Expression, mapping, and genetic variability of Brassica napus disease resistance gene analogues.

Numerous sequences analogous to resistance (R) genes exist in plant genomes and could be involved in resistance traits. The aim of this study was to identify a large number of Brassica napus sequences related to R genes and also to test the adequacy of specific PCR-based tools for studying them. Different consensus primers were compared for their efficiency in amplifying resistance-gene analogues (RGAs) related to the nucleotide-binding-site subgroup of R genes. Specific primers were subsequently designed to fine-study the different RGAs and we tested their efficiency in three species related to B. napus: Brassica oleracea, Brassica rapa, and Arabidopsis thaliana. Forty-four B. napus RGAs were identified. Among 29 examined, at least one-third were expressed. Eighteen RGAs were mapped on 10 of the 19 B. napus linkage groups. The high variability within these sequences permitted discrimination of each genotype within a B. napus collection. The RGA-specific primers amplified RGAs in the B. oleracea and B. rapa genomes, but the sequences appear to be poorly conserved in A. thaliana. Specific RGA primers are a precise tool for studying known-sequence RGAs. These sequences represent interesting markers that could be correlated with resistance traits in B. napus or related Brassica genomes.     

Resistance gene analogues of chickpea ( Cicer arietinum L.): isolation,genetic mapping and association with a Fusarium resistance gene cluster

Resistance gene analogues (RGAs) of Cicer were isolated by different PCR approaches and mapped in an inter-specific cross segregating for fusarium wilt by RFLP and CAPS analysis. Initially, two pairs of degenerate primers targeting sequences encoded at nucleotide-binding sites (NBS), which are conserved in plant disease resistance genes such as RPS2, L6 and N, were selected for amplification. Cloning and sequence analysis of amplified products from C. arietinum DNA revealed eight different RGAs. Additionally, five RGAs were identified after characterisation of the presumptive RGA alleles from C. reticulatum. Therefore, a total of 13 different RGAs were isolated from Cicer and classified through pair-wise comparison into nine distinct classes with sequence similarities below a 68% amino acid identity threshold. Sequence comparison of seven RGA alleles of C. arietinum and C. reticulatum revealed polymorphisms in four RGAs with identical numbers of synonymous and non-synonymous substitutions. An NlaIII site, unique in the RGA-A allele of C. arietinum, was exploited for CAPS analysis. Genomic organisation and map position of the NBS-LRR candidate resistance genes was probed by RFLP analysis. Both single-copy as well as multi-copy sequence families were present for the selected RGAs, which represented eight different classes. Five RGAs were mapped in an inter-specific population segregating for three race-specific Fusarium resistances. All RGAs mapped to four of the previously established eight linkage groups for chickpea. Two NBS-LRR clusters were identified that could not be resolved in our mapping population. One of these clusters, which is characterised by RFLP probe CaRGA-D, mapped to the linkage group harbouring two of three Fusarium resistance genes characterised in the inter-specific population. Our study provides a starting point for the characterisation and genetic mapping of candidate resistance genes in Cicer that is useful for marker-assisted selection and as a pool for resistance genes of Cicer.     

Potato homologs of Arabidopsis thaliana genes functional in defense signaling--identification, genetic mapping, and molecular cloning

Defense against pests and pathogens is a fundamental process controlled by similar molecular mechanisms in all flowering plants. Using Arabidopsis thaliana as a model, steps of the signal transduction pathways that link pathogen recognition to defense activation have been identified and corresponding genes have been characterized. Defense signaling (DS) genes are functional candidates for controlling natural quantitative variation of resistance to plant pathogens. Nineteen Arabidopsis genes operating in defense signaling cascades were selected. Solanaceae EST (expressed sequence tag) databases were employed to identify the closest homologs of potato (Solanum tuberosum). Sixteen novel DS potato homologs were positioned on the molecular maps. Five DS homologs mapped close to known quantitative resistance loci (QRL) against the oomycete Phytophthora infestans causing late blight and the bacterium Erwinia carotovora subsp. atroseptica causing blackleg of stems and tuber soft rot. The five genes are positional candidates for QRL and are highly sequence related to Arabidopsis genes AtSGT1b, AtPAD4, and AtAOS. Full-length complementary DNA and genomic sequences were obtained for potato genes StSGT1, StPAD4, and StEDS1, the latter being a putative interactor of StPAD4. Our results form the basis for further studies on the contributions of these candidate genes to natural variation of potato disease resistance.     

Molecular cloning and chromosomal mapping of type one serine/threonine protein phosphatases in Arabidopsis thaliana

Type one serine/threonine protein phosphatases (PP1s) have been implicated in various processes of plant growth and development. In all plant species studied, PP1s are encoded by multigene families. Previous studies in our laboratory identified five Arabidopsis thaliana PP1 genes (TOPP1, TOPP2, TOPP3, TOPP4 and TOPP5). In the present study, we report the isolation of three additional PP1 genes (TOPP6, TOPP7 and TOPP8). Southern blot analyses indicate that these three newly isolated genes are single-copy genes in A. thaliana genome. All the three genes are expressed in roots, rosettes and flowers, although their expression levels appear to be lower than those of the five previously identified TOPP genes. Six of the eight TOPP genes were mapped to different positions on four of five A. thaliana chromosomes. Sequence comparison revealed that TOPP genes belong to different subgroups of plant PP1 genes, suggesting that they may encode proteins with distinct functions.     

Barley disease resistance gene analogs of the NBS-LRR class: identification and mapping

The majority of verified plant disease resistance genes isolated to date are of the NBS-LRR class, encoding proteins with a predicted nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region. We took advantage of the sequence conservation in the NBS motif to clone, by PCR, gene fragments from barley representing putative disease resistance genes of this class. Over 30 different resistance gene analogs (RGAs) were isolated from the barley cultivar Regatta. These were grouped into 13 classes based on DNA sequence similarity. Actively transcribed genes were identified from all classes but one, and cDNA clones were isolated to derive the complete NBS-LRR protein sequences. Some of the NBS-LRR genes exhibited variation with respect to whether and where particular introns were spliced, as well as frequent premature polyadenylation. DNA sequences related to the majority of the barley RGAs were identified in the recently expanded public rice genomic sequence database, indicating that the rice sequence can be used to extract a large proportion of the RGAs from barley and other cereals. Using a combination of RFLP and PCR marker techniques, representatives of all barley RGA gene classes were mapped in the barley genome, to all chromosomes except 4H. A number of the RGA loci map in the vicinity of known disease resistance loci, and the association between RGA S-120 and the nematode resistance locus Ha2 on chromosome 2H was further tested by co-segregation analysis. Most of the RGA sequences reported here have not been described previously, and represent a useful resource as candidates or molecular markers for disease resistance genes in barley and other cereals.     

Microdissection and molecular manipulation of single chromosomes in woody fruit trees with small chromosomes using pomelo (<Emphasis Type="Italic">Citrus grandis</Emphasis>) as a model. II. Cloning of resistance gene analogs from single chromosomes

Amplification of resistance gene analogs (RGAs) is both a useful method for acquiring DNA markers closely linked to disease resistance (R) genes and a potential approach for the rapid cloning of R genes in plants. However, the screening of target sequences from among the numerous amplified RGAs can be very laborious. The amplification of RGAs from specific chromosomes could greatly reduce the number of RGAs to be screened and, consequently, speed up the identification of target RGAs. We have developed two methods for amplifying RGAs from single chromosomes. Method 1 uses products of Sau3A linker adaptor-mediated PCR (LAM-PCR) from a single chromosome as the templates for RGA amplification, while Method 2 directly uses a single chromosomal DNA molecule as the template. Using a pair of degenerate primers designed on the basis of the conserved nucleotide-binding-site motifs in many R genes, RGAs were successfully amplified from single chromosomes of pomelo using both these methods. Sequencing and cluster analysis of RGA clones obtained from single chromosomes revealed the number, type and organization of R-gene clusters on the chromosomes. We suggest that Method 1 is suitable for analyzing chromosomes that are unidentifiable under a microscope, while Method 2 is more appropriate when chromosomes can be clearly identified.Communicated by P. Langridge     

Genetic Analysis of Resistance Gene Analogues from a Sugarcane Cultivar Resistant to Red Rot Disease

One of the important approaches for disease control in sugarcane is to develop a disease‐resistant variety; this may be accomplished through identification of resistance genes in sugarcane. In this study, PCR primers targeting the conserved motifs of the nucleotide‐binding site (NBS) class and kinase class of the resistance gene analogues (RGAs) were used to amplify the RGAs from a red rot‐resistant sugarcane cultivar (Saccharum spp. hybrid) HSF 240. Upon subcloning and sequencing, fifteen putative RGAs were identified. These RGAs shared 63–98% identity to the reported disease‐resistant genes in the NCBI GenBank database. Deduced amino acid sequences also showed the presence of expected conserved domains characteristic of RGAs. Phylogenetic analysis indicated that these RGAs clustered with R genes from other plant species. The findings will be useful for studying disease‐resistant genes in sugarcane.     

Genome-wide isolation of resistance gene analogs in maize (Zea mays L.)

Conserved domains or motifs shared by most known resistance (R) genes have been extensively exploited to identify unknown R-gene analogs (RGAs). In an attempt to isolate all potential RGAs from the maize genome, we adopted the following three methods: modified amplified fragment length polymorphism (AFLP), modified rapid amplification of cDNA ends (RACE), and data mining. The first two methods involved PCR-based isolations of RGAs with degenerate primers designed based on the conserved NBS domain; while the third method involved mining of RGAs from the maize EST database using full-length R-gene sequences. A total of 23 and 12 RGAs were obtained from the modified AFLP and RACE methods, respectively; while, as many as 109 unigenes and 77 singletons with high homology to known R-genes were recovered via data-mining. Moreover, R-gene-like ESTs (or RGAs) identified from the data-mining method could cover all RACE-derived RGAs and nearly half AFLP-derived RGAs. Totally, the three methods resulted in 199 non-redundant RGAs. Of them, at least 186 were derived from putative expressed R-genes. RGA-tagged markers were developed for 55 unique RGAs, including 16 STS and 39 CAPS markers.     

Frequent sequence exchanges between homologs of RPP8 in Arabidopsis are not necessarily associated with genomic proximity

Disease resistance (R) genes are often clustered in plant genomes and may exhibit heterogeneous rates of evolution. Some (type I R genes) have evolved rapidly through frequent sequence exchanges, while others (type II R genes) have evolved independently and tend to be conserved in different genotypes or related species. The RPP8 resistance gene in Arabidopsis thaliana is located at a complex locus that also harbors the sequence-related resistance genes HRT and RCY1 in different ecotypes. We sequenced 98 homologs of RPP8 from A. thaliana, Arabidopsis arenosa and Arabidopsis lyrata. Three lineages of type II and one lineage of type I RPP8 homologs were identified. Two of the three lineages of type II genes are each represented by a single-copy locus on either chromosomes I or V. Chromosome V contains two small clusters of RPP8 paralogs. One cluster contains both type I and type II genes and the other comprises only type I genes. These multi-copy loci have expanded and contracted through unequal crossovers, which have generated chimeric genes as well as variations in copy number. Sequence exchanges, most likely gene conversions, were detected between RPP8 homologs that are spatially separated by 2.2 Mb and 12 cM. The sequence exchanges between type I homologs within a locus have been more frequent than sequence exchanges between homologs from two different loci, indicating the influence of chromosomal position on the evolution of these R genes. However, physical distance was not the only factor determining the frequency of sequence exchange, because some closely linked paralogs exhibited little sequence exchange. At least two distinct lineages of type II RPP8 homologs were identified in different species, with obvious allelic/orthologous relationships within each lineage. Therefore, the differentiation of type I and type II RPP8 homologs seems to have occurred before speciation of A. thaliana, A. arenosa and A. lyrata.     

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, genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine ( Pinus monticola Dougl. ex. D. Don.)

Western white pine ( Pinus monticola Dougl. ex. D. Don., WWP) shows genetic variation in disease resistance to white pine blister rust ( Cronartium ribicola). Most plant disease resistance (R) genes encode proteins that belong to a superfamily with nucleotide-binding site domains (NBS) and C-terminal leucine-rich repeats (LRR). In this work a PCR strategy was used to clone R gene analogs (RGAs) from WWP using oligonucleotide primers based on the conserved sequence motifs in the NBS domain of angiosperm NBS-LRR genes. Sixty-seven NBS sequences were cloned from disease-resistant trees. BLAST searches in GenBank revealed that they shared significant identity to well-characterized R genes from angiosperms, including L and M genes from flax, the tobacco N gene and the soybean gene LM6. Sequence alignments revealed that the RGAs from WWP contained the conserved motifs identified in angiosperm NBS domains, especially those motifs specific for TIR-NBS-LRR proteins. Phylogenic analysis of plant R genes and RGAs indicated that all cloned WWP RGAs can be grouped into one major branch together with well-known R proteins carrying a TIR domain, suggesting they belong to the subfamily of TIR-NBS-LRR genes. In one phylogenic tree, WWP RGAs were further subdivided into fourteen clusters with an amino acid sequence identity threshold of 75%. cDNA cloning and RT-PCR analysis with gene-specific primers demonstrated that members of 10 of the 14 RGA classes were expressed in foliage tissues, suggesting that a large and diverse NBS-LRR gene family may be functional in conifers. These results provide evidence for the hypothesis that conifer RGAs share a common origin with R genes from angiosperms, and some of them may play important roles in defense mechanisms that confer disease resistance in western white pine. Ratios of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) in the WWP NBS domains were greater than 1 or close to 1, indicating that diversifying selection and/or neutral selection operate on the NBS domains of the WWP RGA family.     

Hybrid incompatibility in Arabidopsis is determined by a multiple-locus genetic network

The cross between Arabidopsis thaliana and the closely related species Arabidopsis arenosa results in postzygotic hybrid incompatibility, manifested as seed death. Ecotypes of A. thaliana were tested for their ability to produce live seed when crossed to A. arenosa. The identified genetic variation was used to map quantitative trait loci (QTLs) encoded by the A. thaliana genome that affect the frequency of postzygotic lethality and the phenotypes of surviving seeds. Seven QTLs affecting the A. thaliana component of this hybrid incompatibility were identified by crossing a Columbia × C24 recombinant inbred line population to diploid A. arenosa pollen donors. Additional epistatic loci were identified based on their pairwise interaction with one or several of these QTLs. Epistatic interactions were detected for all seven QTLs. The two largest additive QTLs were subjected to fine-mapping, indicating the action of at least two genes in each. The topology of this network reveals a large set of minor-effect loci from the maternal genome controlling hybrid growth and viability at different developmental stages. Our study establishes a framework that will enable the identification and characterization of genes and pathways in A. thaliana responsible for hybrid lethality in the A. thaliana × A. arenosa interspecific cross.     

Genetic diversity of NBS–LRR class disease-resistance gene analogs in cultivated and wild eggplants

The genes encoding the nucleotide-binding site (NBS) and leucine-rich repeat (LRR) motifs constitute a large gene family in plants and have attracted much interest, because most of the plant disease-resistance genes that have been cloned are from this gene family. In this study, degenerate oligonucleotide primers, designed on the basis of conserved regions of the NBS domains from known plant resistance genes, were used to isolate resistance gene analogs (RGAs) from cultivated and wild eggplants, i.e., S. melongena, S. aethiopicum gr. Gilo, S. linnaeanum, S. integrifolium, S. sisymbriifolium, and S. khasianum. Sequence analysis indicated that the cloned eggplant RGAs belong to the non-TIR–NBS–LRR type, which are very similar to the R genes or the RGAs identified in other plant species, especially Solanaceae plants, suggesting the existence of common ancestors. Wide genetic diversity of eggplant RGAs was observed both in interspecific and intraspecific sequences, and eight distinct families of eggplant RGAs were identified. Further studies revealed a high average ratio of synonymous to non-synonymous substitution and a low level of recombination. These results suggest that NBS-encoding sequences of RGAs in cultivated and wild eggplants are subject to gradual accumulation of mutations leading to purifying selection. This is the first report of NBS–LRR class RGAs in eggplants.     

Comparative pangenome analyses provide insights into the evolution of Brassica rapa resistance gene analogues (RGAs)

Brassica rapa is grown worldwide as economically important vegetable and oilseed crop. However, its production is challenged by yield-limiting pathogens. The sustainable control of these pathogens mainly relies on the deployment of genetic resistance primarily driven by resistance gene analogues (RGAs). While several studies have identified RGAs in B. rapa, these were mainly based on a single genome reference and do not represent the full range of RGA diversity in B. rapa. In this study, we utilized the B. rapa pangenome, constructed from 71 lines encompassing 12 morphotypes, to describe a comprehensive repertoire of RGAs in B. rapa. We show that 309 RGAs were affected by presence-absence variation (PAV) and 223 RGAs were missing from the reference genome. The transmembrane leucine-rich repeat (TM-LRR) RGA class had more core gene types than variable genes, while the opposite was observed for nucleotide-binding site leucine-rich repeats (NLRs). Comparative analysis with the B. napus pangenome revealed significant RGA conservation (93%) between the two species. We identified 138 candidate RGAs located within known B. rapa disease resistance QTL, of which the majority were under negative selection. Using blackleg gene homologues, we demonstrated how these genes in B. napus were derived from B. rapa. This further clarifies the genetic relationship of these loci, which may be useful in narrowing-down candidate blackleg resistance genes. This study provides a novel genomic resource towards the identification of candidate genes for breeding disease resistance in B. rapa and its relatives.     

Characterization of expressed genes in the SLL2 region of self-compatible Arabidopsis thaliana.

Self-incompatibility in Brassica species is regulated by a set of S-locus genes: SLG, SRK, and SP11/SCR. In the vicinity of the S-locus genes, several expressed genes, SLL2 and SP2/ClpP, etc., were identified in B. campestris. Arabidopsis thaliana is a self-compatible Brassica relative, and its complete genome has been sequenced. From comparison of the genomic sequences between B. campestris and A. thaliana, microsynteny between gene clusters of Arabidopsis and Brassica SLL2 regions was observed, though the S-locus genes, SLG, SRK, and SP11/SCR were not found in the region of Arabidopsis. Almost all genes predicted in this region of Arabidopsis were expressed in both vegetative and reproductive organs, suggesting that the genes in the SLL2 region might not be related to self-incompatibility. Considering the recent speculation that the S-locus genes were translocated as a single unit between Arabidopsis and Brassica, the translocation might have occurred in the region between the SLL2 and SP7 genes.     

The Absence of TIR-Type Resistance Gene Analogues in the Sugar Beet (Beta vulgaris L.) Genome

The majority of known plant resistance genes encode proteins with conserved nucleotide-binding sites and leucine-rich repeats (NBS-LRR). Degenerate primers based on conserved NBS-LRR motifs were used to amplify analogues of resistance genes from the dicot sugar beet. Along with a cDNA library screen, the PCR screen identified 27 genomic and 12 expressed NBS-LRR RGAs (nlRGAs) sugar beet clones. The clones were classified into three subfamilies based on nucleotide sequence identity. Sequence analyses suggested that point mutations, such as nucleotide substitutions and insertion/deletions, are probably the primary source of diversity of sugar beet nlRGAs. A phylogenetic analysis revealed an ancestral relationship among sugar beet nlRGAs and resistance genes from various angiosperm species. One group appeared to share the same common ancestor as Prf, Rx, RPP8, and Mi, whereas the second group originated from the ancestral gene from which 12C1, Xa1, and Cre3 arose. The predicted protein products of the nlRGAs isolated in this study are all members of the non-TIR-type resistance gene subfamily and share strong sequence and structural similarities with non-TIR-type resistance proteins. No representatives of the TIR-type RGAs were detected either by PCR amplification using TIR type-specific primers or by in silico screening of more than 16,000 sugar beet ESTs. These findings suggest that TIR type of RGAs is absent from the sugar beet genome. The possible evolutionary loss of TIR type RGAs in the sugar beet is discussed. These authors (Yanyan Tian, Longjiang Fan) contributed equally to this work.