Localization of<Emphasis Type="Italic"> jointless-2</Emphasis> gene in the centromeric region of tomato chromosome 12 based on high resolution genetic and physical mapping

Abstract Abscission is a universal process whereby plants shed their organs, such as flowers, fruit and leaves. In tomato, the non-allelic mutations jointless and jointless-2 have been discovered as recessive mutations that completely suppress the formation of pedicel abscission zones. A high resolution genetic map of jointless-2 was constructed using 1,122 jointless F₂ plants. Restriction fragment length polymorphism (RFLP) marker RPD140 completely co-segregated with the jointless-2 locus and mapped in a 2.4 cM interval between RFLP markers CD22 and TG618. To chromosome walk to jointless-2, all three markers were used to screen a bacterial artificial chromosome (BAC) library and contigs were developed. Intensive efforts to expand and merge the BAC contigs were unsuccessful because of the highly repetitive sequence content on the distal ends of each contig. To determine the physical distance between and the orientation of the three contigs, we used high resolution pachytene fluorescence in situ hybridization (FISH) mapping. The RPD140 contig was positioned in the centromeric region of chromosome 12 between two large pericentric heterochromatin blocks, about 50 Mb from the TG618 contig on the short arm and 10 Mb from the CD22 contig on the long arm, respectively. Based on high resolution genetic and physical mapping, we conclude that the jointless-2 gene is located within or near the chromosome 12 centromere where 1 cM is approximately 25 Mb in length.Communicated by Q. ZhangM.A. Budiman, S-B. Chang and S. Lee contributed equally to the work.     

Map-based cloning in crop plants. Tomato as a model system: I. Genetic and physical mapping of jointless

A map-based cloning scheme is being used to isolate the jointless (j) gene of tomato. The jointless locus is defined by a single recessive mutation that completely suppresses the formation of the fruit and flower pedicel and peduncle abscission zone. jointless was mapped in an F₂ population of an interspecific cross between Lycopersicon esculentum and Lycopersicon pennellii to a 7.1 cM interval between two restriction fragment length polymorphism (RFLP) markers TG523 and TG194. Isogenic DNA pools were then constructed from a subset of the mapping population and screened with 800 random decamers for random amplification of polymorphic DNA (RAPD) polymorphisms. Five new RAPD markers were isolated and mapped to chromosome 11, two of which were mapped within the targeted interval. One marker, RPD158, was mapped 1.5 cM to the opposite side of jointless relative to TG523 and thus narrowed the interval between the closest flanking markers to 3.0 cM. Physical mapping by pulse-field gel electrophoresis using TG523 and RPD158 as probes demonstrated that both markers hybridize to a common 600 kb SmaI restriction fragment. This provided an estimate of 200 kb/cM for the relationship between physical and genetic distances in the region of chromosome 11 containing the j locus. The combined results provide evidence for the feasibility of the next step toward isolation of the jointless gene by map-based cloning — a chromosome walk or jump to jointless.     

Map-based cloning in crop plants. Tomato as a model system: I. Genetic and physical mapping of jointless

A map-based cloning scheme is being used to isolate the jointless (j) gene of tomato. The jointless locus is defined by a single recessive mutation that completely suppresses the formation of the fruit and flower pedicel and peduncle abscission zone. jointless was mapped in an F₂ population of an interspecific cross between Lycopersicon esculentum and Lycopersicon pennellii to a 7.1 cM interval between two restriction fragment length polymorphism (RFLP) markers TG523 and TG194. Isogenic DNA pools were then constructed from a subset of the mapping population and screened with 800 random decamers for random amplification of polymorphic DNA (RAPD) polymorphisms. Five new RAPD markers were isolated and mapped to chromosome 11, two of which were mapped within the targeted interval. One marker, RPD158, was mapped 1.5 cM to the opposite side of jointless relative to TG523 and thus narrowed the interval between the closest flanking markers to 3.0 cM. Physical mapping by pulse-field gel electrophoresis using TG523 and RPD158 as probes demonstrated that both markers hybridize to a common 600 kb SmaI restriction fragment. This provided an estimate of 200 kb/cM for the relationship between physical and genetic distances in the region of chromosome 11 containing the j locus. The combined results provide evidence for the feasibility of the next step toward isolation of the jointless gene by map-based cloning — a chromosome walk or jump to jointless.     

Map-based cloning in crop plants: tomato as a model system II. Isolation and characterization of a set of overlapping yeast artificial chromosomes encompassing the jointless locus

A map-based cloning technique for crop plants is being developed using tomato as a model system. The target gene jointless is a recessive mutation that completely suppresses the formation of flower and fruit pedicel abscission zones. Previously, the jointless locus was mapped to a 3 cM interval between the two molecular markers TG523 and RPD158. Physical mapping of the jointless region by pulsed-field gel electrophoresis demonstrated that TG523 and RPD158 reside on a 600 kb SmaI fragment. In this study, TG523 was used as a probe to screen a tomato yeast artificial chromosome (YAC) library. Six tomato YAC (TY) clones were isolated, ranging from 220 to 380 kb in size. Genetic mapping of YAC ends demonstrated that this set of overlapping YACs encompasses the jointless locus. Two YAC ends, TY159L (L indicates left end) and TY143R (R indicates right end), cosegregate with the jointless locus. Only one of the six YACs (TY142) contained single-copy DNA sequences at both ends that could be mapped. The two ends of TY142 were mapped to either side of the jointless locus, indicating that TY142 contains a contiguous 285 kb tomato DNA fragment that probably includes the jointless locus. Physical mapping of the TY142 clone revealed that TY159L and TY143R reside on a 55 kb SalI fragment. Southern blot hybridization analysis of the DNAs of tomato lines nearly isogenic for the jointless mutation has allowed localization of the target locus to a region of less than 50 kb within the TY142 clone.Communicated by H. Saedler     

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.     

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.     

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.     

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.     

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.     

fs8.1, a major QTL, sets the pattern of tomato carpel shape well before anthesis

fs8.1 is a major fruit-shape QTL differentiating fresh-market and processing tomatoes. Mature fruits from plants with the wild-type fresh-market alleles are round, whereas those with alleles from processing variety E6203 are elongated (sometimes referred to as blocky or square tomatoes). Fine mapping was undertaken to determine whether the effect is due to a single gene or several tightly linked genes. RAPD and RFLP linkage analysis, and substitution mapping of nearly isogenic lines (NILs) segregating for the 22.8 cM-TG176-CT92 interval at the top of chromosome 8 in tomato were used for high-resolution mapping. For the 1212 gametes screened in F₂ and F₃ families, it was determined that fs8.1 maps as a single locus near the centromere of chromosome 8. A comparative developmental study of fs8.1 NILs revealed that fs8.1 alleles exert their effects on fruit shape early in carpel development at least 6 days before anthesis. Field evaluations of the NILs indicate that fs8.1 affects not only fruit shape, fruit length, and fruit weight but also the number of flowers and fruits per inflorescence, and the harvest index. The date of first flower and fruit diameter were not significantly affected. Received: 19 July 1999 / Accepted: 16 December 1999     

Development of diagnostic PCR markers closely linked to the tomato powdery mildew resistance gene Ol-1 on chromosome 6 of tomato

Lycopersicon hirsutum G1.1560 is a wild accession of tomato that shows resistance to Oidium lycopersicum, a frequently occurring tomato powdery mildew. This resistance is largely controlled by an incompletely dominant gene Ol-1 near the Aps-1 locus in the vicinity of the resistance genes Mi and Cf-2/Cf-5. Using a new F₂ population (n=150) segregating for resistance, we mapped the Ol-1 gene more accurately to a location between the RFLP markers TG153 and TG164. Furthermore, in saturating the Ol-1 region with more molecular markers using bulked segregant analysis, we were able to identify five RAPDs associated with the resistance. These RAPDs were then sequenced and converted into SCAR markers: SCAB01 and SCAF10 were L. hirsutum-specific; SCAE16, SCAG11 and SCAK16 were L. esculentum-specific. By linkage analysis a dense integrated map comprising RFLP and SCAR markers near Ol-1 was obtained. This will facilitate a map-based cloning approach for Ol-1 and marker-assisted selection for powdery mildew resistance in tomato breeding. Received: 21 June 1999 / Accepted: 1 December 1999     

The genetic basis of pear-shaped tomato fruit

Molecular-marker analysis of a cross between yellow pear, a tomato variety bearing small, pear-shaped fruit, and the round-fruited, wild species, Lycopersicon pimpinellifolium LA1589, revealed that pear-shaped fruit is determined largely by a major QTL on chromosome 2 and, to a lesser extent, a minor QTL on chromosome 10. The locus on chromosome 2 was also detected in a cross between yellow pear and the round-fruited introgression line (IL2–5) which carried the distal portion of chromosome 2 from the Lycopersicon pennellii genome. Based on its map position, we propose that the locus detected on chromosome 2 is the same as a locus referred to as ovate in the early tomato literature (Linstrom 1926, 1927). The fruit-shape index (length/diameter) and neck constriction were highly correlated in both populations suggesting that ovate exerts control over both traits or that the genes for these traits are tightly linked on chromosome 2. Using two-way ANOVA test, the minor QTL on chromosome 10 showed no significant interaction with the ovate locus on chromosome 2 with respect to the fruit-shape index. For ovate round fruit was dominant to elongated fruit in the L. pimpinellifolium populations, but additive in the IL2–5 population. Thus far, no genes controlling fruit shape have been cloned. The molecular mapping of the ovate locus may ultimately lead to its isolation via map-based cloning. Received: 8 January 1999 / Accepted: 30 January 1999     

The Asc locus for resistance to Alternaria stem canker in tomato does not encode the enzyme aspartate carbamoyltransferase

The fungal disease resistance locus Alternaria stem canker (Asc) in tomato has been suggested to encode the enzyme aspartate carbamoyltransferase (AC Tase). To test this hypothesis a segment of the tomato ACTase gene was amplified by the polymerase chain reaction (PCR) using degenerate primers. The PCR product obtained was subsequently used to isolate an ACTase cDNA clone. Restriction fragment length polymorphism (RFLP) linkage analysis showed that the ACTase gene and the Asc locus do not cosegregate. RFLP mapping positioned the ACTase gene on chromosome 11, while the Asc locus is located on chromosome 3. These results exclude the possibility that the ACTase protein is encoded by the Asc locus.     

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.     

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     

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.     

Genetic characterization of the polycotyledon locus in tomato

Developmental mutants serve as a useful material to unravel the mechanisms necessary for organ development. The polycotyledon (poc) mutant of tomato, with multiple cotyledons in the seedling and varied phenotypic effects in the adult plant is one such mutant. Studies using physiological and anatomical methods in our lab suggest that POC is involved in the negative regulation of polar auxin transport, which is likely the reason for the pleiotropic phenotype in the mutant. Because of the physiological significance of the polycotyledon mutant described in this paper and also being first of its kind in tomato and also other plant species, we are using a map-based cloning approach to map the polycotyledon gene. Molecular mapping of this locus using segregating interspecific F₂ mapping population localized polycotyledon gene close to TG424 marker on the long arm of chromosome 9. The closest marker mapped was a PCR marker identified in this study, E8A2 at a distance of 7.4 cM from the poc locus. The absence of tightly linked RAPD markers and the non-availability of more mapped markers in this region led us to initiate chromosome walk to polycotyledon gene. Both the flanking markers TG248 and E8A2 were used to screen the BAC library and a contig was developed for TG248 marker. The BAC-end sequences were analyzed for their use as RFLP markers to enrich this region for markers. Analysis of the BAC-end sequences revealed that poc is localized in the region surrounded by copia-like retrotransposon elements explaining the absence of markers in the euchromatin region on long arm of chromosome 9. Further studies identified two BAC-end sequences which mapped around the poc locus and also indicated very low physical versus genetic distance ratio in this region. The double mutant analyses of poc with the other two known polycotyledon mutants of tomato, pct and dem revealed allelism with pct; therefore, the poc mutant was named as pct1-2, and also the original pct mutant was renamed as pct1-1.     

The genetic and functional analysis of flavor in commercial tomato: the FLORAL4 gene underlies a QTL for floral aroma volatiles in tomato fruit

Tomato (Solanum lycopersicum L.) has become a popular model for genetic studies of fruit flavor in the last two decades. In this article we present a study of tomato fruit flavor, including an analysis of the genetic, metabolic and sensorial variation of a collection of contemporary commercial glasshouse tomato cultivars, followed by a validation of the associations found by quantitative trait locus (QTL) analysis of representative biparental segregating populations. This led to the identification of the major sensorial and chemical components determining fruit flavor variation and detection of the underlying QTLs. The high representation of QTL haplotypes in the breeders’ germplasm suggests that there is great potential for applying these QTLs in current breeding programs aimed at improving tomato flavor. A QTL on chromosome 4 was found to affect the levels of the phenylalanine‐derived volatiles (PHEVs) 2‐phenylethanol, phenylacetaldehyde and 1‐nitro‐2‐phenylethane. Fruits of near‐isogenic lines contrasting for this locus and in the composition of PHEVs significantly differed in the perception of fruity and rose‐hip‐like aroma. The PHEV locus was fine mapped, which allowed for the identification of FLORAL4 as a candidate gene for PHEV regulation. Using a gene‐editing‐based (CRISPR‐CAS9) reverse‐genetics approach, FLORAL4 was demonstrated to be the key factor in this QTL affecting PHEV accumulation in tomato fruit.     

Characterization and mapping of a gene controlling shoot regeneration in tomato

The superior regeneration capacity of Lycopersicon peruvianum was introduced into the cultivated tomato Lycopersicon esculentum by backcrossing hybrid material with the tomato genotype VF11. In segregating material derived from these backcrosses, the ability to regenerate shoots on root explants cultured on a zeatin-containing medium, was highly correlated with the ability to regenerate shoots on established callus cultures. The efficient shoot-regenerating root explant system permitted us to study the genetics of this trait and to locate the genes involved, using a set of morphological markers defining all 12 tomato chromosomes. Depending on the tomato genotype, mono, -di- or trigenic ratios were observed. It is concluded that a dominant L. peruvianum allele at a locus (Rg-1) near the middle of chromosome 3 determines efficient shoot regeneration on root explants in tomato in combination with dominant alleles at one or two other loci of either L. peruvianum or L. esculentum origin. The map location of the Rg-1 locus was refined further using a number of chromosome-3-specific RFLPs. The addition of new classical and RFLP linkage data to existing literature data and subsequent processing resulted in a revised and integrated map of tomato chromosome 3. From a morphological and physiological analysis of genotypes differing in Rg phenotype, it is concluded that the genetic component associated with regeneration determines the maintenance of morphogenetic competence and not the sensitivity to hormones.