Isolation of a family of resistance gene analogue sequences of the nucleotide binding site (NBS) type fromLensspecies

Abstract

Most known plant disease-resistance genes (R genes) include in their encoded products domains such as a nucleotide-binding site (NBS) or leucine-rich repeats (LRRs). Sequences with unknown function, but encoding these conserved domains, have been defined as resistance gene analogues (RGAs). The conserved motifs within plant NBS domains make it possible to use degenerate primers and PCR to isolate RGAs. We used degenerate primers deduced from conserved motifs in the NBS domain of NBS-LRR resistance proteins to amplify genomic sequences from Lens species. Fragments from approximately 500-850 bp were obtained. The nucleotide sequence analysis of these fragments revealed 32 different RGA sequences in Lens species with a high similarity (up to 91%) to RGAs from other plants. The predicted amino acid sequences showed that lentil sequences contain all the conserved motifs (P-loop, kinase-2, kinase-3a, GLPL, and MHD) present in the majority of other known plant NBS-LRR resistance genes. Phylogenetic analyses grouped the Lens NBS sequences with the Toll and interleukin-1 receptor (TIR) subclass of NBS-LRR genes, as well as with RGA sequences isolated from other legume species. Using inverse PCR on one putative RGA of lentil, we were able to amplify the flanking regions of this sequence, which contained features found in R proteins.

Legume Crops Phylogeny and Genetic Diversity for Science and Breeding

“…The DNA-based markers, such as RFLP (Havey and Muehlbauer, 1989;Muench et al, 1991;Rajora and Mahon, 1994), RAPD (Sharma et al, 1995;Ferguson et al, 2000;Sonnante and Pignone, 2001;Toklu et al, 2009;Tewari et al, 2012), AFLP (Sharma et al, 1996;Zavodna et al, 2000;Duran and Perez de le Vega, 2004;Kahraman et al, 2004;Rubeena et al, 2006;Fiocchetti et al, 2009;Toklu et al, 2009), microsatellite markers (Duran and Perez de le Hamwieh et al, 2009;Liu et al, 2008;Babayeva et al, 2009;Reddy et al, 2010;Gupta et al, 2012, Tewari et al, 2012Zaccardelli et al, 2012), inter simple sequence repeat (ISSR) (Zavodna et al, 2000;Sonnante and Pignone, 2001;Toklu et al, 2009), internal transcribed spacers (ITS) (Fernandez et al, 2000;Mayer and Bagga, 2002;Sonnante et al, 2003), non-transcribed spacer (NTS) (Fernandez et al, 2005), sequenced tagged microsatellite site (STMS) (Inder et al, 2008;Datta et al, 2011), single-nucleotide polymorphisms (SNPs) (Alo et al, 2011), and resistance gene analogue (RGA) (Yaish et al, 2004;Sari et al, 2013) were used to analyze phylogenetic relationships among taxa in the genus Lens, as well as the genetic diversity of cultivated lentil.…”

Identification and Characterization of Nucleotide-Binding Site-Leucine-Rich Repeat Genes in the Model Plant Medicago truncatula

“…Mechanisms like duplication, unequal crossing over, ectopic recombination, gene conversion, and diversifying selection have been proposed to contribute to the structure of R gene clusters and the evolution of resistance specificities (Michelmore and Meyers, 1998;Young, 2000;Sun et al, 2001). Moreover, the presence of conserved motifs primarily within the NBS domain has been used extensively to identify resistance genes homologs in model and crop species (Kanazin et al, 1996;Aarts et al, 1998;Penuela et al, 2002;Zhu et al, 2002;Ferrier-Cana et al, 2003;Yaish et al, 2004;Palomino et al, 2006). In species where R genes have been studied, this pattern of widespread multigene clusters is common (Young, 2000;Hulbert et al, 2001;Meyers et al, 2003).…”

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

“…In this study, of the 10 RGAs identified, seven were characterized as belonging to the non-TIR-NBS-LRR subclass. In contrast, relatively more TIR-NBS class RGAs have been reported in the cowpea (Bhavani et al, 2002), lentil (Yaish et al, 2004), and soybean (Kanazin et al 1996). …”

“…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).…”

“…This primer set has previously allowed the isolation of eight RGAs from two other pea varieties (Timmerman-Vaughan et al, 2000) and three RGAs from the lentil (Yaish et al, 2004). However, additional studies conducted on the faba bean and chickpea have found that set III is more efficient, as it allowed the identification of 55 RGAs among the 149 isolated from these two Fabaceae species (Palomino et al, 2006).…”