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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.

The present work compiles the results of three-year expeditions organized between 2011 and 2013 aimed at plant crop collection in the area of Lithuania, an Eastern European country. Accessions of fruit trees, vegetables, cereals, forage, industrial, fibrous, medical, and spice crops as well as accompanying segetal plants were collected in 5 ethnographic regions of Lithuania. In total, 1010 samples of seeds, bulbs, and plant grafts were obtained. The majority of the collected samples belonged to the Cucurbitaceae, Fabaceae, Solanaceae, Alliaceae, and Rosaceae families. The accessions were described and deposited in the long-term storage in Lithuanian and Polish Gene Banks. Almost all collected cucumbers, pumpkins, squashes, and oat plants were morphologically characterized in field experiments. Cucurbita pepo accessions showed high morphological diversity, while limited diversity of cucumber accessions was observed. Oat plants were characterized by high morphological diversity and resistance to diseases present in some of the investigated accessions. Further characterization of collected apple trees and other accessions is underway. Obtaining germplasm in the area of Lithuania fulfills a gap in current plant crop collections. Collected material could be valuable for pre-breeding evaluation and further breeding programs as well as the study of genetic diversity.

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.

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.

Although many Pseudomonas syringae strains have already been determined, only a few genomes of strains belonging to pathovar lachrymans have been sequenced so far. In this study we report the genome sequence of P. syringae pv. lachrymans strain 814/98, which is highly virulent to cucumber. The genome size was estimated to be 6.58 Mb, with 57.97% GC content. In total, 6024 genes encoding proteins and 92 genes encoding RNAs were identified in this genome. Comparisons with the available sequenced genomes of pathovar lachrymans as well as with other P. syringae pathovars were conducted, revealing the presence of three unique plasmids and 24 type III effector proteins (TTEs) in strain 814/98. The phylogenetic analyses of MLST loci and TTEs clearly showed the existence of two distinct clusters of strains within pathovar lachrymans, which were grouped into either phylogroup 1 or 3, supporting non-monophyly within this pathovar.

Molecular marker examinations of genetic diversity are valuable for assessment of within-species diversity and relationships, genotype identification, to assist selection for crop improvement programs, and to handle coherently germplasm collections. Cucurbita maxima is a highly diverse cucurbit crop, but its genetic diversity at the molecular level is inadequately characterized. Presently, genomic simple sequence repeat (SSR) markers that were described and employed for Cucurbita moschata and Cucurbita pepo were screened on C. maxima. A set of 23 highly informative SSRs was selected to evaluate genetic diversity within a collection of 85 C. maxima accessions. This collection contained cultivars and breeding lines from Europe, North America, Asia, Australia, and New Zealand, of six horticultural groups. Based on the genotyping results and statistical analyses, a fundamental division was observed in C. maxima above the level of horticultural groups. One major cluster was comprised between accessions from the Banana, Buttercup, and Hubbard horticultural groups. The other major cluster was divided in two sub-clusters, the larger of which included all accessions of the Mammoth (Show) Pumpkin horticultural group and the smaller, more outlying cluster was comprised of accessions of the Australian Blue horticultural group, with the one representative of the Turban group most outlying. Generally, the horticultural groups formed sub-sub-clusters, as most accessions within these respective groups showed close affinity. The selected set of 23 Cucurbita-conserved SSR markers is expected to be of great value for further germplasm characterization in this species and for taxonomical identifications within the genus Cucurbita.

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.

Styrian hull-less pumpkins are valued for their use in health-promoting foods such as oils and snacks. Although deriving from Styria, they are now cultivated globally. Seeds of Styrian oil pumpkins are rich in lipids and represent a high-value product. Thus, seed yield and quality are traits of economic importance. These seed characteristics depend mostly on the cultivated variety and plant growing conditions. This study aimed to assess the variation in hull-less seed lipids of new hybrids of Styrian oil pumpkin developed recently for cultivation in moderate/colder climate zones of the north-eastern part of Poland. The study showed that the newly-developed hybrids have a relatively high content of total lipids in seeds, with a substantially higher share of linoleic acid (up to 63%). However, sterols, tocopherols, and squalene content and composition were similar to pumpkin seed oils produced from plants cultivated in other, warmer regions. This study provides insights into the chemical composition of Styrian pumpkin oil produced from hybrids grown under the relatively severe climatic conditions of north-eastern Poland.

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.

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.

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. Electronic supplementary material The online version of this article (10.1007/s11032-018-0866-2) contains supplementary material, which is available to authorized users.

The high content of carotenoids, sugars, dry matter, vitamins and minerals makes the fruit of winter squash (

This study provides first results of transcriptome data for Cucumis sativus under sodium silicate (Si) supplementation, in order to identify differentially expressed genes (DEGs). In vitro-generated clonal C. sativus line B10 material, derived from leaf microexplants, was established and the plants were cultivated in Murashige and Skoog medium, non-supplemented and Si-supplemented with Na2(SiO2)xxH2O, respectively. The mRNA enrichment of pooled leaf and stem material from both the control and sodium Si-supplemented plants was performed, followed by RNA-Seq. Analysis of the cucumber transcriptome of the control and the Si-treated plants allowed for the determination of 18,957 and 18,882 transcripts referring to 19,896 genes. In total, 1,136 DEGs were determined and 522 (46 %) were assigned to biological processes, in most instances related to primary metabolism (photosynthesis, transport, biosynthesis), thereby supporting previous reports about the impact of Si on plant development, while some transcripts belong to secondary metabolism enabling subsequent analyses including stresses. Some transcripts were slightly up- or down-regulated and might be related to NaCl traces, due to the form of Si used in this instance. These transcriptome data provide the first insights into intermediate Si-accumulating cucumber tissue culture under sodium Sitreatment, with prior analyses on abiotic and biotic stresses.

W pracy podjęto próbę identyfikacji markerów SSR użytecznych do konstrukcji mapy genetycznej ogórka dla populacji mapującej 110 rekombinacyjnych linii wsobnych (RIL). Populację uzyskano z krzyżowania amerykańskiej linii Gy14 wykazującej tolerancję na bakteryjną kanciastą plamistość z polską linią B10 wrażliwą na tę chorobę. Analiza bioinformatyczna zróżnicowania sekwencji loci mikrosatelitarnych pozwoliła wskazać markery SSR potencjalnie przydatne do mapowania w tej populacji. Wytypowano 222 markery SSR, spośród których 160 przetestowano na liniach rodzicielskich i wybranych RIL. Potwierdzono polimorfizm dla 103 markerów SSR (64,4%), zaś dla 52 z nich wykazano przydatność do konstrukcji mapy genetycznej populacji Gy14 × B10 i mapowania genów odporności na kanciastą plamistość liści ogórka. Wstępnie wskazano, że marker SSR00398 zlokalizowany na chromosomie 5 może być sprzężony z genem odporności na tę chorobę.

Przeprowadzono ocenę fenotypową populacji dyni olbrzymiej pod względem cech ilościowych takich jak: średnia masa owocu, liczba owoców na roślinę oraz plon handlowy. W eksperymencie wykorzystano populację mapującą składającą się ze 112 rekombinacyjnych linii wsobnych (RIL) pokolenia F6 dyni olbrzymiej uzyskanych z mieszańca dwóch linii wsobnych 802 × 801 o odmien¬nym pochodzeniu i skrajnie zróżnicowanych pod względem badanych cech. Doświadczenie polowe założono w układzie losowym w trzech powtórzeniach w 2013 i 2014 roku. Linie rodzicielskie wykazywały istotne statystycznie różnice w odniesieniu do wartości cech. Zakres zmienności cech w populacji mapującej przekraczał wartości cech dla linii rodzicielskich. Zakres zmienności dla średniej masy owocu wynosił od 0,6 kg do 9,0 kg, dla średniej liczby owoców na roślinie od 1,0 do 7,5 (2013) i od 1,0 do 11,5 (2014), dla plonu handlowego (kg/roślinę) 1,7–11,8 (2013) i 1,26–30,0 (2014). Odziedziczalność cech wynosiła 71% dla plon handlowego, 70% dla średniej masy owoców i 82% dla średniej liczby owoców na roślinę.

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.

Trichoderma strains that are beneficial to both the growth and health of plants can be used as plant growth-promoting fungi (PGPF) or biological control agents (BCA) in agricultural and horticultural practices. In order to select PGPF or BCA strains, their biological properties and taxonomy must be carefully studied. In this study, 104 strains of Trichoderma collected at geographically different locations in Poland for selection as PGPF or BCA were identified by DNA barcoding, based on the sequences of internal transcribed spacers 1 and 2 (ITS1 and 2) of the ribosomal RNA gene cluster and on the sequences of translation elongation factor 1 alpha (tef1), chitinase 18-5 (chi18-5), and RNA polymerase II subunit (rpb2) gene fragments. Most of the strains were classified as: T. atroviride (38%), T. harzianum (21%), T. lentiforme (9%), T. virens (9%), and T. simmonsii (6%). Single strains belonging to T. atrobrunneum, T. citrinoviride, T. crassum, T. gamsii, T. hamatum, T. spirale, T. tomentosum, and T. viridescens were identified. Three strains that are potentially pathogenic to cultivated mushrooms belonging to T. pleuroticola and T. aggressivum f. europaeum were also identified. Four strains: TRS4, TRS29, TRS33, and TRS73 were classified to Trichoderma spp. and molecular identification was inconclusive at the species level. Phylogeny analysis showed that three of these strains TRS4, TRS29, and TRS33 belong to Trichoderma species that is not yet taxonomically established and strain TRS73 belongs to the T. harzianum complex, however, the species could not be identified with certainty.

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. Electronic supplementary material The online version of this article (doi:10.1007/s11105-015-0883-9) contains supplementary material, which is available to authorized users.

Genetically-modified (GM) tomatoes, carrying thaumatin gene encoding sweet-tasting protein may be a component of diet with high sensory values, constituting a valuable source of nutrients and substances with a health-promoting role. Good utilization and a lack of the effect on animal growth, value of hematological parameters, concentration of immunoglobulins and most of chemical blood parameters of laboratory rats were demonstrated in the nutritional studies on fruits of tomato GM plants. The biological response of the rats receiving GMO or its isogenic equivalent in the diet was recognized as similar. However, the unfavourable effect of the diets containing addition of tomatoes with the recombined thaumatin on the degree of oxidative degradation of DNA of rats liver was recorded. At the same time, the discussed dietary component had no effect on values of the remaining parameters of the oxidative status of tissue of the above mentioned organ and its histological image.

The nuclear male sterility gene ms8 is expected to facilitate the production of sweet pepper (Capsicum annuum L.) hybrids as it provides means for hybridization without the labor-intensive hand emasculation of female inbred lines. The development of molecular markers linked to ms8 locus will help the breeding practice for the selection of hybrid parental lines. In this study, F2 population resulting from a cross between the sweet pepper male sterile line 320 and the male fertile variety Elf was used to identify DNA markers linked to the ms8 locus. With the use of RAPD–BSA technique, seven markers linked to the ms8 locus were found. Four of them were converted into SCAR markers. In addition, two COSII/CAPS markers linked to the ms8 locus were identified. Comparative mapping with reference pepper maps indicated that the ms8 locus is located on the lower arm of the pepper chromosome P4. Identified markers are useful for molecular breeding, however, at present markers tightly linked to ms8 locus are still lacking. Identification of molecular markers linked to the ms8 locus and determination of its chromosomal localization are useful for fine mapping and also provide the perspective for ms8 gene cloning.

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.

Angular leaf spot is a common disease of cucumber (Cucumis sativus) caused by Pseudomonas syringae pv. lachrymans. Genetics of resistance to this disease was investigated using two sets of parameters: (i) disease severity, i.e. the number and size of necrotic and chlorotic lesions on the infected leaves, and (ii) presence or absence of a chlorotic halo around the necrotic spots on the infected leaves. Disease severity appears to be controlled by multiple genes and the heritability of the resistance was estimated to be 53%. The presence or absence of the chlorotic halo was determined to be governed by a single gene, with the presence of the halo (the susceptible phenotype) being a dominant character. A RAPD marker linked to the gene conferring the chlorotic halo was identified. Genetic distance between this marker, OP-AO07, a polymorphic 420 bp amplicon in the DNA of the susceptible plants, and the locus encoding the chlorotic halo was estimated to be 13 cM.

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.

International audience; The PPR-B gene is responsible for male-fertility restoration of the Ogura-type male-sterile radish plants, and it is located in the complex Rfo locus in the vicinity of similar PPR-A gene and PPR-C pseudogene. The aim of this study was to identify PPR-B alleles and understand the structure of the Rfo locus in radish breeding lines. Five lines of radish with normal male-fertile cytoplasm were tested. The entire PPR-B gene was amplified, sequenced and allelic PPR-B sequences were identified. The results indicated that the maintainer lines 7, 15 and 21 contained a non-restoring form of PPR-B protein. A unique PPR-B was found in lines 24/15 and 31 that are restorer and maintainer lines, respectively. The substitutions might be responsible for the loss of a restoring function of the PPR-B-31 allele. Amplification of the PPR-A/PPR-B and PPR-B/PPR-C intergenic regions allowed to identify rearrangements within Rfo locus. Obtained results confirm the wide allelic variation within the Rfo locus, as well as high genetic complexity of the fertility restoration mechanism in radish

Several strains of Pseudomonas syringae pathovar (pv.) lachrymans and related bacterial pathogens were isolated from cucumber (Cucumis sativus) leaves collected in central and southern Poland in 2001 and 2002. Twenty five original strains, together with five reference strains of P. syringae pv. lachrymans, pv. syringae and pv. tomato, were genetically characterized by PCR-RFLP (polymerase chain reaction − restriction fragment length polymorphism), ADSRRS (amplification of DNA fragments surrounding rare restriction sites), and PCR-MP (PCR − melting profiles) fingerprinting techniques. Genetic similarity analyses of the PCR-RFLP and ADSRRS fingerprints showed that strains of P. syringae pv. lachrymans form distinct clusters. The results also indicated that the ADSRRS and the PCR-MP fingerprinting techniques may serve as more efficient tools for evaluating genetic similarity among pathovars and strains of P. syringae than PCR-RFLP. The 25 strains showed diverse pathogenicity to cucumber seedlings and biochemical tests were varied. The syrB gene was identified in four cucumber strains, characterized as P. syringae pv. syringae.

The cucumber MSC16 mutant was obtained by regeneration from cell cultures of the inbred line B and it is associated with complex mitochondrial genome rearrangements causing DNA deletions and duplications. We compared cell suspensions of cucumber MSC16 and the control wild type line B. MSC16 growth intensity in cell cultures was the same as a control line B. There were differences, however, in ability to undergo somatic embryogenesis. In auxin-dependent cell suspension (ADS) the MSC16 formed abnormal embryos not able to convert into the plantlets, however, 14% of MSC16 somatic embryos were normal after application of the cytokinin-dependent cell suspension (CDS) procedure. There were no differences between MSC16 and wild type line B observed in liquid culture of meristematic clumps (LMC). An electron microscopy revealed long fasciated mitochondria present in MSC16 ADS culture and mitochondria organized in clumps present in MSC16 CDS culture, with a control showing typical mitochondria appearance. An accumulation of large starch grains in chloroplasts appeared in cell cultures of the MSC16. These differences are discussed in the context of MSC16 mutant formation and its unique features.

The mitochondrial genome of cucumber shows paternal transmission and there are no reports of variation for mitochondrial transmission in cucumber. We used a mitochondrially encoded mosaic (MSC) phenotype to reveal phenotypic variation for mitochondrial-genome transmission in cucumber. At least 10 random plants from each of 71 cucumber plant introductions (PIs) were crossed as the female with an inbred line (MSC16) possessing the MSC phenotype. Nonmosaic F1 progenies were observed at high frequencies (greater than 50%) in F1 families from 10 PIs, with the greatest proportions being from PI 401734. Polymorphisms near the mitochondrial cox1 gene and JLV5 region revealed that nonmosaic hybrid progenies from crosses of PI 401734 with MSC16 as the male possessed the nonmosaic-inducing mitochondrial DNA (mtDNA) from the paternal parent. F2 ,F 3, and backcross progenies from nonmosaic F1 plants from PI 401734 3 MSC16 were testcrossed with MSC16 as the male parent to reveal segregation of a nuclear locus (Psm for Paternal sorting of mitochondria) controlling sorting of mtDNA from the paternal parent. Psm is a unique locus at which the maternal genotype affects sorting of paternally transmitted mtDNA.

A Lycopersicon esculentum Mill. (tomato) cDNA clone with high similarity to a Nicotiana plumbaginifolia Viv. (tobacco) cytochrome P450 gene was isolated using 5' and 3' rapid amplification of cDNA ends (RACE). The isolated cDNA (GenBank Accession No. AF249329) has an open reading frame of 1494 base pairs (bp) and encodes a protein of 498 amino acids with 75% identity to the N. plumbaginifolia cytochrome P450 (CYP72A2) and 45% to a Catharanthus roseus G. Don (Madagaskar periwinkle) CYP72A1 protein sequence. By Southern-blot analysis, one or two highly homologous genes were detected in the L. esculentum genome. Expression of the cloned P450 gene was regulated by circadian rhythm and enhanced by wounding. Leaf transcripts were detected in the light but not dark. Highest transcript levels were observed 3 hours after mechanical wounding. No increase in expression was seen in response to applications of zeatin as with the N. plumbaginifolia gene. Of the tissues analyzed, shoot tips and young leaves and fruit had the highest detectable transcript levels. Attempts to transform more than 1400 cotyledon explants of L. esculentum with sense or antisense CYP72A2 gene constructs produced no transgenic plants.

The parameters of chlorophyll a fluorescence induction were measured: Fv/Fm, Sc/Fm, Rfd and coefficient of Ld delayed luminescence decay kinetics, related with a course of primary photosynthesis reactions on leaves of strawberry plants, cultured in vitro by means of the micropropagation methods. Strawberry plants cv. Ananasowa from in vitro cultures in optimal condition show significantly higher values of luminescence parameters indicating better condition of plants of this variety in comparison with the variety Senga Sengana. After temperature lowering, however, these values were more reduced than for plants of Senga Sengana, which can be interpreted as higher susceptibility of this variety to chill. Addition of BAP caused disturbance of primary photosynthesis reactions rate, particularly in lower temperature. Auxin 2,4-D had no effect on the luminescence parameters in comparison with control cultures. Dehydration stress strongly diminished the values of measured parameters for Ananasowa variety what indicates the inhibition of primary photosynthesis reaction in leaves. The old culture of Senga Sengana variety showed higher tolerance on linuron in comparison with the new one.

cDNA clone sharing high similarity to Nicotiana plumbaginifolia putative cytochrome P450 monooxygenase was isolated from Lycopersicon esculentum cv. ‘Rutgers’. Isolated cDNA has a full open reading frame and its protein sequence identity to Nicotiana. plumbaginifolia P450 (CYP72A2) evaluated at 75%. Tomato genomic P450 sequence contains 3 short introns. Expression on mRNA level of tomato P450 was checked. The highest level of tomato P450 transcript was observed in young tissues. The transcript level of P450 was increased after mechanical wounding in tomato leaves. Zeatin spray and zeatin uptake do not affect tomato P450 expression.

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 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.

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.

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.

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.

Plants possess three major genomes, carried in the chloroplast, mitochondrion, and nucleus. The chloroplast genomes of higher plants tend to be of similar sizes and structure. In contrast both the nuclear and mitochondrial genomes show great size differences, even among closely related species. The largest plant mitochondrial genomes exist in the genus Cucumis at 1500 to 2300 kilobases, over 100 times the sizes of the yeast or human mitochondrial genomes. Biochemical and molecular analyses have established that the huge Cucumis mitochondrial genomes are due to extensive duplication of short repetitive DNA motifs. The organellar genomes of almost all organisms are maternally transmitted and few methods exist to manipulate these important genomes. Although chloroplast transformation has been achieved, no routine method exists to transform the mitochondrial genome of higher plants. A mitochondrial-transformation system for a higher plant would allow geneticists to use reverse genetics to study mitochondrial gene expression and to establish the efficacy of engineered mitochondrial genes for the genetic improvement of the mitochondrial genome. Cucumber possesses three unique attributes that make it a potential model system for mitochondrial transformation of a higher plant. Firstly, its mitochondria show paternal transmission. Secondly, microspores possess relatively few, huge mitochondria. Finally, there exists in cucumber unique mitochondrial mutations conditioning strongly mosaic (msc) phenotypes. The msc phenotypes appear after regeneration of plants from cell culture and sort with specific rearranged and deleted regions in the mitochondrial genome. These mitochondrial deletions may be a useful genetic tool to develop selectable markers for mitochondrial transformation of higher plants.

The cucumber mitochondrial genome is unique because of its large size, paternal transmission, and the existence of a paternally transmitted mosaic (MSC) phenotype spontaneously appearing after independent tissue culture experiments. Transmission studies eliminated paternal imprinting as the genetic basis for the MSC phenotype. We identified a 13 kb region (JLV5-DEL) in the wild-type mitochondrial genome that was absent from three MSC lines. This deleted region was paternally transmitted with the MSC phenotype through the F3 and test-cross generations. Southern hybridizations and PCR amplifications using primers within the JLV5 region revealed that rare wild-type sorters possessed the wild-type region. MSC plants possess the wild-type region at levels (approximately 0.002) below detection by standard PCR reactions and Southern hybridizations, using genomic DNA. Sequence analysis of the wild-type contig revealed no homologies to mitochondrial genes and no open reading frames within JLV5-DEL. PCR amplifications across JLV5-DEL using MSC mtDNA revealed that the loss of this region was not a simple deletion and may be associated with a rearrangement. Because no genic regions were identified within the wild-type JLV5 region, the specific lesion conditioning the MSC phenotype may be associated with the putative rearrangement or another mutation that occurred during tissue culture or exists substoichiometrically in the parental line and is transmitted together with JLV5 within the same mitochondrion.

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.

This work was done to determine the diversity of pathogenic Pseudomonas syringae pv. lachrymans and pv. syringae strains, isolated from cucurbit plants. Pathogenicity tests performed in this work on cucumber indicated that the 22 tested strains differed dramatically in virulence level. Genetic characterization using MLST loci, as well as ITS1, ERIC and REP fingerprinting, allowed the grouping of strains into four phylogenetic groups. After amplicon sequencing and BLAST alignment the collected strains comply with P. syringae pathovars: lachrymans and syringae and with saprophytic P. fluorescens. The strains of Pseudomonas syringae pv. lachrymans may be categorized into at least two types differing in both disease symptoms and molecular characteristics. We confirmed that MLST-loci-based grouping of strains corresponds well to grouping based on pathogenicity tests.

Significant yield losses in commercial tomato production caused by tomato spotted wilt virus (TSWV) are the reason why we have undertaken studies on resistance to this pathogen. One of the possible sources of resistance can be the incorporation of the nucleoprotein N viral gene by Agrobacterium transformation. The N gene was introduced into three Lycopersicon esculentum forms. Out of the total of 3044 cotyledon explants 14.7% regenerated shoots, but only a few were rooted on medium containing kanamycin. The preliminary analysis indicated that 18 plants are putative transformants.

This study was undertaken to remedy significant yield losses in commercial tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana tabacum L.) production caused by tomato spotted wilt virus (TSWV). One of the possible sources of resistance can be incorporation into the host plant of a viral nucleoprotein (N) gene by Agrobacterium-mediated transformation. Twelve primary transformants of tomato and 141 of tobacco were analyzed for the expression of the N gene and for resistance to the TSWV infection. The tests have demonstrated that transgenic plants were protected against virus infection irrespective of whether or not they contained detectable levels of the translational product.