A comparative linkage map of oilseed rape and its use for QTL analysis of seed oil and erucic acid content.

We have developed a new DH mapping population for oilseed rape, named TNDH, using genetically and phenotypically diverse parental lines. We used the population in the construction of a high stringency genetic linkage map, consisting of 277 loci, for use in quantitative genetic analysis. A proportion of the markers had been used previously in the construction of linkage maps for Brassica species, thus permitting the alignment of maps. The map includes 68 newly developed Sequence Tagged Site (STS) markers targeted to the homologues of defined genes of A. thaliana. The use of these markers permits the alignment of our linkage map with the A. thaliana genome sequence. An additional 74 loci (31 newly developed STS markers and 43 loci defined by SSR and RFLP markers that had previously been used in published linkage maps) were added to the map. These markers increased the resolution of alignment of the newly constructed linkage map with existing Brassica linkage maps and the A. thaliana genome sequence. We conducted field trials with the TNDH population at two sites, and over 2 years, and identified reproducible QTL for seed oil content and erucic acid content. The results provide new insights into the genetic control of seed oil and erucic acid content in oilseed rape, and demonstrate the utility of the linkage map and population.

Does the efficacy of supplemental oxygen for the prevention of postoperative nausea and vomiting depend on the measured outcome, observational period or site of surgery?

High intra-operative oxygen concentration reportedly reduces postoperative nausea and vomiting (PONV), but recent data are conflicting. Therefore, we tested whether the effectiveness of supplemental oxygen depends on the endpoint (nausea vs. vomiting), observation interval (early vs. late) or surgical field (abdominal vs. non-abdominal). We randomly assigned 560 adult patients undergoing various elective procedures with a PONV risk of at least 40% to intra-operative 80% (supplemental) or 30% oxygen (control). Potential confounding factors were similar between groups. Incidences of nausea were similar in the groups during early (12% (supplemental) vs. 10% (control), p = 0.43) and late intervals, 26%vs. 20%, p = 0.09, as were the incidences of vomiting (early: 2%vs. 3%, p = 0.40; late: 8%vs. 9%, p = 0.75). Supplemental oxygen was no more effective at reducing PONV in abdominal (40%vs. 31%, p = 0.37) than in non-abdominal surgery (25%vs. 21%, p = 0.368). Thus, supplemental oxygen was unable to reduce PONV independent of the endpoint, observational period or site of surgery.

Efficient large-scale development of microsatellites for marker and mapping applications in Brassica crop species.

A set of 398 simple sequence repeat markers (SSRs) have been developed and characterised for use with genetic studies of Brassica species. Small-insert (250-900 bp) genomic libraries from Brassica rapa, B. nigra, B. oleracea and B. napus, highly enriched for dinucleotide and trinucleotide SSR motifs, were constructed. Screening the clones with a mixture of oligonucleotide repeat probes revealed positive hybridisation to between 75% and 90% of the clones. Of these, 1230 were sequenced. Primer pairs were designed for 398 SSR clones, and of these, 270 (67.8%) amplified a PCR product of the expected size in their focal and/or closely related species. A further screen of 138 primers pairs that produced a PCR product in B. napus germplasm found that 86 (62.3%) revealed length polymorphisms within at least one line of a test array representing the four Brassica species. The results of this screen were used to identify 56 SSRs and were combined with 41 SSRs that had previously shown polymorphism between the parents of a B. napus mapping population. These 97 SSR markers were mapped relative to a framework of RFLP markers and detected 136 loci over all 19 linkage groups of the oilseed rape genome.

[Calibration of a room air gas monitor with certified reference gases]. / Kalibrierung eines Raumluft-Gasmonitors mit zertifizierten Prüfgasen.

Photo-acoustic infrared spectrometry is considered to be the gold standard for on-line measurement of anesthetic waste gas in room air. For maintenance of the precision of the measurements, the manufacturer recommends calibration of the gas monitor monitor every 3-12 months. We investigated whether the use of reference gases with analysis certificate could serve as a feasible alternative to commercial recalibration. We connected a multi-gas monitor type1302 (Bruel & Kjaer, Naerum, Denmark) to compressed air bottles containing reference gases with analysis certificate. Using a T-piece with a flow-meter, we avoided the entry of room air during the calibration phase. Highly purified nitrogen was used for zero calibration. The reference concentrations for desflurane, enflurane, halothane, isoflurane, and sevoflurane ranged from 41.6-51.1 ml/m(3) (ppm) in synthetic air. Since there is an overlap of the infrared absorption spectra of volatile anesthetics with alcohol used in operating rooms, we performed a cross-compensation with iso-propanol (107.0 ppm). A two-point calibration was performed for N(2)O (96.2 and 979.0 ppm), followed by cross-compensation with CO(2). Nafion tubes were used in order to avoid erroneous measurements due to molecular relaxation phenomena. The deviation of the measurement values ranged initially from 0-2.0% and increased to up to 4.9% after 18 months. For N(2)O, the corresponding values were 4.2% and 2.7%, respectively. Thus, our calibration procedure using certified reference gases yielded precise measurements with low deterioration over 18 months. It seems to be advantageous that the precision can be determined whenever deemed necessary. This allows for an individual decision, when the gas monitor needs to be calibrated again. The costs for reference gases and working time as well as logistic aspects such as storage and expiration dates must be individually balanced against the costs for commercial recalibration.

The development of multiplex simple sequence repeat (SSR) markers to complement distinctness, uniformity and stability testing of rape (Brassica napus L.) varieties.

To assess the potential of multiplex SSR markers for testing distinctness, uniformity and stability of rape (Brassica napus L.) varieties, we developed three multiplex SSR sets composed of five markers each. These were used to measure the extent of diversity within and between a set of ten varieties using a fluorescence-based semi-automated detection technology. Also, we evaluated the significance of any correlation between SSRs, pedigree and five of the morphological characters currently used for statutory distinctness, uniformity and stability testing of rape varieties. An assignment test was allowed to identify 99% of the plants examined, with the correct variety based on the analysis of 48 individual plants for each variety. Principal coordinate analysis confirmed that a high degree of separation between varieties could be achieved. Varieties were separated in three groups corresponding to winter, spring and forage types. These results suggested that it should be possible to select a set of markers for obtaining a suitable separation. Diversity within varieties varied considerably, according to the variety and the locus examined. No significant correlation was found between SSR and morphological data. However, genetic distances measured by SSRs were correlated to pedigree. These results suggested that SSRs could be used for pre-screening or grouping of existing and candidate varieties, allowing the number of varieties that need to be grown for comparison to be reduced. Multiplex SSR sets gave high-throughput reproducible results, thus reducing the costs of SSR assessment. Multiplex SSR sets are a promising way forward for complementing the current variety testing system in B. napus.

Assessing the level of collinearity between Arabidopsis thaliana and Brassica napus for A. thaliana chromosome 5.

This study describes a comprehensive comparison of chromosome 5 of the model crucifer Arabidopsis with the genome of its amphidiploid crop relative Brassica napus and introduces the use of in silico sequence homology to identify conserved loci between the two species. A region of chromosome 5, spanning 8 Mb, was found in six highly conserved copies in the B. napus genome. A single inversion appeared to be the predominant rearrangement that had separated the two lineages leading to the formation of Arabidopsis chromosome 5 and its homologues in B. napus. The observed results could be explained by the fusion of three ancestral genomes with strong similarities to modern-day Arabidopsis to generate the constituent diploid genomes of B. napus. This supports the hypothesis that the diploid Brassica genomes evolved from a common hexaploid ancestor. Alignment of the genetic linkage map of B. napus with the genomic sequence of Arabidopsis indicated that for specific regions a genetic distance of 1 cM in B. napus was equivalent to 285 Kb of Arabidopsis DNA sequence. This analysis strongly supports the application of Arabidopsis as a tool in marker development, map-based gene cloning, and candidate gene identification for the larger genomes of Brassica crop species.

UK CropNet: a collection of databases and bioinformatics resources for crop plant genomics.

The UK Crop Plant Bioinformatics Network (UK CropNet) was established in 1996 in order to harness the extensive work in genome mapping in crop plants in the UK. Since this date we have published five databases from our central UK CropNet WWW site (http://synteny.nott.ac.uk/) with a further three to follow shortly. Our resource facilitates the identification and manipulation of agronomically important genes by laying a foundation for comparative analysis among crop plants and model species. In addition, we have developed a number of software tools that facilitate the visualisation and analysis of our data. Many of our tools are made freely available for use with both crop plant data and with data from other species.

Pollen-stigma adhesion in Brassica spp involves SLG and SLR1 glycoproteins.

The adhesion of pollen grains to the stigma is the first step of pollination in flowering plants. During this step, stigmas discriminate between pollen grains that can and cannot be permitted to effect fertilization. This selection is operated by various constituents of the cell walls of both partners. Several genes structurally related to the self-incompatibility system that prevents self-pollination in Brassica spp are known to target their products into the stigma cell wall. We proposed previously that one of these genes, the one encoding the S locus glycoprotein (SLG)-like receptor 1 (SLR1), which is coexpressed with that encoding SLG, may participate in pollen-stigma adhesion. Here, we exploit a biomechanical assay to measure the pollen adhesion force and show that it is reduced both by transgenic suppression of SLR1 expression and by pretreatment of wild-type stigmas with anti-SLR1 antibodies, anti-SLG antibodies, or pollen coat-protein extracts. Our results indicate a common adhesive function for the SLR1 and SLG proteins in the pollination process.

Visualization of the Brassica self-incompatibility S-locus on identified oilseed rape chromosomes.

Seventy years after Karpechenko [15] first reported the accurate chromosome number of oilseed rape (Brassica napus L., 2n=38), we have developed a quantitative chromosome map of rape using computer imaging technology. The capacity to identify individual rape chromosomes will facilitate a wide range of genetic studies. Here we demonstrate the use of imaging methods in combination with fluorescence in situ hybridization to localize, on identified chromosomes, the single copy S-locus glycoprotein and S-locus-related genes involved in the self-incompatibility system of Brassica. These techniques have a broader application in plant genome research involving the mapping of single-copy genes and markers, irrespective of the plant species.

Collinearity between a 30-centimorgan segment of Arabidopsis thaliana chromosome 4 and duplicated regions within the Brassica napus genome.

Arabidopsis thaliana (the model dicotyledonous plant) is closely related to Brassica crop species. Genome collinearity, or conservation of marker order, between Brassica napus (oilseed rape) and A. thaliana was assessed over a 7.5-Mbp region of the long arm of A. thaliana chromosome 4, equivalent to 30 cM. Estimates of copy number indicated that sequences present in a single copy in the haploid genome of A. thaliana (n = 5) were present in 2-8 copies in the haploid genome of B. napus (n = 19), while sequences present in multiple copies in A. thaliana were present in over 10 copies in B. napus. Genetic mapping in B. napus of DNA markers derived from a segment of A. thaliana chromosome 4 revealed duplicated homologous segments in the B. napus genome. Physical mapping in A. thaliana of homologues of Brassica clones derived from these regions confirmed the identity of six duplicated segments with substantial homology to the 7.5-Mbp region of chromosome 4 in A. thaliana. These six duplicated Brassica regions (on average 22 cM in length) are collinear, except that two of the six copies contain the same large internal inversion. These results have encouraging implications for the feasibility of shuttling between the physical map of A. thaliana and genetic maps of Brassica species, for identifying candidate genes and for map based gene cloning in Brassica crops.

Comparison of flowering time genes in Brassica rapa, B. napus and Arabidopsis thaliana.

The major difference between annual and biennial cultivars of oilseed Brassica napus and B. rapa is conferred by genes controlling vernalization-responsive flowering time. These genes were compared between the species by aligning the map positions of flowering time quantitative trait loci (QTLs) detected in a segregating population of each species. The results suggest that two major QTLs identified in B. rapa correspond to two major QTLs identified in B. napus. Since B. rapa is one of the hypothesized diploid parents of the amphidiploid B. napus, the vernalization requirement of B. napus probably originated from B. rapa. Brassica genes also were compared to flowering time genes in Arabidopsis thaliana by mapping RFLP loci with the same probes in both B. napus and Arabidopsis. The region containing one pair of Brassica QTLs was collinear with the top of chromosome 5 in A. thaliana where flowering time genes FLC, FY and CO are located. The region containing the second pair of QTLs showed fractured collinearity with several regions of the Arabidopsis genome, including the top of chromosome 4 where FRI is located. Thus, these Brassica genes may correspond to two genes (FLC and FRI) that regulate flowering time in the latest flowering ecotypes of Arabidopsis.

An analysis of the relative activities of a number of promoter constructs from genes which are expressed during late pollen development as determined by particle bombardment.

The promoters of a tobacco actin gene, a tobacco pectate lyase, a tobacco and maize polygalacturonase and aBrassica S-locus related gene have been fused to theß-glucuronidase reporter gene and their activities determined by biolistic transient assay in tobacco pollen. In stably transformed tobacco all the transgenes with the exception of Cauliflower Mosaic Virus-35S-ß-glucuronidase appear to express efficiently in maturing pollen. Transient assay analysis showed that the tobacco pectate lyase and the polygalacturonase constructs were 8x more active than the tobacco actin construct, and that the tobacco polygalacturonase construct was some 33x more active than the maize polygalacturonase construct. Constructional manipulations that altered the lengths of the 5'-untranslated leaders including one which resulted in the removal of a 490 bp leader intron had little effect on the observed level of expression. However, the alteration of the context of the ATG from A/TnnATGG to CnnATGT resulting in a 70% reduction in the observed levels of activity, was obtained with the pectate lyase and polygalacturonase promoters. An identical reductional was also observed in transgenic plant populations transformed with the polygalacturonase transgenes.

Expression of the S-locus receptor kinase multigene family in Brassica oleracea.

The S-receptor kinase (SRK) gene which is implicated in the self-incompatibility system of Brassica oleracea is one member of a large and complex family of similar sequences. Genomic and cDNA clones were isolated for the authentic, S-linked SRK29 gene and its DNA sequence determined. Reverse transcriptase PCR (RT-PCR) was used to detect the expression of SRK29 and other members of the family in stigma, leaf and root tissues. The SRK was found to be stigma-specific whereas, for instance, K3 transcripts appeared in all three tissues. The RT-PCR analysis also demonstrated the existence of partially processed intermediates for several of the kinase transcripts and, in the case of SRK29, a product apparently resulting from the splicing of a cryptic intron. RFLP analysis of an F2 family segregating for the S29 allele was used to show S-linkage for the SRK and possibly for the K2 sequence. The K8 kinase probe also revealed a minor RFLP which segregated with the S-locus.

Sequence and expression of endogenous S-locus glycoprotein genes in self-compatible Brassica napus.

Brassica napus is an amphidiploid plant which is self-compatible even though it is derived from hybridisation of the self-incompatible species B. oleracea and B. campestris. Experiments were undertaken to establish if S-locus glycoprotein (SLG) genes exist in B. napus and whether these are expressed as in self-incompatible Brassica species. Two different stigma-specific cDNA sequences homologous to SLG genes were obtained from the B. napus cultivar Westar. One of these sequences, SLGWS1, displayed highest homology to class I SLG alleles, whereas the other, SLGWS2, showed greatest homology to class II SLG genes. Both were expressed at high levels in Westar stigmas following a developmental pattern typical of SLG genes in the self-incompatible diploids. We infer that they represent the endogenous SLG genes at the two homologous S-loci. The occurrence of normally expressed SLG genes and its relevance to the self-compatible phenotype of B. napus is discussed.

Sequence complexity of the S receptor kinase gene family in Brassica.

A genomic library from an S29/S29 self-incompatible genotype of Brassica oleracea was screened with a probe carrying part of the catalytic domain of a Brassica S-receptor kinase (SRK)-like gene. Six positive phage clones with varying hybridisation intensities (K1 to K6) were purified and characterised. A 650-700 bp region corresponding to the probe was excised from each clone and sequenced. DNA and predicted protein sequence comparisons based on a multiple alignment identified K5 as a pseudogene, whereas the others could encode functional proteins. K3 was found to have lost an intron from its genomic sequence. The six genes display different degrees of sequence similarity and form two distinct clusters in a dendrogram. The 98% similarity between K4 and K6, which extends across intron sequences, suggests that these might be very recently diverged alleles or daughters of a duplication. In addition, K2 showed a comparably high similarity to the probe. Clones K1, K3 and K5 cross-hybridised with an SLG29 cDNA probe, indicating the presence of upstream receptor domains homologous to the Brassica SLG gene. This suggests that the previously reported S sequence complexity may be ascribed to a large receptor kinase gene family.

PCR detection of transcripts homologous to the self-incompatibility gene in anthers ofBrassica.

The polymerase chain reaction (PCR) is particularly well suited for the detection of rare sequences. Taking advantage of the recent isolation of sequences associated with stigma self-incompatibility inBrassica oleracea, we used PCR amplifications with primers synthesized to the S6 cDNA sequence, to demonstrate the presence of mRNA homologous to stigmaS-locus gene (SLG) in anthers during early microsporogenesis. In addition, otherS-locus-related (SLR) sequences were shown to be transcribed in sexual as well as in vegetative tissues (roots, leaves), suggesting that the SLG family might be involved not only in pollen-stigma recognition, but more generally in various forms of plant cell signalling processes. This information corroborates the recent discovery of a cDNA-deduced protein kinase from maize roots, whose extracellular receptor displays high homology withBrassica S-locus-specific glycoproteins.

A homozygous S genotype of Brassica oleracea expresses two S-like genes.

The sporophytic self-incompatibility system of Brassica species is controlled by a single locus, S. Recognition of self between pollen and stigma is probably mediated by S locus-specific glycoproteins (SLSGs). We describe the isolation, from an S29 homozygote of Brassica oleracea, of two different cDNA clones for transcripts which are equally abundant in stigmas competent for self-incompatibility and each of which is homologous to previously reported SLSG sequences. Extensive DNA sequence divergence between the two clones precludes their cross-hybridisation and each acts as a gene-specific probe. All S genotypes appear to have a single copy of each gene but there are significantly different levels of polymorphism associated with each. The clear structural homology between the two indicates a gene duplication involving the S locus and, perhaps, related to the evolution of self-incompatibility.