Stability of the cleistogamous trait during the flowering period of oilseed rape

At the field scale, the co-existence of different farming production systems requires strategies to prevent gene flow between adjacent crops. Oilseed rape produces pollen dispersed by wind and insects and the risks of pollen mediated gene flow are significant for this crop. Cleistogamy, the trait of non-opening flowers, could be used to reduce pollen flow. Cleistogamous oilseed rape genotypes were obtained by INRA in France and were bred in order to improve the stability of this trait. In this paper, we examine the reliability of the cleistogamous trait for two inbred lines. The flower opening level was measured at different dates during the flowering period in six field experiments (three sites × 2 years). The results showed that some flowers were partially opened with rates varying from 0.5% to 33% principally depending on genotypes, trials (site and year) and recording dates. Given that other studies have shown that cleistogamy could reduce pollen dispersal, we consider that, even when partially unstable, cleistogamy could be beneficially used in combination with other means in a containment strategy.     

Nectar secretion in winter oilseed rape, Brassica napus— quantitative and qualitative variability among 71 genotypes

The genotypic variability in nectar secretion among 71 genotypes of winter oilseed rapes was tested for floral nectar volume, sugar composition and concentration. Differences were found between seed quality genotypes: double low [99] oilseed rape varieties tended to secrete much more nectar than single low [0 +] and double high [++] varieties. Male-sterile lines (Ogura CMS) secreted one-half to one-third the amount of their isogenic male-fertile counterparts. Restored lines behaved as conventional rape varieties. On average, amounts of nectar were 2 μl/flower and ranged from 0.7 to 6 μl/flower. Among the genotypes under test, the male-sterile lines were not the least productive. The analysis of (Samourai) winter oilseed rape showed that total sugar concentrations in nectar were highest at the beginning of the flowering period (30g/100 ml) and lowest towards the end (10g/100 ml). At a given date, total sugar contents in nectar of [0 +] varieties were significantly higher than those of [++] varieties and were due to higher fructose contents. Differences between genotypes within the same type also occurred, depending on the day of sampling. Oilseed rape nectar was confirmed to be predominantly composed of hexose (glucose and fructose). From the results of these investigations, it is concluded that the opportunity exists to select oilseed rape genotypes, including male-sterile lines, that secrete sufficient amounts of nectar to attract honey-bees to ensure pollination of male-sterile lines for hybrid seed production.     

Evaluating the effectiveness of isolation distances for field plots of oilseed rape (Brassica napus) using a herbicide-resistance transgene as a selectable marker

The effectiveness of 200- and 400-m isolation distances were evaluated for small-scale trials of oilseed rape (Brassica napus L.). These distances have been used for previous transgenic release experiments and are commonly adopted for production of basic and certified breeders seed. A 400-m² donor plot contained plants with a dominant transgene conferring resistance to the herbicide glufosinate-ammonium. This character was used as a selectable marker to measure the frequency of hybrid formation in 400-m² target plots of non-transgenic rape. Seeds produced by the non-transgenic plants were permitted to fall onto the plots and allowed to germinate. At the first true-leaf stage, the plants were sprayed with glufosinate-ammonium. Surviving plants were resprayed, and a subsample assayed for the presence of the transgene using a colorimetric assay and Southern-blot analysis. The average frequency of hybridization over two replicates was 0.0156% at 200 m and 0.0038% at 400 m. These estimates are within the limits established for the production of basic seed (0.1%). Results indicated that bees were the most likely agent for long-distance pollen dispersal.     

Identification of a Brassica juncea-derived recessive gene conferring resistance to Leptosphaeria maculans in oilseed rape

Screening of 212 spring type Brassica napus lines carrying B genome chromosome additions and introgressions from B. nigra, B. juncea and B. carinata resulted in the identification of one line segregating for resistance to Leptosphaeria maculans (anamorph Phoma lingam) at the seedling (cotyledon) stage. This line was derived from an interspecific hybrid containing the B genome of B. juncea. Trypan blue staining of cotyledons from resistant individuals demonstrated a hypersensitive response which is delayed in plants with intermediate lesion size. Genetic analysis supported the hypothesis of a monogenic recessive inheritance of resistance. The resistance gene, termed r_jlm2, is effective in spring and winter type oilseed rape backgrounds against all tested virulent pathotypes, including two isolates which have been shown to overcome two dominant (race‐specific) B genome‐derived resistance genes in B. napus.     

Genetic variation for saturated fatty acid content in a collection of European winter oilseed rape material (Brassica napus)

Oil quality is an important issue in oilseed rape breeding programmes. Recently, the seed oil content of saturated fatty acids has received substantial interest, because in a number of industrialized countries dietary recommendations not only limit total fat intake but also intake of saturated fatty acids. The aim of the present study was to analyse the genetic variation for saturated fatty acid content in two sets of modern winter oilseed rape cultivars ( Brassica napus L.) tested in field experiments under typical German growing conditions. The results showed highly significant genetic differences among the cultivars for total saturated fatty acid content, which ranged from 6.8% to 8.1%, with a mean of 7.4%. For both sets medium to high genotypic heritabilities were found for most fatty acids and for oil and protein content. Close negative correlations between palmitic acid, the predominant saturated fatty acid, and oleic acid as well as oil content were found.     

Combination of resistance to Verticillium longisporum from zero erucic acid Brassica oleracea and oilseed Brassica rapa genotypes in resynthesized rapeseed (Brassica napus) lines

Resynthesized (RS) forms of rapeseed (Brassica napus L.; genome AACC, 2n = 38) generated from interspecific hybridization between suitable genotypes of its diploid progenitors Brassica rapa L. (syn. campestris; genome AA, 2n = 20) and Brassica oleracea L. (CC, 2n = 18) represent a potentially useful resource to introduce resistance against the fungal pathogen Verticillium longisporum into the gene pool of oilseed rape. Numerous cabbage (B. oleracea) accessions are known with resistance to V. longisporum; however, B. oleracea generally has high levels of erucic acid and glucosinolates in the seed, which reduces the suitability of resulting RS rapeseed lines for oilseed rape breeding. In this study resistance against V. longisporum was identified in the cabbage accession Kashirka 202 (B. oleracea convar. capitata), a zero erucic acid mutant, and RS rapeseed lines were generated by crossing the resistant genotype with two spring turnip rape accessions (B. rapa ssp. olerifera) with zero erucic acid. One of the resulting zero erucic acid RS rapeseed lines was found to have a high level of resistance to V. longisporum compared with both parental accessions and with B. napus controls. A number of other zero erucic acid RS lines showed resistance levels comparable to the parental accessions. In the most resistant RS lines the resistance and zero erucic acid traits were combined with variable seed glucosinolate contents. Erucic acid‐free RS rapeseed with moderate seed glucosinolate content represents an ideal basic material for introgression of quantitative V. longisporum resistance derived from B. oleracea and B. rapa into elite oilseed rape breeding lines.     

Identification of AFLP markers linked to one recessive genic male sterility gene in oilseed rape, Brassica napus

The commercial utilization of heterosis in seed yield by means of hybrid varieties is of great importance for increasing oilseed rape production in China. This requires a functional system for the production of hybrid seed. The Brassica napus oilseed rape line 9012AB is a recessive epistatic genic male sterility (GMS) two‐type line, in which the sterility is controlled by two pairs of recessive duplicate sterile genes (ms1 and ms2) interacting with one pair of a recessive epistatic inhibitor gene (rf). Homozygosity at the rf locus (rfrf) inhibits the expression of the recessive male sterility trait in homozygous ms1ms1ms2ms2 plants. This study was conducted to identify molecular markers for one of the male fertility/sterility loci in the B. napus male sterility line 9012AB. Sterile bulk (BS) and fertile bulk (BF) DNA samples prepared from male sterile and male fertile plants of the homozygous two‐type line 9012AB were subjected to amplified fragment length polymorphic (AFLP) analysis. A total of 256 primer combinations were used and seven markers tightly linked to one recessive genic male sterile gene (ms) were identified. Among them, six fragments co‐segregated with the target gene in the tested population, and the other one had a genetic distance of 4.3 cM. The markers identified in this study will greatly enhance the utilization of recessive GMS for the production of hybrid seed in B. napus oilseed rape in China.     

Genetic diversity,population structure,pollen morphology and cross‐compatibility among Chinese Cymbidiums

A total of 85 Chinese Cymbidiums and one Zygopetalum were collected and analysed using 29 simple sequence repeats (SSRs) and 12 intersimple sequence repeats (ISSRs). These Chinese Cymbidiums are some of the most popular cultivars in market. The pairwise genetic distance between these accessions averaged 0.897, ranging from 0.259 to 1.000. A model‐based clustering analysis revealed five genetic groups with serious admixture ancestry, as indicated by a fixation index of 0.156 and only 10.67% genetic variation among groups. By analysing the cross‐compatibility and pollen morphology, Chinese Cymbidiums are revealed to have high compatibility for intra‐ and interspecific crosses. Notably, Zygopetalum mackayi had high compatibility with Chinese Cymbidiums and shared similar morphologies in terms of pollinia and pollen grain. This study is the first to report a successful cross between Zygopetalum and Chinese Cymbidiums. The high compatibility offers the potential for introducing Zygopetalum mackayi's superior traits into Chinese Cymbidiums. The similar pollen morphology as well as the low genetic differentiation might be related to the high cross‐compatibility among these accessions.     

Pollen storage at low temperature as a procedure for the improvement of cold tolerance in spring rape, Brassica napus L.

The possibility of selecting spring rape for cold tolerance at the mature pollen grain stage was studied by investigating the effects of pollen storage at low temperatures on the quality of pollen grains and on the cold tolerance of the plants generated from them. Pollen treatments of F¹ hybrids affected fertilization ability much more than viability and even after 10 days storage at 3 or 10°C the pollen germination percentage was reasonably high. Pollen storage for 7 or 10 days at 3 or 10°C significantly increased the cold tolerance of F² seed germination, with 3°C being more effective. Pollen storage for a shorter time had no effect upon the number of resulting genotypes tolerant to low temperature. This approach may be successfully applied in plant breeding to enrich segregating plant populations with cold-tolerant genotypes.     

Genetic variation for secondary seed dormancy and seed longevity in a set of black‐seeded European winter oilseed rape cultivars

Secondary seed dormancy in oilseed rape is a phenomenon that allows seeds to survive in the soil for many years without germination. Following soil cultivation, dormant seeds may germinate in subsequent years, and they are the main reason for the occurrence of volunteer oilseed rape plants in successive crops. Inheritance of secondary dormancy may be related to seed longevity (SL) in the soil. Genetic reduction in secondary dormancy and SL could provide a mean to reduce the frequency of volunteer plants and especially the dispersal of transgenic oilseed rape. The aim of the present study was to analyse secondary dormancy, germination rate and SL of 28 black‐seeded winter oilseed rape cultivars using in vitro laboratory tests. The material was tested in field experiments at six different locations in Germany in 2008/2009. Significant effects of the genotype and the location on all traits were found. Heritability was high for secondary dormancy (0.97) and moderate for germination rate (0.70) and SL (0.71). Results indicate that a selection for low secondary dormancy would be effective.     

Proteomic alteration of a thermo‐sensitive male sterility SP2S in rapeseed (Brassica napus) in response to mild temperature stress

We conducted a proteomic comparison between thermo‐sensitive genic male sterility (TGMS) SP2S and its near‐isogenic line SP2F grown at 22°C. The proteomes at microsporocyte meiosis and uninucleate microspore stages were profiled using 2‐dimensional gel electrophoresis. Twenty‐five well‐reproducible spots (10 spots at microsporocyte meiosis stage and 15 spots at uninucleate microspore stage) containing 28 proteins were successfully identified by MALDI‐TOF/TOF mass spectrometry. An elongation factor EF‐2 at microsporocyte stage and 4 proteins (aconitate hydratase, triosephosphate isomerase, serine/arginine‐rich mRNA splicing factor and glutathione S‐transferase) at uninucleate microspore stage accumulated in SP2S, but more proteins were lost or reduced, including those associated with amino acid metabolism, photosynthesis, synthesis and degradation of protein, lipid metabolism, cytoskeleton, RNA modification, oxidoreductase and defence response. The dramatic decrease of tubulin, actin and Translationally controlled tumour protein (TCTP) crucial for microtubule and cell division and three enzymes for amino acid metabolite at early stage indicated a serious defect on the cytokinesis. They were important clues for us to search the TGMS genes and its interacting genes.