Structural and functional analysis of RGAs in coffea arabica. Resistance gene screening

Physiological based differentiation of SH genes and H. vastatrix races is one of the principal methods routinely employed in characterizing coffee leaf rust resistance. Molecular techniques like BAC clone sequencing and functional characterization of target gene is believed to enhance precisions to overcome the daunting limitations in classical breeding. In the present work, RGAs conferring coffee leaf rust resistance were sequenced and characterized from a BAC library screened by functional marker. Among the 13 predicted ORFs, five RGAs were annotated and mapped to chromosome 0 of C. canephora. Four of the RGAs are actively expressed during C. arabica-H. vastatrix incompatible interaction. Based on the result, it could be inferred that at least one (gene 11) of the RGAs sequenced confers SH6 gene. This result challenges the previous report of SH1 in 128/2-Dilla & Alghe differential clone. We also report an SH gene (SH6) in differential host clone 644/18 H. Kawisari for the first time. Moreover, comparative analysis of two RGAs belonging to the CC-NBS-LRR gene family showed intense diversifying selection due to nonsynonymous substitution and gene recombination/conversion, all resulting in the evolution of specific resistance. Moreover, phylogenetic analysis of orthologous genes showed high interaspecies variability among the two genes in related species than in coffee. The CC-NBS-LRR genes sequenced in this work are the largest and most complete sequence ever reported in Arabica coffee, making the work extremely important for molecular breeding of coffee rust resistance.