the Poaceae.
* This work is partly funded by the EU Framework V project "Sustainable grasslands withstanding
environmental stresses" (SAGES) which is part of the Quality of Life and Management of Living Resources
Key Action 5.1.1 Sustainable Agriculture Programme. For further information see
http://www.iger.bbsrc.ac.uk/igerweb/SAGES/Welcome.html
3
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
4
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Genome adjustment in hybrids of the Lolium-Festuca complex
I. PAŠAKINSKIENĖ
Lithuanian Institute of Agriculture, 5051 Dotnuva - Akademija, Kėdainiai, Lithuania
Genetic resources of forage grasses are enriched by making Lolium x Festuca hybrids using
different species and genera - principally Italian ryegrass (Lolium multiflorum Lam.),
perennial ryegrass (L. perenne L.), tall fescue (Festuca arundinacea Schreb.) and meadow
fescue (F. pratensis Huds). Understanding the degree of closeness of these species is an
important knowledge base for dealing with the hybrids, and for guiding the direction of our
experimental and breeding work. Chromosome painting by GISH allows us to reveal new
phylogenetic features of the natural allohexaploid F. arundinacea species and provides us
with the means for studying genome rearrangements in the newly formed L. multiflorum x F.
arundinacea allopolyploids.
The role of Lolium in the formation of allohexaploid F. arundinacea
Festuca arundinacea (2n=6x=42) is a natural allohexaploid which originated as a hybrid
between F. pratensis and F. glaucescens (HUMPHREYS et al. 1995). It is known that F.
arundinacea has a close relationship to Lolium multiflorum, and that this closeness is due
mainly to the affinity of the L. multiflorum genome to F. pratensis (PAŠAKINSKIENĖ et al.
1998). GISH was used in order to compare the chromosomes of F. pratensis present as a
constitutive genome in F. arundinacea (FpFa) with the homologous ones in the original
diploid species (Fporign). A rhodamine labeled probe of Lolium was applied onto three types of
mitotic chromosome spreads: F. arundinacea (FpFaFpFaFgFgFgFg), F. arundinacea x F.
pratensis hybrid (FpFaFporignFgFg) and L. multiflorum x F. arundinacea hybrid
(LmFpFaFgFg). The L. multiflorum probe shows an intermediate cross-hybridization over the
chromosomes belonging both to Fporign and FpFa. This allows the chromosomes of F. pratensis
to be distinguished from F. glaucescens, without being confused with the strong signal on L.
multiflorum itself. In addition, segmental regions of strong hybridization appear on the FpFa
chromosomes. We refer to these regions as ‘GISH bands’. However, no corresponding GISHbanding was found within the chromosome set of the original F. pratensis species (Fporign).
This leads us to propose new theories about how the genome of F. pratensis may have
evolved, and diverged from its natural diploid status, since becoming included as part of the
allopolyploid F. arundinacea. We assume that F. arundinacea had accommodated number of
genomic ‘blocks’ of L. multiflorum through some introgression events of species evolution
leading to the hybrid between two fescues, F. pratensis and F. glaucescens.
Genome adjustment and separation through somatic instability in L. multiflorum x F.
arundinacea hybrids
The use of GISH has proved to be a powerful tool to monitor the stability of genomes
brought together in hybrid nuclear environments. L. multiflorum and F. arundinacea were
found to be in conflict, and presented us with numerous examples of genome rearrangements
at the somatic level. Firstly, a new phenomenon of instability was discovered in colchicine
doubled F1C0 hybrids of L. multiflorum x F. arundinacea (2n=8x=56) manifesting itself in
5
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
terms of chromosome elimination and somatic recombination followed by genome
diploidisation (PAŠAKINSKIENĖ et al. 1997). We call these somatically segregated diploids
‘novel diploids’. Chromosome painting by GISH demonstrated them to be new genomic
variants derived from F. pratensis, L. multiflorum and F. glaucescens. The genomes of the
three species are represented in different proportions, and as variable patterns, but in any case
F. pratensis makes the genomic basis of the ‘novel diploids’.
Recently, similar events were discovered in a selected F2C1 hybrid. The hexaploid
genotype F2 3-18 (2n=6x=42), a ‘super-recombinant’, was characterised by a phenotypic
combination of a Festuca type inflorescence and Lolium type leaves. GISH painting revealed
its particular genome composition: an excess of Lolium (24 chromosomes) and a high number
of recombinant chromosomes with 2 out of 10 of them represented as constructs composed of
three species, Lm-Fp-Fg. This genotype was observed for a number of years since it was of
interest from the breeding point, as the plant vigour was complemented by good fertility. The
most phenomenal effect was that it clearly showed phenotypic segregation as it was
multiplied vegetatively. The initial allohexaploid genome give rise to a few somatic
segregants with a chromosome number of 2n=2x=14, of both Festuca and Lolium type. This
‘super-recombinant’ had a wide spectrum of PGI alleles, a+abcd, while this pattern in the
segregated diploids was only partly represented, as abc and bc respectively. GISH is being
carried on to discriminate the genomic structure of the segregated diploids, and inter-SSR
fingerprinting may also provide some insights into this. Preliminary results show a tendency
for F. pratensis genome separation, as it was described above in the F1C0 hybrids.
References
HUMPHREYS M.W., THOMAS H.M., MORGAN W.G., MEREDITH M.R., HARPER J.A.., THOMAS H.,
ZWIERZYKOWSKI Z., GHESQUIÈRE M. (1995). Discriminating the ancestral progenitors of hexaploid
Festuca arundinacea using genomic in situ hybridization. Heredity 75: 171 – 174.
PAŠAKINSKIENĖ I., ANAMTHAWAT-JÓNSSON K., HUMPHREYS M.W., JONES R.N. (1997). Novel
diploids following chromosome elimination and somatic recombination in Lolium multiflorum x
Festuca arundinacea hybrids. Heredity 78: 464-469.
PAŠAKINSKIENĖ I., ANAMTHAWAT-JÓNSSON K., HUMPHREYS M.W., PAPLAUSKIENE V., JONES R.N.
(1998). New molecular evidence on genome relationships and chromosome identification in Festuca
and Lolium. Heredity 81: 659-665.
6
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
DNA and proteins of plant centromeres
A. HOUBEN1, S. HUDAKOVA2, R. TEN HOOPEN2, R. D. DEMIDOV1, D. GERNAND1,
R. MANTEUFFEL3, G. PRESTING2, T. SCHLEKER2, I. SCHUBERT2*
1
Chromosome Structure and Function Group, 2Karyotype Evolution Group, 3Serology Group,
Institute of Plant Genetics and Crop Plant Research, 06466 Gatersleben, Germany
*Schubert@ipk-gatersleben.de
The centromere of monocentric chromosomes is morphologically recognizable as the primary
constriction. Centromeres are essential for correct segregation into daughter cells of sister
chromatids during mitosis and meiosis II and of homologous chromosomes during meiosis I
and are responsible for chromatid cohesion, spindle fibre attachment and chromosome
movement during nuclear division in all eukaryotes. At centromeres a proteinaceous structure,
the kinetochore, is assembled mediating most of the centromeric functions (Choo 1997).
DNA composition of plant centromeres
In contrast to telomeres, centromeres are not specified by highly conserved DNA sequences.
Centromeric DNA sequences have been described for several eukaryotes. However, except
for budding yeasts (Clarke, Carbon 1985), their functional importance is at least controversial.
For several species including plants, neocentromeric activities at non-centromeric positions
have been reported (Manzanero et al. 2002), supporting the idea that the centromere location
might be regulated epigenetically (Vig 1994; Karpe, Allshire 1997).
While for Vicia faba and Tradescantia paludosa no centromere-specific repeats could
be detected (Houben et al. 1996), such sequences have been found in other plants including
Arabidopsis thaliana (Martinez-Zapater et al. 1986, Heslop-Harrison et al. 1999). For cereal
centromeres, two conserved centromere-specific repeats (CCS1, Aragon-Alcaide et al. 1996
and Sau 3A9 Jiang et al. 1996) were reported and later on found to represent parts of a
Ty3/gypsy-like retroelement (Presting et al. 1998, Hudakova et al. 2001). By sequencing,
fingerprinting and in situ hybridisation of a centromere-specific large insert clone (BAC 7),
the sequence organization of barley centromeres could be elucidated. The 23 kb insert of BAC
7 contained three copies of the apparently complete gypsy-like retroelement ‘cereba’ (~7 kb).
Parts of its LTRs (~1 kb) correspond to CCS1 and parts of the integrase region of its polygene
to Sau3A9. Together with a G/C-rich satellite sequence (AGGAG)n, cereba-elements
constitute the major DNA components of barley centromeres (Hudakova et al. 2001). Each
barley centromere contains ~200 ‘cereba’-elements corresponding at least 1.4 Mbp (Presting
et al. 1998). Cereba-related elements are conserved within the centromeres of all cereals since
their radiation ~60 million years ago. They show the highest density and completeness in
centromeres of barley. However, the centromeric satellite sequences of cereals are speciesspecific (for references see Hudakova et al. 2001). Although both types of centromeric
sequences are preferentially immunoprecipitated together with CENH3, a maize homologue
of the human centromeric histone H3 variant CENP-A (Zhong et al. 2002), their functional
importance is still uncertain because some barley telosomes which apparently lack these
sequences are still mitotically and meiotically stable (T.R. Endo pers. communication).
7
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Protein composition of plant kinetochores
Contrary to centromeric DNA, structural and functional kinetochore proteins are highly
conserved between yeasts and metazoa. More than 20 different proteins of the kinetochore are
known for human neocentromeres, which are free of detectable centromeric alphoid satellite
sequences (Saffery et al. 2000). This supports the assumption that the centromere is regulated
epigenetically and, once established, remains stable (Karpen, Allshire 1997). Autoantibodies
from sera of scleroderma (CREST) patients which are frequently directed against human
kinetochores, were found to recognize plant kinetochores (Houben et al. 1995). In order to
study the evolutionary conservation of kinetochore proteins, the cross-reactivity of antibodies
against human kinetochores was tested with mitotic chromosomes of monocots and/or dicots.
Antibodies directed against human CENP-E and CENP-F recognize barley centromeres and
those against CENP-C and CENP-E recognize field bean centromeres on metaphase
chromosomes suggesting the presence of proteins with similar antigenic features in plant and
mammalian kinetochores (ten Hoopen et al. 2000).
Genes encoding putative kinetochore proteins were found for plant species, based on
sequence similarity with non-plant kinetochore protein genes. Putative homologues of the
proteins SKP1 (suppressor of kinetochore protein 1p of yeast), CBF5p (centromere binding
factor 5 of yeast) and of CENP-E (human kinesin-like kinetochore motor protein) were
identified. Antibodies against these proteins were produced and recognized the centromeric
regions on mitotic chromosomes of barley and field bean as shown by indirect
immunofluorescence (ten Hoopen et al. 2000, 2002). Although putative homologues of the
kinetochore proteins SKP1 and CPF5 are apparently present in plant kinetochores, differences
were found with respect to their location in comparison with mammals. For instance, SKP1 in
yeast is part of the CBF2 complex, interacts with CDEII of the centromeric DNA, and thus
represents a kinetochore component, while in mammals it is located within the centrosome.
Antibodies against CBF5-like proteins of barley labelled plant centromeres, while the human
CBF5 homolog dyskerin is located within the nucleolus. The CENP-C homologue of maize
apparently represents a hybrid protein partially homologous to human CENP-C (Dawe et al.
1999) and to another protein located within human centrosomes (ten Hoopen et al. 2000).
Altogether six kinetochore proteins from yeast/metazoa: CENP-C (Dawe et al. 1999), MAD2
(Yu et al. 1999), SKP1, CBF5, CENP-E (ten Hoopen et al. 2000, 2002) and CENP-A (Talbert
et al. 2002, Zhong et al. 2002) are already confirmed for plants. Other ones such as ZW10,
BUB1, BUB3 and MCAK are now under investigation. Furthermore, we generated 6
monoclonal antibodies which recognize constitutive and transient plant kinetochore proteins,
respectively, by immunizing mice with isolated plant chromosomes. The antigenic proteins
have to be identified.
In addition to specific proteins, a pericentromere-specific post-translational
modification of histone H3 has been demonstrated for plant centromeres. In plants, the
pericentromerically localized histone H3 becomes phosphorylated at serines 10 and 28 from
prophase until telophase. A ‘semi-dicentric’ barley chromosome revealed
hyperphosphorylated H3 only at the functional centromere (Houben et al. 1999). During the
first meiotic division, phosphorylated H3Ser10 is detectable along the entire chromosomes.
At second meiotic division only the pericentromeric regions are phosphorylated at H3Ser10 as
during mitosis (Manzanero et al. 2000; Manzanero et al. 2002). In order to answer the
question whether or not phosphorylation of H3 is involved in sister chromatid cohesion, we
will manipulate the activity of histone H3-specific kinases. Aurora/Ipl-kinases are involved in
regulation of histone H3 phosphorylation in C. elegans, S. cerevisiae, Drosophila and
8
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
mammals. Based on sequence comparison we have isolated centromere-localized Aurora-like
kinases in A. thaliana.
References
ARAGÓN-ALCAIDE, L., MILLER, T., SCHWARZACHER, T., READER, S., MOORE, G. (1996). A
cereal centromeric sequence. Chromosoma 105: 261-268.
CHOO, K.H.A. (1997). The centromere. Oxford Univ. Press, Oxford, 304 p.
CLARKE, L., CARBON, J. (1985). Structure and function of yeast centromeres. Annu. Rev.
Genet. 19: 29-56.
DAWE, R.K., REED, L.M., YU, H-G., MUSZYNSKI, M.G., HIATT, E.N. (1999). A maize
homolog of mammalian CENPC is a constitutive component of the inner kinetochore. Plant
Cell 11: 1227-1238.
HESLOP-HARRISON, J.S., MURATA, M., OGURA, Y., SCHWARZACHER, T., MOTOYOSHI, F.
(1999). Polymorphisms and genomic organization of repetitive DNA from centromeric
regions of Arabidopsis chromosomes. Plant Cell 11: 31-42.
HOUBEN, A., BRANDES, A., PICH, U., MANTEUFFEL, R., SCHUBERT, I. (1996). Molecularcytogenetic characterization of a higher plant centromere/kinetochore complex. Theor. Appl.
Genet. 93: 477-484.
HOUBEN, A., GUTTENBACH, M., KREß, W., PICH, U., SCHUBERT, I., SCHMID, M. (1995).
Immunostaining and interphase arrangement of field bean kinetochores. Chromosome Res. 3:
27-31.
HOUBEN, A., WAKO, T., FURUSHIMA-SHIMOGAWARA, R., PRESTING, G., KÜNZEL, G.,
SCHUBERT, I., FUKUI, K. (1999). The cell cycle dependent phosphorylation of histone H3 is
correlated with the condensation of plant mitotic chromosomes. Plant J. 18: 675-679.
HUDAKOVA, S., MICHALEK, W., PRESTING, G.G., TEN HOOPEN, R., DOS SANTOS, K.,
JASENCAKOVA, Z., SCHUBERT, I. (2001). Sequence organization of barley centromeres.
Nucleic Acids Res. 29: 5029-5035.
JIANG, J., NASUDA, S., DONG, F., SCHERRER, C.W., WOO, S-S., WING, R.A., GILL, B.S., WARD,
D.C. (1996). A conserved repetitive DNA element located in the centromeres of cereal
chromosomes. Proc. Natl. Acad. Sci. USA 93: 14210-14213.
KARPEN, G.H., ALLSHIRE, R.C. (1997). The case for epigenetic effects on centromere identity
and function. Trends Genet. 13: 489-496.
MANZANERO, S., ARANA, P., PUERTAS, M.J., HOUBEN, A. (2000). The chromosomal
distribution of phosphorylated histone H3 differs between plants and animals at meiosis.
Chromosoma 109: 308-317.
MANZANERO, S., RUTTEN, T., KOTSERUBA, V., HOUBEN, A. (2002). Alterations in the
distribution of histone H3 phosphorylation in mitotic plant chromosomes in response to cold
treatment and the protein phosphatase inhibitor cantharidin. Chromosome Res. 10: 467-476.
MANZANERO, S., VEGA, J.M., HOUBEN, A., PUERTAS, M.J. (2002). Characterization of the
constriction with neocentric activity of 5RL chromosome in wheat. Chromosoma 111: 228235.
MARTINEZ-ZAPATER, J.M., ESTELLE, M.A., SOMERVILLE, C.R. (1986). A highly repeated
DNA sequence in Arabidopsis thaliana. Mol. Gen. Genet. 204: 417-423.
PRESTING, G.G., MALYSHEVA, L., FUCHS, J., SCHUBERT, I. (1998). A TY3/GYPSY
retrotransposon-like sequence localizes to the centromeric regions of cereal chromosomes.
Plant J. 16: 721-728.
9
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
SAFFERY, R., IRVINE, D.V., GRIFFITHS, B., KALITSIS, P., WORDEMAN, L., CHOO, K.H.A.
(2000). Human centromeres and neocentromeres show identical distribution patterns of >20
functionally important kinetochore-associated proteins. Human Mol. Genet. 9: 175-185.
TALBERT, P.B., MASUELLI, R., TYAGI, A.P., COMAI, L., HENIKOFF, S. (2002). Centromeric
localization and adaptive evolution of an Arabidiosps histone H3 variant. Plant Cell 14: 10531066.
TEN HOOPEN, R., MANTEUFFEL, R., DOLEZEL, J., MALYSHEVA, L., SCHUBERT, I. (2000).
Evolutionary conservation of kinetochore protein sequences in plants. Chromosoma 109: 482489.
TEN HOOPEN, R., SCHLEKER, T., MANTEUFFEL, R., SCHUBERT, I. (2002). Transient CENP-Elike kinetochore proteins in plants. Chromosome Res. 10: 561-570.
VIG, B.K. (1994). Do specific nucleotide bases constitute the centromere? Mutat. Res. 309: 110.
YU, H-G., MUSZYNSKI, M.G., DAWE, R.K. (1999). The maize homologue of the cell cycle
checkpoint protein MAD2 reveals kinetochore substructure and contrasting mitotic and
meiotic localization patterns. J. Cell Biol. 145: 425-435.
ZHONG, C.X., MARSHALL, J.B., TOPP, C., MROCZEK, R., KATO, A., NAGAKI, K., BIRCHLER,
J.A., JIANG, J., DAWE, R.K. (2002). Centromeric retroelements and satellites interact with
maize kinetochore protein CENH3. Plant Cell 14: 2825-2836.
10
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Genome recombination in early generations of
Festuca pratensis × Lolium perenne hybrids
Z. ZWIERZYKOWSKI1, E. ZWIERZYKOWSKA1, A. KOSMALA1, M. ŁUCZAK1, W. JOKŚ2
1
Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań,
Poland; 2 Szelejewo Plant Breeding Ltd., 63-820 Piaski, Poland
Grasses of the Lolium-Festuca complex, and especially four agriculturally important species –
Lolium multiflorum (Italian ryegrass), L. perenne (perennial ryegrass), Festuca pratensis
(meadow fescue) and F. arundinacea (tall fescue) – offer a range of desirable and
complementary traits. Lolium species have high productivity and forage quality, and Festuca
species express better persistency and tolerance to abiotic stresses. Species of Lolium and
Festuca genera are closely related, they can be hybridised with relative ease, and their
chromosomes pair and recombine freely in the hybrids. On the other hand, the chromosomes
of parental species in Lolium  Festuca hybrids and their derivatives can be differentiated by
genomic in situ hybridisation (GISH).
Many recent studies of Lolium-Festuca hybrids and their derivatives using GISH
demonstrated that homoeologous chromosome pairing resulted in extensive recombination
between chromosomes of the parental genomes. For example, extensive recombination was
observed in four F8 breeding populations derived from reciprocal hybrids of tetraploid L.
multiflorum with F. pratensis (ZWIERZYKOWSKI et al. 1998); all of the analysed populations
have been registered in Poland as commercial cultivars (Felopa, Sulino, Rakopan and Agula).
A high level of recombination was also showed in F8 genotypes derived from the amphiploid
L. perenne  F. pratensis cv. Prior (CANTER et al. 1999). The recombination events were
distributed along the whole lengths of the chromosome arms. These observations support
earlier conclusions that the gene pools of the two genera are completely accessible for
manipulation in breeding.
In this study, we present preliminary results of genome recombination using GISH in
early generations (F2-F4) of amphiploid hybrids, F. pratensis (2n=4x=28) x L. perenne
(2n=4x=28).
References
ZWIERZYKOWSKI Z., TAYYAR R., BRUNELL M., LUKASZEWSKI A.J. (1998). Genome recombination in
intergeneric hybrids between tetraploid Festuca pratensis and Lolium multiflorum. J. Heredity 89:
324-328.
CANTER P.H., PAŠAKINSKIENĖ I., JONES R.N., HUMPHREYS M.W. (1999). Chromosome substitutions
and recombination in the amphiploid Lolium perenne × Festuca pratensis cv Prior (2n=4x=28). Theor.
Appl. Genet. 98: 809-814.
11
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
12
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Brachypodium distachyon – a new model grass?
G. JENKINS1, R. HASTEROK2, J. DRAPER1
1
Institute of Biological Sciences, Edward Llwyd Building, University of Wales, Penglais,
Aberystwyth, Ceredigion, SY23 3DA, UK; 2Department of Plant Anatomy and Cytology,
University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland
For the dicots and monocots respectively, Arabidopsis thaliana and rice are well established
model organisms for functional genomics. Both of them possess compact and fully sequenced
genomes, which together with some other desirable features have promoted their extensive
and successful use in a wide range of studies. Despite this, neither of them can be regarded as
an ideal model for the functional genomic and comparative genomic studies of the temperate
cereals and forage grasses, where the utility and effectiveness of the potential model is
primarily dependent upon the degree of conservation of gene order between the organisms
being compared. The relatively far phylogenetic distance to temperate cereals and forage
grasses brings inevitably the risk of breakdown of microsynteny in orthologous chromosome
regions.
Against this background, the Pooidae subfamily was re-examined in order to identify a
grass species which could be developed into a useful model representative of key temperate
cereals and forage grasses. The result of these investigations identified Brachypodium
distachyon, a small temperate grass with a genome as compact and as economical as the
genome of A. thaliana, and four times smaller than the genome of rice, a short 30-80 day life
cycle, undemanding growth requirements, favourable phylogenetic position as a “bridge
species” between tropical and temperate cereals, and many other useful features. Thanks to
this unique combination of features, B. distachyon has the potential to fulfil its role as
a”niche” model for functional genomics, both in terms of comparative genomic studies,
understanding the molecular basis of the response of plants to pathogen attack, and its utility
in metabolomic research.
With respect to cytogenetics, B. distachyon is so far, largely unstudied. In this study we
present the results of novel cytogenetic techniques which have revealed some intriguing
aspects of the organisation of the genome of this species.
13
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
14
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Location of specific nucleotide sequences of DNA on mitotic and meiotic
chromosomes of Secale vavilovii Grossh. by means of FISH
S. M. ROGALSKA, M. ACHREM, A. KALINKA
Chair of Cell Biology, University of Szczecin, Wąska 13, 71-415 Szczecin, Poland
The presence of specific repetitive sequence of DNA named pScJNK1 was identified on
mitotic and meiotic chromosomes of three lines of Secale vavilovii Grossh (225, 52 and 109)
by means of in situ fluorescent hybridisation (FISH). The pScJNK1 DNA sequence was
isolated from chromosome 2R of rye (Secale cereale L.) and it was used as a probe (NAGAKI
et al. 1999 ).
Two sub-lines with one 2R chromosome with an extra C-band and with two 2R
chromosomes with extra C-bands were isolated from the 225 line. Only one 2R with an extra
band was present in the 52 line, while in line 109 – no extra C-band was observed on 2R.
Following hybridisation with the pScJNK1 respectively, 1 and 2 fluorescent signals were
obtained; while in line 109 very small, dispersed signals were visible. Hybridisation signals
were also obtained in meiotic chromosomes, which allowed the authors to monitor the
behaviour of chromosomes with the extra band during meiosis. The extra heterochromatic
band on 2R chromosomes did not disturbed the course of meiosis in PMC’s of investigated
lines.
15
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
16
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Use of GISH and AFLP techniques to locate genes for drought
resistance and winter hardiness transferred from
Festuca sp. into Lolium multiflorum
A. KOSMALA1, M. SKIBIŃSKA1, E. ZWIERZYKOWSKA1, M. ŁUCZAK1, A. LEŚNIEWSKABOCIANOWSKA1, Z. ZWIERZYKOWSKI1, M. RAPACZ2, W. JOKŚ3, M.W. HUMPHREYS4
Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań,
Poland; 2Agricultural University of Cracow, Faculty of Agriculture and Economics,
Department of Plant Physiology, Podłużna 3, 30-239 Kraków, Poland; 3Szelejewo Plant
Breeding Ltd., 63-820 Piaski, Poland; 4Institute of Grassland and Environmental Research,
Plas Gogerddan, Aberystwyth, SY23 3EB, UK
1
In the last decade breeding programmes in grasses of the Lolium-Festuca complex are mainly
focused on introgression procedures, which allow a limited number of genes to be introduced
from one species into the reconstituted genome of the recurrent species. One of principal
objectives is the transfer of genes conferring resistance to abiotic stresses (drought resistance
and winter hardiness) from Festuca species (F. pratensis, F. arundinacea and F. glaucescens)
into Lolium multiflorum and L. perenne germplasm.
In our experiments, two different hybrids: L. multiflorum (2n=4x=28) × F. pratensis
(2n=2x=14) and F. arundinacea (2n=6x=42) × L. multiflorum (2n=4x=28) were backcrossed
onto L. multiflorum cultivars, and numerous BC1, BC2 and BC3 progenies were generated.
BC2/BC3 introgression plants from both combinations were tested in field and/or simulated
conditions for winter hardiness and drought resistance. Genomic in situ hybridization (GISH)
analyses were then performed on the most winter hardy and drought resistant plants to locate
putative genes for stress resistance. Using resistant L. multiflorum genotypes with a single
Festuca chromosome segment, it was possible to allocate AFLP (amplified fragment length
polymorphism) markers specific to that segment. Markers associated with genes conferring
stress resistance facilitate marker-assisted selection programmes to obtain new, more
persistent grass cultivars.
In this work, which is funded by the EU Framework V project "Sustainable grasslands
withstanding environmental stresses" (SAGES), we present the results of GISH analysis to
identify Festuca chromosome segments in L. multiflorum introgression lines and to find
segment-specific AFLP markers.
17
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
18
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Genome size in the genus Phleum
E. ŚLIWIŃSKA1, A. KULA2, A. JOACHIMIAK3, A. STEWART4
1
Department of Genetics and Plant Breeding, University of Technology and Agriculture, Kaliskiego 7,
85-789 Bydgoszcz, Poland; 2Department of Plant Breeding and Seed Science, Agricultural University,
Łobzowska 24, 31-140 Krakow, Poland; 3Laboratory of Plant Cytology and Embryology, Jagiellonian
University, Grodzka 52, 31-044 Krakow, Poland; 4Pyne Gould Guinness Ltd., PO Box 3100,
Christchurch 8015, New Zealand
Nuclear DNA content in 23 Phleum lines belonging to seven species and two sections was
estimated. Flow cytometric analysis revealed a genome size of 3.45 pg/2C for P. nodosum
(2x), 6.79, 9.08 and 12.24 pg/2C for P. pratense (4x, 6x and 8x, respectively), 2.93 pg/2C for
P. rhaeticum (2x), 6.15 pg/2C for P. commutatum (4x) (section Phleum); 3.56 pg/2C for P.
boehmeri (2x), 3.85 pg/2C for P. hirsutum (2x) and 2.89 pg/2C for P. arenarium (section
Chilochloa). The results showed that haploid genomes of P. nodosum and P. rhaeticum which
are supposed to be the parental components of hexaploid P. pratense (JOACHIMIAK, KULA
1996), differ in size (1.73 and 1.46 pg/2C, respectively). Tetraploid forms of P. pratense
originating from the Mediterranean area have a 2C genome twice the size of P. nodosum,
which suggests that they are autotetraploids. DNA content in most of the hexa- and octoploid
lines of P. pratense is not a multiple of P. nodosum or any sum of DNA content of the
hypothetical parental species but is lower than expected. This points to the possibility of
elimination of a part of the DNA in hexa- and octoploids as an effect of hybridisation process.
It is suggested that such phenomenon can be common in allopolyploids, as an effect of
functional and structural match of two different genomes existing in one and the same nucleus
(OZKAN et al. 2001). Higher genome size of perennial (P. boehmeri and P. hirsutum) than of
annual (P. arenarium) species from section Chilochloa seems to confirm that shortening of
life cycle length is followed by decrease of DNA content (BENNETT 1987).
References
BENNETT M.D. (1987). New Phytol. 106 (Suppl.), 177-200.
JOACHIMIAK A., KULA A. (1996). Plant Syst. Evol. 203:11-25.
OZKAN et al. (2001). Plant Cell 13: 1735-1747.
19
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
20
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Evaluation of mating compatibility in intergeneric crosses
Triticale × Zea by observing pollen tube germination
A. WOJCIECHOWSKI, M. ROKICKI, A. KATAŃSKA
Department of Genetics and Plant Breeding, A. Cieszkowski Agricultural University,
Wojska Polskiego 71c, 60-625 Poznań, Poland
Wide crossing is a valuable technique both in fundamental plant genetics and in practical
plant breeding programs. The works concerning wide crosses within the family Gramineae
can be found in literature. The behaviour of plants from different genera varied in particular
crosses. In spite of in many interspecific crosses mating compatibility occurred, the results of
interspecific hybridization depended on a selection of crossed forms.
The objective of the present work was to evaluate a level of cross-compatibility between
the species Triticale and Zea mays. Next, depending on the results we intend to obtain the
triticale haploids and to explain whether they are either result of maize chromosome
elimination or pseudogamy. Two genotypes of hexaploid triticale (Triticale, 2n=6x=42,
strains CHD 37 and CHD 400) were used as female parents in crosses with maize. Three
diploid maize (Zea mays, 2n=2x=20) genotypes were used as male parents, namely, Trophy
(sweet corn), Baccara and Janna. Degree of mating compatibility between the species under
investigation was determined by means of fluorescence microscope. The material used for the
study consisted of pistils after cross-pollination of two triticale genotypes and pistils of
triticale after pollination with maize pollen. Pistils for the microscopic observation were fixed
from 10 minutes to 48 hours after pollination.
In the case of cross pollination of two triticale genotypes the beginning of pollen grains
germination was observed 10’ after pollination and about 1 hour later pollen tubes were
present in ovary. Pollen grains of maize began to germinate on the stigma of triticale about 3
hours after pollination and first pollen tubes in ovary were visible about 4 – 5 hours after
pollination. There were many pollen tubes close to an ovule 24 – 48 hours after pollination
but there were not pollen tubes entering into an ovule.
21
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
22
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Comparative genomics in the grasses: Taking fodder grasses
into the genome age
O. A. ROGNLI
Department of Chemistry and Biotechnology, Agricultural University of Norway,
P.O. Box 5040, N-1432 Ås, Norway
Grasses cover about 40% of the agricultural land in Europe, and are very important both as
meadows and pastures for dairy and meat production, and as permanent grasslands, lawns and
turfs. Breeding of improved cultivars of forage grasses is challenging, and this is due to a
number of factors, i.e. perennial growth, breeding system (outcrossing), complex target traits,
and strong influence of environment and management practices. Although a number of grass
species is being utilized for meadows and grasslands, by far the most important belong to the
closely related genera Lolium and Festuca. Despite the problems of obtaining proper funding
for genomics programmes for these species, at least in Europe, a remarkable progress has
been achieved during the last 10-15 years. The first step in the development of genomic tools
has been the establishment of molecular markers, genetic linkage maps and QTL-mapping
projects both in Lolium and Festuca. The high degree of conservation of linkage blocks in the
genomes of fodder grasses in relation to other grass species like rice and the Triticeae,
demonstrated through the alignment of conserved chromosomal segments using common
RFLP-markers, has made it possible to use comparative genomics to rapidly identify
candidate genes for important traits in forage grasses. In order to use these tools efficiently, a
whole range of resources and technologies that have been developed in other crops will be
needed, e.g. BAC-libraries, cDNA-libraires for specific tissues, developmental stages or stress
factors, sequences for the development of SNP-markers, and transformation technologies to
verify the function of genes and to develop transgenic cultivars in the future. Several groups
around the world are currently developing these resources and technologies, there are for
example large genomics programmes involving EST-sequencing and partial sequencing of
Lolium in Australia, New Zealand and USA, and many groups have established
transformation techniques that work routinely for a number of grass species. These
programmes are largely funded by the private sector and access to these resources for the
public sector is uncertain. Although EU recently has funded genome research in Lolium
through the SAGES and GRASP projects, it is quite clear that this is not enough for Europe to
be competitive.
In this talk I will illustrate how comparative genomics can be used to speed up the
progress in understanding the genetic architecture of traits and the breeding of forage grasses,
using examples from our research in meadow fescue (Festuca pratensis Huds.).
The comparative map of meadow fescue that we have developed show a high degree of
conservation with Lolium and the Triticeae species on the megabase level. However, the
configuration of chromosomal segments of meadow fescue and Lolium is more similar to rice
than to the Triticeae, i.e. more ancestral, confirming the evolutionary divergence of Lolium
and Festuca from the Triticeae species prior to the divergence within the Triticeae. The
comparative map of meadow fescue makes it possible to compare the positions of QTLs
across all the major cereal species, and major, conserved QTLs and candidate genes can be
23
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
identified. This will be illustrated by our QTL mapping of stress-tolerance, seed production and
reproductive traits in meadow fescue. Vice versa, the forage grasses are certainly containing
genes, e.g. disease resistance and stress-tolerance genes, that could be interesting to utilize for
the cereal breeders.
As regards genomic research, grasses have both disadvantages and advantages. An
individual grass plant, whether selected from a cultivar or a natural population, will be highly
heterozygous due to the operation of self-incompatibility, which is securing nearly complete
outcrossing. This means that mapping populations can be constructed that will segregate for a
number of traits simultaneously, and very cost-effective trait mapping can be performed using
the same population and by collecting phenotypic data for a number of traits. In addition most
forage grasses can be clonally propagated, and clonal trials can be performed in diverse
environments making it possible to study genotype x environment interactions. However,
precise location of genes and QTLs is difficult using heterozygous plant materials. The strong
inbreeding depression, which makes it very difficult to develop inbred and recombinant inbred
lines, is a major drawback for forage grass genomics. Other strategies will have to be developed
to tackle the fine-mapping and isolation of genes and QTLs. Among the most promising is
association genetics based on LD mapping. In order to use this strategy, much more sequence
information is needed in order to develop and validate high-throughput SNP markers.
24
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Mapping tetraploid introgression population
within the Festuca/Lolium complex
M. GHESQUIÈRE
Unité de Génétique et d’Amélioration des Plantes Fourragères (UGAPF),
Institut National de la Recherche Agronomique (INRA), 86600 Lusignan, France
Polyploidy is frequently encountered in grasses which enables to develop amphiploid hybrids
usefully combining traits from their parent species. Festulolium varieties illustrates well this
prospect in forage grass breeding. In addition to strict Festulolium derived from crosses
between Italian ryegrass (Lolium multiflorum) and meadow fescue (Festuca pratensis), many
other hybrid combinations are possible involving, for instance, perennial ryegrass (L. perenne)
or F. glaucescens. Also, the hexaploid tall fescue (F. arundinacea) can be used for
amphiploidization with Lolium although not so straightforward as its two ancestor species,
related to present F. pratensis and F. glaucescens, have not the same affinity for homeologous
pairing towards Lolium.
However, genetics in amphiploids is complicate especially when parent species are
outbreeders and hybrids are not of strict disomic inheritance. Alternatively, backcrossing into
recurrent diploid species makes easier the genetic dissection of traits of interest while it limits
the number of independent loci which can be simultaneously transferred from Festuca into
Lolium. Thus, introgression traits may be difficult to be phenotypically detected unless they are
controlled by genes of major additive effect and/or which are closely linked onto the same
chromosome.
Releasing a high rate of suitable Festuca diploid recombinants could be also achieved
by using amphiploids from advanced generation or deliberate tetraploid backcross derivatives
and then, by monitoring through molecular markers the introgression process into diploid level.
That implies molecular mapping and QTL research to be early carried out in tetraploid
introgression population of partial tetrasomic inheritance.
Mapping requires in general to have many loci which are simultaneously heterozygous,
to score how many times recombination has occurred, to estimate accordingly genetic distance
and to finally order them onto linkage groups, i.e. chromosomes. In this aim, autopolyploidy
introduces two major difficulties: i) up to four alleles may be encountered at any locus so that
heterozygosity is complex and that segregating loci may involve not only one-dose allele
(Simplex), as in diploids, but also two-dose alleles (Duplex); ii) estimation of recombination
rate is not the same depending on linkage phasis, linkage in repulsion has much larger error
than linkage in coupling, which leads to perform mapping of any population of a given size at
various Lod scores.
This lecture briefly reviews specificity in mapping tetraploid populations compared to
conventional mapping of diploids. Experience is also reported in the frame of a BC2 tetraploid
population of L. multiflorum where mapping of AFLP and STS/SSR loci enabled to
quantitatively detect introgression of F. glaucescens.
25
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
26
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Bulked segregant analysis (BSA) and interval mapping (IM) as a methods for
identification of molecular markers in cereals
P. MASOJĆ
Agricultural University, Department of Genetics and Plant Breeding, Słowackiego 17,
71-434 Szczecin, Poland
The most commonly used strategy for detection of molecular markers in cereals is bulked
segregant analysis (BSA). A population with different alleles underlying quantitative trait can
be split into two groups of segregates representing opposite edges of the variation range. DNA
pools of these two bulks are subsequently analysed for any differences on a molecular level.
Different banding pattern reflects linkage between marker locus and a gene of interest. This
simple way of marker identification is not applicable to loci linked with the target gene in
repulsion. It is also difficult to identify markers if the trait under consideration is polygenic. A
modification of BSA can be used for detection of marker loci in such cases. First of all, the
number of individuals evaluated in respect to the trait should be adjusted to the expected
number of underlying loci i.e. for a four loci systems, population of at least 2000 plants should
be analysed to be able to select several multiple homozygotes. Secondly, segregants
representing opposite edges of the variation range cannot not be bulked but DNA should be
extracted from individual plants. Thirdly, measurements should be made with precision in
order to minimize environmental error which could make it impossible to distinguish true
homozygotes. Electrophoretic analysis of six to eight DNA samples from individuals of each
opposite phenotypic group is sufficient to reveal significant differences in marker distribution.
Experimental prove that this modification of the BSA method might be useful for marker
identification was obtained through analysis of RAPD markers linked to genes controlling
sprouting resistance and alpha-amylase activity in rye. Although a population originating from
only 350 F2 plants was used for analysis of these two multiloci systems, it was sufficient to
detect several markers each for different gene. Tight linkage of these markers to appropriate
QTL was further proved by interval mapping (IM) using newly built genetic map of rye
genome. Eight independent QTLs for sprouting resistance and ten for alpha-amylase activity in
kernels were detected along linkage groups. Two of these QTLs were common for both traits,
showing that the two genetic systems partially overlap. The studied population was divided into
two genotypic classes within each marker locus - one class with individuals expressing bands
presence and opposite class showing bands absence (null). Differences between genotypic
classes in the mean values of each trait were calculated. This procedure revealed major QTLs,
which strongly affected sprouting or alpha-amylase activity, and those of minor effects. It was
also shown that selection based only on single major QTL was not efficient. The substancial
decrease of sprouting or alpha-amylase activity could only be imposed by pyramiding of 4-5
favourable genes in a single genotype.
Comparison of modified BSA and IM as a two different methods for identification of
valuable gene and its molecular marker showed that in multilocus systems the latter strategy is
much more powerfull. This is because more QTLs and markers can be distinguished and more
information can be gained on linkage and relationship of these loci.
27
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
28
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Acclimation of photosynthetic apparatus to cold: new aspects of
the role in winter hardiness of grasses
M. RAPACZ1*, D. GĄSIOR1, Z. ZWIERZYKOWSKI2, A. LEŚNIEWSKA-BOCIANOWSKA2,
M.W. HUMPHREYS3, A.P. GAY3
1
Agricultural University of Cracow, Faculty of Agriculture and Economics, Department of
Plant Physiology, Podłużna 3, 30-239 Kraków, Poland; 2Institute of Plant Genetics, Polish
Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland; 3Institute of Grassland and
Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion, SY23 3EB, UK
An important aspect of cold tolerance is the avoidance of photoinhibition of photosynthesis that
can occur during periods of relatively high light and low temperature in autumn and winter.
Photoinhibition can occur if the rate of light harvesting by PSII exceeds the capacity of electron
transport, which is reduced by low temperatures. Many studies have shown that resistance to
photoinhibition is related to freezing tolerance and winter hardiness. Several alternative
mechanisms for dissipation of excess energy harvested by PSII have been demonstrated which
reduce the potential for photoinhibition. Evergreen plants dissipate excess light by increasing
capacity and activity of the xanthophyll cycle (increasing NPQ). Green algae reduce their lightharvesting capacity by reducing chlorophyll and by maintaining lower levels of Lhcb
polypeptides. On the other hand cold tolerant herbaceous plants are suggested to decrease
energy imbalance by increasing photosynthetic capacity and accumulation of nonphotosynthetic pigments, including anthocyanins.
In contrast to previous reports for herbaceous Poaceae the recent studies on androgenic
plants of Festulolium shows that in these Lolium and Festuca hybrids, the most common reason
for higher resistance to cold induced photoinhibition was an increase in NPQ. However, in one
of the androgenic genotypes reduced NPQ was compensated for by increased electron transport,
a different mechanism of avoidance of photoinhibition to that observed in the parent material.
The obtained results show that different mechanisms of photoprotection can’t be
uncritically attributed to different plant groups.
The further question is also the potential factor triggering photosynthetic acclimation in
the Lolium-Festuca complex.
29
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
30
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Peroxidase polymorphism in Calamagrostis epigejos (Poaceae)
M. KRZAKOWA¹ J. JAŃCZYK-WĘGLARSKA², E. ŚLIWIŃSKA¹
1
Department of Genetics, Adam Mickiewicz University, Międzychodzka 5, 60-371 Poznań, Poland;
2
Botanical Garden of Adam Mickiewicz University, Dąbrowskiego 165, 61-594 Poznań, Poland
In recent years, the species of Calamagrostis genus and their most frequent representative, C.
epigejos in particular evoke significant interest (FREY, PASZKO 1999). The interest reflects high
plasticity of morphological traits in the species and its broad ecological tolerance, expressed by
its vast manifestation both in natural conditions, e.g., in pine forests, and by its rapid growth in
biotopes destroyed by human activities and polluted with industrial waste, i.e. in conditions
which eliminate other species (JAŃCZYK-WĘGLARSKA 1997). The species exhibits high
propensity to form hybrids with other species of the genus and even the interspecific hybrid is
known (Ammophila arenaria x Calamagrostis epigejos), termed Ammocalamagrostis baltica
(Flugge ex Schrader) P.Fourn.
C. epigejos is an amphimictic polyploid complex with three main cytotypes: the tetraploid
(2n=28), the hexaploid (2n=42) and the octoploid (2n=56) ones; however, cytotypes with
2n=35, and 2n=49 have been also found. In Poland and in Central Europe, the tetraploid
cytotype, 2n=48, is the most frequent (LEHMANN, REBELE 1994). Studies on genetic variability
of natural populations and populations growing on soils contaminated with heavy metals have
demonstrated differences in frequencies of genotypes (LEHMANN 1997). Both C. epigejos and
some of its hybrids can scarcely be distinguished morphologically. Peroxidase of C. epigejos
exhibits cathodal migration. Since cathodic peroxidases seem to represent good markers in
determination of interspecific differences (KRZAKOWA 1993) and of intraspecific variability
(KRZAKOWA 1996), their application for determination of variability level in C. epigejos seems
rational.
In the study, variability in 8 populations of C. epigejos is described. Three of the
populations have proven to be monomorphic while the five remaining ones have demonstrated
certain extent of polymorphism, resulting from gene flow from other species and, thus,
suggesting their hybrid character.
References
FREY L., PASZKO B. (1999). Remarks on the distribution, taxonomy and karyology of Calamagrostis
species (Poaceae) with special reference to their representatives in Poland. Frag. Flor. Geobot. Suppl. 7:
33-45
JAŃCZYK-WĘGLARSKA J. (1997). An ex situ ecological experiment on the morphological and
developmental variation of Calamagrostis epigejos (Poaceae). Frag. Flor. Geobot. 42(2): 239-247.
KRZAKOWA M. (1993). The significance of cathodic peroxidases in the taxonomy of Bryophytes. In:
K.G. Welinder., S.K. Rasmussen., C. Penel., H. Greppin (eds.): Plant Peroxidases: Biochemistry and
Physiology. University of Geneva 1993 pp. 213-219.
KRZAKOWA M. (1996). Genetic diversity of Phragmites australis (Cav.) Trin. ex Stued revealed by
electrophoretically detected differences in peroxidases. In: Obinger C., Burner U., Penel C., Greppin H.
(eds.). Plant Peroxidases: Biochemistry and Physiology University of Geneva 1996 pp. 184-189.
LEHMANN C., REBELE F. (1994). Zum Potential sexueller Fortpflanzung bei Calamagrostis epigejos (L.)
Roth.-Verh. Ges. Ökol. 23: 445-450
LEHMANN C. (1997). Clonal diversity of populations of Calamagrostis epigejos in relation to
environmental stress and habitat heterogeneity. Ecography 20: 483-490.
31
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
32
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
PCR-RFLP analysis of mitochondrial DNA of Secale species
L. SKUZA AND S.M. ROGALSKA
Chair of Cell Biology, University of Szczecin, Wąska 13, 71-415 Szczecin, Poland
Genus Secale include of several species which differed with morphological, physiological and
cytogenetic features. There is a few literature data about mitochondrial genome organization in
Secale. On the other hand there is a lot of information about mitochondrial genome of other
cereals. According to this restriction analysis of mtDNA of rye species was undertaken in Chair
of Cell Biology. Two mitochondrial genes atpA and atp9 were treated with three endonucleases
EcoRI, HindIII and XhoI by means of PCR-RFLP method in five rye species. From the
research were no differences between species. Restriction enzymes HindIII and XhoI didn’t
recognized any restriction sites, however EcoRI recognized one restriction site in atpA only in
all investigated species.
33
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
34
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Cytological and molecular analysis of doubled haploids (DH) of wheat
(Triticum aestivum ssp. vulgare)
Z. BRODA AND A. WOJCIECHOWSKA
Department of Genetics and Plant Breeding, A. Cieszkowski Agricultural University,
Wojska Polskiego 71c, 60-625 Poznań
Doubled haploids have particular and important applications in basic research and plant
breeding. Random amplified polymorphic DNA (RAPD) banding patterns are useful in
identyfying genotypes of various crops.
The investigations were undertaken with the aim to evaluate the genetic similarity
among doubled haploids of wheat. DNA polymorphism was evaluated by PCR-RAPD method
with the use of 18 primers from Operon Technologies. 86 different markers were formed.
Genetic similarity between doubled haploid lines ranged from 35% to 98%.
Cytological analysis showed that in progeny DH2 among investigated lines DH 242,
243, 246, 278, 284 were found delated and univalent chromosomes in metaphase I and
anaphase I about 20% and 15%, respectively.
35
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
36
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Intra- and intervarietal polymorphism in wheat generated
by RAPD primers
M. RĘBARZ, A. KUCZYŃSKA, K. KRYSTKOWIAK, J. BOCIANOWSKI
Institute of Plant Genetics, Polish Academy of Sciences,
Strzeszyńska 34, 60-479 Poznań, Poland
RAPD (random amplified polymorphic DNA) polymorphism was studied in chosen spring and
winter wheat cultivars. The aim of this study was to evaluate genetic similarity between
cultivars with regard to polymorphism within cultivars. Material for the studies covered 12
wheat cultivars: Lavett, Melon, Quatro, Sandrine (spring), and Finezja, Mewa, Pegassos, Roma,
Rysa, Turnia and DED315/89 (winter). Five individuals randomly chosen from each cultivar
were subjected to PCR-RAPD analysis. Additionally, bulk samples of DNA extracted from 5
individuals of each cultivar were analysed. Eight 10-mer primers, selected on the basis of their
performance and reproducibility in previous experiment were used. The primers generated 192
amplification products, 83,85% of which were polymorphic. Genetic similarity was estimated
according to the formula given by Nei and Lee (1979) and then these results were used for the
hierarhical grouping of genotypes. The highest similarity was found between cultivar Rysa and
breeding line DED315/89 and between Pegassos and Quatro. The lowest similarity was
evaluated for Finezja and Lavett, and DED315/89 and Pegassos. The intravarietal diversity was
observed for all the studied cultivars. Among examined cultivars the best homogeneous
appeared to be DED315/89 and Rysa; similarity between individuals within these cultivars
ranged from 0.78 to 0.87 for DED315/89, and 0.71 to 0.82 for Rysa.
37
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
38
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Dynamic of changes in water relation and ABA content during first hours of
cold acclimation of androgenic lines of Festulolium
D. GĄSIOR1, M. RAPACZ1, F. JANOWIAK2, Z. ZWIERZYKOWSKI3,
A. LEŚNIEWSKA-BOCIANOWSKA3, M.W. HUMPHREYS4
1
Agricultural University of Cracow, Faculty of Agriculture and Economics, Department of
Plant Physiology, Podłużna 3, 30-239 Kraków, Poland; 2Department of Plant Physiology,
Polish Academy of Sciences, Podłużna 3, 30-239 Kraków, Poland; 3Institute of Plant Genetics,
Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland; 4Institute of Grassland
and Environmental Research, Plas Gogerddan, Aberystwyth, SY23 3EB, UK
Androgenesis can be the source of differential stress resistance, also in winter hardiness,
however physiological basis of this phenomenon is unclear.
The aim of this experiment was to verify if winter hardiness and frost persistence of
androgenic lines of Festulolium are related to physiological changes in relative water content,
osmotic potential and abscisic acid (ABA) content during first days of cold acclimation. Such
changes in the concentration of ABA which is known as one of potential signal involved in
gene expression during cold acclimation, was related before to winter hardiness of wheat and
barley cultivars.
Three groups of androgenic and parent materials were used in the experiment: lines of
high freezing tolerance (HF-105, HS-212, HS-262, Felopa-18), of medium hardiness (HS-211,
Sulino-4) and frost susceptible HF-58. In the case of frost hardy plants temporary decrease in
ABA concentration was recorded during the first 6 hours of cold acclimation. Next the
concentration increased back and the maximum was observed at 12th hour and then ABA
contents declined again in HS-212 and Felopa. This decrease can’t be confirmed in other winter
hardy plants due to the lack of data recorded after 54 hours of cold hardening (HF-105) or to the
very low concentration of ABA (HS-262). In medium hardy plants the highest increase in ABA
level was observed in the 30th hour of cold hardening. In the case of winter susceptible HF 58
such increase in ABA content was not observed.
The obtained results were consistent with those obtained for wheat and barley cultivars.
On the other hand the changes in ABA concentrations during first hours of cold acclimation
didn’t correlate with water relations parameters – osmotic potential and relative water content.
39
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
40
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Studies of soluble carbohydrate and phenolic accumulation
and winter hardiness of Lolium-Festuca hybrid derivatives
A. PŁAŻEK1, M. WĘDZONY2, E. NIEMCZYK2, Z. ZWIERZYKOWSKI3, A. KOSMALA3,
A. LEŚNIEWSKA-BOCIANOWSKA3, M.W. HUMPHREYS4
1
Agricultural University of Cracow, Faculty of Agriculture and Economics, Department of
Plant Physiology, Podłużna 3, 30-239 Kraków, Poland; 2Department of Plant Physiology,
Polish Academy of Sciences, Podłużna 3, 30-239 Kraków, Poland; 3Institute of Plant Genetics,
Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland; 4Institute of Grassland
and Environmental Research, Plas Gogerddan, Aberystwyth, SY23 3EB, UK
Winter hardiness of grasses depends mainly on their frost tolerance and resistance to snow
mould pathogens. Many authors reported the main role of soluble carbohydrates in coldinduced resistance to frost or fungi. The accumulation of hexoses activates hexokinase, key
sugar-sensing enzyme that initiates a signal transduction process that could be a signal to
production of PR (pathogenesis-related) proteins. High levels of sugars influence the osmotic
potential of plant tissues and can this way limit their colonization by fungi. Under cold
treatment changes in cell wall structure and accumulation of polysaccharides and lignin in cell
walls can also develop. The strengthening of cell walls limits tissue penetration by fungal
mycelium and decreases efflux of water from cells under osmotic stresses.
In performed studies accumulation of soluble carbohydrates and phenolics in LoliumFestuca introgression forms and androgenic forms derived from two Festulolium cultivars
‘Felopa’ and ‘Sulino’ was analysed. The plants were chosen to studies after field selection in
winter conditions in Łopuszna. On the basis of the observed changes in sugar content the
studied forms were compared with parent species and divided into three groups: groupaccumulating soluble carbohydrates like Lolium and like Festuca; the third group was called
‘mixed type’. The winter hardiness and resistance to snow mould in the field conditions,
laboratory frost tolerance and changes in the sugar content were analysed. The introgression
forms of Festuca type were characterised by better winter hardiness and higher frost tolerance
than plants of Lolium type. The forms derived from Festulolium cv. ‘Sulino’ were more tolerant
to frost and winter conditions than genotypes derived from cv. ‘Felopa’. Large differences in
phenolic compound accumulation were observed among the analysed forms. However, this
parameter was not correlated with the winter hardiness of plants. Similarly, no direct influence
of soluble carbohydrate and phenolic compound content on plant resistance to snow mould was
found.
41
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
42
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
Genetic diversity in Middle-East barley and wheat landraces
and cultivars using AFLP markers
A. SHOAIB1, H. MIGDADI2, A. JANIAK3, J. GUZY-WRÓBELSKA3, I. SZAREJKO3
1
Syrian Atomic Energy Commission, P.O. Box 6091, Damascus, Syria; 2 National Centre for
Agriculture Research and Technology Transfer (NCARTT), P.O. Box 639, Baqa’a 19381,
Amman, Jordan; 3 Department of Genetics, Silesian University, Jagiellońska 28,
40-032 Katowice, Poland
A detailed knowledge of the genetic variation and relationships among different genotypes in
the main crops as wheat or barley is an important factor for the success of breeding
programmes, and for the effective sampling and utilisation of available germplasm resources.
Thus, comparative assessments are necessary in order to obtain the knowledge concerning
genetic variability in these crops. The aim of this study was to determine the extent of the
genetic diversity among Jordanian and Syrian wheat and barley genotypes with the use of
AFLP markers.
Altogether, ten barley (Hordeum vulgare) and fifteen wheat (Triticum aestivum and T.
durum) genotypes were analysed using a set of 6 and 11 AFLP primer combinations,
respectively. A total of 250 and 525 bands were scored as binary data, with an average
polymorphism percentage of 36% for barley and 46.67% for wheat genotypes. In all three
species the average genetic diversity was low, and did not exceeded 0.15 among all analysed
genotypes. The dissimilarity matrices were then used to cluster the data using the Unweighted
Pair Group Method with an arithmetic average (UPGMA) algorithm. In barley, the resulting
phenograms indicated a clear distinction between the Jordanian and Syrian landraces as well as
between the improved cultivars from both origins. In wheat, two main clusters were identified,
grouping separately T. aestivum and the T. durum cultivars. In the second cluster the landraces
and improved cultivars of T. durum were also subgrouped separately. All the phenetic clusters
of analysed genotypes were supported with high bootstrap values, confirming the reliability of
the AFLP data used for the genetic diversity analysis.
43
“Application of Novel Cytogenetic and Molecular Techniques in Genetics and Breeding of the Grasses”,
International Workshop, 1-2 April 2003, Poznań, Poland
44