Selection of Genes Related to Rust Resistance for Recombinant Inbred Line Expression Analysis

Differentially expressed genes (DEGs) related to rust resistance were selected from the L. cicera RIL parental lines RNAseq leaf-transcriptome in response to U. pisi infection previously obtained by Santos et al. (2018). The gene selection criteria were: (1) non-redundant DEGs with the log₂ of the ratio between BGE023542 (partially resistant) and BGE008277 (susceptible) inoculated reads higher than 2 and lower than –2; (2) DEGs not directly involved in specific resistance mechanisms, such as oxidative, metabolic, and transporter activities; (3) DEGs involved in defence response and categorised in pathogen recognition, antifungal proteins, cell wall modification proteins or involved in the regulation of other defence related processes and (4) nucleotide sequences of DEGs suitable for primer design, as described in the next section “Primer design” and resulting in a single amplification (primer specificity).

Differentially expressed gene annotation was revised and updated from the previous study (Santos et al., 2018) by BLASTn search against the pea reference genome v1a¹ (Kreplak et al., 2019) and against genomic sequences of other legume species deposited at NCBI databases. The molecular function and biological process from each DEG related to rust resistance were also investigated using InterPro (Hunter et al., 2009) and UniProt² databases.