Non-heading Chinese Cabbage Research Articles

Genome-wide identification of turnip mosaic virus-responsive microRNAs in non-heading Chinese cabbage by high-throughput sequencing

Turnip mosaic virus (TuMV) is the most prevalent viral pathogen infecting most cruciferous plants. MicroRNAs (miRNAs) are around 22 nucleotides long non-protein-coding RNAs that play key regulatory roles in plants. Recent research findings show that miRNAs are involved in plant–virus interaction. However we know little about plant defense and viral offense system networks throughout microRNA regulation pathway. In this study, two small RNA libraries were constructed based on non-heading Chinese cabbage (Brassica campestris ssp. chinensis L. Makino, NHCC) leaves infected by TuMV and healthy leaves, and sequenced using the Illumina-Solexa high-throughput sequencing technology. A total of 86 conserved miRNAs belonging to 25 known miRNA families and 45 novel ones were identified. Among them, twelve conserved and ten new miRNAs were validated by real-time fluorescence quantitative PCR (qPCR). Differential expression analysis showed that 42 miRNAs were down-regulated and 27 miRNAs were up-regulated in response to TuMV stress. A total of 271 target genes were predicted using a bioinformatics approach, these genes are mainly involved in growth and resistance to various stresses. We further selected 13 miRNAs and their corresponding target genes to explore their expression pattern under TuMV and/or cold (4°C) stresses, and the results indicated that some of the identified miRNAs could link TuMV response with cold response of NHCC. The characterization of these miRNAs could contribute to a better understanding of plant–virus interaction throughout microRNA regulation pathway. This can lead to finding new approach to defend virus infection using miRNA in Chinese cabbage.

Characterization and classification of one new cytoplasmic male sterility (CMS) line based on morphological, cytological and molecular markers in non-heading Chinese cabbage (Brassica rapa L.).

A new non-heading Chinese cabbage CMS line M119A was characterized and specific molecular markers were developed to classify different CMS types. One new non-heading Chinese cabbage (Brassica rapa L.) cytoplasmic male sterile (CMS) line M119A was obtained by interspecific crosses between the recently discovered hau CMS line of Brassica juncea and B. rapa. Furthermore, the line was characterized and compared with other five isonuclear-alloplasmic CMS lines. The M119A line produced six stamens without pollen and only two stamen fused together in fewer flowers. Tissue section indicated that anther abortion in M119A may have occurred during differentiation of the archesporial cells without pollen sac. All the six CMS lines were grouped into three types based on the presence of three PCR fragments of 825, 465 and 772 bp amplified with different mitochondrial genes specific primers. The 825-bp fragment was amplified both in 09-10A and H201A using the specific primer pair P-orf224-atp6, and showed 100 % identity with the mitochondrial gene of pol CMS. The 465-bp fragment was amplified in 30A and 105A using the primer pair P-orf138 and shared 100 % identity with the mitochondrial gene of ogu CMS. The 772-bp fragment was amplified in M119A and H203A using the primer pair P-orf288 and showed 100 % identity with the mitochondrial gene of hau CMS. Therefore, these markers could efficiently distinguish different types of isonuclear-alloplasmic CMS lines of non-heading Chinese cabbage, which were useful for improving the efficiency of cross-breeding and heterosis utilization in cruciferous vegetables.

Genome-wide identification of SSR and SNP markers from the non-heading Chinese cabbage for comparative genomic analyses.

BackgroundNon-heading Chinese cabbage (NHCC), belonging to Brassica, is an important leaf vegetable in Asia. Although genetic analyses have been performed through conventional selection and breeding efforts, the domestication history of NHCC and the genetics underlying its morphological diversity remain unclear. Thus, the reliable molecular markers representative of the whole genome are required for molecular-assisted selection in NHCC.ResultsA total of 20,836 simple sequence repeats (SSRs) were detected in NHCC, containing repeat types from mononucleotide to nonanucleotide. The average density was 62.93 SSRs/Mb. In gene regions, 5,435 SSRs were identified in 4,569 genes. A total of 5,008 primer pairs were designed, and 74 were randomly selected for validation. Among these, 60 (81.08%) were polymorphic in 18 Cruciferae. The number of polymorphic bands ranged from two to five, with an average of 2.70 for each primer. The average values of the polymorphism information content, observed heterozygosity, Hardy-Weinberg equilibrium, and Shannon’s information index were 0.2970, 0.4136, 0.5706, and 0.5885, respectively. Four clusters were classified according to the unweighted pair-group method with arithmetic average cluster analysis of 18 genotypes. In addition, a total of 1,228,979 single nucleotide polymorphisms (SNPs) were identified in the NHCC through a comparison with the genome of Chinese cabbage, and the average SNP density in the whole genome was 4.33/Kb. The number of SNPs ranged from 341,939 to 591,586 in the 10 accessions, and the average heterozygous SNPs ratio was ~42.53%. All analyses showed these markers were high quality and reliable. Therefore, they could be used in the construction of a linkage map and for genetic diversity studies for NHCC in future.ConclusionsThis is the first systematic and comprehensive analysis and identification of SSRs in NHCC and 17 species. The development of a large number of SNP and SSR markers was successfully achieved for NHCC. These novel markers are valuable for constructing genetic linkage maps, comparative genome analysis, quantitative trait locus (QTL) mapping, genome-wide association studies, and marker-assisted selection in NHCC breeding system research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1534-0) contains supplementary material, which is available to authorized users.

Effect of light and temperature on seed germination of selected African leafy vegetables

Using laboratory incubation, the response of seed germination and emergence to variability in temperature and light was examined for spider flower (Cleome gynandra L.), amaranth (Amaranthus cruentus L.), non-heading Chinese cabbage (Brassica rapa L. subsp. chinensis), nightshade (Solanum retroflexum Dun.), pumpkin (Cucurbita maxima Duchesne), tsamma melon (Citrullus lanatus Thunb.), Jew's mallow (Corchorus olitorius L.) and cowpea (Vigna unguiculata (L.) Walp.). Effect of temperature on seed germination and emergence was monitored under continuous darkness and at constant temperatures that ranged from 4°C to 44°C with 4°C increments. The effect of light on seed germination was measured at 25°C. Temperature affected germination rate and final germination percentage of all species tested in ways that were characteristic for each of the species tested. Generally, optimum germination occurred at temperatures ranging between 29°C and 32°C but at higher temperatures for V. unguiculata (36°C) and C. olitorius (35°C). The minimum temperature for germination ranged between 8°C and 15°C, and the maximum between 36°C and 44°C. Optimum temperatures for seedling emergence ranged from 25°C to 31°C, the maximum between 32°C and 40°C and minimum between 2°C and 13°C. Light positively (p<0.01) affected onset of germination in A. cruentus, B. rapa subsp. chinensis and C. olitorius, and final germination percentage of B. rapa subsp. chinensis, C. lanatus and S. retroflexum. The results suggested that under South African conditions, seeds of the eight species will typically germinate optimally as temperatures rise during spring before the occurrence of very hot temperatures in summer. Due to their positive response to light, germination of B. rapa subsp. chinensis, C. lanatus and S. retroflexum seeds is expected to be optimal when sown at or close to the soil surface.

Overexpression of BcGS2 gene in non-heading Chinese cabbage (Brassica campestris) enhanced GS activity and total amino acid content in transgenic seedlings

BackgroundGlutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of nitrogen (N) assimilation, catalyzing the synthesis of glutamine from ammonium and glutamate. Plants have two types of GS isoenzymes that localize in different compartments: one in cytosol (GS1) and another in plastids/chloroplasts (GS2). GS2 is expressed abundantly in leaf mesophyll cells and converts ammonium taken up by plant leaves to glutamine. ResultsThe GS2 gene cDNA of non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) cultivar ‘Suzhouqing’ was isolated by RT-PCR and (5′/3′)-RACE techniques from the leaf. It was classified as a GS2 homolog by sequence alignment and motif search, and named BcGS2. However, there are no reports on regulation of GS2 from NHCC. The biological characterization of a full-length cDNA encoding GS2 is described in this paper. The objective is to investigate the expression, overexpression and regulation of BcGS2 in non-heading Chinese cabbage. The results suggest that BcGS2 is a novel member of the GS family, possibly involved in the N metabolism of non-heading Chinese cabbage. The BcGS2 expression level of transgenic plants was significantly higher than that of wild type plants. The glutamine synthetase activity and free amino acid content were all significantly improved compared to non-transgenic plants. ConclusionThe above results suggested that BcGS2 is a novel member of the GS family in plants. BcGS2 significantly related to the N assimilation of non-heading Chinese cabbage, demonstrating that this gene plays an important role in NHCC nitrogen metabolism.

Karyotype variation and conservation in morphotypes of non-heading Chinese cabbage

Non-heading Chinese cabbage encompasses a wide diversity of morphotypes, like the well-known Pak-choi, Wu ta cai, Cai xin, Tai cai, and Fen nei cai. Despite recent developments in re-sequencing which results in the detection of SNPs, insertions, deletions and copy number variants, there has been no systematical cytogenetic characterization of different non-heading Chinese cabbage morphotypes, aiming to detect large structural variations. To tackle this issue, the karyotypes of five genotypes belonging to five non-heading Chinese cabbage morphotypes were analyzed using fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA) and blend DAPI band (BDB) on mitotic chromosomes. The ideograms of these five non-heading Chinese cabbage genotypes were produced by exploring and comparing the distributional patterns in terms of rDNA (45S and 5S) and constitutive heterochromatin regions between morphotypes. This study revealed variation in the distributional patterns of ribosomal genes and heterochromatin regions, which point to chromosomal rearrangements in non-heading Chinese cabbage. The 45S rDNA site on chromosome A5 and 5S rDNA signal on chromosome A10 were conserved between all five morphotypes, suggesting that these sites were presented in the ancestor karyotype of non-heading Chinese cabbage. Karyotypic variation and conservation facilitates accurate estimation of the genetic diversity, to better understanding of the evolution, and insight in this variation may facilitate breeding of non-heading Chinese cabbage.

BcRISP1, isolated from non-heading Chinese cabbage, decreases the seed set of transgenic Arabidopsis.

Mitochondria are the energy sources of plant cells and are involved in regulating cell development. Ubiquinol–cytochrome c reductase iron-sulfur protein, which is necessary for mitochondrial respiration, is a subunit of mitochondrial electron transport chain multimeric enzyme complexes. To better understand the biological function of the ubiquinol–cytochrome c reductase iron–sulfur protein, the full-length cDNA of BcRISP1 was cloned; it was found to contain 810 base pairs and encode 269 amino acids. Unusually, high expression of the BcRISP1 gene in the archesporial cell stages was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of cytoplasmic male sterile lines and maintainer lines. The seed set was affected by the overexpression of BcRISP1, and shorter siliques with lower seed sets were observed in 35S::BcRISP1 Arabidopsis plants. These characteristics may have resulted from the reduced formation of pollen and impaired pollen tube growth. qRT-PCR results revealed that in 35S::BcRISP1 plants, the expression levels of the mitochondrial respiratory chain-related genes, COX10 and RIP1, were enhanced, whereas the expression levels of QCR7 and SDH2-1 were reduced. This result implies that overexpression of BcRISP1 in transgenic Arabidopsis plants may disrupt the mitochondrial electron transport chain by affecting the expression of mitochondrial respiratory chain-related genes and therefore, reducing the seed set.

C-repeat binding factor gene family identified in non-heading Chinese cabbage is functional in abiotic and biotic stress response but different from that in Arabidopsis

C-repeat binding factor (CBF) signaling pathway is involved in cold acclimation responsive to low temperature and some other stresses. CBF transcription factor family is the key component of this pathway. In this study, eight CBF-like genes, BrCBF1, BrCBF2, BrCBF3, BrCBF4, BrCBF5, and BrCBF6A/B/C were isolated from non-heading Chinese cabbage (Brassicacampestris ssp. chinensis L. Makino, NHCC). The deduced CBF proteins shared high similarity with their Arabidopsis orthologs and localized to the nucleus. Furthermore, quantitative real-time PCR (qPCR) analysis showed that BrCBF1~3 were induced by cold (4 °C) but not drought or abscisic acid (ABA), indicating that they are involved in an ABA-independent pathway; however, BrCBF4~6 were regulated by both drought and ABA, suggesting that they were involved in an ABA-dependent pathway. Nevertheless, unlike Arabidopsis, BrCBF4~6 showed response to both cold and ABA, indicates ABA-independent and ABA-dependent parts of CBF pathway in NHCC might not be completely separate, and these genes may act as the connection points in the network. BrCBFs were also accumulated in response to salicylic acid (SA), methyljasmonate (MeJA), and ethylene (ET), indicating that BrCBF genes might participate in the response to biotic stresses. Taken together, eight CBF genes were isolated from NHCC which compose a functional CBF signaling pathway by participating in response to multiple stresses and performing roles from Arabidopsis to some extent.

Overexpression of the Monodehydroascorbate Reductase Gene from Non-heading Chinese Cabbage Reduces Ascorbate Level and Growth in Transgenic Tobacco

l-Ascorbic acid is an important antioxidant in both animals and plants. Monodehydroascorbate reductase is an enzyme involved in regulating the l-ascorbic acid level in plants. Here, we report the cloning of the reductase gene from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) and negative regulation of l-ascorbic acid and dehydroascorbate levels in transgenic tobacco plants expressing the gene under the control of the 35S promoter. The transgenic plants had reduced growth. The transgenic plants had changed the expression of the genes in the d-mannose/l-galactose pathway, degradation and recycling pathway and the activity of enzymes involved in degradation (ascorbate oxidase and ascorbate peroxidase) or recycling of ascorbic acid (dehydroascorbate reductase and glutathione reductase).

Basic helix-loop-helix transcription factor BcbHLHpol functions as a positive regulator of pollen development in non-heading Chinese cabbage.

Cytoplasmic male sterility (CMS) is a common trait in higher plants, and several transcription factors regulate pollen development. Previously, we obtained a basic helix-loop-helix transcription factor, BcbHLHpol, via suppression subtractive hybridization in non-heading Chinese cabbage. However, the regulatory function of BcbHLHpol during anther and pollen development remains unclear. In this study, BcbHLHpol was cloned, and its tissue-specific expression profile was analyzed. The results of real-time polymerase chain reaction showed that BcbHLHpol was highly expressed in maintainer buds and that the transcripts of BcbHLHpol significantly decreased in the buds of pol CMS. A virus-induced gene silencing vector that targets BcbHLHpol was constructed and transformed into Brassica campestris plants to further explore the function of BcbHLHpol. Male sterility and short stature were observed in BcbHLHpol-silenced plants. The degradation of tapetal cells was inhibited in BcbHLHpol-silenced plants, and nutrients were insufficiently supplied to the microspore. These phenomena resulted in pollen abortion. This result indicates that BcbHLHpol functions as a positive regulator in pollen development. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that BcbHLHpol interacted with BcSKP1 in the nucleus. This finding suggests that BcbHLHpol and BcSKP1 are positive coordinating regulators of pollen development. Quantitative real-time PCR indicated that BcbHLHpol and BcSKP1 can be induced at low temperatures. Thus, we propose that BcbHLHpol is necessary for meiosis. This study provides insights into the regulatory functions of the BcbHLHpol network during anther development.

Molecular cloning and expression analysis of a Cu/Zn SOD gene (BcCSD1) from Brassica campestris ssp. chinensis

Superoxide dismutases (SODs) are a family of metalloproteins extensively exists in eukaryote, which plays an essential role in stress-tolerance of higher plants. A full-length cDNA encoding Cu/Zn SOD (BcCSD1) was isolated from young seedlings of non-heading Chinese cabbage (Brassica campestris ssp. chinensis) by rapid amplification of cDNA ends (RACE). Bioinformatics analysis revealed that BcCSD1 belonged to the plant SOD super family and had the closest relationship with SOD from Brassica napus. Tissue expression pattern analysis revealed that the BcCSD1 was constitutively expressed in all the tested tissues, and strongest in leaf, moderate in stem, lowest in root. The expression profiles under different stress treatments such as drought, NaCl, high temperature and ABA were also investigated, and the results revealed that BcCSD1 was a stress-responsive gene, especially to ABA. These results provide useful information for further understanding the role of BcCSD1 resistant to abiotic stress in Brassica campestris in the future.

Copper stress induces the differential expression of microRNAs in non-heading Chinese cabbage

To gain a deep understanding of the regulatory mechanism of Cu-responsive microRNAs (miRNAs) in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino), the transcription of 10 annotated stress-inducible miRNAs and their target genes were investigated in two cultivars Suzhouqing and Wutacai exposed to excess of copper. Results show that these miRNAs were negatively correlated with their target genes under the Cu stress and showed different transcriptions in different tissues and cultivars. The transcriptions of bra-miR1530a and bra-miR1533v were highest in petioles and lowest in roots. Bra-miR1533ah, bra-miR1533m, bra-miR1533t, bra-miR414a, and bra-miR398b had the highest and lowest transcriptions in leaves and roots, respectively. In contrast, the transcription of bra-miR172f was highest in roots and lowest in leaves. Bra-miR1533aj and bra-miR1533d had similar transcriptions in petioles and leaves. The promoter analysis further revealed that seven miRNAs contained the Cu-response element (CuRE). In addition, miRNAs with more CuREs in the 5'-flanking sequences showed a lower expression following the Cu treatment. It imply that CuREs likely played a role in increasing the response to Cu in non-heading Chinese cabbage.

Chloroplast elongation factor BcEF-Tu responds to turnip mosaic virus infection and heat stress in non-heading Chinese cabbage

Eukaryotic elongation factor Tu has been implicated in responses to heat stress and viral infection. In this study, the turnip mosaic virus (TuMV)-response gene BcLRK01, which encodes a leucine-rich repeat receptor-like kinase, was probed using the cDNA library of TuMV-infected leaves of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). The BcEF-Tu gene, which encodes chloroplast elongation factor Tu, was obtained and verified by a yeast two-hybrid system to interact with the BcLRK01 gene. TuMV infection depressed the expression of this gene, whereas a heat stress induced its expression. Overexpression of BcEF-Tu enhanced the viability of Escherichia coli transformants under the heat stress. These results demonstrate that elongation factor BcEF-Tu responded to the TuMV infection and heat stress. This is the first report on chloroplast EF-Tu in non-heading Chinese cabbage which provides a theoretical basis for the functional research of EF-Tu.

Genes associated with agronomic traits in non-heading Chinese cabbage identified by expression profiling.

BackgroundThe genomes of non-heading Chinese cabbage (Brassica rapa ssp. chinensis), heading Chinese cabbage (Brassica rapa ssp. pekinensis) and their close relative Arabidopsis thaliana have provided important resources for studying the evolution and genetic improvement of cruciferous plants. Natural growing conditions present these plants with a variety of physiological challenges for which they have a repertoire of genes that ensure adaptability and normal growth. We investigated the differential expressions of genes that control adaptability and development in plants growing in the natural environment to study underlying mechanisms of their expression.ResultsUsing digital gene expression tag profiling, we constructed an expression profile to identify genes related to important agronomic traits under natural growing conditions. Among three non-heading Chinese cabbage cultivars, we found thousands of genes that exhibited significant differences in expression levels at five developmental stages. Through comparative analysis and previous reports, we identified several candidate genes associated with late flowering, cold tolerance, self-incompatibility, and leaf color. Two genes related to cold tolerance were verified using quantitative real-time PCR.ConclusionsWe identified a large number of genes associated with important agronomic traits of non-heading Chinese cabbage. This analysis will provide a wealth of resources for molecular-assisted breeding of cabbage. The raw data and detailed results of this analysis are available at the website http://nhccdata.njau.edu.cn.

BcPMI2, isolated from non-heading Chinese cabbage encoding phosphomannose isomerase, improves stress tolerance in transgenic tobacco

Phosphomannose isomerase (PMI) is an enzyme that catalyses the first step of the L-galactose pathway for ascorbic acid (AsA) biosynthesis in plants. To clarify the physiological roles of PMI in AsA biosynthesis, the cDNA sequence of PMI was cloned from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) and overexpressed in tobacco transformed with Agrobacterium tumefaciens. The AsA and soluble sugar contents were lower in 35S::BcPMI2 tobacco than in wild-type tobacco. However, the AsA level in BcPMI2-overexpressing plants under stress was significantly increased. The T1 seed germination rate of transgenic plants was higher than that of wild-type plants under NaCl or H2O2 treatment. Meanwhile, transgenic plants showed higher tolerance than wild-type plants. This finding implied that BcPMI2 overexpression improved AsA biosynthetic capability and accumulation, and evidently enhanced tolerance to oxidative and salt stress, although the AsA level was lower in transgenic tobacco than in wild-type tobacco under normal condition.

Major Gene Plus Polygene Inheritance of Vitamin C Content in Non-heading Chinese Cabbage

The mixed major gene plus polygene inheritance model was used to investigate the inheritance law of vitamin C in non-heading Chinese cabbage(Brassica campestris ssp. chinensis Makino) in six generations(P1, P2, F1, B1, B2, and F2) derived from Wutacai line(with high vitamin C content) × Erqing line(with low vitamin C content). The results showed that the trait of vitamin C was controlled by one additive major gene plus additive-dominant polygene in the joint analysis of six generations. The result in 2011 showed that the additive effect was 13.15. Heritabilities of major genes in B1, B2, and F2 populations were 54.38%, 38.58%, and 18.69%, respectively, while those of polygenes in the three populations were 24.69%, 36.92%, and 40.7%, respectively. The result in 2013 showed that the additive effect was 6.04. Heritabilities of major genes in B1, B2, and F2 populations were 1.88%, 6.41%, and 45.04%, respectively, while those of polygenes in B1, B2, and F2 populations were 39.67%, 16.57%, and 16.91%, respectively. The results from two years indicated that environmental factors could play roles in inheritance of vitamin C in non-heading Chinese cabbage. Thus, the marker assisted selection method could be used to screen high vitamin C cultivars of non-heading Chinese cabbage. In addition, environmental conditions should also be considered in the breeding process.

Expression analysis of self-incompatibility-associated genes in non-heading Chinese cabbage.

In Brassicaceae, a self-incompatibility (SI) system mediates pollen-pistil interactions. Self-pollen could be recognized and rejected by incompatible pistils. Several components involved in the SI response have been determined, including S-locus receptor kinase (SRK), S-locus cysteine-rich protein/S-locus protein 11, and arm repeat-containing protein 1 (ARC1). However, the components involved in the SI system of Brassicaceae are not fully understood. Here, we detected expression patterns of 24 SI-related genes in non-heading Chinese cabbage (Brassica campestris ssp chinensis Makino) after compatible and incompatible pollination, and potential interaction relationships of these genes were predicted. SRK and ARC1 transcripts increased initially 0.25 h after incompatible pollination, while kinase-associated protein phosphatase had an expression pattern that was opposite that of SRK transcripts during self-pollination. Plant U-box 8 was not required in the SI response of non-heading Chinese cabbage. Our results showed that the SI signal of non-heading Chinese cabbage could occur within 0.25 h after self-pollination. The hypothetical interaction relationships indicated that plastid-lipid-associated protein and malate dehydrogenase could be negatively regulated by chaperonin 10, glutathione transferase, cytidylate kinase/uridylate kinase, and methionine synthase by indirect interactions. Our findings could be helpful to better understand potential roles of these components in the SI system of non-heading Chinese cabbage.

Induction of tetraploidy in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) by colchicine treatment increases the ascorbic acid concentration

SummaryNon-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) is one of the most commonly consumed vegetables in China, and has recently been introduced into other countries in Asia, North America, and Europe. In this study, we obtained tetraploid, non-heading Chinese cabbage by treating the apical portion of diploid seedlings with various concentrations of colchicine for different treatment times. Treatments included a total of two, four, or six applications of 1.5 g l–1 or 2.0 g l–1 colchicine, performed twice a day at 09.00 h and 15.00 h. Tetraploid plants were selected and identified based on their morphological, anatomical, agronomic, and cytological characteristics, and on their DNA content, as measured by flow cytometry. The highest rate of tetraploidy (8.6%) occurred in seedlings treated six-times with 1.5 g l–1 colchicine. Compared to diploid plants (untreated controls), the tetraploid plants had wider leaves, larger petals, longer siliques, larger seeds, larger stomata, and fewer chloroplasts per guard cell. HPLC showed that tetraploidisation could increase the level of ascorbic acid (AsA) in this crop. Quantitative real-time PCR (qRT-PCR) showed that the levels of expression of genes encoding enzymes involved in the AsA-L-galactose biosynthetic pathway increased in tetraploid plants. This is the first study to compare levels of expression of genes involved in the biosynthetic pathway of AsA between diploid and tetraploid non-heading Chinese cabbage plants..

Production of intergeneric allotetraploid between autotetraploid non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) and autotetraploid radish (Raphanus sativus L.)

Intergeneric hybrids between non-heading Chinese cabbage (&lt;em&gt;Brassica campestris&lt;/em&gt; ssp. &lt;em&gt;chinensis&lt;/em&gt; Makino; 2n = 4x = 40) and radish (&lt;em&gt;Raphanus sativus&lt;/em&gt; L.; 2n = 4x = 36) were obtained through ovary culture and embryo rescue. Some hybrid embryos (0.11 per ovary) were produced, but only 4 of them germinated. As most hybrid embryos failed to develop into plantlets directly, plants were regenerated by inducing shoots on the cultured cotyledon and inducing roots on the root induction medium. All hybrid plants were morphologically uniform. They resembled the non-heading Chinese cabbage in the long-lived habit, the plant status, the vernalization requirement and the petiole color, while the petiole shape, leaf venation pattern and flowers were more similar to those of radish. Upon examination of the flowers, these were found to have normal pistil, but rudimentary anthers with non-functional pollen grains. The somatic chromosome number of F1 plants was 38. Analysis of SSR banding patterns provided additional confirmation of hybridity.

Karyotype analysis of autotetraploidy in Brassica rapa ssp. chinensis

SummaryBrassica rapa ssp. chinensis (non-heading Chinese cabbage) is one of the most important vegetables grown in southern China. Autotetraploidy, induced artificially by colchicine treatment, has increased its nutritional value. For the first time, we report the distribution of repeat DNA sequences in autotetraploid B. rapa ssp. chinensis using fluorescence in situ hybridisation (FISH) with ribosomal DNA (rDNA) probes. Comparisons of inbred lines of a diploid (10P15) and its induced autotetraploid (10P11) of B. rapa ssp. chinensis used genomic in situ hybridisation (GISH) by labelling the total genomic DNA as a probe, and using unlabelled genomic DNA of B. oleracea L. var. italica as the blocking reagent. GISH signals appeared at the centromere and/or adjacent regions of each chromosome; therefore, these could be used as alternative centromere markers. In addition, variations in the number and loci of repeat sequences between the diploid and its autotetraploid were observed, suggesting that some deletions or insertions had occurred in the regions of repeat sequences during formation of the autotetraploid. These findings increase our knowledge of the karyotypes of the mitotic chromosomes in diploid and autotetraploid B. rapa ssp. chinensis, and will facilitate further research in Brassica evolution and marker-assisted breeding.