Resistance Gene Analogs from Vitis cinerea, Vitis rupestris, and Vitis Hybrid Horizon

Abstract

Resistance gene analogs (RGAs) characterized by the presence of nucleotide-binding sites (NBS) were cloned from Vitis cinerea, V. rupestris, and V. hybrid Horizon. Two degenerate PCR primer pairs were designed from conserved regions of NBS motifs within known resistance (R) genes and used for PCR amplification of putative RGAs. A total of 122 putative RGA sequences were cloned from all three genotypes by P-loop/GLPLAL-1 primers. Based on nucleic acid sequence-identity of 90% or greater, RGA clones were subdivided into eight, four, and seven groups for V. cinerea, V. rupestris, and Horizon, respectively. All of these clones showed similarity of nucleotide sequences to other known R genes or NBS-type nucleotide sequences, and seven clones showed high similarity. Thirty sequences were cloned from V. cinerea by P-loop/Rev loop and subdivided into four sequence groups, none of which were similar to nucleotide sequences of other R genes. Nineteen representative RGA clones were classified into 13 TIR- (Drosophila Toll and mammalian Interleukin-1 Receptors) NBS-leucine rich repeat (LRR)-like genes and six non-TIR-NBS-LRR-like genes based primarily on nucleotide sequences of kinase-2 motifs and phylogenetic analysis with known TIR or non-TIR proteins. Twenty-three sequence tagged site (STS) and three cleaved amplified polymorphic sequence (CAPS) markers developed from RGAs were checked for segregation among 179 seedlings from Horizon x Illinois (Ill.) 547-1, and 18 showed goodness-of-fit using a chi-square test. Marker stkVa011 correlated with segregation for downy mildew resistance in this population. These STS markers are currently being investigated for their potential in molecular breeding for disease resistance.