Resistance to powdery mildew (Oidium lycopersicum) in Lycopersicon hirsutum is controlled by an incompletely-dominant gene Ol-1 on chromosome 6

The inheritance of resistance to powdery mildew (Oidium lycopersicum) in Lycopersicon hirsutum was investigated by disease tests in segregating populations obtained by hybridising tomato (L. esculentum) cv Moneymaker with the wild relative L. hirsutum G1.1560. One incompletely dominant gene Ol-1 was found to largely control resistance to the disease. To map Ol-1, DNA pools from seven resistant and ten susceptible F₂ plants were analyzed for random amplified polymorphic DNA (RAPD). With 32 primers tested, one RAPD, primed with the sequence 5-GACGTGGTGA-3, was observed between the susceptible and the resistant bulks, which cosegregated with resistance in the F₂ population of L. esculentum × L. hirsutum G1.1560. This RAPD was mapped on chromosome 6 by using an F₂ (L. esculentum × L. pennellii) already mapped for 49 RFLPs. RFLP analysis of the F₂ from L. esculentum cv Moneymaker × L. hirsutum G1.1560 demonstrated that Ol-1 maps near the Aps-1 region on chromosome 6, in the vicinity of the resistance genes to Meloidogyne spp. (Mi) and to Cladosporium fulvum (Cf-2/Cf-5).     

RFLP linkage analysis of the Cf-4 and Cf-9 genes for resistance toCladosporium fulvum in tomato

Four different populations segregating for one of the two closely linked (possibly allelic) tomato disease resistance genes to the fungusCladosporium fulvum,Cf-4 andCf-9, were generated and analysed for recombination frequencies between theCf-genes and restriction fragment length polymorphism (RFLP) loci. The population consisting of F₂ progeny from the interspecific crossLycopersicon esculentum carryingCf-9 ×L. pennellii was identified as the most useful for RFLP mapping of theCf-4/9 locus and an RFLP map around this locus was constructed mainly using this population. The two closest markers identified were CP46, 2.6 cM distal, and a group of 11 markers including TG236, 3.7 cM proximal toCf-4/9. A polymerase chain reaction (PCR)-based procedure for the rapid identification of recombination events between these two markers was developed. The regions of foreign DNA introgression surroundingCf-4 andCf-9 in near-isogenic lines were delimited.     

Molecular characterization of the SCAR markers tightly linked to the Tm-2 locus of the genus Lycopersicon

The Tm-2 gene and its alleles conferring tomato mosaic virus resistance in tomato originate from Lycopersicon peruvianum, a wild relative of tomato. DNA fragments of several RAPD markers tightly linked with the Tm-2 locus in tomato were successfully cloned and sequenced. Subsequently, the 24-mer oligonucleotide primer pairs of the SCAR markers corresponding to the RAPD markers were designed based on the 5’-endmost sequences. A fragment of the same size as that of a SCAR marker was amplified in the ToMV-susceptible tomato line with no Tm-2, but the digests of the PCR fragments by AccI exhibited polymorphism in fragment length between the two lines. We chose three SCAR markers and three RAPD markers tightly linked with the Tm-2 locus, and examined whether the same-sized fragments corresponding to these markers were also present in three other lines carrying Tm-2a or one of the other Tm-2 alleles. The fragments corresponding to the three SCAR markers were present in all of the three lines, but the other markers (three RAPDs ) were absent in one or two lines, suggesting that the three SCAR markers are closer to Tm-2 than the other markers. Comparison of the nucleotide sequences of these fragments revealed that they are all homologous to the corresponding SCAR markers. Received: 8 November 1999 / Accepted: 15 November 1999     

Linked, if not the same, Mi-1 homologues confer resistance to tomato powdery mildew and root-knot nematodes

On the short arm of tomato chromosome 6, a cluster of disease resistance (R) genes have evolved harboring the Mi-1 and Cf genes. The Mi-1 gene confers resistance to root-knot nematodes, aphids, and whiteflies. Previously, we mapped two genes, Ol-4 and Ol-6, for resistance to tomato powdery mildew in this cluster. The aim of this study was to investigate whether Ol-4 and Ol-6 are homologues of the R genes located in this cluster. We show that near-isogenic lines (NIL) harboring Ol-4 (NIL-Ol-4) and Ol-6 (NIL-Ol-6) are also resistant to nematodes and aphids. Genetically, the resistance to nematodes cosegregates with Ol-4 and Ol-6, which are further fine-mapped to the Mi-1 cluster. We provide evidence that the composition of Mi-1 homologues in NIL-Ol-4 and NIL-Ol-6 is different from other nematode-resistant tomato lines, Motelle and VFNT, harboring the Mi-1 gene. Furthermore, we demonstrate that the resistance to both nematodes and tomato powdery mildew in these two NIL is governed by linked (if not the same) Mi-1 homologues in the Mi-1 gene cluster. Finally, we discuss how Solanum crops exploit Mi-1 homologues to defend themselves against distinct pathogens.     

Chromosomal localization and molecular-marker tagging of the powdery mildew resistance gene (Lv) in tomato

We report the tagging of a powdery mildew [Leveillula taurica (Lév.) Arnaud.] resistance gene (Lv) in tomato using RAPD and RFLP markers. DNA from a resistant (cv Laurica) and a susceptible cultivar were screened with 300 random primers that were used to amplify DNA of resistant and susceptible plants. Four primers yielded fragments that were unique to the resistant line and linked to the resistance gene in an F₂ population. One of these amplified fragments, OP248, with a molecular weight of 0.7 kb, was subsequently mapped to chromosome 12, 1 cM away from CT134. Using RFLP markers located on chromosome 12, it was shown that approximately one half of chromosome 12 (about 42 cM), in the resistant variety is comprised of foreign DNA, presumably introgressed with the resistance gene from the wild species L. chilense. Further analysis of a backcross population revealed that the Lv gene lies in the 5.5-cM interval between RFLP markers, CT211 and CT219. As a prelude to map-based cloning of the Lv gene, we are currently enriching the density of markers in this region by a combination of RAPD primers and other techniques.     

Naturally occurring broad-spectrum powdery mildew resistance in a Central American tomato accession is caused by loss of mlo function

The resistant cherry tomato (Solanum lycopersicum var. cerasiforme) line LC-95, derived from an accession collected in Ecuador, harbors a natural allele (ol-2) that confers broad-spectrum and recessively inherited resistance to powdery mildew (Oidium neolycopersici). As both the genetic and phytopathological characteristics of ol-2-mediated resistance are reminiscent of powdery mildew immunity conferred by loss-of-function mlo alleles in barley and Arabidopsis, we initiated a candidate-gene approach to clone Ol-2. A tomato Mlo gene (SlMlo1) with high sequence-relatedness to barley Mlo and Arabidopsis AtMLO2 mapped to the chromosomal region harboring the Ol-2 locus. Complementation experiments using transgenic tomato lines as well as virus-induced gene silencing assays suggested that loss of SlMlo1 function is responsible for powdery mildew resistance conferred by ol-2. In progeny of a cross between a resistant line bearing ol-2 and the susceptible tomato cultivar Moneymaker, a 19-bp deletion disrupting the SlMlo1 coding region cosegregated with resistance. This polymorphism results in a frameshift and, thus, a truncated nonfunctional SlMlo1 protein. Our findings reveal the second example of a natural mlo mutant that possibly arose post-domestication, suggesting that natural mlo alleles might be evolutionarily short-lived due to fitness costs related to loss of mlo function.     

Identification and mapping on chromosome 9 of RAPD markers linked to Sw-5 in tomato by bulked segregant analysis

Bulked segregant analysis was used to identify random amplified polymorphic DNA (RAPD) markers linked to the Sw-5 gene for resistance to tomato spotted wilt virus (TSWV) in tomato. Using two pools of phenotyped individuals from one segregating population, we identified four RAPD markers linked to the gene of interest. Two of these appeared tightly linked to Sw-5, whereas another, linked in repulsion phase, enabled the identification of heterozygous and susceptible plants. After linkage analysis of an F₂ population, the RAPD markers were shown to be linked to Sw-5 within a distance of 10.5 cM. One of the RAPD markers close to Sw-5 was used to develop a SCAR (sequence characterized amplified region) marker. Another RAPD marker was stabilized into a pseudo-SCAR marker by enhancing the specificity of its primer sequence without cloning and sequencing. RAPD markers were mapped to chromosome 9 on the RFLP tomato map developed by Tanksley et al. (1992). The analysis of 13 F₃ families and eight BC₂ populations segregating for resistance to TSWV confirmed the linkage of the RAPD markers found. These markers are presently being used in marker-assisted plant breeding.     

Tomato defense to Oidium neolycopersici: dominant Ol genes confer isolate-dependent resistance via a different mechanism than recessive ol-2

Tomato powdery mildew caused by Oidium neolycopersici has become a globally important disease of tomato (Lycopersicon esculentum). To study the defense responses of tomato triggered by tomato powdery mildew, we first mapped a set of resistance genes to O. neolycopersici from related Lycopersicon species. An integrated genetic map was generated showing that all the dominant resistance genes (Ol-1, Ol-3, Ol-4, Ol-5, and Ol-6) are located on tomato chromosome 6 and are organized in three genetic loci. Then, near-isogenic lines (NIL) were produced that contain the different dominant Ol genes in a L. esculentum genetic background. These NIL were used in disease tests with local isolates of O. neolycopersici in different geographic locations, demonstrating that the resistance conferred by different Ol genes was isolate-dependent and, hence, may be race-specific. In addition, the resistance mechanism was analyzed histologically. The mechanism of resistance conferred by the dominant Ol genes was associated with hypersensitive response, which varies in details depending on the Ol-gene in the NIL, while the mechanism of resistance governed by the recessive gene ol-2 on tomato chromosome 4 was associated with papillae formation.     

Mapping <Emphasis Type="Italic">Ol-4</Emphasis>, a gene conferring resistance to <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> and originating from <Emphasis Type="Italic">Lycopersicon peruvianum</Emphasis> LA2172, requires multi-allelic,single-locus markers

Lycopersicon peruvianum LA2172 is completely resistant to Oidium neolycopersici, the causal agent of tomato powdery mildew. Despite the large genetic distance between the cultivated tomato and L. peruvianum, fertile F₁ hybrids of L. esculentum cv. Moneymaker × L. peruvianum LA2172 were produced, and a pseudo-F₂ population was generated by mating F₁ half-sibs. The disease tests on the pseudo-F₂ population and two BC₁ families showed that the resistance in LA2172 is governed by one dominant gene, designated as Ol-4. In the pseudo-F₂ population, distorted segregation was observed, and multi-allelic, single-locus markers were used to display different marker-allele configurations per locus. Parameters for both distortion and linkage between genetic loci were determined by maximum likelihood estimation, and the necessity of using multi-allelic, single-locus markers was illustrated. Finally, a genetic linkage map of chromosome 6 around the Ol-4 locus was constructed by using the pseudo-F₂ population.     

Correlation of genetic and physical structure in the region surrounding the I2Fusarium oxysporum resistance locus in tomato

Summary The dominant gene I ₂ confers on tomato (Lycopersicon esculentum) resistance against the fungus Fusarium oxysporum f. sp. lycopersici race 2. A restriction fragment length polymorphism (RFLP) marker, TG105, has recently been found to be tightly linked to I ₂. The potential for cloning this gene by a reverse genetics approach prompted us to describe in both genetic and physical detail the region surrounding the I ₂ locus on chromosome 11. We have analyzed patterns of segregation of RFLP markers on chromosome 11 and Fusarium resistance in 140 F₂ plants from a cross between Fusarium-resistant and susceptible parental lines. Marker TG105 mapped 0.4 centi-Morgan (CM) from I ₂. Physical analysis of TG105 and its flanking RFLP markers, TG26 and TG36, by pulsed field gradient gel electrophoresis (PFGE) yielded a restriction map for this region encompassing at least 620 kb of the tomato genome. TG105 and TG26 hybridized to the same 175 kb MluI-NruI restriction fragment. We have therefore linked two genetically distinct RFLP markers. Based on the 4.1 cM distance between them, we have assigned a mean value of 43 kb for each cM recombination distance in the vicinity of I ₂. This local ratio between physical and genetic distances is more than 10-fold below the average for the tomato genome. It should therefore be possible to clone I ₂ by chromosome walking from TG105.     

QTLs for tomato powdery mildew resistance (Oidium lycopersici) in Lycopersicon parviflorum G1.1601 co-localize with two qualitative powdery mildew resistance genes

Tomato (Lycopersicon esculentum) is susceptible to the powdery mildew Oidium lycopersici, but several wild relatives such as Lycopersicon parviflorum G1.1601 are completely resistant. An F2 population from a cross of Lycopersicon esculentum cv. Moneymaker x Lycopersicon parviflorum G1.1601 was used to map the O. lycopersici resistance by using amplified fragment length polymorphism markers. The resistance was controlled by three quantitative trait loci (QTLs). Ol-qtl1 is on chromosome 6 in the same region as the Ol-1 locus, which is involved in a hypersensitive resistance response to O. lycopersici. Ol-qtl2 and Ol-qtl3 are located on chromosome 12, separated by 25 cM, in the vicinity of the Lv locus conferring resistance to another powdery mildew species, Leveillula taurica. The three QTLs, jointly explaining 68% of the phenotypic variation, were confirmed by testing F3 progenies. A set of polymerase chain reaction-based cleaved amplified polymorphic sequence and sequence characterized amplified region markers was generated for efficient monitoring of the target QTL genomic regions in marker assisted selection. The possible relationship between genes underlying major and partial resistance for tomato powdery mildew is discussed.     

Molecular mapping of the py-1 gene for resistance to corky root rot (Pyrenochaeta lycopersici) in tomato

 We report the molecular mapping of the py-1 gene for resistance to corky root rot [Pyrenochaeta lycopersici (Schneider and Gerlach)] in tomato using RAPD and RFLP marker analysis. DNA from near-isogenic lines (NILs) of tomato differing in corky root rot resistance was screened with 575 random oligonucleotide primers to detect polymorphic DNAs linked to py-1. Three primers (OPW-04, OPC-02, OPG-19) revealed polymorphisms between the NILs. Twelve resistant and eight susceptible DNA pools derived from segregating F₃ families were used to confirm that the RAPD markers were linked to the py-1 gene. Two of the linked amplified fragments, corresponding to OPW-04 and OPC-02, were subsequently cloned and mapped on the tomato molecular linkage map as RFLPs. These clones were located between TG40 and CT31 on the short arm of chromosome 3. Further analysis with selected RFLP markers showed that 7% (8.8 cM) of chromosome 3 of the resistant line ‘Moboglan’ was introgressed from the L. peruvianum donor parent. Three RFLP markers (TG40, TG324, and TG479) from the introgressed part of chromosome 3 were converted to cleaved amplified polymorphism (CAP) markers for use in a polymerase chain reaction (PCR) assay. These PCR markers will allow rapid large-scale screening of tomato populations for corky root rot resistance. Received: 2 January 1998 / Accepted: 12 January 1998     

Development of SCAR markers linked to powdery mildew (<Emphasis Type="Italic">Uncinula necator</Emphasis>) resistance in grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L. and <Emphasis Type="Italic">Vitis</Emphasis> sp.)

Sequence-characterized amplified regions markers (SCARs) were developed from six randomly amplified polymorphic DNA (RAPD) markers linked to the major QTL region for powdery mildew (Uncinula necator) resistance in a test population derived from the cross of grapevine cultivars “Regent” (resistant) × “Lemberger”(susceptible). RAPD products were cloned and sequenced. Primer pairs with at least 21 nucleotides primer length were designed. All pairs were tested in the F1 progeny of “Regent” × “Lemberger”. The SCAR primers resulted in the amplification of specific bands of expected sizes and were tested in additional genetic resources of resistant and susceptible germplasm. All SCAR primer pairs resulted in the amplification of specific fragments. Two of the SCAR markers named ScORA7-760 and ScORN3-R produced amplification products predominantly in resistant individuals and were found to correlate to disease resistance. ScORA7-760, in particular, is suitable for marker-assisted selection for powdery mildew resistance and to facilitate pyramiding powdery mildew resistance genes from various sources.     

Polymorphic change of appressoria by the tomato powdery mildew Oidium neolycopersici on host tomato leaves reflects multiple unsuccessful penetration attempts

The appressorial shapes of the powdery mildews are an important clue to the taxonomy of the powdery mildew fungi, but the conidia of the tomato powdery mildew Oidium neolycopersici KTP-01 develop non-lobed, nipple-shaped, and moderately lobed or multilobed appressoria on the same leaves. To remove this ambiguity, we performed consecutive observations of sequential appressorial development of KTP-01 conidia with a high-fidelity digital microscope. Highly germinative conidia of KTP-01, collected from conidial pseudochains formed on the tomato leaves, were inoculated into host tomato and nonhost barley leaves or an artificial hydrophobic membrane (Parafilm). Events from germination initiation to appressorium formation were synchronous in all conidia on all materials used for inoculation, but post-appressorial behaviors varied among the materials. Appressoria on the membrane-stuck glass slide formed several projections at different portions of the appressoria to repeat unsuccessful penetration attempts. Similar unsuccessful penetration behavior by KTP-01 conidia was observed in the inoculations into leaves of barley plants, wild tomato species Lycopersicon peruvianum LA2172 (carrying the Ol-4 gene for powdery mildew resistance), and a susceptible host tomato (Lycopersicon esculentum) that had been inoculated with the barley powdery mildew (Blumeria graminis f. sp. hordei, race 1) conidia. On the barley leaves, all penetrations of KTP-01 were impeded by the papillae formed beneath the sites of the appressorial projections. On both the wild tomato and the race 1-inoculated cultivated tomato plants, KTP-01 conidia were prevented from forming functional haustoria by hypersensitive epidermal cell death; this hypersensitive reaction involved the Ol-4 gene in the wild tomato plants or the 'induced resistance' acquired by the nonpathogenic conidia previously inoculated into the cultivated tomato plants. All these KTP-01 conidia produced several projections on the appressoria during the repeated unsuccessful penetration attempts and eventually exhibited multilobed appressoria. On the host tomato leaves inoculated singly with KTP-01 conidia, fewer than 20% of the conidia located appressoria on the central part of target epidermal cells and succeeded in forming functional haustoria at the first penetration attempt without forming an appressorial projection. These conidia exhibited non-lobed appressoria. The remaining conidia, however, whose appressoria were located on/near the border of the target epidermal cells, were more likely to fail to penetrate at the first penetration, and then to develop additional projections for subsequent penetrations. Most conidia succeeded in forming functional haustoria at the second to fourth penetration attempts, but a few conidia failed to produce haustoria at all attempted penetrations. Eventually, the conidia that succeeded at the second penetration possessed a single appressorial projection (exhibiting the nipple-shaped appressoria), whereas the remaining conidia exhibited moderately lobed appressoria with two to four appressorial projections and multilobed appressoria, with more projections. Thus, the present study revealed that the basic shape of appressoria of KTP-01 was the non-lobed type, and that polymorphic changes of the appressoria occurred as a result of successive production of projections during repeated unsuccessful penetration attempts.     

Identification of two AFLP markers linked to bacterial wilt resistance in tomato and conversion to SCAR markers

Tomato bacterial wilt (BW) incited by Ralstonia solanacearum is a constraint on tomato production in tropical, subtropical and humid regions of the world. In this paper, we present the results of a research aimed at the identification of PCR-based markers amplified fragment length polymorphism (AFLP) linked to the genes that confer resistance to tomato BW. To this purpose, bulked segregant analysis was applied to an F₂ population segregating for the BW resistant gene and derived from the pair-cross between a BW resistant cultivar T51A and the susceptible cultivar T9230. Genetic analysis indicated that tomato BW was conferred by two incomplete dominant genes. A CTAB method for total DNA extraction, developed by Murray and Thompson with some modifications was used to isolation the infected tomato leaves. Thirteen differential fragments were detected using 256 primer combinations, and two AFLP markers were linked to the BW resistance. Subsequently, the AFLP markers were converted to co-dominant SCAR markers, named TSCAR_AAT/CGA and TSCAR_AAG/CAT. Linkage analysis showed that the two markers are on the contralateral side of TRSR-1. Genetic distance between TSCAR_AAT/CGA and TRS-1 was estimated to 4.6 cM, while 8.4 cM between TSCAR_AAG/CAT and TRS-1.     

Linkage analysis of er-1, a recessive Pisum sativum gene for resistance to powdery mildew fungus (Erysiphe pisi D.C.)

Linkage analysis was used to determine the genetic map location of er-1, a recessive gene conditioning resistance to powdery mildew, on the Pisum sativum genome. Genetic linkage was demonstrated between er-1 and linkage group 6 markers after analyzing the progeny of two crosses, an F₂ population and a set of recombinant inbred lines. The classes of genetic markers surrounding er-1 include RFLP, RAPD and allozyme markers as well as the morphological marker Gty. A RAPD marker tightly linked to er-1 was identified by bulked segregant analysis. After DNA sequence characterization, specific PCR primers were designed to convert this RAPD marker into a sequence characterized amplified region (SCAR).     

Molecular characterization of RAPD and SCAR markers linked to the Tm-1 locus in tomato

We have cloned and sequenced six RAPD fragments tightly linked to the Tm-1 gene which confers tomato mosaic virus (ToMV) resistance in tomato. The terminal ten bases in each of these clones exactly matched the sequence of the primer for amplifying the corresponding RAPD marker, except for one in which the 5-endmost two nucleotides were different from those of the primer. These RAPD clones did not cross-hybridize with each other, suggesting that they were derived from different loci. From Southern-hybridization experiments, five out of the six RAPD clones were estimated to be derived from middle- or high-repetitive sequences, but not from any parts of the ribosomal RNA genes (rDNA), which are known to be tightly linked with the Tm-1 locus. The remaining clone appeared to be derived from a DNA family consisting of a few copies. These six RAPD fragments were converted to sequence characterized amplified region (SCAR) markers, each of which was detectable using a pair of primers having the same sequence as that at either end of the corresponding RAPD clone. All pairs of SCAR primers amplified distinct single bands whose sizes were the same as those of the RAPD clones. In four cases, the SCAR markers were present in the line with Tm-1 but absent in the line without it, as were the corresponding RAPD markers. In the two other cases, the products of the same size were amplified in both lines. When these SCAR products were digested with different restriction endonucleases which recognize 4-bp sequences, however, polymorphisms in fragment length were found between the two lines. These co-dominant markers are useful for differentiating heterozygotes from both types of homozygote.     

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.     

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.