Genetic characterization of the Pto locus of tomato: semi-dominance and cosegregation of resistance to Pseadomonas syringae pathovar tomato and sensitivity to the insecticide Fenthion

The Pto locus governs resistance to bacterial speck disease in tomato caused by race 0 strains of Pseudomonas syringae pathovar tomato (Pst). Large populations segregating for the Pto locus were generated and genetically characterized. Analysis of the locus has revealed that Pto acts in a semi-dominant manner and cosegegrates with sensitivity to an organophosphorous insecticide, Fenthion, suggesting that Pto may be a complex locus responsible for both phenotypes. We have redefined its map position on chromosome five of the classical genetic map and assigned its position on the molecular map, thus facilitating the alignment of the two genetic maps of the short arm of chromosome five of tomato. Furthermore, we have screened random amplified polymorphic (RAPD) markers for their ability to differentiate near-isogenic lines that differ only with respect to Pto and have identified and mapped seven of these markers. Our results suggest that Pto may be located in a euchromatic region on chromosome five which will be advantageous for the cloning of this locus by one of several molecular strategies.     

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).     

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.     

Characterisation and genetic mapping of resistance and defence gene analogs in cocoa (Theobroma cacao L.)

Disease resistance and defence gene analog (RGA/DGA) sequences were isolated in cocoa using a PCR approach with degenerate primers designed from conserved domains of plant resistance and defence genes: the NBS (nucleotide binding site) motif present in a number of resistance genes such as the tobacco N, sub-domains of plant serine/threonine kinases such as the Pto tomato gene, and conserved domains of two defence gene families: pathogenesis-related proteins (PR) of classes 2 and 5. Nucleotide identity between thirty six sequences isolated from cocoa and known resistance or defence genes varied from 58 to 80%. Amino acid sequences translated from corresponding coding sequences produced sequences without stop codons, except for one NBS –like sequence. Most of the RGAs could be mapped on the cocoa genome and three clusters of genes could be observed : NBS-like sequences clustered in two regions located on chromosomes 7 and 10, Pto-like sequences mapped in five genome regions of which one, located on chromosome 4, corresponded to a cluster of five different sequences. PR2-like sequences mapped in two regions located on chromosome 5 and 9 respectively. An enrichment of the genetic map with microsatellite markers allowed us to identify several co-localisations of RGAs, DGAs and QTL for resistance to Phytophthora detected in several progenies, particularly on chromosome 4 where a cluster of Pto-like sequences and 4 QTL for resistance to Phytophthora were observed. Many other serious diseases affect cocoa and the candidate genes, isolated in this study, could be of broader interest in cocoa disease management.     

巴西橡胶Pto类抗病同源序列的克隆与系统发育重建

番茄Pto基因是一类可以编码丝氨酸/苏氨酸激酶(STK)序列的广谱抗性候选基因,其序列克隆与鉴定为深入了解番茄的抗病机制奠定了基础.在该研究中,一对依据Pto基因的保守序列设计的简并引物被用来扩增巴西橡胶中Pto基因抗病同源序列,扩增得到了一个约550 bp的基因片段,其随后被克隆并测序.序列分析发现,其中的7个抗病同源序列与Pto基因高度同源(BLASTX E value <3e-53),所以其被认为是Pto基因抗病同源序列(Pto-RGCs).通过巴西橡胶的Pto-RGCs多序列比对表明,这些序列包含了多个STKs保守的次级结构域.此外,系统发育分析也表明,巴西橡胶的Pto-RGCs属于Pto基因同源的R基因.该研究结果中Pto-RGCs可为巴西橡胶抗病的发展提供一个有效的基因资源.     

Position of the reduced mycorrhizal colonisation (<Emphasis Type="Italic">Rmc</Emphasis>) locus on the tomato genome map

Our research aims to investigate the molecular communication between land plants and arbuscular mycorrhizal (AM) fungi in the establishment of symbiosis. We have identified a mutation in the facultative AM host tomato, which we named rmc. Plants that are homozygous for rmc no longer host most AM fungi. The mutation also affects the interaction of tomato with root knot nematode and Fusarium wilt. However, the function/s encoded by the intact Rmc locus is/are unknown. To clone and sequence the gene or genes that comprise the Rmc locus, we have initiated a positional cloning project. In this paper, we report the construction of mapping populations and use of molecular markers from the published genome map to identify the location of Rmc on tomato chromosome 8. Nucleotide binding site-leucine rich repeat resistance genes, reported to reside in the same region of that chromosome, provided insufficient differences to develop cleaved amplified polymorphic sequence markers. Therefore, we were unable to map these sequences in relation to rmc. Our results potentiate future work to identify the Rmc function and to determine the genetic basis for the multiple plant-microbe interaction functions that the rmc mutation has defined.     

Tomato chromosome 6: a high resolution map of the long arm and construction of a composite integrated marker-order map

Integration of molecular and classical genetic maps is an essential requirement for marker-assisted breeding, quantitative trait locus mapping and map-based cloning. With respects to tomato, such maps are only available for the top part of chromosome 1, for chromosome 3 and for the short arm and the centromere proximal part of the long arm of chromosome 6. Employing an L. esculentum line carrying an L. hirsutum introgression we constructed an integrated linkage map for the telomere proximal part of the long arm of tomato chromosome 6, thereby completing the integrated map published previously. With an average map distance of only 0.6 cM the map provides detailed information on the relative position of molecular markers and several traits of economical importance, such as the fruit color marker B. Furthermore, two additional crosses using lines containing L. pennellii introgressions were performed to address the question as to how the recombination frequency in a marked interval on the long arm of chromosome 6 is affected by introgressed segments from different origins. It is concluded that recombination is not merely affected by the local level of homology but also by surrounding sequences. Combination of all the linkage data generated in various crosses described in this and other studies enabled the construction of the first integrated map of an entire tomato chromosome. This map carries 42 loci and shows the position of 15 classical genes relative to 59 molecular markers.     

Molecular mapping of the centromeres of tomato chromosomes 7 and 9

The centromeres of two tomato chromosomes have been precisely localized on the molecular linkage map through dosage analysis of trisomic stocks. To map the centromeres of chromosomes 7 and 9, complementary telo-, secondary, and tertiary trisomic stocks were used to assign DNA markers to their respective chromosome arms and thus to localize the centromere at the junction of the short and long arms. It was found that both centromeres are situated within a cluster of cosegregating markers. In an attempt to order the markers within the centric clusters, genetic maps of the centromeric regions of chromosomes 7 and 9 were constructed from F₂ populations of 1620Lycopersicon esculentum × L. pennellii (E × P) plants and 1640L. esculentum × L. pimpinellifolium (E × PM) plants. Despite the large number of plants analyzed, very few recombination events were detected in the centric regions, indicating a significant suppression of recombination at this region of the chromosome. The fact that recombination suppression is equally strong in crosses between closely related (E × PM) and remotely related (E × P) parents suggests that centromeric suppression is not due to DNA sequence mismatches but to some other mechanism. The greatest number of centromeric markers was resolved in theL. esculentum × L. pennellii F₂ population. The centromere of chromosome 7 is surrounded by eight cosegregating markers: three on the short arm, five on the long arm. Similarly, the centric region of chromosome 9 contains ten cosegregating markers including one short arm marker and nine long arm markers. The localization of centromeres to precise intervals on the molecular linkage map represents the first step towards the characterization and ultimate isolation of tomato centromeres.     

Construction of a yeast artificial chromosome library of tomato and identification of cloned segments linked to two disease resistance loci

Summary We have constructed a yeast artificial chromosome (YAC) library of tomato for chromosome walking that contains the equivalent of three haploid genomes (22 000 clones). The source of high molecular weight DNA was leaf protoplasts from the tomato cultivars VFNT cherry and Rio Grande-PtoR, which together contain loci encoding resistance to six pathogens of tomato. Approximately 11 000 YACs have been screened with RFLP markers that cosegregate withTm-2a andPto — loci conferring resistance to tobacco mosaic virus andPseudomonas syringae pv.tomato, respectively. Five YACs were identified that hybridized to the markers and are therefore starting points for chromosome walks to these genes. A subset of the library was characterized for the presence of various repetitive sequences and YACs were identified that carried TGRI, a repeat clustered near the telomeres of most tomato chromosomes, TGRII, an interspersed repeat, and TGRIIl, a repeat that occurs primarily at centromeric sites. Evaluation of the library for organellar sequences revealed that approximately 10% of the clones contain chloroplast sequences. Many of these YAC clones appear to contain the entire 155 kb tomato chloroplast genome. The tomato cultivars used in the library construction, in addition to carrying various disease resistance genes, also contain the wild-type alleles corresponding to most recessive mutations that have been mapped by classical linkage analysis. Thus, in addition to its utility for physical mapping and genome studies, this library should be useful for chromosome walking to genes corresponding to virtually any phenotype that can be scored in a segregating population.     

Molecular genetic analysis of theripening-inhibitor andnon-ripening loci of tomato: A first step in genetic map-based cloning of fruit ripening genes

Ripening represents a complex developmental process unique to plants. We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype. We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato,ripening-inhibitor (rin), andnon-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process. A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning therin andnor loci, respectively. To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci. Based on this analysis, the relationship in the region spanning therin locus is estimated to be 200–300 kb/cM, while thenor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked torin andnor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA. We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains thenot locus.     

Determining resistance to Pseudomonas syringae in tomato, a comparison of different molecular markers

Pseudomonas syringae pv. tomato (Pst) is the causal agent of bacterial speck disease in tomato. Resistance to Pst is determined by Pto, a single resistance gene that belongs to a multi-gene family clustered on chromosome 5. Pst-resistant phenotypes in cultivated tomato are determined by a semi-dominant allele of Solanum pimpinellifolium, which was introgressed into Solanum lycopersicum in the past century. Seed companies, which are continuously interested in producing resistant varieties, can benefit from genetic markers closely linked to the Pto locus in breeding programs based on marker-assisted selection. In this research, three sequence characterised amplified region markers have been developed for identification of resistant and susceptible genotypes of Solanum lycopersicum. A cleaved amplified polymorphic sequence marker has been adapted to a real-time polymerase chain reaction platform using high-resolution melting curve analysis. Application to a tomato population for breeding programs is described. Advantages and disadvantages of the different markers are discussed.     

Genetic analysis and FISH mapping of the Colourless non-ripening locus of tomato

Cnr (Colourless non-ripening) is a dominant pleiotropic ripening mutation of tomato (Lycopersicon esculentum) which has previously been mapped to the proximal region of tomato chromosome 2. We describe the fine mapping of the Cnr locus using both linkage analysis and fluorescence in situ hybridisation (FISH). Restriction fragment length polymorphism (RFLP)-, amplified restriction fragment polymorphism (AFLP)-, and cleaved amplified polymorphic sequence (CAPS)-based markers, linked to the Cnr locus were mapped onto the long arm of chromosome 2. Detailed linkage analysis indicated that the Cnr locus was likely to lie further away from the top of the long arm than previously thought. This was confirmed by FISH, which was applied to tomato pachytene chromosomes in order to gain an insight into the organisation of hetero- and euchromatin and its relationship to the physical and genetic distances in the Cnr region. Three molecular markers linked to Cnr were unambiguously located by FISH to the long arm of chromosome 2 using individual BAC probes containing these single-copy sequences. The physical order of the markers coincided with that established by genetic analysis. The two AFLP markers most-closely linked to the Cnr locus were located in the euchromatic region 2.7-cM apart. The physical distance between these markers was measured on the pachytene spreads and estimated to be approximately 900 kb, suggesting a bp:cM relationship in this region of chromosome 2 of about 330 kb/cM. This is less than half the average value of 750 kb/cM for the tomato genome. The relationship between genetic and physical distances on chromosome 2 is discussed. Received: 11 January 2001 / Accepted: 30 April 2001     

Genetic mapping of jointless-2 to tomato chromosome 12 using RFLP and RAPD markers

Abscission zones are specialized regions in plants, usually located at the base of most plant parts, such as flowers, fruit and leaves, where organs are shed. Although a great deal of information is known about the physiological and biochemical events that lead to organ shedding, very little is known of the molecular events that lead to the formation of the abscission zone itself. In tomato, two recessive mutations have been discovered that completely suppress the formation of flower and fruit pedicel abscission zones, i.e., jointless (j) and jointless-2 (j-2), both tentatively localized to chromosome 11 about 30 cM apart. Because the study of the control of abscission zone development is important for both basic and applied research we are using a map-based cloning approach to identify the jointless genes. The first step in any positional cloning experiment is to establish segregating mapping populations for the target gene and identify closely linked molecular markers that flank the locus. In this study, bulked segregant analysis was used to identify a RAPD marker associated with the j-2 locus, RPD140. To determine the chromosome location of RPD140, we converted it to an RFLP marker that was then mapped on the Cornell reference tomato map in a marker-dense region of chromosome 12. To verify that the j-2 locus was located on tomato chromosome 12, we used nine chromosome 12 RFLP markers linked with RPD140 to map the j-2 gene in an interspecific F₂ mapping population of 151 plants segregating for j-2. The j-2 gene was localized to a 3.0-cM interval between RPD140 and TG618 on tomato chromosome 12. Received: 29 March 1999 / Accepted: 13 October 1999     

Genetics of actin-related sequences in tomato

Summary The genomic distribution of actin-related sequences in tomato was investigated using a cloned actin gene from soybean. Ten actin loci account for most of the hybridizing fragments observed with Southern analysis. Single loci were found on chromosomes 1, 3 and 10 and two loci on chromosome 4. One locus is linked to an unmapped isozyme marker, Sod-1. The four remaining actin loci are independent of each other and of any of the other markers tested. The number of actin loci in tomato (10) is greater than that estimated for soybean (8). As soybean is apparently a tetraploid and tomato a diploid, these results suggest that the number of actin loci has not been stable during the evolution of dicots. A number of these mapped loci lie in regions of the genome previously devoid of molecular markers and thus may be useful in basic and applied genetic research.     

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.     

Tomato resistance to Alternaria stem canker: localization in host genotypes and functional expression compared to non-host resistance

Summary The Alternaria stem canker resistance locus (Asc-locus), involved in resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici and in insensitivity to host-specific toxins (AAL-toxins) produced by the pathogen, was genetically mapped on the tomato genome. Susceptibility and resistance were assayed by testing a segregating F₂ population for sensitivity to AAL-toxins in leaf bioassays. Linkage was observed to phenotypic markers solanifolium and sunny, both on chromosome 3. For the Asc-locus, a distance of 18 centiMorgan to solanifolium was calculated, corresponding to position 93 on chromosome 3. This map position of the resistance locus turned out to be the same in three different resistant tomato accessions, one Dutch and two American, that are at least 40 years apart. AAL-toxin sensitivity in susceptible and resistant tomato genotypes was compared with AAL-toxin sensitivity in a non-host Nicotiana tabacum during different levels of plant cell development. In susceptible and resistant tomato genotypes, inhibitory effects were demonstrated at all levels, except for leaves of resistant genotypes. However, during pollen and root development, inhibitory effects on susceptible genotypes were larger than on resistant genotypes. In the non-host Nicotiana tabacum, hardly any effects of AAL-toxins were demonstrated. Apparently, a cellular target site is present in tomato, but not in Nicotiana tabacum. It was concluded that three levels of AAL-toxin sensitivity exist: (1) a susceptible host sensitivity, (2) a resistant host sensitivity, (3) a non-host sensitivity, and that the resistance mechanism operating in tomato is different from that operating in Nicotiana tabacum.     

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.     

Mapping the Sw-5 locus for tomato spotted wilt virus resistance in tomatoes using RAPD and RFLP analyses

The Sw-5 locus confers dominant resistance to tomato spotted wilt virus (TSWV). To map the location and facilitate the identification of markers linked to Sw-5 we developed a pair of near-isogenic lines (NILs) and an F₂ Lycopersicon esculentum x L. pennellii population segregating for resistance to TSWV. DNA from the NILs was analyzed using 748 random 10-mer oligonucleotides to discern linked molecular markers using a random amplified polymorphic DNA (RAPD) approach. One random primer (GAGCACGGGA) was found to produce a RAPD band of about 2200 bp that demonstrates linkage to Sw-5. Data from co-segregation of resistance and restriction fragment length polymorphisms (RFLPs) in a F₂ interspecific population position Sw-5 between the markers CT71 and CT220 near the telomere of the long arm of chromosome 9.     

Genetic and physical mapping of the lateral suppressor (ls) locus in tomato

Tomato plants homozygous for the recessive lateral suppressor (ls) mutation show a number of phenotypic abnormalities among which the lack of lateral meristem initiation during vegetative growth and the absence of petals on the flower are the most prominent. As a first step towards the isolation of the Ls gene by means of map-based cloning, we have determined its position on the restriction fragment length polymorphism (RFLP) map of tomato. RFLP analysis of 527 F2 plants segregating for the ls allele allowed us to define an interval of 0.8 cM in which the Ls gene is located. Analysis of the physical distance between the two flanking RFLP markers by pulsed field gel electrophoresis revealed that they lie no further than 375 kb apart. Knowledge of the physical distance together with the availability of a tomato yeast artificial chromosome (YAC) library, makes it feasible to isolate the Ls gene by a map-based cloning approach.     

The Pik m gene, conferring stable resistance to isolates of Magnaporthe oryzae , was finely mapped in a crossover-cold region on rice chromosome 11

The Pik ^m gene in rice confers a high and stable resistance to many isolates of Magnaporthe oryzae collected from southern China. This gene locus was roughly mapped to the long arm of rice chromosome 11 with restriction fragment length polymorphic (RFLP) markers in the previous study. To effectively utilize the resistance, a linkage analysis was performed in a mapping population consisting of 659 highly susceptible plants collected from four F₂ populations using the publicly available simple sequence repeat (SSR) markers. The result showed that the locus was linked to the six SSR markers and defined by RM254 and RM144 with ≈13.4 and ≈1.2 cM, respectively. To fine map this locus, additional 10 PCR-based markers were developed in a region flanked by RM254 and RM144 through bioinformatics analysis (BIA) using the reference sequence of cv. Nipponbare. The linkage analysis with these 10 markers showed that the locus was further delimited to a 0.3-cM region flanked by K34 and K10, in which three markers, K27, K28, and K33, completely co-segregated with the locus. To physically map the locus, the Pik ^m -linked markers were anchored to bacterial artificial chromosome clones of the reference cv. Nipponbare by BIA. A physical map spanning ≈278 kb in length was constructed by alignment of sequences of the clones anchored by BIA, in which only six candidate genes having the R gene conserved structure, protein kinase, were further identified in an 84-kb segment.