Candidate-gene cloning and targeted marker enrichment of wheat chromosomalregions using RNA fingerprinting - differential display

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.