Identification and characterization of NBS–LRR class resistance gene analogs in faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.)

A PCR approach with degenerate primers designed from conserved NBS–LRR (nucleotide binding site – leucine-rich repeat) regions of known disease-resistance (R) genes was used to amplify and clone homologous sequences from 5 faba bean (Vicia faba) lines and 2 chickpea (Cicer arietinum) accessions. Sixty-nine sequenced clones showed homologies to various R genes deposited in the GenBank database. The presence of internal kinase-2 and kinase-3a motifs in all the sequences isolated confirm that these clones correspond to NBS-containing genes. Using an amino-acid sequence identitiy of 70% as a threshold value, the clones were grouped into 10 classes of resistance-gene analogs (RGA01 to RGA10). The number of clones per class varied from 1 to 30. RGA classes 1, 6, 8, and 9 were comprised solely of clones isolated from faba bean, whereas classes 2, 3, 4, 5, and 7 included only chickpea clones. RGA10, showing a within-class identity of 99%, was the only class consisting of both faba bean and chickpea clones. A phylogenetic tree, based on the deduced amino-acid sequences of 12 representative clones from the 10 RGA classes and the NBS domains of 6 known R genes (I2 and Prf from tomato, RPP13 from Arabidopsis, Gro1–4 from potato, N from tobacco, L6 from flax), clearly indicated the separation between TIR (Toll/interleukin-1 receptor homology: Gro1–4, L6, N, RGA05 to RGA10)- and non-TIR (I2, Prf, RPP13, RGA01 to RGA04)-type NBS–LRR sequences. The development of suitable polymorphic markers based on cloned RGA sequences to be used in genetic mapping will facilitate the assessment of their potential linkage relationships with disease-resistance genes in faba bean and chickpea. This work is the first to report on faba bean RGAs.

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Identifying Resistance Gene Analogs Associated With Resistances to Different Pathogens in Common Bean

Phytopathology ◽

10.1094/phyto.2003.93.1.88 ◽

2003 ◽

Vol 93 (1) ◽

pp. 88-95 ◽

Cited By ~ 74

Author(s):

Camilo E. López ◽

Iván F. Acosta ◽

Carlos Jara ◽

Fabio Pedraza ◽

Eliana Gaitán-Solís ◽

...

Keyword(s):

Common Bean ◽

Resistance Gene ◽

Plant Disease Resistance ◽

Amino Acid Sequences ◽

Yellow Mosaic Virus ◽

Resistance Gene Analogs ◽

R Genes ◽

Degenerate Primers ◽

Golden Yellow ◽

Major Cluster

A polymerase chain reaction approach using degenerate primers that targeted the conserved domains of cloned plant disease resistance genes (R genes) was used to isolate a set of 15 resistance gene analogs (RGAs) from common bean (Phaseolus vulgaris). Eight different classes of RGAs were obtained from nucleotide binding site (NBS)-based primers and seven from not previously described Toll/Interleukin-1 receptor-like (TIR)-based primers. Putative amino acid sequences of RGAs were significantly similar to R genes and contained additional conserved motifs. The NBS-type RGAs were classified in two subgroups according to the expected final residue in the kinase-2 motif. Eleven RGAs were mapped at 19 loci on eight linkage groups of the common bean genetic map constructed at Centro Internacional de Agricultura Tropical. Genetic linkage was shown for eight RGAs with partial resistance to anthracnose, angular leaf spot (ALS) and Bean golden yellow mosaic virus (BGYMV). RGA1 and RGA2 were associated with resistance loci to anthracnose and BGYMV and were part of two clusters of R genes previously described. A new major cluster was detected by RGA7 and explained up to 63.9% of resistance to ALS and has a putative contribution to anthracnose resistance. These results show the usefulness of RGAs as candidate genes to detect and eventually isolate numerous R genes in common bean.

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Identification and Mapping of Nucleotide Binding Site–Leucine-rich Repeat Resistance Gene Analogs in Bermudagrass

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.135.1.74 ◽

2010 ◽

Vol 135 (1) ◽

pp. 74-82 ◽

Cited By ~ 3

Author(s):

Karen R. Harris ◽

Brian M. Schwartz ◽

Andrew H. Paterson ◽

Jeff A. Brady

Keyword(s):

Disease Resistance ◽

Amino Acid ◽

Binding Site ◽

Resistance Gene ◽

Resistance Genes ◽

Nucleotide Binding ◽

Amino Acid Sequences ◽

Resistance Gene Analogs ◽

Leucine Rich Repeat ◽

Linkage Groups

Thirty-one partial bermudagrass (Cynodon spp.) disease-resistance gene analogs (BRGA) were cloned and sequenced from diploid, triploid, tetraploid, and hexaploid bermudagrass using degenerate primers to target the nucleotide binding site (NBS) of the NBS–leucine-rich repeat (LRR) resistance gene family. Alignment of deduced amino acid sequences revealed that the conserved motifs of the NBS are present and all sequences have non-Drosophila melanogaster Toll and mammalian interleukin-1 receptor (TIR) motifs. Using a neighbor-joining algorithm, a dendrogram was created and nine groups of deduced amino acid sequences from bermudagrass could be identified from those sequences that span the NBS. Four BRGA markers and 15 bermudagrass expressed sequence tags (ESTs) with similarity to resistance genes or resistance gene analogs were placed on a bermudagrass genetic map. Multiple BRGA and EST markers mapped on T89 linkage groups 1a and 5a and clusters were seen on T89 19 and two linkage groups previously unidentified. In addition, three primers made from BRGA groups and ESTs with similarity to NBS-LRR resistance genes amplify NBS-LRR analogs in zoysiagrass (Zoysia japonica or Z. matrella) or seashore paspalum (Paspalum vaginatum). This gives evidence of conservation of NBS-LRR analogs among the subfamilies Chloridoideae and Panicoideae. Once disease resistance genes are identified, these BRGA and EST markers may be useful in marker-assisted selection for the improvement of disease resistance in bermudagrass.

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Isolation, Sequence Analysis, and Linkage Mapping of Nucleotide Binding Site–Leucine-rich Repeat Disease Resistance Gene Analogs in Watermelon

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.134.6.649 ◽

2009 ◽

Vol 134 (6) ◽

pp. 649-657 ◽

Cited By ~ 8

Author(s):

Karen R. Harris ◽

W. Patrick Wechter ◽

Amnon Levi

Keyword(s):

Disease Resistance ◽

Resistance Gene ◽

Linkage Mapping ◽

Citrullus Lanatus ◽

Nucleotide Binding ◽

Disease Resistance Gene ◽

Resistance Gene Analogs ◽

Leucine Rich Repeat ◽

Degenerate Primers ◽

Sequence Tagged Sites

Sixty-six watermelon (Citrullus lanatus var. lanatus) disease resistance gene analogs were cloned from ‘Calhoun Gray’, PI 296341, and PI 595203 using degenerate primers to select for the nucleotide binding sites (NBS) from the NBS–leucine-rich repeat (LRR) resistance gene family. After contig assembly, watermelon resistance gene analogs (WRGA) were identified and amino acid sequence alignment revealed that these groups contained motifs characteristic of NBS-LRR resistance genes. Using cluster analysis, eight groups of WRGA were identified and further characterized as having homology to Drosophila Toll and mammalian interleukin-1 receptor (TIR) and non-TIR domains. Three of these WRGA as well as three disease-related watermelon expressed sequence tag homologs were placed on a test-cross map. Linkage mapping placed the WRGA on linkage group XIII, an area on the watermelon map where resistance gene analogs cluster. In addition, these WRGA sequence-tagged sites (STS) were amplified from various genera of the Cucurbitaceae indicating that conservation of resistance gene analogs exists among cucurbits. These WRGA-STS markers may be useful in marker-assisted selection for the improvement for disease resistance in watermelon.

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Isolation, Characterization and Phylogenetic Analysis of Nucleotide Binding Site-encoding Disease-resistance Gene Analogues from European Aspen (Populus tremula)

Silvae Genetica ◽

10.1515/sg-2010-0009 ◽

2010 ◽

Vol 59 (1-6) ◽

pp. 68-77 ◽

Cited By ~ 1

Author(s):

Yong Zhang ◽

Shougong Zhang ◽

Liwang Qi ◽

Tao Zhang ◽

Chunguo Wang ◽

...

Keyword(s):

Disease Resistance ◽

Binding Site ◽

Resistance Gene ◽

Nucleotide Binding ◽

Nucleotide Binding Site ◽

Populus Tremula ◽

R Gene ◽

R Genes ◽

Degenerate Primers ◽

European Aspen

Abstract The majority of verified plant disease resistance genes (R genes) isolated to date was of the nucleotide binding site-leucine rich repeat (NBS-LRR) class. The conservation between different NBS-LRR R genes opens the avenue for the use of PCR based strategies in isolating and cloning other R gene family members or analogs (resistance gene analogue, RGA) using degenerate primers for these conserved regions. In this study, to better understand the R gene in European aspen (Populus tremula), a perennial tree, we used degenerate primers to amplify RGA sequences from European aspen. Cloning and sequence characterization identified 37 European aspen RGAs, which could be phylogenetically classified into seven subfamilies. Deduced amino acid sequences of European aspen RGAs showed strong identity, ranging from 30.41 to 46.63%, to toll interleukin receptor (TIR) R gene subfamily. BLAST searches with reference to the genomic sequence of P. trichocarpa found 209 highly homologous regions distributed in 28 genomic loci, suggesting the abundance and divergence of NBS-encoding R genes in European aspen genome. Although, numerous studies have reported that plant R genes are under diversifying selection for specificity to evolving pathogens, non-synonymous to synonymous nucleotide substitution (dN/dS) ratio were <1 for NBS domains of European aspen RGA, showing the evidence of purifying selection in this perennial tree. In further analysis, many intergenic exchanges were also detected among these RGAs, indicating a probable role in homogenising NBS domains. The present study permits insights into the origin, diversification, evolution and function of NBS-LRR R genes in perennial species like European aspen and will be useful for further R gene isolation and exploitation.

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Isolation and linkage analysis of expressed disease-resistance gene analogues of sugar beet (Beta vulgaris L.)

Genome ◽

10.1139/g02-106 ◽

2003 ◽

Vol 46 (1) ◽

pp. 70-82 ◽

Cited By ~ 47

Author(s):

Sandra Hunger ◽

Gabriele Di Gaspero ◽

Silke Möhring ◽

Diana Bellin ◽

Ralf Schäfer-Pregl ◽

...

Keyword(s):

Disease Resistance ◽

Sugar Beet ◽

Resistance Gene ◽

Beta Vulgaris ◽

Nematode Resistance ◽

Kinase Domain ◽

Disease Resistance Gene ◽

R Genes ◽

Degenerate Primers ◽

Organ Specific

Sequence conservation among resistance genes (R genes) was exploited to identify 47 R gene analogues (RGAs) from sugar beet (Beta vulgaris L.). Using degenerate primers, 11 RGAs were amplified from genomic DNA and 7 from leaf or beet cDNA. Twenty-nine were selected from an EST sequencing program. Twenty-one RGAs contained structures similar to the nucleotide binding site (NBS) leucine rich repeat (LRR) domain, a motif commonly found in several R genes. Among the remaining RGAs, 19 revealed similarity to the serine (threonine) protein kinase domain of R genes, 4 showed features related to the LRR region of the rice disease resistance gene Xa21, 1 RGA resembled the sugar beet nematode resistance gene Hs1pro-1, and 2 had homologies to other gene products associated with disease resistance. For 20 EST-derived RGAs, transcript levels were compared in leaf and root tissue revealing organ-specific transcription in 7 cases. Thirty-three RGAs were spread over all nine sugar beet chromosomes, except for a cluster of nine closely linked RGAs on chromosome 7. The analysis of linkage between RGAs and loci for rhizomania and Cercospora resistance identified alleles associated with resistance in both cases.Key words: RGA, Beta vulgaris, NBSLRR, genetic linkage map, molecular marker.

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Development of a New DNA Marker for Fusarium Yellows Resistance in Brassica rapa Vegetables

Plants ◽

10.3390/plants10061082 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1082

Author(s):

Naomi Miyaji ◽

Mst Arjina Akter ◽

Chizuko Suzukamo ◽

Hasan Mehraj ◽

Tomoe Shindo ◽

...

Keyword(s):

Disease Resistance ◽

Amino Acid ◽

Resistance Gene ◽

Brassica Rapa ◽

Dna Marker ◽

Amino Acid Sequences ◽

Inoculation Test ◽

Causative Locus ◽

Resistant Lines ◽

Susceptible Allele

In vegetables of Brassica rapa L., Fusarium oxysporum f. sp. rapae (For) or F. oxysporum f. sp. conglutinans (Foc) cause Fusarium yellows. A resistance gene against Foc (FocBr1) has been identified, and deletion of this gene results in susceptibility (focbr1-1). In contrast, a resistance gene against For has not been identified. Inoculation tests showed that lines resistant to Foc were also resistant to For, and lines susceptible to Foc were susceptible to For. However, prediction of disease resistance by a dominant DNA marker on FocBr1 (Bra012688m) was not associated with disease resistance of For in some komatsuna lines using an inoculation test. QTL-seq using four F2 populations derived from For susceptible and resistant lines showed one causative locus on chromosome A03, which covers FocBr1. Comparison of the amino acid sequence of FocBr1 between susceptible and resistant alleles (FocBr1 and FocBo1) showed that six amino acid differences were specific to susceptible lines. The presence and absence of FocBr1 is consistent with For resistance in F2 populations. These results indicate that FocBr1 is essential for For resistance, and changed amino acid sequences result in susceptibility to For. This susceptible allele is termed focbr1-2, and a new DNA marker (focbr1-2m) for detection of the focbr1-2 allele was developed.

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Cloning and Analysis of NBS-LRR Type Disease Resistance Gene Analogs from Rosa rubus in Yunnan

Plant Diversity and Resources ◽

10.3724/sp.j.1143.2012.11057 ◽

2012 ◽

Vol 34 (1) ◽

pp. 56

Author(s):

Ling CHEN ◽

Hao ZHANG ◽

Xian-Qin QIU ◽

Hui-Jun YAN ◽

Qi-Gang WANG ◽

...

Keyword(s):

Disease Resistance ◽

Resistance Gene ◽

Disease Resistance Gene ◽

Resistance Gene Analogs

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Organization, Expression and Evolution of a Disease Resistance Gene Cluster in Soybean

Genetics ◽

10.1093/genetics/162.4.1961 ◽

2002 ◽

Vol 162 (4) ◽

pp. 1961-1977

Author(s):

Michelle A Graham ◽

Laura Fredrick Marek ◽

Randy C Shoemaker

Keyword(s):

Disease Resistance ◽

Resistance Gene ◽

Resistance Genes ◽

Developmental Stages ◽

Gene Evolution ◽

Pcr Amplification ◽

Disease Resistance Genes ◽

Disease Resistance Gene ◽

R Genes ◽

Specific Primers

Abstract PCR amplification was previously used to identify a cluster of resistance gene analogues (RGAs) on soybean linkage group J. Resistance to powdery mildew (Rmd-c), Phytophthora stem and root rot (Rps2), and an ineffective nodulation gene (Rj2) map within this cluster. BAC fingerprinting and RGA-specific primers were used to develop a contig of BAC clones spanning this region in cultivar “Williams 82” [rps2, Rmd (adult onset), rj2]. Two cDNAs with homology to the TIR/NBD/LRR family of R-genes have also been mapped to opposite ends of a BAC in the contig Gm_Isb001_091F11 (BAC 91F11). Sequence analyses of BAC 91F11 identified 16 different resistance-like gene (RLG) sequences with homology to the TIR/NBD/LRR family of disease resistance genes. Four of these RLGs represent two potentially novel classes of disease resistance genes: TIR/NBD domains fused inframe to a putative defense-related protein (NtPRp27-like) and TIR domains fused inframe to soybean calmodulin Ca2+-binding domains. RT-PCR analyses using gene-specific primers allowed us to monitor the expression of individual genes in different tissues and developmental stages. Three genes appeared to be constitutively expressed, while three were differentially expressed. Analyses of the R-genes within this BAC suggest that R-gene evolution in soybean is a complex and dynamic process.

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