Ks. Gill et D. Sandhu, Candidate-gene cloning and targeted marker enrichment of wheat chromosomalregions using RNA fingerprinting - differential display, GENOME, 44(4), 2001, pp. 633-639
The usefulness of the RNA fingerprinting - differential display technique i
n gene cloning and targeted marker enrichment in wheat is demonstrated. A s
mall region of chromosome 5BL was targeted that contains Ph1, a chromosome-
pairing regulator gene. The cultivar Chinese Spring (CS) and mutant ph1b ar
e almost identical except for chromosome 5BL, which, in the mutant line, ca
rries an interstitial deletion encompassing the Ph1 gene. Poly(A)(+) RNA of
the two lines from anthers at developmental stages ranging from pre-meioti
c mitosis to anaphase II was PCR-amplified using 38 pairwise combinations o
f 19 primers. The S-35-labeled amplified products were size-separated on de
naturing polyacrylamide-urea gels. A total of 3154 fragment bands were obse
rved, of which 43 were present in CS but absent in the ph1b mutant. These 4
3 fragment bands were eluted, re-amplified, and used as probes in gel-blot
DNA analyses of wheat group 5 nullisomic-tetrasomic lines and the ph1b muta
nt. Twenty-four of these 43 probes were single- or few-copy sequences. Eigh
t of the 24 probes mapped to wheat group 5 and five mapped to the deletion
of the ph1b mutant. Three of these five probes were further localized to th
e submicroscopic region containing the Ph1 gene, by using two deletion line
s flanking the region. Northern-blot analysis revealed that the gene corres
ponding to one of these three probes expresses mainly during meiosis and is
from the B genome.