ABSTRACT. Pea (Pisum sativum) is one of the most cultivated legumes in the world, and its yield and seed quality are affected by a variety of pathogens. In plants, NBS-LRR (nucleotide binding siteleucine-rich repeat) is the main class of disease resistance genes. Using degenerate primers deduced from conserved motifs in the NBS domain of known resistance genes, we identified 10 NBS sequences in three varieties of P. sativum. The deduced amino acid sequences of the identified resistance gene analogues (RGAs) exhibited the typical motifs of the NBS domain (P-loop, kinase-2, kinase-3a, and the hydrophobic domain, GLPL) present in the majority of plant proteins belonging to the NBS-LRR class. Phylogenetic analysis showed that seven RGAs belonged to the non-TIR-NBS-LRR subclass and three to the TIR-S. Djebbi et al. 6420©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (2): 6419-6428 (2015) NBS-LRR subclass. The results of this study provide insights into the structure of this class of resistance genes in the pea, and their evolutionary relationships with those of other plant species.

Host resistance is the most economical, effective and ecologically sustainable method of controlling diseases in crop plants. In bread wheat, despite the high number of resistance loci that have been cataloged to date, only few have been cloned, underlying the need for genomics-guided investigations capable of providing a prompt and acute knowledge on the identity of effective resistance genes that can be used in breeding programs. Proteins with a nucleotide-binding site (NBS) encoded by the major plant disease resistance (R) genes play an important role in the responses of plants to various pathogens. In this study, a comprehensive analysis of NBS-encoding genes within the whole wheat genome was performed, and the genome scale characterization of this gene family was established. From the recently published wheat genome sequence, we used a data mining and automatic prediction pipeline to identify 580 complete ORF candidate NBS-encoding genes and 1,099 partial-ORF ones. Among complete gene models, 464 were longer than 200 aa, among them 436 had less than 70 % of sequence identity to each other. This gene models set was deeply characterized. (1) First, we have analyzed domain architecture and identified, in addition to typical domain combinations, the presence of particular domains like signal peptides, zinc fingers, kinases, heavy-metal-associated and WRKY DNA-binding domains. (2) Functional and expression annotation via homology searches in protein and transcript databases, based on sufficient criteria, enabled identifying similar proteins for 60 % of the studied gene models and expression evidence for 13 % of them. (3) Shared orthologous groups were defined using NBS-domain proteins of rice and Brachypodium distachyon. (4) Finally, alignment of the 436 NBS-containing gene models to the full set of scaffolds from the IWGSC's wheat chromosome survey sequence enabled high-stringence anchoring to chromosome arms. The distribution of the R genes was found balanced on the three wheat sub-genomes. In contrast, at chromosome scale, 50 % of members of this gene family were localized on 6 of the 21 wheat chromosomes and ~22 % of them were localized on homeologous group 7. The results of this study provide a detailed analysis of the largest family of plant disease resistance genes in allohexaploid wheat. Some structural traits reported had not been previously identified and the genome-derived data were confronted with those stored in databases outlining the functional specialization of members of this family. The large reservoir of NBS-type genes presented and discussed will, firstly, form an important framework for marker-assisted improvement of resistance in wheat, and, secondly, open up new perspectives for a better understanding of the evolution dynamics of this gene family in grass species and in polyploid systems.

In this study, a systematic analysis of Nucleotide-Binding Site (NBS) disease resistance (R) gene family in the barley, Hordeum vulgare L. cv. Bowman, genome was performed. Using multiple computational analyses, we could identify 96 regular NBS-encoding genes and characterize them on the bases of structural diversity, conserved protein signatures, genomic distribution, gene duplications, differential expression, selection pressure, codon usage, regulation by microRNAs and phylogenetic relationships. Depending on the presence or absence of CC and LRR domains; the identified NBS genes were assigned to four distinct groups; NBS-LRR (53.1%), CC-NBS-LRR (14.6%), NBS (26%), and CC-NBS (6.3%). NBS-associated domain analysis revealed the presence of signal peptides, zinc fingers, diverse kinases, and other structural features. Eighty-five of the identified NBS-encoding genes were mapped onto the seven barley chromosomes, revealing that 50% of them were located on chromosomes 7H, 2H, and 3H, with a tendency of NBS genes to be clustered in the distal telomeric regions of the barley chromosomes. Nine gene clusters, representing 22.35% of total mapped barley NBS-encoding genes, were found, suggesting that tandem duplication stands for an important mechanism in the expansion of this gene family in barley. Phylogenetic analysis determined 31 HvNBS orthologs from rice and Brachypodium. 87 out of 96 HvNBSs were supported by expression evidence, exhibiting various and quantitatively uneven expression patterns across distinct tissues, organs, and development stages. Fourteen potential miRNA-R gene target pairs were further identified, providing insight into the regulation of NBS genes expression. These findings offer candidate target genes to engineer disease-resistant barley genotypes, and promote our understanding of the evolution of NBS-encoding genes in Poaceae crops. Keywords Hordeum vulgare • Nucleotide-binding site • Disease-resistance genes • Genome analysis Abbreviations CC Coiled-coil (domain) IBSC The International Barley Genome Sequencing Consortium LRR Leucine-rich repeat (domain) NBS Nucleotide-binding site (domain) NLR NOD-like receptors miRNA MicroRNAs TIR Toll-interleukin-1 receptor (domain)