High-resolution genetical and physical mapping of the Rx gene for extreme resistance to potato virus X in tetraploid potato

The Rx locus in potato confers extreme resistance to PVX. In the F₁ progeny of crosses between the PVX-susceptible cultivar Huinkel and the cultivar Cara (Rx genotype) there was a 1?:?1 segregation of PVX resistance, indicating that Rx in Cara is present in the simplex condition. Using potato and tomato RFLP markers, we mapped Rx in Cara to the distal end of chromosome XII at a different position to the previously mapped Rx1 locus. To generate a high-resolution linkage map in the vicinity of Rx a total 728 AFLP primer combinations were screened using DNA of bulked resistant and susceptible segregants. We also screened segregating populations for chromosomal recombination events linked to the Rx locus and identified 82 plants with recombination events close to Rx. Using these recombinant plants we have identified AFLPs that flank Rx and span an interval of 0.23 cM in a region of the genome where 1 cM corresponds to approximately 400?kb.     

Development of a CAPS marker for the Pvr4 locus: a tool for pyramiding potyvirus resistance genes in pepper.

The Pvr4 resistance gene in pepper confers a complete resistance to the three pathotypes of potato virus Y (PVY) and to pepper mottle virus (PepMoV). In order to use this gene in a marker-assisted selection (MAS) program and to permit the pyramiding of several potyvirus resistance genes in the same cultivar, tightly linked amplified fragment length polymorphism (AFLP) markers were obtained by the bulked segregant analysis method. Eight linked AFLP markers were mapped in an interval from 2.1 +/- 0.8 to 13.8 +/- 2.9 cM around this locus. The closest codominant AFLP marker was converted into a codominant CAPS (cleaved amplified polymorphic sequence) marker using data from the alignment of the two allele sequences. We have further characterized the relevance of the CAPS marker for MAS programs in different pepper breeding lines.     

A high-resolution map of the H1 locus harbouring resistance to the potato cyst nematode Globodera rostochiensis

The resistance gene H1 confers resistance to the potato cyst nematode Globodera rostochiensis and is located at the distal end of the long arm of chromosome V of potato. For marker enrichment of the H1 locus, a bulked segregant analysis (BSA) was carried out using 704 AFLP primer combinations. A second source of markers tightly linked to H1 is the ultra-high-density (UHD) genetic map of the potato cross SH × RH. This map has been produced with 387 AFLP primer combinations and consists of 10,365 AFLP markers in 1,118 bins (). Comparing these two methods revealed that BSA resulted in one marker/cM and the UHD map in four markers/cM in the H1 interval. Subsequently, a high-resolution genetic map of the H1 locus has been developed using a segregating F₁ SH × RH population consisting of 1,209 genotypes. Two PCR-based markers were designed at either side of the H1 gene to screen the 1,209 genotypes for recombination events. In the high-resolution genetic map, two of the four co-segregating AFLP markers could be separated from the H1 gene. Marker EM1 is located at a distance of 0.2 cM, and marker EM14 is located at a distance of 0.8 cM. The other two co-segregating markers CM1 (in coupling) and EM15 (in repulsion) could not be separated from the H1 gene.Communicated by J.G. Wenzel     

Identification of AFLP molecular markers for resistance against Melampsora larici-populina in Populus

We have identified AFLP markers tightly linked to the locus conferring resistance to the leaf rust Melampsora larici-populina in Populus. The study was carried out using a hybrid progeny derived from an inter-specific, controlled cross between a resistant Populus deltoides female and a susceptible P. nigra male. The segregation ratio of resistant to susceptible plants suggested that a single, dominant locus defined this resistance. This locus, which we have designated Melampsora resistance (Mer), confers resistance against E1, E2, and E3, three different races of Melampsora larici-populina. In order to identify molecular markers linked to the Mer locus we decided to combine two different techniques: (1) the high-density marker technology, AFLP, which allows the analysis of thousands of markers in a relatively short time, and (2) the Bulked Segregant Analysis (BSA), a method which facilitates the identification of markers that are tightly linked to the locus of interest. We analyzed approximately 11,500 selectively amplified DNA fragments using 144 primer combinations and identified three markers tightly linked to the Mer locus. The markers can be useful in current breeding programs and are the basis for future cloning of the resistance gene.     

The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a non-virion form of the coat protein.     

A potato hypersensitive resistance gene against potato virus X maps to a resistance gene cluster on chromosome 5

 The dominant Nb gene of potato confers strain-specific hypersensitive resistance against potato virus X (PVX). A population segregating for Nb was screened for resistance by inoculating with PVX strain CP2, which is sensitive to Nb. Through a combination of bulked segregant analysis and selective restriction fragment amplification, several amplified fragment length polymorphism (AFLP) markers linked to Nb were identified. These were cloned and converted into dominant cleaved amplified polymorphic sequence (CAPS) markers. The segregation of these markers in a Lycopersicon esculentum×L. pennellii mapping population suggested that Nb is located on chromosome 5. This was confirmed by examining resistant and susceptible potato individuals with several tomato and potato chromosome-5-specific markers. Nb maps to a region of chromosome 5 where several other resistance genes– including R1, a resistance gene against Phytophthora infestans, Gpa, a locus that confers resistance against Globodera pallida, and Rx2, a gene that confers extreme resistance against PVX–have previously been identified. Received: 2 January 1997/Accepted: 7 February 1997     

High-resolution genetic analysis of the Sd-1 aphid resistance locus in Malus spp

Aphids cause serious physical and economic damage to most major crops throughout the world through feeding damage, with consequent symptom development and virus transmission. The rosy leaf-curling aphid ( Dysaphis devecta Wlk.) is a pest of apple ( Malus spp.) which displays an exceptionally clear phenotype with respect to susceptible and resistant symptoms. The Sd-1 locus for resistance to D. devecta biotypes 1 and 2 is present in Cox's Orange Pippin and its progeny and had previously been mapped to the top of linkage group 7. Detailed fine mapping of the locus was initiated with AFLP bulked segregant analysis of both pedigree and segregating bulks, which identified three new marker loci. Preliminary marker order in the Sd-1 region was established through mapping in a family derived from Prima x Fiesta, with additional segregation analysis on a Fiesta x Golden Delicious family. Previous recombinant data was re-evaluated and corrected. Two co-segregating AFLP fragments were found to contain a common (GA)(23) repeat, from which a PCR-based simple sequence repeat (SSR) assay was developed. A high-resolution map around the Sd-1 region was established by analysing a large meta-population of Sd-1 recombinants using 759 additional individuals from different families. The Sd-1 gene has been located within a 1.3-cM interval flanked by the molecular markers SdSSRa and 2B12a and co-locates with the RFLP marker MC064. Allelism between Sd-1 and Sd-2 resistant sources was tested. Molecular markers tightly linked to Sd-1 were shown to be co-segregating with the Sd-2 locus, which indicated that Sd-1 and Sd-2 loci are at least tightly linked and, probably, allelic.     

Generation and mapping of SCAR and CAPS markers linked to the seed coat color gene in Brassica napus using a genome-walking technique.

The yellow seed coat trait in No. 2127-17, a resynthesized purely yellow Brassica napus line, is controlled by a single partially dominant gene, Y. A double-haploid population derived from the F1 of No. 2127-17 x 'ZY821' was used to map the seed coat color phenotype. A combination of AFLP analysis and bulked segregant analysis identified 18 AFLP markers linked to the seed coat color trait. The 18 AFLP markers were mapped to a chromosomal region of 37.0 cM with an average of 2.0 cM between adjacent markers. Two markers, AFLP-K and AFLP-H, bracketed the Y locus in an interval of 1.0 cM, such that each was 0.5 cM away from the Y locus. Two other markers, AFLP-A and AFLP-B, co-segregated with the seed color gene. For ease of use in breeding programs, these 4 most tightly linked AFLP markers were converted into reliable PCR-based markers. SCAR-K, which was derived from AFLP-K, was assigned to linkage group 9 (N9) of a B. napus reference map consisting of 150 commonly used SSR (simple sequence repeat) markers. Furthermore, 2 SSR markers (Na14-E08 and Na10-B07) linked to SCAR-K on the reference map were reversely mapped to the linkage map constructed in this study, and also showed linkage to the Y locus. These linked markers would be useful for the transfer of the dominant allele Y from No. 2127-17 to elite cultivars using a marker-assisted selection strategy and would accelerate the cloning of the seed coat color gene.     

High-resolution genetic and physical mapping of the region containing the Bs2 resistance gene of pepper

The Bs2 resistance gene of pepper confers resistance against the bacterial pathogen Xanthomonas campestris pv. vesicatoria. As a first step toward isolation of the Bs2 gene, molecular markers tightly linked to the gene were identified by randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analysis of near-isogenic lines. Markers flanking the locus were identified and a high-resolution linkage map of the region was developed. One AFLP marker, A2, was found to cosegregate with the locus, while two others, F1 and B3, flank the locus and are within 0.6 cM. Physical mapping of the A2 and F1 markers indicates that these markers may be within 150 kb of each other. Together, these results indicate that the Bs2 region may be cloned either by chromosome walker or landing. The linked markers were also used to characterize gamma-irradiation-induced mutants at the Bs2 locus. Received: 15 January 1999 / Accepted: 11 May 1999     

AFLP markers linked to resistance against Striga gesnerioides race 1 in cowpea (Vigna unguiculata).

Amplified fragment length polymorphism (AFLP) analysis was used in combination with bulked segregant analysis (BSA) to identify molecular markers linked to two cowpea (Vigna unguiculata (L.) Walp.) genes conferring resistance to Striga gesnerioides race 1. After AFLP analysis of an F2 population derived from a cross between the resistant cultivar Gorom and the susceptible cultivar Tvx 3236, seven AFLP markers were identified that are linked to Rsg3, the gene conferring race I resistance in 'Gorom'. The distances between these markers and Rsg3 ranged from 9.9 to 2.5 cM, with two markers, E-AGA/M-CTA460 and E-AGA/M-CAG300, flanking Rsg3 at 2.5 and 2.6 cM, respectively. Analysis of a second F2 population derived from the cross between 'Tvx 3236' and the resistant cultivar IT81D-994 identified five AFLP markers linked to the race 1 resistance gene 994-Rsg present in 'IT81D-994'. The two markers showing the tightest linkage to the 994-Rsg locus were E-AAG/M-AAC450 and E-AAG/M-AAC150 at 2.1 and 2.0 cM, respectively. Two of the markers linked to 994-Rsg, E-AGA/M-CAG300 and E-AGA/M-CAG450, were also linked to Rsg3. The identification of molecular markers in common between the two sources of race 1 resistance suggests that either Striga resistance genes are clustered in these plants or that these loci are allelic. Mapping of the resistance loci within the cowpea genome revealed that three markers linked to Rsg3 and (or) 994-Rsg are located on linkage group 6.     

Chromosome landing at the Mla locus in barley (Hordeum vulgare L.) by means of high-resolution mapping with AFLP markers

 The complex Mla locus of barley determines resistance to the powdery mildew pathogen Erysiphe graminis f. sp. hordei. With a view towards gene isolation, a population consisting of 950 F₂ individuals derived from a cross between the near-isogenic lines ‘P01’ (Mla1) and ‘P10’ (Mla12) was used to construct a high-resolution map of the Mla region. A fluorescence-based AFLP technique and bulked segregant analysis were applied to screen for polymorphic, tightly linked AFLP markers. Three AFLP markers were selected as suitable for a chromosome-landing strategy. One of these AFLP markers and a closely linked RFLP marker were converted into sequence-specific PCR markers. PCR-based screening of approximately 70 000 yeast artificial chromosome (YAC) clones revealed three identical YACs harbouring the Mla locus. Terminal insert sequences were obtained using inverse PCR. The derived STS marker from the right YAC end-clone was mapped distal to the Mla locus. Received: 17 July 1998 / Accepted: 9 August 1998     

Genetic characterization and fine mapping of the blast resistance locus Pigm(t) tightly linked to Pi2 and Pi9 in a broad-spectrum resistant Chinese variety

The identification and utilization of broad-spectrum resistance genes have been proven the most effective and economical approach to control rice blast disease. To understand the molecular mechanism of broad-spectrum resistance to rice blast, we conducted genetic and fine mapping analysis of the blast resistance gene in a Chinese rice variety: Gumei 4 (GM4) identified with broad-spectrum resistance and used in rice breeding for blast resistance for more than 20 years. Genetic and mapping analysis indicated that blast resistance to nine isolates of different Chinese races in GM4 was controlled by the same dominant locus designated as Pigm(t) that was finely mapped to an approximately 70-kb interval between markers C5483 and C0428 on chromosome 6, which contains five candidate NBS--LRR disease resistance genes. The allelism test showed that Pigm(t) was either tightly linked or allelic to Pi2 and Pi9, two known blast resistance genes. Mapping information also indicated that another blast resistance gene Pi26(t) might also be located at the same region. Candidate genes were identified by sequence analysis of the Nipponbare and Pi9 locus and the corresponding region in GM4. Sequence divergence of candidate genes was observed between GM4 and model varieties Nipponbare and 9311, and Pi9. Our current study provides essential information and new genetic resource for the cloning of functional resistance gene(s) and for marker-assisted selection in rice breeding for broad-spectrum blast resistance.Yiwen Deng and Xudong Zhu contributed equally to this work.     

Dissection of the fusarium I2 gene cluster in tomato reveals six homologs and one active gene copy.

The I2 locus in tomato confers resistance to race 2 of the soil-borne fungus Fusarium oxysporum f sp lycopersici. The selective restriction fragment amplification (AFLP) positional cloning strategy was used to identify I2 in the tomato genome. A yeast artificial chromosome (YAC) clone covering approximately 750 kb encompassing the I2 locus was isolated, and the AFLP technique was used to derive tightly linked AFLP markers from this YAC clone. Genetic complementation analysis in transgenic R1 plants using a set of overlapping cosmids covering the I2 locus revealed three cosmids giving full resistance to F. o. lycopersici race 2. These cosmids shared a 7-kb DNA fragment containing an open reading frame encoding a protein with similarity to the nucleotide binding site leucine-rich repeat family of resistance genes. At the I2 locus, we identified six additional homologs that included the recently identified I2C-1 and I2C-2 genes. However, cosmids containing the I2C-1 or I2C-2 gene could not confer resistance to plants, indicating that these members are not the functional resistance genes. Alignments between the various members of the I2 gene family revealed two significant variable regions within the leucine-rich repeat region. They consisted of deletions or duplications of one or more leucine-rich repeats. We propose that one or both of these leucine-rich repeats are involved in Fusarium wilt resistance with I2 specificity.     

Efficient targeting of plant disease resistance loci using NBS profiling

The conserved sequences in the nucleotide-binding sites of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of disease resistance (R) genes have been used for PCR-based R-gene isolation and subsequent development of molecular markers. Here we present a PCR-based approach (NBS profiling) that efficiently targets R genes and R-gene analogs (RGAs) and, at the same time, produces polymorphic markers in these genes. In NBS profiling, genomic DNA is digested with a restriction enzyme, and an NBS-specific (degenerate) primer is used in a PCR reaction towards an adapter linked to the resulting DNA fragments. The NBS profiling protocol generates a reproducible polymorphic multilocus marker profile on a sequencing gel that is highly enriched for R genes and RGAs. NBS profiling was successfully used in potato with several restriction enzymes, and several primers targeted to different conserved motifs in the NBS. Across primers and enzymes, the NBS profiles contained 50–90% fragments that were significantly similar to known R-gene and RGA sequences. The protocol was similarly successful in other crops (including tomato, barley, and lettuce) without modifications. NBS profiling can thus be used to produce markers tightly linked to R genes and R-gene clusters for genomic mapping and positional cloning and to mine for new alleles and new sources of disease resistance in available germplasm.Communicated by H.F. Linskens     

Characterization and high-resolution mapping of a late blight resistance locus similar to R2 in potato

Identification of resistance (R) genes to Phytophthora infestans is an essential step in molecular breeding of potato. We identified three specific R genes segregating in a diploid mapping population. One of the R genes is located on chromosome 4 and proved phenotypically indistinguishable from the Solanum demissum-derived R2, although S. demissum is not directly involved in the pedigree of the population. By bulked segregant analysis combined with a resistance assay, a genetic linkage map of the R2-like locus was constructed with 30 coupling and 23 repulsion phase AFLP markers. Two markers flanking the R2-like locus were applied to screen an extended population of 1,586 offspring. About 103 recombinants were selected, and an accurate high-resolution map was constructed. The R2-like resistance was localized in a 0.4 cM interval and was found co-segregating with four AFLP markers, which can be used to isolate the R2-like gene by map-based gene cloning. By analyzing race-specificity and R gene-specific molecular markers, we also found that an R1-like gene and an additional unknown R gene are segregating in the population.     

Marker-assisted combination of major genes for pathogen resistance in potato

Closely linked PCR-based markers facilitate the tracing and combining of resistance factors that have been introgressed previously into cultivated potato from different sources. Crosses were performed to combine the Ry _adg gene for extreme resistance to Potato virus Y (PVY) with the Gro1 gene for resistance to the root cyst nematode Globodera rostochiensis and the Rx1 gene for extreme resistance to Potato virus X (PVX), or with resistance to potato wart (Synchytrium endobioticum). Marker-assisted selection (MAS) using four PCR-based diagnostic assays was applied to 110 F1 hybrids resulting from four 2× by 4× cross-combinations. Thirty tetraploid plants having the appropriate marker combinations were selected and tested for presence of the corresponding resistance traits. All plants tested showed the expected resistant phenotype. Unexpectedly, the plants segregated for additional resistance to pathotypes 1, 2 and 6 of S. endobioticum, which was subsequently shown to be inherited from the PVY resistant parents of the crosses. The selected plants can be used as sources of multiple resistance traits in pedigree breeding and are available from a potato germplasm bank.     

Occurrence of potato virus X strain group 1 in seed stocks of potato cultivars lacking resistance genes

Potato virus X (PVX) isolates were obtained from a simple seed potato production scheme or from ware potatoes produced by seed potatoes obtained from it. In this scheme, PVX infection is widespread in seed stocks and most of the potatoes grown lack PVX resistance genes. Thirteen PVX isolates were typed to strain group by inoculation to potato cultivars containing different combinations of hypersensitivity genes Nx and Nb. Six failed to overcome either gene and therefore belonged to strain group 1, four overcame Nb only and were placed in strain group 3 and three were mixtures of the two. All 13 isolates failed to overcome extreme resistance/immunity gene Rx. Naturally infected cultivars of genotype nx.nb contained strain group 1 alone or strain groups 1 and 3, while those of genotype nx:Nb contained only strain group 3. The widespread occurrence of strain group 1 contrasts with the predominant occurrence of strain group 3 in potatoes in the UK. However, it resembles the UK situation before sophisticated seed potato production schemes were introduced and before PVX hypersensitivity genes Nx and Nb were deliberately exploited in potato breeding. Prior infection with potato leafroll virus (PLRV) did not affect expression of hypersensitivity to PVX in inoculated leaves of an nx:Nb genotype.     

Identification and mapping of molecular markers linked to rust resistance genes located on chromosome 1RS of rye using wheat-rye translocation lines

The short arm of rye (Secale cereale) chromosome 1 has been widely used in breeding programs to incorporate new disease resistance genes into wheat. Using wheat-rye translocation and recombinant lines, molecular markers were isolated and mapped within chromosomal regions of 1RS carrying rust resistance genes Lr26, Sr31, Yr9 from 'Petkus' and SrR from 'Imperial' rye. RFLP markers previously mapped to chromosome 1HS of barley - flanking the complex Mla powdery mildew resistance gene locus - and chromosome 1DS of Aegilops tauschii - flanking the Sr33 stem rust resistance gene - were shown to map on either side of rust resistance genes on 1RS. Three non cross-hybridising Resistance Gene Analog markers, one of them being derived from the Mla gene family, were mapped within same region of 1RS. PCR-based markers were developed which were tightly linked to the rust resistance genes in 'Imperial' and 'Petkus' rye and which have potential for use in marker-assisted breeding.     

Delimitation of the fertility restorer locus Rfk1 to a 43-kb contig in Kosena radish (Raphanus sativus L.)

We are pursuing a positional cloning strategy to isolate the fertility restoration gene Rfk1 from radish. Random polymorphic DNA-sequence-tagged site (RAPD-STS) markers tightly linked to the gene in radish were isolated, and a RAPD map surrounding the Rfk1 locus was constructed. We surveyed 948 F2 plants with adjacent RAPD-STS markers to isolate recombinants for bulk segregant analysis. This analysis was effective in isolating tightly linked amplification fragment length polymorphism (AFLP) markers surrounding the gene of interest. Ten tightly linked AFLP markers were obtained and used to construct a high-resolution map of the region. The closest AFLP-STS markers flanking Rfk1 were 0.1 cM and 0.2 cM away. Using the four adjacent AFLP markers, we screened lambda and cosmid libraries. The lambda and cosmid clones were aligned by examination of end sequences and restriction fragment length polymorphism (RFLP) patterns for each clone, and by hybridization to the DNA isolated from recombinants. Finally, we constructed a 198-kb contig encompassing the Rfk1 gene and comprising 20 lambda and two cosmid clones. By analysis of the breakpoints in recombinants with the rfk1/rfk1 or Rfk1/- genotype, the Rfk1 locus could be assigned to a 43-kb region comprising four lambda clones and one cosmid clone. This pinpoint localization in the radish genome has made it possible for us to identify the gene by sequence analysis and genetic transformation of cytoplasmic male-sterile Brassica napus plants.     

Molecular markers for rapidly identifying candidate genes in Chlamydomonas reinhardtii. Ery1 and ery2 encode chloroplast ribosomal proteins

To take advantage of available expressed sequence tags and genomic sequence, we have developed 64 PCR-based molecular markers in Chlamydomonas reinhardtii that map to the 17 linkage groups. These markers will allow the rapid association of a candidate gene sequence with previously identified mutations. As proof of principle, we have identified the genes encoded by the ERY1 and ERY2 loci. Mendelian mutations that confer resistance to erythromycin define three unlinked nuclear loci in C. reinhardtii. Candidate genes ribosomal protein L4 (RPL4) and L22 (RPL22) are tightly linked to the ERY1 locus and ERY2 locus, respectively. Genomic DNA for RPL4 from wild type and five mutant ery1 alleles was amplified and sequenced and three different point mutations were found. Two different glycine residues (G(102) and G(112)) are replaced by aspartic acid and both are in the unstructured region of RPL4 that lines the peptide exit tunnel of the chloroplast ribosome. The other two alleles change a splice site acceptor site. Genomic DNA for RPL22 from wild type and three mutant ery2 alleles was amplified and sequenced and revealed three different point mutations. Two alleles have premature stop codons and one allele changes a splice site acceptor site.