Locus In Tomato Research Articles

Genetic mapping of ripening and ethylene-related loci in tomato

The regulation of tomato fruit development and ripening is influenced by a large number of loci as demonstrated by the number of existing non-allelic fruit development mutations and a multitude of genes showing ripening-related expression patterns. Furthermore, analysis of transgenic and naturally occurring tomato mutants confirms the pivotal role of the gaseous hormone ethylene in the regulation of climacteric ripening. Here we report RFLP mapping of 32 independent tomato loci corresponding to genes known or hypothesized to influence fruit ripening and/or ethylene response. Mapped ethylene-response sequences fall into the categories of genes involved in either hormone biosynthesis or perception, while additional ripening-related genes include those involved in cell-wall metabolism and pigment biosynthesis. The placement of ripening and ethylene-response loci on the tomato RFLP map will facilitate both the identification and exclusion of candidate gene sequences corresponding to identified single gene and quantitative trait loci contributing to fruit development and ethylene response.

Dissection of the fusarium I2 gene cluster in tomato reveals six homologs and one active gene copy.

The I2 locus in tomato confers resistance to race 2 of the soil-borne fungus Fusarium oxysporum f sp lycopersici. The selective restriction fragment amplification (AFLP) positional cloning strategy was used to identify I2 in the tomato genome. A yeast artificial chromosome (YAC) clone covering approximately 750 kb encompassing the I2 locus was isolated, and the AFLP technique was used to derive tightly linked AFLP markers from this YAC clone. Genetic complementation analysis in transgenic R1 plants using a set of overlapping cosmids covering the I2 locus revealed three cosmids giving full resistance to F. o. lycopersici race 2. These cosmids shared a 7-kb DNA fragment containing an open reading frame encoding a protein with similarity to the nucleotide binding site leucine-rich repeat family of resistance genes. At the I2 locus, we identified six additional homologs that included the recently identified I2C-1 and I2C-2 genes. However, cosmids containing the I2C-1 or I2C-2 gene could not confer resistance to plants, indicating that these members are not the functional resistance genes. Alignments between the various members of the I2 gene family revealed two significant variable regions within the leucine-rich repeat region. They consisted of deletions or duplications of one or more leucine-rich repeats. We propose that one or both of these leucine-rich repeats are involved in Fusarium wilt resistance with I2 specificity.

Novel disease resistance specificities result from sequence exchange between tandemly repeated genes at the Cf-4/9 locus of tomato : Parniske M, Hammond-Kosack KE, Golstein C, Thomas CM, Jones DA, Harrison K, Wulff BHW, Jones JDG: Cell 1997, 91:821–832

Novel disease resistance specificities result from sequence exchange between tandemly repeated genes at the Cf-4/9 locus of tomato : Parniske M, Hammond-Kosack KE, Golstein C, Thomas CM, Jones DA, Harrison K, Wulff BHW, Jones JDG: Cell 1997, 91:821–832

High-resolution genetic map of the Lv resistance locus in tomato

Bulked segregant analysis and high-resolution mapping were used to pinpoint the position of the Lv gene for resistance to Leveillula taurica in tomato. Mapping in an F2, corresponding to more than 3800 gametes, indicates that Lv is positioned within the 0.84-cM interval defined by the RFLP markers CT121 and CT129, with the closest marker, CT121, being only 0.16 cM from the gene. The tight linkage of these markers demonstrates their usefulness in marker-assisted breeding for Lv, and the high-resolution map provides a starting a starting point for positional cloning of this resistance gene.

High-resolution mapping and isolation of a yeast artificial chromosome contig containing fw2.2: a major fruit weight quantitative trait locus in tomato.

A high-resolution physical and genetic map of a major fruit weight quantitative trait locus (QTL), fw2.2, has been constructed for a region of tomato chromosome 2. Using an F(2) nearly isogenic line mapping population (3472 individuals) derived from Lycopersicon esculentum (domesticated tomato) x Lycopersicon pennellii (wild tomato), fw2.2 has been placed near TG91 and TG167, which have an interval distance of 0.13 +/- 0.03 centimorgan. The physical distance between TG91 and TG167 was estimated to be </= 150 kb by pulsed-field gel electrophoresis of tomato DNA. A physical contig composed of six yeast artificial chromosomes (YACs) and encompassing fw2.2 was isolated. No rearrangements or chimerisms were detected within the YAC contig based on restriction fragment length polymorphism analysis using YAC-end sequences and anchored molecular markers from the high-resolution map. Based on genetic recombination events, fw2.2 could be narrowed down to a region less than 150 kb between molecular markers TG91 and HSF24 and included within two YACs: YAC264 (210 kb) and YAC355 (300 kb). This marks the first time, to our knowledge, that a QTL has been mapped with such precision and delimited to a segment of cloned DNA. The fact that the phenotypic effect of the fw2.2 QTL can be mapped to a small interval suggests that the action of this QTL is likely due to a single gene. The development of the high-resolution genetic map, in combination with the physical YAC contig, suggests that the gene responsible for this QTL and other QTLs in plants can be isolated using a positional cloning strategy. The cloning of fw2.2 will likely lead to a better understanding of the molecular biology of fruit development and to the genetic engineering of fruit size characteristics.

Less-than-additive epistatic interactions of quantitative trait loci in tomato.

Epistasis plays a role in determining the phenotype yet quantitative trait loci (QTL) mapping has uncovered little evidence for it. To address this apparent contradiction, we analyzed interactions between individual Lycopersicon pennellii chromosome segments introgressed into an otherwise homogeneous genetic background of L. esculentum (cv. M82). Ten different homozygous introgression lines, each containing from 4 to 58 cM of introgressed DNA, were crossed in a half diallele scheme. The 45 derived double heterozygotes were evaluated in the field for four yield-associated traits, along with the 10 single heterozygotes and M82. Of 180 (45 x 4) tested interactions, 28% were epistatic (P < 0.05) on both linear and geometric scales. The detected epistasis was predominantly less-than-additive, i.e., the effect of the double heterozygotes was smaller than the sum of the effects of the corresponding single heterozygotes. Epistasis was also found for homozygous linked QTL affecting fruit mass and total soluble solids. Although the frequency of epistasis was high, additivity was the major component in the interaction of pairs of QTL. We propose that the diminishing additivity of QTL effects is amplified when more loci are involved; this mode of epistasis may be an important factor in phenotype canalization and in breeding.

Analysis of phytochrome‐deficient yellow‐green‐2 and aurea mutants of tomato

Two alleles of the yellow‐green‐2 (yg‐2) and eight different alleles of the aurea (au) locus of tomato (Lycopersicon esculentum Mill.) were compared. All are characterized by a paler green colour compared with wild‐type (WT), an elongated hypocotyl in red light, and low or below detection limits of spectrophotometrically active phytochrome. Hypocotyl length was variable in white light, ranging from that of WT to more elongated. Immunochemical analysis revealed that etiolated seedlings of the yg‐2 mutant have approximately 25% of the WT level of phytochrome A protein (PHYA), whereas that of phytochrome B protein (PHYB) is normal. In this it resembles the au mutant. The au,yg‐2 double mutant has a more extreme chlorophyll deficiency than either parent. Since the yg‐2 and au mutants have a less severe phenotype at the adult stage, that is, are leaky, the additive effect can be explained by assuming that the mutants control two steps in the chromophore biosynthesis pathway. Combination, by crossing, of the yg‐2 and au mutants with a transgenic tomato line that overexpresses oat phytochrome A3 (PhyA‐3) essentially failed to restore the WT phenotype under white fluorescent light conditions, although under greenhouse conditions some evidence for increased sensitivity to light was observed. Immunochemically, oat PHYA‐3 protein is detectable in both the yg‐2,PhyA‐3 and au,PhyA‐3‘double’ mutants. Spectrophotometrical analysis, however, revealed that holophytochrome was undetectable in the yg‐2,PhyA‐3 and au,PhyA‐3‘double’ mutants. These results are compatible with both mutants being disturbed in phytochrome chromophore biosynthesis.

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.

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.

Identification of RAPD markers linked to the Tm-2 locus in tomato

Tm-2 and Tm-2a are genes conferring resistance to tomato mosaic virus in Lycopersicon esculentum. They are allelic and originated from different lines of L. peruvianum, a wild relative of tomato. In this study, random amplified polymorphic DNA (RAPD) markers linked to these genes were screened in nearly isogenic lines (NILs). To detect RAPDs differentiating NILs, 220 different 10-base oligonucleotide primers were examined by the polymerase chain reaction (PCR), and 43 of them generated 53 consistent polymorphic fragments among the NILs. Out of these 53 fragments, 13 were arbitrarily chosen and examined in respect of whether they were linked to the netted virescent (nv) gene, since nv is tightly linked to the Tm-2 locus and its phenotype is more easily distinguishable. As a result, all 13 markers were shown to be linked to nv, and hence to the Tm-2 locus. Among them, two fragments specific to the NIL carrying Tm-2 three specific to the NIL carrying Tm-2a, and four specific to both of these NILs were closely linked to nv.

Germinal transpositions of the maize element Dissociation from T-DNA loci in tomato.

We have analyzed the pattern of germinal transpositions of artificial Dissociation (Ds) transposons in tomato. T-DNA constructs carrying Ds were transformed into tomato, and the elements were trans-activated by crossing to lines transformed with a stabilized Activator (sAc) that expressed the transposase gene. The sAc T-DNA carried a GUS gene to monitor its segregation. The Ds elements were inserted in a marker gene so that excision from the T-DNA could be monitored. The Ds elements also carried a genetic marker that was intended to be used for reinsertion selection of the elements after excision. Unfortunately, this gene was irreversibly inactivated on crossing to sAc. Germinal excision frequencies of Ds averaged 15-40%, but there was large variation between and within plants. Southern hybridization analysis of stable transposed Ds elements indicated that although unique transpositions predominate, early transposition events can lead to large clonal sectors in the germline of developing plants and to sibling offspring carrying the same transposition event. Multiple germinal transpositions from three different loci were examined for uniqueness, and 15 different transpositions were identified from each of three T-DNA loci that carried a single independent Ds. These were mapped relative to the donor T-DNA loci, and for each locus 70-80% of the transposed elements were closely linked to the donor site.

A re-investigation of the instability at the yv locus in tomato

The instability at the yv locus on chromosome 6 of Lycopersicon esculentum has been re-investigated. Cytogenetic analysis of green, green-yellow variegated and yellow plants showed that the instability is not caused by the somatic segregation of extra chromosomal fragments as was proposed earlier by Hagemann. Instead, it is postulated that the variegated line carries an unstable recessive allele of yv (yvmut) that mutates frequently from dominant green to recessive yellow on the basis of the following observations: (i) variegated plants produced variegated and yellow offspring in aberrant ratios, and (ii) yellow male sterile plants appeared in populations derived from crosses of the Yvms line with wild-type plants and the line LA 780, being recessive yv. Using the isozyme marker Aps-1 linked to yv, it was shown that the deficit in variegated and yellow mutants, which frequently occurs in F2 populations derived from crosses between the variegated mutant and wild-type yv+ plants, was due to certation. From the analysis of these F2 populations, it is concluded that the instability at yv is likely to be controlled autonomously. No evidence was obtained showing germinal reversion events to occur, as inferred from the absence of green plants among the selfed offspring. Variegated offspring were produced by a yellow plant which is interpreted to represent a somatic reversion event from yellow to variegated. Remarkably, when variegated plants were crossed to wild-type plants, F1 plants with a mutant phenotype appeared. On the basis of analogies with mutable alleles from maize and Drosophila, alternative mechanisms underlying the mutability of yv are discussed.

A PCR-based marker tightly linked to the nematode resistance gene, Mi, in tomato

A PCR-based codominant marker has been developed which is tightly linked to Mi, a dominant genetic locus in tomato that confers resistance to several species of root-knot nematode. DNA from tomato lines differing in nematode resistance was screened for random amplified polymorphic DNA markers linked to Mi using decamer primers. Several markers were identified. One amplified product, REX-1, obtained using a pair of decamer primers, was present as a dominant marker in all nematode-resistant tomato lines tested. REX-1 was cloned and the DNA sequences of its ends were determined and used to develop 20-mer primers. PCR amplification with the 20-mer primers produced a single amplified band in both susceptible and resistant tomato lines. The amplified bands from susceptible and resistant lines were distinguishable after cleavage with the restriction enzyme Taq I. The linkage of REX-1 to Mi was verified in an F2 population. This marker is more tightly linked to Mi than is Aps-1, the currently-used isozyme marker, and allows screening of germplasm where the linkage between Mi and Aps-1 has been lost. Homozygous and heterozygous individuals can be distinguished and the procedure can be used for rapid, routine screening. The strategy used to obtain REX-1 is applicable to obtaining tightly-linked markers to other genetic loci. Such markers would allow rapid, concurrent screening for the segregation of several loci of interest.

Close linkage between the Cf-2/Cf-5 and Mi resistance loci in tomato.

Analysis of restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers in tomato plants segregating for resistance to the fungus Cladosporium fulvum was used to localize the resistance genes Cf-2 and Cf-5 to the same region of chromosome 6. This region, between GP79 and Aps-1, is the same as that reported for the Mi gene, which confers resistance to root-knot nematodes (meloidogyne spp.). Recombination values based on F2 populations from crosses between near-isogenic lines of L. esculentum 'Moneymaker' carrying Cf-2 or Cf-5 and Lycopersicon pennellii, indicate that this region occupies 4-5 centiMorgans (cM). However, in F2 populations from crosses between the L. esculentum stock LA1190 carrying yv and these lines, this value is 1-2 cM. The Cf-2 gene, introduced into L. esculentum from L. pimpinellifolium, is on an introgressed segment that extends from a point distal to GP79 to a point between TG232 and H2D1. The origin of Cf-5 was found to be L. esculentum var. cerasiforme rather than L. pimpinellifolium as previously reported. No RFLP markers and only one RAPD marker showed a polymorphism between Moneymaker and the near-isogenic line carrying Cf-5.

AAL toxin-induced physiological changes in Lycopersicon esculentum Mill: Differential sucrose transport in tomato lines isogenic for the Asc locus

Susceptibility to Alternaria alternata f. sp. lycopersici and sensitivity to AAL toxin are regulated by the Asc locus in tomato. Toxin-induced patterns in leaflet necrosis, defined by cell location and relative tissue age, correspond to developmental patterns in capacity to retrieve exogenous sucrose in leaves. Uptake of AAL toxin by detached leaflets of sensitive asc / asc and resistant Asc / Asc lines induced interveinal necrosis within 48 h, the magnitude of which was dependent on the age of the tissue. The youngest leaflets were the most sensitive. In the absence of the toxin, the youngest asc / asc leaflets tested exhibited a three-fold higher endogenous sucrose transport capacity compared to equivalent Asc / Asc leaflets. Exposure to toxin resulted in a strong temporal decrease in sucrose influx within 4 h in both lines, although the magnitude of this effect was greater in asc / asc than in Asc / Asc . Recovery in sucrose influx was observed in Asc / Asc by 8 h but not in asc / asc . In intermediate-aged leaflets, differential genotype-specific changes in sucrose influx were observed within 1 h following toxin treatment. Treatment of leaflets of each genotype with an inhibitor of sucrose transport, p-chloromercuribenzene-sulphonic acid, or with lithium, an inhibitor of sucrose transport via perturbation of signal transduction pathways, caused genotype-specific necrosis similar to AAL toxin. These inhibitors enhanced necrosis in the presence of AAL toxin in both genotypes. These observations suggest: (1) a relationship between sucrose transport capacity, initial response to the toxin, and the Asc locus, and (2) a correlation between high capacity for sucrose influx in the asc / asc isoline and sensitivity to AAL toxin.

High-Resolution Linkage Analysis and Physical Characterization of thePtoBacterial Resistance Locus in Tomato

High-Resolution Linkage Analysis and Physical Characterization of the<i>Pto</i>Bacterial Resistance Locus in Tomato

Correlation of genetic and physical structure in the region surrounding the I2 Fusarium oxysporum resistance locus in tomato.

The dominant gene I2 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 I2. 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 I2 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 centiMorgan (CM) from I2. 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 I2. 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 I2 by chromosome walking from TG105.

RFLP mapping of I1, a new locus in tomato conferring resistance against Fusarium oxysporum f. sp. lycopersici race 1

The inheritance and linkage relationships of a gene for resistance to Fusarium oxysporum f. sp. lycopersici race 1 were analyzed. An interspecific hybrid between a resistant Lycopersicon pennellii and a susceptible L. esculentum was backcrossed to L. esculentum. The genotype of each backcross-1 (BC1) plant with respect to its Fusarium response was determined by means of backcross-2 progeny tests. Resistance was controlled by a single dominant gene, I1, which was not allelic to I, the traditional gene for resistance against the same fungal pathogen that was derived from L. pimpinellifolium. Linkage analysis of 154 molecular markers that segregated in the BC1 population placed I1 between the RFLP markers TG20 and TG128 on chromosome 7. The flanking markers were used to verify the assignment of the I1 genotype in the segregating population. The results are discussed with reference to the possibility of cloning Fusarium resistance genes in tomato.

Genetic and physical mapping of the patatin genes in potato and tomato

Genes for the major storage protein of potato, patatin, have been mapped genetically and physically in both the potato and tomato genomes. In potato, all patatin genes detected by the cDNA clone pGM01 map to a single locus at the end of the long arm of chromosome 8. By means of pulsed field gel electrophoresis (PFGE) it was possible further to delimit this locus, containing 10-15 copies of the gene, to a maximum size of 1.4 million base pairs. Hybridizations with class-specific clones suggest that the locus is at least partially divided into domains containing the two major types of patatin genes, class I and II. In tomato, patatin-homologous sequences were found to reside at the orthologous locus at the end of chromosome 8. The approximately three copies in tomato were localized by PFGE to a single fragment of 300 kilobases. Whereas the class II-specific 5' promoter sequences reside in tomato at the same locus as the coding sequences, the single class I-specific copy of the 5' promoter sequences was localized on chromosome 3 with no coding sequence attached to it. A clone from this chromosome 3 locus of tomato was isolated and by restriction fragment length polymorphism mapping it could be further shown that a similar class I-specific sequence also exists on chromosome 3 of potato. As in tomato, this copy on chromosome 3 is not linked to a coding sequence for patatin. The results are discussed with respect to genome evolution and PFGE analysis of complex gene families.

Molecular analysis of a U3 RNA gene locus in tomato: transcription signals, the coding region, expression in transgenic tobacco plants and tandemly repeated pseudogenes

By screening a tomato genomic library with a tomato U3 RNA probe, we detected a U3 genomic locus whose coding region was determined by primer extension (5' end) and direct RNA sequencing of purified U3 RNA from tomato (3' end). Tomato U3 RNA is 216 nucleotides long, contains all the four evolutionarily highly conserved sequence blocks (Boxes A to D), has at its 5' end a cap not precipitable with anti-m3G antibodies and can be folded into a peculiar secondary structure with two stem-loops at its 5' end. A tagged derivative of the U3 gene was faithfully expressed in transgenic tobacco plants. In the 5' flanking region both plant-specific UsnRNA transcription signals [the TATA-like sequence and the upstream sequence element (USE)] were present, but were positioned closer to each other and also to the cap site in the U3 gene than in the genes for the plant spliceosomal UsnRNAs studied so far. The 3' flanking region of the tomato U3 gene lacked the consensus sequence of the putative termination signal established for the plant spliceosomal UsnRNA genes and contained a pyrimidine-rich tract (R1) followed by four tandemly repeated U3 pseudogenes (U3.1 ps to U3.4 ps) flanked by slightly altered forms (R2 to R5) of R1 and most probably generated by DNA-mediated events. Our results are in line with the conjecture that the enzyme transcribing the tomato U3 gene has different structural requirements for transcriptional activity than the enzyme transcribing plant U1, U2 and U5 genes.