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

Abstract

The usefulness of the RNA fingerprinting – differential display technique in gene cloning and targeted marker enrichment in wheat is demonstrated. A small region of chromosome 5BL was targeted that contains Ph1, a chromosome-pairing regulator gene. The cultivar Chinese Spring (CS) and mutant ph1b are almost identical except for chromosome 5BL, which, in the mutant line, carries an interstitial deletion encompassing the Ph1 gene. Poly(A)+ RNA of the two lines from anthers at developmental stages ranging from pre-meiotic mitosis to anaphase II was PCR-amplified using 38 pairwise combinations of 19 primers. The 35S-labeled amplified products were size-separated on denaturing polyacrylamide-urea gels. A total of 3154 fragment bands were observed, of which 43 were present in CS but absent in the ph1b mutant. These 43 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 mutant. Twenty-four of these 43 probes were single- or few-copy sequences. Eight 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 the submicroscopic region containing the Ph1 gene, by using two deletion lines flanking the region. Northern-blot analysis revealed that the gene corresponding to one of these three probes expresses mainly during meiosis and is from the B genome.Key words: RNA fingerprinting – differential display, wheat, gene cloning, marker enrichment, Ph1 gene.