Use of 5´-anchored primers for the enhanced recovery of specific microsatellite markers in Brassica napus L.

Microsatellite markers have assumed great significance in biological research. The isolation and characterisation of microsatellites involves DNA library construction and screening, DNA sequencing, primer design and PCR optimisation. When a microsatellite is situated close to the beginning or end of a cloned fragment, specific primers cannot be designed for one of the flanking sequences, thus hindering the utilisation of such microsatellites as markers. The present approach was to use one 5′-anchored primer complementary to the microsatellite sequence in combination with one specific Cy5- labelled primer with a view to retrieving useful microsatellites, which would otherwise be lost. Six pairs of a 5′ anchored primer and a specific primer were used across a set of 31 Brassica napus winter cultivars and one accession each of five additional Brassica species. Using laser fluorometry a single labelled product was observed after amplification with each of four primer pairs, and one primer pair gave two labelled products. Three products corresponded in size with the products expected if 5′ anchoring was effective, indicating the amplification of locus-specific full-length products including all of the microsatellite repeats. All six primer pairs showed polymorphisms across the Brassica species examined, but only one primer pair showed polymorphisms within B. napus, making it useful for genetic analysis in rapeseed cultivars. The other primer pairs could be useful in studying gene introgression into B. napus or for investigating interspecific crosses involving different Brassica species. Received: 5 August 1999 / Accepted: 1 November 1999     

Computational analysis of storage synthesis in developing Brassica napus L. (oilseed rape) embryos: flux variability analysis in relation to ¹³C metabolic flux analysis

Plant oils are an important renewable resource, and seed oil content is a key agronomical trait that is in part controlled by the metabolic processes within developing seeds. A large‐scale model of cellular metabolism in developing embryos of Brassica napus (bna572) was used to predict biomass formation and to analyze metabolic steady states by flux variability analysis under different physiological conditions. Predicted flux patterns are highly correlated with results from prior ¹³C metabolic flux analysis of B. napus developing embryos. Minor differences from the experimental results arose because bna572 always selected only one sugar and one nitrogen source from the available alternatives, and failed to predict the use of the oxidative pentose phosphate pathway. Flux variability, indicative of alternative optimal solutions, revealed alternative pathways that can provide pyruvate and NADPH to plastidic fatty acid synthesis. The nutritional values of different medium substrates were compared based on the overall carbon conversion efficiency (CCE) for the biosynthesis of biomass. Although bna572 has a functional nitrogen assimilation pathway via glutamate synthase, the simulations predict an unexpected role of glycine decarboxylase operating in the direction of NH₄⁺ assimilation. Analysis of the light‐dependent improvement of carbon economy predicted two metabolic phases. At very low light levels small reductions in CO₂ efflux can be attributed to enzymes of the tricarboxylic acid cycle (oxoglutarate dehydrogenase, isocitrate dehydrogenase) and glycine decarboxylase. At higher light levels relevant to the ¹³C flux studies, ribulose‐1,5‐bisphosphate carboxylase activity is predicted to account fully for the light‐dependent changes in carbon balance.     

油菜简单重复序列SSR(simple sequence repeat)研究进展

简单重复序列(simple sequence repeat,SSR)是重复单元少于6个核苷酸重复序列,广泛分布于动植物基因组中,呈孟德尔遗传,已被作为一种理想的共显性标记应用于动植物遗传研究中。本文重点介绍了SSR分类、特点,及近几年来油菜SSR标记的开发和SSR技术在油菜基因定位、品种鉴定中的应用,并对SSR标记在油菜中的应用进行了探讨。     

Poly(β-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus

Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally sustainable alternative to petroleum-based plastics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(β-hydroxybutyrate) (PHB) has previously been produced in leaves of Arabidopsis thaliana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91: 12760–12764). However, Brassica napus oilseed may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize PHB: a β-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate 12-hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform B. napus. Poly(β-hydroxybutyrate) accumulated in leukoplasts to levels as high as 7.7% fresh seed weight of mature seeds. Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation. Received: 26 May 1999 / Accepted: 16 June 1999     

The after-effects of temperature and irradiance during early growth of winter oilseed rape (Brassica napus L. var. oleifera, cv. Górczański) seedlings on the progress of their cold acclimation

Experiments performed under controlled conditions showed that level of PPFD (photosynthetic photon flux density) during early seedlings growth (preceding cold acclimation at +2 °C) was not the key factor for the development of frost resistance. It did not modify the beneficial effects of prehardening (Rapacz 1997, in this issue) at moderately low (+12 °C) day temperature. Now I have shown that the increase of PPFD may replace to some extent prehardening in the development of frost resistance. It was particularly seen in non-prehardened plants, which had been grown under warm-day (+20 °C) conditions. Prehardening performed under controlled conditions, as well as seedlings growth under natural autumn conditions in the field, allowed to maintain a high net-photosynthesis rate at chilling temperatures. A net-photosynthesis rate during cold acclimation at +2 °C corresponded well with higher frost resistance. As a result, seedlings non subjected to prehardening and grown before cold acclimation under low PPFD acclimated better, if the cold treatment was applied only at nights (+20/2 °C day/night). Only under such conditions the photosynthetic rate was sufficiently high to allow plants to reach a higher level of frost resistance. All other plants acclimated better when they were exposed to the hardening temperature continuously during days and nights (+2/2 °C day/night).     

The effects of day and night temperatures during early growth of winter oilseed rape (Brassica napus L. var. oleifera cv. Górczański) seedlings on their morphology and cold acclimation responses

Studies on the effects of temperature during the early stage of growth on frost resistance of winter rape seedlings under controlled conditions were performed. It was found that cold acclimation responses of plants were affected to a great extent by the conditions of the seedlings early growth. During this period, when the day temperatures were reduced to the range from +10 °C to +15 °C, a process termed “prehardening” was observed. During prehardening plants formed leaf rosettes. Their ability to develop frost resistance during acclimation at +2 °C also increased. Frost resistance of these plants was comparable with the resistance of plants growing in autumn under field conditions.     

Genetic load and transgenic mitigating genes in transgenic Brassica rapa (field mustard) × Brassica napus (oilseed rape) hybrid populations

Background

Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics.

Results

In this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-γ-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation.

Conclusion

We here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.     

The transfer of ‘Polima’ cytoplasmic male sterility from oilseed rape (Brassica napus) to broccoli (B. oleracea) by protoplast fusion

Protoplast fusion was utilised to transfer Polima type cytoplasmic male sterility (CMS) from Brassica napus, canola cv. Polima Karat (Pol-Karat) to B. oleracea, broccoli, var. Green Comet. Southern and RFLP analysis confirmed that four cybrids possessed nuclear genomes of broccoli with Polima mitochondria and chloroplasts. A fifth cybrid was a nuclear hybrid between broccoli and Pol-Karat, with Polima mitochondria and chloroplasts of broccoli. The broccoli type cybrids were morphologically similar to Green Comet, while the hybrid type was an intermediate of the two fusion parents. Flowers on the cybrids were distinctive in that although they possessed a morphology typical of Polima, they had very reduced petals. The broccoli type cybrids exhibited some female fertility, albeit low, establishing potential for F₁ hybrid production.     

Molecular characterization of Platonia insignis Mart. (“Bacurizeiro”) using inter simple sequence repeat (ISSR) markers

Platonia insignis Mart. (Clusiaceae) is widespread throughout the Amazon and adjacent areas. The fruits (known locally as “bacuri”) have significant commercial potential, but the species is under threat from agro-industrial expansion. The genetic variability within 72 genotypes of P. insignis belonging to ten populations collected in the Brazilian states of Maranhão and Piauí, and maintained in the germplasm collection of Embrapa Meio-Norte, has been determined, and the organization of genetic diversity within populations, between populations and among geographic groups verified. Eighteen selected inter simple sequence repeat primers allowed amplification of 236 loci of which 221 (93.64 %) were polymorphic, indicating a high level of genetic diversity. At the population level, the Shannon and Nei diversity indices ranged from 0.082 to 0.323 and from 0.120 to 0.480, respectively. The global coefficient of genetic differentiation (G _ST ) was 0.4730 indicating that differentiation between populations was significant, a finding that was confirmed by analysis of molecular variance (Φ _ST  = 0.28). UPGMA cluster analysis revealed that the genotypes could be stratified into groups that were well defined and consistent with those identified in the dendrogram constructed using pair wise Φ _ST values. The high genetic diversity established in this study may facilitate the management and conservation of the germplasm of P. insignis.     

Elucidation of origin of the present day hybrid banana cultivars using the 5′ETS rDNA sequence information

In the present study, our intention was to elucidate the genetic relation of M. acuminata subspecies and analyse the diversity of the M. balbisiana gene-pool using nuclear ribosomal gene loci based marker system. Additionally the obtained information allowed elucidating the structure and ancestry of the nuclear genomes of diploid and triploid cultivars. By establishing the nucleotide sequence of the rDNA locus for M. acuminata and partially for M. balbisiana and their comparative analysis revealed that the 5′ETS region was the most divergent between acuminata and balbisiana genomes. Based on the SNP sites identified in this region a PCR based system was built, which revealed four gene-pools among M. acuminata wild types, while M. balbisiana showed no sequence divergence. The developed markers proved to be a powerful tool in the identification of the acuminata component of diploid and triploid hybrid cultivars and discovery of unexpected genotypes.     

Does genotypic variation in nitrogen remobilisation efficiency contribute to nitrogen efficiency of winter oilseed-rape cultivars (Brassica napus L.)?

Aims

Winter oilseed-rape production is characterized by a low N efficiency, due to low N uptake and insufficient N remobilisation to the seeds. In particular, a reduction of leaf N losses might be one way to improve N efficiency of this crop. It was tested if variations in leaf N losses and in stem residual N amounts at maturity exist between cultivars differing in N efficiency.

Methods

In a 3-year field experiment, four oilseed rape cultivars were cultivated at limiting, medium, and high N supply.

Results

N harvest indices in this study were comparatively high (around 0.79) and leaf N losses amounted to at most 13 kg N ha^?1. 86 % of the leaf N present at the beginning of flowering was remobilised, irrespective of N rate or cultivar. Nevertheless, genotypic variation in leaf N loss existed. They were mainly due to differences in leaf N accumulation until flowering. Residual N in stems (up to 33 kg N ha^?1) was higher than leaf N losses and varied more between treatments but was not related to genotypic variation in yield.

Conclusions

N uptake after flowering was more important than N remobilisation from vegetative biomass for genotypic variation in seed yield both at low and high N supply.     

Regional distribution of genetically modified organisms (GMOs)—Up-scaling the dispersal and persistence potential of herbicide resistant oilseed rape (Brassisca napus)

Most genetically modified (GM) crop plants are designed to be grown on large areas. However, empirical investigations for risk assessment are limited in their temporal and spatial extent. In the case of GM crop plants it is difficult to test the relevance of anticipated risks on the same spatial scale as the intended use. Processes which are difficult to assess experimentally include combinatory effects, interactions between different integration levels, persistence, long distance dispersal and occurrence of rare events. To a limited extent, it is possible to combine results of investigations on small spatial scales in a way that large-scale and long-term implications on the regional scale can be analysed by using modelling and extrapolation approaches. It is thus possible to indicate some of the involved risks which are not accessible otherwise.In this paper we present the results of an extrapolation methodology comprising several scales from the field size up to the landscape level. This methodology aimed at analysing the implications of a large-scale release of genetically modified oilseed rape (GM OSR). The approach consisted of an extrapolation scheme beginning with a landscape analysis which generated representative scenarios considering climate and OSR cultivation characteristics. For the spatial extent of several fields this information was applied in an individual-based model representing ontogeny, dispersal and persistence of cultivated, volunteers and feral oilseed rape. In a final step, simulation results were extrapolated to the region of Northern Germany.Here we focus on the model results which were extrapolated to the regional level by applying a set of ecological indicators which allowed to assess potential implications on this level. These indicators included the number and distribution of flowering GM plants and the dynamics of GM OSR seeds in the soil seedbank. Specific results related to the long-term dynamics in the seedbank and volunteer development. Model results emphasise the long-term consequences of GM OSR cultivation and the explicit necessity to regard high variability in potential GMO admixture. This has to be considered when developing landscape management schemes for co-existence.The extrapolation approach presented here, integrates different traits to assess effects of GMOs on large spatial scales with respect to persistence and dispersal. The developed methodology is equally applicable for other crops, regions and different agricultural conditions.     

Uridine 5′-diphospho-D-glucose-dependent vectorial sucrose synthesis in tonoplast vesicles of the storage hypocotyl of red beet (Beta vulgaris L. ssp. conditiva)

Tonoplast vesicles were prepared from red-beet (Beta vulgaris L. ssp. conditiva) hypocotyl tubers (beetroot) known to store sucrose. Uptake experiments, employing uridine 5-diphospho-[¹⁴C]glucose (UDP-[¹⁴C]glucose) showed the operation of an UDP-glucose-dependent group translocator for vectorial synthesis and accumulation of sucrose, recently described for sugarcane and red-beet vacuoles and for tonoplast vesicles prepared from sugarcane suspension cells. Characterization of the kinetic properties yielded the following results. Uptake of UDP-glucose was linear for 15 min. The apparent K _m was 0.75 mM for UDP-glucose (at pH 7.2, 1 mM Mg²⁺), V _max was 32 nmol·(mg protein)^-1·min^-1. The incorporation of UDP-glucose exhibited a sigmoidal substrate-saturation curve in the absence of Mg²⁺, the Hill coefficient (n _H) was 1.33; Michaelis-Menten kinetics were obtained, however, in the presence of 1 mM MgCl₂. For the reaction sequence under the control of the group translocator a dual pH optimum was found at pH 7.2 and 7.9, respectively. All reaction intermediates and the end product sucrose could be identified by two-dimensional high-performance thin-layer chromatography and autoradiography. The distribution pattern of radioactivity showed almost uniformly high labeling of all intermediates and sucrose. The physiological relevance of the results is discussed in the light of the fact that the tonoplast of red-beet storage cells accommodates two mechanisms of sucrose uptake (i) vectorial sucrose synthesis and (ii) direct ATP-dependent sucrose assimilation.Abbreviations HPTLC High-performance thin-layer chromatography - UDP uridine 5-diphosphate - SDS sodium dodecyl sulfate     

Cloning and sequence analysis of the X-Prolyl-dipeptidyl-aminopeptidase gene (pepX) from Lactobacillus delbrückii ssp. lactis DSM7290

Lactobacillus delbrückii ssp. lactis DSM7290 possesses an X-prolyl-dipeptidyl-aminopeptidase, designated PepX, which catalyses the hydrolytic removal of N-terminal dipeptidyl residues from peptides containing proline in the penultimate position. Using the specific substrate L-Ala-L-Pro-p-nitroanilide, PepX was purified by a four-step procedure including ammonium sulphate fractionation, hydrophobic interaction chromotography, ion exchange chromotography, and affinity chromotography. The N-terminus of the purified protein was sequenced. Screening of a gene library of chromosomal Lactobacillus delbrückii ssp. lactis DSM7290 DNA in the low-copy-number vector pLG339 resulted in the identification of the pepX gene in Escherichia coli using a specific plate assay with Gly-L-Pro--naphthalamide as substrate. Nucleotide sequence analysis revealed an open reading frame of 2376 bp, coding for a protein of 792 amino acids with a molecular mass of 88449 Da. Correspondence to: E. C. Meyer-Barton     

Erwinia carotovora ssp.carotovora Infection Induced“Defense Lignin”Accumulation and Lignin Biosynthetic Gene Expression in Chinese Cabbage(Brassica rapa L.ssp.pekinensis)

Erwinia carotovora subsp. carotovora (Ecc) infects and causes soft rot disease in hundreds of crop species includingvegetables, flowers and fruits. Lignin biosynthesis has been implicated in defensive reactions to injury and pathogeninfection in plants. In this work, variations of lignin content and gene expression in the molecular interaction betweenChinese cabbage and Ecc were investigated. H_2O_2 accumulation and peroxidase activity were detected by 3, 3-Dimethoxybenzidine staining at mocked and Ecc-inoculated sites of Chinese cabbage leafstalks. Mason lignin contentin inoculated plants increased by about 7.84%, 40.37%, and 43.13% more than that of the mocked site at 12, 24 and 72 hafter inoculation, respectively. Gas chromatography detected more p-coumaryl (H) and less coniferyl (G) and sinapyl (S)monolignins in leafstalks of Chinese cabbage. All three monomers increased in Ecc-infected leafstalks, and the Ecc-induced"defense lignin" were composed of more G and H monolignins, and less S monolignin. After searching the expressedsequence tags (EST) data of Chinese cabbage, 12 genes putatively encoding enzymes involved in lignin biosynthesis wereselected to study their expression. All of these genes could be induced by mock inoculation and Ecc infection, while thegene expression lasted for several more hours in the infected samples than in mocked and untreated plants. Our resultsindicated that "defense lignin" was different from the developmental lignin in composition; G and S monolignins weresignificantly induced in plants in response to the soft rot Ecc; thus, lignin biosynthesis was differentially regulated andplayed a role in plant response to the soft rot Ecc.     

芸苔属(Brassica,L.)植物的新种合成及其细胞遗传学研究 Ⅲ.胜利油菜(B.napus,L.var.oleifera,DC.)C组染色体的人工置换

从以白花芥蓝(B.alboglabra,2n(2x)=18,组型为c′c′)为母本,胜利油菜同源异源八倍体(B.napus,4n(8x)=76,组型为aaaacccc)为父本的杂交组合中,获得了一个染色体数为4x=38的F_1植株(组型为aaac′),实现了c 组染色体的人工置换。F_1杂种在形态、生理上与普通胜利油菜(aacc)相同。但花冠粉黄色,为双亲的中间类型。F_2花色从黄到白,广泛分离。对F_1及F_2进行了形态、生理及细胞遗传学诸特征的研究,并对F_1aacc′染色体组型形成的机理提出了模型。F_3群体对龙头病具有免疫力,十分引人注目。