Abstract: 【Objective】Isolating NBS-type （Nucleotide binding site） resistance gene analogs (RGAs) from walnut (Juglans regia L.) using homology-based method would provide a foundation for molecular-assisted selection and for cloning R gene during walnut breeding. 【Method】Thirty-five walnut superiors were used as plant materials in the study and their resistance to anthracnose was identified with inoculation. NBS-type RGAs were isolated by a PCR strategy using degenerate primers specific to P-loop and GLPL, conserved motifs of NBS domain in plant R gene. The relationship between NBSs and walnut anthracnose resistance was analyzed. Sequence identification of obtained sequences was performed against known RGAs deposited in GenBank using BLASTN/X algorithms. Similar and phylogenetic analysis were elaborated using MEGA 5.2 and DNAman 7.0 software.【Result】Out of 35 tested walnut superiors, 20 superiors were identified as resistant (R) with a relative resistance index (RRI) ranging from 0.63 to 0.82; 5 of them were medium resistant (M), whose RRI ranged 0.27-0.56; the rest 10 superiors as susceptible (S) with the RRI ranging 0.00-0.21. NBSs were amplified only in 20 resistant superiors and no bands were found in the remaining 15 superiors (5 medium resistant and 10 susceptible), indicating that the NBS-RGAs were associated with the resistance to walnut anthracnose (Colletotrichum gloeosporioides). BLASTN showed the obtained NBSs shared high similarities to cloned jrRGAPGs with a identity ranging 89%-100%; and they shared than 69% homology to other species from GenBank; BLASTX revealed 77%-99% and 49%-66% similarity to the NBS proteins from jrRGAPGs and other species, respectively. Multiple alignment analysis revealed that these NBS-type RGAs contained some well-characteristics motifs of NBS genes, including P-loop, kinase-2, kinase-3 and GLPL. The nucleotide polymorphism and diversity (Pi) were highly conserved at each motif than non-conservative fragments, indicating their conservative structures. Phylogenetic analysis revealed that the NBSs were clustered into seven subgroups at nucleotide level. They were grouped into two clades (TIR and non-TIR) and were subdivided into 7 groups based on their amino acid sequence similarity. Ratio of non-synonymous to synonymous nucleotide substitution (dN/dS) among NBS-RGAs varied from 0.00 to 0.95 (lower than one) for different classes, suggesting a purifying selection. Similarity percentages of deduced amino acid among these 7 NBS subgroups ranged from 28.3% to 63.5% with identifies to R genes ranging from 22.0% to 48.5%. 【Conclusion】 NBS-type RGAs isolated in the study were associated with anthracnose resistance, they were highly similar to cloned R genes and contained some conserved motifs. Walnut NBS-type RGAs undergo a purifying selection.

Key words: walnut (Juglans regia L.) , walnut anthracnose (Colletotrichum gloeosporioides) , resistance gene analogs (RGA) , nucleotide polymorphism and diversity (Pi) , phylogenetic analysis