This is the author’s final, peer-reviewed manuscript as accepted for publication. The
publisher-formatted version may be available through the publisher’s web site or your
institution’s library.
This item was retrieved from the K-State Research Exchange (K-REx), the institutional
repository of Kansas State University. K-REx is available at http://krex.ksu.edu
Single-copy gene fluorescence in situ hybridization and
genome analysis: Acc-2 loci mark evolutionary chromosomal
rearrangements in wheat
Tatiana V. Danilova, Bernd Friebe, Bikram S. Gill
How to cite this manuscript
If you make reference to this version of the manuscript, use the following information:
Danilova, T. V., Friebe, B., & Gill, B. S. (2012). Single-copy gene fluorescence in situ
hybridization and genome analysis: Acc-2 loci mark evolutionary chromosomal
rearrangements in wheat. Retrieved from http://krex.ksu.edu
Published Version Information
Citation: Danilova, T. V., Friebe, B., & Gill, B. S. (2012). Single-copy gene fluorescence
in situ hybridization and genome analysis: Acc-2 loci mark evolutionary chromosomal
rearrangements in wheat. Chromosoma, 121(6), 597-611.
Copyright: © Springer-Verlag Berlin Heidelberg 2012
Digital Object Identifier (DOI): doi:10.1007/s00412-012-0384-7
Publisher’s Link: http://link.springer.com/article/10.1007%2Fs00412-012-0384-7
1
Single-copy gene fluorescence in situ hybridization and genome analysis: Acc-
2 loci mark evolutionary chromosomal rearrangements in wheat
Tatiana V. Danilova, Bernd Friebe, Bikram S. Gill
Wheat Genetic and Genomic Resources Center, Department of Plant Pathology, Kansas State
University, Manhattan, KS 66506
Abstract Fluorescent in situ hybridization (FISH) is a useful tool for physical mapping of
chromosomes and studying evolutionary chromosome rearrangements. Here we report a robust
method for single-copy gene FISH for wheat. FISH probes were developed from cDNA of
cytosolic acetyl-CoA carboxylase gene (Acc-2) and mapped on chromosomes of bread wheat,
Triticum aestivum L. (2n=6x=42, AABBDD), and related diploid and tetraploid species. Another
nine full-length cDNA FISH probes were mapped and used to identify chromosomes of wheat
species. The Acc-2 probe was detected on the long arms of each of the homoeologous group-3
chromosomes (3A, 3B, and 3D), on 5DL and 4AL of bread wheat, and on homoeologous and
nonhomoeologous chromosomes of other species. In the species tested, FISH detected more Acc-
2 gene or pseudogene sites than previously found by PCR and Southern hybridization analysis
and showed presence/absence polymorphism of Acc-2 sequences. FISH with the Acc-2 probe
revealed the 4A-5A translocation, shared by several related diploid and polyploid species and
inherited from an ancestral A-genome species, and the T. timopheevii specific 4A
t
-3A
t
translocation.
Key words wheat, single-copy gene fluorescence in situ hybridization, acetyl-CoA carboxylase,
karyotype evolution
2
Introduction
Bread wheat, Triticum aestivum L., is an allohexaploid (2n=6x=42, AABBDD) containing
genomes of three species. Tetraploid wheats, 2n=4x=28, with AABB (Emmer group) and
A
t
A
t
GG (Timopheevii group) genomes are the result of two separate hybridization events
involving T. urartu Tumanian ex Gandilyan, the A-genome donor (Dvorak et al. 1993), and an
outcrossing species closely related to the S genome of Aegilops speltoides Tausch, the ancestor
of B and G genomes (Dvorak and Zhang 1990; Kilian et al. 2007). Hexaploid wheat arose from
hybridization of cultivated emmer T. turgidum L. (AABB) (Nesbitt and Samuel 1996; Dvorak et
al. 2004) and the D-genome donor Ae. tauschii Coss. (Kihara 1944; McFadden and Sears 1946;
Dvorak et al. 1998).
The C- and N-banding karyotyping of bread wheat and its cultivated and wild relatives
provided a foundation for analyzing the cytogenetic structure of the Triticeae (Gill et al. 1991;
Friebe and Gill 1996). In situ hybridization with probes containing rye repeats (pSc119) were
used to identify B-genome chromosomes (Rayburn and Gill 1985), and the Afa-family repeat
from Ae. tauschii was used to identify the D-genome chromosomes (Rayburn and Gill 1987;
Mukai et al. 1993). Fluorescence in situ hybridization (FISH) with probes containing a GAA-
satellite sequence produced patterns similar to N-banding or C-banding on chromosomes of
barley, rye, wheat, and Aegilops species (Gerlach 1977; Pedersen et al. 1996). Chromosome
identification using repeated DNA probes is difficult because the abundance and distribution of
repetitive elements can vary among homologous chromosomes within a species (Friebe and Gill
1994), or among chromosomes of closely related species (Badaeva et al. 1994; Dedkova et al.
2007).
A series of wheat aneuploid, deletion, and substitution lines have been used for gene
mapping (Sears 1954; Endo and Gill 1996). The chromosome bin physical map of about 6,000
expressed sequence tags (ESTs) has been established using deletion stocks (Qi et al. 2004).
3
However, the deletion bins are large in size and loci within the bins cannot be ordered
physically.
Using single-copy gene FISH has the potential of fine physical mapping and ordering of
genes along the chromosomes, including chromosomal regions with low recombination rates. In
wheat, gene-specific probes and probes for tandem repeats, including oligonucleotide probes,
were used in indirect FISH with blocking to detect tandem repeats and gene clusters (Mukai et
al. 1993; Pedersen and Langridge 1997; Turner et al. 1999; Li et al. 2003; Turnbull et al. 2003;
Szakacs and Molnar-Lang 2007; Cuadrado et al. 2008 a, b). Indirect FISH uses nonfluorescent
chemicals for labeling and needs an additional detection step for visualizing the hybridization
site and amplifying of the signal. Single gene probes also were used in FISH with tyramide
signal amplification (Perez et al. 2009). These methods need blocking, detection, and signal
amplification steps and have not been used widely.
BAC clones were used as FISH probes for genic regions in species with small genomes and
a low content of repetitive elements, such as Arabidopsis (Lysak et al. 2001), Brachypodium
(Febrer et al. 2010; Ma et al. 2010), rice (Jiang et al. 1994), and sorghum (Woo et al. 1994; Kim
et al. 2002). Bread wheat has one of the largest plant genomes (17 Gb), containing about 90%
repetitive sequences, 70% of which are transposable elements and a large fraction of
microsatellites and tandem repeats (Li et al. 2004). BAC-FISH in wheat painted either all
chromosomes over their entire length, produced a genome-specific painting, or painted clusters
of tandem repeats depending on the content of repetitive sequences in a BAC clone (Zhang et al.
2004 a, b).
For genomes enriched with repeats, a repeat-free probe for a particular genic region either
can be produced from cDNA or developed from genomic DNA by sequence analysis and PCR
amplification of the repeat-free region, and used in direct FISH as was shown in maize (Wang et
al. 2006; Lamb et al. 2007; Danilova and Birchler 2008) and barley (Ma et al. 2010). In direct
4
FISH, fluorochromes are incorporated directly into DNA probes and the procedure does not need
detection step.
As a model for probe development, we selected the wheat gene Acc-2 encoding cytosolic
acetyl-CoA carboxylase (ACCase). Plants have two forms of ACCase; one is present in plastids,
catalyzing the de novo synthesis of fatty acids and another in the cytosol, involved in the
synthesis of very long-chain fatty acids and secondary metabolites such as flavonoids and
anthocyanins. Distinct from other species, where plastid ACCase is composed of four subunits
some of which are encoded by chloroplast genes, in grasses both plastid and cytosolic forms of
ACCase are encoded by nuclear genes, Acc-1 and Acc-2, respectively (Sasaki and Nagano 2004).
In wheat, the Acc-1 genes were mapped on the short arms of the group-2 chromosomes
(Gornicki et al. 1997) and Acc-2 in the distal region of the long arm of the group-3 chromosomes
and chromosome arm 5DL (Faris et al. 2001). A partly processed pseudogene Ψ-Acc-2 with
more than 90% identity to the Acc-2 coding sequence was found in wheat, T. urartu, and Ae.
tauschii. Six different sequences of the Acc-2 gene and pseudogene are present in the wheat
genome (Podkowinski et al. 1996; Huang et al. 2002); at least three of the Acc-2 genes are
expressed equally in young leaves (Podkowinski et al. 2003) and their coding sequences (6.3 kb)
have 98% identity (Gornicki et al. 1994; Chalupska et al. 2008). The genes and pseodogenes are
likely arranged in tandem repeats (Faris et al. 2001).
The objective of this research was to develop an Acc-2-specific probe and map Acc-2 on
chromosomes of wheat and its diploid and tetraploid progenitor species using multicolor direct
FISH. For individual chromosome identification, probes for tandem repeats and microsatellites
were used. For chromosomes lacking these markers, chromosome-specific full-length cDNA
FISH markers were developed. The present work demonstrates the usefulness of direct FISH for
physical mapping of genic sequences and studying chromosome rearrangements and will have
broad applications in genome analysis of the Triticeae.
