(go to
KOMUGI Home) (go
to WIS List) (go to NO.86
Contents)
Materials and methods
Source of the probe
Aneuploid analysis was done using the NcoI-EcoRI 335 bp
DNA fragment encoding amino acid residues 67-178 from the deduced
open reading frame of wheat protein disulfide isomerase (PDI) cDNA
(Shimoni et al. 1995). In addition, for RFLP studies of wheat and
related species, we have also used the 3'untranslated region (3'UTR)
of the same cDNA which was a 234 bp SacI-XhoI fragment,
from the first nucleotide 5' of the stop codon to the cloning
site.
Results and discussion
Chromosomal location, deletion mapping and copy number
Hybridization experiments conducted under high stringency using the
PDI coding probe revealed three fragments of similar signal intensity
on Southern blots of the common hexaploid wheat cv. Chinese Spring
(CS). Southern analysis of wheat nullisomic-tetrasomic and
ditelosomic lines of CS allowed us to assign these fragments to
chromosome arm 4AL, 4BS and 4DS (Fig.
1). Wheat
chromosome 4A is known to have undergone complex rearrangements
(Naranjo et al. 1987; Devos et al. 1995). We inferred that PM maps
proximal to the 4AL/5AL translocation breakpoint, since it remained
on chromosome 4A.
The physical location of the PDI genes on chromosome 4D was studied
by deletion mapping, using DNA isolated from Endo's (1988) deletion
stocks. The sequence from the 4DS chromosome arm (Fig.
2) was missing in
lines 4DS-1 and 4DS-3, with fraction length (FL, the chromosome arm
length in the deletion line relative to the standard length) of 0.53
and 0.67 respectively, whereas it was present in line 4DS-2, with FL
of 0.82. This indicates that the corresponding PDI locus resides on
the distal third of chromosome arm 4DS, between FL values 0.67 and
0.82. Inspection of the cytological map of wheat chromosome 4D
(Mickelson- Young et al. 1995) indicates that this sub-chromosomal
region, encompassing 15% of the arm length, currently lacks any
molecular markers.
Comparison of the hybridization intensities of CS total DNA and known
amounts of plasmid DNA corresponding to 5-40 copies of PDI
(Fig.
3) suggests that
this sequences are present in ca. 30 copies in hexaploid wheat, or
5-6 copies per wheat constituent genome. This indicates that the
number of PDI loci in wheat is similar to that in alfalfa (Shorrosh
and Dixon 1991), barley (Chen and Hayes 1994) and maize (Li and
Larkins 1996).
Gene nomenclature (McIntosh et al. 1993)
Xpdi (4A, 4B, 4D)
Polymorphism and presence of PDI sequences in wheat and related
species
Southern analyses of DNA from 34 cultivars of bread wheat digested
with four restriction enzymes (EcoRI, BamHL
HindIII and DraI) did not detect any restriction
fragment polymorphism, with both coding region and 3'UTR probes (data
not shown). When the PDI coding probe was hybridized to DNAs
extracted from wheat and related species of various origins and
ploidy, digested with BamHI (Fig.
4), 1-3 bands
were detected. Hexaploid wheats (Panel A, lanes 1- 5) presented three
monomorphic bands. Tetraploid wheats with genome AABB (lanes 6-11)
showed two bands. With the exception of durum. wheat, they shared a
B-genome fragment of higher mobility than the one from hexaploid
wheat. T. timopheevi var. araraticum (lanes 12-14)
presented an additional band and showed no intraspecific variation.
Among the diploids, one band was observed in all accessions except
for T. monococcum var. monococcum accession TMM04
(Panel B, lane 17) and T. monococcum var. boeoticum.,
accession TMB01 (Panel B, lane 20), which showed three and two
fragments, respectively. T. tauschii accessions (Panel A,
lanes 15-18) were monomorphic, with restriction fragments similar in
size to the CS D-genome bands. Polymorphisms were evident between and
within species of the S- genome Sitopsis group (Panel B, lanes 2-12),
which is related to the B genome of hexaploid wheat. The A genome
diploids (lanes 13-20) also showed interspecific and intraspecific
variation, with no variant closely matching the gel mobility of the
A-genome M band in CS.
Despite limited polymorphism between bread wheat cultivars, the
cytological location of the PDI locus, the absence of nearby cloned
markers and observed variation in tetraploid wheat could make PDI an
useful marker when analyzing DNA transfer from emmer wheat into
hexaploid wheat.
<--Back | -->Next
(go to
KOMUGI Home) (go
to WIS List) (go to NO.86
Contents)