Identification and mapping of the leaf stripe resistance gene Rdg1a in Hordeum spontaneum

Leaf stripe of barley, caused by Pyrenophora graminea, is an important seed-borne disease in organically grown as well as in conventionally grown Nordic and Mediterranean barley districts. Two barley segregating populations represented by 103 recombinant inbred lines (RILs) of the cross L94 (susceptible) × Vada (resistant) and 194 RILs of the cross Arta (susceptible) × Hordeum spontaneum 41-1 (resistant) were analysed with two highly virulent leaf stripe isolates, Dg2 and Dg5, to identify loci for P. graminea resistance. A major gene with its positive allele contributed by Vada and H. spontaneum 41-1 was detected in both populations and for both pathogen isolates on chromosome 2HL explaining 44.1 and 91.8% R ², respectively for Dg2 and Dg5 in L94 × Vada and 97.8 and 96.1% R ², respectively for Dg2 and Dg5 in Arta × H. spontaneum 41-1. Common markers in the gene region of the two populations enabled map comparison and highlighted an overlapping for the region of the resistance locus. Since the map position of the resistance locus identified in this report is the same as that for the leaf stripe resistance gene Rdg1a, mapped earlier in Alf and derived from the ‘botanical’ barley line H. laevigatum, we propose that leaf stripe resistance in Vada and H. spontaneum 41-1 is governed by the same gene, namely by Rdg1a, and that Rdg1a resistance could be traced back to H. spontaneum, the progenitor of cultivated barley. PCR-based molecular markers that can be used for marker-assisted selection (MAS) of Rdg1a were identified. An Rdg1a syntenic interval with the rice chromosome arm 4L was identified on the basis of rice orthologs of EST-based barley markers. Analysis of the rice genes annotated into the syntenic interval did not reveal sequences strictly belonging to the major class (nucleotide-binding site plus leucine-rich repeat) of the resistance genes. Nonetheless, four genes coding for domains that are present in the major disease-resistance genes, namely receptor-like protein kinase and ATP/GTP-binding proteins, were identified together with a homolog of the barley powdery mildew resistance gene mlo. Three (out of five) homologs of these genes were mapped in the Rdg1a region in barley and the mlo homolog map position was tightly associated with the LOD score peak in both populations.     

Resistance gene analogues from rice: cloning, sequencing and mapping

 Degenerate oligonucleotide primers were designed on the basis of nucleotide-binding-site (NBS) motifs conserved between resistance genes of Arabidopsis, flax and tobacco and subsequently used as PCR primers to amplify resistance gene analogues (RGA) in rice. Primers amplified a major band of approximately 500 bp. Restriction analysis of the amplified product revealed that the band was made up of several different fragments. Many of these fragments were cloned. Sixty different cloned fragments were analysed and assigned to 14 categories based on Southern blot analysis. Fourteen clones, each representing one of the 14 categories of RGAs were mapped onto the rice genetic map using a Nipponbare ( japonica)בKasalath’ (indica) mapping population consisting of 186 F₂ lines. Of the 14 clones representing each class 12 could be mapped onto five different chromosomes of rice with a major cluster of 8 RGAs on chromosome 11. Our results indicate that it is possible to use sequence homology from conserved motifs of known resistance genes to amplify candidate resistance genes from diverse plant taxa. Received: 23 September 1998 / Accepted: 28 November 1998     

Identification and mapping of a new leaf stripe resistance gene in barley (Hordeum vulgare L.)

Pyrenophora graminea is the seed-borne pathogen causal agent of barley leaf stripe disease. Near-isogenic lines (NILs) carrying resistance of the cv ”Thibaut” against the highly virulent isolate Dg2 were obtained by introgressing the resistance into the genetic background of the susceptible cv ”Mirco”. The segregation of the resistance gene was followed in a F₂ population of 128 plants as well as on the F₃ lines derived from the F₂ plants; the segregation fitted the 1:2:1 ratio for a single gene. By using NILs, a RAPD marker associated with the resistance gene was identified; sequence-specific (STS) primers were designed on the basis of the amplicon sequence and a RILs mapping population with an AFLP-based map were used to position this molecular marker to barley chromosome 1 S (7HS). STS and CAPS markers were developed from RFLPs mapped to the telomeric region of barley chromosome 7HS and three polymorphic PCR-based markers were developed. The segregation of these markers was followed in the F₂ population and their map position with respect to the resistance gene was determined. Our results indicate that the Thibaut resistance gene, which we designated as Rdg2a, maps to the telomeric region of barley chromosome 7HS and is flanked by the markers OPQ-9₇₀₀ and MWG 2018 at distances of 3.1 and 2.5 cM respectively. The suitability of the PCR-based marker MWG2018 in selection- assisted barley breeding programs is discussed. Received: 22 June 2000 / Accepted: 16 October 2000     

Fine genetic mapping localizes cucumber scab resistance gene <Emphasis Type="Italic">Ccu</Emphasis> into an <Emphasis Type="Italic">R</Emphasis> gene cluster

Scab, caused by Cladosporium cucumerinum, is an important disease of cucumber, Cucumis sativus. In this study, we conducted fine genetic mapping of the single dominant scab resistance gene, Ccu, with 148 F₉ recombinant inbred lines (RILs) and 1,944 F₂ plants derived from the resistant cucumber inbred line 9110Gt and the susceptible line 9930, whose draft genome sequence is now available. A framework linkage map was first constructed with simple sequence repeat markers placing Ccu into the terminal 670 kb region of cucumber Chromosome 2. The 9110Gt genome was sequenced at 5× genome coverage with the Solexa next-generation sequencing technology. Sequence analysis of the assembled 9110Gt contigs and the Ccu region of the 9930 genome identified three insertion/deletion (Indel) markers, Indel01, Indel02, and Indel03 that were closely linked with the Ccu locus. On the high-resolution map developed with the F₂ population, the two closest flanking markers, Indel01 and Indel02, were 0.14 and 0.15 cM away from the target gene Ccu, respectively, and the physical distance between the two markers was approximately 140 kb. Detailed annotation of the 180 kb region harboring the Ccu locus identified a cluster of six resistance gene analogs (RGAs) that belong to the nucleotide binding site (NBS) type R genes. Four RGAs were in the region delimited by markers Indel01 and Indel02, and thus were possible candidates of Ccu. Comparative DNA analysis of this cucumber Ccu gene region with a melon (C. melo) bacterial artificial chromosome (BAC) clone revealed a high degree of micro-synteny and conservation of the RGA tandem repeats in this region.     

The gibberellic-acid insensitive dwarfing gene<Emphasis Type="Italic"> sdw3</Emphasis> of barley is located on chromosome 2HS in a region that shows high colinearity with rice chromosome 7L

In this study, comparative high resolution genetic mapping of the GA-insensitive dwarfing gene sdw3 of barley revealed highly conserved macrosynteny of the target region on barley chromosome 2HS with rice chromosome 7L. A rice contig covering the sdw3-orthologous region was identified and subsequently exploited for marker saturation of the target interval in barley. This was achieved by (1) mapping of rice markers from the orthologous region of the rice genetic map, (2) mapping of rice ESTs that had been physically localized on the rice contig, or (3) mapping of barley ESTs that show strong sequence similarity to coding sequences present in the rice contig. Finally, the sdw3 gene was mapped to an interval of 0.55 cM in barley, corresponding to a physical distance of about 252 kb in rice, after employing orthologous EST-derived rice markers. Three putative ORFs were identified in this interval in rice, which exhibited significant sequence similarity to known signal regulator genes from different species. These ORFs can serve as starting points for the map-based isolation of the sdw3 gene from barley.Communicated by R. Hagemann     

High-resolution genetic mapping at the <Emphasis Type="Italic">Bph15</Emphasis> locus for brown planthopper resistance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Resistance to the brown planthopper (BPH), Nilaparvata lugens Stål, a devastating sucking insect pest of rice, is an important breeding objective in rice improvement programs. Bph15, one of the 17 major BPH resistance genes so far identified in both cultivated and wild rice, has been identified in an introgression line, B5, and mapped on chromosome 4 flanked by restriction fragment length polymorphism markers C820 and S11182. In order to pave the way for positional cloning of this gene, we have developed a high-resolution genetic map of Bph15 by positioning 21 DNA markers in the target chromosomal region. Mapping was based on a PCR-based screening of 9,472 F₂ individuals derived from a cross between RI93, a selected recombinant inbred line of B5 bearing the resistance gene Bph15, and a susceptible variety, Taichung Native 1, in order to identify recombinant plants within the Bph15 region. Recombinant F₂ individuals with the Bph15 genotype were determined by phenotype evaluation. Analysis of recombination events in the Bph15 region delimited the gene locus to an interval between markers RG1 and RG2 that co-segregated with the M1 marker. A genomic library of B5 was screened using these markers, and bacterial artificial chromosome clones spanning the Bph15 chromosome region were obtained. An assay of the recombinants using the sub-clones of these clones in combination with sequence analysis delimited the Bph15 gene to a genomic segment of approximately 47 kb. This result should serve as the basis for eventual isolation of the Bph15 resistance gene.     

High-resolution mapping and gene prediction of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> resistance gene <Emphasis Type="Italic">Xa7</Emphasis>

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F₂ mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F₂ individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes. Shen Chen and Zhanghui Huang are contributed equally to this work.     

Identification and mapping of resistance gene analogs and a white rust resistance locus in<Emphasis Type="Italic"> Brassica rapa</Emphasis> ssp.<Emphasis Type="Italic"> oleifera</Emphasis>

The objective of this investigation was to tag a locus for white rust resistance in a Brassica rapa ssp. oleifera F₂ population segregating for this trait, using bulked segregant analysis with random amplified polymorphic DNA (RAPD) markers, linkage mapping and a candidate gene approach based on resistance gene analogs (RGAs). The resistance source was the Finnish line Bor4109. The reaction against white rust races 7a and 7v was scored in 20 seedlings from each self-pollinated F₂ individual. The proportion of resistant plants among these F₃ families varied from 0 to 67%. Bulked segregant analysis did not reveal any markers linked with resistance and, therefore, a linkage map with 81 markers was created. A locus that accounted for 18.4% of the variation in resistance to white rust was mapped to linkage group (LG) 2 near the RAPD marker Z19a. During the study, a bacterial resistance gene homologous to Arabidopsis RPS2 and six different RGAs were sequenced. RPS2 and five of the RGAs were mapped to linkage groups LG1, LG4 and LG9. Unfortunately, none of the RGAs could be shown to be associated with white rust resistance.Communicated by H.C. BeckerThe nucleotide sequence data reported has been deposited in the Genbank under the accession numbers AF315081–AF315087.     

Fine genetic mapping fails to dissociate durable stem rust resistance gene Sr2 from pseudo-black chaff in common wheat (Triticum aestivum L.)

The broad-spectrum stem rust resistance gene Sr2 has provided protection in wheat against Puccinia graminis Pers. f. sp. tritici for over 80 years. The Sr2 gene and an associated dark pigmentation trait, pseudo-black chaff (PBC), have previously been localized to the short arm of chromosome 3B. In a first step towards the positional-based cloning of Sr2, we constructed a high-resolution map of this region. The wheat EST (wEST) deletion bin mapping project provided tightly linked cDNA markers. The rice genome sequence was used to infer the putative gene order for orthologous wheat genes and provide additional markers once the syntenic interval in rice was identified. We used this approach to map six wESTs that were collinear with the physical order of the corresponding genes on rice chromosome 1 suggesting there are no major re-arrangements between wheat and rice in this region. We were unable to separate by recombination the tightly linked morphological trait, PBC from the stem rust resistance gene suggesting that either a single gene or two tightly linked genes control both traits.     

High-resolution mapping, cloning and molecular characterization of the Pi-k h gene of rice, which confers resistance to Magnaporthe grisea

In order to understand the molecular mechanisms involved in the gene-for-gene type of pathogen resistance, high-resolution genetic and physical mapping of resistance loci is required to facilitate map-based cloning of resistance genes. Here, we report the molecular mapping and cloning of a dominant gene (Pi-k ^h ) present in the rice line Tetep, which is associated with resistance to rice blast disease caused by Magnaporthe grisea. This gene is effective against M. grisea populations prevalent in the Northwestern Himalayan region of India. Using 178 sequence tagged microsatellite, sequence-tagged site, expressed sequence tag and simple sequence repeat (SSR) markers to genotype a population of 208 F₂ individuals, we mapped the Pi-k ^h gene between two SSR markers (TRS26 and TRS33) which are 0.7 and 0.5 cM away, respectively, and can be used in marker-assisted-selection for blast-resistant rice cultivars. We used the markers to identify the homologous region in the genomic sequence of Oryza sativa cv. Nipponbare, and a physical map consisting of two overlapping bacterial artificial chromosome and P1 artificial chromosome clones was assembled, spanning a region of 143,537 bp on the long arm of chromosome 11. Using bioinformatic analyses, we then identified a candidate blast-resistance gene in the region, and cloned the homologous sequence from Tetep. The putative Pi-k ^h gene cloned from Tetep is 1.5 kbp long with a single ORF, and belongs to the nucleotide binding site-leucine rich repeat class of disease resistance genes. Structural and expression analysis of the Pi-k ^h gene revealed that its expression is pathogen inducible.     

Identification and fine mapping of <Emphasis Type="Italic">Pi33</Emphasis>, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene <Emphasis Type="Italic">ACE1</Emphasis>

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) × Azucena (susceptible) and Azucena × Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F₂ populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.Communicated by D.J. Mackill     

Genetic and physical mapping of <Emphasis Type="Italic">Pi36</Emphasis>(t), a novel rice blast resistance gene located on rice chromosome 8

Blast resistance in the indica cultivar (cv.) Q61 was inherited as a single dominant gene in two F₂ populations, F₂-1 and F₂-2, derived from crosses between the donor cv. and two susceptible japonica cvs. Aichi Asahi and Lijiangxintuanheigu (LTH), respectively. To rapidly determine the chromosomal location of the resistance (R) gene detected in Q61, random amplified polymorphic DNA (RAPD) analysis was performed in the F₂-1 population using bulked-segregant analysis (BSA) in combination with recessive-class analysis (RCA). One of the three linked markers identified, BA1126₅₅₀, was cloned and sequenced. The R gene locus was roughly mapped on rice chromosome 8 by comparison of the BA1126₅₅₀ sequence with rice sequences in the databases (chromosome landing). To confirm this finding, seven known markers, including four sequence-tagged-site (STS) markers and three simple-sequence repeat (SSR) markers flanking BA1126₅₅₀ on chromosome 8, were subjected to linkage analysis in the two F₂ populations. The locus was mapped to a 5.8 cM interval bounded by RM5647 and RM8018 on the short arm of chromosome 8. This novel R gene is therefore tentatively designated as Pi36(t). For fine mapping of the Pi36(t) locus, five additional markers including one STS marker and four candidate resistance gene (CRG) markers were developed in the target region, based on the genomic sequence of the corresponding region of the reference japonica cv. Nipponbare. The Pi36(t) locus was finally localized to an interval of about 0.6 cM flanked by the markers RM5647 and CRG2, and co-segregated with the markers CRG3 and CRG4. To physically map this locus, the Pi36(t)-linked markers were mapped by electronic hybridization to bacterial artificial chromosome (BAC) or P1 artificial chromosome (PAC) clones of Nipponbare, and a contig map was constructed in silico through Pairwise BLAST analysis. The Pi36(t) locus was physically delimited to an interval of about 17.0 kb, based on the genomic sequence of Nipponbare.     

Genetic and physical mapping of the S<Subscript>H</Subscript>3 region that confers resistance to leaf rust in coffee tree (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

Resistance to coffee leaf rust is conferred by S_H3, a major dominant gene that has been introgressed from a wild coffee species Coffea liberica (genome L) into the allotetraploid cultivated species, Coffea arabica (genome C^aE^a). As the first step toward the map-based cloning of the S_H3 gene, using a bacterial artificial chromosome (BAC) library, we describe the construction of a physical map in C. arabica spanning the resistance locus. This physical map consists in two homeologous BAC-contigs of 1,170 and 1,208 kb corresponding to the subgenomes C^a and E^a, respectively. Genetic analysis was performed using a single nucleotide polymorphism detection assay based on Sanger sequencing of amplicons. The C. liberica-derived chromosome segment that carries the S_H3 resistance gene appeared to be introgressed on the sub-genome C^a. The position of the S_H3 locus was delimited within an interval of 550 kb on the physical map. In addition, our results indicated a sixfold reduction in recombination frequency in the introgressed S_H3 region compared to the orthologous region in Coffea canephora.     

High-resolution mapping of <Emphasis Type="Italic">Rsn1</Emphasis>, a locus controlling sensitivity of rice to a necrosis-inducing phytotoxin from <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG1-IA

Rhizoctonia solani is a necrotrophic fungal pathogen that causes disease on many crop-plant species. Anastomosis group 1-IA is the causal agent of sheath blight of rice (Oryza sativa L.), one of the most important rice diseases worldwide. R. solani AG1-IA produces a necrosis-inducing phytotoxin and rice cultivar’s sensitivity to the toxin correlates with disease susceptibility. Unlike genetic analyses of sheath blight resistance where resistance loci have been reported as quantitative trait loci, phytotoxin sensitivity is inherited as a Mendelian trait that permits high-resolution mapping of the sensitivity genes. An F₂ mapping population derived from parent cultivars ‘Cypress’ (toxin sensitive) and ‘Jasmine 85’ (toxin insensitive) was used to map Rsn1, the necrosis-inducing locus. Initial mapping based on 176 F₂ progeny and 69 simple sequence repeat (SSR) markers located Rsn1 on the long arm of chromosome 7, with tight linkage to SSR marker RM418. A high-resolution genetic map of the region was subsequently developed using a total of 1,043 F₂ progeny, and Rsn1 was mapped to a 0.7 cM interval flanked by markers NM590 and RM418. Analysis of the corresponding 29 Kb genomic sequences from reference cultivars ‘Nipponbare’ and ‘93-11’ revealed the presence of four putative genes within the interval. Two are expressed cytokinin-O-glucosyltransferases, which fit an apoptotic pathway model of toxin activity, and are individually being investigated further as potential candidates for Rsn1.     

The CC-NB-LRR-Type Rdg2a Resistance Gene Confers Immunity to the Seed-Borne Barley Leaf Stripe Pathogen in the Absence of Hypersensitive Cell Death

Background

Leaf stripe disease on barley (Hordeum vulgare) is caused by the seed-transmitted hemi-biotrophic fungus Pyrenophora graminea. Race-specific resistance to leaf stripe is controlled by two known Rdg (Resistance to Drechslera graminea) genes: the H. spontaneum-derived Rdg1a and Rdg2a, identified in H. vulgare. The aim of the present work was to isolate the Rdg2a leaf stripe resistance gene, to characterize the Rdg2a locus organization and evolution and to elucidate the histological bases of Rdg2a-based leaf stripe resistance.

Principal Findings

We describe here the positional cloning and functional characterization of the leaf stripe resistance gene Rdg2a. At the Rdg2a locus, three sequence-related coiled-coil, nucleotide-binding site, and leucine-rich repeat (CC-NB-LRR) encoding genes were identified. Sequence comparisons suggested that paralogs of this resistance locus evolved through recent gene duplication, and were subjected to frequent sequence exchange. Transformation of the leaf stripe susceptible cv. Golden Promise with two Rdg2a-candidates under the control of their native 5′ regulatory sequences identified a member of the CC-NB-LRR gene family that conferred resistance against the Dg2 leaf stripe isolate, against which the Rdg2a-gene is effective. Histological analysis demonstrated that Rdg2a-mediated leaf stripe resistance involves autofluorescing cells and prevents pathogen colonization in the embryos without any detectable hypersensitive cell death response, supporting a cell wall reinforcement-based resistance mechanism.

Conclusions

This work reports about the cloning of a resistance gene effective against a seed borne disease. We observed that Rdg2a was subjected to diversifying selection which is consistent with a model in which the R gene co-evolves with a pathogen effector(s) gene. We propose that inducible responses giving rise to physical and chemical barriers to infection in the cell walls and intercellular spaces of the barley embryo tissues represent mechanisms by which the CC-NB-LRR-encoding Rdg2a gene mediates resistance to leaf stripe in the absence of hypersensitive cell death.     

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.     

Microcolinearity between a 2-cM region encompassing the grain protein content locus<Emphasis Type="Italic"> Gpc-6B1</Emphasis> on wheat chromosome 6B and a 350-kb region on rice chromosome 2

The conservation of the linear order (colinearity) of genetic markers along large chromosome segments in wheat and rice is well established, but less is known about the microcolinearity between both genomes at subcentimorgan distances. In this study we focused on the microcolinearity between a 2.6-cM interval flanked by markers Xcdo365 and Xucw65 on wheat chromosome 6B and rice chromosome 2. A previous study has shown that this wheat segment includes the Gpc-6B1 locus, which is responsible for large differences in grain protein content (GPC) and is the target of a positional cloning effort in our laboratories. Twenty-one recombination events between Xcdo365 and Xucw65 were found in a large segregating population (935 gametes) and used to map 17 genes selected from rice chromosome 2 in the wheat genetic map. We found a high level of colinearity between a 2.1-cM region flanked by loci Xucw75 and Xucw67 on wheat chromosome 6B and a 350-kb uninterrupted sequenced region in rice chromosome arm 2S. Colinearity between these two genomes was extended to the region proximal to Xucw67 (eight colinear RFLP markers), but was interrupted distal to Xucw75 (six non-colinear RFLP markers). Analysis of different comparative studies between rice and wheat suggests that microcolinearity is more frequently disrupted in the distal region of the wheat chromosomes. Fortunately, the region encompassing the Gpc-6B1 locus showed an excellent conservation between the two genomes, facilitating the saturation of the target region of the wheat genetic map with molecular markers. These markers were used to map the Gpc-6B1 locus into a 0.3-cM interval flanked by PCR markers Xucw79 and Xucw71, and to identify five candidate genes within the colinear 64-kb region in rice.     

Characterisation and genetic mapping of resistance and defence gene analogs in cocoa (Theobroma cacao L.)

Disease resistance and defence gene analog (RGA/DGA) sequences were isolated in cocoa using a PCR approach with degenerate primers designed from conserved domains of plant resistance and defence genes: the NBS (nucleotide binding site) motif present in a number of resistance genes such as the tobacco N, sub-domains of plant serine/threonine kinases such as the Pto tomato gene, and conserved domains of two defence gene families: pathogenesis-related proteins (PR) of classes 2 and 5. Nucleotide identity between thirty six sequences isolated from cocoa and known resistance or defence genes varied from 58 to 80%. Amino acid sequences translated from corresponding coding sequences produced sequences without stop codons, except for one NBS –like sequence. Most of the RGAs could be mapped on the cocoa genome and three clusters of genes could be observed : NBS-like sequences clustered in two regions located on chromosomes 7 and 10, Pto-like sequences mapped in five genome regions of which one, located on chromosome 4, corresponded to a cluster of five different sequences. PR2-like sequences mapped in two regions located on chromosome 5 and 9 respectively. An enrichment of the genetic map with microsatellite markers allowed us to identify several co-localisations of RGAs, DGAs and QTL for resistance to Phytophthora detected in several progenies, particularly on chromosome 4 where a cluster of Pto-like sequences and 4 QTL for resistance to Phytophthora were observed. Many other serious diseases affect cocoa and the candidate genes, isolated in this study, could be of broader interest in cocoa disease management.     

Fine genetic mapping and physical delimitation of the lesion mimic gene <Emphasis Type="Italic">spotted leaf 5</Emphasis> (<Emphasis Type="Italic">spl5</Emphasis>) in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Spotted leaf 5 (spl5), a lesion mimic mutant, was first identified in rice (Oryza sativa L.) japonica cv. Norin8 in 1978. This mutant exhibits spontaneous disease-like lesions in the absence of any pathogens and resistance to rice blast and bacterial blight; however, the target gene has not yet been isolated. In the present study, we employed a map-based cloning strategy to finely map the spl5 gene. In an initial mapping with 100 F₂ individuals (spl5/spl5) derived from a cross between the spl5 mutant and indica cv. 93-11, the spl5 gene was located in a 3.3-cM region on chromosome 7 using six simple sequence repeat (SSR) markers. In a high-resolution genetic mapping, two F₂ populations with 3,149 individuals (spl5/spl5) were derived from two crosses between spl5 mutant and two indica cvs. 93-11 and Zhefu802 and six sequence-tagged site (STS) markers were newly developed. Finally, the spl5 gene was mapped to a region of 0.048 cM between two markers SSR7 and RM7121. One BAC/PAC contig map covering these markers’ loci and the spl5 gene was constructed through Pairwise BLAST analysis. Our bioinformatics analysis shows that the spl5 gene is located in the 80-kb region between two markers SSR7 and RM7121 with a high average ratio of physical to genetic distance (1.67 Mb/cM) and eighteen candidate genes. The analysis of these candidate genes indicates that the spl5 gene represents a novel class of regulators controlling cell death and resistance response in plants.     

Isolation and mapping of resistance gene analogs from the Avena strigosa genome

Degenerate primers based on conserved regions of the nucleotide binding site (NBS) domain (encoded by the largest group of cloned plant disease resistance genes) were used to isolate a set of 15 resistance gene analogs (RGA) from the diploid species Avena strigosa Schreb. These were grouped into seven classes on the basis of 60% or greater nucleic acid sequence identity. Representative clones were used for genetic mapping in diploid and hexaploid oats. Two RGAs were mapped at two loci of the linkage group AswBF belonging to the A. strigosa × A. wiestii Steud map, and ten RGAs were mapped at 15 loci in eight linkage groups belonging to the A. byzantina C. Koch cv. Kanota × A. sativa L. cv. Ogle map. A similar approach was used for targeting genes encoding receptor-like kinases. Three different sequences were obtained and mapped to two linkage groups of the hexaploid oat map. Associations were explored between already known disease resistance loci mapped in different populations and the RGAs. Molecular markers previously linked to crown rust and barley yellow dwarf resistance genes or quantitative trait loci were found in the Kanota × Ogle map linked to RGAs at a distance ranging from 0 cM to 20 cM. Homoeologous RGAs were found to be linked to loci either conferring resistance to different isolates of the same pathogen or to different pathogens. This suggests that these RGAs identify genome regions containing resistance gene clusters.