Introgression into tomato (Lycopersicon esculentum) of the L. chmielewskii sucrose accumulator gene (sucr) controlling fruit sugar composition.

High sucrose concentration in fruit of Lycopersicon chmielewskii is governed by the recessive sucrose accumulator gene (sucr) that is situated in the pericentromeric region of chromosome 3. The sucr gene was introgressed into the genetic background of the hexose-accumulating cultivated tomato (L. esculentum cv 'Hunt 100') by marker-assisted selection using tightly linked RFLP markers and a tomato acid invertase cDNA as probes for sucr. RFLP mapping indicated that the segment containing sucr comprised over 43.2 cM in the BC1F2 generation, representing over one-third of the total length of chromosome 3. By selecting for crossovers between sucr and the flanking visual marker r (yellow fruit flesh) and RFLP marker TG288, we were able to reduce the size of the sucr introgression fragment to 0.8-7.1 cM by the BC5 generation. Smaller recombinant fragments were not obtained despite screening a large BC6F2 population. The smallest sucr introgression reduced recombination between the flanking visual markers sy (sunny) and bls (baby lea syndrome) by 38%. To facilitate future introgression and recombination experiments, a PCR-based test for the sucr gene was developed using primers specific to the tomato invertase gene. This assay takes advantage of a small deletion that maps to the second intron of the L. chmielewskii nvertase gene. The assay detected significant allelic variation both within and between hexose- and sucrose-accumulating Lycopersicon spp.

Effects of the Lycopersicon chmielewskii sucrose accumulator gene (sucr) on fruit yield and quality parameters following introgression into tomato.

A gene controlling fruit sucrose accumulation, sucr, was introgressed from the wild tomato species Lycopersicon chmielewskii into the genetic background of a hexose-accumulating cultivated tomato, L. esculentum. During introgression, the size of the L. chmielewskii chromosomal segment containing sucr was reduced by selection for recombination between RFLP markers for the sucr gene and flanking loci. The effects of sucr on soluble solids content, fruit size, yield and other fruit parameters were studied in the genetic background of the processing tomato cultivar 'Huntl00'. In a segregating BC5F2 generation, the smallest introgression containing sucr-associated markers was necessary and sufficient to confer high-level sucrose accumulation, the effects of which were completely recessive. Fruit of sucr/sucr genotypes were smaller than those of +/sucr or +/+ genotypes at all stages of development. The timing of sugar accumulation and total sugar concentration were unaffected by sugar composition. No differences in total fruit biomass (fresh weight of red and green fruit) at harvest were observed between the genotypes, and sucrose accumulators produced greater numbers of fruit than hexose accumulators in one family. However, the proportion of ripe fruit at harvest, and hence yield of ripe fruit, as well as average ripe fruit weight and seed set were reduced in sucr/sucr genotypes. Sucrose accumulation was also associated with increased soluble solids content, consistency, serum viscosity, predicted paste yield and acidity, and decreased color rating. In the first backcross to L. chmielewskii, hexose accumulators (+/sucr) had larger fruit than sucrose accumulators (sucr/sucr), while no difference in soluble solids was detected.

Sink Metabolism in Tomato Fruit : IV. Genetic and Biochemical Analysis of Sucrose Accumulation.

Fruit of domesticated tomato (Lycopersicon esculentum) accumulate primarily glucose and fructose, whereas some wild tomato species, including Lycopersicon chmielewskii, accumulate sucrose. Genetic analysis of progeny resulting from a cross between L. chmielewskii and L. esculentum indicated that the sucrose-accumulating trait could be stably transferred and that the trait was controlled by the action of one or two recessive genes. Biochemical analysis of progeny resulting from this cross indicated that the sucrose-accumulating trait was associated with greatly reduced levels of acid invertase, but normal levels of sucrose synthase. Invertase from hexose-accumulating fruit was purified and could be resolved into three isoforms by chromatofocusing, each with isoelectric points between 5.1 and 5.5. The invertase isoforms showed identical polypeptide profiles on sodium dodecyl sulfate polyacrylamide gel electrophoresis, consisting of a primary 52 kilodalton polypeptide and two lower molecular mass polypeptides that appear to be degradation products of the 52 kilodalton polypeptide. The three invertase isoforms were indistinguishable based on pH, temperature, and substrate concentration dependence. Immunological detection of invertase indicated that the low level of invertase in sucrose-accumulating fruit was due to low levels of invertase protein rather than the presence of an invertase inhibitor. Based on comparison of genetic and biochemical data we speculate that a gene either encoding tomato fruit acid invertase or one required for its expression, plays an important role in determining sucrose accumulation.

Expression of unilateral incompatibility in pollen of Lycopersicon pennellii is determined by major loci on chromosomes 1, 6 and 10.

We have previously described gene introgression from the wild nightshade Solanum lycopersicoides into tomato (Lycopersicon esculentum) through the use of either diploid or sesquidiploid hybrids (the latter consisting of two genomes of L. esculentum and one genome of S. lycopersicoides). Both types of intergeneric hybrids display pollen sterility, but workable ovule fertility. Unilateral incompatibility prevents their direct hybridization with staminate L. esculentum. Pollen of a self-compattible form of the related wild species L. pennellii is compatible with pistils of L. esculentum x S. lycopersicoides hybrids. This trait was backcrossed from L. pennellii to L. esculentum in order to develop bridging lines that could be used to obtain progeny from the intergeneric hybrids and to study the inheritance of bridging ability. In progeny of L. esculentum x S. lycopersicoides hybrids pollinated with L. pennellii-derived bridging lines, preferential transmission of L. pennellii alleles was observed for certain isozyme and RFLP markers on chromosomes 1, 6 and 10. The skewed segregations suggest linkage to three major pollen-expressed compatibility loci. This was confirmed by observations of pollen tube growth, which indicated that compatibility with pistils of the diploid intergeneric hybrid occurred only in bridging lines at least heterozygous for the L. pennellii markers on chromosomes 1, 6 and 10. Compatibility with the sesquidiploid hybrid required only the chromosome 1 and 6 loci, indicating an apparent effect of gene dosage on expression of incompatibility in the pistil. In an F2 L. esculentum x L. pennellii population, preferential transmission of L. pennellii alleles was observed for the same markers on chromosomes 1 and 10, as well as other markers on chromosomes 3, 11, and 12, but not 6. The chromosome 1 pollen compatibility locus maps to or near the S-locus, which determines S-allele specificity. The results are discussed in relation to existing genetic models for unilateral incompatibility, including the possible involvement of the S-locus.

Sexual hybridization of Lycopersicon esculentum and Solanum rickii by means of a sesquidiploid bridging hybrid.

A sesquidiploid hybrid having two genomes of Lycopersicon esculentum and one of Solanum lycopersicoides served as a pistillate bridging parent in crosses with Solanum rickii to produce L. esculentum x S. rickii hybrid progeny. Of the four progeny obtained, one (GH2754) was diploid and three were aneuploid with extra S. lycopersicoides chromosomes. The hybrids had morphological features of both parents, but attributes of the wild parent dominated. The hybrid nature of the four progeny was confirmed by isozyme, restriction fragment length polymorphism, and cytological analyses. A mean of 9.15 bivalents was observed in pollen mother cells of GH2754. A high level of pollen abortion was seen in all hybrids. Crosses of the hybrids with staminate S. rickii yielded one backcross individual, revealing a very low, but certain level of female fertility. Colchicine treatment of GH2754 generated one promising amphidiploid hybrid, which exhibited strong preferential chromosome pairing (94% of the examined cells had 24 bivalents) and appreciable pollen fertility (43% stainable). Chromosome pairing, isozyme, and restriction fragment length polymorphism data support a very close relationship between the two Solanum spp. and a much greater distance between them and L. esculentum, but the data do not discriminate between them in respect to their distances from the latter. The cytological and molecular observations, previous reports of successful transfer of traits from S. lycopersicoides to L. esculentum, and our hybridization of L. esculentum x S. rickii suggest good prospects for gene transfer from S. rickii to L. esculentum.

Fine mapping of quantitative trait loci using selected overlapping recombinant chromosomes, in an interspecies cross of tomato.

Quantitative trait loci (QTLs) have been mapped to small intervals along the chromosomes of tomato (Lycopersicon esculentum), by a method we call substitution mapping. The size of the interval to which a QTL can be mapped is determined primarily by the number and spacing of previously mapped genetic markers in the region surrounding the QTL. We demonstrate the method using tomato genotypes carrying chromosomal segments from Lycopersicon chmielewskii, a wild relative of tomato with high soluble solids concentration but small fruit and low yield. Different L. chmielewskii chromosomal segments carrying a common restriction fragment length polymorphism were identified, and their regions of overlap determined using all available genetic markers. The effect of these chromosomal segments on soluble solids concentration, fruit mass, yield, and pH, was determined in the field. Many overlapping chromosomal segments had very different phenotypic effects, indicating QTLs affecting the phenotype(s) to lie in intervals of as little as 3 cM by which the segments differed. Some associations between different traits were attributed to close linkage between two or more QTLs, rather than pleiotropic effects of a single QTL: in such cases, recombination should separate desirable QTLs from genes with undesirable effects. The prominence of such trait associations in wide crosses appears partly due to infrequent reciprocal recombination between heterozygous chromosomal segments flanked by homozygous regions. Substitution mapping is particularly applicable to gene introgression from wild to domestic species, and generally useful in narrowing the gap between linkage mapping and physical mapping of QTLs.

Recombination in sesquidiploid hybrids of Lycopersicon esculentum × Solanum lycopersicoides and derivatives.

Sesquidiploid hybrids of L. esculentum (L) x S. lycopersicoides (S) were backcrossed to L via L. pennellii (P) as a bridging species in order to detect and measure recombination. Although use of P injected its traits into the populations, the investigated traits were proven to originate from S. The appearance of S traits in diploids in the immediate progeny of sesquidiploids but mainly of derived alien addition types proved the occurrence of recombination at rates varying from 1.6% to 16%. In subsequent BC's, these traits were inherited in dominant Mendelian fashion, except for deviations favoring recurrent parent alleles, sometimes with highly significant deviations from 1∶1. Inheritance was investigated in BC and F2 ex BC for 13 traits with strong phenotypic modifications of morphological, physiological, and isozymic nature. Monogenic determination was confirmed in most instances by tight linkages. For most of the traits, small progenies allowed only rough estimates of linkage intensities, but for Wa (gene for White anthers, universal in S), a test cross with four markers on chromosome 8 established its locus 2 cM distal to dl, proximally on 8L. Also noteworthy is the linkage of Dls, a gene determining sensitivity of flowering to long days, close to sp, situated subterminally on 6L. For the majority of traits, these manifestations of linkage proved that the appearance of S traits resulted from recombination, not alien chromosome substitution - a conclusion also reinforced by observations of chromosome pairing in alien addition types and diploid derivatives. Recombined S alleles have loci in various chromosome positions. Although they were discovered on the shorter chromosomes (nos. 6-12), hybridization barriers precluded tests with the longer chromosomes. Thus, no evidence was found for restriction of recombination to certain chromosomes or chromosomal regions. The prospects therefore appear favorable for deriving valuable traits from the S parent.

Bypassing prefertilization barriers to hybridization in Nicotiana using in vitro pollination and fertilization.

In vitro pollination of placenta attached ovules was useful in bypassing unilateral incongruity barriers for several Nicotiana interspecific hybrid combinations (N. tabacum cv. 'Ky 17' X N. amplexicaulis, 'Ky 17' X N. benthamiana, and 'Ky 17' X N. repanda). By measuring the pollen tube growth over time, prefertilization barriers were determined to be the cause of the incongruity. Seedling necrosis was a problem in the development of the N. amplexicaulis hybrid and it prevented maturation of the N. repanda hybrid. Callus produced from cotyledons of the N. amplexicaulis hybrid eventually resulted in plants that survived to maturity. This procedure was not successful for the N. repanda materials. The N. amplexicaulis and N. benthamiana hybrids were sterile but following chromosome doubling by midrib culture, male and female fertile plants were produced.Conventional hybridization, fertilized ovule culture, and in vitro pollination were unsuccessful in obtaining hybrids of 'Ky 17' crossed with N. arentsii or N. bonariensis. Apparently, strong postfertilization barriers prevent the production of viable seed of these hybrids. Each of the N. repanda - N. tabacum reciprocal hybrids could not be rescued using callus culture; this adds support to the existence of strong sexual postfertilization barriers. A recent report, however, showed that it was possible to obtain this hybrid using the technique of somatic hybridization. Thus, it appears that it may also be possible to obtain asexual hybrids of N. arentsii and N. bonariensis with N. tabacum.

Maternal haploids of Petunia axillaris (Lam.) B.S.P. via culture of placenta attached ovules.

Hybridization of Petunia axillaris and P. parodii with Nicotiana tabacum was attempted using the method of in vitro pollination and fertilization. Seedlings were produced when the Petunia species and N. tabacum were used as the maternal parents; however, most of these had the identical somatic chromosome complement of the maternal parent. With crosses involving P. axillaris as the maternal parent, a low frequency of haploids was also produced. Due to the potential of haploids in basic and applied genetic research, additional experiments were carried out to determine whether in vitro pollination was necessary to stimulate haploid production and to more closely define the optimal time for ovule excision and culture. Four treatments were applied to accomplish these objectives. They were: placentas cultured prior to the time of anthesis, with and without pollination, and placentas cultured after the time of anthesis, with and without pollination. In vitro pollination had no effect on the frequency of haploids produced. Placenta attached ovules cultured prior to the time of anthesis produced significantly more haploids than those cultured after anthesis. The preanthesis treatment produced a frequency of 6.5 haploids per 100 ovaries cultured. The culture of placenta attached ovules provides an alternative to anther culture as a means for haploid production.