Transcriptome Analysis of White- and Red-Fleshed Apple Fruits Uncovered Novel Genes Related to the Regulation of Anthocyanin Biosynthesis.

The red flesh coloration of apples is a result of a biochemical pathway involved in the biosynthesis of anthocyanins and anthocyanidins. Based on apple genome analysis, a high number of regulatory genes, mainly transcription factors such as MYB, which are components of regulatory complex MYB-bHLH-WD40, and several structural genes (PAL, 4CL, CHS, CHI, F3H, DFR, ANS, UFGT) involved in anthocyanin biosynthesis, have been identified. In this study, we investigated novel genes related to the red-flesh apple phenotype. These genes could be deemed molecular markers for the early selection of new apple cultivars. Based on a comparative transcriptome analysis of apples with different fruit-flesh coloration, we successfully identified and characterized ten potential genes from the plant hormone transduction pathway of auxin (GH3); cytokinins (B-ARR); gibberellins (DELLA); abscisic acid (SnRK2 and ABF); brassinosteroids (BRI1, BZR1 and TCH4); jasmonic acid (MYC2); and salicylic acid (NPR1). An analysis of expression profiles was performed in immature and ripe fruits of red-fleshed cultivars. We have uncovered genes mediating the regulation of abscisic acid, salicylic acid, cytokinin, and jasmonic acid signaling and described their role in anthocyanin biosynthesis, accumulation, and degradation. The presented results underline the relationship between genes from the hormone signal transduction pathway and UFGT genes, which are directly responsible for anthocyanin color transformation as well as anthocyanin accumulation during apple-fruit ripening.

The genetic basis of cold tolerance in cucumber (Cucumis sativus L.)-the latest developments and perspectives.

Cold stress is one of the main causes of yield losses in plant production in temperate climate areas. Cold stress slows down and even stops plant growth and development and causes injuries that may result in the plant's death. Cucumber (Cucumis sativus L.), an economically important vegetable, is sensitive to low temperatures, thus improving cold tolerance in cucumber would benefit cucumber producers, particularly those farming in temperate climates and higher altitude areas. So far, single cucumber accessions showing different degrees of cold tolerance have been identified, and genetic studies have revealed biparentally and maternally inherited genetic factors responsible for chilling tolerance. Paternally transmitted chilling tolerance has also been suggested. Quantitative trait loci (QTL) associated with seed germination ability at low temperature and seedling recovery from chilling have been described. Several transgenic attempts have been made to improve cold tolerance in cucumber. Despite numerous studies, the molecular mechanisms of cold tolerance in cucumber have still not been sufficiently elucidated. In this review, we summarise the results of research focused on understanding the genetic basis of cold tolerance in cucumber and their implications for cucumber breeding.

Transcriptome Profiling of Cucumber (Cucumis sativus L.) Early Response to Pseudomonas syringae pv. lachrymans.

Bacterial angular leaf spot disease (ALS) caused by Pseudomonas syringae pv. lachrymans (Psl) is one of the biological factors limiting cucumber open-field production. The goal of this study was to characterize cytological and transcriptomic response of cucumber to this pathogen. Plants of two inbred lines, B10 (susceptible) and Gy14 (resistant), were grown, and leaves were inoculated with highly virulent Psl strain 814/98 under growth chamber conditions. Microscopic and transcriptional evaluations were performed at three time points: before, 1 and 3 days post inoculation (dpi). Investigated lines showed distinct response to Psl. At 1 dpi bacterial colonies were surrounded by necrotized mesophyll cells. At 3 dpi, in the susceptible B10 line bacteria were in contact with degraded cells, whereas cells next to bacteria in the resistant Gy14 line were plasmolyzed, but apparently still alive and functional. Additionally, the level of H2O2 production was higher in resistant Gy14 plants than in B10 at both examined time points. In RNA sequencing more than 18,800 transcripts were detected in each sample. As many as 1648 and 2755 differentially expressed genes (DEGs) at 1 dpi as well as 2992 and 3141 DEGs at 3 dpi were identified in B10 and Gy14, respectively. DEGs were characterized in terms of functional categories. Resistant line Gy14 showed massive transcriptomic response to Psl at 1 dpi compared to susceptible line B10, while a similar number of DEGs was detected for both lines at 3 dpi. This suggests that dynamic transcriptomic response to the invading pathogen may be related with host resistance. This manuscript provides the first transcriptomic data on cucumber infected with the pathovar lachrymans and helps to elucidate resistance mechanism against ALS disease.

Identification of Fruit-Associated QTLs in Winter Squash (Cucurbita maxima Duchesne) Using Recombinant Inbred Lines.

Cucurbita maxima Duchesne squash and pumpkins are cultivated world-wide. Cucurbita maxima fruits are produced for fresh market and are valuable for food processing. Therefore, fruit characteristics and yield are the traits of high economic importance for breeders. To date, the genetic basis of fruit-associated traits in C. maxima have been poorly understood. In the present study, we evaluated fruit-associated traits and conducted quantitative trait locus (QTL) analysis using recombinant inbred lines (RILs) derived from a cross of two inbred lines with different fruit morphotypes. Phenotypic data for nine fruit traits (earliness, weight, number per plant, yield per plant, length and diameter, shape index, flesh thickness, sucrose content and dry matter content) were collected for RILs in two open-field experiments. Pairwise analysis of the phenotypic data revealed correlations among the fruit and yield-associated traits. Using a previously developed genetic map, we identified 26 QTLs for eight traits. The QTLs were found in 10 locations on eight chromosomes of C. maxima. The QTLs were detected across experiments and explained up to 41.4% of the observed phenotypic variations. Major-effect QTLs for multiple fruit-associated traits were clustered on chromosome 4, suggesting that this genomic region has been under selection during diversification and/or domestication of C. maxima.

Genetic mapping of psl locus and quantitative trait loci for angular leaf spot resistance in cucumber (Cucumis sativus L.).

One of the most important cucumber diseases is bacterial angular leaf spot (ALS), whose increased occurrence in open-field production has been observed over the last years. To map ALS resistance genes, a recombinant inbred line (RIL) mapping population was developed from a narrow cross of cucumber line Gy14 carrying psl resistance gene and susceptible B10 line. Parental lines and RILs were tested under growth chamber conditions as well as in the field for angular leaf spot symptoms. Based on simple sequence repeat and DArTseq, genotyping a genetic map was constructed, which contained 717 loci in seven linkage groups, spanning 599.7 cM with 0.84 cM on average between markers. Monogenic inheritance of the lack of chlorotic halo around the lesions, which is typical for ALS resistance and related with the presence of recessive psl resistance gene, was confirmed. The psl locus was mapped on cucumber chromosome 5. Two major quantitative trait loci (QTL) psl5.1 and psl5.2 related to disease severity were found and located next to each other on chromosome 5; moreover, psl5.1 was co-located with psl locus. Identified QTL were validated in the field experiment. Constructed genetic map and markers linked to ALS resistance loci are novel resources that can contribute to cucumber breeding programs.

Genetic mapping of ovary colour and quantitative trait loci for carotenoid content in the fruit of Cucurbita maxima Duchesne.

The high content of carotenoids, sugars, dry matter, vitamins and minerals makes the fruit of winter squash (Cucurbita maxima Duchesne) a valuable fresh-market vegetable and an interesting material for the food industry. Due to their nutritional value, long shelf-life and health protective properties, winter squash fruits have gained increased interest from researchers in recent years. Despite these advantages, the genetic and genomic resources available for C. maxima are still limited. The aim of this study was to use the genetic mapping approach to map the ovary colour locus and to identify the quantitative trait loci (QTLs) for high carotenoid content and flesh colour. An F6 recombinant inbred line (RIL) mapping population was developed and used for evaluations of ovary colour, carotenoid content and fruit flesh colour. SSR markers and DArTseq genotyping-by-sequencing were used to construct an advanced genetic map that consisted of 1824 molecular markers distributed across linkage groups corresponding to 20 chromosomes of C. maxima. Total map length was 2208 cM and the average distance between markers was 1.21 cM. The locus affecting ovary colour was mapped at the end of chromosome 14. The identified QTLs for carotenoid content in the fruit and fruit flesh colour shared locations on chromosomes 2, 4 and 14. QTLs on chromosomes 2 and 4 were the most meaningful. A correlation was clearly confirmed between fruit flesh colour as described by the chroma value and carotenoid content in the fruit. A high-density genetic map of C. maxima with mapped loci for important fruit quality traits is a valuable resource for winter squash improvement programmes.

Transcriptome Analyses of Mosaic (MSC) Mitochondrial Mutants of Cucumber in a Highly Inbred Nuclear Background.

Cucumber (Cucumis sativus L.) has a large, paternally transmitted mitochondrial genome. Cucumber plants regenerated from cell cultures occasionally show paternally transmitted mosaic (MSC) phenotypes, characterized by slower growth, chlorotic patterns on the leaves and fruit, lower fertility, and rearrangements in their mitochondrial DNAs (mtDNAs). MSC lines 3, 12, and 16 originated from different cell cultures all established using the highly inbred, wild-type line B. These MSC lines possess different rearrangements and under-represented regions in their mtDNAs. We completed RNA-seq on normalized and non-normalized cDNA libraries from MSC3, MSC12, and MSC16 to study their nuclear gene-expression profiles relative to inbred B. Results from both libraries indicated that gene expression in MSC12 and MSC16 were more similar to each other than MSC3. Forty-one differentially expressed genes (DEGs) were upregulated and one downregulated in the MSC lines relative to B. Gene functional classifications revealed that more than half of these DEGs are associated with stress-response pathways. Consistent with this observation, we detected elevated levels of hydrogen peroxide throughout leaf tissue in all MSC lines compared to wild-type line B. These results demonstrate that independently produced MSC lines with different mitochondrial polymorphisms show unique and shared nuclear responses. This study revealed genes associated with stress response that could become selection targets to develop cucumber cultivars with increased stress tolerance, and further support of cucumber as a model plant to study nuclear-mitochondrial interactions.

Next generation sequencing and omics in cucumber (Cucumis sativus L.) breeding directed research.

In the post-genomic era the availability of genomic tools and resources is leading us to novel generation methods in plant breeding, as they facilitate the study of the genotype and its relationship with the phenotype, in particular for complex traits. In this study we have mainly concentrated on the Cucumis sativus and (but much less) Cucurbitaceae family several important vegetable crops. There are many reports on research conducted in Cucurbitaceae plant breeding programs on the ripening process, phloem transport, disease resistance, cold tolerance and fruit quality traits. This paper presents the role played by new omic technologies in the creation of knowledge on the mechanisms of the formation of the breeding features. The analysis of NGS (NGS-next generation sequencing) data allows the discovery of new genes and regulatory sequences, their positions, and makes available large collections of molecular markers. Genome-wide expression studies provide breeders with an understanding of the molecular basis of complex traits. Firstly a high density map should be created for the reference genome, then each re-sequencing data could be mapped and new markers brought out into breeding populations. The paper also presents methods that could be used in the future for the creation of variability and genomic modification of the species in question. It has been shown also the state and usefulness in breeding the chloroplastomic and mitochondriomic study.

Persistence and Protection of Mitochondrial DNA in the Generative Cell of Cucumber is Consistent with its Paternal Transmission.

Plants predominantly show maternal transmission of mitochondrial DNA (mtDNA). One known exception is cucumber, in which the mtDNA is paternally inherited. However, the mechanisms regulating this unique mode of transmission are unclear. Here we monitored the amounts of mtDNA throughout the development of cucumber microspores into pollen and observed that mtDNA decreases in the vegetative cell, but persists in the generative cell that ultimately produces the sperm cells. We characterized the cucumber homolog (CsDPD1) of the Arabidopsis gene defective in pollen organelle DNA degradation 1 (AtDPD1), which plays a direct role in mtDNA degradation. CsDPD1 rescued an Arabidopsis AtDPD1 mutant, indicating the same function in both plants. Expression of CsDPD1 coincided with the decrease of mtDNA in pollen, except in the generative cell where both the expression of CsDPD1 and mtDNA levels remained high. Our cytological results confirmed that the persistence of mtDNA in the cucumber generative cell is consistent with its paternal transmission. Our molecular analyses suggest that protection of mtDNA in the generative cell may be the critical factor for paternal mtDNA transmission, rather than mtDNA degradation mediated by CsDPD1. Taken together, these findings indicate that a mechanism may protect paternal mtDNA from degradation and is likely to be the genetic basis of paternal mtDNA transmission.

The Mosaic Mutants of Cucumber: A Method to Produce Knock-Downs of Mitochondrial Transcripts.

Cytoplasmic effects on plant performance are well-documented and result from the intimate interaction between organellar and nuclear gene products. In plants, deletions, mutations, or chimerism of mitochondrial genes are often associated with deleterious phenotypes, as well as economically important traits such as cytoplasmic male sterility used to produce hybrid seed. Presently, genetic analyses of mitochondrial function and nuclear interactions are limited because there is no method to efficiently produce mitochondrial mutants. Cucumber (Cucumis sativus L.) possesses unique attributes useful for organellar genetics, including differential transmission of the three plant genomes (maternal for plastid, paternal for mitochondrial, and bi-parental for nuclear), a relatively large mitochondrial DNA in which recombination among repetitive motifs produces rearrangements, and the existence of strongly mosaic (MSC) paternally transmitted phenotypes that appear after passage of wild-type plants through cell cultures and possess unique rearrangements in the mitochondrial DNA. We sequenced the mitochondrial DNA from three independently produced MSC lines and revealed under-represented regions and reduced transcription of mitochondrial genes carried in these regions relative to the wild-type parental line. Mass spectrometry and Western blots did not corroborate transcriptional differences in the mitochondrial proteome of the MSC mutant lines, indicating that post-transcriptional events, such as protein longevity, may compensate for reduced transcription in MSC mitochondria. Our results support cucumber as a model system to produce transcriptional "knock-downs" of mitochondrial genes useful to study mitochondrial responses and nuclear interactions important for plant performance.

Genetic diversity of Trichoderma atroviride strains collected in Poland and identification of loci useful in detection of within-species diversity.

Molecular markers that enable monitoring of fungi in their natural environment or assist in the identification of specific strains would facilitate Trichoderma utilization, particularly as an agricultural biocontrol agent (BCA). In this study, sequence analysis of internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of the ribosomal RNA (rRNA) gene cluster, a fragment of the translation elongation factor 1-alpha (tef1) gene, and random amplified polymorphic DNA (RAPD) markers were applied to determine the genetic diversity of Trichoderma atroviride strains collected in Poland, and also in order to identify loci and PCR-based molecular markers useful in genetic variation assessment of that fungus. Although tef1 and RAPD analysis showed limited genetic diversity among T. atroviride strains collected in Poland, it was possible to distinguish major groups that clustered most of the analyzed strains. Polymorphic RAPD amplicons were cloned and sequenced, yielding sequences representing 13 T. atroviride loci. Based on these sequences, a set of PCR-based markers specific to T. atroviride was developed and examined. Three cleaved amplified polymorphic sequence (CAPS) markers could assist in distinguishing T. atroviride strains. The genomic regions identified may be useful for further exploration and development of more precise markers suitable for T. atroviride identification and monitoring, especially in environmental samples.

Suppression of NGB and NAB/ERabp1 in tomato modifies root responses to potato cyst nematode infestation.

Plant-parasitic nematodes cause significant damage to major crops throughout the world. The small number of genes conferring natural plant resistance and the limitations of chemical control require the development of new protective strategies. RNA interference or the inducible over-expression of nematicidal genes provides an environment-friendly approach to this problem. Candidate genes include NGB, which encodes a small GTP-binding protein, and NAB/ERabp1, which encodes an auxin-binding protein, which were identified as being up-regulated in tomato roots in a transcriptome screen of potato cyst nematode (Globodera rostochiensis) feeding sites. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization confirmed the localized up-regulation of these genes in syncytia and surrounding cells following nematode infection. Gene-silencing constructs were introduced into tomato, resulting in a 20%-98% decrease in transcription levels. Nematode infection tests conducted on transgenic plants showed 57%-82% reduction in the number of G. rostochiensis females in vitro and 30%-46% reduction in pot trials. Transmission electron microscopy revealed a deterioration of cytoplasm, and degraded mitochondria and plastids, in syncytia induced in plants with reduced NAB/ERabp1 expression. Cytoplasm in syncytia induced in plants with low NGB expression was strongly electron translucent and contained very few ribosomes; however, mitochondria and plastids remained intact. Functional impairments in syncytial cytoplasm of silenced plants may result from NGB's role in ribosome biogenesis; this was confirmed by localization of yellow fluorescent protein (YFP)-labelled NGB protein in nucleoli and co-repression of NGB in plants with reduced NAB/ERabp1 expression. These results demonstrate that NGB and NAB/ERabp1 play important roles in the development of nematode-induced syncytia.

Cucumber Possesses a Single Terminal Alternative Oxidase Gene That is Upregulated by Cold Stress and in the Mosaic (MSC) Mitochondrial Mutants.

Alternative oxidase (AOX) is a mitochondrial terminal oxidase which is responsible for an alternative route of electron transport in the respiratory chain. This nuclear-encoded enzyme is involved in a major path of survival under adverse conditions by transfer of electrons from ubiquinol instead of the main cytochrome pathway. AOX protects against unexpected inhibition of the cytochrome c oxidase pathway and plays an important role in stress tolerance. Two AOX subfamilies (AOX1 and AOX2) exist in higher plants and are usually encoded by small gene families. In this study, genome-wide searches and cloning were completed to identify and characterize AOX genes in cucumber (Cucumis sativus L.). Our results revealed that cucumber possesses no AOX1 gene(s) and only a single AOX2 gene located on chromosome 4. Expression studies showed that AOX2 in wild-type cucumber is constitutively expressed at low levels and is upregulated by cold stress. AOX2 transcripts and protein were detected in leaves and flowers of wild-type plants, with higher levels in the three independently derived mosaic (MSC) mitochondrial mutants. Because cucumber possesses a single AOX gene and its expression increases under cold stress and in the MSC mutants, this plant is a unique and intriguing model to study AOX expression and regulation particularly in the context of mitochondria-to-nucleus retrograde signaling.

The genome sequence of the North-European cucumber (Cucumis sativus L.) unravels evolutionary adaptation mechanisms in plants.

Cucumber (Cucumis sativus L.), a widely cultivated crop, has originated from Eastern Himalayas and secondary domestication regions includes highly divergent climate conditions e.g. temperate and subtropical. We wanted to uncover adaptive genome differences between the cucumber cultivars and what sort of evolutionary molecular mechanisms regulate genetic adaptation of plants to different ecosystems and organism biodiversity. Here we present the draft genome sequence of the Cucumis sativus genome of the North-European Borszczagowski cultivar (line B10) and comparative genomics studies with the known genomes of: C. sativus (Chinese cultivar--Chinese Long (line 9930)), Arabidopsis thaliana, Populus trichocarpa and Oryza sativa. Cucumber genomes show extensive chromosomal rearrangements, distinct differences in quantity of the particular genes (e.g. involved in photosynthesis, respiration, sugar metabolism, chlorophyll degradation, regulation of gene expression, photooxidative stress tolerance, higher non-optimal temperatures tolerance and ammonium ion assimilation) as well as in distributions of abscisic acid-, dehydration- and ethylene-responsive cis-regulatory elements (CREs) in promoters of orthologous group of genes, which lead to the specific adaptation features. Abscisic acid treatment of non-acclimated Arabidopsis and C. sativus seedlings induced moderate freezing tolerance in Arabidopsis but not in C. sativus. This experiment together with analysis of abscisic acid-specific CRE distributions give a clue why C. sativus is much more susceptible to moderate freezing stresses than A. thaliana. Comparative analysis of all the five genomes showed that, each species and/or cultivars has a specific profile of CRE content in promoters of orthologous genes. Our results constitute the substantial and original resource for the basic and applied research on environmental adaptations of plants, which could facilitate creation of new crops with improved growth and yield in divergent conditions.

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.

The selection of mosaic (MSC) phenotype after passage of cucumber (Cucumis sativus L.) through cell culture - a method to obtain plant mitochondrial mutants.

Mosaic (MSC) mutants of cucumber (Cucumis sativus L.) appear after passage through cell cultures. The MSC phenotype shows paternal transmission and is associated with mitochondrial DNA rearrangements. This review describes the origins and phenotypes of independently produced MSC mutants of cucumber, including current knowledge on their mitochondrial DNA rearrangements, and similarities of MSC with other plant mitochondrial mutants. Finally we propose that passage of cucumber through cell culture can be used as a unique and efficient method to generate mitochondrial mutants of a higher plant in a highly homozygous nuclear background.

Organization of repetitive DNAs and the genomic regions carrying ribosomal RNA, cob, and atp9 genes in the cucurbit mitochondrial genomes.

Plants in the genus Cucumis (cucumber and melon) have the largest mitochondrial genomes known among all plants, due in part to the accumulation of repetitive DNAs of varying complexities. Recombination among these repetitive DNAs should produce highly rearranged mitochondrial genomes relative to the smaller mitochondrial genomes of related plants. We cloned and sequenced mitochondrial genomic regions near the rRNA, atp9 and cob genes from cucumber, melon, squash and watermelon (all members of the Cucurbitaceae family), and compared to the previously sequenced mitochondrial genomes of Arabidopsis thaliana and sugar beet to study the distribution and arrangement of coding and repetitive DNAs. Cucumber and melon had regions of concentrated repetitive DNAs spread throughout the sequenced regions; few repetitive DNAs were revealed in the mitochondrial genomes of A. thaliana, sugar beet, squash and watermelon. Recombination among these repetitive DNAs most likely produced unique arrangements of the rrn18 and rrn5 genes in the genus Cucumis. Cucumber mitochondrial DNA had more pockets of dispersed direct and inverted repeats than melon and the other plants, and we did not reveal repetitive sequences significantly contributing to mitochondrial genome expansion in both cucumber and melon.

Transcriptional expression of a Solanum sogarandium pGT::Dhn10 gene fusion in cucumber, and its correlation with chilling tolerance in transgenic seedlings.

The expression pattern of a Solanum sogarandinum pGT::Dhn10 gene fusion encoding a dehydrin DHN10 protein and the potential role of that protein in cold tolerance in cucumber were analysed in three T1transgenic lines. An accumulation of Dhn10 mRNA was detected in the leaves, cotyledons, hypocotyls and roots of the transgenic seedlings both under the control conditions and after a cold treatment at 6 degrees C for 24 h. This was confirmed by RT-PCR. However, no DHN10 protein was detected by the alkaline phosphatase-conjugated antibody. The transgenic lines exhibited different levels of chilling tolerance. The TCC5/1 line showed a significant increase in its chilling tolerance compared to the non-transgenic line. No chilling injury was observed when the cold hardened (6 degrees C, 24 h) TCC5/1 plants were subsequently exposed to a temperature of 2 degrees C for 6 h. The other two transgenic lines, TCC2/1 and TCC3/2, exhibited a comparable level of chilling tolerance to that of the non-transgenic control. The transgenic lines showed similar or significantly decreased freezing tolerance compared to the non-transgenic control, as evaluated by an electrolyte leakage test. We concluded that the S. sogarandnium GT promoter is functional in the chilling sensitive species Cucumis sativus L., and that the pGT::Dhn10 gene fusion is expressed at the transcriptional level.

Mosaic (MSC) cucumbers regenerated from independent cell cultures possess different mitochondrial rearrangements.

Passage of the highly inbred cucumber ( Cucumis sativus L.) line B through cell culture produces progenies with paternally transmitted, mosaic (MSC) phenotypes. Because the mitochondrial genome of cucumber shows paternal transmission, we evaluated for structural polymorphisms by hybridizing cosmids spanning the entire mitochondrial genome of Arabidopsis thaliana L. to DNA-gel blots of four independently generated MSC and four wild-type cucumbers. Polymorphisms were identified by cosmids carrying rrn18, nad5-exon2, rpl5, and the previously described JLV5 deletion. Polymorphisms revealed by rrn18 and nad5-exon2 were due to one rearrangement bringing together these two coding regions. The polymorphism revealed by rpl5 was unique to MSC16 and was due to rearrangement(s) placing the rpl5 region next to the forward junction of the JLV5 deletion. The rearrangement near rpl5 existed as a sublimon in wild-type inbred B, but was not detected in the cultivar Calypso. Although RNA-gel blots revealed reduced transcription of rpl5 in MSC16 relative to wild-type cucumber, Western analyses revealed no differences for the RPL5 protein and the genetic basis of the MSC16 phenotype remains enigmatic. We evaluated 17 MSC and wild-type lines regenerated from independent cell-culture experiments for these structural polymorphisms and identified eight different patterns, indicating that the passage of cucumber through cell culture may be a unique mechanism to induce or select for novel rearrangements affecting mitochondrial gene expression.

Chromosome number variation in somatic hybrids between transgenic tomato (Lycopersicon esculentum) and Solanum lycopersicoides.

Leaf mesophyll protoplasts of Lycopersicon esculentum were fused with suspension-culture-derived protoplasts of Solanum lycopersicoides by a PEG treatment. Both species have the same chromosome number (2n = 2x = 24). The hybrid calli were selected using the full selection method - kanamycin resistance and culture conditions critical for L. esculentum protoplast divisions. The genomic in situ hybridization analyses indicated a hypo- and hypertetraploid character of the hybrid plant with a majority of S. lycopersicoides chromosomes and a variation in chromosome number from 46 to 53. The hybrids contained a transgene derived from L. esculentum, as shown by Southern blot hybridization and PCR analyses. Their mitochondria were derived from the wild species, S. lycopersicoides. More than 60 regenerated plants were transferred into the greenhouse. They grew very slowly and were not able to flower for almost one year. The main morphological characters of the hybrids included a single shoot and small, dark-green leaves with strongly wrinkled blades. The reasons for nuclear genome asymmetry between hybrids and the possibilities of using them in a genetic and breeding programme are discussed in this paper.