ALLOZYME VARIATION WITHIN SOLANUM SECT. PETOTA,SER. ETUBEROSA (SOLANACEAE)

Enzyme electrophoresis was employed to measure genetic variation within and divergence among 32 populations of three species in Solanum sect. Petota (S. brevidens, S. etuberosum, and S. fernandezianum). These species are self-compatible, diploid (2n = 2x = 24), and members of the monophyletic series Etuberosa. Solanum etuberosum is distributed in southern Chile, S. brevidens occurs in southern Chile and adjacent southern Argentina, and S. fernandezianum is endemic to Masatierra Island in the Juan Fernández Archipelago, 650 km west of continental Chile. Very low levels of observed heterozygosity (0.00–0.04) are found within populations of all three species. Interspecific mean genetic identities between S. brevidens and S. etuberosum (0.854) were similar to their intraspecific values (0.923, 0.865, respectively), with both species monomorphic for alleles at nine of the 12 loci examined. Solanum fernandezianum shows no heterozygosity and is more divergent to both S. brevidens (0.780) and S. etuberosum (0.698) than either is to each other. The divergence of S. fernandezianum to S. brevidens and S. etuberosum results from novel alleles at two of the 12 isozyme loci; in addition, it possesses only a subset of the variability found in S. brevidens and S. etuberosum at three other loci.     

Interactions of the Solanum spp. of the Etuberosa group and nine potato-infecting viruses and a viroid

All 26 accessions of Solanum brevidens, one accession of S. etuberosum and one accession of S. fernandezianum tested were all extremely resistant to potato leafroll virus (PLRV) and potato viruses Y (PVY) and A (PVA). S. brevidens and S. etuberosum were also resistant to Andean potato mottle virus (APMV) and moderately resistant to potato virus X (PVX), whereas S. fernandezianum was susceptible to these viruses. Additionally, S. brevidens was resistant to sap-inoculated potato viruses M (PVM) and S (PVS). All the Etuberosa accessions were susceptible by graft-inoculation to PVM, PVS, potato virus T (PVT) and Andean potato latent virus (APLV). Infections by the above mentioned viruses were symptomless in all of the Etuberosa spp. S. etuberosum and S. fernandezianum were infected by mechanical inoculation with potato spindle tuber viroid, S. etuberosum developing severe stunting and leaf-curl symptoms, but S. brevidens was infected only by graft-inoculation. The genes conferring resistance to PVY and PVX in S. brevidens and S. etuberosum appeared to be different from those currently utilised by plant breeders.     

Somatic hybrids between Solanum etuberosum and diploid,tuber bearing Solanum clones

Electrofusion was used to obtain somatic hybrids between Solanum etuberosum (2n=2x=24) and two diploid potato lines. These hybridizations were conducted to determine if haploidxwild species hybrids are better fusion partners than conventional S. tuberosumGp. Tuberosum haploids. Restriction fragment length polymerase (RFLP) analyses of the putative somatic hybrids confirmed that each parental genome was present. The somatic hybrids between S. etuberosum and a haploid S. tuberosum clone, US-W730, were stunted and had curled, purple leaves. In contrast, somatic hybrids between S. etuberosum and a haploidxwild species hybrid (US-W 730 haploidx S. berthaultii), were vigorous and generally tuberized under field conditions. These hybrids were designated as E+BT somatic hybrids. Analyses of 23 E+BT somatic hybrids revealed a statistically significant bias towards the retention of S. etuberosum chloroplasts. Stylar incompatibilities were observed when the E+BT somatic hybrids were used as pollen donors in crosses with S. tuberosum cultivars. Reciprocal crosses did not show this incompatibility. The progeny were vigorous and had improved tuber traits when compared to the maternal E+BT parent. RFLP analyses of three sexual progeny lines confirmed the presence of all 12 S. etuberosum chromosomes. In two of these lines, RFLPs that marked each of the 24 chromosome arms of S. etuberosum were present. However, RFLP markers specific for regions on chromosomes 2, 7, and 11 were missing from the third clone. Because other markers for these chromosomes were present in the progeny line, these results indicated the likelihood of pairing and recombination between S. etuberosum and S. tuberosum chromosomes.     

Protoplast-fusion-derived Solanum cybrids: application and phylogenetic limitations

Summary We established interspecific Solanum cybrids in order to study the intrageneric nuclear-organelle compatibility and the introgression of advantageous plasmone-coded breeding traits into potato. Cybridization was performed by the donor-recipient protoplast-fusion procedure. We found that the plastomes of S. chacoense, S. brevidens, and S. etuberosum could be transferred into the cybrids having S. tuberosum nuclear genomes; chondriome components were likewise transferred from the former species into these cybrids. The combination with S. chacoense as organelle donor and potato as recipient resulted in green fertile plants with potato morphology. By using S. etuberosum as an organelle donor and potato as recipient, male-sterile cybrid plants, most of them having pigmentation abnormalities, were obtained. The combination of S. brevidens with potato resulted in palegreen (almost albino) regenerants. The latter albino plantlets had both the chloroplast DNA and the mitochondrial DNA of the donor (S. brevidens) and did not survive the transfer into the greenhouse. An immediately applicative result of this study is the de novo establishment of male-sterile plants in a potato cultivar. Such plants should be useful as seed parents in the production of hybrid, true-potato seeds.     

Comparison of four molecular markers in measuring relationships among the wild potato relatives Solanum section Etuberosum (subgenus Potatoe)

We evaluated chloroplast DNA (cpDNA), isozymes, single to low-copy nuclear DNA (RFLPs), and random amplified polymorphic DNAs (RAPDs) in terms of concordance for genetic distance of 15 accessions each of Solanum etuberosum and S. palustre, and 4 accessions of S. fernandezianum. These self-compatible, diploid (2n=24), and morphologically very similar taxa constitute all species in Solanum sect. Etuberosum, a group of non-tuber-bearing species closely related to Solanum sect. Petota (the potato and its wild relatives). Genetic distance and multidimentional scaling results show general concordance of isozymes, RFLPs and RAPDs between all three taxa; cpDNA shows S. etuberosum and S. palustre to be more similar to each other than to S. fernandezianum. Interspecific sampling variance shows a gradation of resolution from allozyme (low) to RAPD to RFLP (high); while intraspecific comparisons graded from RFLPs (low) to RAPDs (high; lack of sufficient allozyme variability within species precluded comparisons for allozymes). Experimental error was low in RFLPs and RAPDs.Names are necessary to report factually and available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable     

Recombination of Solanum brevidens chromosomes in the second backcross generation from a somatic hybrid with S. tuberosum

Solanum brevidens synteny groups were examined with 47 widely-distributed RFLP markers in 17 BC₂ progeny from six fertile BC₁ plants. The BC₁ plants were derived from a single S. brevidens + S. tuberosum somatic hybrid backcrossed with S. tuberosum (potato). Probes which were linked in potato and tomato were also found to be syntenic along each of the 12 S. brevidens chromosomes. More than half of the S. brevidens synteny groups had lost one or more S. brevidens-specific RFLPs in the BC₂, suggesting that recombination had occurred. For 8 of the 12 S. brevidens RFLP synteny groups, the frequency of recombinant chromosomes exceeded that of intact parental chromosomes. Using the RFLP data, 161 RAPD markers were tentatively located throughout the S. brevidens genome. Further analyses with 39 of these 161 RAPD markers generally showed that RAPD and RFLP results were comparable, but some inconsistencies were noted with 14 of the 39 RAPD markers. The extent of marker loss and the high frequency of synteny groups which were marked by a single S. brevidens-specific RFLP marker suggest that the S. brevidens chromosomes have some pairing affinity with potato chromosomes. This interaction should facilitate the transfer of novel disease-resistance traits into potato breeding lines. One plant was recovered with the chromosome number of S. tuberosum (2n=48) that carried a single S. brevidens RFLP marker, suggesting transfer of this S. brevidens marker into the genome of S. tuberosum.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Analysis of the mitochondrial DNA of the somatic hybrids of Solanum brevidens and S. tuberosum using non-radioactive digoxigenin-labelled DNA probes

Summary Mitochondrial (mt) DNAs of somatic hybrids obtained by electrical and chemical fusion of mesophyll protoplasts of S. brevidens and a dihaploid line of S. tuberosum PDH 40 were analysed by Southern hybridization using the digoxigenin-labelled mtDNA sequences nad5 or orf25. In the Southern analysis of the hybrid mtDNA probed with nad5, most of the 19 hybrids analyzed had an RFLP pattern similar, but not identical, to one of the parents, S. tuberosum, PDH40. Nineteen percent of the hybrids had most of the S. brevidens fragments. Five of the hybrids had an identical RFLP pattern to either one of the parents while another two hybrids had novel RFLP patterns. Similar results were obtained by Southern analysis with orf25. These results clearly show that mtDNA rearrangements had occurred at a high frequency in the somatic hybrids. There were no differences in the frequencies of rearrangements observed between the hybrids regenerated from chemical and electrical fusions.     

Somatic hybrids produced by protoplast fusion between S. tuberosum and S. brevidens: phenotypic variation under field conditions

Summary Phenotypic and flowering characteristics of hybrid plants generated by protoplast fusion between a tetraploid S. tuberosum line and diploid S. brevidens were assessed under field conditions. Hybrids were compared to both clonal parental material and protoplast-derived plants of each parent. Almost all of the hybrids were hexaploid. A wide range of variation in morphological characters was observed for hybrids and protoclones. Flowering was markedly reduced in protoclones. The majority of hybrids flowered, had viable pollen and set tubers. Tuber and pollen characteristics of hybrids produced from individual fusion calli also varied. The potential usefulness of fusion hybrids in potato improvement is discussed.     

Nuclear genomic composition of asymmetric fusion products between irradiated transgenic Solanum brevidens and S. tuberosum: limited elimination of donor chromosomes and polyploidization of the recipient genome

Summary The production of asymmetric somatic hybrid calli after fusion between gamma-irradiated protoplasts from transgenic Solanum brevidens and protoplasts from S. tuberosum are reported. Transgenic (kanamycin-resistant, GUS-positive) S. brevidens plants and hairy root clones were obtained after transformation with Agrobacterium tumefaciens LBA 1060 (pRi1855) (pBI121) and LBA 4404 (pRAL4404) (pBI121), and A. rhizogenes LBA 9402 (pRi1855) (pBI121), respectively. Leaf protoplasts isolated from the transgenic plants or root protoplasts from the hairy root clones were fused with S. tuberosum leaf protoplasts, and several calli were selected on kanamycin-containing medium. The relative nuclear DNA content of the hybrid calli was measured by flow cytometry (FCM), and the percentages of DNA of the S. brevidens and S. tuberosum genomes in the calli were determined by dot blot analysis using species-specific DNA probes. Chromosome-specific restriction fragment length polymorphism (RFLP) markers were used to investigate the elimination of specific S. brevidens chromosomes in the hybrids. The combined data on FCM, dot blot and RFLP analysis revealed that 18–62% of the S. brevidens DNA was eliminated in the hybrid calli and that the RFLP marker for chromosome 7 was absent in seven out of ten calli. The absence of RFLP markers for chromosomes 5 and 11 hardly ever occurred. In most of the hybrids the ploidy level of the S. tuberosum genome had increased considerably.     

Resistance to potato virus Y in somatic hybrids between Solanum etuberosum and S. tuberosum x S. berthaultii hybrid

Somatic hybrids between a potato virus Y (PVY) resistant Solanum etuberosum clone and a susceptible diploid potato clone derived from a cross between S. tuberosum Gp. Tuberosum haploid US-W 730 and S. berthaultii were evaluated for resistance to PVY. All but one of the tested somatic hybrids were significantly more resistant than cultivars Atlantic and Katahdin. However, none was as resistant as the S. etuberosum parent. One hexaploid somatic hybrid, possibly the product of a triple-cell fusion involving one S. etuberosum protoplast and two haploid x S. berthaultii protoplasts, was as susceptible to PVY infection as the cultivars. Tetraploid progeny of the somatic hybrids, obtained from crosses with Gp. Tuberosum cultivars, were neither as resistant as the maternal somatic hybrid parent, nor as susceptible as the paternal cultivar parent. It appears that the introgression of PVY resistance from (1EBN) S. etuberosum into (4EBN) S. tuberosum (EBN-endosperm balance number) will be successful through the use of somatic hybridization and subsequent crosses of the somatic hybrids back to S. tuberosum.     

Production of asymmetric hybrids between Solanum tuberosum and irradiated S. brevidens

Summary Asymmetric somatic hybrids were obtained by fusion of Solanum tuberosum (PDH40) protoplasts with 300- or 500-Gy irradiated protoplasts of S. brevidens. These radiation doses were sufficient to prevent the growth of the S. brevidens protoplasts. Putative hybrids were selected on the basis of phenotype from regenerated shoots and identified with a S. brevidens-specific probe. From these, 31 asymmetric hybrids were confirmed by morphological characteristics, isoenzyme patterns and RFLP analysis. The morphology of the asymmetric hybrids was intermediate between that of S. tuberosum and symmetric hybrids of both species (obtained without irradiation treatment). Chromosome counts from 17 asymmetric hybrids showed that the chromosome number of the hybrids ranged from 31 to 64. The asymmetric hybrids probably had one or two genome complements (i.e. either 24 or 48 chromosomes) from S. tuberosum and 7–22 chromosomes from S. brevidens. There was no clear correlation between the radiation dose and the degree of elimination of the S. brevidens genome.     

Transfer of resistance to potato leaf roll virus from Solanum brevidens into Solanum tuberosum by somatic fusion

Tetraploid somatic hybrids were produced by protoplast fusion between Solanum brevidens, a diploid non-tuber-bearing wild species and a diploid tuber-bearing potato line derived from an S. tuberosum Gp. Phureja-Stenotumum population. S. brevidens has resistance to potato leaf roll virus (PLRV) and frost but is difficult to cross sexually with cultivated potato. Hybridity was verified by morphological characteristics and cytological observations. Nine of ten hybrids tested showed resistance to PLRV. Hybrids produced fertile pollen and eggs which may allow beneficial traits of S. brevidens to be incorporated into a conventional potato breeding programme.     

Resistance to potato leaf roll virus and potato virus Y in somatic hybrids between dihaploid Solanum tuberosum and S. brevidens

Summary Many somatic fusion hybrids have been produced between a dihaploid potato Solanum tuberosum and the sexually-incompatible wild species S. brevidens using both chemical and electrical fusion techniques. S. brevidens was resistant to both potato leaf roll virus (PLRV) and potato virus Y (PVY), the viruses being either at low (PLRV) or undetectable (PVY) concentrations as determined by enzyme-linked immunosorbent assay (ELISA). The S. tuberosum parent was susceptible to both viruses. A wide range of resistance, expressed as a decrease in virus concentration to both viruses was found amongst fusion hybrids, four of which were especially resistant. The practicality of introducing virus resistance from S. brevidens into cultivated potatoes by somatic hybridisation is discussed.     

Low night temperatures have a differential effect on the diurnal cycling of gene expression in cold–sensitive and tolerant tomatoes*

Abstract. Comparisons were made between the changes in mRNA levels induced by low night temperatures in the cold–sensitive tomato and two altitudinal ecotypes of the wild species L. hirsutum. Changes in mRNA levels were detected by resolving in vitro translation products of poly(A)⁺ RNA by 2-D PAGE. The treatment was applied by first growing plants in a thermoperiod of 25/18°C and then switching to 25/6°C. All tomatoes displayed a diurnal cycling in which a set of mRNAs accumulated at the end of the 18°C nights, whereas another accumulated at the end of the 25°C days. The accumulation of night specific mRNAs was inhibited by 6°C nights in the cold sensitive tomatoes while that of the tolerant one was only marginally affected. All tomatoes showed a similar reduction in the apparent turnover rate of the day specific mRNAs during the 6°C nights. Finally, low night temperatures induced the accumulation of six to eight mRNAs in all genotypes. This number increased by 15 in L. esculentum after the seventh night and are likely involved in stress response rather than acclimation/tolerance. The tomato is proposed as a genetic model to discriminate genes involved in acclimation/tolerance from those involved in stress response.     

Characterisation of somatic hybrids between Solanum tuberosum and its frost-tolerant relative Solanum commersonil

 Somatic hybrids between three dihaploid Solanum tuberosum (2n=2x=24) genotypes and the frost-tolerant, diploid, relative Solanum commersonii (2n=2x=24) were analysed for variation in morphological traits, fertility and frost tolerance. The somatic hybrids were more vigorous than their parents and in many ways resembled a tetraploid S. tuberosum. All of the hybrids flowered profusely, although the male fertility was largely dependent on the S. tuberosum genotype used. In one hybrid combination all plants were both male- and female-fertile, while in the other two combinations the majority of plants were male-sterile but female-fertile. The somatic hybrids showed an increase in direct frost tolerance when compared with the dihaploid S. tuberosum parents, and to a varying extent had gained the capacity to cold acclimate. These somatic hybrids will be used in breeding programmes involving repeated cycles of anther culture and somatic hybridisation. Received: 20 May 1997 / Accepted: 12 June 1997     

Molecular,cytogenetic and morphological characterization of somatic hybrids of dihaploid Solanum tuberosum and diploid S. brevidens

Summary Fifty-eight somatic hybrid plants, produced both by chemical (11) and electrical fusion (47) of protoplasts of dihaploid Solanum tuberosum and S. brevidens, have been analysed by molecular, cytological and morphological methods. The potentially useful euploid plants constituted 34% of the total, of which 20% were tetraploid and 14% hexaploid; the remainder were aneuploid at the tetraploid, hexaploid and octoploid levels. Analysis of chloroplast DNA showed that 55% of hybrids contained chloroplasts from S. brevidens and 45% from S. tuberosum. Hexaploids, the products of three protoplasts fusing together, were analyzed with specific DNA probes, and this revealed that nuclear genome dosages could be either 21 S. tuberosumS. brevidens, or vice-versa. Chloroplast types of hexaploids were not influenced by nuclear genome dosage, and all six possible combinations of genome dosage and chloroplast types were found amongst tetraploids and hexaploids. To examine the morphology of the hybrid population and its possible relation to the chromosome number and chloroplast DNA type, 18 morphological characteristics were measured on greenhouse-grown plants and analyzed by principal component and canonical variate analyses. Both analyses showed that nuclear ploidy has the most prominent influence on the overall morphology of the hybrids. Differential parental genome expression in the morphology of the hybrids is discussed. These results provide useful data on the range of genetic combinations that can be expected to occur amongst somatic hybrid plants.     

Cytogenetic characterization and nuclear DNA content of three North African endemic Centaurea species

Three endemic Centaurea species from North Africa are investigated for the first time by chromomycin fluorochrome banding for GC-rich DNA distribution, fluorescence in situ hybridization for physical mapping of rRNA genes, and flow cytometry for genome-size assessment. Investigated species belong to three different sections and possess three basic chromosome numbers: C. tougourensis subsp. tougourensis 2n = 4x = 36 (x = 9), C. musimonum 2n = 2x = 20 (x = 10), and C. maroccana 2n = 2x = 24 (x = 12). The number and distribution of chromomycin positive bands (CMA⁺) and 18S-5.8S-26S (35S) rDNA loci were different among investigated species and ranged from 6 to 80 chromomycin bands and from 2 to 6 35S rDNA loci. The three species have just one 5S rDNA locus at intercalary position on a separate chromosome pairs, except in the case of C. musimonum in which both rDNA loci were localized on the same chromosome. All rDNA loci were co-localized with CMA⁺ bands, except three 35S in C. musimonum. Genome size ranged from 2C = 1.66 to 2C = 2.86 pg in diploid species (C. musimonum and C. maroccana, respectively) and to 2C = 4.51 pg in tetraploid C. tougourensis subsp. tougourensis.     

Characterization of the expression and inheritance of potato leafroll virus (PLRV) and potato virus Y (PVY) resistance in three generations of germplasm derived from Solanum etuberosum

Potato virus Y (PVY) and potato leafroll virus (PLRV) are two of the most important viral pathogens of potato. Infection of potato by these viruses results in losses of yield and quality in commercial production and in the rejection of seed in certification programs. Host plant resistance to these two viruses was identified in the backcross progeny of a Solanum etuberosum Lindl. somatic hybrid. Multiple years of field evaluations with high-virus inoculum and aphid populations have shown the PVY and PLRV resistances of S. etuberosum to be stably expressed in two generations of progeny. However, while PLRV resistance was transmitted and expressed in the third generation of backcrossing to cultivated potato (Solanum tuberosum L. subsp. tuberosum), PVY resistance was lost. PLRV resistance appears to be monogenic based on the inheritance of resistance in a BC₃ population. Data from a previous evaluation of the BC₂ progeny used in this study provides evidence that PLRV resistance was partly conferred by reduced PLRV accumulation in foliage. The field and grafting data presented in this study suggests that resistance to the systemic spread of PLRV from infected foliage to tubers also contributes to the observed resistance from S. etuberosum. The PLRV resistance contributed by S. etuberosum is stably transmitted and expressed through sexual generations and therefore would be useful to potato breeders for the development of PLRV resistant potato cultivars.     

Improved isolation of dihaploidsolanum tubersoum protoplasts and the production of somatic hybrids between dihaploidS. tuberosum andS. brevidens

Summary A modified protoplast isolation technique, applicable to a range of dihaploidSolanum tuberosum genotypes, has been developed. A combination of high calcium and high pH was used to fuse mesophyl protoplasts of dihaploidS. tuberosum (PDH40) and the diploid wild speciesS. brevidens. Large numbers of colonies were obtained after fusion and putative hybrids selected on the basis of phenotype from regenerated shoots. From these, 11 somatic hybrid plants have been identified by their isoenzyme patterns and morphologic characteristics. Four of these hybrids had the expected chromosome number of 48. The approach of mass culture after fusion followed by selection of hybrids from regenerated shoots and the application of somatic hybridization to potato breeding are discussed.     

Analysis of chloroplast and mitochondrial DNA in asymmetric somatic hybrids betweenSolanum tuberosum and irradiatedS. brevidens

Organellar DNA of asymmetric somatic hybrids betweenSolanum tuberosum and irradiatedS. brevidens were analysed by DNA hybridization methods using the spinach chloroplast probepSBD, wheat mitochondrial genenad5 and petunia mitochondrial geneorf25. Eight of the 12 asymmetric hybrid plants hadS. tuberosum chloroplast DNA and the remaining fourS. brevidens chloroplast DNA. A novel mitochondrial hybridization pattern was present in eight out of the 17 hybrids tested. In six hybrids, novel combinations of chloroplasts and mitochondria were found, indicating that both organelle types sorted out independently.