FORWARD AND REVERSE GENETIC APPROACHES FOR CHARACTERISATION OF TILLERING GENES IN CEREALS

Recessive mutations in Cul4 locus was described as causing low tillering phenotype in barley. The locus was previously mapped to 0.22 cM genetic interval and Candidate Gene (CG) was proposed. In this study, we mapped the locus into 0.09 cM interval and proved the correspondence between the CG and cul4 phenotype by showing the recombination between CG and adjacent annotated genes. Also, we isolated three independent mutant alleles for Cul4 in the available mutant stocks, and we showed their correlation to cul4 phenotype. Detailed phenotyping of cul4 mutant lines revealed various effects on plant vegetative, reproductive, and developmental parameters. The Cul4 gene was identified as encoding BTB/ANK domain protein highly related to Arabidopsis BOP1 and BOP2 genes. To assess the role of Cul4/BOP-like genes in other plant species, protein sequences similar to Cul4 were used to carry out phylogenetic analysis. Cul4/BOP-like proteins appear to be organised in two main clades corresponding to monocot and dicot divergence. Two sub-clades exist within monocot clade, representing relatively ancient duplication after monocot and dicot separation; Cul4 orthologs and paralogs. Recent duplications appear within Cul4 monocot paralogs, as well as for Arabidopsis BOP1 and BOP2 proteins. Publicly available transcriptomics data for Cul4 rice ortholog support involvement in axillary meristem development, leaf morphogenesis, and floral organ development. Rice mutant lines carrying T-DNA inserts in Cul4-like genes were identified and initially characterised. Screening of mutagenized TILLMore barley collection identified 12 lines with significant defect in tillering. Further histological characterisation of 3 lines displaying the most severe low tillering phenotype, showed inhibition in tiller outgrowth rather than axillary bud formation. Initial steps in constructing mapping populations for selected mutant lines were performed by crossing them to different wild-type parents.