Two additive QTLs conferring broad-spectrum resistance in potato to Globodera pallida are localized on resistance gene clusters

Broad-spectrum resistance in potato to the potato cyst nematode (PCN) is commonly regarded as a complex inherited trait. Yet, in this paper we show that, by use of a selected set of PCN test populations, broad-spectrum resistance to the species Globodera pallida can be fully ascribed to the action of two loci: Gpa5 and Gpa6. These loci were readily mapped by means of a strategy based on two steps. Firstly, the chromosomal localization of both loci was assessed by use of an online catalogue of AFLP markers covering a substantial part of the potato genome (http://www.spg.wau.nl/pv/aflp/catalog.htm). Subsequently the chromosomal regions of both loci were identified by means of CAPS markers based on RFLP insert sequences. Locus Gpa5 explains at least 61% of the genetic variation. This locus maps to chromosome 5 on a region which has previously been shown to harbor resistance factors to viral (Nb, Rx2), fungal (R1) and nematodal (Gpa, Grp1) pathogens. The Gpa6 locus exhibits a minor effect on the resistance (24%) and acts additively to Gpa5. Interestingly, the Gpa6 locus maps to a region on chromosome 9 where, in the homoeologous tomato genome, the virus resistance gene Sw-5 resides as part of a resistance gene cluster. In potato, resistance to potato virus X has been reported in the vicinity of this region. The map location of Gpa6 indicates the presence of a resistance gene cluster at the end of the long arm of chromosome 9 of potato. Received: 10 January 2000 / Accepted: 31 January 2000     

A QTL for broad-spectrum resistance to cyst nematode species (Globodera spp.) maps to a resistance gene cluster in potato

 Broad-spectrum resistance in potato to the potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida is commonly regarded as a polygenically inherited trait. Yet, by use of QTL analysis and a selected set of PCN populations, resistance to both PCN species could be ascribed to the action of locus Grp1. Grp1 confers major resistance to G. rostochiensis line Ro₅-22 and G. pallida population Pa₂-D383 and partial resistance to G. pallida population Pa₃-Rookmaker. Grp1 was mapped on chromosome 5 using previously characterized AFLP markers. Cleaved amplified polymorphic sequence (CAPS) markers available for RFLP loci GP21 and GP179 revealed that Grp1 maps on a genomic region harboring other resistance factors to viral, fungal and nematodal pathogens. The present data indicate that Grp1 is a compound locus which contains multiple genes involved in PCN resistance. Received: 10 September 1997 / Accepted: 6 October 1997     

Marker enrichment and high-resolution map of the segment of potato chromosome VII harbouring the nematode resistance gene Gro1

The dominant allele Gro1 confers on potato resistance to the root cyst nematode Globodera rostochiensis. The Gro1 locus has been mapped to chromosome VII on the genetic map of potato, using RFLP markers. This makes possible the cloning of Gro1 based on its map position. As part of this strategy we have constructed a high-resolution genetic map of the chromosome segment surrounding Gro1, based on RFLP, RAPD and AFLP markers. RAPD and RFLP markers closely linked to Gro1 were selected by bulked segregant analysis and mapped relative to the Gro1 locus in a segregating population of 1105 plants. Three RFLP and one RAPD marker were found to be inseparable from the Gro1 locus. Two AFLP markers were identified that flanked Gro1 at genetic distances of 0.6 cM and 0.8 cM, respectively. A genetic distance of 1 cM in the Gro1 region corresponds to a physical distance of ca. 100 kb as estimated by long-range restriction analysis. Marker-assisted selection for nematode resistance was accomplished in the course of constructing the high-resolution map. Plants carrying the resistance allele Gro1 could be distinguished from susceptible plants by marker assays based on the polymerase chain reaction (PCR).     

Mapping of resistance to the potato cyst nematode Globodera rostochiensis from the wild potato species Solanum vernei

A population of diploid potato (Solanum tuberosum) was used for the genetic analysis and mapping of a locus for resistance to the potato cyst nematode Globodera rostochiensis, introgressed from the wild potato species Solanum vernei. Resistance tests of 108 genotypes of a F₁ population revealed the presence of a single locus with a dominant allele for resistance to G. rostochiensis pathotype Ro1. This locus, designated GroV1, was located on chromosome 5 with RFLP markers. Fine-mapping was performed with RAPD and SCAR markers. The GroV1 locus was found in the same region of the potato genome as the S. tuberosum ssp. andigena H1 nematode resistance locus. Both resistance loci could not excluded to be allelic. The identification of markers flanking the GroV1 locus offers a valuable strategy for marker-assisted selection for introgression of this nematode resistance.Abbreviations BSA bulked segregant analysis - RAPD random-amplified polymorphic DNA - RFLP restriction fragment length polymorphism - SCAR sequence-characterized amplified region     

Marker enrichment and high-resolution map of the segment of potato chromosome VII harbouring the nematode resistance gene Gro1

The dominant allele Gro1 confers on potato resistance to the root cyst nematode Globodera rostochiensis. The Gro1 locus has been mapped to chromosome VII on the genetic map of potato, using RFLP markers. This makes possible the cloning of Gro1 based on its map position. As part of this strategy we have constructed a high-resolution genetic map of the chromosome segment surrounding Gro1, based on RFLP, RAPD and AFLP markers. RAPD and RFLP markers closely linked to Gro1 were selected by bulked segregant analysis and mapped relative to the Gro1 locus in a segregating population of 1105 plants. Three RFLP and one RAPD marker were found to be inseparable from the Gro1 locus. Two AFLP markers were identified that flanked Gro1 at genetic distances of 0.6 cM and 0.8 cM, respectively. A genetic distance of 1 cM in the Gro1 region corresponds to a physical distance of ca. 100 kb as estimated by long-range restriction analysis. Marker-assisted selection for nematode resistance was accomplished in the course of constructing the high-resolution map. Plants carrying the resistance allele Gro1 could be distinguished from susceptible plants by marker assays based on the polymerase chain reaction (PCR).     

Localization by restriction fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to the potato cyst nematode Globodera rostochiensis

Summary A major dominant locus conferring resistance against several pathotypes of the root cyst nematode Globodera rostochiensis was mapped on the linkage map of potato using restriction fragment length polymorphism (RFLP) markers. The assessment of resistance versus susceptibility of the plants in the experimental population considered was based on an in vivo (pot) and an in vitro (petri dish) test. By linkage to nine RFLP markers the resistance locus Gro1 was assigned to the potato linkage group IX which is homologous to the tomato linkage group 7. Deviations from the additivity of recombination frequencies between Gro1 and its neighbouring markers in the pot test led to the detection of a few phenotypic misclassifications of small plants with poor root systems that limited the observation of cysts on susceptible roots. Pooled data from both tests provided better estimates of recombination frequencies in the linkage interval defined by the markers flanking the resistance locus.     

A molecular genetic map of the long arm of chromosome 6R of rye incorporating the cereal cyst nematode resistance gene, CreR

 A genetic map of the long arm of chromosome 6R of rye was constructed using eight homoeologous group-6 RFLP clones and five PCR markers derived from the rye-specific dispersed repetitive DNA family, R173. The map was developed using a novel test-cross F₁ (TC-F₁) population segregating for resistance to the cereal cyst nematode. Comparisons were made between the map generated with other rye and wheat group-6 chromosome maps by the inclusion of RFLP clones previously mapped in those species. Co-linearity was observed for common loci. This comparison confirmed a dramatic reduction in recombination for chromosome 6R in the TC-F₁ population. The CreR locus was included in the linkage map via progeny testing of informative TC-F₁ individuals. CreR mapped 3.7 cM distal from the RFLP locus, XksuF37. Comparative mapping should allow the identification of additional RFLP markers more closely linked to the CreR locus. Received: 14 April 1998 / Accepted: 29 April 1998     

Autotetraploids and genetic mapping using common AFLP markers: the R2 allele conferring resistance to Phytophthora infestans mapped on potato chromosome 4

 Due to the complexity of tetrasomic inheritance, mapping studies in potato (Solanum tuberosum L.) are generally conducted at the diploid level. In the present study we tested the feasibility of Bulked Segregant Analysis (BSA) using a tetraploid offspring for the identification of AFLP markers linked to the R2 allele, which confers race-specific resistance to Phytophthora infestans. Eleven bulk-specific AFLP markers, detected in fingerprints of 205 AFLP primer combinations, could be mapped in a linkage group encompassing the R2 locus. The efficiency of BSA at the tetraploid level, determined by the frequency of single-dose restriction fragments (SDRF), was much higher than expected on the basis of overall genetic dissimilarity between the parental clones. The fortuitous detection of AFLPs with linkage to the R2 allele is explained on the basis of specific genetic dissimilarity between cultivated potato and the chromosomal segment introgressed from S. demissum carrying the resistant R2 allele. AFLP markers common to those with linkage to R2 were visually recognized by their electrophoretic mobility in the AFLP fingerprint in a parental clone of a reference mapping population. Using these common AFLP markers we anchored the linkage group comprising the R2 allele to potato chromosome 4. Received: 30 October 1997 / Accepted: 6 November 1997     

The R1 gene conferring race-specific resistance to Phytophthora infestans in potato is located on potato chromosome V.

Summary Late blight in potato is caused by the fungusPhytophthora infestans and can inflict severe damage on the potato crop. Resistance toP. infestans is either based on major dominantR genes conferring vertical, race-specific resistance or on minor genes inducing horizontal, unspecific resistance. A dihaploid potato line was identified which carried theR1 gene, conferring vertical resistance to allP. infestans races, with the exception of those homozygous for the recessive virulence allele of the locusV1. The F₁ progeny of a cross between this resistant parent P(R1) and P(r), a line susceptible to all races, was analysed for segregation ofR1 and of restriction fragment length polymorphism (RFLP) markers distributed on the potato RFLP map comprising more than 300 loci. TheR1 locus was mapped to chromosome V in the interval between RFLP markers GP21 and GP179. The map position ofR1 was found to be very similar to the one ofRx2, a dominant locus inducing extreme resistance to potato virus X.     

Identification of AFLP markers in soybean linked to resistance to Meloidogyne javanica and conversion to Sequence Characterized Amplified Regions (SCARs)

Meloidogynejavanica is the most widely spread nematode pest on soybean in SouthAfrica. Only a few registered commercial South African cultivars are poor hostsof this nematode species and there is an urgent need for an efficient breedingprogramme for resistant cultivars of all maturity groups. However, breeding ishampered by laborious screening procedures for selection of poor host cultivarsand/or lines. The objective of this study was to develop an economically viablemolecular marker system for application in selection procedures. BothRestriction Fragment Length Polymorphism (RFLP) and Amplified Fragment LengthPolymorphism (AFLP) screening techniques identified markers linked togall-indexvariation in a segregating population of 60 F₂ progeny from a crossbetween a resistant cultivar (Gazelle) and a highly susceptible variety(Prima).A codominant RFLP marker( B212) was linked significantly to M.javanica resistance and explained 62% of the variation ingall-index.Seven AFLP markers were linked significantly to the resistance trait, of whichfour were linked in repulsion phase and three in coupling phase. All seven AFLPmarkers mapped to LG-F (Linkage Group F) on the public soybean molecular map.The major quantitative trait locus (QTL) for resistance mapped between markersE-ACC/M-CTC2(SOJA6) (linked in coupling phase), B212 and E-AAC/M-CAT1(SOJA7)(linked in repulsion phase). These two AFLP markers bracketing the majorresistance QTL were successfully converted to SCARs (Sequence CharacterizedAmplified Regions). Marker E-ACC/M-CTC2 was converted to a codominant SCARmarker SOJA6, which accounted for 41% of variation in gall-index in the mappingpopulation. Marker E-AAC/M-CAT1 was converted to a dominant SCAR marker (SOJA7)and explained 42% of gall-index variation in the mapping population. These twomarkers mapped approximately 3.8 cM and 2.4 cMrespectively from the resistance QTL. This study represents the first report ofthe development of PCR-based sequence specific markers linked to M.javanica resistance in soybean.     

RFLP analysis of resistance to Columbia root-knot nematode derived from Solanum bulbocastanum in a BC2 population

The mapping of resistance toMeloidogyne chitwoodi derived from Solarium bulbocastanum is reported. A population suitable for mapping was developed as follows. A somatic hybrid of nematode-resistant S. bulbocastanum and cultivated tetraploid potato was produced. This was backcrossed to tetraploid potato, and a single resistant BC₁ was selected and backcrossed again to the same recurrent tetraploid parent. The mapping population consisted of 64 BC₂ progeny scored for restriction fragment length polymorphic (RFLP) markers and 62 of these were evaluated for the reproductive efficiency of race 1 of M. chitwoodi. Forty-eight polymorphic RFLP markers, originally derived from tomato and mapped in diploid cultivated potato, were assigned to 12 chromosomes of S. bulbocastanum. Of the 62 progeny screened for nematode resistance, 18 were non-hosts and four were poor hosts. The rest were highly susceptible (good hosts). Analysis of the resistance (including non-hosts and poor hosts) as both a qualitative trait and as a meristic trait on which QTL analysis was applied supported the same genetic hypothesis. Genetic control was localized solely to factor(s) lying at one end of chromosome 11. The level of expression of resistance in the S. bulbocastanum parent and the resistant portion of the BC₂ was essentially the same. This fact, together with the highly significant LOD scores for one end of the chromosome-11 marker array, supports a genetic model equivalent to monogenic dominant control.     

Identification of RFLP markers closely linked to the H1 gene conferring resistance to Globodera rostochiensis in potato

Summary Resistance to the root cyst nematode Globodera rostochiensis is an agronomic trait that is at present incorporated into most new potato varieties. Major dominant genes are available that originate from wild and cultivated Solanum species closely related to the cultivated European potato (Solanum tuberosum ssp. tuberosum). One of those genes, H1, from S. Tuberosum ssp. andigena, was mapped to a distal position on potato chromosome V using restriction fragment length polymorphism (RFLP) markers. The H1 locus segregates independently from Gro1, a second dominant gene presumably from S. Spegazzinii that confers resistance to G. Rostochiensis and which has been mapped to chromosome VII. One marker, CP113, was linked without recombination to the H1 locus.     

A major gene mapped on chromosome XII is the main factor of a quantitatively inherited resistance to Meloidogyne fallax in Solanum sparsipilum

Meloidogyne fallax is an emerging pest in Europe and represents a threat for potato production. We report the mapping of genetic factors controlling a quantitative resistance against M. fallax identified in the Solanum sparsipilum genotype 88S.329.15. When infected, this genotype develops a necrotic reaction at the feeding site of the juveniles and totally prevents their development to the female stage. A “F1” diploid progeny consisting of 128 individuals was obtained using the potato (S. tuberosum) dihaploid genotype BF15 H1 as female progenitor. Sixty-eight hybrid genotypes displayed necrosis at the feeding site of the juveniles and 60 other genotypes showed no defence reaction. This suggested a monogenic control of the resistance. However, when considering the number of nematode females developed in their roots, a continuous distribution was observed for both “necrotic” and “non-necrotic” hybrid genotypes, indicating a polygenic control of the resistance. A linkage map of each parental genotype was constructed using AFLP markers. The necrotic reaction (NR) was mapped as a qualitative trait on chromosome XII of the resistant genotype 88S.329.15. Quantitative trait locus (QTL) analysis for the number of nematode females developed per “F1” plant genotype was performed using the QTL cartographer software. No QTL was detected on the linkage map of the susceptible parent. A QTL explaining 94.5% of the phenotypic variation was mapped on chromosome XII of the resistant progenitor. This QTL, named MfaXIIspl, was mapped in a genomic region collinear to the map position of the Mi-3 gene conferring resistance to Meloidogyne incognita in tomato. It corresponds to the NR locus.     

Molecular mapping of the blast resistance gene, Pi44(t), in a line derived from a durably resistant rice cultivar

 A recombinant inbred line derived from a cross between CO39 and ‘Moroberekan’, RIL276, was found to be resistant to lineage 44 isolates of Pyricularia grisea in the Philippines. One hundred F₂ individuals were obtained from a backcross of RIL276 and CO39. Phenotypic analysis showed that RIL276 carries a single locus, tentatively named Pi44(t), conferring complete resistance to lineage 44 isolates of P. grisea. RFLP probes, STS primers and AFLP markers were applied to identify DNA markers linked to Pi44(t). Neither RFLP nor STS-PCR analysis gave rise to DNA markers linked to the locus. Using bulk segregant AFLP analysis, however, two dominant AFLP markers (AF₃₄₈ and AF₃₄₉) linked to Pi44(t) were identified. AF₃₄₉ and AF₃₄₈ were located at 3.3±1.5 cM and 11±3.5 cM from Pi44(t), respectively. These markers were mapped on chromosome 11 using an F₂ population derived from a cross between ‘Labelle’ and ‘Black Gora’. The location of AF₃₄₈ on chromosome 11 was confirmed using another F₂ mapping population derived from IR40931-26-3-3-5/ PI543851. DNA products at the loci linked to Pi44(t) were amplified from RIL276, ‘Labelle’ and PI543851 using the same primer pairs used to amplify AF₃₄₉ and AF₃₄₈. Sequence analysis of these bands showed 100% identity between lines. This result indicates that these AFLP markers could be used for the comparison of maps or assignment of linkage groups to chromosomes. Received: 12 May 1998 / Accepted: 13 November 1998     

Mapping of loci involved in quantitatively inherited resistance to the potato cyst-nematode Globodera rostochiensis pathotype Ro1

We report the identification and mapping of two quantitative trait loci (QTLs) of Solanum spegazzinii BGRC, accession 8218-15, involved in resistance to the potato cyst-nematode Globodera rostochiensis pathotype Ro1, by means of restriction fragment length polymorphisms (RFLPs). For this purpose we crossed a susceptible diploid S. tuberosum with the resistant S. spegazzinii, and tested the F₁ population for resistance to the Ro1 pathotype. Since the F₁ segregated for the resistance, the S. spegazzinii parent was concluded to be heterozygous at the nematode resistance loci. For the mapping of the resistance loci we made use of RFLP markers segregating for S. spegazzinii alleles in the F₁. One hundred and seven RFLP markers were tested in combination with four different restriction enzymes; 29 of these displayed a heterozygous RFLP pattern within S. spegazzinii and were used for mapping. Analysis of variance (ANOVA) was applied to test the association of the RFLP patterns of these markers with nematode resistance. Two QTLs involved in disease resistance to Globodera rostochiensis pathotype Ro1 were identified and mapped to chromosomes 10 and 11 respectively.     

Isolation and FISH mapping of Yeast Artificial Chromosomes (YACs) encompassing an allele of the Gm2 gene for gall midge resistance in rice

 Ten yeast artificial chromosomes (YACs) spanning the Gm2 locus have been isolated by screening high-density filters containing a total of approximately 7000 YAC (representing six genome equivalents) clones derived from a japonica rice, Nipponbare. The screening was done with five RFLP markers flanking a gall midge resistance gene, Gm2, which was previously mapped onto chromosome 4 of rice. This gene confers resistance to biotype 1 and 2 of gall midge (Orseolia oryzae), a major insect pest of rice in South and Southeast Asia. The RFLP markers RG214, RG329 and F8 hybridized with YAC Y2165. Two overlapping YAC clones (Y5212 and Y2165) were identified by Southern hybridization, with Gm2-flanking RFLP markers, and their inserts isolated. The purified YACs and RFLP markers flanking Gm2 were labeled and physically mapped by the fluorescence in situ hybridization (FISH) technique. All of them mapped to the long arm of chromosome 4 of the resistant variety of rice, ‘Phalguna’, confirming the previous RFLP mapping data. Received: 15 December 1997 / Accepted: 5 March 1998     

Mapping QTLs for resistance against <Emphasis Type="Italic">Globodera pallida</Emphasis> (Stone) Pa2/3 in a diploid potato progeny originating from <Emphasis Type="Italic">Solanum spegazzinii</Emphasis>

A "F1" diploid population between Solanum tuberosum 2 x and the wild Solanum spegazzinii was studied. It segregated for resistance against the potato cyst nematode Globodera pallida derived from the wild species. The inheritance had a quantitative nature. Linkage maps of AFLP and RFLP markers were constructed for both parents. Three QTLs were identified on the map of the resistant parent on chromosomes V, VI and XII, respectively. The first one had a major effect and explained more than 50% of the total variance of resistance. It is located in a cluster of resistance genes and may be the same locus as Gpa which has been described formerly. The two others explained about 20% of the total variance each. The QTL on chromosome XII is also in a cluster of resistance genes, and in an orthologous position with resistance genes against nematodes in tomato and pepper.     

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     

A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers

The R1 allele confers on potato a race-specific resistance to Phytophthora infestans. The corresponding genetic locus maps on chromosome V in a region in which several other resistance genes are also located. As part of a strategy for cloning R1, a high-resolution genetic map was constructed for the segment of chromosome V that is bordered by the RFLP loci GP21 and GP179 and includes the R1 locus. Bulked segregant analysis and markers based on amplified fragment length polymorphisms (AFLP markers) were used to select molecular markers closely linked to R1. Twenty-nine of approximately 3200 informative AFLP loci displayed linkage to the R1 locus. Based on the genotypic analysis of 461 gametes, eight loci mapped within the GP21–GP179 interval. Two of those could not be seperated from R1 by recombination. For genotyping large numbers of plants with respect to the flanking markers GP21 and GP179 PCR based assays were also developed which allowed marker-assisted selection of plants with genotypes Rr and rr and of recombinant plants.     

Combined mapping of AFLP and RFLP markers in barley

AFLP marker technology allows efficient DNA fingerprinting and the analysis of large numbers of polymorphic restriction fragments on polyacrylamide gels. Using the doubled haploids from the F₁ of the cross Proctor × Nudinka, 118 AFLP markers were mapped onto a barley (Hordeum vulgare L.) RFLP map, also including five microsatellite and four protein marker loci. The AFLP markers mapped to all parts of the barley chromosomes and filled in the gaps on barley chromosomes 2L, 4L and 6 in which no RFLP loci had been mapped. Interestingly, the AFLP markers seldom interrupted RFLP clusters, but grouped next to them. The combined map covers 1873 cM, with a total of 282 markers. The merging of AFLP and RFLP markers increased the total map length; 402 cM were added to the map at the tips of chromosomes or in regions corresponding to earlier gaps. Another 375 cM resulted from mapping AFLP markers near to RFLP clusters or in between non-clustered RFLP markers.