Molecular mapping of chromosome segments introgressed from Solanum lycopersicoides into cultivated tomato (Lycopersicon esculentum)

The wild nightshade Solanum lycopersicoides (accessionLA2951) was backcrossed to the cultivated tomato (Lycopersicon esculentum cv ’VF36’), then inbred through single-seed descent for several generations. Over 300 backcross-inbred families thereby derived were genotyped at 139 marker loci, consisting of RFLPs, allozymes, and monogenic morphological markers, to identify introgressed S. lycopersicoides chromosomes and segments thereof. The pattern of genotypes observed in the lines indicated a high degree of overall synteny between the S. lycopersicoides genome and that of tomato. Two putative single-copy RFLP probes revealed secondary loci in this wide cross. Recovery of the L. esculentum genome was more rapid than expected, with an average value in the BC₂ generation of 97.8%, versus the expected value of 87.5%. This was due to widespread segregation distortion that favored L. esculentum alleles as well as a tendency for plants homozygous for in- trogressed segments to be partially or completely male-sterile, thereby preventing the fixation of S. lycopersicoides markers in many lines. Despite these difficulties, nearly every S. lycopersicoides marker (or approximately 98% of the genome, measured in centi Morgans) was represented in at least 1 backcross-inbred line, with only a region on chromosome 4L missing from the population as a whole. Although the extent of transmission and fixation of introgressed segments varied according to chromosome, overall approximately 66% of the S. lycopersicoides genome was represented by homozygous in- trogressions with sufficient fertility to reproduce by self-pollination. An excess of terminal (vs. interstitial) segments was noted, and putative heterozygous substitutions for chromosomes 6, 7, 8, and 10 were found. Recombination within certain introgressed regions was reduced over 100-fold. These backcross-inbred lines are expected to facilitate the genetic analysis of traits identified in S. lycopersicoides and their transfer into horticultural tomatoes. Received: 16 March 1999 / Accepted: 22 June 1999     

Regeneration of intergeneric somatic hybrid plants between Lycopersicon esculentum and Solanum muricatum

Summary Mesophyll protoplasts of tomato (Lycopersicon esculentum) and pepino (Solanum muricatum) were fused by using an electrofusion method and cultured in modified MS medium supplemented with naphthaleneacetic acid and kinetin, in which only pepino and somatic hybrid protoplasts could divide. Somatic hybrid plants showing intermediate characteristics in morphology were regenerated from the calli exhibiting vigorous growth in contrast with those of pepino. The hybrid nature of these plants was confirmed by cytological observation and biochemical analyses of phosphoglucomutase isozymes and the fraction-1-protein. The regenerated somatic hybrids grew to flowering stage and set fruits.     

Marker-assisted introgression of blackmold resistance QTL alleles from wild Lycopersicon cheesmanii to cultivated tomato (L. esculentum) and evaluation of QTL phenotypic effects

Blackmold, caused by the fungus Alternaria alternata, is a major ripe fruit disease of processing tomatoes. Previously, we found blackmold resistance in a wild tomato (Lycopersicon cheesmanii) and quantitative trait loci (QTL) for resistance were mapped in an interspecific population. Five QTLs were selected for introgression from L. cheesmanii into cultivated tomato using marker-assisted selection (MAS). Restriction fragment length polymorphism and PCR-based markers flanking, and within, the chromosomal regions containing QTLs were used for MAS during backcross and selfing generations. BC₁ plants heterozygous at the QTLs, and subsequent BC₁S₁ and BC₁S₂ lines possessing different homozygous combinations of alleles at the target QTLs, were identified using DNA markers. Field experiments were conducted in 1998 (with 80 marker-selected BC₁S₂ lines) and 1999 (with 151 marker-selected BC₁S₂ and BC₁S₃ lines) at three California locations. Blackmold resistance was assessed during both years, and horticultural traits were evaluated in 1999. The BC₁S₂ and BC₁S₃ lines containing L. cheesmanii alleles at the QTLs were associated with a large genetic variance for resistance to blackmold and moderate heritability, suggesting that significant genetic gain may be achieved by selection in this genetic material. L. cheesmanii alleles at three of the five introgressed QTLs showed a significant, positive effect on blackmold resistance. A QTL on chromosome 2 had the largest positive effect on blackmold resistance, alone and in combination with other QTLs, and was also associated with earliness, a positive horticultural trait. The other four QTLs were associated primarily with negative horticultural traits. Fine mapping QTLs using near isogenic lines could help determine if such trait associations are due to linkage drag or pleiotropy.     

Marker-assisted transfer of acylsugar-mediated pest resistance from the wild tomato,Lycopersicon pennellii,to the cultivated tomato,Lycopersicon esculentum

Acylsugars exuded from type IV trichomes mediate the multiple pest resistance found in the wild tomato species, Lycopersicon pennellii. A marker-assisted selection breeding program was used to attempt the transfer of the ability to accumulate acylsugars to cultivated tomato. RFLP and PCR-based markers were used through three backcross generations to select plants containing 5 target regions associated by QTL analysis with acylsugar accumulation. The BC1F1 plant selected possessed all 5 target regions and accumulated acylsugars at a moderate level similar to that of the interspecific F1 control. The BC2F1 and BC3F1 selections contained complementary subsets of the 5 target regions and did not accumulate acylsugars. BC3F1 plants with complementary subsets of the 5 target regions were intermated to produce populations segregating for the 5 target regions. From 1000 BC3F1-intermated plants, three plants were found which accumulated acylsugars at low levels and contained 3 to 5 of the target regions. The recovery of acylsugar accumulation in progeny of the intermated BC3F1 plants supports the involvement of at least some of the 5 target regions in acylsugar biosynthesis. However, since the levels of acylsugars accumulated by these plants were lower than that of the interspecific F1, it is likely that another, as of yet unidentified, region is necessary for accumulation of higher levels of acylsugars.     

Specificity of induced resistance in the tomato, Lycopersicon esculentum

Specificity in the induced responses of tomato foliage to arthropod herbivores was investigated. We distinguished between two aspects of specificity: specificity of effect (the range of organisms affected by a given induced response), and specificity of elicitation (ability of the plant to generate distinct chemical responses to different damage types). Specificity of effect was investigated by examining the effect of restricted feeding by Helicoverpa zea on the resistance of tomato plants to an aphid species (Macrosiphum euphorbiae), a mite species (Tetranychus urticae), a noctuid species (Spodoptera exigua), and to a phytopathogen, Pseudomonas syringae pv. tomato. Prior H. zea feeding was found to increase the resistance of tomato plants to all four organisms. Specificity in elicitation was investigated by examining the effect of aphid feeding on the activities of four defense-related proteins and on the suitability of foliage for S. exigua. Aphid feeding was found to induce peroxidase and lipoxygenase activities but not polyphenol oxidase and proteinase inhibitor activities; this response is distinct from the response to H. zea feeding, which induces polyphenol oxidase and proteinase inhibitors but not peroxidase. Leaflets which had been fed upon by aphids were better sources of food for S. exigua than were leaflets which had not been fed upon by aphids. Studies of both these aspects of specificity are needed to understand the way in which plants coordinate and integrate induced responses against insects with other physiological processes. Received: 20 December 1996 / Accepted: 2 July 1997     

Asymmetric somatic hybridization between tomato (Lycopersicon esculentum Mill) and gamma-irradiated potato (Solanum tuberosum L.): a quantitative analysis

We analyzed 110 asymmetric fusion products, obtained by fusion of hygromycin-resistant tomato protoplasts and gamma-irradiated kanamycin-resistant potato protoplasts that expressed -glucuronidase (GUS). The fusion products were selected for resistance to both antibiotics, and were subsequently analyzed for their shoot regeneration potential, GUS activity, expression of two potato isoenzymes, chloroplast type, total genomic DNA content, and relative genomic composition. No viable plants could be obtained and the calli were highly polypoid. All hybrids expressed GUS activity, whereas they displayed a large variation with respect to the other traits.     

Characterization of the subtilase gene family in tomato (Lycopersicon esculentum Mill.)

The gene family of subtilisin-like serine proteases (subtilases, SBTs) in tomato (Lycopersicon esculentum Mill.) comprises at least 15 members, 12 of which have been characterized in this study. Sequence comparison revealed that tomato subtilases fall into 5 distinct subfamilies. Single genes were shown to exist for LeSBT1, LeSBT2 and tmp, while 5 and 6 genes were found in the LeSBT3/4 and P69 subfamilies, respectively. With the exception of tmp, tomato subtilase genes were found to lack introns. Expression of subtilase genes was confirmed at the mRNA level by northern blot analysis and/or by primer extension experiments. For each of the 5 subtilase subfamilies, a distinctive pattern of expression was observed in tomato organs. At least one of the subtilases was found to be expressed in each organ analysed. Structural features evident from deduced amino acid sequences are discussed with reference to the related mammalian proprotein convertases.     

Cytogenetic analysis of Lycopersicon esculentum (+) Solanum etuberosum somatic hybrids and their androgenetic regenerants

The aim of the study was to characterize genomic relationships among cultivated tomato (Lycopersicon esculentum Mill.) (2n=2x=24) and diploid (2n=2x=24) non-tuberous wild Solanum species (S. etuberosum Lindl.). Using genomic in situ hybridization (GISH) of mitotic and meiotic chromosomes, we analyzed intergeneric somatic hybrids between tomato and S. etuberosum. Of the five somatic hybrids, two plants were amphidiploids (2n=4x=48) mostly forming intragenomic bivalents in their microsporocytes, with a very low frequency of multivalents involving the chromosomes of tomato and S. etuberosum (less than 0.2 per meiocyte). Tomato chromosomes showed preferential elimination during subsequent meiotic divisions of the amphidiploids. Transmission of the parental chromosomes into microspores was also evaluated by GISH analysis of androgenic plants produced by direct embryogenesis from the amphidiploid somatic hybrids. Of the four androgenic regenerants, three were diploids (2n=2x=24 or 2n=2x+1=25) derived from reduced male gametes of the somatic hybrids, and one plant was a hypertetraploid (2n=4x+4=52). GISH revealed that each anther-derived plant had a unique chromosome composition. The prospects for introgression of desirable traits from S. etuberosum into the gene pool of cultivated tomato are discussed. Received: 2 August 2000 / Accepted: 4 December 2000     

Stomatal Response to Light of Solanum pennellii, Lycopersicon esculentum, and a Graft-induced Chimera

To learn how species differences in stomatal behavior are regulated, the response of epidermal and leaf diffusive resistance to light was investigated in Lycopersicon esculentum Mill., Solanum pennellii Corr., and a periclinal chimera having an S. pennellii epidermis and an L. esculentum mesophyll that was produced from a graft of the two species. S. pennellii has about 23% fewer stomata per square millimeter than does L. esculentum, and the two species have contrasting stomatal sensitivities to light. The abaxial stomata of L. esculentum open in dimmer light and to a greater extent than the adaxial stomata. The abaxial and adaxial stomata of S. pennellii respond similarly to light incident on the adaxial epidermis and are less open at all quantum flux densities than comparable stomata of L. esculentum. The patterns of response to light of the abaxial and adaxial stomata of the chimera were practically identical to those of L. esculentum, and quite unlike those of S. pennellii. Thus, the pattern of stomatal light response in the chimera was regulated by the L. esculentum mesophyll. The reduction in stomatal frequency of the chimera, which was regulated by the epidermis of S. pennellii, contributed to the 40% difference in leaf diffusive resistance between the plants in moderate light.     

Cloning and characterization of salicylic acid-induced, intracellular pathogenesis-related gene from tomato (Lycopersicon esculentum)

Intracellular pathogenesis-related gene (IPR) from tomato was cloned from a salicylic acid (SA) induced cDNA library and was designated asTSI-1 (tomato stress induced-1). The deduced amino acid sequence ofTSI-1 codes for a 178 amino acid polypeptide of molecular weight 20.4 kDa.TSI-1 is highly homologous to the potatoSTH-2 andSTH-21 IPR and tree pollen allergens which cause type I allergic reactions in humans.TSI-1 lacks a signal peptide like other IPR members. It is organized as a multigene family and is inducible by SA andFusarium oxysporum infection.     

Interspecific protoplast fusion for the transfer of atrazine resistance from Solanum nigrum to tomato (Lycopersicon esculentum. L)

TAES Technical Article #23184     

Use of leaky nitrate reductase-deficient mutants of tomato (Lycopersicon esculentum Mill.) for selection of somatic hybrid cell lines with wild type potato (Solanum tuberosum L.)

Protoplasts of two leaky nitrate reductase deficient and thus nitrate auxotrophic (NAR) mutants of tomato and their wild types, were fused with protoplasts of monoploid potato. In all four combinations hybrid calli grew more vigorously than parental calli and this somatic hybrid vigour as such provided a useful enrichment for somatic hybrids. Selection against nitrate auxotrophy further increased the efficiency of the enrichment, particularly if a molybdenum cofactor mutation was used as the basis for the selection. It is concluded that the nitrate auxotrophy of these NAR mutants is sufficiently expressed at the level of the cell, to allow its use in somatic hybridization experiments with potato.     

Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-22 from Lycopersicon esculentum

In tomato, infections by tomato mosaic virus are controlled by durable Tm-2² resistance. In order to gain insight into the processes underlying disease resistance and its durability, we cloned and analysed the Tm-2² resistance gene and the susceptible allele, tm-2. The Tm-2² gene was isolated by transposon tagging using a screen in which plants with a destroyed Tm-2² gene survive. The Tm-2² locus consists of a single gene that encodes an 861 amino acid polypeptide, which belongs to the CC-NBS-LRR class of resistance proteins. The putative tm-2 allele was cloned from susceptible tomato lines via PCR with primers based on the Tm-2² sequence. Interestingly, the tm-2 gene has an open reading frame that is comparable to the Tm-2² allele. Between the tm-2 and the Tm-2² polypeptide 38 amino acid differences are present of which 26 are located in the second half of the LRR-domain. Susceptible tomato plants, which were transformed with the Tm-2² gene, displayed resistance against ToMV infection. In addition, virus specificity, displayed by the Tm-2² resistance was conserved in these transgenic lines. To explain the durability of this resistance, it is proposed that the Tm-2²-encoded resistance is aimed at the Achilles' heel of the virus.     

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.     

Introgression and DNA marker analysis of Lycopersicon peruvianum, a wild relative of the cultivated tomato, into Lycopersicon esculentum, followed through three successive backcross generations

 Segregation of the Lycopersicon peruvianum genome was followed through three generations of backcrossing to the cultivated tomato L. esculentum cv ‘E6203’ using molecular markers. Thirteen BC₁ plants were genotyped with 113 markers, 67 BC₂ plants with 84 markers, and finally 241 BC₃ plants were genotyped with 177 markers covering the entire genome and a BC₃ map constructed. Several segments of the genome, including parts of chromosomes 3, 4, 6, and 10, quickly became fixed for esculentum alleles, possibly due to sterility problems encountered in the BC₁. Observed overall heterozygosity and chromosome segment lengths at each generation were very near the expected theoretical values. Markers located near the top telomeric region of chromosome 9 showed segregation highly skewed towards the wild allele through all generations, suggesting the presence of a gamete promoter gene. One markers, TG9, mapped to a new position on chromosome 9, implying an intrachromosomal translocation event. Despite the great genetic distance between the two parents, overall recombination was only 25% less than that observed in a previous tomato cross, indicating that L. peruvianum genes may be more readily introgressed into cultivated germplasm than originally believed. Received: 9 April 1997 / Accepted : 20 May 1997     

Organization of a Solatium brevidens repetitive sequence related to the TGRI subtelomeric repeats of Lycopersicon esculentum

A species-specific repetitive DNA fragment has been isolated from a genomic library of Solanum brevidens. Sequence analysis revealed a regular organization of three non-homologous subrepeats forming tandemly-arranged composite repetitive units. Interpretation of Southern hybridization patterns based on the known sequence data suggests that the isolated sequence element represents an abundant organization type, although the presence of simple tandem arrays of the subrepeats is also indicated. Seventy-four percent sequence similarity was found between one of the S. brevidens subrepeats (Sb4AX) and a satellite DNA (TGRI) localized as a subtelomeric repeat on almost all Lycopersicon esculentum chromosomes. Insitu hybridization indicated that, similarly to TGRI, the S. brevidens-specific repeats are located at the ends of the arms of several chromosomes. On the basis of the data obtained, a common ancestral sequence can be proposed for the tomato (TGRI) and the S. brevidens (Sb4AX) repeat however, the molecular organization of this element in these two species evolved in a basically different manner.     

Cloning and characterization of the gene for phytoene desaturase (Pds) from tomato (Lycopersicon esculentum)

The gene Pds encodes phytoene desaturase, a key enzyme in carotenoid biosynthesis that converts phytoene to -carotene. We have cloned and analyzed the genomic DNA sequence of Pds from tomato. In tomato Pds is comprised of 15 exons that, together with the introns occupy over 8 kb. A putative promoter sequence has been identified by comparison with the cDNA sequence of Pds. A consensus nucleotide sequence around intron splicing sites in tomato genes was determined by compiling data on 137 introns in 34 genes. This consensus sequence generally agrees with the consensus sequence of other higher plants with only minor differences that are unique to tomato.     

Amount of introgression in flycatcher hybrid zones reflects regional differences in pre and post-zygotic barriers to gene exchange

Introgression is the incorporation of alleles from one species or semispecies into the gene pool of another through hybridization and backcrossing. The rate at which this occurs depends on the frequency of hybridization and the fitness of hybrids and backcrosses compared to 'pure' individuals. The collared flycatcher (Ficedula albicollis) and the pied flycatcher (F. hypoleuca) co-exist and hybridize at low to moderate frequencies in a clinal hybrid zone in Central Europe and on the islands of Gotland and Oland off the Swedish east coast. Data on hatching success suggest that hybrids are less fertile in Central Europe compared to on the islands. Direct fitness estimates using molecular markers to infer paternity are consistent with the demographic data. Applying a tag-array-based minisequencing assay to genotype interspecific substitutions and single nucleotide polymorphisms we demonstrate that the amount of introgression from the pied to the collared flycatcher is higher in the two island populations (Gotland and Oland) than in two geographically distinct areas from the Central European hybrid zone (Czech Republic and Hungary). In all areas the amount of introgression from collared to pied flycatchers is very low or seemingly absent. The different patterns of introgression are consistent with regional differences in rates of hybridization and fitness of hybrids. We suggest that barriers to gene exchange may have been partly broken down on the islands due to asymmetric gene flow from allopatry. Alternatively, or in addition, more pronounced reinforcement of prezygotic isolation in Central Europe might have increased post-zygotic isolation through hitchhiking, since genes affecting pre and post-zygotic isolation are both sex-linked in these birds. One of our genetic markers appears to introgress from pied to collared flycatchers at a much higher rate than the other markers. We discuss the possibility that the introgressed marker may be linked to a gene which is under positive selection in the novel genetic background.