1 3 Mol Genet Genomics DOI 10.1007/s00438-014-0834-4 ORIGINAL PAPER Characterization of novel wheat NBS domain-containing sequences and their utilization, in silico, for genome-scale R-gene mining Dhia Bouktila · Yosra Habachi-Houimli · Yosra Khalfallah · Maha Mezghani-Khemakhem · Mohamed Makni · Hanem Makni Received: 6 August 2013 / Accepted: 21 February 2014 © Springer-Verlag Berlin Heidelberg 2014 second step, sequences were used as initial seeds to walk up and downstream the NBS domain. This procedure ena- bled identifying 8 loci ranging in size between 2,115 and 7,653 bp. Ab initio gene prediction identified 8 gene mod- els, among which two had complete ORFs. While Gen- Bank survey confirmed the belonging of sequences to two groups, subsequent characterization using IWGSC genomic and proteomic data showed that the 8 gene models, reported in this study, were unique and their loci matched scaffolds on chromosome arms 1AS, 1BS, 4BS and 1DS. The gene model located on 1DS is a pseudo-Lr21 that was shown to have an NBS-LRR domain structure, while the potential association of the RGAs, here reported, is discussed. This study has produced novel R-gene-like loci and models in the wheat genome and provides the first steps toward fur- ther elucidation of their role in wheat disease resistance. Keywords Triticum aestivum · Nucleotide-binding site (NBS) · Plant · Disease · R-gene · Genome Introduction In their struggle against attacks of viruses, bacteria, fungi, protozoa, nematodes and insects, plants have evolved a wide range of defense mechanisms. While some of these resistance strategies rely on simple physical or chemical barriers, modern concepts in plant immunity focus on the role and evolution of plant protein receptors corresponding to specific pathogen effectors. To explain this interaction, at least three models are currently widely endorsed. The first, called gene-for-gene model (Flor 1971), involves the direct effect of a plant receptor that recognizes a specific patho- gen effector. The second, an extension of the gene-for-gene model, called the guard model (Jones and Dangl 2006), Abstract In crop improvement, the isolation, cloning and transfer of disease resistance genes (R-genes) is an ultimate goal usually starting from tentative R-gene analogs (RGAs) that are identified on the basis of their structure. For bread wheat, recent advances in genome sequencing are sup- porting the efforts of wheat geneticists worldwide. Among wheat R-genes, nucleotide-binding site (NBS)-encoding ones represent a major class. In this study, we have used a polymerase chain reaction-based approach to amplify and clone NBS-type RGAs from a bread wheat cultivar, ‘Salambo 80.’ Four novel complete ORF sequences show- ing similarities to previously reported R-genes/RGAs were used for in silico analyses. In a first step, where analyses were focused on the NBS domain, these sequences were phylogenetically assigned to two distinct groups: a first group close to leaf rust Lr21 resistance proteins; and a sec- ond one similar to cyst nematode resistance proteins. In a Communicated by S. Hohmann. Nucleotide sequence data reported are available in the GenBank database under the accession numbers JX566982 to JX566985. D. Bouktila (*) · Y. Habachi-Houimli · Y. Khalfallah · M. Mezghani-Khemakhem · M. Makni · H. Makni Unité de Recherche Génomique des Insectes Ravageurs des Cultures d’intérêt agronomique (GIRC, UR11ES10), Faculté des Sciences de Tunis, Université de Tunis El-Manar, El-Manar, 2092 Tunis, Tunisia e-mail: dhia_bouktila2000@yahoo.fr D. Bouktila Institut Supérieur de Biotechnologie de Béja (ISBB), Université de Jendouba, 9000 Béja, Tunisia H. Makni Institut Supérieur de l’Animation pour la Jeunesse et la Culture (ISAJC), Université de Tunis, Bir-El-Bey, Tunisia