Resistance Gene Analogs Sequences Research Articles

Gene isolation using degenerate primers targeting protein motif: A laboratory exercise.

Structures and functions of protein motifs are widely included in many biology-based course syllabi. However, little emphasis is placed to link this knowledge to applications in biotechnology to enhance the learning experience. Here, the conserved motifs of nucleotide binding site-leucine rich repeats (NBS-LRR) proteins, successfully used for the isolation and characterization of many plant resistance gene analogues (RGAs), is featured in the development of a series of laboratory experiments using important molecular biology techniques. A set of previously isolated RGA sequences is used as the model for performing sequence alignment and visualising 3D protein structure using current bioinformatics programs (Clustal Omega and Argusdock software). A pair of established degenerate primer sequences is provided for the prediction of targeted amino acids sequences in the RGAs. Reverse transcription-polymerase chain reaction (RT-PCR) is used to amplify RGAs from total RNA samples extracted from the tropical wild relative of black pepper, Piper colubrinum (Piperaceae). This laboratory exercise enables students to correlate specific DNA sequences with respective amino acid codes and the interaction between conserved motifs of resistance genes with putatively targeted proteins. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):47-53, 2018.

Adaptive response of Pinus monticola driven by positive selection upon resistance gene analogs (RGAs) of the TIR-NBS-LRR subfamily

Abstract: Western white pine (Pinus monticola Dougl.) is an important forest tree species, which is intensively plagued by the fungus Cronartium ribicola. Resistance gene analogs (RGAs) are the most highly abundant class of potential resistance (R) genes sharing greatly conserved domains and structures. Hence RGAs are crucial components for disease resistance breeding programs on P. monticola serving as useful functional markers. A total of 33 P. monticola RGAs gene homologues were mined from GenBank, encoding for R gene members of the TIR-NBS-LRR subfamily. The existence of positive selection acting upon RGAs was determined using a series of maximum likelihood analyses. Robust evidence of positive selection was showed to be acting widely in three clades across RGA gene phylogeny, both on terminal and ancestral lineages. Furthermore, our analysis revealed that the majority of positively selected residues sites are localized widely across these RGAs sequences, putatively affecting the structures of their ligand-binding domains and offering novel specificities. These results may find immediate application in ongoing disease resistance breeding programs.

Evidence of extensive positive selection acting on cherry (Prunus avium L.) resistance gene analogs (RGAs)

The cherry tree (Prunus avium L.), is an important tree species which is intensively plagued by many phytopathogenic fungal species. Resistance gene analogs (RGAs) are the largest class of resistance (R) genes and are pivotal components at breeding projects, serving as useful functional markers linked to R genes. In order to assess the evolutionary pressures acting upon P. avium RGAs candidates, their 173 homologues that have previously been deposited in GenBank were mined. Their proteins were clustered according to their blast(p) similarities in 12 MCL (Markov Cluster Algorithm) tribes, resulting in unique and well supported paralogous gene groups (PGGs). The extent to which these genes exhibit evidence of adaptive, positive selective pressures, which are causing excessive fixation of non-synonymous mutations, was determined using a series of maximum likelihood analyses using the PAML package. The results postulate existence of robust evidence of positive selection, acting in almost all of the clustered PGGs across their phylogenies. Furthermore, analyses revealed that the majority of the positively selected amino acid residues sites are localized widely across these RGAs sequences. We speculate that the clustered distribution of these RGAs might also be pronounced of high birth and death genes rates with diversifying episodes acting on their NB-ARC domains, putatively affecting their ligandbinding specificities. Such evolutionary insights shed light on how these NBS-encoding RGAs in P. avium are being evolved, assigning them as the foremost surveillance mechanism against rapidly evolving fungal pathogens, and providing breeders with effective tools for fast-tracking the development of varieties with more durable resistance.

Identification and evidence of positive selection upon resistance gene analogs in cotton (Gossypium hirsutum L.).

Upland cotton (Gossypium hirsutum L.) is an important fiber crop species, which is intensively plagued by a plethora of phytopathogenic fungi such as Fusarium oxysporum f. sp. vasinfectum (Fov) causing severe wilt disease. Resistance gene analogs (RGAs) are the largest class of potential resistance (R) genes depicting highly conserved domains and structures in plants. Additionally, RGAs are pivotal components of breeding projects towards host disease resistance, serving as useful functional markers linked to R genes. In this study, a cloning approach based on conserved RGAs motifs was used in order to amplify 38 RGAs from two upland cotton cultivars differing in their Fov susceptibility. Besides, we assessed the phylogenetic expansion and the evolutionary pressures acting upon 127 RGA homologues, which were previously deposited in GenBank along with the 38 RGAs from this study. A total of 165 RGAs sequences were clustered according to their BLAST(P) similarities in ten paralogous genes groups (PGGs). These RGAs exhibited intensive signs of positive selection as it was revealed by inferring various maximum likelihood analyses. The results showed robust signs of positive selection, acting in almost all PGGs across the phylogeny. The evolutionary analysis revealed the existence of 42 positively selected residue sites across the PGG lineages, putatively affecting their ligand-binding specificities. As RGAs derived markers are in close linkage to R genes, these results could be used in ongoing breeding programs of upland cotton.

Isolation of NBS-LRR RGAs from invasive Wedelia trilobata and the calculation of evolutionary rates to understand bioinvasion from a molecular evolution perspective

Supports for the molecular evolution of host–pathogen interactions on enemy release hypothesis are rare. According to the theory of plant immunity and the coevolution of hosts and pathogens, we hypothesized that the evolutionary rate (dN/dS) of resistance genes (R-genes) in invasive plants would be greater than in non-invasive plants, assuming that based on the enemy release hypothesis, the former would suffer less selection stress from co-evolutionary specialist pathogens. To test our hypothesis, we isolated and analyzed the conserved nucleotide-binding sites (NBS) of resistance gene analogues (RGAs) of an invasive weed, Wedelia trilobata (WTRGA). We then used the information in GenBank to compare the dN/dS of the NBS R-gene/RGAs in invasive and homologous non-invasive plants. Three W. trilobata NBS RGA sequences were obtained, belonging to the Toll/Interleukin-1 receptor (TIR) (WTRGA1 and WTRGA2) and non-TIR subclasses (WTRGA3). Compared with the homologous non-invasive plants, the invasive plants showed a significantly greater dN/dS for TIR NBS R-gene/RGAs (p < 0.0001), supporting our hypothesis. Future research should include an examination of R-genes/RGAs from more invasive plants on a population level to understand diversity and R-gene functions in invasive plant species, as well as to explore how disease resistance allows plants to adapt to changing pathogen stresses.

Identification of expressed resistance gene analog sequences in coconut leaf transcriptome and their evolutionary analysis

Coconut, an important crop of the tropics and subtropics, is susceptible to a variety of diseases and enhancing disease resistance has been the major goal of coconut breeding programs all over the world. Information on the presence and distribution of disease resistance ® genes, which play a primary role in the detection of pathogens and the initiation of specific plant defenses, is scarce in coconut. In this study, RNA-Seq was used to generate the transcriptome of leaf samples of coconut root (wilt) disease-resistant cultivar Chowghat Green Dwarf. Comprehensive bioinformatics analysis identified 243 resistance gene analog (RGA) sequences, comprising 6 classes of RGAs. Domain and conserved motif predictions of clusters were performed to analyze the architectural diversity. Phylogenetic analysis of deduced amino acid sequences revealed that coconut NBS-LRR type RGAs were classified into distinct groups based on the presence of TIR or CC motifs in the N-terminal regions. Furthermore, qRT-PCR analysis validated the expression of randomly selected NBS-LRR type RGAs. The results of this study provide a sequence resource for development of RGA-tagged markers in coconut, which would aid mapping of disease-resistant candidate genes. In addition, we hope that this study will provide a genomic framework for isolation of additional RGAs in coconut via comparative genomics and also contribute to the deciphering of mode of evolution of RGAs in Arecaceae.

PCR-based approach for mining of resistant gene analogues in taro (Colocasia esculenta)

Primers based on the conserved motifs were used to isolate nucleotide-binding sites (NBS) type sequences in taro (Colocasia esculenta). Cloning and sequencing identified three taro NBS-type sequences called resistance gene analogues (RGAs) that depicted similarity to other cloned RGA sequences. The deduced amino acid sequences of the RGAs detected the presence of conserved domains, viz. P-loop, categorising them with the NBS–leucine-rich repeat class gene family. Phylogenetic characterisation of the taro RGAs along with RGAs of other plant species grouped them with the non-toll interleukin receptor subclasses of the NBS sequences. The isolation and characterisation of taro RGAs have been reported for the first time in this study. This will provide a starting point towards characterisation of candidate resistance genes in taro and can act as a reference guide for future studies.

Isolation and characterization of resistant gene analogs in cassava, wild Manihot species, and castor bean (Ricinus communis)

Cassava, Manihot esculenta , is one of the major food crops in sub-Saharan Africa (SSA) providing the bulk of dietary calories to hundreds of millions of households. Two viral diseases, namely cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) pose a serious threat to cassava production. The emergence of new virus species and strains that overcome the existing resistant/tolerant cultivars entails identification and pyramiding of new sources of resistance using marker-aided selection. The isolation of resistance gene analogues (RGAs) using a homology-based approach can provide useful resources towards this goal. Degenerate primers based on the conserved motif of the nucleotide binding site (NBS) domain from resistance (R) genes were used to isolate RGAs from genomic DNA and cDNA in cassava, wild Manihot species, and castor bean ( Ricinus communis ). A total of 552 RGAs sequences were identified and deposited in GenBank. Conserved motifs such as P-loop, Kinase-2a and GLPL were present in the NBS domain. This study sheds light on the nature of NBS- leucine-rich repeat (LRR) R genes in cassava and closely related taxa in the family Euphorbiaceae. These candidate sequences mapped to the draft cassava genome with high sequence similarity to predicted NBS-LRR genes. These novel sequences may serve as a stepping stone for further characterization and experimental validation of predicted R genes in the draft cassava genome, ultimately leading to the development of functional gene-targeted markers that can be used in molecular resistance breeding aimed at combating CBSD and CMD. Key words: Cassava, resistance, Manihot , castor bean, resistance gene analog, nucleotide binding site.

Non-TIR-NBS-LRR resistance gene analogs in apricot (Prunus armeniacaL.)

Genes encoding for proteins with nucleotide-binding site and leucine-rich repeat motifs (NBS-LRR) have been suggested to play a general role in plant defence mechanism. In Prunus species, many TIR (Toll / Interleukin-1 Receptor), and only very few non-TIR sequences were identified, which was explained either by the unequal distribution of TIR/non-TIR sequences in the Prunus genome or by the incapability of primers in the amplification of non-TIR RGAs. The objective of this work was to check whether a new semi-nested PCR strategy can be developed for the targeted isolation of non-TIR-NBS-LRR Resistance Gene Analog (RGA) sequences from apricot. Three primers (CUB-P-loop F, CUB-Kin2 F and CUB-HD R) were designed, from which CUB-Kin2 F and CUB-HD R were constructed to anneal selectively to the non-TIR sequences. A colony Polymerase Chain Reaction (PCR) indicated that out of the 96 clones tested 28 showed amplification using the newly developed primers, while no amplification occurred when using the formerly described primers. Half of the 28 positive clones were sequenced and they turned out to represent 11 different non-TIR RGA sequences. A phylogenetic analysis was carried out based on an alignment containing 293 Rosaceae and 21 non-Rosaceaa sequences. A significantly higher ratio (91%) of non-TIR sequences were arranged in multi-genera clades than that of (57%) the TIR groups confirming that non-TIR sequences might be of more ancient origin than TIR sequences.

Construction of a Linkage Map and Identification of Resistance Gene Analog Markers for Root-knot Nematodes in Wild Peach, Prunus kansuensis

The root-knot nematode (RKN) Meloidogyne incognita can cause severe crop loss in economically important Prunus species like peach (P. persica), almond (P. communis), plum (P. salicina), and apricot (P. armeniaca). Some peach rootstock, including Nemaguard (P. persica), Nemared (P. persica), and Myrobalan plum (P. cerasifera), display significant resistance to RKN. We present a genetic linkage map constructed by using simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) for a peach backcross population (190 individuals) of RKN-resistant ‘Honggengansutao’ (P. kansuensis) and susceptible ‘Bailey’ (P. persica). Degenerate primers designed from conserved motifs of known plant resistance gene (R) products were used to amplify genomic DNA sequences. Twenty-two resistance gene analog (RGA) sequences were selected from 48 RGAs with open-reading frames to design sequence-tagged site markers. The linkage map of ‘Honggengansutao’ is composed of 138 loci (30 SSRs, 102 SRAPs, five RGAs, and one morphological marker for RKN resistance) assigned to eight linkage groups. The map covers 616 cM of the peach genome with an average marker spacing of 4.9 cM. The five RGAs were mapped to Groups 2, 7, and 8. One gene (designated PkMi) involved in resistance to RKN was mapped to Group 2 (which also includes the known RKN-resistance RMia gene). BLASTN analysis mapped all RGAs to the peach genome sequence. The map constructed in the study will aid future rootstock breeding with marker-assisted selection to identify additional candidate RGA sequences.

Isolation and characterization of resistance gene analogues from Psilanthus species that represent wild relatives of cultivated coffee endemic to India

Biotic or abiotic stress can cause considerable damage to crop plants that can be managed by building disease resistance in the cultivated gene pool through breeding for disease resistance genes (R-genes). R-genes, conferring resistance to diverse pathogens or pests share a high level of similarity at the DNA and protein levels in different plant species. This property of R-genes has been successfully employed to isolate putative resistance gene analogues (RGAs) using a PCR-based approach from new plant sources. Using a similar approach, in the present study, we have successfully amplified putative RGAs having nucleotide-binding-site leucine-rich repeats (NBS-LRR-type RGAs) from seven different sources: two cultivated coffee species (Coffea arabica L. and Coffea canephora Pierre ex. A. Froehner), four related taxa endemic to India (wild tree coffee species: Psilanthus bengalensis (Roem. & Schuttles) J.-F. Leroy, Psilanthus khasiana , Psilanthus travencorensis (Wight & Arn.) J.-F. Leroy, Psilanthus weightiana (Wall. ex Wight & Arn.) J.-F. Leroy), and a cDNA pool originally prepared from light- and drought-stressed Coffea arabica L. leaves. The total PCR amplicons obtained using NBS-LRR-specific primers from each source were cloned and transformed to construct seven independent libraries, from which 434 randomly picked clones were sequenced. In silico analysis of the sequenced clones revealed 27 sequences that contained characteristic RGA motifs, of which 24 had complete uninterrupted open reading frames. Comparisons of these with published RGAs showed several of these to be novel RGA sequences. Interestingly, most of such novel RGAs belonged to the related wild Psilanthus species. The data thus suggest the potential of the secondary gene pool as possible untapped donors of resistance genes to the present day cultivated species of coffee.

Isolation of resistance gene analogs from grapevine resistant and susceptible to downy mildew and anthracnose

Downy mildew and anthracnose are major diseases of the grapevine ( Vitis spp.) cultivars grown in Thailand. Due to the deleterious effects of fungicides frequently used for disease management in grapevine, disease resistance genes have been sought after with the ultimate goal of developing new cultivars with improved disease resistance levels. In this study, nucleotide-binding site (NBS)-leucine rich repeat (LRR) class of resistance gene analogs (RGAs) were cloned by PCR amplification using degenerate primers specific to P-loop and GLPL, conserved regions of NBS. Ninety-one clones containing putative RGA sequences were obtained from a downy mildew and anthracnose resistant hybrid ‘NY88.0507.01’ and a susceptible cultivar ‘Black Queen’. These cloned sequences were subdivided into 14 groups based on their nucleotide sequence similarity of 90% or greater. BLASTx of fourteen selected clones showed the highest amino acid sequence similarity with known NBS-LRR proteins or putative resistance (R) protein candidates. Multiple alignments of these representative RGAs with 5 known R proteins revealed conserved P-loop, kinase-2, RNBS and GLPL motifs which are typical components of the NBS-LRR proteins. Four RGAs had at least 40% identity with known R proteins. Phylogenetic analysis demonstrated that the representative RGAs from both resistant and susceptible grapevines dispersed along the phylogram on the two major branches of either TIR ( Drosophila Toll and mammalian Interleukin- 1 Receptor) or non-TIR type of the NBS-LRR proteins.

Characterization of resistance gene analogs from Gossypium arboreum and their evolutionary relationships with homologs from tetraploid cottons

Four cotton species (genus Gossypium) produce spinable fiber. The two diploid species of Asiatic origin, Gossypium arboreum and G. herbaceum, have been largely replaced by G. hirsutum. However, these diploid species are potentially a rich source of genes for the improvement of G. hrisutum, particularly in terms of providing resistance against biotic and abiotic stresses. As a first step towards understanding the mechanisms of resistance in cotton, we designed 24 non-degenerate primers based on resistance gene analogs (RGAs) cloned from G. hirsutum for screening a number of cotton species with the A and D genomes. Most of these RGAs are conserved on the A genome (G. arboreum), suggesting a bias towards this genome. The amplified RGAs from G. arboreum were cloned and their nucleotide and amino acid sequences compared with RGA sequences available in public databases. The majority of the RGAs identified were homologous to those isolated from G. hirsutum and G. barbadense, but their diversity was greater than expected at both the nucleotide and amino acid levels. These RGAs provide useful tools for the identification of full-length resistance genes from bacterial artificial chromosome and cDNA libraries.

Importance and Use of Resistance Gene Analogs

The identification, isolation and subsequent cloning of effective disease resistance genes for their use to enhance the protection level against various groups of pathogens in plants is an important aspect of crop improvement. Disease resistance genes (R-genes) have been characterized and cloned from many plant species. The resistance gene analogs (RGAs) are putative or tentative disease resistance genes which are identified on the basis of their structure. The RGAs are an efficient tool in identifying and isolating disease resistance genes and have got efficiency in building a durable resistance. In order to know the exact function of RGAs they need to be characterized and linked with the genes actually conferring resistance phenotype. Regions of amino acid conservation in resistance gene encoded proteins have facilitated the isolation of RGA sequences from genomic DNA in many crop plants using polymerase chain reaction based techniques.

Mapping of STS markers obtained from oat resistance gene analog sequences

Two previously isolated resistance gene analogs (RGAs) of oat have been located as RFLPs in the reference map of Avena byzantina 'Kanota' x Avena sativa 'Ogle' in regions either homologous or homoeologous to loci for resistance to Puccinia coronata, the causal agent of crown rust. In this study, the RGAs were mapped in two recombinant inbred line (RIL) populations that segregate for crown rust resistance: the diploid Avena strigosa x Avena wiestii RIL population (Asw), which has been used for mapping the complex locus PcA, and the hexaploid MN841801-1 x Noble-2 RIL population (MN), in which QTLs have been located. To obtain single-locus markers, RGAs were converted to sequence tagged site (STS) markers using a procedure involving extension of the original RGA sequence lengths by PCR genome walking, amplification and cloning of the parental fragments, and identification of single nucleotide polymorphisms. The procedure successfully obtained STSs from different members of the L7M2 family of sequences, the initial NBS of which have nucleotide similarities of >83%. However, for RGA III2.18, the parental lines were not polymorphic for the STSs assayed. A sequence characterized amplified region (SCAR) marker with features of an RGA had been previously identified for gene Pc94. This marker was also mapped in the above RIL populations. Markers based on RGA L7M2 co-localized with markers defining the QTL Prq1a in linkage group MN3, and were located 15.2 cM from PcA in linkage group AswAC. The SCAR marker for Pc94 was also located in the QTL Prq1a but at 39.5 cM from PcA in AswAC, indicating that the NBS-LRR sequence represented by this marker is not related to PcA. L7M2 was also excluded as a member of the PcA cluster, although it could be an appropriate marker for the Prq1a cluster if chromosome rearrangements are postulated.

Isolation and sequence analysis of NBS–LRR disease resistance gene analogues from hop Humulus lupulus L.

Improvement of disease resistance is one of the most important aims in hop breeding and there is a need for the development of marker assisted resistant breeding. In the present work, a PCR strategy was used to clone resistance gene analogues (RGAs) using degenerative primers designed at the conserved motif of cloned plant NBS–LRR R genes. Fifty-six sequenced PCR clones showed homologies to various R genes deposited in the GenBank database and these clones were further divided into 17 RGA groups. Phylogenetic analysis of hop RGA groups with cloned R genes revealed a separation of hop RGAs into TIR (7 RGA groups with 16 sequences) and non-TIR (10 RGA groups with 40 sequences) NBS–LRR protein classes. The amino acid identity of hop RGAs to various R genes ranged from 41% (TIR–NBS–LRR) to 81% (non-TIR–NBS–LRR). RGA primer sets, designed on 17 reference RGAs, amplified additional RGA sequences from two hop genotypes which served for the development of 11 RGA markers of which eight amplified PCR products of expected sizes also in various hop cultivars, indicating a high conservation level of RGA sequences within the hop genome and two RGA markers segregated in the mapping family. The structure of RGA sequences and similarity to other R gene or R-gene-like sequences is discussed. This is the first report on RGAs in hop and it provides useful data for further breeding manipulations.

Isolation of Resistance Gene Analogues from Flowering Dogwood (Cornus florida L.)

AbstractCloned resistance (R) genes from a broad range of plant species are known to share similarities in DNA sequences and structural motifs. Oligonucleotide primers, designed to conserved regions of nucleotide binding site (NBS) motifs within previously cloned pathogen resistance genes, were used to amplify resistance gene analogues (RGAs) from dogwood. Twenty eight unique dogwood RGA sequences were identified and subdivided into 11 groups on the basis of nucleic acid sequence‐identity of approximately 70% or greater. Phylogenetic analysis of the predicted amino acid sequences grouped the RGAs into three distinct classes from which several subgroups were delineated based on nucleic acid sequences. Gene database searches with the consensus protein sequences of each of the three classes and respective subgroups of dogwood RGAs revealed their conserved NBS domains and homology to RGAs and known resistance genes from a variety of plant genera. Given the complete lack of knowledge regarding molecular organization of R genes in dogwood, the cloned RGAs described here may be useful as probes to map, characterize, and manipulate R genes of the dogwood genome.

Genetic mapping and transcription analyses of resistance gene loci in potato using NBS profiling

NBS profiling is a method for the identification of resistance gene analog (RGA) derived fragments. Here we report the use of NBS profiling for the genome wide mapping of RGA loci in potato. NBS profiling analyses on a minimal set of F1 genotypes of the diploid mapping population previously used to generate the ultra dense (UHD) genetic map of potato, allowed us to efficiently map polymorphic RGA fragments relative to 10,000 existing AFLP markers. In total, 34 RGA loci were mapped, of which only 13 contained RGA sequences homologous to RGAs genetically positioned at approximately similar positions in potato or tomato. The remaining RGA loci mapped either at approximate chromosomal regions previously shown to contain RGAs in potato or tomato without sharing homology to these RGAs, or mapped at positions not yet identified as RGA-containing regions. In addition to markers representing RGAs with unknown functions, segregating markers were detected that were closely linked to four functional R genes that segregate in the UHD mapping population. To explore the potential of NBS profiling in RGA transcription analyses, RNA isolated from different tissues was used as template for NBS profiling. Of all the fragments amplified approximately 15% showed putative intensity or absent/present differences between different tissues suggesting putative tissue specific RGA or R gene transcription. Putative absent/present differences between individuals were also found. In addition to being a powerful tool for generating candidate gene markers linked to R gene loci, NBS profiling, when applied to cDNA, can be instrumental in identifying those members of an R gene cluster that are transcribed, and thus putatively functional.

Isolation and linkage mapping of NBS-LRR resistance gene analogs in red raspberry (Rubus idaeus L.) and classification among 270 Rosaceae NBS-LRR genes

Plant R genes confer resistance to pathogens in a gene-for-gene mode. Seventy-five putative resistance gene analogs (RGAs) containing conserved domains were cloned from Rubus idaeus L. cv. ‘Latham’ using degenerate primers based on RGAs identified in Rosaceae species. The sequences were compared to 195 RGA sequences identified from five Rosaceae family genera. Multiple sequence alignments showed high similarity at multiple nucleotide-binding site (NBS) motifs with homology to Drosophila Toll and mammalian interleukin-1 receptor (TIR) and non-TIR RNBSA-A motifs. The TIR sequences clustered separately from the non-TIR sequences with a bootstrap value of 76%. There were 11 clusters each of TIR and non-TIR type sequences of multiple genera with bootstrap values of more than 50%, including nine with values of more than 75% and seven of more than 90%. Polymorphic sequence characterized amplified region and cleaved amplified polymorphic sequence markers were developed for nine Rubus RGA sequences with eight placed on a red raspberry genetic linkage map. Phylogenetic analysis indicated four of the mapped sequences share sequence similarity to groupTIR I, while three others were spread in non-TIR groups. Of the 75 Rubus RGA sequences analyzed, members were placed in five TIR groups and six non-TIR groups. These group classifications closely matched those in 12 of 13 studies from which these sequences were derived. The analysis of related DNA sequences within plant families elucidates the evolutionary relationship and process involved in pest resistance development in plants. This information will aid in the understanding of R genes and their proliferation within plant genomes.

Analysis of non-TIR NBS-LRR resistance gene analogs in Musa acuminata Colla: isolation, RFLP marker development, and physical mapping.

BackgroundMany commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.ResultsA computational strategy was developed for unbiased conserved motif discovery in NBS and LRR domains in R-genes and homologues in monocotyledonous plant species. Degenerate PCR primers targeting conserved motifs were tested on the wild cultivar Musa acuminata subsp. burmannicoides, var. Calcutta 4, which is resistant to a number of fungal pathogens and nematodes. One hundred and seventy four resistance gene analogs (RGAs) were amplified and assembled into 52 contiguous sequences. Motifs present were typical of the non-TIR NBS-LRR RGA subfamily. A phylogenetic analysis of deduced amino-acid sequences for 33 RGAs with contiguous open reading frames (ORFs), together with RGAs from Arabidopsis thaliana and Oryza sativa, grouped most Musa RGAs within monocotyledon-specific clades. RFLP-RGA markers were developed, with 12 displaying distinct polymorphisms in parentals and F1 progeny of a diploid M. acuminata mapping population. Eighty eight BAC clones were identified in M. acuminata Calcutta 4, M. acuminata Grande Naine, and M. balbisiana Pisang Klutuk Wulung BAC libraries when hybridized to two RGA probes. Multiple copy RGAs were common within BAC clones, potentially representing variation reservoirs for evolution of new R-gene specificities.ConclusionThis is the first large scale analysis of NBS-LRR RGAs in M. acuminata Calcutta 4. Contig sequences were deposited in GenBank and assigned numbers ER935972 – ER936023. RGA sequences and isolated BACs are a valuable resource for R-gene discovery, and in future applications will provide insight into the organization and evolution of NBS-LRR R-genes in the Musa A and B genome. The developed RFLP-RGA markers are applicable for genetic map development and marker assisted selection for defined traits such as pest and disease resistance.