Genome Organization and Comparative Genomics - Wheat Science and Trade - page 328

genes present in the region spanned by those two
markers were mapped in the population of infor-
mative recombinants. The recombinant progeny
were also test-crossed to fully classify their geno-
type at the VRN-1 locus. Two genes, AP1 and
AGLG1 , were completely linked to VRN-1 and
were used to screen a T. monococcum DV92 BAC
library. Additional screens were carried out with
markers that closely fl anked VRN-1 , resulting in
three T. monococcum contigs. One of the contigs
contained AP1 and two other markers that fl anked
VRN-1 proximally at 0.02 cM; a second contig
contained AGLG1 ; and the third contig contained
markers that fl anked VRN-1 distally, with the
closest marker located 0.02 cM from VRN-1 . The
two gaps in the T. monococcum contig were bridged
by contigs in rice and sorghum. Comparative
sequence analysis of the orthologous wheat, rice,
and sorghum regions showed, with the exception
of a single gene duplication in sorghum and wheat
compared to rice, perfect colinearity. No new
genes were identifi ed and AP1 and AGLG1
remained the best candidates for VRN-1 . Expres-
sion profi ling and sequence variation identifi ed in
different VRN-A m 1 alleles identifi ed AP1 as the
most likely candidate for VRN-1 (Yan et al.,
2003).
A similar methodology was used for cloning
the VRN-A m 2 gene in the distal region of 5A m L.
Two markers that fl anked the VRN-2 gene in a
low-resolution map were used to identify recom-
binant plants from a large (5,698 gametes)
mapping population (Yan et al., 2004). Physical
mapping was conducted simultaneously in T.
monoccoccum , barley, and rice. Markers developed
from the BAC clones were mapped in the recom-
binant population, which was also phenotyped for
vernalization response. This resulted in VRN-2
being delimited to a 0.04-cM interval, spanned in
T. monococcum by four BAC clones that totaled
some 440 kb. Sequencing and annotation of these
BAC clones identifi ed one pseudogene and eight
genes, three of which were completely linked to
VRN-2 . Two of the genes, designated ZCCT1
and ZCCT2 , were the result of a duplication that
occurred some 14 MYA and showed homology to
the constans ( CO ) and CO -like proteins in Arabi-
dopsis; CO is the key gene in the Arabidopsis

photoperiod pathway, making these two genes
good candidates for VRN-2 . No function could
be assigned to the third gene. A phylogenetic
analysis of the ZCCT genes and their homologues
in the A genome of tetraploid wheat, in barley,
rice, and Arabidopsis suggested that the lineage
that gave rise to the ZCCT genes originated in the
grasses and that the ZCCT genes most likely arose
as a cold-adaptation response in temperate cereals.
Messenger RNA of ZCCT1 was detected in the
leaves and apices of unvernalized plants and grad-
ually decreased during vernalization; ZCCT2 had
a similar expression pattern in the leaves but
could not be detected in the apices, which are the
critical meristems for transition from vegetative
to reproductive phase. This made ZCCT1 the
preferred candidate for VRN-2 (Yan et al.,
2004).
Sequence analysis of the ZCCT1 gene in winter
and spring T. monococcum accessions showed that
some of the spring ZCCT1 alleles carried a single
base-pair mutation that resulted in the substitu-
tion of an amino acid that was conserved in all
ZCCT and CO-like proteins analyzed. In other
spring accessions, the entire ZCCT1 gene had
been deleted. The deletion of the ZCCT1 gene
also appeared to be the cause of differentiation
between winter and spring habit at the barley
VRN-H2 locus. The identity of ZCCT1 as
VRN-2 was validated using an RNAi transgenic
approach. Down-regulation of ZCCT1 in hexa-
ploid wheat by transformation with an RNAi con-
struct that contained part of the T. monococcum
ZCCT1 gene accelerated fl owering. Transgenic
plants had reduced ZCCT1 and enhanced AP1
levels, consistent with the hypothesis that ZCCT1
is a repressor that is derepressed by vernalization
(Yan et al., 2004).
The cloning of VRN-3 represents an example
of how comparative information can be used to
identify candidate genes for the trait of interest
(Yan et al., 2006). The VRN-B3 locus was mapped
to a 6-cM interval in a population of 82 recombi-
nant substitution lines. The most closely linked
marker was ABC158, which mapped 1 cM proxi-
mal to VRN-B3 . The ABC158 orthologue in rice
was located 50 kb proximal to Hd3a ; Hd3a is
orthologous to the Arabidopsis gene FLOWER-