Characterization of novel wheat NBS domain-containing sequences and their utilization, in silico, for genome-scale R-gene mining

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 supporting 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 showing 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 second one similar to cyst nematode resistance proteins. In a second step, sequences were used as initial seeds to walk up and downstream the NBS domain. This procedure enabled identifying 8 loci ranging in size between 2,115 and 7,653 bp. Ab initio gene prediction identified 8 gene models, among which two had complete ORFs. While GenBank 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 further elucidation of their role in wheat disease resistance.

Identification and characterization of novel NBS-LRR resistance gene analogues from the pea

“…Using degenerate primers to isolate resistance gene analogs from different plant species has so far been an effective strategy (Yaish et al, 2004;Palomino et al, 2006;Bouktila et al, 2014). In the present study, we identified and characterized 10 RGAs in three pea varieties, using four primer combinations designated from G. max (Kanazin et al, 1996) and S. tuberosum (Leister et al, 1996).…”

“…Using this approach, several RGAs have been isolated from various species, including wheat (Zhang et al, 2011;Bouktila et al, 2014), A. thaliana (Meyers et al, 2003), faba bean and chickpea (Palomino et al, 2006), soybean (Graham et al, 2000), pea (Timmerman-Vaughan et al, 2000), and Medicago truncatula (Zhu et al, 2002). The identified RGAs correspond to different disease specificities, and, in some cases, their mapping has provided evidence that they co-segregate with resistance markers related to disease (Meyers et al, 1999).…”

In Silico Characterization of Meloidogyne Genus Nematode Cellulose Binding Proteins

“…This type of experimentation represents a low cost alternative for studies on biological functions with very precise computational models when compared with laboratory conditions. Computational analyzes have been used in the characterization of several proteins and enzymes in both eukaryotes and prokaryotes, in search of genes linked to parasitism and resistance in host species [25][26][27][28][29][30][31] . The identification of proteins involved in the relationship of parasitism of nematodes and plants can provide important tools for research that develop plant species with resistant genes and in the search for mechanisms that avoid the breakdown of this resistance by phytonematodes.…”

Análisis de secuencias NBS-LRR en el género lens = Analysis of NBS-LRR sequences en the genus Lens

“…La mayor parte de los genes R clonados pertenecen a la familia NBS-LRR. Esta familia tiene unos motivos muy conservados en su secuencia, lo que ha permitido el diseño de cebadores degenerados basados en los motivos conservados de genes R conocidos para aislar secuencias análogas a genes de resistencia o RGAs (Resistance Gene Analogs) en otras especies vegetales mediante técnicas que utilizan la reacción en cadena de la polimerasa (PCR) (Bouktila et al, 2014;Deng et al, 2000;Donald et al, 2002;Graham et al, 2000;Meyers et al, 2003;Palomino et al, 2006;Timmerman-Vaughan et al, 2000;Wan et al, 2010;Xu et al, 2005;Zhang et al, 2011;Zhu et al, 2002), ya sea con ADN total y utilizando geles de agarosa (Djebbi et al, 2015;Kanazin et al, 1996;Leister et al, 1996;Reddy et al, 2015;Wan et al, 2012;Yaish et al, 2004;Yu et al, 1996) o geles de poliacrilamida (Chen et al, 1998) para la separación de los fragmentos, o utilizando ADNc en la técnica llamada "NBS profiling" que combina la digestión del ADN con enzimas de restricción con una posterior amplificación por PCR (Brugmans et al,2008;Sanz et al, 2013;van der Linden et al, 2004).…”

“…La mayor parte de los genes R clonados en plantas pertenecen a la familia NBS-LRR. Esta familia tiene unos motivos muy conservados en su secuencia, lo que ha permitido el diseño de cebadores degenerados basados en dichos motivos para aislar secuencias análogas a genes de resistencia o RGAs (Resistance Gene Analogs) en un gran número de especies vegetales (Bouktila et al, 2014;Djebbi et al, 2015;Graham et al, 2000;Kanazin et al, 1996;Leister et al, 1996;Meyers et al, 2003;Palomino et al, 2006;Timmerman-Vaughan et al, 2000;Wan et al, 2012;Yaish et al, 2004;Yu et al, 1996;Zhang et al, 2011;Zhu et al, 2002) Meyers et al (1999) y Lukasik y Takken (2009) describieron una serie de motivos conservados que caracterizan las secuencias de estos como TIR o TNL (siglas del dominio Toll interleukin receptor) y No-TIR o CNL.…”