Cloning of cer-zv, a Novel Gene Responsible for Cutin Formation in the Barley Leaf Cuticle

The recessive mutation cer-zv induces drought hypersensitivity in barley. Its presence is associated with a decreased deposition of cutin monomer and a significant reduction in the thickness of the leaf cuticle. The mutation was initially mapped to a 1.7 cM interval in the pericentromeric region of chromosome 4H, a location which was later narrowed, using a fine mapping procedure with a progeny equivalent to 7490 gametes, to a 0.02 cM interval on the short arm. Although genetically small, this interval still represents more than half of the chromosome arm, as shown by an analysis of a set of markers positioned in a barley cytological map based on chromosome dissection lines. As a result, a map-based cloning approach to isolate the gene is unlikely to be feasible. As an alternative, we have compared the RNA-seq based transcriptome of the mutant with its wild type, and have been able to identify 16 genes located in the expected genomic region which feature mis-sense mutations (SNPs or indels). Two of these genes are likely to be functionally related with cutin formation, so these were targeted for re-sequencing among wild type lines and eight carriers of cer-zv. The procedure has led to the identification of cer-zv, a novel cutin synthase gene, which encodes a member of the Gly-Asp-Ser-Leu (GDSL) esterase/lipase family of plant proteins. The gene is strongly transcribed in the leaf elongation zone where cutin deposition occurs most strongly. The indication is that cer-zv is involved in the formation of cutin in the barley leaf cuticle.