Mapping a novel heat-stable resistance to Meloidogyne in Lycopersicon peruvianum

 The root-knot nematode heat-stable resistance locus from L. peruvianum LA2157 was mapped on chromosome 6. All wild tomato LA2157 entries and the LA2157 S₁ progeny tested were resistant to Mi-avirulent Meloidogyne spp. isolates at 32°C, indicating that the self-compatible accession is homozygous for heat-stable nematode resistance. The novel resistance locus was mapped on a RFLP linkage map; this map was based on a segregating F₂ population obtained from the interspecific F₁ between L. esculentum cv ‘Solentos’ and L. peruvianum LA2157. The inheritance of the heat-stable resistance was evaluated in 100 F₃ lines derived from one F₁ interspecific hybrid. The genotype of the resistance locus of the individual F₂ plants was based on the phenotypic classification of their F₃ lines, and the data were used to map the resistance locus on the arm of chromosome 6 with the closest linkage to TG178. The position of the novel heat-stable resistance of LA2157 was localized in the resistance genes’ cluster close to the location of gene Mi-1. Cuttings of the F₃ lines expressed resistance to Mi-1-avirulent M. incognita and M. javanica biotypes at 25°C and at 32°C (a temperature at which Mi-1 resistance is not expressed). There was no difference in the segregating population for expression of heat-unstable resistance and heat-stable resistance to Mi-1-avirulent Meloidogyne spp. However, LA2157 and cuttings of the above F₃ lines were susceptible to a Mi-1-virulent M. incognita isolate at 30°C and to a M. hapla isolate at 25°C. Received: 6 July 1998 / Accepted: 28 July 1998     

Mapping of a Heat-Stable Gene for Resistance to Southern Root-Knot Nematode in Solanum lycopersicum

Root-knot nematodes (Meloidogyne spp.) can cause severe problems in tomato production in warm climates. To date, Mi-1 is the only gene that has been used widely to develop cultivars for controlling disease caused by nematodes around the world. However, Mi-1 does not provide resistance to the pest when the soil temperature is above 28 °C. Tomato breeding line ZN17 has been reported to possess resistance to Meloidogyne incognita at high temperatures (32 °C). To identify markers linked tightly to resistance, an F₂ population was developed by crossing the resistance line ZN17 to susceptible line 09C84. Analysis of F₂ individuals by inoculating M. incognita suggested that resistance in ZN17 is conditioned by a single dominant gene temporarily designated as Mi-HT. Linkage analysis suggested that Mi-HT is located on the short arm of chromosome 6. One marker, W737, co-segregated with Mi-HT. Comparisons of map positions of Mi-HT, Mi-1, and Mi-9, as well as marker genotypes in LA2157, Motelle, and ZN17 suggested that Mi-HT might be a homologue of Mi-1 and Mi-9 or a new gene. The results obtained in this study will facilitate fine-mapping and cloning of the gene as well as marker-assisted breeding for heat-stable resistance to southern root-knot nematodes in tomato.     

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.     

Heterologous expression of the Mi-1.2 gene from tomato confers resistance against nematodes but not aphids in eggplant

The Mi-1.2 gene in tomato (Solanum lycopersicum) is a member of the nucleotide-binding leucine-rich repeat (NBLRR) class of plant resistance genes, and confers resistance against root-knot nematodes (Meloidogyne spp.), the potato aphid (Macrosiphum euphorbiae), and the sweet potato whitefly (Bemisia tabaci). Mi-1.2 mediates a rapid local defensive response at the site of infection, although the signaling and defensive pathways required for resistance are largely unknown. In this study, eggplant (S. melongena) was transformed with Mi-1.2 to determine whether this gene can function in a genetic background other than tomato. Eggplants that carried Mi-1.2 displayed resistance to the root-knot nematode Meloidogyne javanica but were fully susceptible to the potato aphid, whereas a susceptible tomato line transformed with the same transgene was resistant to nematodes and aphids. This study shows that Mi-1.2 can confer nematode resistance in another Solanaceous species. It also indicates that the requirements for Mi-mediated aphid and nematode resistance differ. Potentially, aphid resistance requires additional genes that are not conserved between tomato and eggplant.     

Ectopic Expression of the Tomato <Emphasis Type="Italic">Mi-1</Emphasis> Gene Confers Resistance to Root Knot Nematodes in Lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis>)

The full genomic region of the root knot nematode (Meloidogyne spp.) resistance gene Mi-1 was cloned from tomato and transformed into lettuce to investigate its function in a heterologous system. Transgenic lettuce lines containing the Mi-1 gene were developed using Agrobacterium-mediated transformation. Ectopic expression of the Mi-1 gene was observed in transgenic lines, and resistance to root knot nematode was improved.     

The tomato Rme1 locus is required for Mi-1-mediated resistance to root-knot nematodes and the potato aphid

The tomato Mi-1 gene confers resistance against root-knot nematodes (Meloidogyne spp.) and a biotype of the potato aphid (Macrosiphum euphorbiae). Four mutagenized Mi-1/Mi-1 tomato populations were generated and screened for altered root-knot nematode resistance. Four independent mutants belonging to two phenotypic classes were isolated. One mutant was chosen for further analyzes; rme1 (for resistance to Meloidogyne) exhibited levels of infection comparable with those found on susceptible controls. Molecular and genetic data confirmed that rme1 has a single recessive mutation in a locus different from Mi-1. Cross-sections through galls formed by feeding nematodes on rme1 roots were identical to sections from galls of susceptible tomato roots. In addition to nematode susceptibility, infestation of rme1 plants with the potato aphid showed that this mutation also abolished aphid resistance. To determine whether Rme1 functions in a general disease-resistance pathway, the response against Fusarium oxysporum f.sp. lycopersici race 2, mediated by the I-2 resistance gene, was studied. Both rme1 and the wild type plants were equally resistant to the fungal pathogen. These results indicate that Rme1 does not play a general role in disease resistance but may be specific for Mi-1-mediated resistance.     

Three QTLs from Lycopersicon peruvianum confer a high level of resistance to Clavibactermichiganensis ssp. michiganensis

Lycopersicon peruvianum LA2157 originates from 1650 m above sea level and harbours several beneficial traits for cultivated tomatoes such as cold tolerance, nematode resistance and resistance to bacterial canker (Clavibacter michiganensis ssp. michiganensis). In order to identify quantitative trait loci (QTLs) for bacterial canker resistance, a QTL mapping approach was carried out in an F₂ population derived from the interspecific F₁ between Lycopersicon esculentum cv Solentos and L. peruvianum LA2157. Three QTLs for resistance mapped to chromosomes 5, 7 and 9 respectively. The resistance loci were additive and co-dominant with the QTL on chromosome 7 explaining the largest part of the variation for resistance in the F₂ population. The combination of this QTL with either of the other two QTLs conferred a resistance similar to the level in the resistant parent L. peruvianum. Some RFLP markers flanking this QTL on chromosome 7 were converted into SCAR markers allowing efficient marker-assisted selection of plants with high resistance to bacterial canker. Received: 26 February 1999 / Accepted: 12 March 1999     

Fine mapping of the nematode resistance gene <Emphasis Type="Italic">Mi-3</Emphasis> in <Emphasis Type="Italic">Solanum peruvianum</Emphasis> and construction of a <Emphasis Type="Italic">S. lycopersicum</Emphasis> DNA contig spanning the locus

Currently, the only genetic resistance against root-knot nematodes in the cultivated tomato Solanum lycopersicum (Lycopersicon esculentum) is due to the gene Mi-1. Another resistance gene, Mi-3, identified in the related wild species Solanum peruvianum (Lycopersicon peruvianum) confers resistance to nematodes that are virulent on tomato lines that carry Mi-1, and is effective at temperatures at which Mi-1 is not effective (above 30°C). Two S. peruvianum populations segregating for Mi-3 were used to develop a high-resolution map of the Mi-3 region of chromosome 12. S. lycopersicum BACs carrying flanking markers were identified and used to construct a contig spanning the Mi-3 region. Markers generated from BAC-end sequences were mapped in S. peruvianum plants in which recombination events had occurred near Mi-3. Comparison of the S. peruvianum genetic map with the physical map of S. lycopersicum indicated that marker order is conserved between S. lycopersicum and S. peruvianum. The 600 kb contig between Mi-3-flanking markers TG180 and NR18 corresponds to a genetic distance of about 7.2 cM in S. peruvianum. We have identified a marker that completely cosegregates with Mi-3, as well as flanking markers within 0.25 cM of the gene. These markers can be used to introduce Mi-3 into cultivated tomato, either by conventional breeding or cloning strategies.     

Flanking Microsatellite Markers for Breeding Varieties Against Asian Rice Gall Midge

The Asian rice gall midge, Orseolia oryzae Wood-Mason (Cecidomyiidae: Diptera) is a serious pest of wet season rice in South and Southeast Asia. Due to internal feeding habit and presence of biotypes of the pest, the most feasible way to control is breeding varieties resistant against multiple biotypes through marker-assisted breeding (MAB). But very few versatile co-dominant markers linked to the gall midge resistance genes are available. We used a set of F₉ recombinant inbred lines (RILs) of the cross TN1/PTB10 and identified microsatellite markers for the gall midge resistance gene in cv. PTB10 on short arm of rice chromosome 8. Markers RM22550 and RM547 flank the gene at a distance of 0.9 and 1.9 cM, respectively. Amplification of the markers in gall midge resistant and susceptible cultivars showed that these markers can be successfully used in MAB for development of gall midge resistant varieties.     

Identification of flanking SSR markers for a major rice gall midge resistance gene <Emphasis Type="Italic">Gm1</Emphasis> and their validation

Host-plant resistance is the preferred strategy for management of Asian rice gall midge (Orseolia oryzae), a serious pest in many rice-growing countries. The deployment of molecular markers linked to gall midge resistance genes in breeding programmes can accelerate the development of resistant cultivars. In the present study, we have tagged and mapped a dominant gall midge resistance gene, Gm1, from the Oryza sativa cv. W1263 on chromosome 9, using SSR markers. A progeny-tested F₂ mapping population derived from the cross W1263/TN1 was used for analysis. To map the gene locus, initially a subset of the F₂ mapping population consisting of 20 homozygous resistant and susceptible lines each was screened with 63 parental polymorphic SSR markers. The SSR markers RM316, RM444 and RM219, located on chromosome 9, are linked to Gm1 at genetic distances of 8.0, 4.9 and 5.9 cM, respectively, and flank the gene locus. Further, gene/marker order was also determined. The utility of the co-segregating SSR markers was tested in a backcross population derived from the cross Swarna/W1263//Swarna, and allelic profiles of these markers were analysed in a set of donor rice genotypes possessing Gm1 and in a few gall midge-susceptible, elite rice varieties.     

The Effects of Root-knot Nematode Infection and Mi-mediated Nematode Resistance in Tomato on Plant Fitness

The Mi-1.2 resistance gene in tomato (Solanum lycopersicum) confers resistance against several species of root-knot nematodes (Meloidogyne spp.). This study examined the impact of M. javanica on the reproductive fitness of near-isogenic tomato cultivars with and without Mi-1.2 under field and greenhouse conditions. Surprisingly, neither nematode inoculation or host plant resistance impacted the yield of mature fruits in field microplots (inoculum=8,000 eggs/plant), or fruit or seed production in a follow-up greenhouse bioassay conducted with a higher inoculum level (20,000 eggs/plant). However, under heavy nematode pressure (200,000 eggs/plant), greenhouse-grown plants carrying Mi-1.2 had more than ten-fold greater fruit production than susceptible plants and nearly forty-fold greater estimated lifetime seed production, confirming prior reports of the benefits of Mi-1.2. In all cases Mi-mediated resistance significantly reduced nematode reproduction. These results indicated that tomato can utilize tolerance mechanisms to compensate for moderate levels of nematode infection, but that the Mi-1.2 resistance gene confers a dramatic fitness benefit under heavy nematode pressure. No significant cost of resistance was detected in the absence of nematode infection.     

The MI-1-mediated pest resistance requires Hsp90 and Sgt1

The tomato (Solanum lycopersicum) Mi-1 gene encodes a protein with putative coiled-coil nucleotide-binding site and leucine-rich repeat motifs. Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum euphorbiae), and sweet potato whitefly (Bemisia tabaci). To identify genes required in the Mi-1-mediated resistance to nematodes and aphids, we used tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to repress candidate genes and assay for nematode and aphid resistance. We targeted Sgt1 (suppressor of G-two allele of Skp1), Rar1 (required for Mla12 resistance), and Hsp90 (heat shock protein 90), which are known to participate early in resistance gene signaling pathways. Two Arabidopsis (Arabidopsis thaliana) Sgt1 genes exist and one has been implicated in disease resistance. Thus far the sequence of only one Sgt1 ortholog is known in tomato. To design gene-specific VIGS constructs, we cloned a second tomato Sgt1 gene, Sgt1-2. The gene-specific VIGS construct TRV-SlSgt1-1 resulted in lethality, while silencing Sgt1-2 using TRV-SlSgt1-2 did not result in lethal phenotype. Aphid and root-knot nematode assays of Sgt1-2-silenced plants indicated no role for Sgt1-2 in Mi-1-mediated resistance. A Nicotiana benthamiana Sgt1 VIGS construct silencing both Sgt1-1 and Sgt1-2 yielded live plants and identified a role for Sgt1 in Mi-1-mediated aphid resistance. Silencing of Rar1 did not affect Mi-1-mediated nematode and aphid resistance and demonstrated that Rar1 is not required for Mi-1 resistance. Silencing Hsp90-1 resulted in attenuation of Mi-1-mediated aphid and nematode resistance and indicated a role for Hsp90-1. The requirement for Sgt1 and Hsp90-1 in Mi-1-mediated resistance provides further evidence for common components in early resistance gene defense signaling against diverse pathogens and pests.     

Histological response of resistant tomato cultivars to infection of virulent Tunisian root-knot nematode (Meloidogyne incognita) populations

The response of a susceptible tomato cultivar (Solanum lycopersicum cv. Rio Grande) to infection by three populations of root-knot nematode (Meloidogyne incognita) was compared histologically with that of Lycopersicon esculentum cv. Monita, L. esculentum cv. VFN8 and Solanum lycopersicum cv. Nemador possessing the Mi-1 resistance gene and accession PI126443 of L. peruvianum possessing the Mi-3 gene. The resistant cultivars showed susceptibility to the Tunisian Meloidogyne populations. Feeding sites were characterised by the development of giant cells that contained granular cytoplasm and several hypertrophied nuclei. The cytoplasm of giant cells was aggregated along their thickened cell walls and consequently the vascular tissues within galls appeared disrupted and disorganised. Feeding site formed on resistant L. esculentum lines and susceptible cultivar Rio Grande are similar according to cell and nucleus number, and the nurse superficies. Resistant accession L. peruvianum PI126443, known to possess heat-stable nematode resistance, also showed susceptible reaction to Tunisian Meloidogyne incognita populations; however, nematode development was reduced in comparison with susceptible plants and less developed feeding cells were observed.     

The nematode-resistance gene,<Emphasis Type="Italic"> Mi-1</Emphasis>, is associated with an inverted chromosomal segment in susceptible compared to resistant tomato

The gene Mi-1 confers effective resistance in tomato (Lycopersicon esculentum) against root-knot nematodes and some isolates of potato aphid. This locus was introgressed from L. peruvianum into the corresponding region on chromosome 6 in tomato. In nematode-resistant tomato, Mi-1 and six homologs are grouped into two clusters separated by 300 kb. Analysis of BAC clones revealed that the Mi-1 locus from susceptible tomato carried the same number and distribution of Mi-1 homologs, as did the resistant locus. Molecular markers flanking the resistant and susceptible loci were in the same relative orientation, but markers between the two clusters were in an inverse orientation. The simplest explanation for these observations is that there is an inversion between the two clusters of homologs when comparing the Mi-1 loci from L. esculentum and L. peruvianum. Such an inversion may explain previous observations of severe recombination suppression in the region. Two Mi-1 homologs identified from the BAC library derived from susceptible tomato are not linked to the chromosome 6 locus, but map to chromosome 5 in regions known to contain resistance gene loci in other solanaceous species.Communicated by J.S. Heslop-Harrison     

Differentiation of Meloidogyne incognita and M. arenaria novel resistance phenotypes in Lycopersicon peruvianum and derived bridge-lines

Lycopersicon peruvianum PI 270435 clone 2R2 and PI 126443 clone 1MH were crossed reciprocally with three L. esculentum-L. peruvianum bridge-lines. The incongruity barrier between the two plant species was overcome; F₁ progeny were obtained from crosses between four parental combinations without embryo-rescue culture. Hybridity was confirmed by leaf and flower morphology and by the production of nematode-resistant F₁ progeny on homozygous susceptible parents. Clones of the five F₁ bridgeline hybrids were highly resistant to Mi-avirulent root-knot nematode (Meloidogyne incognita) at both 25°C and 30°C soil temperatures. However, only clones from PI 270435-3MH and PI 126443-1MH, and hybrids from PI 126443-1MH, were resistant to Mi-virulent M. incognita isolates at high soil temperature. Clones and hybrids from PI 270435-2R2 were not resistant to two Mi-virulent M. incognita isolates at high soil temperature. A source of heat-stable resistance was identified in bridge-line EPP-2, and was found to be derived from L. peruvianum LA 1708. Accessions of the L. peruvianum Maranon races, LA 1708 and LA 2172, and bridge-line EPP-2, segregated for heat-stable resistance to Mi-avirulent M. incognita, but were susceptible to Mi-virulent M. incognita isolates. Clone LA 1708-I conferred heat-stable resistance to M. arenaria isolate W, which is virulent to heat-stable resistance genes in L. peruvianum PI 270435-2R2, PI 270435-3MH, and PI 126443-1MH. Clone LA 1708-I has a distinct heat-stable factor for resistance to Mi-avirulent M. arenaria isolate W, for which the gene symbol Mi-4 is proposed. A Mi-virulent M. arenaria isolate Le Grau du Roi was virulent on all Lycopersicon spp. accessions tested, including those with novel resistance genes.     

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.