The largest group of plant disease resistance (R) genes that share similar structures contains a predicted nucleotide-binding site (NBS) domain. NBS domains of this class of R genes show highly conserved amino acid motifs, which makes it possible to isolate resistance gene analogs (RGAs) by PCR with degenerate primers and homology searches from public databases. Multiple combinations of degenerate primers were designed from three conserved motifs (one motif was used for a subgroup-specific primer design) in the NBS regions of R genes of various plants. All combinations of primer pairs were used to amplify genomic DNA from sorghum. TIR-specific primer combinations showed no PCR amplification in sorghum. Homology searches identified many NBS-encoding sequences among the expressed or genomic molecular database entries for sorghum. Motif analysis of the sorghum NBS sequences that were identified in this study revealed eight major conserved motifs plus two additional highly conserved motifs, but no TIR-specific motifs. Phylogenetic analysis of sorghum NBS sequences showed tree topology typical of NBS-LRR genes, including clustered nodes and longbranch lengths. Eleven distinct families of NBS sequences, representing a highly diverse sample, were isolated from Sorghum bicolor. With two exceptions, sorghum RGA families appeared to be closely related in sequence to at least one R-gene cloned from other species. In addition, deduced amino acid sequences of sorghum RGAs showed strong sequence similarity to almost all known non-TIR (Toll/Interleukin 1 Receptor)- type R-genes. Mapping with sorghum RGA markers revealed one linkage group containing four out of ten randomly selected markers, suggesting non-random distribution of NBS sequences in the sorghum genome. Rice sequences homologous to sorghum NBS sequences were found from two-way BLAST searches. Some of them were shown to be orthologs, when determined by using phylogenetic approaches which combined five different evolution models and tree-building methods.

In this study, it was aimed to identify chickpea (Cicer arietinum) genes or gene fragments expressed upon Ascochyta rabiei infection using a tolerant chickpea cultivar ILC195 and fungal isolates with varying level of pathogenicity. PCR amplification of resistance gene analogs (RGA) and disease related genes, and mRNA differential display reverse transcription (DDRT) were used to get these expressed gene fragments in chickpea. The constitutively or differentially expressed PCR product fragments were cloned and sequenced. Out of nearly 300 clones, 160 sequences (expressed sequence tags, ESTs) could be analyzed and these sequences were disclosed in this study. About 100 of these ESTs were classified according to predicted &ldquo / molecular function&rdquo / , &ldquo / biological process&rdquo / and &ldquo / cellular component&rdquo / . The most common ppredicted functions of the products coded by these ESTs were &ldquo / Protein Fate&rdquo / , &ldquo / Metabolism&rdquo / , &ldquo / Cell Rescue, Defense and Virulence&rdquo / , &ldquo / Transcription&rdquo / , &ldquo / Transport&rdquo / , &ldquo / Energy&rdquo / , and &ldquo / Cell Fate&rdquo / . Six ESTs were subjected to Real-Time quantitative RT-PCR analysis to compare the response of ILC195 infected by one A.rabiei isolate with another resistant chickpea genotype (FLIP84-92C)/A.rabiei pathotype system. Some of these genes were differentially expressed among different chickpea/A.rabiei isolate combinations. Highly upregulated ESTs in all these combinations were a formate dehydrogenase (metabolism and detoxification), a serine carboxypeptidase (protein fate and communication) and a hypothetical protein probably similar to acyl-CoA synthetases. A genetic mapping study was carried out with EST specific primers and two EST markers were assigned in the current chickpea genetic map. However, no genetic linkage of them was detected with known chickpea quantitative trait loci for A.rabiei resistance.