Freezing Tolerance in the Triticeae - Translational Genomics for Crop Breeding: Abiotic Stress, Yield and Quality - page 108

cDNA sequencing strategies. Even the genomic
clones having the different sequenced CBF2A -
CBF4B genome segments were indistinguish-
able when they were RFLP(restriction fragment
length polymophism)-fingerprinted with a stan-
dard set of enzymes and coding sequence probes.

Winter Hardiness and Association
between VRN-1Allelic State and
CBFCopy-Numbers

The relationship in these four barley geno-
types between the allelic state at VRN-H1 / FR-
H1 and the copy-numbers of the CBF2A - CBF4B
genomic segment at FR-H2 raised the question
of whether this association occurs in other bar-
leys, or in wheat. To address these questions, a
wider sampling of barley and surveys of wheat
were carried out using DNA blot hybridization
strategies. The wider sample of barley was scored
with a CBF2 gene-specific probe (Knox et al.
2010). This CBF2 probe cross hybridizes to both
CBF2A and CBF2B , the latter a single copy
gene, and as such the copy-numbers of CBF2A
can be estimated by determining the ratio of
CBF2A to CBF2B signal intensity. While this
is by no means an exhaustive survey of barley,
the data strongly supported the notion that there
are two CBF2 paralogs in the genomes of vrn-H1
winter allele genotypes, one of which is ampli-
fied to multiple copies, and that there is only a
single CBF2 paralog in the genomes of spring
allele genotypes (Figure 7.2). Just as analysis of
CBF s in barley revealed that CBF2 and CBF4
expression levels correlated with gene copy-
numbers, a similar strategy in wheat revealed that
CBF14 expression levels seem to correlate with
copy-numbers (Knox et al. 2010). Using single
chromosome recombinant lines Vagujfalvi and
colleagues (Vagujfalvi et al. 2005) detected sig-
nificantly higher transcript levels of CBF14 ,
CBF15 , and CBF16 ( CBF7 , CBF1A , and
CBF1C , respectively) in recombinants having
the 'Cheyenne' 5A homoeolog segment than in
recombinants having the 'Chinese Spring' 5A
homoeolog segment. In separate experiments

Fig. 7.3. CBF14 expression in eight winter wheat culti-
vars. CBF14 transcript levels are normalized to actin tran-
script levels. Wheats are grouped by their functional class.
Hard red winter: 'Karl 92' (Krl92), 'Jagger' (Jggr), 'Overly'
(Ovrly), TAM101 (T101), TAM105 (T105); soft red win-
ter: 'Freedom' (Frdm), 'McCormick' (McCk), and 'Pioneer
2545' (2545).

in which a panel of nine winter wheats and
three spring wheats were assayed, transcripts for
CBF1 , CBF2 , CBF9, and CBF14 accumulated
to much higher levels in winter wheats than in
spring wheats (Stockinger et al. 2007). How-
ever, across the group of nine winter wheats
large differences in CBF14 transcript levels
were detected (Figure 7.3). Scoring this wheat
germplasm with a CBF14 gene-specific probe
revealed that CBF14 copy-numbers did indeed
vary (Knox et al. 2010). Moreover, CBF14 copy-
number differences parallel expression level dif-
ferences; that is, the wheats expressing CBF14
at higher levels had greater signal intensity for
two of the CBF14 cross hybridizing fragments
relative to levels detected in lines having greater
signal intensity for only one of the CBF14 cross
hybridizing fragments (Knox et al. 2010). At this
time it is unclear whether the other CBF s exhibit-
ing expression-level differences across the differ-
ent wheat lines also differ in copy-number.
Copy-number variable regions are thought
to be a source of allelic diversity in terms of
base pair differences within a species that is
greater than that of single nucleotide polymor-
phisms (Alkan et al. 2011). In Triticeae cere-
als the genomic regions encompassing the CBF