Dynamic adaptation of the extremophilic red microalga Cyanidioschyzon merolae to high nickel stress.

The order of Cyanidiales comprises seven acido-thermophilic red microalgal species thriving in hot springs of volcanic origin characterized by extremely low pH, moderately high temperatures and the presence of high concentrations of sulphites and heavy metals that are prohibitive for most other organisms. Little is known about the physiological processes underlying the long-term adaptation of these extremophiles to such hostile environments. Here, we investigated the long-term adaptive responses of a red microalga Cyanidioschyzon merolae, a representative of Cyanidiales, to extremely high nickel concentrations. By the comprehensive physiological, microscopic and elemental analyses we dissected the key physiological processes underlying the long-term adaptation of this model extremophile to high Ni exposure. These include: (i) prevention of significant Ni accumulation inside the cells; (ii) activation of the photoprotective response of non-photochemical quenching; (iii) significant changes of the chloroplast ultrastructure associated with the formation of prolamellar bodies and plastoglobuli together with loosening of the thylakoid membranes; (iv) activation of ROS amelioration machinery; and (v) maintaining the efficient respiratory chain functionality. The dynamically regulated processes identified in this study are discussed in the context of the mechanisms driving the remarkable adaptability of C. merolae to extremely high Ni levels exceeding by several orders of magnitude those found in the natural environment of the microalga. The processes identified in this study provide a solid basis for the future investigation of the specific molecular components and pathways involved in the adaptation of Cyanidiales to the extremely high Ni concentrations.

Migration of repetitive DNAs during evolution of the permanent translocation heterozygosity in the oyster plant (Tradescantia section Rhoeo).

Due to translocation heterozygosity for all chromosomes in the cell complement, the oyster plant (Tradescantia spathacea) forms a complete meiotic ring. It also shows Rabl-arrangement at interphase, featured by polar centromere clustering. We demonstrate that the pericentromeric regions of the oyster plant are homogenized in concert by three subtelomeric sequences: 45S rDNA, (TTTAGGG)n motif, and TSrepI repeat. The Rabl-based clustering of pericentromeric regions may have been an excellent device to combine the subtelomere-pericentromere sequence migration (via inversions) with the pericentromere-pericentromere DNA movement (via whole arm translocations) that altogether led to the concerted homogenization of all the pericentromeric domains by the subtelomeric sequences. We also show that the repetitive sequence landscape of interstitial chromosome regions contains many loci consisting of Arabidopsis-type telomeric sequence or of TSrepI repeat, and it is extensively heterozygous. However, the sequence arrangement on some chromosomal arms suggest segmental inversions that are fully or partially homozygous, a fact that could be explained if the inversions started to create linkages already in a bivalent-forming ancestor. Remarkably, the subterminal TSrepI loci reside exclusively on the longer arms that could be due to sharing sequences between similarly-sized chromosomal arms in the interphase nucleus. Altogether, our study spotlights the supergene system of the oyster plant as an excellent model to link complex chromosome rearrangements, evolution of repetitive sequences, and nuclear architecture.

Proteomic Studies of Roots in Hypoxia-Sensitive and -Tolerant Tomato Accessions Reveal Candidate Proteins Associated with Stress Priming.

Tomato (Solanum lycopersicum L.) is a vegetable frequently exposed to hypoxia stress induced either by being submerged, flooded or provided with limited oxygen in hydroponic cultivation systems. The purpose of the study was to establish the metabolic mechanisms responsible for overcoming hypoxia in two tomato accessions with different tolerance to this stress, selected based on morphological and physiological parameters. For this purpose, 3-week-old plants (plants at the juvenile stage) of waterlogging-tolerant (WL-T), i.e., POL 7/15, and waterlogging-sensitive (WL-S), i.e., PZ 215, accessions were exposed to hypoxia stress (waterlogging) for 7 days, then the plants were allowed to recover for 14 days, after which another 7 days of hypoxia treatment was applied. Root samples were collected at the end of each time-point and 2D-DIGE with MALDI TOF/TOF, and expression analyses of gene and protein-encoded alcohol dehydrogenase (ADH2) and immunolabelling of ADH were conducted. After collating the obtained results, the different responses to hypoxia stress in the selected tomato accessions were observed. Both the WL-S and WL-T tomato accessions revealed a high amount of ADH2, which indicates an intensive alcohol fermentation pathway during the first exposure to hypoxia. In comparison to the tolerant one, the expression of the adh2 gene was about two times higher for the sensitive tomato. Immunohistochemical analysis confirmed the presence of ADH in the parenchyma cells of the cortex and vascular tissue. During the second hypoxia stress, the sensitive accession showed a decreased accumulation of ADH protein and similar expression of the adh2 gene in comparison to the tolerant accession. Additionally, the proteome showed a greater protein abundance of glyceraldehyde-3-phosphate dehydrogenase in primed WL-S tomato. This could suggest that the sensitive tomato overcomes the oxygen limitation and adapts by reducing alcohol fermentation, which is toxic to plants because of the production of ethanol, and by enhancing glycolysis. Proteins detected in abundance in the sensitive accession are proposed as crucial factors for hypoxia stress priming and their function in hypoxia tolerance is discussed.

Photosynthetic efficiency, growth and secondary metabolism of common buckwheat (Fagopyrum esculentum Moench) in different controlled-environment production systems.

Light-emitting diodes (LEDs) and high-pressure sodium lamps (HPS) are among the most commonly used light sources for plant cultivation. The objective of this study was to evaluate the effect of two controlled-environment production systems differing in light sources on growth, photosynthetic activity, and secondary metabolism of common buckwheat. We hypothesized that LED light with the majority of red and blue waves would increase physiological and biochemical parameters compared to sunlight supplemented with HPS lamps. The experiment was performed in a phytotronic chamber (LEDs) and in a greenhouse (solar radiation supplemented with HPS lamps as a control). The effects were analyzed at the flowering phase with biometric measurements, leaf chlorophyll index, the kinetics of chlorophyll a fluorescence, content of soluble carbohydrates and phenolics in the leaves. Applied LED light decreased the biomass but stimulated the production of phenolics compared to control plants. In control plants, a positive correlation between flavonoid content and energy dissipation from photosystem II (DIo/CSm) was found, while in plants under LEDs total pool of phenolic content correlated with this parameter and the quantum yield of electron transport (φ Ro and ψ Ro) was lower than that of the control, probably affecting buckwheat biomass.

Physiological and Biochemical Response to Fusarium culmorum Infection in Three Durum Wheat Genotypes at Seedling and Full Anthesis Stage.

Fusarium culmorum is a worldwide, soil-borne plant pathogen. It causes diseases of cereals, reduces their yield, and fills the grain with toxins. The main direction of modern breeding is to select wheat genotypes the most resistant to Fusarium diseases. This study uses seedlings and plants at the anthesis stage to analyze total soluble carbohydrates, total and cell-wall bound phenolics, chlorophyll content, antioxidant activity, hydrogen peroxide content, mycotoxin accumulation, visual symptoms of the disease, and Fusarium head blight index (FHBi). These results determine the resistance of three durum wheat accessions. We identify physiological or biochemical markers of durum wheat resistance to F. culmorum. Our results confirm correlations between FHBi and mycotoxin accumulation in the grain, which results in grain yield decrease. The degree of spike infection (FHBi) may indicate accumulation mainly of deoxynivalenol and nivalenol in the grain. High catalase activity in the infected leaves could be considered a biochemical marker of durum sensitivity to this fungus. These findings allowed us to formulate a strategy for rapid evaluation of the disease severity and the selection of plants with higher level, or resistance to F. culmorum infection.

Haploid and Doubled Haploid Plant Production in Brassica rapa L. subsp. pekinensis Via Microspore Culture.

The production of haploid and doubled haploid plants is a biotechnological tool that shortens the breeding process of new cultivars in many species. Doubled haploid plants are homozygous at every locus and they can be utilized as parents to produce F1 hybrids. In this chapter, we describe a protocol for the production of doubled haploid plants in Brassica rapa L. subsp. pekinensis using androgenesis induced by isolated microspore cultures.

VCF2CAPS-A high-throughput CAPS marker design from VCF files and its test-use on a genotyping-by-sequencing (GBS) dataset.

Next-generation sequencing (NGS) is a powerful tool for massive detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertions/deletions (indels). For routine screening of numerous samples, these variants are often converted into cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within PCR products. Current computational tools for SNP to CAPS conversion are limited and usually infeasible to use for large datasets as those generated with NGS. Moreover, there is no available tool for massive conversion of MNPs and indels into CAPS markers. Here, we present VCF2CAPS-a new software for identification of restriction endonucleases that recognize SNP/MNP/indel-containing sequences from NGS experiments. Additionally, the program contains filtration utilities not available in other SNP to CAPS converters-selection of markers with a single polymorphic cut site within a user-specified sequence length, and selection of markers that differentiate up to three user-defined groups of individuals from the analyzed population. Performance of VCF2CAPS was tested on a thoroughly analyzed dataset from a genotyping-by-sequencing (GBS) experiment. A selection of CAPS markers picked by the program was subjected to experimental verification. CAPS markers, also referred to as PCR-RFLPs, belong to basic tools exploited in plant, animal and human genetics. Our new software-VCF2CAPS-fills the gap in the current inventory of genetic software by high-throughput CAPS marker design from next-generation sequencing (NGS) data. The program should be of interest to geneticists involved in molecular diagnostics. In this paper we show a successful exemplary application of VCF2CAPS and we believe that its usefulness is guaranteed by the growing availability of NGS services.

The use of Callitriche cophocarpa Sendtn. for the reclamation of Cr-contaminated freshwater habitat: benefits and limitations.

This work is the first attempt to evaluate suitability of Callitriche cophocarpa Sendtn. (water-starwort) to remove Cr under real-world conditions. Our earlier laboratory-scale studies demonstrated outstanding hyperaccumulation properties of this aquatic higher plant (macrophyte) toward chromium in solution. We introduced C. cophocarpa plants into the watershed with sediments heavily polluted (on average 1400 mg/kg d.w. of Cr) by a tannery. The plants grew vigorously and exhibited no physiological or anatomical disorders. Based on chemical fractionations of bottom sediments, we found low Cr bioavailability. The element was strongly associated with the sediments and could be classified into the following fractions (%): oxidizable III (68.2) > residual IV (28.8) > reducible II (1.6) > exchangeable I (1.4). Despite this, Cr content in plant organs at the contaminated sites was 33 up to 83 times greater than in the control leaf/stem and roots, respectively. Altering redox potential during, i.e., sediment deposition on land may change chemical forms of bound metals in a solid phase, and thus further increase Cr phytoextraction by plants. With this in mind, we concluded that the species, being an outstanding Cr accumulator under laboratory conditions, can be useful in the reclamation of Cr-polluted sediments under controlled, oxidizing conditions.

Stabilized nanosystem of nanocarriers with an immobilized biological factor for anti-tumor therapy.

OBJECTIVE: The inadequate efficiency of existing therapeutic anti-cancer regiments and the increase in the multidrug resistance of cancer cells underscore the need to investigate novel anticancer strategies. The induction of apoptosis in tumors by cytotoxic agents produced by pathogenic microorganisms is an example of such an approach. Nevertheless, even the most effective drug should be delivered directly to targeted sites to reduce any negative impact on other cells. Accordingly, the stabilized nanosystem (SNS) for active agent delivery to cancer cells was designed for further application in local anti-tumor therapy. A product of genetically modified Escherichia coli, listeriolysin O (LLO), was immobilized within the polyelectrolyte membrane (poly(ethylenimine)|hyaluronic acid) shells of 'LLO nanocarriers' coupled with the stabilizing element of natural origin. METHODS AND RESULTS: The impact of LLO was evaluated in human leukemia cell lines in vitro. Correspondingly, the influence of the SNS and its elements was assessed in vitro. The viability of targeted cells was evaluated by flow cytometry. Visualization of the system structure was performed using confocal microscopy. The membrane shell applied to the nanocarriers was analyzed using atomic force microscopy and Fourier transform infrared spectroscopy techniques. Furthermore, the presence of a polyelectrolyte layer on the nanocarrier surface and/or in the cell was confirmed by flow cytometry. Finally, the structural integrity of the SNS and the corresponding release of the fluorescent solute listeriolysin were investigated. CONCLUSION: The construction of a stabilized system offers LLO release with a lethal impact on model eukaryotic cells. The applied platform design may be recommended for local anti-tumor treatment purposes.

Analysis of the mitochondrial proteome in cytoplasmic male-sterile and male-fertile beets.

The reported analyses were aimed at identification of the mitochondrial proteome features which were associated with cytoplasmic male sterility (CMS) in beets. The set of analyzed accessions included CMS, maintainer and restored lines. Mitochondrial preparations were subjected to blue-native electrophoresis followed either by in-gel activity assays or separation in denaturing conditions. The CMS condition was associated with decreased activity of complex V and enhancement of additional complexes with the ATPase activity. This was accompanied by accumulation of heptamer HSP60, preSATP6 and an increase in the fraction of the free ATP9 oligomer (not bound to complex V). The ATP9 effect was reversed upon fertility restoration. BIOLOGICAL SIGNIFICANCE: The reported work provides one of very few comprehensive comparisons of the mitochondrial proteomes from cytoplasmic male-sterile (CMS) and male-fertile plants. It shows that in beets the CMS trait is associated with altered functioning of the mitochondrial ATP synthase complex. The presence of CMS-specific ATP synthase subcomplexes resembles deficiencies of this enzyme reported for mammalian cells.

Mitochondrial atp9 genes from petaloid male-sterile and male-fertile carrots differ in their status of heteroplasmy, recombination involvement, post-transcriptional processing as well as accumulation of RNA and protein product.

KEY MESSAGE: Petaloid cytoplasmic male-sterile carrots exhibit overexpression of the mitochondrial atp9 genes which is associated with specific features in organization and expression of these sequences. In carrots, the Sp-cytoplasm causes transformation of stamens into petal-like organs, while plants carrying normal N-cytoplasm exhibit normal flower morphology. Our work was aimed at characterization of distinct features both cytoplasms display with respect to organization and expression of the mitochondrial atp9 genes. We show that two carrot atp9 genes, previously reported as cytoplasm-specific, in fact occur in heteroplasmic condition. In the Sp-cytoplasm the atp9-1 version dominates over atp9-3, while in N-cytoplasmic plants this proportion is reversed. Herein, we also indicate the presence and recombination activity of a 130-/172-bp sequence repeat which likely shaped the present organization of carrot atp9 loci. Furthermore, cDNA sequence examination revealed that the atp9 open reading frames (ORFs) were C to U edited in 4 nucleotide positions. One of the editing events turns a glutamine triplet into the stop codon, thereby equalizing ORFs of atp9-1 and atp9-3. A certain fraction of partially edited molecules was identified-they all represented the atp9-3 sequence. In either Sp- or N-cytoplasmic plants multiple 5' transcript termini were observed. Of these, the ones mapping more distantly from the atp9 ORF were more pronounced in case of petaloid accessions. It was also shown that despite comparable copy number of the genomic atp9 sequences, the level of the respective mRNAs was approximately 3 times higher in case of petaloid carrots. The latter fact corresponded to the elevated content of the ATP9 protein in plants carrying Sp-cytoplasm. The semi-fertile phenotype of such plants is associated with a drop in ATP9 accumulation.

De novo assembly of the carrot mitochondrial genome using next generation sequencing of whole genomic DNA provides first evidence of DNA transfer into an angiosperm plastid genome.

BACKGROUND: Sequence analysis of organelle genomes has revealed important aspects of plant cell evolution. The scope of this study was to develop an approach for de novo assembly of the carrot mitochondrial genome using next generation sequence data from total genomic DNA. RESULTS: Sequencing data from a carrot 454 whole genome library were used to develop a de novo assembly of the mitochondrial genome. Development of a new bioinformatic tool allowed visualizing contig connections and elucidation of the de novo assembly. Southern hybridization demonstrated recombination across two large repeats. Genome annotation allowed identification of 44 protein coding genes, three rRNA and 17 tRNA. Identification of the plastid genome sequence allowed organelle genome comparison. Mitochondrial intergenic sequence analysis allowed detection of a fragment of DNA specific to the carrot plastid genome. PCR amplification and sequence analysis across different Apiaceae species revealed consistent conservation of this fragment in the mitochondrial genomes and an insertion in Daucus plastid genomes, giving evidence of a mitochondrial to plastid transfer of DNA. Sequence similarity with a retrotransposon element suggests a possibility that a transposon-like event transferred this sequence into the plastid genome. CONCLUSIONS: This study confirmed that whole genome sequencing is a practical approach for de novo assembly of higher plant mitochondrial genomes. In addition, a new aspect of intercompartmental genome interaction was reported providing the first evidence for DNA transfer into an angiosperm plastid genome. The approach used here could be used more broadly to sequence and assemble mitochondrial genomes of diverse species. This information will allow us to better understand intercompartmental interactions and cell evolution.

Against the traffic: The first evidence for mitochondrial DNA transfer into the plastid genome.

Transfer of DNA between different compartments of the plant cell, i.e., plastid, mitochondrion and nucleus, is a well-known phenomenon in plant evolution. Six directions of inter-compartmental DNA migration are possible in theory, however only four of them have been previously reported. These include frequent cases of mitochondrion and plastid to nucleus transfer, plastid to mitochondrion transfer, and rare nucleus to mitochondrion migrations. The connection between the plastid and mitochondrial genomes in flowering plants has been viewed as a one way road. Contrary to these observations we found that a sequence widespread in the carrot mitochondrial genome, designated as DcMP, was transferred to the plastid genome of a carrot ancestor. Interestingly, DcMP was integrated into a tRNA promoter of the plastid trnV gene, replacing the original promoter sequence. The rearrangement of the plastid genome is specific for carrot and closely related species belonging Scandiceae clade. The structure of the sequence and the presence of a 6 nt target site duplication led us to speculate that the transfer was a result of a transposition event of a non-LTR retrotransposon. These findings open interesting questions about the evolution of organellar genomes and mobile genetic elements and provide a useful plastid marker to phylogenetically delineate species relationships within the Scandiceae clade.

Dynamics of Vulmar/VulMITE group of transposable elements in Chenopodiaceae subfamily Betoideae.

Transposable elements are important factors driving plant genome evolution. Upon their mobilization, novel insertion polymorphisms are being created. We investigated differences in copy number and insertion polymorphism of a group of Mariner-like transposable elements Vulmar and related VulMITE miniature inverted-repeat transposable elements (MITEs) in species representing subfamily Betoideae. Insertion sites of these elements were identified using a modified transposon display protocol, allowing amplification of longer fragments representing regions flanking insertion sites. Subsequently, a subset of TD fragments was converted into insertion site-based polymorphism (ISBP) markers. The investigated group of transposable elements was the most abundant in accessions representing the section Beta, showing intraspecific insertion polymorphisms likely resulting from their recent activity. In contrast, no unique insertions were observed for species of the genus Beta section Corollinae, while a set of section-specific insertions was observed in the genus Patellifolia, however, only two of them were polymorphic between P. procumbens and P. webbiana. We hypothesize that Vulmar and VulMITE elements were inactivated in the section Corollinae, while they remained active in the section Beta and the genus Patellifolia. The ISBP markers generally confirmed the insertion patterns observed with TD markers, including presence of distinct subsets of TE insertions specific to Beta and Patellifolia.

Ribosomal DNA, tri- and bi-partite pericentromeres in the permanent translocation heterozygote Rhoeo spathacea.

High- and low-stringency FISH and base-specific fluorescence were performed on the permanent translocation heterozygote Rhoeo spathacea (2n = 12). Our results indicate that 45S rDNA arrays, rDNA-related sequences and other GC-rich DNA fraction(s) are located within the pericentromeric regions of all twelve chromosomes, usually colocalizing with the chromomycin A(3)-positive bands. Homogenization of the pericentromeric regions appears to result from the concerted spread of GC-rich sequences, with differential amplification likely. We found new 5S rDNA patterns, which suggest a variability in the breakpoints and in the consequent chromosome reorganizations. It was found that the large 5S rDNA locus residing on each of the 8E and 9E arms consisted of two smaller loci. On each of the two chromosome arms 3b and 4b, in addition to the major subtelomeric 5S rDNA locus, a new minor locus was found interstitially about 40% along the arm length. The arrangement of cytotogenetic landmarks and chromosome arm measurements are discussed with regard to genome repatterning in Rhoeo.

A one-megabase physical map provides insights on gene organization in the enormous mitochondrial genome of cucumber.

Cucumber (Cucumis sativus) has one of the largest mitochondrial genomes known among all eukaryotes, due in part to the accumulation of short 20 to 60 bp repetitive DNA motifs. Recombination among these repetitive DNAs produces rearrangements affecting organization and expression of mitochondrial genes. To more efficiently identify rearrangements in the cucumber mitochondrial DNA, we built two nonoverlapping 800 and 220 kb BAC contigs and assigned major mitochondrial genes to these BACs. Polymorphism carried on the largest BAC contig was used to confirm paternal transmission. Mitochondrial genes were distributed across BACs and physically distant, although occasional clustering was observed. Introns in the nad1, nad4, and nad7 genes were larger than those reported in other plants, due in part to accumulation of short repetitive DNAs and indicating that increased intron sizes contributed to mitochondrial genome expansion in cucumber. Mitochondrial genes atp6 and atp9 are physically close to each other and cotranscribed. These physical contigs will be useful for eventual sequencing of the cucumber mitochondrial DNA, which can be exploited to more efficiently screen for unique rearrangements affecting mitochondrial gene expression.

Characterization of a deep-coverage carrot (Daucus carota L.) BAC library and initial analysis of BAC-end sequences.

Carrot is the most economically important member of the Apiaceae family and a major source of provitamin A carotenoids in the human diet. However, carrot molecular resources are relatively underdeveloped, hampering a number of genetic studies. Here, we report on the synthesis and characterization of a bacterial artificial chromosome (BAC) library of carrot. The library is 17.3-fold redundant and consists of 92,160 clones with an average insert size of 121 kb. To provide an overview of the composition and organization of the carrot nuclear genome we generated and analyzed 2,696 BAC-end sequences (BES) from nearly 2,000 BACs, totaling 1.74 Mb of BES. This analysis revealed that 14% of the BES consists of known repetitive elements, with transposable elements representing more than 80% of this fraction. Eleven novel carrot repetitive elements were identified, covering 8.5% of the BES. Analysis of microsatellites showed a comparably low frequency for these elements in the carrot BES. Comparisons of the translated BES with protein databases indicated that approximately 10% of the carrot genome represents coding sequences. Moreover, among eight dicot species used for comparison purposes, carrot BES had highest homology to protein-coding sequences from tomato. This deep-coverage library will aid carrot breeding and genetics.

Identification of sterility-inducing cytoplasms in rye using the plasmotype-genotype interaction test and newly developed SCAR markers.

A series of 25 rye (Secale cereale L.) inbred lines was tested with respect to three mitochondrial sequence-characterized amplified region (SCAR) polymorphisms. The analysis revealed a close association between the marker-determined mitotypes and plasmotypes (cytoplasm types known from breeding data) represented by the inbreds. The mitochondrial markers also confirmed normal (N-) cytoplasmic character of three wild rye species: Secale montanum, S. vavilovii and S. kuprijanovii. For 186 plants from open-pollinated cultivars of Turkish and South American origin, cytoplasm identification was performed with the use of crossing with double non-restoring tester. In 77 plants the normal (N) cytoplasm was detected, and for 63 of these the PCR analysis was performed producing results which were consistent with the genetic data based on testcrossing and phenotype assessment. The mitochondrial markers also confirmed a presence of sterility-inducing cytoplasm in the remaining 109 plants. Moreover, the markers allowed for differentiation between Pampa (P-) and Vavilovii (V-) cytoplasmic individuals. For 60 plants the latter results were verified using crosses with a line maintaining P-cytoplasmic sterility and acting as a restorer of the V-cytoplasm. For two of these plants contradicting results were produced with the applied methods of cytoplasm identification and the basis of this discrepancy is discussed. Regardless of the identification method, widespread occurrence of a sterility-inducing cytoplasm was revealed, especially in South American populations.

Selection of tomato plants resistant to a local Polish isolate of tomato spotted wilt virus (TSWV).

We found that the Sw-5 gene confers resistance to one of the Polish isolates of tomato spotted wilt virus (TSWV). A series of tomato breeding accessions was analysed along with standards of resistance and susceptibility to TSWV. The presence of the Sw-5 gene was determined using the available PCR marker. Subsequently plants from these accessions were grown in the presence of the TSWV isolate from Poland. Some of them developed severe symptoms of the TSWV disease. Expression of the virus proteins was also assayed in tissues of the investigated plants. We found general agreement between either lack or presence of the disease symptoms, virus proteins and resistance gene. Some observed discrepancies of these data are also discussed. Our results indicate that marker-assisted selection can be used for breeding of the TSWV-resistant tomato in Poland.

The use of cytoplasmic markers in onion hybrid breeding.

We applied the RFLP approach to identify the cytoplasmic genotypes of selected onion breeding materials from Poland. For this purpose, mitochondrial DNA from cytoplasmic male-sterile (CMS) and male-fertile onions were hybridized with the probes for the following mitochondrial genes: atpA, atp6, atp9, cob, cox1, nad3, nad4 and nad6. S-, T- or C-cytoplasm was represented in each analyzed sterile accession. Some new polymorphisms shared by S- and C-cytoplasmic onions were identified. We also used currently available PCR markers to test if cytoplasmic heterogenity occurs within onion inbreds. A fraction of the plants bearing S-cytoplasm were found within two male-fertile lines, but such plants were not detected in the open-pollinated cultivars Sochaczewska, Wolska and Zytawska. Both the RFLP and PCR approaches gave some proof of existing mitochondrial heteroplasmy in onions