Nucleotide Binding Site Profiling Research Articles

Novel applications of motif-directed profiling to identify disease resistance genes in plants

BackgroundMolecular profiling of gene families is a versatile tool to study diversity between individual genomes in sexual crosses and germplasm. Nucleotide binding site (NBS) profiling, in particular, targets conserved nucleotide binding site-encoding sequences of resistance gene analogs (RGAs), and is widely used to identify molecular markers for disease resistance (R) genes.ResultsIn this study, we used NBS profiling to identify genome-wide locations of RGA clusters in the genome of potato clone RH. Positions of RGAs in the potato RH and DM genomes that were generated using profiling and genome sequencing, respectively, were compared. Largely overlapping results, but also interesting discrepancies, were found. Due to the clustering of RGAs, several parts of the genome are overexposed while others remain underexposed using NBS profiling. It is shown how the profiling of other gene families, i.e. protein kinases and different protein domain-coding sequences (i.e., TIR), can be used to achieve a better marker distribution. The power of profiling techniques is further illustrated using RGA cluster-directed profiling in a population of Solanum berthaultii. Multiple different paralogous RGAs within the Rpi-ber cluster could be genetically distinguished. Finally, an adaptation of the profiling protocol was made that allowed the parallel sequencing of profiling fragments using next generation sequencing. The types of RGAs that were tagged in this next-generation profiling approach largely overlapped with classical gel-based profiling. As a potential application of next-generation profiling, we showed how the R gene family associated with late blight resistance in the SH*RH population could be identified using a bulked segregant approach.ConclusionsIn this study, we provide a comprehensive overview of previously described and novel profiling primers and their genomic targets in potato through genetic mapping and comparative genomics. Furthermore, it is shown how genome-wide or fine mapping can be pursued by choosing different sets of profiling primers. A protocol for next-generation profiling is provided and will form the basis for novel applications. Using the current overview of genomic targets, a rational choice can be made for profiling primers to be employed.

Identification of RFLP and NBS/PK profiling markers for disease resistance loci in genetic maps of oats

Two of the domains most widely shared among R genes are the nucleotide binding site (NBS) and protein kinase (PK) domains. The present study describes and maps a number of new oat resistance gene analogues (RGAs) with two purposes in mind: (1) to identify genetic regions that contain R genes and (2) to determine whether RGAs can be used as molecular markers for qualitative loci and for QTLs affording resistance to Puccinia coronata. Such genes have been mapped in the diploid A. strigosa × A. wiestii (Asw map) and the hexaploid MN841801-1 × Noble-2 (MN map). Genomic and cDNA NBS-RGA probes from oat, barley and wheat were used to produce RFLPs and to obtain markers by motif-directed profiling based on the NBS (NBS profiling) and PK (PK profiling) domains. The efficiency of primers used in NBS/PK profiling to amplify RGA fragments was assessed by sequencing individual marker bands derived from genomic and cDNA fragments. The positions of 184 markers were identified in the Asw map, while those for 99 were identified in the MN map. Large numbers of NBS and PK profiling markers were found in clusters across different linkage groups, with the PK profiling markers more evenly distributed. The location of markers throughout the genetic maps and the composition of marker clusters indicate that NBS- and PK-based markers cover partly complementary regions of oat genomes. Markers of the different classes obtained were found associated with the two resistance loci, PcA and R-284B-2, mapped on Asw, and with five out of eight QTLs for partial resistance in the MN map. 53 RGA-RFLPs and 187 NBS/PK profiling markers were also mapped on the hexaploid map A. byzantina cv. Kanota × A. sativa cv. Ogle. Significant co-localization was seen between the RGA markers in the KO map and other markers closely linked to resistance loci, such as those for P. coronata and barley yellow dwarf virus (Bydv) that were previously mapped in other segregating populations.

Assessment of Genetic Diversity in Zingiberaceae Through Nucleotide Binding Site-Based Motif-Directed Profiling

Functional motif-directed profiling was performed with 15 nucleotide binding site (NBS) primer-enzyme combinations to identify and elucidate the phylogenetic relationships among 15 genotypes of the family Zingiberaceae. We retrieved 167 polymorphic bands (24.85 %), with an average of 11.13 bands per primer. Mean polymorphism rates were detected using MseI (26 %), RsaI (21 %), and AluI (28 %) as restriction enzymes. The polymorphism information content (PIC) for each NBS primer-enzyme combination ranged from 0.48 to 0.76 with a mean value of 0.65. The 38 NBS profiling markers had PIC values ranging from 0.3 to 0.6 and exhibited good power to discriminate between genotypes. Comparison of NBS profiling with microsatellite data for the same set of genotypes exhibited a correlation value of 0.78, P ≤ 0.001. Our study suggests that genetic variability assessment could be more efficient if it targeted genes that exhibit functionally relevant variation, rather than random markers.

Distribution of genetic diversity in wild European populations of prickly lettuce (Lactuca serriola): implications for plant genetic resources management

Genetic variation inLactuca serriola, the closest wild relative of cultivated lettuce, was studied across Europe from the Czech Republic to the United Kingdom, using three molecular marker systems, simple sequence repeat (SSR, microsatellites), AFLP and nucleotide-binding site (NBS) profiling. The ‘functional’ marker system NBS profiling, targeting disease resistance genes of the NBS/LRR family, did not show marked differences in genetic diversity parameters to the other systems. The autogamy of the species resulted in low observed heterozygosity and high population differentiation. Intra-population variation ranged from complete homogeneity to nearly complete heterogeneity. The highest genetic diversity was found in central Europe. The SSR results were compared to SSR variation screened earlier in the lettuce collection of the Centre for Genetic Resources, the Netherlands (CGN). In the UK, practically only a single SSR genotype was found. This genotype together with a few other common SSR genotypes comprised a large part of the plants sampled on the continent. Among the ten most frequent SSR genotypes observed, eight were already present in the CGN collection. Overall, the CGN collection appears to already have a fair representation of genetic variation from NW Europe. The results are discussed in relation to sampling strategies for improving genebank collections of crop wild relatives.

DISCRIMINATING CAPACITY OF NBS AND MYB GENE PROFILING FOR GENETIC ANALYSES OF CAMPANULA SPP.

Two new DNA-based marker methodologies, the nucleotide binding site (NBS) and myb gene profiling, were evaluated to assess their discriminating capacity and efficiency in genetic analyses of 11 Campanula populations (C. latifolia, C. rapunculoides, C. spicata and C. trachelium). Three restriction enzymes (MseI, RsaI and HaeIII) with two NBS primers (GLPL6 and NBS2) and one myb primer (MYB2) were used. Looking at the number of banding pattern, both techniques discriminated the genotypes very effectively. On an individual assay basis, NBS profiling completely distinguished all the genotypes of one population of C. latifolia and C. trachelium. To test the usefulness of the overall information provided by these markers for establishing genetic variability and distances, AMOVA and two Principal Coordinate Analyses were performed. Both NBS and myb gene profiling resulted to be remarkably effective for group discrimination and genetic diversity studies. However, some differences could be highlighted. The myb marker resulted to be more efficient to differentiating species than populations, while NBS profiling appeared to better discriminate the populations. In general, the plots reflected the geographical distributions. The use of these techniques is discussed in terms of the appropriate choice for studying different aspects of Campanula species and populations evaluation.

Consequences of Geographical Habitats on Population Structure and Genetic Diversity in Campanula spp.

Characterization of populations by means of DNA techniques provides a tool for precise identification and a quantitative estimate of genetic diversity, crucial in evaluation of genetic fragmentation within and among populations. NBS profiling are PCR-based approaches that sample genetic variation in resistance genes (R-gene), and R gene analogs (RGA). To date, myb patterns have not been used for evaluating genetic diversity in other species. NBS primers are homologous to the conserved sequences in the Nucleotide-Binding-Site of the NBS-LRR class of R-genes. A total of 12 populations from five Campanula species (C. barbata L., C. latifolia L., C. rapunculoides L., C. spicata L. and C. trachelium L.), autochthonous of the West Italian Alps, were genotyped via nucleotide-binding site (NBS) and myb gene profiling. The selected markers produced a total of 361 bands, showing high levels of polymorphism. Genetic diversity among and within species and population structure was evaluated by different statistical analyses performed using TREECON software, Mantel Nonparametric Test, NTSYS package, AMOVA and STRUCTURE. The correlation between genetic variability and geographical location suggests that the five Campanula species have been subjected to long-term evolutionary processes consistent with the natural fragmentation of continuous mountains areas.

SPECIFIC MAPPING OF DISEASE RESISTANCE GENES IN TETRAPLOID CUT ROSES

Control of fungal diseases is a major constraint of cut-rose cultivation in greenhouses and in transportation around the world. Therefore, development of resistant cultivars is a promising way to reduce the use of chemicals required for controlling the diseases. Genetic analyses and breeding for resistance, however, are hampered by the high degree of heterozygosity and the polyploid nature of cultivated roses. Nucleotide-binding site (NBS) profiling of Van der Linden et al. (2004) was used as a tool enabling a more directed way of studying the genetics of resistance to pathogens responsible for diseases such as powdery mildew. NBS profiling is a multiplex screening technique, producing amplified resistance gene (R-gene) and resistance gene analogue (RGA) fragments by using degenerated primers based on the conserved motifs present in the NBS domain of resistance genes. Since RGAs are abundantly distributed and highly polymorphic within the plant genome, NBS profiling generates multiple markers of putative resistance genes. Twelve NBS degenerated primer/ restriction enzyme combinations were used to genotype the whole rose tetraploid K5 population (Yan, 2005) and its parents. To generate RGA profiles, the restriction enzymes: AluI, HaeIII, Mse and RsaI were used in combination with degenerated primers NBS1, NBS3, and NBS5a6. The profiles were dominantly scored resulting in 106 polymorphic RGA markers which segregated in a 1:1 or 3:1 ratio. Uni-and bi-parental simplex markers will be mapped on the two available AFLP/SSR K5 maps (Yan, 2005) with Joinmap 4.0. The resulting parental tetraploid maps will be used to dissect the genetic variation for resistance to powdery mildew resistance. Additional Rosaceae SSRs mentioned in the literature are currently tested on the K5 population to obtain allelic bridges between the tetraploid and diploid genetic maps in rose and related species in order to align them. These bridges will improve cross-ploidy comparisons in roses in order to strengthen cut rose breeding

A candidate for Lr19, an exotic gene conditioning leaf rust resistance in wheat

Lr19, one of the few widely effective genes conferring resistance to leaf rust in wheat, was transferred from the wild relative Thinopyrum ponticum to durum wheat. Since Lr19 confers a hypersensitive response to the pathogen, it was considered likely that the gene would be a member of the major nucleotide-binding site (NBS)-leucine-rich repeat (LRR) plant R gene family. NBS profiling, based on PCR amplification of conserved NBS motifs, was applied to durum wheat-Th. ponticum recombinant lines involving different segments of the alien 7AgL chromosome arm, carrying or lacking Lr19. Differential PCR products were isolated and sequenced. From one such sequence (AG15), tightly linked to Lr19, a 4,121-bp full-length cDNA was obtained. Its deduced 1,258 amino acid sequence has the characteristic NBS-LRR domains of plant R gene products and includes a coiled-coil (CC) region typical of monocots. The genomic DNA sequence showed the presence of two exons and a short intron upstream of the predicted stop codon. Homology searches revealed considerable identity of AG15 with the cloned wheat resistance gene Pm3a and a lower similarity with wheat Lr1, Lr21, and Lr10. Quantitative PCR on leaf-rust-infected and non-infected Lr19 carriers proved AG15 to be constitutively expressed, as is common for R genes.

The utility of NBS profiling for plant systematics: a first study in tuber-bearing Solanum species

Systematic relationships are important criteria for researchers and breeders to select materials. We evaluated a novel molecular technique, nucleotide binding site (NBS) profiling, for its potential in phylogeny reconstruction. NBS profiling produces multiple markers in resistance genes and their analogs (RGAs). Potato (Solanum tuberosum L.) is a crop with a large secondary genepool, which contains many important traits that can be exploited in breeding programs. In this study we used a set of over 100 genebank accessions, representing 49 tuber-bearing wild and cultivated Solanum species. NBS profiling was compared to amplified fragment length polymorphism (AFLP). Cladistic and phenetic analyses showed that the two techniques had similar resolving power and delivered trees with a similar topology. However, the different statistical tests used to demonstrate congruency of the trees were inconclusive. Visual inspection of the trees showed that, especially at the lower level, many accessions grouped together in the same way in both trees; at the higher level, when looking at the more basal nodes, only a few groups were well supported. Again this was similar for both techniques. The observation that higher level groups were poorly supported might be due to the nature of the material and the way the species evolved. The similarity of the NBS and AFLP results indicate that the role of disease resistance in speciation is limited.

Nucleotide-binding site (NBS) profiling of genetic diversity in durum wheat

Molecular markers are effective tools to investigate genetic diversity for resistance to pathogens. NBS (nucleotide-binding site) profiling is a PCR (polymerase chain reaction)-based approach to studying genetic variability that specifically targets chromosome regions containing R-genes and R-gene analogues. We used NBS profiling to measure genetic diversity among 58 accessions of durum wheat. Mean polymorphism rates detected using MseI and AluI as restriction enzymes were 34% and 22%, respectively. Mean number of polymorphisms per enzyme-primer combination was equal to 23.8 +/- 5.9, ranging from 13 to 31 polymorphic bands. In total, 96 markers over 190 indicated a good capacity to discriminate between accessions (the polymorphic index content ranging from 0.30 to 0.50). The results obtained with NBS profiling were compared with simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) data of the same set of accessions. The genetic distances computed with 190 NBS profiling markers were in close agreement with those obtained with AFLP and SSR markers (r = 0.73 and 0.76, respectively). Our results indicate that NBS profiling provides an effective means to investigate genetic diversity in durum wheat.

Resistance gene analogues identified through the NBS-profiling method map close to major genes and QTL for disease resistance in apple

We used a new method called nucleotide-binding site (NBS) profiling to identify and map resistance gene analogues (RGAs) in apple. This method simultaneously allows the amplification and the mapping of genetic markers anchored in the conserved NBS-encoding domain of plant disease resistance genes. Ninety-four individuals belonging to an F1 progeny derived from a cross between the apple cultivars 'Discovery' and 'TN10-8' were studied. Two degenerate primers designed from the highly conserved P-loop motif within the NBS domain were used together with adapter primers. Forty-three markers generated with NBS profiling could be mapped in this progeny. After sequencing, 23 markers were identified as RGAs, based on their homologies with known resistance genes or NBS/leucine-rich-repeat-like genes. Markers were mapped on 10 of the 17 linkage groups of the apple genetic map used. Most of these markers were organized in clusters. Twenty-five markers mapped close to major genes or quantitative trait loci for resistance to scab and mildew previously identified in different apple progenies. Several markers could become efficient tools for marker-assisted selection once converted into breeder-friendly markers. This study demonstrates the efficiency of the NBS-profiling method for generating RGA markers for resistance loci in apple.