Cryptic introgressions contribute to transgressive segregation for early blight resistance in tomato.

KEY MESSAGE: We identified cryptic early blight resistance introgressions in tomato breeding lines and demonstrated efficient genotypic selection for resistance in the context of a tomato breeding program. Early blight is a widespread and problematic disease affecting tomatoes (Solanum lycopersicum). Caused by the fungal pathogen Alternaria linariae (syn. A. tomatophila), symptoms include lesions on tomato stems, fruit, and foliage, often resulting in yield losses. Breeding tomatoes with genetic resistance would enhance production sustainability. Using cross-market breeding populations, we identified several quantitative trait loci (QTL) associated with early blight resistance. Early blight resistance putatively derived from 'Campbell 1943' was confirmed in modern fresh market tomato breeding lines. This resistance offered substantial protection against early blight stem lesions (collar rot) and moderate protection from defoliation. A distinctive and potentially novel form of early blight foliar resistance was discovered in a processing tomato breeding line and is probably derived from S. pimpinellifolium via 'Hawaii 7998'. Additional field trials validated the three most promising large-effect QTL, EB-1.2, EB-5, and EB-9. Resistance effects for EB-5 and EB-9 were consistent across breeding populations and environments, while EB-1.2's effect was population specific. Using genome-wide marker-assisted backcrossing, we developed fresh market tomato lines that were near-isogenic for early blight QTL. Resistance in these lines was largely mediated by just two QTL, EB-5 and EB-9, that together captured 49.0 and 68.7% of the defoliation and stem lesion variance, respectively. Our work showcases the value of mining cryptic introgressions in tomato lines, and across market classes, for use as additional sources of disease resistance.

Identification of quantitative trait loci associated with acylsugar accumulation using intraspecific populations of the wild tomato, Lycopersicon pennellii.

Lycopersicon pennellii LA716, a wild relative of tomato, is resistant to a number of insect pests due to the accumulation of acylsugars exuded from type IV trichomes. These acylsugars are a class of compounds including both acylglucoses and acylsucroses. Intraspecific populations between L. pennellii LA716 and L. pennellii LA1912, the latter an accession that assorts for low-level acylsugar accumulation, were created to study the inheritance of type IV trichome density, acylsugar accumulation levels, percentage of acylsugars that are acylglucoses, and leaf area. The F2 population was subsequently used to determine genomic regions associated with these traits. The relative proportion of acylglucoses and acylsucroses was found to be largely controlled by a single locus near TG549 on chromosome 3. One locus on chromosome 10 showed significant associations with acylsugar levels. In addition, 1 locus on chromosome 4 showed significant associations with leaf area. Ten additional loci showed modest associations with one or more of the traits examined, 5 of which have been previously reported.

QTL analysis of pest resistance in the wild tomato Lycopersicon pennellii: QTLs controlling acylsugar level and composition.

Some accessions of Lycopersicon pennellii, a wild relative of the tomato Lycopersicon esculentum, are resistant to a number of important pests of cultivated tomato due to the accumulation of acylsugars, which constitute 90% of the exudate of type-IV trichomes in L. pennellii LA716. An interspecific F2 population, created by the cross L. esculentum x L. pennellii LA 716, was surveyed for acylsugar accumulation and subjected to RFLP/QTL analysis to determine the genomic regions associated with the accumulation of acylglucoses, acylsucroses, and total acylsugars, as well as with acylglucoses as a percentage of total acylsugars (mole percent acylglucoses). Data were analyzed using MAPMAKER/QTL with and without a log10 transformation. A threshold value of 2.4 (default value for MAPMAKER/QTL) was used, as well as 95% empirically derived threshold values. Five genomic regions, two on chromosome 2 and one each on chromosomes 3, 4 and 11, were detected as being associated with one or more aspects of acylsugar production. The L. esculentum allele is partially dominant to the L. pennellii allele in the regions on chromosomes 2 and 11, but the L. pennellii allele is dominant in the region on chromosome 3. Throughout this study, we report the comparative effects of analytical methodology on the identification of acylsugar QTLs. Similarities between our results and published results for the genus Solanum are also discussed.

A novel anther-expressed adh-homologous gene in Lycopersicon esculentum.

Two novel tandemly-oriented open reading frames (ORFs) with homology to alcohol dehydrogenase (ADH) were isolated from tomato. The predicted amino acid composition for each of the two tandem adh genes indicates the presence of 22 and 21, respectively, of 22 amino acids conserved in ADH proteins from plants and animals. However, comparison to known plant adh genes reveals a significantly lower similarity indicating that they belong to a novel class of ADHs. According to mapping data, the adh-homologous ORFs do not represent either of the previously studied adh1 or adh2 genes of tomato. The tandem genes, termed adh3a and adh3b, mapped to a distal region of the long arm of chromosome 4, unlike adh1, which maps closer to the centromere. Adh3a and adh3b have over 90% similarity to each other at the nucleotide and putative peptide levels. The adh3a gene has ten exons and nine introns with the transcription initiation site 57 bp upstream of the translation start. A putative TATA box and polyadenylation site have been identified. Adh3a is transcribed and, according to cDNA sequence analysis, fully processed in the late stages of anther development. According to transformation analysis, tissue-specific regulatory elements reside within the -448 to +724 region. The termination codon of adh3a is separated from the putative adh3b translation start site by 789 bp of intervening sequence. The 5' untranscribed sequences of each gene contain a stretch of 68 bp with 78% similarity. Within this stretch are sequences which are homologous to sequences found in anaerobically-induced or pollen-expressed genes from various plant species.

The effect of linkage on sample size determination for multiple trait selection.

Sufficient sample sizes are needed in breeding programs to be confident, with a specified probability α, of obtaining a specified number of plants of a desired genotype in segregating populations. We develop a method of determining the minimum sample size needed to produce, with specified probability α, at least m individuals of a desired genotype. This method takes into consideration factors affecting differential selection of gametes, segregation at a single locus, and linkage among the loci of interest. We first consider the effects in the gametophyte (haploid level) of fitness and linkage on the frequencies of alleles at two linked loci, then at three or more linked loci. The probability of obtaining at least m successes, or occurrences of the desired allele, among n gametes is given by a formula based on the binomial distribution. This probability is affected by fitness and linkage through their impact on the probability that a single randomly chosen gamete is of the desired type. Using an extension of this approach, we examine the effects of the altered allelic frequencies on the likelihood of obtaining the desired genotype from a randomly chosen pair of gametes in the sporophyte (diploid level). A table and a figure show the sample size required to produce, with probability 0.95, m individuals of the desired g enotype or phenotype, as a function of m and the probability that a randomly selected individual is of the desired type.

Novel mitochondrial genomes in Brassica napus somatic hybrids.

The mitochondrial genomes of nine male-fertile and two Ogura cytoplasmic male-sterile (cms) Brassica napus somatic hybrids were probed with 46 mitochondrial DNA fragments. The distribution of information obtained from each fusion partner was not random. Several regions, including the coxI gene and a major recombination repeat sequence, were always derived from the Brassica campestris fusion partner, and some regions were always derived from the Ogura mitochondrial genome. Novel fragments occurred in seven distinct regions. Some of the rearrangement breakpoints were located near the evolutionary breakpoints relating the mitochondrial genomes of the Brassica species. The sizes of the mitochondrial genomes in the somatic hybrids ranged from 224.8 to 285.3 kb. A direct correlation between a specific gene and the cms phenotype was not observed; however, a possible cms-associated region was identified. It corresponds to a region that was identified through analysis of fertile revertants from a cms B. napus cybrid.

Protoplast fusion-derived Ogura male sterile cauliflower with cold tolerance.

Cauliflower (Brassica oleracea L. ssp. botrytis) protoplasts with Ogura male sterile and fertile B. oleracea cytoplasms were fused, producing plants with an array of organellar types. Plants with Ogura mitochondria were male sterile; those with B. oleracea chloroplasts were cold tolerant. In some fusions, unfused parental protoplasts were eliminated by double inactivation with iodoacetate and gamma-irradiation; in others, fused protoplasts were physically isolated by micromanipulation or by cell sorting. Double inactivation fusions produced the most plants, including many which were male sterile, female fertile, cold tolerant and diploid.

Chimeric tomato plants show that aphid resistance and triacylglucose production are epidermal autonomous characters.

Graft chimeras were generated using Lycopersicon pennellii and L. esculentum to determine the contribution of the three meristem layers (L1, L2, and L3) to trichome density, sugar ester production, and aphid resistance. Sugar esters, in the form of triacylglucoses, have been implicated in the aphid resistance of pennellii. One chimera possessed the epidermal layer (L1) of pennellii and the internal tissues (L2 and L3) of the aphid-susceptible esculentum. The second chimera had both the L1 and L2 of pennellii and the L3 of esculentum. Type IV trichome densities did not differ significantly among the chimeras and pennellii. Both chimeras accumulated sugar esters with similar sugar and fatty acid composition as pennellii. The concentration of epicuticular sugar ester on the chimeras was also comparable with that of pennellii. Leaf cage and feeding studies demonstrated that both chimeras are as resistant to aphids as is pennellii. The resistance could be reduced similarly on all three types of plants by removal of the type IV trichome exudate. These results indicate that the presence and density of the type IV trichomes and the amount and type of sugar esters produced are features determined by the genotype of the epidermis. These epidermal features are sufficient to account for the aphid resistance observed in pennellii.

Mapping of ripening-related or -specific cDNA clones of tomato (Lycopersicon esculentum).

Nineteen ripening-related or -specific clones from Lycopersicon esculentum were mapped via RFLP analysis using an F2 population from the cross L. esculentum x L. pennellii and cDNA or genomic clones of known map location. The map produced using cDNA and genomic clones of known map location corresponded well with previously published maps of tomato. The number of loci detected for each ripening-related or-specific clone varied from one to seven. These loci were located on all 12 chromosomes of the tomato genome. There was no significant clustering of ripening-related or-specific genes. Regions of very low recombination were observed. The clone for polygalacturonase (TOM6) mapped to a single region on chromosome 10, the same chromosome as the nor and alc ripening mutants. To fine map this chromosome, two backcross populations were produced from the cross of L. esculentum x L. pimpenillifolium, in which the esculentum parents used were homozygous for either the alc or the nor. The coding region for polygalacturonase is functionally unlinked to either of these two ripening mutants.

Inheritance of potato aphid resistance in hybrids between Lycopersicon esculentum and L. pennellii.

The potato aphid, Macrosiphum euphorbiae Thomas, is an important pest of tomato, Lycopersicon esculentum Mill., because it transmits tomato viruses and directly reduces crop yields by its feeding. This study was conducted to determine whether the wild tomato species, Lycopersicon pennellii (Corr.) D'Arcy, would be useful as a source of potato aphid resistance for tomato. Type IV trichome density and aphid resistance were assessed in six generations (P1, P2, F1, F2, BC1P1, and BC1P2) from crosses between L. pennellii (LA 716) and two tomato cultivars, New Yorker and VF Vendor. Weighted leastsquares were used in joint scaling tests to estimate the relative importance of gene effects on type IV trichome density and potato aphid resistance of the hybrids. A simple additive-dominance model adequately explained the variation in type IV trichome density. Models which included digenic epistatic effects were required to explain the variation in aphid resistance. Standard unit heritability estimates of aphid resistance in the backcross to L. esculentum were obtained by regression of BC1F2 off-spring families on BC1F1 parents. Regression coefficients and heritability estimates varied between years with the level and uniformity of the aphid infestation. In the 1985-1986 growing seasons, when aphid infestations were uniform, aphid resistance exhibited a moderate level of heritability (29.8% ± 14.1% and 47.1% ± 11.5% in New Yorker and VF Vendor backcross populations, respectively). The non-uniform aphid infestation of 1984 resulted in lower heritability estimates in the 1984-1985 growing seasons (16.1% ± 15.7% and 21.9% ± 14.8% in the New Yorker and VF Vendor backcross populations, respectively). Selection for potato aphid resistance would probably be most efficient if it were delayed until gene combinations are fixed in later generations, because of the large epistatic effects and the low heritability of this trait in seasons with variable aphid infestations.

Atrazine-resistant cauliflower obtained by somatic hybridization between Brassica oleracea and ATR-B. napus.

Somatic hybridization between Brassica oleracea ssp. botrytis (cauliflower, 2n=18), carrying the Ogura (R1) male-sterile cytoplasm and B. napus (2n= 38), carrying a male-fertile, atrazine-resistant (ATR) cytoplasm, yielded three hybrids (2n=56) and six cauliflower cybrids (2n=18), which were selected for resistance to the herbicide in vitro. The hybrids and cybrids were male fertile and self-compatible. They contained both chloroplasts and mitochondria from the ATR cytoplasm. We found no evidence for mtDNA recombination in any of the regenerated plants. Selfed progeny of the B. oleracea atrazine-resistant cybrids were evaluated for tolerance to the herbicide in the field. Resistant plants exposed to 0.56-4.48 kg/ha (0.5-4.0 pounds/acre) atrazine in the soil showed no damage at any herbicide level, whereas plants of a susceptible alloplasmic line were severely damaged at the lowest level of herbicide application and killed at all higher levels. These atrazine-resistant cauliflower may have potential horticultural use, especially in fields where atrazine carry over is a serious problem.

Synthesis of male sterile, triazine-resistant Brassica napus by somatic hybridization between cytoplasmic male sterile B. oleracea and atrazine-resistant B. campestris.

Fusion of leaf protoplasts from an inbred line of Brassica oleracea ssp. botrytis (cauliflower, n=9) carrying the Ogura (R1) male sterile cytoplasm with hypocotyl protoplasts of B. campestris ssp. oleifera (cv "Candle", n=10) carrying an atrazine-resistant (ATR) cytoplasm resulted in the production of synthetic B. napus (n=19). Thirty-four somatic hybrids were produced; they were characterized for morphology, phosphoglucose isomerase isoenzymes, ribosomal DNA hybridization patterns, chromosome numbers, and organelle composition. All somatic hybrids carried atrazine-resistant chloroplasts derived from B. campestris. The mitochondrial genomes in 19 hybrids were examined by restriction endonuclease and Southern blot analyses. Twelve of the 19 hybrids contained mitochondria showing novel DNA restriction patterns; of these 12 hybrids, 5 were male sterile and 7 were male fertile. The remaining hybrids contained mitochondrial DNA that was identical to that of the ATR parent and all were male fertile.

Aphid deterrence by glucose esters in glandular trichome exudate of the wild tomato,Lycopersicon pennellii.

Settling of the potato aphid,Macrosiphum euphorbiae, on feeding membranes was deterred by methanolic leaf rinses ofLycopersicon pennellii, or of its F1 with tomato,L. esculentum. The active compounds in theL. pennellii rinsates were identified as 2,3,4-tri-O-acylglucoses bearing short to medium chain length fatty acids. These compounds are localized in the glandular exudate of the type IV trichomes and may accumulate to levels in excess of 400 µg/cm(2). In choice assays, purified glucose esters fromL. pennellii reduced aphid settling at concentrations as low as 25 µg/cm(2); at concentrations of 150 µg/cm(2) or more, all aphids avoided treated areas. Glucose esters were also active in deterring aphid settling in no-choice assays. At 100 µg/ cm(2), these esters resulted in increased levels of mortality after 48 hr.

Polyamine content of long-keeping alcobaca tomato fruit.

Fruit of tomato landrace Alcobaca, containing the recessive allele alc, ripen more slowly, with a reduced level of ethylene production, and have prolonged keeping qualities. The levels of polyamines in pericarp tissues of alc and ;wild type' Alc (cv Rutgers and Alcobaca-red) fruit were measured by HPLC in relation to ripening. Putrescine was the predominant polyamine with a lower content of spermidine, while spermine was just detectable. The level of putrescine was high at the immature green stage and declined in the mature green stage. In Alc fruit the decline persisted but in alc fruit the putrescine level increased during ripening to a level similar to that present at the immature green stage. There was no pronounced change or difference in spermidine levels. The enhanced polyamine level in alc fruit may account for their ripening and storage characteristics.

Analysis of organelle genomes in a somatic hybrid derived from cytoplasmic male-sterile Brassica oleracea and atrazine-resistant B. campestris.

An atrazine-resistant, male-fertile Brassica napus plant was synthesized by fusion of protoplasts from the diploid species B. oleracea and B. campestris. Leaf protoplasts from B. oleracea var. italica carrying the Ogura male-sterile cytoplasm derived from Raphanus sativus were fused with etiolated hypocotyl protoplasts of atrazine-resistant B. campestris. The selection procedure was based on the inability of B. campestris protoplasts to regenerate in the media used, and the reduction of light-induced growth of B. oleracea tissue by atrazine. A somatic hybrid plant that differed in morphology from both B. oleracea and B. campestris was regenerated on medium containing 50 µM atrazine. Its chromosome number was 36-38, approximately that of B. napus. Furthermore, nuclear ribosomal DNA from this hybrid was a mixture of both parental rDNAs. Southern blot analyses of chloroplast DNA and an assay involving tetrazolium blue indicated that the hybrid contained atrazine-resistant B. campestris chloroplasts. The hybrid's mitochondrial genome was recombinant, containing fragments unique to each parent, as well as novel fragments carrying putative crossover points. Although the plant was female-sterile, it was successfully used to pollinate B. napus.

Variation in the Accumulation of Seed Storage Protein Among Genotypes of Phaseolus vulgaris (L.).

Differences in the accumulation of total seed protein and globulin-1 (G1) protein were detected among three inbred lines of common bean. Total protein accumulation ranged from 2.3 to 3.7 milligrams per cotyledon pair per day among lines. In all lines the dry weight and protein accumulation ceased and a loss of chlorophyll in the cotyledons occurred when the moisture content had fallen to 50% of fresh weight. G1 was first detected and rapid accumulation began 14 days after flowering in two lines, whereas in the cultivar Endogava zurundi namame, rapid accumulation was delayed until 20 days after flowering. Rates of G1 accumulation ranged from 1.0 to 1.8 milligrams G1 per cotyledon pair per day among lines. G1 accumulation ceased 6 days before the end of total protein accumulation in Sanilac. A steady rate of protein accumulation was observed in Sanilac, but pauses in the accumulation of G1 and of total protein were documented in Endogava zurundi namame. The rate of G1 accumulation preceding and following the pause in Endogava zurundi namame was 2.7 milligrams G1 per cotyledon pair per day, nearly double that of the other lines.

Protein Synthesis and Accumulation in Bean Cotyledons during Growth.

Analysis of total protein, of specific proteins by gel electrophoresis and immunoelectrophoresis, and of protein synthetic activity in vitro confirmed that intense protein synthesis and accumulation occurred as the French bean (Phaseolus vulgaris L). seed grew from 12 to 20 millimeters. These techniques showed that there was no globulin-1 (G1) fraction (requiring high salt for solubility) present in 6-millimeter seeds, and only very small amounts were synthesized in seeds less than 9 millimeters long. The 7- to 9-millimeter stages represent a 2-day transition period over which genetic information for the G1 protein becomes actively expressed, accounting for at least 50% of all protein synthesized in this tissue during the following 14 days. At maturity, the electrophoretic analysis confirmed that G1 globulin was the major storage protein, representing some 50% of the dry seed protein. Cell-free protein synthesis assays, including immunoprecipitation of the in vitro products, clearly showed G1 polypeptides to be among the polysome-directed products.