Rdr3, a novel locus conferring black spot disease resistance in tetraploid rose: genetic analysis, LRR profiling, and SCAR marker development

Black spot disease of rose, incited by the fungus Diplocarpon rosae, is found worldwide and is the most important disease of garden roses. A gene-for-gene interaction in this pathosystem is evidenced by the presence of pathogenic races of D. rosae and the previous discovery of a dominant resistance allele at the Rdr1 locus in the diploid Rosa multiflora. The objective of the present study was to genetically analyze resistances to North American black spot races 3, 8, and 9 previously reported in tetraploid roses. Resistance to North American races 3 and 8 segregated 1:1 in multiple F₁ populations, indicating that both are conferred by dominant alleles at single loci and are present in simplex (Rrrr) configuration. Gene pyramiding was demonstrated by combining both resistances into single genotypes. Resistance to race 9 was partial and segregated in a quantitative fashion. Analysis of these populations with microsatellite markers previously developed for Rdr1 revealed that the gene conferring race 3 resistance resides within the same R gene cluster as Rdr1. Race 8 resistance segregated independently and is, therefore, a novel locus for black spot resistance in rose which we have named Rdr3. NBS and LRR profiling were used in a bulked segregant analysis to identify a marker 9.1 cM from Rdr3, which was converted to a SCAR marker form for marker-assisted breeding.     

Construction of a BAC library and a physical map of a major QTL for CBB resistance of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

A major quantitative trait loci (QTL) conditioning common bacterial blight (CBB) resistance in common bean (Phaseolus vulgaris L.) lines HR45 and HR67 was derived from XAN159, a resistant line obtained from an interspecific cross between common bean lines and the tepary bean (P. acutifolius L.) line PI319443. This source of CBB resistance is widely used in bean breeding. Several other CBB resistance QTL have been identified but none of them have been physically mapped. Four molecular markers tightly linked to this QTL have been identified suitable for marker assisted selection and physical mapping of the resistance gene. A bacterial artificial chromosome (BAC) library was constructed from high molecular weight DNA of HR45 and is composed of 33,024 clones. The size of individual BAC clone inserts ranges from 30 kb to 280 kb with an average size of 107 kb. The library is estimated to represent approximately sixfold genome coverage. The BAC library was screened as BAC pools using four PCR-based molecular markers. Two to seven BAC clones were identified by each marker. Two clones were found to have both markers PV-tttc001 and STS183. One preliminary contig was assembled based on DNA finger printing of those positive BAC clones. The minimum tiling path of the contig contains 6 BAC clones spanning an estimated size of 750 kb covering the QTL region.     

Construction of a BAC library of <Emphasis Type="Italic"> Rosa rugosa </Emphasis>Thunb. and assembly of a contig spanning<Emphasis Type="Italic"> Rdr1</Emphasis>, a gene that confers resistance to blackspot

A BAC library to serve as a general tool for the physical mapping and positional cloning of rose genes has been constructed from Rosa rugosa DNA. With 27,264 clones the library contains 5.2 genome equivalents. The library was used to assemble a contig of BAC clones spanning Rdr1, a locus that confers resistance to blackspot. For this purpose fine-scale mapping of the target locus was achieved by bulked segregant analysis using 816 AFLP primer combinations. The target region around Rdr1 comprises about 400 kb and is covered by a minimum of six BAC clones. Furthermore, the detection of at least five resistance gene analogs of the TIR-NBS-LRR family on the contig indicates the presence of a cluster of resistance genes around Rdr1. These results will not only allow the isolation and identification of Rdr1 in the near future, but also provide the tools for the physical mapping and positional cloning of other horticulturally interesting genes in roses.     

Identification of an 85-kb DNA fragment containing pms1, a locus for photoperiod-sensitive genic male sterility in rice

Photoperiod-sensitive genic male-sterile rice has a number of desirable characteristics for hybrid rice production. Previous studies identified pms1, located on chromosome 7, as a major locus for photoperiod-sensitive genic male sterility. The objective of this study was to localize the pms1 locus to a specific DNA fragment by genetic and physical mapping. Using 240 highly sterile individuals and a random sample of 599 individuals from an F2 population of over 5000 individuals from a cross between Minghui 63 and 32001S, we localized the pms1 locus by molecular marker analysis to a genetic interval of about 4 cM, 0.25 cM from RG477 on one side and 3.8 cM from R1807 on the other side. A contig map composed of seven BAC clones spanning approximate 500 kb in length was constructed for the pms1 region by screening a BAC library of Minghui 63 DNA using RFLP markers and chromosomal walking. Analysis of recombination events in the pms1 region among the highly sterile individuals reduced the length of the contig map to three BAC clones. Sequencing of one BAC clone, 2109, identified two SSR markers located 85 kb apart in the clone that flanked the pms1 locus on both sides, as indicated by the distribution of recombination events. We thus concluded that the pms1 locus was located on the fragment bounded by the two SSR markers.     

Genetic and physical mapping of a high recombination region on chromosome 7H(1) in barley

Approaches utilizing microlinearity between related species allow for the identification of syntenous regions and orthologous genes. Within the barley Chromosome 7H(1) is a region of high recombination flanked by molecular markers cMWG703 and MWG836. We present the constructed physical contigs linked to molecular markers across this region using bacterial artificial chromosomes (BAC) from the cultivar Morex. Barley expressed sequence tags (EST), identified by homology to rice chromosome 6 between the rice molecular markers C425A and S1434, corresponded to the barley syntenous region of Chromosome 7H(1) Bins 2–5 between molecular markers cMWG703-MWG836. Two hundred and thirteen ESTs were genetically mapped yielding 267 loci of which 101 were within the target high recombination region while 166 loci mapped elsewhere. The 101 loci were joined by 43 other genetic markers resulting in a highly saturated genetic map. In order to develop a physical map of the region, ESTs and all other molecular markers were used to identify Morex BAC clones. Seventy-four BAC contigs were formed containing 2–102 clones each with an average of 19 and a median of 13 BAC clones per contig. Comparison of the BAC contigs, generated here, with the Barley Physical Mapping Database contigs, resulted in additional overlaps and a reduction of the contig number to 56. Within cMWG703-MWG836 are 24 agriculturally important traits including the seedling spot blotch resistance locus, Rcs5. Genetic and physical analysis of this region and comparison to rice indicated an inversion distal of the Rcs5 locus. Three BAC clone contigs spanning the Rcs5 locus were identified. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of SNP markers linked to the <Emphasis Type="Italic">L</Emphasis> locus in <Emphasis Type="Italic">Capsicum</Emphasis> spp. by a comparative genetic analysis

In pepper, the TMV resistance locus L is syntenic to the tomato I2 and the potato R3 loci on chromosome 11. In this report, we identified pepper bacterial artificial chromosome (BAC) clones corresponding to the I2 and R3 loci and developed L-linked markers using the BAC sequence information. A BAC library was screened using the tomato I2C-1 gene as a probe. The resulting clones were sorted further by PCR screening, sequencing, and genetic mapping. A linkage analysis revealed that BAC clone 082F03 could be anchored to the target region near TG36 on chromosome 11. Using the 082F03 sequence, more BAC clones were identified and a BAC contig spanning 224 kb was constructed. Gene prediction analysis showed that there were at least three I2/R3 R gene analogs (RGAs) in the BAC contig. Three DNA markers closely linked (about 1.2 cM) to the L ⁴ gene were developed by using the BAC contig sequence. The single nucleotide polymorphism marker 087H3T7 developed in this study was subjected to linkage analysis in L ⁴ - and L ³ -segregating populations together with previously developed markers. The 189D23M marker, which is known to co-segregate with L ³ , was located on the opposite side of 087H3T7, about 0.7 cM away from L ⁴ . This supports the idea that L ³ and L ⁴ may be different genes closely linked within the region instead of different alleles at the same locus. Finally, use of flanking markers in molecular breeding program for introgression of L ⁴ to elite germplasm against most aggressive tobamoviruses pathotype P_1,2,3 is discussed.     

The TNL gene Rdr1 confers broad‐spectrum resistance to Diplocarpon rosae

Black spot disease, which is caused by the ascomycete Diplocarpon rosae, is the most severe disease in field‐grown roses in temperate regions and has been distributed worldwide, probably together with commercial cultivars. Here, we present data indicating that muRdr1A is the active Rdr1 gene, a single‐dominant TIR‐NBS‐LRR (Toll/interleukin‐1 receptor‐nucleotide binding site‐leucine rich repeat) (TNL)‐type resistance gene against black spot disease, which acts against a broad range of pathogenic isolates independent of the genetic background of the host genotype. Molecular analyses revealed that, compared with the original donor genotype, the multiple integrations that are found in the primary transgenic clone segregate into different integration patterns in its sexual progeny and do not show any sign of overexpression. Rdr1 provides resistance to 13 different single‐spore isolates belonging to six different races and broad field mixtures of conidia; thus far, Rdr1 is only overcome by two races. The expression of muRdr1A, the active Rdr1 gene, leads to interaction patterns that are identical in the transgenic clones and the non‐transgenic original donor genotype. This finding indicates that the interacting avirulence (Avr) factor on the pathogen side must be widespread among the pathogen populations and may have a central function in the rose–black spot interaction. Therefore, the Rdr1 gene, pyramided with only a few other R genes by sexual crosses, might be useful for breeding roses that are resistant to black spot because the spread of new pathogenic races of the fungus appears to be slow.     

Genetic and physical mapping of xa13, a recessive bacterial blight resistance gene in rice

 The recessive gene, xa13, confers resistance to Philippine race 6 (PXO99) of the bacterial blight pathogen Xanthomonas oryzae pv oryzae. Fine genetic mapping and physical mapping were conducted as initial steps in an effort to isolate the gene. Using nine selected DNA markers and two F₂ populations of 132 and 230 plants, xa13 was fine-mapped to a genomic region <4 cM on the long arm of rice chromosome 8, flanked by two RFLP markers, RG136 and R2027. Four DNA markers, RG136, R2027, S14003, and G1149, in the target region were used to identify bacterial artificial chromosome (BAC) clones potentially harboring the xa13 locus from a rice BAC library. A total of 11 BACs were identified, forming four separate contigs including a single-clone contig, 29I3, associated with the RG136 STS marker, the S14003 contig consisting of four clones (44F8, 41O2, 12A16, and 12F20), the G1149 contig with two clones, 23D11 and 21H18, and the R2027 contig consisting of four overlapping clones, 42C23, 30B5, 6B7 and 21H14. Genetic mapping indicated that the xa13 locus was contained in the R2027 contig. Chromosomal walking on the R2027 contig resulted in two more clones, 33C7 and 14L3. DNA fingerprinting showed that the six clones of the R2027 contig were overlapping. Clone 44F8 hybridized with a single fragment from the clone 14L3, integrating the R2027 and S14003 contigs into a single contig consisting of ten BAC clones with a total size of approximately 330 kb. The physical presence of the xa13 locus in the contig was determined by mapping the ends of the BAC inserts generated by TAIL-PCR. In an F₂ population of 230 plants, the BAC-end markers 42C23R and 6B7F flanked the xa13 locus. The probes 21H14F and 21H14R derived from BAC clone 21H14 were found to flank xa13 at a distance of 0.5 cM on either side, using a second F₂ population of 132 plants. Thus, genetic mapping indicated that the contig and the 96-kb clone, 21H14, contained the xa13 locus. Received: 15 August 1998 / Accepted: 29 September 1998     

Physical mapping of the <Emphasis Type="Italic">Rf</Emphasis><Subscript><Emphasis Type="Italic">1</Emphasis></Subscript> fertility-restoring gene to a 100 kb region in cotton

Cytoplasmic male sterility (CMS) plays an important role in crop heterosis exploitation. Determining one or more nuclear genes that can restore male fertility to CMS is essential for developing hybrid cultivars. Genetic and physical mapping is the standard technique required for isolating these restoration genes. By screening 2,250 simple sequence repeat (SSR) primer pairs in cotton (Gossypium hirsutum L.), we identified five new SSR markers that are closely linked to the Rf ₁ gene, a fertility restorer gene of cotton for CMS-D2. Based on our previous fine mapping of the Rf ₁ gene and assemblage of three published STS markers, we constructed a high-resolution genetic map of Rf ₁ containing 13 markers in a genetic distance of 0.9 cM. The 13 molecular markers were used to screen a bacterial artificial chromosome (BAC) library from a restorer line 0-613-2R containing Rf ₁ gene, which yielded 50 single positive clones. There was an average of 3.8 clones ranging from 1 to 12 BAC clones per PCR marker. These 50 clones produced an average insert size of 120 kb (ranging between 80 and 225 kb). Thirty-five primer pairs were designed based on 38 sequences of BAC ends, and two new STS markers tightly linked to Rf ₁ gene have been tagged and integrated into this map. The physical map for the Rf ₁ gene was constructed by fingerprinting the positive clones digested with the HindIII enzyme. We were able to delimit the possible location of the Rf ₁ gene to a minimum of two BAC clones spanning an interval of approximately 100 kb between two clones designated 081-05K and 052-01N. Further work using these two BAC clones will lead to isolation of the Rf ₁ gene in cotton.     

Construction of a 550 kb BAC contig spanning the genomic region containing the apple scab resistance gene Vf

A positional cloning project was started in apple with the aim of isolating the Vf resistance gene of Malus floribunda 821. Vf confers resistance against apple scab, the most important disease in apple orchards. A chromosome walk starting from two molecular markers (M18-CAPS and AM19-SCAR) flanking Vf was performed, using a bacterial artificial chromosome (BAC) library containing inserts of the cultivar Florina, which is heterozygous for Vf. Thirteen BAC clones spanning the region between the two markers were identified in nine chromosome walking steps. The size of the resulting contig is approximately 550 kb. In order to map the Vf region in more detail, we analyzed over 2000 plants from different populations segregating for Vf with markers produced from BAC end sequences. In this way, we were able to restrict the possible location of the Vf gene to a minimum of five clones spanning an interval of approximately 350 kb. Received: 4 July 1999 / Accepted: 16 September 1999     

Genomic analysis and marker development for the Tsn1 locus in wheat using bin-mapped ESTs and flanking BAC contigs

The wheat Tsn1 gene confers sensitivity to the host-selective toxin Ptr ToxA produced by the tan spot fungus (Pyrenophora tritici-repentis). The long-term goal of this research is to isolate Tsn1 using a positional cloning approach. Here, we evaluated 54 ESTs (expressed sequence tags) physically mapped to deletion bin 5BL 0.75–0.76, which is a gene-rich region containing Tsn1. Twenty-three EST loci were mapped as either PCR-based single-stranded conformational polymorphism or RFLP markers in a low-resolution wheat population. The genetic map corresponding to the 5BL 0.75–0.76 deletion bin spans 18.5 cM and contains 37 markers for a density of 2 markers/cM. The EST-based genetic map will be useful for tagging other genes, establishing colinearity with rice, and anchoring sequence ready BAC contigs of the 5BL 0.75–0.76 deletion bin. High-resolution mapping showed that EST-derived markers together with previously developed AFLP-derived markers delineated Tsn1 to a 0.8 cM interval. Flanking markers were used to screen the Langdon durum BAC library and contigs of 205 and 228 kb flanking Tsn1 were assembled, sequenced, and anchored to the genetic map. Recombination frequency averaged 760 kb/cM across the 228 kb contig, but no recombination was observed across the 205 kb contig resulting in an expected recombination frequency of more than 10 Mb/cM. Therefore, chromosome walking within the Tsn1 region may be difficult. However, the sequenced BACs allowed the identification of one microsatellite in each contig for which markers were developed and shown to be highly suitable for marker-assisted selection of Tsn1.     

A physical map covering the nsv locus that confers resistance to Melon necrotic spot virus in melon (Cucumis melo L.)

Melon necrotic spot virus (MNSV) is a member of the genus Carmovirus, which produces severe yield losses in melon and cucumber crops. The nsv gene is the only known natural source of resistance against MNSV in melon, and confers protection against all widespread strains of this virus. nsv has been previously mapped in melon linkage group 11, in a region spanning 5.9 cM, saturated with RAPD and AFLP markers. To identify the nsv gene by positional cloning, we started construction of a high-resolution map for this locus. On the basis of the two mapping populations, F₂ and BC1, which share the same resistant parent PI 161375 (nsv/nsv), and using more than 3,000 offspring, a high-resolution genetic map has been constructed in the region around the nsv locus, spanning 3.2 cM between CAPS markers M29 and M132. The availability of two melon BAC libraries allowed for screening and the identification of new markers closer to the resistance gene, by means of BAC-end sequencing and mapping. We constructed a BAC contig in this region and identified the marker 52K20sp6, which co-segregates with nsv in 408 F₂ and 2.727 BC1 individuals in both mapping populations. We also identified a single 100 kb BAC that physically contains the resistance gene and covers a genetic distance of 0.73 cM between both BAC ends. These are the basis for the isolation of the nsv recessive-resistance gene.     

Construction of a BAC contig containing the xa5 locus in rice

 The recessive gene xa5 confers resistance to bacterial blight in rice. To generate a physical map of the xa5 locus, three RFLP markers RG556, RG207 and RZ390, closely linked to xa5, were used to screen a rice bacterial artificial chromosome (BAC) library. The identified overlapping BAC clones formed two small contigs which were extended to both sides by chromosome walking. The final physical map consisted of 14 BAC clones and covered 550 kb. Genetic analysis with an F₂ population showed that two RFLP markers 28N22R and 40F20R, derived from the BAC clones in the contig, flanked the xa5 locus. To further delimit the location of the xa5 locus, RFLP markers RG556 and RG207 were converted to sequence tagged sites and used to perform genetic analysis. The results indicated that the xa5 locus was most likely located between RG207 and RG556. Among the BAC clones in the contig, one clone, 44B4, hybridized to both RG207 and RG556. This suggests that BAC clone 44B4 carried the xa5 locus. Received: 12 January 1998 / Accepted: 27 May 1998     

<Emphasis Type="Italic">Rpp1</Emphasis>, a dominant gene providing race-specific resistance to rose powdery mildew (<Emphasis Type="Italic">Podosphaera pannosa</Emphasis>): molecular mapping,SCAR development and confirmation of disease resistance data

We have previously demonstrated that in the diploid rose population 97/9 resistance to the powdery mildew race 9 is controlled by a major dominant resistance gene, Rpp1. In the study reported here, we isolated several molecular markers closely linked to Rpp1 via bulked segregant analysis, with the gene being tagged in an interval of 5 cM between the two most adjacent markers. It was possible to convert the most closely linked amplified fragment length polymorphic (AFLP) marker into a sequence-characterised amplified region (SCAR) segregating in the same manner. Indirect mapping of Rpp1 in relation to the black spot resistance gene Rdr1 revealed no linkage between the two R genes. Furthermore, the genetic model based on a single dominant resistance gene was supported by the marker data.     

Construction of a 1.5-Mb Bacterial Artificial Chromosome Contig in Xp11.23, a Region of High Gene Content

To generate sequence-ready templates for the gene-rich Xp11.23 region, we have constructed a 1.5-Mb bacterial artificial chromosome (BAC) contig spanning the interval between the DNA markers OATL1 and DXS255. The contig includes 28 BACs, ranging in size from 58 to 285 kb with an average size of 135 kb, which provide 2.5-fold coverage of the region. The BAC contig was constructed based entirely on the content of 40 DNA markers from a previously established YAC contig and 11 new markers developed from BAC-end DNA sequences, 4 of which were required to close gaps in the map. There was no evidence of rearrangement, instability, or chimerism in any of the BAC clones. The BAC cloning system appears to provide robust and total physical coverage of this gene-rich region with clones that are suitable for DNA sequencing.     

AFLP-derived SCARs facilitate construction of a 1.1 Mb sequence-ready map of a region that spans the Vf locus in the apple genome

The availability of high-density anchored markers is a prerequisite for reliable construction of a deep coverage BAC contig, which leads to creation of a sequence-ready map in the target chromosomal region. Unfortunately, such markers are not available for most plant species, including woody perennial plants. Here, we report on an efficient approach to build a megabase-size sequence-ready map in the apple genome for the Vf region containing apple scab resistance gene(s) by targeting AFLP-derived SCAR markers to this specific genomic region. A total of 11 AFLP-derived SCAR markers, previously tagged to the Vf locus, along with three other Vf-linked SCAR markers have been used to screen two apple genome BAC libraries. A single BAC contig which spans the Vf region at a physical distance of approximately 1,100 kb has been constructed by assembling the recovered BAC clones, followed by closure of inter-contig gaps. The contig is 4 ×deep, and provides a minimal tiling path of 16 contiguous and overlapping BAC clones, thus generating a sequence-ready map. Within the Vf region, duplication events have occurred frequently, and the Vf locus is restricted to the ca. 290 kb region covered by a minimum of three overlapping BAC clones.     

Fine genetic mapping and physical delimitation of the lesion mimic gene Spl11 to a 160-kb DNA segment of the rice genome

The rice lesion mimic mutant spl11 was previously found to confer broad-spectrum disease resistance to both Magnaporthe grisea and Xanthomonas oryzae pv. oryzae. To better understand the molecular basis underlying cell death and disease resistance in rice, a map-based cloning strategy has been employed to isolate Spl11. Five Spl11-linked RAPD markers were developed and four of them were mapped to rice chromosome 12. A high-resolution genetic map was developed using a segregating population consisting of 1138 lesion mimic individuals. Recombination suppression was observed in the vicinity of Spl11. Three molecular markers tightly linked to Spl11 were identified and used to screen a BAC library. A contig spanning the Spl11 locus was constructed and physical mapping delimited Spl11 to a 160-kb DNA segment within a single BAC clone. These results provide the essential information for the final isolation of this important gene in the rice defense pathway.     

Resolving the aphid resistance locus Sd-1 on a BAC contig within a sub-telomeric region of Malus linkage group 7.

Aphids cause serious physical and economic damage to most major crops throughout the world, and there is a pressing requirement to isolate genes conferring aphid resistance. The Sd-1 locus in Malus spp. (apple) confers resistance against the rosy leaf-curling aphid (Dysaphis devecta Wlk.), and was recently positioned within a 1.3-cM region on linkage group 7, flanked by molecular markers. These markers were used as a basis for development of a BAC contig spanning the locus, together with adapter-mediated amplification of flanking sequences to obtain BAC insert-end sequences, and fingerprinting of BAC clones. Approximately 800 kb of the Sd-1 genomic region was covered by 19 overlapping BACs, with an average insert size of 75-150 kb. The physical-genetic distance ratio was estimated at 460 kb/cM, although the distribution of recombination events was irregular with respect to estimated physical distance. Recombinant analysis and development of new markers allowed Sd-1 to be positioned within an interval of approximately 180 kb located on either of two overlapping BACs. From one of these, an insert end sequence showed a significant degree of similarity to nucleotide binding site-leucine rich repeat (NBS-LRR) resistance genes. Fluorescent in situ hybridization (FISH) of BAC clones within the contig enabled positioning and orientation of the locus within a euchromatic region, very close to the telomere of linkage group 7.     

High-resolution genetic and physical mapping of the cauliflower high-β-carotene gene<Emphasis Type="Italic"> Or</Emphasis> (<Emphasis Type="Italic">Orange</Emphasis>)

Mutation in the cauliflower gene Or causes high levels of -carotene to accumulate in various tissues of the plant that are normally devoid of carotenoids. To decipher the molecular basis by which Or regulates carotenoid accumulation, we have undertaken the isolation of Or by a map-based cloning strategy. Two previously isolated, locus-specific, sequence-characterized amplified region (SCAR) markers that flank Or were employed for the analysis of a large segregating population consisting of 1632 F₂ individuals, and a high-resolution genetic linkage map of the Or locus region was developed. To facilitate positional cloning, we constructed a cauliflower genomic library in a bacterial artificial chromosome (BAC) vector, using high molecular weight DNA from Or homozygotes. The BAC library comprises 60,288 clones with an average insert size of 110 kb, and represents an estimated 10-fold coverage of the genome. A BAC contig encompassing the Or locus was established by screening the library with a marker that is closely linked to Or and by identifying overlapping BAC clones by chromosome walking. Physical mapping delimited the Or locus to a 50-kb DNA fragment within a single BAC clone, which corresponds to a genetic interval of 0.3 cM.Communicated by R. Hagemann     

Exploitation of a marker dense linkage map of potato for positional cloning of a wart disease resistance gene

A marker-saturated linkage map of potato was used to genetically map a locus involved in the resistance against wart disease Synchytrium endobioticum race 1. The locus mapped on the long arm of chromosome 4 and is named Sen1-4 in contrast to a Sen1 locus on chromosome 11. The AFLP markers from the Sen1-4 interval enabled the isolation of BAC clones from an 11 genome equivalent BAC library. This was achieved via fingerprinting of BAC pools with the AFLP primer pairs that resemble the genetic marker loci. With non-selective AFLP primers, fingerprints of individual BAC clones were generated to analyse the overlap between BAC clones using FPC. This resulted in a complete contig and a minimal tiling path of 14 BAC clones enclosing the Sen1-4 locus. The BAC contig has a genetic length of ~6 cM and a physical length of ~1 Mb. Our results demonstrate that map-based cloning of Sen1-4 can be pursued on the basis of a strategy of marker saturation alone. Genetic resolution achieved by screening large numbers of offspring for recombination events may not be required. Together with the construction of the BAC contig, a physical map with the position of the markers is accomplished in one step. This provides proof of concept for the utility of the marker saturation that is offered by the ultra dense AFLP map of potato for gene cloning.