In silico guided structural and functional analysis of genes with potential involvement in resistance to coffee leaf rust: A functional marker based approach

Abstract

Physiology-based differentiation of S H genes and Hemileia vastatrix races is the principal method employed for the characterization of coffee leaf rust resistance. Based on the genefor-gene theory, nine major rust resistance genes (S H 1-9) have been proposed. However, these genes have not been characterized at the molecular level. Consequently, the lack of molecular data regarding rust resistance genes or candidates is a major bottleneck in coffee breeding. To address this issue, we screened a BAC library with resistance gene analogs (RGAs), identified RGAs, characterized and explored for any S H related candidate genes. Herein, we report the identification and characterization of a gene (gene 11), which shares conserved sequences with other S H genes and displays a characteristic polymorphic allele conferring different resistance phenotypes. Furthermore, comparative analysis of the two RGAs belonging to CC-NBS-LRR revealed more intense diversifying selection in tomato and grape genomes than in coffee. For the first time, the present study has unveiled novel insights into the molecular nature of the S H genes, thereby opening new avenues for coffee rust resistance molecular breeding. The characterized candidate RGA is of particular importance for further biological function analysis in coffee.

Marker-Assisted Pyramiding of Multiple Disease Resistance Genes in Coffee Genotypes (Coffea arabica)

“…For HdT rust resistance genes, a functional molecular marker, CARF005, has been identified and developed [13,47], and this marker is used in direct rust resistance screening. CARF005 tags a resistance gene analog encoding the disease resistance nucleotide binding-ARC (APAF-1, R protein, and CED-4) domain.…”

“…To obtain durable resistance and identify various rust resistance loci, the progenies were also analyzed using the CARF005 marker. This marker amplifies a DNA fragment that corresponds to the nucleotide binding-leucine rich repeat (CC-NBS-LRR) gene [47], which shares conserved sequences with other S H genes and expresses a characteristic polymorphic allele conferring distinct resistance phenotypes [13]. The PCR for CARF005 was performed in a 20 µL reaction mixture containing 50 ng of DNA, 0.1 µM of each primer, 0.15 mM of each dNTP (Promega), 1.0 mM of MgCl 2 , 1.0 U of Taq DNA polymerase (Invitrogen), and 1× PCR reaction buffer.…”

“…CaRHv8 (Locus A), SSR16 (Locus A), CaRHv9 (Locus B), and CaRHv10_CAPS (Locus B), which confer resistance to race I, race II, and pathotype 001 of H. vastatrix, were used to monitor the presence of QTL-LG2 and QTL-LG5. CARF005 (Locus C), a resistance gene analog (RGA) marker [13,46], associated with other loci conferring resistance to CLR was also used. This marker shares conserved sequences with other CLR resistance genes and displays a characteristic polymorphic allele conferring different resistance phenotypes [13].…”

“…CARF005 (Locus C), a resistance gene analog (RGA) marker [13,46], associated with other loci conferring resistance to CLR was also used. This marker shares conserved sequences with other CLR resistance genes and displays a characteristic polymorphic allele conferring different resistance phenotypes [13]. In order to develop coffee genotype with multiple disease resistance genes, the markers CBD-Sat207 and CBD-Sat235 (Locus D) associated with resistance to CBD were included in the MAS approach.…”

“…For instance, the resistance genes SH1, SH2, SH4, and SH5 in C. arabica; SH6, SH7, SH8, and SH9 in C. canephora; and SH3 in C. liberica have been identified. Additional resistance genes have also been detected but remain to be characterized; these genes, alone or together with SH1 to SH9, confer resistance to over 50 physiological races of H. vastatrix [9,[12][13][14]. These resistance genes have been introgressed into coffee plants to develop CLR-resistant cultivars along with other desirable agronomic traits, such as high yield, vegetative vigor, fruit ripening uniformity, plant architecture, resistance to other diseases and pests, and cup quality [15][16][17].…”