A genotype-specific pollen gene associated with self-incompatibility in Lycopersicon peruvianum

Self-incompatibility is a genetically controlled process used to prevent self-pollination. We report here the characterization of pollen cDNA clones of Lycopersicon peruvianum, and the identification of a genotype-specific pollen factor involved in self-incompatibility. To identify the latter, differential mRNA display RT-PCR was performed on pollen cDNAs from S12Sa and S11Sa genotypes. We isolated four cDNA fragments expressed preferentially in S12Sa pollen, and screened a cDNA library from S12Sa pollen with the four cDNA fragments to isolate the corresponding full length cDNAs. One of the four isolated cDNAs encoded part of an actin depolymerizing factor protein that we named LpADF. LpADF is highly homologous to actin depolymerizing factors of Arabidopsis thaliana, Lilium longiflorum, and Zea mays. RNA blot analysis revealed that LpADF is only expressed in mature pollen of the S12Sa genotype, and is therefore a candidate pollen factor in the gametophyte self-incompatibility system of L. peruvianum.     

Molecular diversity of three S-allele cDNAs associated with gametophytic self-incompatibility in Lycopersicon peruvianum

We isolated S allele-associated cDNA clones from each of the stylar cDNA libraries of Lycopersicon peruvianum of two different S genotypes (S ₁₂S_band S ₁₃ S _c) with S ₁₁ S _callele-associated cDNA (LPS11) as a probe. The longest cDNA clones, designated LPS12 and LPS13, which were 779 bp and 853 bp in length, contained open reading frames of 189 and 210 amino acids, respectively. The three S alleleassociated cDNAs (LPS11, LPS12, and LPS13) did not cross-hybridize to each other under highly stringent condition by northern blot analysis. Their average identity to Nicotiana alata S-proteins so far was 49%. The fragments corresponding to LPS11 or LPS12 cosegregated with their respective S alleles in genetic crosses. From these results, we conclude that the three cloned cDNAs were derived from the three different S alleles of L. peruvianum.     

Breakdown of self-incompatibility during pistil development in Lycopersicon peruvianum by modified bud pollination

Summary Stylar self-incompatibility barriers in L. peruvianum can be avoided if pollen germination and growth through immature pistils is promoted under specific environmental conditions approximately 2–3 days before the initiation of anthesis. Since immature stigmas lack sufficient exudate for pollen germination, the sandwiching of a thin layer of pollen germination medium between the stigma and a mineral oil layer containing pollen allows precocious pollen germination and some compatible pollen tube growth through the style. This procedure is rapid, inexpensive, applicable in the field, and makes efficient use of pollen. Consistent though low seed yields have been obtained. A high proportion of aborted seed, seedling lethals, and aberrant seedling phenotypes in selfed progeny indicate the presence of strong post-zygotic barriers to such selfing. No evidence for a reduction in the strength of the SI response with increasing pistil age was observed.     

Breakdown of self-incompatibility in tetraploid Lycopersicon peruvianum: inheritance and expression of S-related proteins

 Lycopersicon peruvianum displays gametophytic self-incompatibility (GSI). We have isolated self-compatible (SC) tetraploids of L. peruvianum from tissue-cultured leaves and have explored the expression and inheritance of their S-related proteins. The Srelated protein profiles of styles of SC tetraploids were indistinguishable from the diploid self-incompatible (SI) explant source based on SDS-PAGE. All progeny obtained from self-fertilization of two tetraploids were SC. Cloned cDNA sequences of the S-related proteins were used to determine the inheritance of this locus in these progeny through Southern hybridization. The allelic ratio, as determined from the intensity of DNA restriction fragments, was consistent with the predicted ratio if only pollen bearing two different alleles was successful in achieving fertilization. All progeny obtained had at least one copy of each allele, and individuals fully homozygous for either allele were not found, indicating that pollen grains bearing two identical alleles were inhibited. In addition, the level of expression of the S-related proteins in the progeny correlated with the allelic dosage at the DNA level. We demonstrate that the observed self-compatibility in the tetraploids was not caused by an alteration in the expression of S-related proteins. Received: 11 September 1996 / Accepted: 21 March 1997     

Style proteins of a wild tomato (Lycopersicon peruvianum) associated with expression of self-incompatibility

The identification, isolation and aminoterminal sequencing of two S-genotype-associated proteins from style extracts of Lycopersicon peruvianum Mill. is reported. There is a high level of homology between these two sequences and with the amino-terminal sequences of other S-allele-associated glycoproteins isolated from Nicotiana alata Link et Otto. These sequences were obtained by a new high-sensitivity method of selected twodimensional gel analysis followed by electroelution and purification of proteins by inverse-gradient high-performance liquid chromatography before sequencing.Abbreviations HPLC high-performance liquid chromatography - Mr relative molecular mass - PTH phenylthiohydrantoin - SDS sodium dodecyl sulphate     

Analysis of the Tissue-SpecificS RNase gene promoter associated with self-incompatibility in tomato (Lycopersicon peruvianum L.)

To understand the expression pattern of theS RNase gene in the floral tissues associated with self-incompatibility (SI), promoter region of S₁₁ RNase gene was serially deleted and fused GUS. Five chimeric constructs containing a deleted promoter region of the S₁₁ RNase gene were constructed, and introduced intoNicotiana tabacum using Agrobacterium-mediated transformation. Northern blot analysis revealed that the GUS gene was expressed in the style, anther, and developing pollen of all stages in each transgenic tobacco plant The developing pollen expressed the same amount of GUS mRNA in all stages in transgenic tobacco plants. In addition, histochemical analysis showed GUS gene expression in vascular bundle, endothecium, stomium, and tapetum cells during pollen development in transgenic plants. From these results, it is speculated that SI ofLycopersicon peruvianum may occur through the interaction ofS RNase expressed in both style and pollen tissues.     

Genetic mapping and protein product diversity of the self-incompatibility locus in wild tomato (Lycopersicon peruvianum)

Phenotypic diversity of self-incompatibility (S) alleles within nine natural populations ofLycopersicon peruvianum was investigated. Only 7 incompatible responses were observed of a total of 276 unique combinations tested, on the basis of controlled pollinations, indicating the large number of alleles that exist within these populations. Molecular weight polymorphism for specific major stylar proteins observed on SDS-PAGE was also evident in two of the populations examined. Five proteins were shown to map to theS locus and to be associated with differentS alleles through controlled pollinations and segregation of the proteins. Two of theseS related proteins had been described previously in terms of spatial and temporal expression consistent with their involvement in self-incompatibility (Mauet al., Planta 169, 184–191, 1986). A mapping population derived from a fully compatible cross was used to establish linkage of theS locus to two DNA markers,CD15 andTG184, that lie on chromosome 1. The order of the markers and estimates of map distances are given.     

Asymmetric somatic hybrids between Lycopersicon esculentum and irradiated Lycopersicon peruvianum

Summary Asymmetric somatic hybrids of Lycopersicon esculentum and Lycopersicon peruvianum were analysed for the retention of genes and alleles specific for L. peruvianum. The hybrids were obtained by fusion of protoplasts from L. esculentum with those of L. peruvianum (the donor), the latter having been irradiated before fusion with 50, 300 or 1,000 Gy of gamma-rays. The retention of three different types of genes or alleles was analysed. (1) The gene coding for kanamycin resistance, which is dominant and had been introduced in most of the L. peruvianum donor plants by transformation. It was present at one locus in 16 L. peruvianum donor plants and at two loci in one donor plant. (2) The genes coding for acid phosphatase, locus Aps-1, and glutamate oxaloacetate transaminase (GOT); different alleles of these genes are co-dominant and were detected by isozyme analysis. (3) Eighteen single gene morphological markers for which most of the L. esculentum genotypes used were homozygous recessive. Kanamycin resistance from donor plants with one locus was retained in about 50% of the asymmetric 30H-hybrids (the donor was irradiated with 300 Gy). L. peruvianum specific alleles of Aps-1 and GOT were present in at least 70% of the hybrids; the retention of donor alleles was lower in 30H- than in 5H-hybrids (donor irradiated with 50 Gy). On average, 73% of the L. peruvianum-specific alleles (one or both) of the morphological markers were detected in the 30H-hybrids. Several of the L. esculentum genotypes used were homozygous recessive for two morphological markers on the same chromosome; in 43% of the 30H-hybrids derived from them, only one of these markers was complemented by the L. peruvianum allele. This is an indication of frequent breakage of the L. peruvianum chromosomes. Several hybrid calli regenerated genotypically different shoots. On the whole, this analyses confirms the conclusion drawn from the cytogenetic and morphological analysis of these asymmetric hybrids, namely that irradiation prior to fusion eliminates the L. peruvianum genome to only a limited extent.     

Asymmetric somatic hybrids between Lycopersicon esculentum and irradiated Lycopersicon peruvianum

Summary Asymmetric somatic hybrids of Lycopersicon esculentum and Lycopersicon peruvianum were obtained by fusion of leaf protoplasts from both species after irradiation of protoplasts or leaf tissue of L. peruvianum with 50, 300, or 1,000 Gy of gamma-rays. These radiation doses were sufficient to abolish the growth of the L. peruvianum protoplasts. The hybrids were selected for their ability to regenerate plants; this regeneration capacity derived from L. peruvianum. All asymmetric hybrid plants were aneuploid. The ploidy level, the morphology, and the regeneration rate were analyzed in relation to the radiation dose applied to L. peruvianum. After a low dose (50 Gy), most hybrids had near-triploid chromosome numbers, whereas after a high dose (300 or 1,000 Gy), most hybrids had near-pentaploid numbers. The morphology of the asymmetric hybrids was intermediate between that of L. esculentum and symmetric somatic hybrids of both species (obtained without irradiation treatment), and approached the morphology of L. esculentum to a greater extent after a high dose of irradiation. The asymmetric hybrids regenerated more slowly than the symmetric hybrids and regeneration proceeded more slowly after a high dose than after a low dose of irradiation. The high-dose hybrids also grew more slowly, flowered less, and set fruits less than the low-dose hybrids. No seeds could be obtained from any asymmetric hybrid.     

An RFLP linkage map of Lycopersicon peruvianum

In order to map genes determining resistance to bacterial canker in tomato, backcrosses were made between a resistant and a susceptible Lycopersicon peruvianum accession. The linkage study with RFLP markers yielded a genetic map of L. Peruvianum. This map was compared to that derived from a L. esculentum x L. pennellii F₂ population, based on 70 shared RFLP markers. The maps showed a good resemblance in both the order of markers and the length of the chromosomes, with the exception of just one relocated marker on chromosome 9. Because backcrosses were made with the F₁, either as the pollen parent or as the pistil parent, linkage maps from male and female meioses could be estimated. It was concluded that recombination at male meiosis was reduced, and that gametophytic selection for parental genotypes at more than one locus per chromosome might be partly responsible for the reduction of the estimated male map length.     

Use of an interspecific hybrid in identifying a new allelic specificity generated at the self-incompatibility locus after inbreeding in Lycopersicon peruvianum

Summary An interspecific hybrid between Lycopersicon esculentum () and L. peruvianum has been raised by embryo rescue in vitro and used to confirm the presence of a new S-allelic specificity in its inbred L. peruvianum parent, a plant derived by enforced bud self-pollination of a self-incompatible clone with the genotype S ₁ S ₂. The inbred plant showed breeding behavior characteristic of both S ₂ and a second specificity which was not S ₁, S ₂, S ₃ or S _f. Two-dimensional gel electrophoresis of stylar proteins, however, showed only a single typical S-associated component with the M_r and pI characteristic of S₂. The alteration in specificity, therefore, was not associated with a detectable change in an S-associated protein. The F₁ interspecific hybrid showed intermediacy of vegetative and reproductive characters, relatively high fertility and full self-incompatibility. Backcrossing to L. esculentum produced only abortive seeds requiring embryo culture. Backcrosses to L. peruvianum produced a very low proportion of filled germinable seeds. Pollen of the hybrid showed superior viability and tube growth rate compared with pollen of the two parent plants.     

Primary structural features of rosaceous S-RNases associated with gametophytic self-incompatibility

We isolated cDNA clones encoding five S-RNases (S_1-,_S3- , S_5-, S_6-, S_7-RNases) from pistils of Pyrus pyrifolia (Japanese pear), a member of the Rosaceae. Their amino acid sequences were aligned with those of other rosaceous S-RNases sequenced so far. A total of 76 conserved amino acid residues were stretched throughout the sequence, but were absent from the 51–66 region which was designated the hypervariable (HV) region. The phylogenetic tree of rosaceous S-RNases showed that S-RNase polymorphism predated the divergence of Pyrus and Malus. Pairwise comparison of these S-RNases detected two highly homologous pairs, P. pyrifolia S_1- and S_4-RNases (90.0%) and P. pyrifolia S_3- and S_5-RNases (95.5%). The positions of amino acid substitutions between S_1- and S_4-RNases were spread over the entire region, but in the pair of S_3- and S_5-RNases, amino acid substitutions were found in the 21–90 region including the HV region. The substitutions in this restricted region appear to be sufficient to discriminate between S₃ and S₅ pollen and to trigger the self-incompatible reaction.     

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.     

Somatic embryogenesis from Lycopersicon peruvianum leaf mesophyll protoplasts

Summary One to five percent of Lycopersicon peruvianum (L.) Mill. leaf mesophyll protoplasts undergo cell division and concomitant organization to form embryogenic-like structures when cultured in Murashige and Skoog medium (1962) containing 3% sucrose, 9% mannitol, 1.0 mg/l kinetin (K) and 1.0 mg/l naphthalene acetic acid (NAA) at pH 5.6–5.8 (medium A). These embryogenic structures, after passing through developmental stages similar to those observed in zygotic embryogeny, are capable of forming shoots on hormone-free medium A. In medium B, wherein 0.5 mg/l of 2,4-dichlorophenoxyacetic (2,4-D) replaced the hormones (K and NAA), embryogenic structures did not develop. However, callus originating in medium B retained morphogenetic capacity as was evidenced by subsequent shoot regeneration when they were transferred to medium A with K and NAA replaced by 1.0 mg/l zeatin (Z). The potential value of incorporating this regeneration trait into Lycopersicon species and cultivated lines for use in tissue culture programs is discussed.Michigan Agricultural Experiment Station Journal No. 9676     

Organelle analysis of symmetric and asymmetric hybrids between Lycopersicon peruvianum and Lycopersicon esculentum

Summary The organelles of somatic hybrids obtained from symmetric and asymmetric fusions between the Lycopersicon species L. peruvianum and L. esculentum were analyzed by DNA hybridization methods. In the asymmetric fusions the L. peruvianum protoplasts were gamma-irradiated at a dose of 50, 300 and 1,000 Gy. The organelles were characterized using the Petunia chloroplast probe pPCY64 and the mitochondrial EcoRI-SalI fragment of the Pcf gene. In all symmetric and asymmetric hybrid plants, a total of 73 being analyzed, only one of the parental chloroplast genomes was present, except for one hybrid plant which harbored both parental chloroplast genomes. No recombination and/or rearrangement in the chloroplast genome could be identified with the pPCY64 probe. Irradiation of the L. peruvianum protoplasts did not significantly reduce the fraction of asymmetric hybrids with L. peruvianum chloroplasts. A novel mitochondrial restriction pattern was present in 5 out of 24 hybrids tested. In 9 hybrids novel combinations of chloroplasts and mitochondria were found, indicating that both organelle types sorted out independently.     

The Pollen Tube Pathway in the Pistil of Lycopersicon peruvianum

The pollen tube pathway has been studied in unpollinated andpollinated pistils of Lycopersicon peruvianum using histochemicalstains for detection of proteins, lipids and arabinogalactansby bright-field microscopy, and decolourized aniline blue fordetection of pollen tubes by epifluoresence microscopy. Thepollen tube pathway is a continuous tract of mucilage from thestigma surface to the ovule micropyles, and is associated witha continuous tract of specialized, protein-rich transmittingcells comprising the stigmatic papillae, vertical files of stylartransmitting cells and the placental epithelium within the ovary.The superficial exudate of the stigma is hydrophobic and richin lipids. The mucilage of the style and ovary is hydrophilicand rich in arabinogalactans but low in proteins. Pollen tubesgrow between cells through the mucilage of the stigma and stylartransmitting tract, and across the surface of the placenta inthe ovarian mucilage. The structure of the junction of the stylartransmitting tract with the top of the ovary placenta assistseffective distribution of pollen tubes within the ovary. Lycopersicon, solanaceae, fertilization, pistil, pollen tube, pollination