Three subtelomeric satellites and one interstitial 5S rDNA were characterized in Cucumis hystrix, and the pericentromeric signals of two C. hystrix subtelomeric satellites along C. sativus chromosomes supported the hypothesis of chromosome fusion in Cucumis.Tandem repeats are chromosome structural fractions consisting of highly repetitive sequences organized in large tandem arrays in most eukaryotes. Differentiation of tandem repeats directly affects the chromosome structure, which contributes to species formation and evolution. Cucumis hystrix (2n = 2x = 24) is the only wild Cucumis species grouped into the same subgenus with C. sativus (2n = 2x = 14), hence its phylogenetic position confers a vital role for C. hystrix to understand the chromosome evolution in Cucumis. However, our knowledge of C. hystrix tandem repeats is insufficient for a detailed understanding of the chromosome evolution in Cucumis. Based on de novo tandem repeat characterization using bioinformatics and in situ hybridization (ISH), we identified and characterized four differentially amplified tandem repeats, Cucumis hystrix satellite 1–3 (CuhySat1–CuhySat3) located at the subtelomeric regions of all chromosomes, and Cucumis hystrix 5S (Cuhy5S) located at the interstitial regions of one single chromosome pair. Comparative ISH mapping using CuhySat1–3 and Cuhy5S revealed high homology of tandem repeats between C. hystrix and C. sativus. Intriguingly, we found signal distribution variations of CuhySat2 and CuhySat3 on C. sativus chromosomes. In comparison to their subtelomeric signal distribution on C. hystrix chromosomes, CuhySat3 showed a pericentromeric signal distribution and CuhySat2 showed both subtelomeric and pericentromeric signal distributions on C. sativus chromosomes. This detailed characterization of four C. hystrix tandem repeats significantly widens our knowledge of the C. hystrix chromosome structure, and the observed signal distribution variations will be helpful for understanding the chromosome evolution of Cucumis.

Cucumis × hytivus (2n = 4× = 38) is a synthetic allotetraploid obtained from interspecific hybridization between the cucumber (2n = 2× = 14) and its wild relative C. hystrix (2n = 2× = 24). The synthesis of this species built a bridge for cucumber improvement through gene introgression. Allotriploid and introgression lines (ILs) have previously been produced and characterized with respect to morphology, cytology, and molecular markers. However, no clear evidence of how the chromosomal segments of C. hystrix were introgressed and inherited was found owing to the small size of chromosomes. In the present study, cucumber-C. hystrix introgression lines were developed by backcrossing the allotriploid to North China cucumber breeding line “P01” followed by self-pollination. The introgressed segments of C. hystrix in the ILs were revealed by meiotic pachytene chromosome analysis. Fluorescence in situ hybridization (FISH) was performed on pachytene chromosomes using fosmid clones from cucumber, which confirmed that introgression occurred in the long arm of chromosome 7. Molecular analysis using a set of 53 simple sequence repeats (SSRs) indicated that the chromosomal segments of C. hystrix were introduced into 4 cucumber chromosomes, the short arms of chromosomes 2 and 6, and long arms of chromosomes 3 and 7. The inheritance of alien sequences in the long arm of chromosome 7 was investigated with 21 SSRs in self-pollinated progenies. C. hystrix-specific bands of several SSRs were still present in some individuals, indicating that the introgressed segment was partially preserved. The first unambiguous identification of alien chromosome segments in cucumber ILs using combined molecular cytogenetics could facilitate the determination of effects of wild alleles and promote cucumber improvement.

Allopolyploidy and homoeologous recombination are two important processes in reshaping genomes and generating evolutionary novelties. Newly formed allopolyploids usually display chromosomal perturbations as a result of pairing errors at meiosis. To understand mechanisms of stabilization of allopolyploid species derived from distant chromosome bases, we investigated mitotic stability of a synthetic Cucumis allotetraploid species in relation to meiosis chromosome behavior. The Cucumis × hytivus is an allotetraploid synthesized from interspecific hybridization between cucumber (Cucumis sativus, 2n = 14) and its wild relative Cucumis hystrix (2n = 24) followed by spontaneous chromosome doubling. In the present study, we analyzed the wild parent C. hystrix and the latest generation of C. hytivus using GISH (genomic in situ hybridization) and cross-species FISH (fluorescence in situ hybridization). The karyotype of C. hystrix was constructed with two methods using cucumber fosmid clones and repetitive sequences. Using repeat-element probe mix in two successive hybridizations allowed for routine identification of all 19 homoeologous chromosomes of allotetraploid C. hytivus. No aneuploids were identified in any C. hytivus individuals that were characterized, and no large-scale chromosomal rearrangements were identified in this synthetic allotetraploid. Meiotic irregularities, such as homoeologous pairing, were frequently observed, resulting in univalent and intergenomic multivalent formation. The relatively stable chromosome structure of the synthetic Cucumis allotetraploid may be explained by more deleterious chromosomal viable gametes compared with other allopolyploids. The knowledge of genetic and genomic information of Cucumis allotetraploid species could provide novel insights into the establishment of allopolyploids with different chromosome bases.

Pakchoi is an important vegetable grown in worldwide that prefers a cool climate, and its growth is severely affected by high temperatures in the summer. Little is known for the molecular regulatory mechanisms of high-temperature stress in pakchoi. The limited availability of gene sequence has greatly affected molecular breeding and functional genomic analysis. In this study, we conducted comprehensive analyses for heat treatment in pakchoi by RNA-Seq. Finally, 64.29 million clean reads containing 32,666 unigenes with an N50 length of 1405 bp, and a total length of 33.39 Mb were identified. Overall, 11,024 SSRs located in 8404 unigenes were obtained; these findings will be very useful for molecular assisted breeding. Totally, 1220 differentially expressed genes (DEGs) were detected between the high-temperature group (TH) and the control (CK), among which 699 DEGs were up-regulated and 521 were down-regulated. The enrichment analysis indicated that 12 GO subcategories and 9 KEGG pathways were enriched significantly (P value <0.05). Finally, six DEGs were validated by qRT-PCR, and the results further verified the reliability of RNA-Seq. Herein, we present the first comprehensive characterization of heat-treated pakchoi using transcriptome analysis. Several important genes that respond to high temperature were identified, and their roles in heat stress responses are discussed. In conclusion, our study represents a fully characterised pakchoi transcriptome. In addition, it provides the important resources for future genomic and genetic studies of pakchoi under heat stress.

Comparative genetic mapping revealed the origin of Xishuangbanna cucumber through diversification selection after domestication. QTL mapping provided insights into the genetic basis of traits under diversification selection during crop evolution.The Xishuangbanna cucumber, Cucumis sativus L. var. xishuangbannanesis Qi et Yuan (XIS), is a semi-wild landrace from the tropical southwest China with some unique traits that are very useful for cucumber breeding, such as tolerance to low light, large fruit size, heavy fruit weight, and orange flesh color in mature fruits. In this study, using 124 recombinant inbred lines (RILs) derived from the cross of the XIS cucumber with a cultivated cucumber inbred line, we developed a linkage map with 269 microsatellite (or simple sequence repeat) markers which covered 705.9 cM in seven linkage groups. Comparative analysis of orders of common marker loci or marker-anchored draft genome scaffolds among the wild (C. sativus var. hardwickii), semi-wild, and cultivated cucumber genetic maps revealed that the XIS cucumber shares major chromosomal rearrangements in chromosomes 4, 5, and 7 between the wild and cultivated cucumbers suggesting that the XIS cucumber originated through diversifying selection after cucumber domestication. Several XIS-specific minor structural changes were identified in chromosomes 1 and 6. QTL mapping with the 124 RILs in four environments identified 13 QTLs for domestication and diversifying selection-related traits including 2 for first female flowering time (fft1.1, fft6.1), 5 for mature fruit length (fl1.1, fl3.1, fl4.1, fl6.1, and fl7.1), 3 for fruit diameter (fd1.1, fd4.1, and fd6.1), and 3 for fruit weight (fw2.1, fw4.1, and fw6.1). Six of the 12 QTLs were consistently detected in all four environments. Among the 13 QTLs, fft1.1, fl1.1, fl3.1, fl7.1, fd4.1, and fw6.1 were major-effect QTLs for respective traits with each explaining at least 10 % of the observed phenotypic variations. Results from this study provide insights into the cytological and genetic basis of crop evolution leading to the XIS cucumber. The molecular markers associated with the QTLs should be useful in exploring the XIS cucumber genetic resources for cucumber breeding.

Plant D-type cyclin genes (CYCDs) are important regulators of cell division. However, little is known on their participation during the early developmental stage of cucumber fruit. In this study, cucumber CYCD genes were identified and characterized. The expression levels of these genes during early fruit development were assessed from 0 to 8 days after anthesis (DAA). The results revealed the presence of 13 different CYCD genes, which were named according to identity percentages of the corresponding orthologs in Arabidopsis thaliana and poplar. The genomic organization of each subgroup CYCD was similar to their orthologs in A. thaliana and poplar. The expression levels of CsCYCD genes were analyzed in cucumber fruits under different treatments including natural parthenocarpic fruit, pollinated fruit, and N-(2-chloro-4-pyidyl)-N′-phenyurea (CPPU)-induced parthenocarpic fruit. The highest expression levels of most CsCYCDs genes were at four DAA in natural parthenocarpic and pollinated fruits. Interestingly, the expression patterns of 8 of 13 CsCYCD genes in natural parthenocarpic fruit were similar to those in pollinated fruit, but different from those in CPPU-induced parthenocarpic fruit. Collectively, the results of this study provide insights on the CYCDs involved in cucumber parthenocarpic fruit development.

Phytohormone auxin plays an important role in fruit development and is perceived by the TIR1/AFB family of F-box proteins as auxin receptors involved in auxin signal pathway. Cucumber (Cucumis sativus L.) fruit development is either parthenocarpic or non-parthenocarpic. However, little is known on TIR1 and AFB participation in the early stage of cucumber fruit development. In present study, TIR1 and AFB2 were isolated from cucumber. CsTIR1 and CsAFB2 were highly expressed in leaves and ovaries. Their transcript levels decreased in parthenocarpic and pollinated fruits, but continuously up-regulated in aborted fruits, indicating that down-regulation of CsTIR1 and CsAFB2 may be in favor of cucumber fruit set and development. The transcript levels of CsTIR1 and CsAFB2 were significantly induced in leaves by NAA, 6-BA, GA3, ABA, and ethephon. The expression levels were up-regulated by ABA and ethephon treatments. This expression patterns was accordant with the aborted fruits. Thus, CsTIR1 and CsAFB2 may be important regulators during cucumber fruit development.

Gummy stem blight (GSB, Didymella bryoniae (Auersw.) Rehm) is a devastating disease occurring worldwide in cucumber (Cucumis sativus L.) production and causing considerable yield loss. No commercially available cultivars are resistant to GSB. By screening 52 introgression lines (ILs) derived from the cross of C. hystrix × C. sativus and eight cucumber cultivar/lines through a whole plant assay, three ILs (HH1-8-1-2, HH1-8-5, HH1-8-1-16) were identified as GSB resistant lines. Six common introgression regions in these three ILs were on Chromosomes 1, 4, and 6. To further map the resistance in the ILs, three mapping populations (2009F2, 2009F2′ and 2010F2) from a cross between resistant IL HH1-8-1-2 and susceptible 8419 were constructed and used for QTL mapping with SSR markers. Two quantitative trait loci (QTLs) were identified; one on Chromosome 4 and the other on Chromosome 6. The interval for Chromosome 4 QTL is 12 cM spanning 3.569 Mbp, and the interval for Chromosome 6 QTL is 11 cM covering 1.299 Mbp. The mapped QTLs provide a foundation for map-based cloning of the genes and establishing an understanding of the associated mechanisms underlying GSB resistance in cucumber.

Haploids and doubled haploids are very important in plant breeding, enabling the time needed to produce homozygous lines to be shortened compared with conventional breeding. In the present review, emphasis is given to haploid induction through unfertilized ovule/ovary culture. Attention is given to induction of haploid plants from female gametophyte culture through analysis of factors in the processes of gynogenesis, including genotype selection, stage of ovule development, pretreatment, and culture media containing nutritional components and phytohormones. The gynogenetic approach may be of great value in discovering novel genetic recombinations. Application of double haploids in genetics and plant breeding is also highlighted. This review also identifies some existing knowledge gaps where work may increase the efficiency of this process in different plant species.

Allopolyploidization is considered an essential evolutionary process in plants that could trigger genomic shock in allopolyploid genome through activation of transcription of retrotransposons, which may be important in plant evolution. Two retrotransposon-based markers, inter-retrotransposon amplified polymorphism and retrotransposon-microsatellite amplified polymorphism and a microsatellite-based marker, inter simple sequence repeat were employed to investigate genomic changes in early generations of a newly synthesized allotetraploid Cucumis × hytivus Chen & Kirkbride (2n = 4x = 38) which was derived from crossing between cultivated cucumber C. sativus L. (2n = 2x = 14) and its wild relative C. hystrix Chakr. (2n = 2x = 24). Extensive genomic changes were observed, most of which involved the loss of parental DNA fragments and gain of novel fragments in the allotetraploid. Among the 28 fragments examined, 24 were lost while four were novel, suggesting that DNA sequence elimination is a relatively frequent event during polyploidization in Cucumis. Interestingly, of the 24 lost fragments, 18 were of C. hystrix origin, four were C. sativus-specific, and the remaining two were shared by both species, implying that fragment loss may be correlated with haploid DNA content (genome size) of diploid parents. Most changes were observed in the first generation after polyploidization (S1) and stably inherited in the subsequent three generations (S2–S4), indicating that genomic changes might be a rapid driving force for the stabilization of allotetraploids. Sequence analysis of 11 of the 28 altered DNA fragments showed that genomic changes in the allotetraploid occurred in both coding and non-coding regions, which might suggest that retrotransposons inserted into genome randomly and had a genome-wide effect on the allotetraploid evolution. Fluorescence in situ hybridization (FISH) analysis revealed a unique distribution of retrotransposon and/or microsatellite flanking sequences in mitotic and meiotic chromosomes, where the preferential FISH signals occurred in the centromeric and telomeric regions, implying that these regions were the possible hotspots for genomic changes.

Quantitative real-time polymerase chain reaction (QRT-PCR) has become one of the most widely used methods for gene expression analysis. However, the expression profile of a target gene may be misinterpreted due to unstable expression of the reference genes under different experimental conditions. Thus, a systematic evaluation of these reference genes is necessary before experiments are performed. In this study, 10 putative reference genes were chosen for identifying expression stability using geNorm, NormFinder, and BestKeeper statistical algorithms in 12 different cucumber sample pools, including those from different plant tissues and from plants treated with hormones and abiotic stresses. EF1α and UBI-ep exhibited the most stable expression across all of the tested cucumber samples. In different tissues, in addition to expression of EF1α and UBI-ep, the expression of TUA was also stable and was considered as an appropriate reference gene. Evaluation of samples treated with different hormones revealed that TUA and UBI-ep were the most stably expressed genes. However, for abiotic stress treatments, only EF1α showed a relatively stable expression level. In conclusion, TUA, UBI-ep, and EF1α will be particularly helpful for reliable QRT-PCR data normalization in these types of samples. This study also provides guidelines for selecting different reference genes under different conditions.

Changes of gene expression played an important role in the evolution of plant allopolyploids. Frequency, time and type of the changes of gene expression between the first two self-pollinated generations (S1 and S2) of a synthesized allotetraploid Cucumis × hytivus Chen et Kirkbride and in its diploid parents was analyzed with cDNA-AFLP and reverse-Northern blot technique. Sequences similarity of genes involved in changed expression were also analyzed with BLAST package. The results from cDNA-AFLP analysis showed that 36 (3.37%) genes showed silencing (27) or activation (9) in allotetraploids. These changes initiated in S1 or S2 generation. The silenced/activated genes included rRNA and protein-coding genes. Further reverse-Northern blot analysis validated the results obtained. Thus four types of changes of gene expression were observed, including silencing of genes from both parents, maternal parent, paternal parent, and genes novel expression. The results indicate rapid changes in gene expression in early generations of C. × hytivus, which contributed to the evolution of this synthetic allotetraploid.

To promote cytogenetical studies on cucumber (Cucumis sativus L., 2n = 2x = 14), the reciprocal crosses were made between autotriploid and diploid for selecting the primary trisomics. Meanwhile, chromosome behavior during meiosis in autotriploid cucumber was investigated to look for cytological evidences for origin of primary trisomics. Many viable F1 seeds were obtained from reciprocal crosses between autotriploid and diploid. The number of chromosomes of 56 surviving progenies varied from 14 to 28, with plants having 2n = 15 occurring at the highest frequency (51.8%). Primary trisomics were firstly obtained in this study. Four types of primary trisomics were isolated and they could be distinguished from each other, as well as diploid. Variable chromosome configurations, e.g. univalent, bivalents and trivalents were observed in many pollen mother cells of the autotriploid at metaphase I. Binomial chromosome distribution was observed at anaphase I and frequency of 8/13 was 6.25%. The meiosis of autotriploid, especially the class of gametes with eight chromosomes, gave the cytological evidence of producing 2x + 1 type gamete and could be induced into primary trisomic plants from progeny of autotriploid–diploid crosses. These studies have established a ground work for selecting a series of primary trisomics, and further using them for associating linkage groups with specific chromosomes in cucumber.

The molecular evolution of the cucumber Por gene in early generations of the synthesized allotetraploid Cucumis × hytivus was investigated. The results from gene expression analysis showed that the cucumber Por gene was silenced in the S1 generation, and re-activated in the S2 generation. In the S3 and S4 generations, the transcripts remained activated but sequence changes were observed. Further analysis indicated that base substitutions, including two transitions and one transversion, occurred in the S1 and S3 generation, respectively, and in the S3 generation, an intron was found to be retained in the transcript. This indicates allopolyploidy induced rapid silencing and mutation of the cucumber Por gene. Further, gene mutations such as base substitution and intron retention are modes of evolution for duplicated genes in newly formed polyploids.

To understand the correlation between chromosomes behavior and fertility in autotriploid cucumber (Cucumis sativus L.), microsporogenesis in pollen mother cells (PMCs) and male gametophyte development were studied using improved staining and chromosome preparation techniques. Meanwhile, for more efficient selection of trisomics from the progeny of autotriploid-diploid crosses, fertilization rates of ovules from reciprocal crosses were counted to observe the transfer rate of gametes in the autotriploid cucumber. Variable chromosome configurations, e.g. multivalents, quadrivalents, trivalents, bivalents and univalents were observed in the most PMCs of the autotriploids at metaphase I. Chromosome lagging and bridges at anaphase in both meiotic divisions resulted from irregular chromosome separation and asynchronization was frequently observed as well, which led to formation of micronuclei and inviable gametes. The frequency of normal PMCs in autotriploids at the stage of tetrad was only 40.6%. Among those normal microspores, most of them (91.2%) could develop into normal gametophytes with 2 cells and 3 germ pores. Stainability and germination rate of pollen grains were only 18.8 and 13.5%, respectively. However, chromosomes separated to form gametes with 8 chromosomes at anaphase I, suggesting a possible method for the production of primary trisomics from the progeny of autotriploid-diploid crosses. Fruit set of 3n × 2n and 2n × 3n were 80 and 70%, respectively. It obtained an average of 6.2 plump seeds per fruit in 3n × 2n, while 4.9 in 2n × 3n crosses. Transfer rates of gametes through the gastrula or the pollen in autotriploids were 13.4 and 10.4%, respectively. Some aneuploid gametes (n + 1 = 8, n + 2 = 9) also have capability of setting seed and sexual reproduction besides normal gametes containing whole chromosome sets (n = 7, 2n = 14). Further, some primary trisomic plants were selected from the progeny of autotriploid-diploid crosses. Based on the results obtained we suggest that abnormal meiosis in PMCs was the cytogenetic reason for low fertility of autotriploid cucumber pollen. 3n × 2n cross was more efficient for selecting primary trisomic plants in cucumber.

Cucumber mosaic virus (CMV) has resulted in much damage to Danshen (Salvia miltiorrhiza Bunge) crops in China. A 5-year survey was conducted in Hebei, Shandong, Sichuan, Shanxi, Henan and Gansu Provinces, all major Danshen-growing areas. A total of 156 Danshen plant samples with CMV symptoms were collected and tested for the presence of CMV by a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using polyclonal antibodies against CMV and a positive control, according to the supplier’s instructions (Agdia). They were confirmed to be CMV by amplification of complete coat protein gene and analysis of the gene sequence. The results showed that 122 samples were infected by CMV and all of these CMV isolates belonged to subgroup IB.

Six experiments (including pretreatment, embryonic callus induction media, preculture conditions, embryo induction media, embryo germination media, and genotypic effects) were conducted to develop an efficient cucumber (Cucumis sativus L., 2n = 2x = 14) anther culture protocol. Pretreatment and embryo induction were key factors for successful anther culture. Suitable temperature stress depended on the ecotype, i.e., cucumbers from cold areas responded well to cold shock whereas those from temperate areas responded well to heat treatment. The best medium for embryonic callus induction was MS medium supplemented with 4.44 μM BA, 2.26 μM 2, 4-D, 4.64 μM KIN, 3% sucrose and 0.8% agar. For embryo induction, MS medium supplemented with 0.54 μM NAA, 13.32 μM BA, 3% sucrose and 0.8% agar was optimal, and for embryo germination MS medium containing 2.22 μM BA, 6% sucrose and 1.2% agar was best. Using this protocol, we produced callus from 16 genotypes and regenerated plants from three of 20 evaluated. Three embryos per anther and 42 DH per 45 anthers (93% success) were obtained for cv. Ningjia No. 1, which was an improved result over a previous report. The origin of regenerants from microspores was determined by cytological, morphological and AFLP analyses.

We used a newly synthesized allotetraploid between C. sativus (2n = 2x = 14, n gametic chromosome number, x haploid chromosome number) and C. hystrix (2n = 2x = 24) to study the genomic events in its early generations. Results from cytological characterization of the F1 and the allotetraploid progenies showed that the rate of bivalents in meiotic metaphase I of the F1 was greatly improved by chromosome doubling, and further improved during the selfing process of allopolyploid resulting into relatively diploid-like meiosis. Extensive genomic changes were detected by amplified fragment length polymorphism analysis. The changes mainly involved loss of parental restriction fragments and gaining of novel fragments. The total detectable changes were from 11.1 to 32.1%, and the frequency of losing parental fragments was much higher than that of gaining novel fragments. Some of the changes were initiated as early as in the F1 hybrid, whereas others occurred after chromosome doubling (polyploid formation). No significant differences were detected in the reciprocal F1 hybrids and S0 generations. But the data showed that the frequency of sequence losing in C. sativus was about two times higher than in the C. hystrix. Our results demonstrated that the sequence elimination was the major event of genomic changes, and it might provide the physical basis for the diploid-like meiotic behavior in the diploidization of the newly formed allopolyploids. Moreover, the results suggest that the sequence elimination was not caused by cytoplasmic factors, and might relate to genomic recombination and to the numbers of parental chromosome.

•Genetics of salinity tolerance in cucumber studied under NaCl stress.•Generation mean analysis revealed heritability and gene effects of cucumber.•Narrow-sense heritability of the traits ranged from low to high.•Advanced selection for salt tolerance is necessary due to low heritability.•Intermating of superior segregants may increase salinity tolerance in cucumber.Cucumber production is greatly reduced by high salinity. Our recent identification of cucumber genotypes with relative tolerance to salinity provides the potential for development of salt tolerant cultivars if the mode of inheritance for this trait is known. In this study, two cucumber parental lines, 11411S (P1, salt tolerant), 11439S (P2, salt sensitive) were crossed to study gene actions responsible for inheritance of salinity tolerance (TOL), relative leaf number (RLN14), area of second largest fully expanded leaf (LA) and vine length (VL) under saline growing conditions. The six populations denoted as P1, P2, F1, B1 (F1 × P1), B2 (F1 × P2) and F2 (F1 × F1) were subjected to 80 mM NaCl stress in greenhouse pot experiments and data subjected to generation mean analysis. Epistatic gene effects on all the traits were not detected by A, B, C scaling test while joint scaling test showed the presence of non-allelic interactions for all the traits. TOL and RLN14 were predominantly under additive gene effect while inheritance LA was largely influenced by dominance and additive gene effects. Dominant gene effect significantly controlled the inheritance of VL. The narrow sense heritability for TOL, RLN14, VL and LA was 0.57, 0.26, 0.74 and 0.66, respectively. VL and LA registered significant positive heterobeltiosis. The presence of dominance gene effect and low heritability of photosynthesis enhancing trait, RLN14 makes it impractical to improve salinity through simple selection or pedigree breeding. Simultaneous selection for RLN14 and TOL in segregating population at advanced filial generations in this cross may be useful in developing cucumber varieties with increased salinity tolerance. Alternatively, intermating of superior segregants may be explored to concentrate the favorable genes for salt tolerance.

Several factors, i.e. the duration of thermal shock pretreatment at 35 °C, the concentrations of TDZ and the silver nitrate were investigated for their effects on embryo formation in a variety of cucumber (Cucumis sativus L) ovary culture. The results showed that a thermal shock for 3 days at 35 °C at the start of the culture resulted in higher frequency of embryo formation than 2 or 4 days. TDZ had a positive effect on the embryo formation. Highest embryo formation frequency (72.7%) was recorded by adding 0.04 mg/L TDZ into the induction medium. The results found that addition of AgNO3 to induction medium had no significant effect on frequency of embryo formation but shortened embryo sprouting period and improved number of embryos formed in each ovary slice. All the experiment materials responded well to ovary culture, and there were no difference among genotypes used in this study. Among the forty regenerated plants obtained, two were identified as haploid plants (2n = x = 7), five were tetraploid plants (2n = 4x = 28), and the rest were diploid plants. Microsatellite markers (SSR) were used to analyze the homozygosity of the diploid plants, the putative chromosome-doubled haploids. Of the 33 diploid plants, 17 (51.5%) were identified as double haploids. Based on the above results, we have established a useful protocol for production of cucumber doubled haploids with ovary culture.

Two stable introgression lines (ILs), IL56 and IL73 with fourteen chromosomes, derived from a cross between the wild relative Cucumis hystrix Chakr. (2n = 24) and the cultivated cucumber “beijingjietou” (Cucumis sativus L., 2n = 14) were characterized using SSR and AFLP markers. Twenty-three SSR primers were used to detect the introgression from C. hystrix to C. sativus, and one locus at 210 bp was revealed and assigned to introgressive fragment of C. hystrix genome in IL56. Substantial genomic changes in these two lines were detected by AFLP analysis with thirty different primer combinations. C. hystrix-specific bands, novel bands for the two parents and deleted bands in C. sativus were observed. One hundred and forty-seven polymorphic bands were observed, but only four and five introgressed bands were found in IL56 and IL73, respectively. Although representing only a small proportion of the C. hystrix genome, these two introgression lines with downy mildew and fusarium wilt resistance will be valuable to resistant genetics and breeding.

•We examine the nucleotide polymorphisms of mitochondria in intraspecific crosses.•The synthetic allotetraploid is from a wide cross between Cucumis species with different numbers of chromosome.•This intraspecific cross also showed differential transmission of the three genomes.Paternal transmission of the mitochondrial (mt) genome in cucumber (Cucumis sativus L.) was revealed previously using intraspecific crosses. The present study reports the first paternal transmission of mtDNA observed between Cucumis species. Mitochondrial primers specific to nad1, cob, and nad7 were used to amplify the DNAs from the female parent (C. hystrix), male parent (C. sativus) and the allotetraploid (C. × hytivus) derived the interspecific hybridization. By sequence alignment, we found that the explicit nucleotide polymorphisms of the allotetraploid were all inherited from male parent, C. sativus. Similar experiment with chloroplast (cp) genome was conducted and the results showed that while mtDNA was paternally inherited, cpDNA was still maternally inherited between Cucumis species. This work provides novel evidence of paternal transmission of the mitochondrial genome between plant species and lays the foundation for further research on the genetic mechanism of mitochondrial transmission in Cucumis.

Ty1-copia retrotransposons are ubiquitous in the plant kingdom and have an important effect on genome structure and evolution. Cucumis hystrix (2n = 2x = 24) is an exotic wild species of Cucumis with a number of valuable traits that are not available in cultivated cucumber. However, lack of the basic genome structure and evolutionary information about C. hystrix has hindered its utilization in cucumber breeding and evolutionary research. In the present study, reverse transcriptase (RT) sequences of Ty1-copia retrotransposons were isolated from genome of C. hystrix by PCR amplification using degenerate oligonucleotide primers. The 24 clones obtained were different from each other and showed high heterogeneity. Of the 24 sequences, 13 are intact whereas the remaining sequences have a stop codon, a frameshift, or both. Southern hybridization revealed that Ty1-copia retrotransposons distributed widely in C. hystrix by multiple copies, and the total number was estimated to be about 5460 as calculated by dot-blot hybridization. The retrotransposons of this kind comprise approximately 8.5% of the C. hystrix genome. No transcription of Ty1-copia retrotransposons was detected by reverse transcriptase PCR (RT-PCR) in leaves of chilling or heat treated plants. Moreover, phylogenetic analysis showed that the 24 RT sequences were divided into five families and had homology with other species, indicating that both vertical transmission and horizontal transmission are the source of Ty1-copia retrotransposons in C. hystrix. FISH with Ty1-copia retrotransposons in C. hystrix revealed the retrotransposons of this kind were widely dispersed over all the chromosomes with clustering in terminal heterochromatin regions. These results contribute to our understanding about the organization and evolution of C. hystrix genome and will provide valuable information for the utilization of retrotransposons in C. hystrix.Research highlights▶ Reverse transcriptase (RT) sequences of Ty1-copia retrotransposons were isolated from genome of C. hystrix by PCR amplification using degenerate oligonucleotide primers. ▶ Southern hybridization revealed that Ty1-copia retrotransposons distributed widely in C. hystrix by multiple copies. ▶ The total number of Ty1-copia retrotransposons was estimated by dot-blot hybridization. ▶ FISH revealed the retrotransposons of this kind were widely dispersed over all the chromosomes with clustering in terminal heterochromatin regions.

•LTR retrotransposons are distributed throughout all the chromosomes of the newly synthesized Cucumis allotetraploid, with clusters on the terminal regions.•The epigenetic changes were mainly present in the early stage of allotetraploid, and it would be stably inherited in the subsequent generation.•The genes flanking LTR retrotransposons with altered expression may be beneficial in the stabilization of Cucumis allotetraploid.Cucumis hytivus is a newly synthesized allotetraploid in which retrotransposons was strongly activated owing to allopolyploidization. In present study, the chromosomal distribution of LTR retrotransposons and their effects on expression of adjacent genes were observed by investigating the first four generations of C. hytivus. Fluorescent in situ hybridization (FISH) analysis revealed that LTR retrotransposons are distributed throughout all the chromosomes. The clusters on terminal regions indicated the high copy number of retrotransposons in C. hytivus. The extensive epigenetic changes (gene silencing and gene activation) were detected by cDNA-SSAP analysis. Totally, twenty transcripts subjected to gene expression alterations were sequenced, including 12 gene silencing and 8 gene activation. However, the silenced/activated transcripts consisted of known genes or putative proteins as well as new sequences that had no similarity to any known genes. Both gene silencing and activation mainly occurred in the early generations of allotetraploid. The interlaced distribution of retrotransposons and genes might lead to the transcriptional interference of retrotransposons on expression of their adjacent genes, which may contribute to the rapid genetic stabilization of newly formed allotetraploid.

Cucumber chilling-resistant cultivar Changchun mici and -sensitive cultivar Beijing jietou were used in this study to investigate the effects of exogenous PAs on protection against chilling injury as well as on changes of physiological features, and the fluctuation of free PAs content in the leaves under chilling stress. Upon chilling treatment, free spermidine (Spd), spermine (Spm) and putrescine (Put) were remarkably induced in the leaves of cv. Changchun mici 1 day after treatment. The induction of Put declined thereafter, whereas Spd and Spm levels increased steadily. In the leaves of cv. Beijing jietou, Put content was increased only at 1 day after chilling while Spd content decreased significantly upon chilling treatment. Chilling reduced soluble protein content, and decreased the activities of antioxidant enzymes, including superoxidase dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) only in Beijing jietou. However, these changes could be renovated by exogenous application of Put and Spd. It was also found that pretreatment with Put and Spd diminished the increased electrolyte leakage and malondialdehyde (MDA) content caused by chilling in the leaves of both cultivars. Pretreatment of methyglyoxal-bis-(guanylhydrazone) (MGBG), the PAs biosynthetic inhibitor cancelled the effects of PAs in most of the treatments. Moreover, histochemical staining and quantitative measurements showed that exogenous application of Put and Spd eliminated but MGBG exaggerated the hydrogen peroxide (H2O2) accumulation caused by chilling stress, especially in leaves of Beijing jietou. Interestingly, Changchun mici was found to contain higher endogenous free PAs contents compared to Beijing jietou. While no significant difference of SOD, POD and CAT activities was found between non-chilling Changchun mici and Beijing jietou seedlings, the former exhibited higher APX activity than the latter. These results suggest that PAs play important roles in the tolerance of cucumber against chilling stress, which is most likely achieved by acting as oxidative machinery against chilling injury.