Allelic variation in the dehydrin gene family of ‘Himalaya’ barley (Hordeum vulgare L.)

Abstract

 Variation in the dehydrin gene family of ‘Himalaya’ barley (Hordeum vulgare L.) was studied using 3′ fragments of dehydrin cDNA clones (DHN1–4) as hybridisation probes. Each fragment specifically hybridised to the corresponding cDNA clone, but the 3′ fragment of DHN1 also hybridised to the very closely related DHN7 genomic clone. The same fragments were hybridised to Southern blots of genomic DNA prepared from a pool of 100 plants and digested with different restriction enzymes. DHN1 and DHN2 probes hybridised to one or two fragments, but only single bands were seen with DHN3 and DHN4 probes. When DNA was prepared from individual plants, the DHN1 and DHN2 probes each hybridised to single bands, representing one or other of the two bands in the pooled DNA sample. The ‘Himalaya’ population is therefore polymorphic, with two types of individual, designated P1 and P2. A cross between P1 and P2 types yielded an F₁ with the same hybridisation profile as the pooled DNA. Segregation observed in the F₂ generation was consistent with a 1 : 2 : 1 ratio. Therefore, P1 and P2 individuals contain RFLP alleles detected by DHN1 and DHN2 probes, with the alleles occurring in specific associations consistent with linkage between Dhn1 and Dhn2 loci. Allelic variation was also detected within the Dhn1 locus. Dehydrin RNA from P1 and P2 individuals was analysed by reverse transcription/polymerase chain reaction using primers specific for DHN1/DHN7 sequences. Sequencing of the amplified and cloned products indicated that P1 individuals expressed only the DHN1 sequence, and P2 individuals only DHN7. These results indicate that the DHN1 and DHN7 clones represent allelic alternatives at the Dhn1 locus (now designated Dhn1-1 and Dhn1-2, respectively). A stock that contained only P2 individuals was generated from a single seed, allowing hormonal regulation of Dhn1 gene expression to be studied without complications arising from polymorphism.