Genetic and physical localization of the root-knot nematode resistance locus Mi in tomato

As part of a map-based cloning strategy designed to isolate the root-knot nematode resistance gene Mi, tomato F2 populations were analyzed in order to identify recombination points close to this economically important gene. A total of 21 089 F2 progeny plants were screened using morphological markers. An additional 1887 F2 were screened using PCR-based flanking markers. Fine-structure mapping of recombinants with newly developed AFLP markers, and RFLP markers derived from physically mapped cosmid subclones, localized Mi to a genomic region of about 550 kb. The low frequency of recombinants indicated that recombination was generally suppressed in these crosses and that crossovers were restricted to particular regions. To circumvent this problem, a population of Lycopersicon peruvianum, the species from which Mi was originally introgressed, that was segregating for resistance was developed. Screening of this population with PCR, RFLP and AFLP markers identified several plants with crossovers near Mi. Recombination frequency was approximately eight-fold higher in the Mi region of the L. peruvianum cross. However, even within the wild species cross, recombination sites were not uniformly distributed in the region. By combining data from the L. esculentum and L. peruvianum recombinant analyses, it was possible to localize Mi to a region of the genome spanning less than 65 kb. Received: 15 July 1997 / Accepted: 1 October 1997     

Tomato chromosome 1: high resolution genetic and physical mapping of the short arm in an interspecific Lycopersicon esculentum×L. peruvianum cross

A detailed map of part of the short arm of chromosome 1 proximal to the Cf-4/Cf-9 gene cluster was generated by using an F₂ population of 314 plants obtained from the cross between the remotely related species Lycopersicon esculentum and L. peruvianum. Six markers that cosegregate in an L. esculentum×L. pennellii F₂ population showed high recombination frequencies in the present interspecific population, spanning an interval of approximately 13?cM. Physical distances between RFLP markers were estimated by pulsed field gel electrophoresis of high-molecular-weight DNA and by identifying YACs that recognized more than one RFLP marker. In this region 1?cM corresponded to 55–110?kb. In comparsion with the value of 730?kb per cM averaged over the entire genome, this reflects the remarkably high recombination frequencies in this region in the hybrid L. esculentum×L. peruvianum progeny population. The present data underline the fact that recombination is not a process that occurs randomly over the entire genome, but can vary dramatically in intensity between chromosomal regions and among populations.     

Mapping a new nematode resistance locus in Lycopersicon peruvianum

Accessions of the wild tomato species L. peruvianum were screened with a root-knot nematode population (557R) which infects tomato plants carrying the nematode resistance gene Mi. Several accessions were found to carry resistance to 557R. A L. peruvianum backcross population segregating for resistance to 557R was produced. The segregation ratio of resistant to susceptible plants suggested that a single, dominant gene was a major factor in the new resistance. This gene, which we have designated Mi-3, confers resistance against nematode strains that can infect plants carrying Mi. Mi-3, or a closely linked gene, also confers resistance to nematodes at 32°C, a temperature at which Mi is not effective. Bulked-segregant analysis with resistant and susceptible DNA pools was employed to identify RAPD markers linked to this gene. Five-hundred-and-twenty oligonucleotide primers were screened and two markers linked to the new resistance gene were identified. One of the linked markers (NR14) was mapped to chromosome 12 of tomato in an L. esculentum/L. pennellii mapping population. Linkage of NR14 and Mi-3 with RFLP markers known to map on the short arm of chromosome 12 was confirmed by Southern analysis in the population segregating for Mi-3. We have positioned Mi-3 near RFLP marker TG180 which maps to the telomeric region of the short arm of chromosome 12 in tomato.     

High resolution RFLP map around the root knot nematode resistance gene (Mi) in tomato

Summary In the 1940's the root-knot nematode resistance gene (Mi) was introgressed into the cultivated tomato from the wild species, L. peruvianum, and today it provides the only form of genetic resistance against this pathogen. We report here the construction of a high resolution RFLP map around the Mi gene that may aid in the future cloning of this gene via chromosome walking. The map covers the most distal nine map units of chromosome 6 and contains the Mi gene, nine RFLP markers, and one isozyme marker (Aps-1). Based on the analysis of more than 1,000 F₂ plants from four crosses, we were able to pinpoint the Mi gene to the interval between two of these markers — GP79 and Aps-1. In crosses containing the Mi gene, this interval is suppressed in recombination and is estimated to be 0.4 cM in length. In contrast, for a cross not containing Mi, the estimated map distance is approximately 5 times greater (ca. 2 cM).Using RFLP markers around Mi as probes, it was possible to classify nematode resistant tomato varieties into three types based on the amount of linked peruvianum DNA still present. Two of these types (representing the majority of the varieties tested) were found to still contain more than 5 cM of peruvianum chromosome — a result that may explain some of the negative effects (e.g. fruit cracking) associated with nematode resistance. The third type (represented by a single variety) is predicted to carry a very small segment of peruvianum DNA (<2 cM) and may be useful in the identification of additional markers close to Mi and in the orientation of clones during a chromosome walk to clone the gene.     

Tomato chromosome 1: high resolution genetic and physical mapping of the short arm in an interspecific Lycopersicon esculentum × L. peruvianum cross

 A detailed map of part of the short arm of chromosome 1 proximal to the Cf-4/Cf-9 gene cluster was generated by using an F₂ population of 314 plants obtained from the cross between the remotely related species Lycopersicon esculentum and L. peruvianum. Six markers that cosegregate in an L. esculentum×L. pennellii F₂ population showed high recombination frequencies in the present interspecific population, spanning an interval of approximately 13 cM. Physical distances between RFLP markers were estimated by pulsed field gel electrophoresis of high-molecular-weight DNA and by identifying YACs that recognized more than one RFLP marker. In this region 1 cM corresponded to 55–110 kb. In comparsion with the value of 730 kb per cM averaged over the entire genome, this reflects the remarkably high recombination frequencies in this region in the hybrid L. esculentum×L. peruvianum progeny population. The present data underline the fact that recombination is not a process that occurs randomly over the entire genome, but can vary dramatically in intensity between chromosomal regions and among populations. Received: 20 May 1996 / Accepted: 10 September 1996     

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.     

Chromosome landing at the Arabidopsis TORNADO1 locus using an AFLP-based strategy

The Arabidopsis tornado1 (trn1) mutation causes severe dwarfism combined with twisted growth of all organs. We present a chromosome landing strategy, using amplified restriction fragment length polymorphism (AFLP) marker technology, for the isolation of the TRN1 gene. The recessive trn1 mutation was identified in a C24 transgenic line and is located 5?cM from a T-DNA insertion. We mapped the TRN1 locus to the bottom half of chromosome 5 relative to visible and restriction fragment length polymorphism (RFLP) markers. Recombinant classes within a 3-cM region around TRN1 were used to build a high-resolution map in this region, using the AFLP technique. Approximately 300 primer combinations have been used to test about 26?000 fragments for polymorphisms. Seventeen of these AFLP markers were identified in the 3-cM region around TRN1. These markers were mapped within this region using individual recombinants. Four of these AFLP markers co-segregate with TRN1 whereas one maps at one recombinant below TRN1. We isolated and cloned three of these AFLP markers. These markers identified two yeast artificial chromosome (YAC) clones, containing the RFLP marker above and the AFLP marker below TRN1, demonstrating that these YACs span the TRN1 locus and that chromosome landing has been achieved, using an AFLP-based strategy.     

Recombination around the Tm2a and Mi resistance genes in different crosses of Lycopersicon peruvianum

The amount of recombination in three different intraspecific crosses of the wild tomato species Lycopersicon peruvianum was investigated for the short arm of chromosome 6 that harbors the Mi nematode resistance gene and the centromeric region of chromosome 9 that contains the Tm2a virus resistance gene. These two genes have been introgressed into the cultivated tomato and are associated with a significant reduction in recombination in the respective region when crossed to other L. esculentum lines. For both regions and all crosses within L. peruvianum significantly more recombination (up to more than ten fold) was observed in the gametes derived from the female parent than in those from the male parent. In general, the differences were more pronounced for chromosome 6 than for chromosome 9. The amount of recombination in the three intraspecific L. peruvianum crosses was compared with the amount of recombination observed in the standard interspecific cross used for the construction of a saturated genetic map of tomato (L. esculentum x L. pennellii). In two of three cases for each region, more recombination was observed in the intraspecific crosses and in one case for each region significantly less recombination was found in the intraspecific cross when compared to the interspecific cross. Specifically for the Mi-carrying region, crosses within L. peruvianum exhibited up to 15-fold more recombination than crosses between resistant and susceptible L. esculentum lines, and such crosses will allow the fine mapping of this gene for the purpose of map-based cloning.     

Long-range physical maps of two loci (Aps-1 and GP79) flanking the root-knot nematode resistance gene (Mi) near the centromere of tomato chromosome 6

The root knot nematode resistance gene Mi in tomato has been mapped in the pericentromeric region of chromosome 6. With the objective of isolating Mi through a map-based cloning approach, we have previously identified and ordered into a high-resolution genetic linkage map a variety of tightly linked molecular markers. Using pulsed-field gelelectrophoresis and various rarely cutting restriction enzymes in single, double and partial digestions, we now report long-range physical maps of the two closest flanking markers, acid phosphatase-1 (Aps-1) and GP79, which span over 400 and 800 kb, respectively. It is concluded that the physical distance between both markers is larger than predicted on the basis of genetic linkage analysis. Furthermore, two RFLP markers (H3F8 and H4H10) which map genetically to the same locus as Aps-1 do not show physical linkage, indicating severe suppression of recombination in this region of the chromosome. Finally, no evidence was obtained showing the presence of a CpG island near Aps-1.     

Reproduction of Mi-Virulent Meloidogyne incognita Isolates on Lycopersicon spp.

Selection of detectable numbers of Mi-virulent root-knot nematodes has necessitated a greater understanding of nematode responses to new sources of resistance. During the course of this research, we compared the reproduction of four geographically distinct Mi-virulent root-knot nematode isolates on three resistant accessions of Lycopersicon peruvianum. Each accession carried a different resistant gene, Mi-3, Mi-7, or Mi-8. All nematode isolates were verified as Meloidogyne incognita using diagnostic markers in the mitochondrial genome of the nematode. Reproduction of Mi-virulent isolates W1, 133 and HM, measured as eggs per g of root, was greatest on the Mi-7 carrying accession and least on the Mi-8 carrying accession. In general, Mi-3 behaved similar to the Mi-8 carrying accession. Reproduction of the four nematode isolates was also compared on both Mi and non-Mi-carrying L. esculentum cultivars and a susceptible L. peruvianum accession. Resistance mediated by Mi in L. esculentum still impacted the Mi-virulent nematodes with fewer eggs per g of root on the resistant cultivar (P ≤ 0.05). Preliminary histological studies suggests that Mi-8 resistance is mediated by a hypersensitive response, similar to Mi.     

RFLP markers linked to the root knot nematode resistance gene Mi in tomato

Summary The Mi gene originating from the wild tomato species Lycopersicon peruvianum confers resistance to all major root knot nematodes (Meloidogyne spp.). This single dominant gene is located on chromosome 6 and is very closely linked to the acid phosphatase-1 (Aps-1) locus. Resistance to nematodes has been introgressed into various cultivars of the cultivated tomato (L. esculentum), in many cultivars along with the linked L. peruvianum Aps-1 ¹ allele. By using a pair of nearly isogenic lines differing in a small chromosomal region containing the Mi and Aps-1 loci, we have identified two RFLP markers, GP79 and H6A2c2, which are located in the introgressed L. peruvianum region. Analysis of a test panel of 51 L. esculentum genotypes of various origins indicated that GP79 is very tightly linked to the Mi gene and allows both homozygous and heterozygous nematode-resistant genotypes to be distinguished from susceptible genotypes, irrespective of their Aps-1 alleles. Marker H6A2c2 is linked to the Aps-1 locus and is capable of discriminating between the L. peruvianum Aps-1 ¹ allele and the L. esculentum Aps-1 ³ and Aps-1 ⁺ alleles. In combination, these RFLP markers may provide a powerful tool in breeding tomatoes for nematode resistance.     

An integrated restriction fragment length polymorphism--amplified fragment length polymorphism linkage map for cultivated sunflower.

Restriction fragment length polymorphism (RFLP) maps have been constructed for cultivated sunflower (Helianthus annuus L.) using three independent sets of RFLP probes. The aim of this research was to integrate RFLP markers from two sets with RFLP markers for resistance gene candidate (RGC) and amplified fragment length polymorphism (AFLP) markers. Genomic DNA samples of HA370 and HA372, the parents of the F2 population used to build the map, were screened for AFLPs using 42 primer combinations and RFLPs using 136 cDNA probes (RFLP analyses were performed on DNA digested with EcoRI, HindIII, EcoRV, or DraI). The AFLP primers produced 446 polymorphic and 1101 monomorphic bands between HA370 and HA372. The integrated map was built by genotyping 296 AFLP and 104 RFLP markers on 180 HA370 x HA372 F2 progeny (the AFLP marker assays were performed using 18 primer combinations). The HA370 x HA372 map comprised 17 linkage groups, presumably corresponding to the 17 haploid chromosomes of sunflower, had a mean density of 3.3 cM, and was 1326 cM long. Six RGC RFLP loci were polymorphic and mapped to three linkage groups (LG8, LG13, and LG15). AFLP markers were densely clustered on several linkage groups, and presumably reside in centromeric regions where recombination is reduced and the ratio of genetic to physical distance is low. Strategies for targeting markers to euchromatic DNA need to be tested in sunflower. The HA370 x HA372 map integrated 14 of 17 linkage groups from two independent RFLP maps. Three linkage groups were devoid of RFLP markers from one of the two maps.     

An AFLP marker tightly linked to apomixis reveals hemizygosity in a portion of the apomixis-controlling locus in Paspalum simplex.

A mapping population of Paspalum simplex segregating for apomixis (asexual reproduction through seeds) was screened with AFLPs to find apomixis-linked markers. Four AFLPs linked to apomixis in coupling phase were found. Three of them did not show recombinants among the 87 individuals of the mapping population, whereas the other was more loosely linked. Integrating the AFLP data with those obtained previously with rice RFLP anchor markers, a map was drawn for the chromosome region of P. simplex encompassing apomixis. We cloned the three AFLPs tightly linked with apomixis into plasmid vectors and used them as probes to hybridize the restriction digested DNA of the mapping population. Two of them revealed RFLP bands linked to apomixis together with other alleles, whereas one was proven to belong to a hemizygous portion of the apomixis locus. The total picture resulting from AFLP and RFLP analyses was that a cluster of markers tightly linked with apomixis was detected in P simplex together with two other markers that were more loosely linked. These two markers enclosed a relatively large chromosome segment characterized by strong repression of recombination. The block of recombination may have caused sequence divergence and, therefore, hemizygosity of some regions belonging to the apomixis-controlling chromosome segment of P. simplex. The potential of developing an apomixis-specific sequence for screening large-fragment libraries for the physical isolation of the locus encompassing apomixis is discussed.     

Aflp markers tightly linked to the sex locus in Asparagus officinalis L.

Nine AFLP markers linked to the sex locus in Asparagus officinalis L. have been identified by non-radioactive AFLP technique and bulked segregant analysis. A composite map of one F₂ and two F₁ populations identified three very tightly linked markers. These markers did not give recombinants in the three different populations and mapped 0.5, 0.7 and 1 cM to the sex locus. Codominant scoring of the markers in the F₂ population from a selfed andromonoecious plant could distinguish the XX, XY and YY asparagus plants. The AFLP markers were isolated from the gel and cloned into plasmid vectors. The marker E41M50, which is a low-copy sequence and did not give any recombinants in the screened populations, detected polymorphism between female and male plants when used as RFLP probe. The AFLP markers we obtained are important to plant breeding, particularly in the development of sex specific PCR primers that could be used in the screening of different asparagus plants at the seedling stage. They are likewise important in the elucidation of the mechanisms underlying sex determination and differentiation in this species.     

Fine mapping QMi-C11 a major QTL controlling root-knot nematodes resistance in Upland cotton

The identification and utilization of a high-level of host plant resistance is the most effective and economical approach to control root-knot nematode (Meloidogyne incognita). In an earlier study, we identified a major quantitative trait locus (QTL) for resistance to root-knot nematode in the M-120 RNR Upland cotton line (Gossypium hirsutum L.) of the Auburn 623 RNR source. The QTL is located in a 12.9-cM interval flanked by the two SSR markers CIR069 and CIR316 on the distal segment of chromosome 11. To construct a fine map around the target region, a bulked segregation analysis was performed using two DNA pools consisting of five individuals, with each being homozygous for the two parental alleles. From a survey of 1,152 AFLP primer combinations, 9 AFLP markers closely linked to the target region were identified. By screening an additional 1,221 F₂ individuals developed from the initial mapping population, the Mi-C11 locus was delimited to a 3.6-cM interval flanked by the SSR marker CIR069 and the AFLP marker E14M27-375. These results further elucidate the genetic fine structure of the Mi-C11 locus and provide the basis for map-based isolation of the nematode resistance gene in M-120 RNR.     

Detection of wheat-alien recombinant chromosomes using co-dominant DNA markers**

A study of homoeologous recombination along almost the complete genetic length of two homoeologous chromosomes in the Triticeae was conducted. Sears' phlb mutant was used to induce homoeologous pairing between chromosomes 7A of common wheat and 7Ai–l of Agropyron intermedium. 390 ph1b ph1b homozygous F₃ progeny were screened using six co-dominant DNA markers (RFLP loci). 63 of the progeny (16%) were putative recombinants, showing dissociation of RFLP markers within the arm(s). Progeny tests of self-fertile putative recombinants confirmed the dissociation phenotypes observed in the F₃ progeny. No recombination could be confirmed in 117 F₃ progeny plants having the Ph1– allele (control population). Frequencies and distribution of chiasmata along the chromosome arm 7AS were analysed using additional RFLP markers. The patterns of recombination between the two homoeologous chromosomes were found similar to those reported for homologous recombination between the same markers on short arms of group 7 chromosomes of Triticeae.     

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.     

Construction of a high-resolution genetic map and YAC-contigs in the tomato Tm-2a region

With the ultimate goal of cloning the Tobacco Mosaic Virus (TMV) resistance gene Tm-2a from tomato by means of positional cloning, a high-resolution map of a 4.3-cM region surrounding the Tm-2a gene has been constructed. In total, 13 RFLP and RAPD markers were mapped in close proximity to Tm-2a using 2112 individuals from an intraspecific Lycopersicon peruvianum backcross. The closest flanking markers were separated from Tm-2a by 0.05 cM on each side. Only one marker, the cDNA clone R12, co-segregated with Tm-2a. In order to physically cover the Tm-2a region, R12 and the flanking DNA marker TG207 were used to select homologous YAC clones. To-date, two YAC-contigs spanning approximately 340 kb and 360 kb have been constructed. The data obtained from these experiments indicate that recombination around the centromere of chromosome 9 is extremely suppressed.     

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).     

Dissection and fine mapping of a major QTL for preharvest sprouting resistance in white wheat Rio Blanco

Preharvest sprouting (PHS) is a major constraint to white wheat production. Previously, we mapped quantitative trait loci (QTL) for PHS resistance in white wheat by using a recombinant inbred line (RIL) population derived from the cross Rio Blanco/NW97S186. One QTL, QPhs.pseru-3A, showed a major effect on PHS resistance, and three simple sequence repeat (SSR) markers were mapped in the QTL region. To determine the flanking markers for the QTL and narrow down the QTL to a smaller chromosome region, we developed a new fine mapping population of 1,874 secondary segregating F₂ plants by selfing an F6 RIL (RIL25) that was heterozygous in the three SSR marker loci. Segregation of PHS resistance in the population fitted monogenic inheritance. An additive effect of the QTL played a major role on PHS resistance, but a dominant effect was also observed. Fifty-six recombinants among the three SSR markers were identified in the population and selfed to produce homozygous recombinants or QTL near-isogenic lines (NIL). PHS evaluation of the recombinants delineated the QTL in the region close to Xbarc57 flanked by Xbarc321 and Xbarc12. To saturate the QTL region, 11 amplified fragment length polymorphism (AFLP) markers were mapped in the QTL region with 7 AFLP co-segregated with Xbarc57 by using the NIL population. Dissection of the QTL as a Mendelian factor and saturation of the QTL region with additional markers created a solid foundation for positional cloning of the major QTL.