Cytoplasmic male sterility in rapeseed (Brassica napus L.)

Summary Restriction patterns of chloroplast (cp) and mitochondrial (mt) DNA in Brassica napus rapeseed reveal the alloplasmic nature of cytoplasmic male sterility in this crop. Both the Shiga and Bronowski systems probably exploit cytoplasmic diversity in B. napus cultivars arising from introgression of cytoplasm from the other rapeseed species, B. campestris. Nuclear genes specific to these systems do not cause sterility in maintainers (Bronowski and Isuzu-natane) because they have a campestris cytoplasm, but give rise to sterility in napus cytoplasms. In the course of hybridization to napus cultivars a line with the triazine resistant cytoplasm (a campestris cytoplasm) has undergone an alteration in the mt genome rendering its restriction pattern more similar than previously to that of napus. The alteration may be an inversion between 7.2 and 3.4 kb in length.     

The identification of quantitative trait loci that control the paternal inheritance of a mitochondrial plasmid in rapeseed (Brassica napus L.)

Some varieties of Brassica napus (rapeseed) and B. rapa contain a liner mitochondrial plasmid that is unique in that it can be inherited from the male parent through the pollen. We found that two rapeseed cultivars, Norin 16 and Westar, showed different rates of plasmid inheritance from the paternal parent (78.8% and 27.5%, respectively). To identify nuclear genes controlling the inheritance of the plasmid, we carried out quantitative trait locus (QTL) analyses using F(2) populations derived from a cross between these two cultivars. The F1 plants transmitted the plasmid from the paternal plant at a frequency of approximately 60%; the transmission rates of the F2 lines varied greatly, from 0 to 100%, with an average of 68.2%. A genetic map was constructed based on the segregation of 175 loci in the 102 F2 plants. A total of 22 linkage groups were obtained, all of which could be assigned to the 19 rapeseed chromosomes. The total map length was 1374.7 cM, with an average distance of 7.9 cM between the markers. We found that three quantitative trait loci for plasmid paternal transfer, qPpt1, qPpt2 and qPpt3, located on chromosomes A5, C2 and C9, respectively, were significantly linked to the transmission frequency, whose the logarithm of odds (LOD) score were 4.97, 3.49 and 3.57, respectively. Their explained phenotypic variances were 25.0%, 22.2% and 37.1%, respectively. These results suggest that the paternal inheritance of the mitochondrial plasmid is controlled by a relatively small number of nuclear genes.     

Molecular analysis and expression of a floral organ-specific polygalacturonase gene isolated from rapeseed (Brassica napus L.)

High throughput screening of stage-specific differentially expressed genes in a Brassica napus two-line Rs1046A/B subtractive library was used to identify the BnQRT3 gene associated with cell wall metabolism. Phylogenetic analysis indicates the protein product of BnQRT3 is polygalacturonase. According to cytological comparisons of Rs1046 sterile and fertile anthers, RT–PCR studies and in situ hybridizations, BnQRT3 is expressed most strongly in floral organs and may play an essential role in pollen maturation. Analysis of the histological staining pattern of BnQRT3 promoter-GUS constructs in transgenic Arabidopsis and Brassica napus revealed that proximal part of 5′-flanking region directed expression in the vascular tissue of filaments, veins in sepal and petals, stigma, branch connective and the floral organ abscission zone during the open flower stage. In the meanwhile, Activity of BnQRT3 was detected in the anthers, which commences at the microsporocyte stage and persists as anther approaches dehiscence. Strong GUS expression also can be observed in the vascular tissue of leaves and stem by compression with forceps or excision, suggesting that the BnQRT3 promoter is responsive to wounding.     

Large scale purification of rapeseed proteins (Brassica napus L.)

Rapeseed (Brassica napus L.) cruciferin (12S globulin), napin (2S albumin) and lipid transfer proteins (LTP) were purified at a multi-g scale. The procedure developed was simple, rather fast and resolutive; it permitted the recovery of these proteins with a good yield, such as 40% for cruciferin and 18% for napin. Nanofiltration eliminated the major phenolic compounds. The remaining protein fraction was fractionated by cation exchange chromatography (CEC) on a streamline SP-XL column in alkaline conditions. The unbound neutral cruciferin was polished by size exclusion chromatography. The alkaline napin isoforms and LTP, adsorbed on the beads, were eluted as a whole fraction and further separated by an other CEC step at acidic pH. Napins were polished by hydrophobic interaction chromatography (HIC). The fractions were characterized by reverse phase HPLC, electrophoresis, N-terminal sequencing and mass spectrometry. All the fractions contained less than 5% of impurities.     

Embryogenesis following cryopreservation in isolated microspores of rapeseed (Brassica napus L.)

A simple procedure is described for cryopreservation of isolated microspores of rapeseed in liquid nitrogen without loss of embryogenic capacity (i.e. embryogenes is can still be induced following freezing). Microspores frozen in Lichter's (1982) medium with 13% sucrose produced ca. 10% of the embryos yielded by an unfrozen control. Microspores frozen in Lichter's medium with 13% sucrose, and supplemented with 0.5 M glycerol and 0.5 M DMSO produced no embryos. Regeneration of embryos obtained from frozen microspores yielded 88% diploid and 12% haploid plants, while embryos from unfrozen controls produced 7% diploids and 93% haploids. The potential to increase the efficiency of the rapeseed haploidy system using cryopreservation is discussed in light of these results.     

Properties of a membrane-bound triglyceride lipase of rapeseed (Brassica napus L.) cotyledons

The properties of the alkaline lipase activity (EC 3.1.1.3) that was recovered almost completely from a microsomal membrane fraction of 4-d-old rapeseed (Brassica napus L.) cotyledons were studied employing a titrimetric test procedure. The apparent K_M was 6.5 mmol l^-1, with emulgated sunflower oil as the substrate. The products of triglyceride hydrolysis in vitro were glycerol, free fatty acids, and minor amounts of mono- and diglycerides. Maximum lipase activity depended on the preincubation of the lipolytic membrane fraction in 0.15 mol l^-1 NaCl and on the presence of at least 0.1 mol l^-1 NaCl in the test mixture. Desoxycholate and up to 0.1 mol l^-1 CaCl₂ also activated the enzyme while EDTA and detergents such as trito x-100, digitonin, tween 85, and sodium dodecylsulfate were inhibitory. The rapeseed lipase displayed a conspicuous substrate selectivity among different plant triglycerides; the activity was inversely correlated with the oleic acid content of the oils. Water-soluble triacetin and the phospholipid lecithin were not hydrolyzed. Increasing amounts of free fatty acids reduced lipase activity; erucic acid, a major component of rapeseed oil, exhibited the strongest effect, suggesting a possible role in the regulation of lipase activity in vivo. The data demonstrate that the lipolytic membrane fraction houses a triglyceride lipase with properties similar to other plant and animal lipases. It can both qualitatively and quantitatively account for the fat degradation in rapeseed cotyledons. The evidence that provides further reason to acknowledge the membranous appendices of the spherosomes as the intracellular site of lipolysis is discussed.     

Association mapping of six yield-related traits in rapeseed (Brassica napus L.)

Yield is one of the most important traits for rapeseed (Brassica napus L.) breeding, but its genetic basis remains largely ambiguous. Association mapping has provided a robust approach to understand the genetic basis of complex agronomic traits in crops. In this study, a panel of 192 inbred lines of B. napus from all over the world was genotyped using 451 single-locus microsatellite markers and 740 amplified fragment length polymorphism markers. Six yield-related traits of these inbred lines were investigated in three consecutive years with three replications, and genome-wide association studies were conducted for these six traits. Using the model controlling both population structure and relative kinship (Q + K), a total of 43 associations (P < 0.001) were detected using the means of the six yield-related traits across 3 years, with two to fourteen markers associated with individual traits. Among these, 18 markers were repeatedly detected in at least 2 years, and 12 markers were located within or close to QTLs identified in previous studies. Six markers commonly associated with correlated traits. Conditional association analysis indicated that five of the associations between markers and correlated traits are caused by one QTL with pleiotropic effects, and the remaining association is caused by linked but independent QTLs. The combination of favorable alleles of multiple associated markers significantly enhances trait performance, illustrating a great potential of utilization of the associations in rapeseed breeding programs.     

Thermal reactivity of rapeseed (Brassica napus L.) under different gas atmospheres

Non-isothermal thermogravimetric method was applied to rapeseed to investigate its thermal reactivity under both individual dynamic atmospheres of nitrogen, steam, carbon dioxide and dry air, and under several mixtures of these gases (nitrogen+steam), (steam+dry air), and (nitrogen+carbon dioxide+steam). In this method, the sample was heated from ambient to 1273 K with a constant heating rate of 20 K/min. The trend of the TGA curves and the derived DTG profiles were interpreted regarding the conversion yields and the reactivity of the samples. Conversion yields of biomass to gaseous products changed in the range of 75-94% on original basis, with respect to the atmosphere under which the experiment was carried out. The maximum rates of the mass losses from the sample were found between 2.6 and 4.3 mg/min. The activation energies were calculated using the method of Coats-Redfern, and varied between 21.3 and 96.0 kJ/mol.     

Subunit composition of the globulin fraction of rapeseed (Brassica napus L.)

Multidimensional gel electrophoretic procedures have been employed to studythe polypeptide composition of the 12 S globulin (cruciferin) from rapeseed (Brassica napus L. cv. Tandem). Cruciferin was shown to consist of three main groups of proteins with M_r in the range of 60-52 (A group), 37-35 and 31-29 (respectively B₁ and B₂ groups) and 24-22 (C group). When reduced the A protein group gave rise to two classes of polypeptide constituents. The larger had M_r and isoelectric points (6.4–7.1) corresponding to those of B protein groups whereas the smaller were essentially composed of M_r 22 and a specific 25 kilodaltons (kD) polypeptide with basic isoelectric points. The initial B and C protein groups were unaltered by reducing conditions. These results support the notion that native cruciferin is composed of subunits with large and small polypeptides linked by disulphide bonds and of similar or closely-related polypeptides which are not covalently bonded.     

Evidence of cross-reactivity between porcine pancreatic and rapeseed (Brassica napus L.) lipases

A cross-reactivity between animal and plant lipases was determined, using immunological techniques. It was shown by ELISA and dot-blotting that these antibodies react with lipases in the rapeseed crude extract and in the different cellular fractions obtained by differential centrifugation. Pre-incubation of the antiserum with the rapeseed crude extract affects the amount of antibodies binding to the porcine pancreatic lipase. Antibodies were able to precipitate lipase activity from 3-day-old rapeseed crude extract. These epitopes seem to be located in the catalytic site, suggesting that a consensus sequence exists in oleaginous lipases and that it will be universal.     

The gene for tRNA(Lys) is encoded in the rapeseed (Brassica napus L.) mitochondrial DNA.

The nucleotide sequence of the gene coding for tRNA(Lys) and its flanking regions from the rapeseed mitochondrial genome are presented and compared with other known tRNA(Lys) genes from plant mitochondria. This tRNA sequence can be folded into the standard cloverleaf structure model. Also, this tRNA sequence shows less similarity with its chloroplast counterparts and therefore appears to be 'native' mitochondrial tRNA.     

Fatty acid inheritance in microspore-derived Populations of spring rapeseed (Brassica napus L.)

Summary Hard seededness in soybean [Glycine max (L.) Merr.] is a quantitative trait that affects the germination rate, viability, and quality of stored seeds. We have used 72 restriction fragment length polymorphisms (RFLPs) to identify genomic regions containing quantitative trait loci (QTL) affecting hard seededness in a segregating population from a G. max by a Glycine soja (Sieb. & Zucc.) cross. Five independent RFLP markers were found to be associated with variation in the hard-seeded trait. These markers and the epistatic interactions between them explain 71% of the variation for hard seededness. A genomic region associated with the i locus accounted for 32% of the variation in this segregating population. This study illustrates one approach to physiological genetic studies in plants.Journal Paper No. J-13557 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 2763     

The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana

The entire mitochondrial genome of rapeseed (Brassica napus L.) was sequenced and compared with that of Arabidopsis thaliana. The 221 853 bp genome contains 34 protein-coding genes, three rRNA genes and 17 tRNA genes. This gene content is almost identical to that of Arabidopsis. However the rps14 gene, which is a pseudo-gene in Arabidopsis, is intact in rapeseed. On the other hand, five tRNA genes are missing in rapeseed compared to Arabidopsis, although the set of mitochondrially encoded tRNA species is identical in the two Cruciferae. RNA editing events were systematically investigated on the basis of the sequence of the rapeseed mitochondrial genome. A total of 427 C to U conversions were identified in ORFs, which is nearly identical to the number in Arabidopsis (441 sites). The gene sequences and intron structures are mostly conserved (more than 99% similarity for protein-coding regions); however, only 358 editing sites (83% of total editings) are shared by rapeseed and Arabidopsis. Non-coding regions are mostly divergent between the two plants. One-third (about 78.7 kb) and two-thirds (about 223.8 kb) of the rapeseed and Arabidopsis mitochondrial genomes, respectively, cannot be aligned with each other and most of these regions do not show any homology to sequences registered in the DNA databases. The results of the comparative analysis between the rapeseed and Arabidopsis mitochondrial genomes suggest that higher plant mitochondria are extremely conservative with respect to coding sequences and somewhat conservative with respect to RNA editing, but that non-coding parts of plant mitochondrial DNA are extraordinarily dynamic with respect to structural changes, sequence acquisition and/or sequence loss.     

Identification of cytoplasm types in rapeseed (Brassica napus L.) accessions by a multiplex PCR assay

Cytoplasmic male sterility (CMS) has widely been used as an efficient pollination control system in rapeseed hybrid production. Identification of cytoplasm type of rapeseed accessions is becoming the most important basic work for hybrid-rapeseed breeding. In this study, we report a simple multiplex PCR method to distinguish the existing common cytoplasm resources, Pol, Nap, Cam, Ogu and Ogu-NWSUAF cytoplasm, in rapeseed. Cytoplasm type of 35 F₁ hybrids and 140 rapeseed open pollinated varieties or breeding lines in our rapeseed breeding programme were tested by this method. The results indicated that 10 of 35 F₁ hybrids are the Nap, and 25 the Pol cytoplasm type, which is consistent with the information provided by the breeders. Out of 140 accessions tested, 100 (71.4%), 21 (15%) and 19 (13.6%) accessions possess Nap, Cam and Pol cytoplasm, respectively. All 19 accessions with Pol cytoplasm are from China. Pedigree analysis indicated that these accessions with Pol cytoplasm were either restorers for Pol CMS, including Shaan 2C, Huiyehui, 220, etc. or derived from hybrids with Pol CMS as female parent. Our molecular results are consistent with those of the classical testcross, suggesting the reliability of this method. The multiplex PCR assay method can be applied to CMS “three-line” breeding, selection and validation of hybrid rapeseed.     

The genetic basis of flowering time and photoperiod sensitivity in rapeseed (Brassica napus L.)

The objective of this study was to dissect the genetic control of days to flowering (DTF) and photoperiod sensitivity (PS) into the various components including the main-effect quantitative trait loci (QTLs), epistatic QTLs and QTL-by-environment interactions (QEs). Doubled haploid (DH) fines were produced from an F1 between two spring Brassica napus cultivars Hyola 401 and Q2. DTF of the DH lines and parents were investigated in two locations, one location with a short and the other with a long photoperiod regime over two years. PS was calculated by the delay in DTF under long day as compared to that under short day. A genetic linkage map was constructed that comprised 248 marker loci including SSR, SRAP and AFLP markers. Further QTL analysis resolved the genetic components of flowering time and PS into the main-effect QTLs, epistatic QTLs and QEs. A total of 7 main-effect QTLs and 11 digenic interactions involving 21 loci located on 13 out of the 19 linkage groups were detected for the two traits. 3 main-effect QTLs and 4 pairs of epistatic QTLs were involved in QEs conferring DTF. One QTL on linkage group (LG) 18 was revealed to simultaneously affect DTF and PS and explain for the highest percentage of the phenotypic variation. The implications of the results for B. napus breeding have been discussed.     

Extent and structure of linkage disequilibrium in canola quality winter rapeseed (Brassica napus L.)

Linkage disequilibrium was investigated in canola quality winter rapeseed to analyze (1) the prospects for whole-genome association analyses and (2) the impact of the recent breeding history of rapeseed on linkage disequilibrium. A total of 845 mapped AFLP markers with allele frequencies ≥0.1 were used for the analysis of linkage disequilibrium in a population of 85 canola quality winter rapeseed genotypes. A low overall level of linkage disequilibrium was found with a mean r ² of only 0.027 over all 356,590 possible marker pairs. At a significance threshold of P = 2.8 × 10⁻⁷, which was derived by a Bonferroni correction from a global α-level of 0.1, only 0.78% of the marker pairs were in significant linkage disequilibrium. Among physically linked marker pairs, the level of linkage disequilibrium was about five times higher with more than 10% of marker pairs in significant linkage disequilibrium. Linkage disequilibrium decayed rapidly with distance between linked markers with high levels of linkage disequilibrium extending only for about 2 cM. Owing to the rapid decay of linkage disequilibrium with distance association analyses in canola quality rapeseed will have a significantly higher resolution than QTL analyses in segregating populations by interval mapping, but much larger number of markers will be necessary to cover the whole genome. A major impact of the recent breeding history of rapeseed on linkage disequilibrium could not be observed.     

Agravitropic behaviour of roots of rapeseed (Brassica napus L.) transformed by Agrobacterium rhizogenes

Transgenic hairy roots of Brassica napus (cv. Omega) have been developed, using Agrobacterium rhizogenes strain AR 25, for use as a model system in the investigation of physiological and morphological differences between transgenic and normal roots. The basic parameters of growth and normal or altered gravitropical behaviour of hairy roots are for the first time presented in this paper together with an ultrastructural and morphological analysis of the root statocytes. The results obtained also represented the basis for the TRANSF0RM-experiment on the IML-2 mission performed onboard the Space Shuttle Columbia. Typical hairy root traits such as hormone-autonomous growth high growth rate, lateral branching, and changed/absence of gravitropism were detected. The transformed nature of the roots was confirmed by Southern blot analyses. The gravitropical behaviour of apices from hairy root cultures of this clone has been compared with root tips from normal seedlings. While the wild type roots curved progressively with increasing stimulation angles, the transformed roots showed no curvature when stimulated at 45 degrees, 90 degrees or 135 degrees on the ground. The morphology and ultrastructure of the root tip regions were examined by light microscopy and transmission electron microscopy. At the ultrastructural level no major differences could be detected between the roots studied. There was, however, a slight reduction in the starch content of most of the amyloplasts of the transgenic root tips, and the root cap was more V-shaped in the transgenic roots than in the wild type. Preliminary results from the Shuttle experiment TRANSFORM show a random distribution of amyloplasts in the root cells of both transformed and wild type root caps after 14 h on a 1xg centrifuge followed by 37 h in microgravity.