Characterization of Brassica nigra chromosomes and of blackleg resistance in B. napus–B. nigra addition lines

Brassica napus-B. nigra addition lines were previously created using the variety ‘Darmor’ as the oilseed rape genetic background. Two isozyme loci and 46 RAPD markers were added on five different B. nigra chromosomes. The oilseed rape variety used was highly susceptible to blackleg at the cotyledon stage and only the addition of chromosome 4 gave the same level of blackleg resistance as B. nigra. This resistance was efficient whatever the isolates used. A significant effect on the development of stem canker under field conditions was observed only for the line carrying chromosome 4 which was more resistant than the susceptible control. The potential effects of two other chromosomes have to be confirmed. F1 hybrids obtained by crosses between two highly susceptible lines and the monosomic addition line carrying chromosome 4 were examined under field conditions. No effect of the oilseed rape genetic background on the expression of resistance was detected. The introduction of this resistance and mapping of the gene(s) into oilseed rape varieties are discussed.     

A study on disease variation in the populations of an interspecific cross of Brassica juncea L. x B. napus L.

Summary F₁ behaviour and F₂ variation in disease reaction were studied in the interspecific cross Brassica juncea x B. napus. Gene(s) for adult resistance to blackleg (Leptosphaeria maculans) were found to be present in the A genome of B. juncea and could be transferred to B. napus. Gene(s) for complete (seedling plus adult) resistance in B. juncea appeared to be located in the B genome. The chance of their transfer to the oilseed rapes (B. napus or B. campestris) would therefore seem to be remote.     

Transfer of disease resistance within the genus Brassica through asymmetric somatic hybridization

Summary Asymmetric somatic hybrid plants between Brassica napus L. (oilseed rape genome AACC) and a transgenic line of Brassica nigra L. Koch (black mustard genome BB) were tested for their resistance against rapeseed pathogens Phoma lingam (black leg disease) and Plasmodiophora brassicae (club root disease). The transgenic B. nigra line used (hygromycin-resistant, donor) is highly resistant to both fungi, whereas B. napus (recipient) is highly susceptible. The asymmetric somatic hybrids were produced using the donor-recipient fusion method (with X-irradiation of donor protoplasts) reported by Zelcer et al. (1978) for the production of cybrids. Using hygromycin-B for selection, a total of 332 hybrid calli were obtained. Regenerants, resistant or susceptible to both diseases, were selected. Many hybrids expressed resistance to only one pathogen. Dot blot experiments showed that the asymmetric hybrid plants contained varying amounts of the donor genomic DNA. Furthermore, a correlation was detected between the radiation dose and the degree of donor DNA elimination.     

Brassilexin accumulation and resistance to Leptosphaeria maculans in Brassica spp. and progeny of an interspecific cross B. juncea × B. napus

Summary Resistance to Leptosphaeria maculans was assessed in Brassica napus, B. juncea, B. carinata, B. nigra and progeny issuing from an interspecific cross B. napus × B. juncea, using a cotyledon-inoculation test. In these individual plants, brassilexin accumulation was determined following an abiotic, non-specific, elicitation. All the tested B. napus cultivars were highly susceptible to the parasite and weakly accumulated brassilexin. In contrast, B. juncea, B. carinata, and B. nigra usually displayed a hypersensitive response to the inoculation and accumulated more brassilexin than B. napus. The same correlation between resistance to L. maculans and phytoalexin accumulation was observed in the interspecific hybrid progeny. The cotyledon-inoculation test allowed the discrimination of plants displaying a hypersensitive response to the inoculation from those highly sensitive to the parasite, but intermediate disease severity classes were not usually representative of resistance or susceptibility. In this respect, brassilexin determination allowed differentiation, within a set of plants presenting an intermediate response to the pathogen, of plants with a high (B. juncea-like), and with a weak (B. napus-like) ability to accumulate brassilexin.Abbreviations IHP interspecific hybrid progeny - JR B. juncea-type complete resistance to blackleg (Roy, 1984) - W&D test cotyledon-inoculation test as described by Williams & Delwiche (1979)     

Association of RFLP markers with trait loci affecting clubroot resistance and morphological characters in Brassica oleracea L.

Summary Resistance to Plasmodiophora brassicae Wor. race 7, the causal agent of the disease clubroot, was examined in an F₂ population of a cross between a clubroot resistant broccoli (Brassica oleracea var. italica) and a susceptible cauliflower (B. oleracea var. botrytis). A genetic linkage map was constructed in the same population based on the segregation of 58 dispersed restriction fragment length polymorphism (RFLP) markers. Associations between the inheritance of RFLP marker genotypes and segregation for disease resistance, morphological and maturity characteristics were examined. For each triat examined, several chromosomal regions marked by RFLP probes appeared to contain trait loci, suggesting that each trait was under polygenic control. RFLP marker linkage to a major factor imparting dominance for clubroot resistance from the broccoli parent was observed in this population. Additionally, RFLP marker linkage to an independently segregating factor contributing clubroot resistance from the cauliflower parent was observed, indicating that it should be possible to use RFLP markers to facilitate selection of transgressive segregants having the combined resistance from both parental sources. In some instances, RFLP markers from the same or closely linked chromosomal regions were associated with both clubroot resistance and morphological traits. Analysis of RFLP marker genotypes at linked loci should facilitate the selection of desired disease resistant morphotypes.     

Development of molecular markers linked to the Leptosphaeria maculans resistance gene Rlm6 and inheritance of SCAR and CAPS markers in Brassica napus × Brassica juncea interspecific hybrids

Leptosphaeria maculans causes blackleg disease on Brassica napus, an economically important oilseed crop. Brassica juncea has high resistance to blackleg and is a source for the development of resistant B. napus varieties. To transfer the Rlm6 resistance gene from B. juncea into B. napus, an interspecific cross between B. napus “Topas DH16516” and B. juncea “Forge” was produced, followed by the development of F₂ and F₃ generations. Sequence characterized amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers linked to the L. maculans resistance gene Rlm6 were developed. Segregation of SCAR and CAPS markers linked to Rlm6 were confirmed by genotyping of F₂ and F₃ progeny. Segregation of CAPS markers and phenotypes for blackleg disease severity in F₂ plants had a Mendelian ratio of 3:1 in resistant vs. susceptible plants, respectively, supporting the assumption that genetic control of resistance was by a single dominant gene. The molecular markers developed in this study, which show linkage with the L. maculans resistance gene Rlm6, would facilitate marker‐assisted backcross breeding in a variety development programme.     

Studies on Resistance to Phoma lingam in Brassies napus-Brassica nigra Addition Lines

B. nigra exhibits high levels of resistance to Phoma lingam. The genetic behaviour of this resistance was investigated using B. napus-B. nigra addition lines. At least 3 different B. nigra chromosomes were found to contribute to the blackleg resistance. Thus, this resistance was suggested to be polygenic. In addition, high levels of P. lingam resistance in euploid offspring led to the assumption that translocations have taken place in early generations after hybridization between B. napus and B. nigra.     

Dissection of the Brassica nigra Genome by Monosomic Addition Lines

Hyperploid derivatives of Diplotaxis erucoides × Brassica nigra hybrids were used to extract seven out of the eight possible monosoinic addition lines for B. nigra (genome B). The fertility and transmission of the lines varied depending on the added chromosome. However, these parameters were high enough to assure the maintenance of the addition lines. Although no phenotypic changes were observed, the plants carrying extra chromosomes were slower in development than diploid D. emcoides. Each of the B. nigra chromosomes was recognizable cytologically by size and heterochromatin distribution. Seven of these were characterized by a series of isozymes and RFLP markers. Ribosotnal DNA sequences were detected in two independent B. nigra chromosomes. Two probes disclosed fragments dispersed in more than one chromosome.     

Production of yellow-seeded Brassica napus through interspecific crosses

Yellow‐seeded Brassica napus was developed from interspecific crosses between yellow‐seeded Brassica rapa var.‘yellow sarson’ (AA), black‐seeded Brassica alboglabra (CC), yellow‐seeded Brassica carinata (Bbcc) and black‐seeded B. napus (AACC). Three different interspecific crossing approaches were undertaken. Approaches 1 and 2 were designed directly to develop yellow‐seeded B. napus while approach 3 was designed to produce a yellow‐seeded CC genome species. Approaches 1 and 2 differed in the steps taken after trigenomic interspecific hybrids (ABC) were generated from B. carinata×B. rapa crosses. The aim of approach 1 was to transfer the yellow seed colour genes from the A to the C genome as an intermediate step in developing yellow‐seeded B. napus. For this purpose, the ABC hybrids were crossed with black‐seeded B. napus and the three‐way interspecific hybrids were self‐pollinated for a number of generations. The F₇ generation resulted in the yellowish‐brown‐seeded B. napus line, No. 06. Crossing this line with the B. napus line No. 01, resynthesized from a black‐seeded B. alboglabra x B. rapa var.‘yellow sarson’ cross (containing the yellow seed colour genes in its AA genome), yielded yellow‐seeded B. napus. This result indicated that the yellow seed colour genes were transferred from the A to the C genome in the yellowish‐brown seed colour line No. 06. In approach 2, trigenomic diploids (AABBCC) were generated from the above‐mentioned trigenomic haploids (ABC). The seed colour of the trigenomic diploid was brown, in contrast to the yellow seed colour of the parental species. Trigenomic diploids were crossed with the resynthesized B. napus line No. 01 to eliminate the B genome chromosomes, and to develop yellow‐seeded B. napus with the AA genome of ‘yellow sarson’ and the CC genome of B. carinata with yellow seed colour genes. This interspecific cross failed to generate any yellow‐seeded B. napus. Approach 3 was to develop yellow‐seeded CC genome species from B. alboglabra×B. carinata crosses. It was possible to obtain a yellowish‐brown seeded B. alboglabra, but crossing this B. alboglabra with B. rapa var.‘yellow sarson’ failed to produce yellow seed in the resynthesized B. napus. The results of approaches 2 and 3 demonstrated that yellow‐seeded B. napus cannot be developed by combining the yellow seed colour genes of the CC genome of yellow‐seeded B. carinata and the AA genome of ‘yellow sarson’.     

Detecting and estimating segregation distortion and linkage between glufosinate tolerance and blackleg resistance in Brassica napus L.

Summary The segregation and linkage between glufosinate (transgenes ‘Rf3’ and ‘T177’) and blackleg resistance genes in canola (Brassica napus L.) were assessed using F₁ microspore-derived doubled haploid (DH) populations from four crosses including reciprocals, two involving the transgene ‘Rf3’ and the other two involving the transgene ‘T177’. To relax the assumption of no segregation distortion required for the conventional analysis of segregation and linkage, we employed Bailey's analysis that allows detecting segregation distortion at linked loci. The significant departures from the 1:1 segregation were detected in the crosses involving the transgene ‘T177’ but not in the crosses involving the transgene ‘Rf3’. The apparent deficit of the herbicide tolerant DH lines in the crosses with the transgene ‘T177’ is likely due to differential selection against the gametes carrying ‘T177’ during microspore culture. The linkage was strong between blackleg resistance and the transgene ‘Rf3’ but weak or absent between blackleg resistance and the transgene ‘T177’, suggesting that the two transgenes are probably inserted into distant regions of the genome. The observed linkage offers an opportunity to develop new canola cultivars with both glufosinate tolerance conferred by transgene ‘Rf3’ and blackleg resistance.     

Interspecific transfer of Brassica juncea-type high blackleg resistance to Brassica napus

Summary Complete resistance to Leptosphaeria maculans, the cause of blackleg of oilseed rape (Brassica napus), was transferred from B. juncea to B. napus through an interspecific cross. B. juncea-type complete resistance (JR) was recognized first in one F₃ progeny (Onap^JR) by the absence of leaf-lesions on seedlings and canker-free adult plants. The commercially important characters of B. napus were retained in advanced lines of Onap^JR, which combined JR with low erucic acid levels (<0.5%), high seed yield and variable maturity dates.JR appeared to be inherited as a major gene or genes. Segregation for resistance and susceptibility contintied to occur during later generations of selection of Onap^JR. JR was readily transferred from Onap^JR to other suitable B. napus cultivars or lines with partial resistance to blackleg and resulted in highly vigorous carly generation selections adapted to cold, wet situations along with complete resistance to blackleg.     

Genetic dissection of intersubgenomic heterosis in Brassica napus carrying genomic components of B. rapa

Although strong intersubgenomic heterosis for seed production has been observed between “natural” domesticated Brassica napus (rapeseed, AACC) and a new type of rapeseed into which subgenomic components of Brassica rapa (AA) have been introgressed, the molecular genetic mechanism of this intersubgenomic heterosis is not understood. In this study, a recombinant inbred line population of new type rapeseed derived from a cross between B. napus and B. rapa, together with a population from a backcross with the parental line of B. napus, was used to identify single-locus quantitative trait locus (QTL) and interacting QTL pairs for yield and nine yield-related traits. More than half of single-locus QTLs and interacting QTL pairs detected were involved with the novel alleles induced by the introgression of B. rapa. The alleles directly from B. rapa A genome played a secondary role in contributing to intersubgenomic heterosis. Allelic and nonallelic interactions of both novel alleles generated by B. rapa introgression and the alleles directly from B. rapa A genome contributed to the intersubgenomic heterosis between “natural” domesticated rapeseed and new type rapeseed into which B. rapa had been introgressed. Six loci for fixed heterosis were identified and their possible applications are also discussed.     

Genetic effects on biomass yield in interspecific hybrids between Brassica napus and B. rapa

This study was conducted to estimate the genetic effects on biomass yield in the interspecific hybrids between Brassica napus and B. rapa, and to evaluate the relationship between parental genetic diversity and its effect on biomass yield of interspecific hybrids. Six cultivars and lines of oilseed B. napus and 20 cultivars of oilseed B. rapa from different regions of the world were chosen to produce interspecific hybrids using NC design II. Obvious genetic differences between B. rapa and B. napus were detected by RFLP. In addition, Chinese B. rapa and European B. rapa were shown genetically differences. Plant biomass yield from these interspecific hybrids were measured at the end of flowering period. Significant differences were detected among general combining ability (GCA) effects over two years and specific combining ability (SCA) effects differences were detected in 2000. The ratios of mean squares, (σ² _GCA(f) + σ² _GCA(m)) / (σ² _GCA(f) + σ² _GCA(m) + σ² _SCA), were 89% and 88% in 1999 and 2000, respectively. This indicates that both additive effects and non-additive effects contributed to the biomass yield of interspecific hybrids and the former played more important role. Some European B. rapa had significant negative GCA effects while many of Chinese B. rapa had significant positive GCA effects, indicating that Chinese B. rapa may be a valuable source for transferring favorable genes of biomass yield to B. napus. Significant positive correlation between parental genetic distance and biomass yield of interspecific hybrids implies that larger genetic distance results in higher biomass yield for the interspecific hybrids. A way to utilize interspecific heterosis for seed yield was discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of the cytoplasms of Brassica napus and B. juncea on some characteristics of B. carinata, including flower morphology

This study was conducted to assess the cytoplasm effects of Brassica napus and B. juncea on the some characteristics of B. carinata, as well as the phylogenetic distances separating the three species. Alloplasmic lines of B. carinata were developed from B. napus × B. carinata and B. juncea × B. carinata hybrids by recurrent backcrossing to the BC₇ generation. Sixteen populations from three generations were compared for a number of characteristics. Plants with the cytoplasm of B. napus flowered later, had shorter filaments and longer pistils, lower pollen amount, lower seed set, lower petal length and width and different petal color; plants with the cytoplasm of B. juncea had shorter pistils and filaments, and lower petal length and width than their corresponding euplasmic sibs, respectively. The results suggest that the cytoplasm is involved in the development of flower organs. The natural species, B. carinata showed a balance between the nucleus and cytoplasm. The cytoplasm from B. napus showed a stronger disturbing effect than that of B. juncea, suggesting that B. carinata might be genetically closer to B. juncea than to B. napus. The significant difference in the alloplasmic effect of the cytoplasms of B. napus and B. juncea also suggests that in B. carinata the B genome may play a greater role than the C genome. An erratum to this article can be found at     

Different Behavior of Brassica juncea and B. carinata as Sources of Phoma lingam Resistance in Experiments of Interspecific Transfer to B. napus

With the aim to transfer Phoma lingam resistance into rape, successful interspecific crosses were made between three oilseed rape varieties (Brassica napus) and the resistant species B. carinata and B. carinata. Although both hybrid types B. napus×B. juncea and B. napus×B. carinata showed the same high level of resistance as the respective resistant parent, the resistance could be only transferred from juncea crosses. After three backcross generations, lines morphologically undistinguishable from rape, fertile, and with a high degree of resistance were obtained. The resistance of B. carinata was practically lost in the first backcross. A possible explanation of this different behavior could be a higher recombination between the genomes B and C (juncea crosses) than between B and A (carinata crosses). The: applied embryo culture increased the yield of hybrids and first backcross plants and reduced considerably the generation time.     

Quantitative trait analysis of flowering time in spring rapeseed (B. napus L.)

The inheritance of flowering time trait in spring-type rapeseed (Brassica napus L.) is poorly understood, and the investigations on mapping of quantitative trait loci (QTL) for the trait are only few. We identified QTL underlying variation for flowering time in a doubled haploid (DH) mapping population of nonvernalization-responsive canola (B. napus L.) cultivar 465 and line 86 containing introgressions from Houyou11, a Chinese early-flowering cultivar in Brassica rapa L. Significant genetic variation in flowering time and response to photoperiod were observed among the DH lines from 465/86. A molecular linkage map was generated comprising three types of markers loci. QTL analysis indicated that flowering time is a complex trait and is controlled by at least 4 major loci, localized on four different linkage groups A6, A7, C8 and C9. These loci each accounted for between 9.2 and 12.56 % of the total genotypic variation for first flowering. The published high-density maps for flowering time mapping used different marker systems, and the parents of our crosses have different genetic origins, with either spring-type B. napus or B. rapa. So we cannot determine whether the QTL on the same linkage groups were in the same region or not. There was evidence of additive × additive epistatic effects for flowering time in the DH population. Epistasis existed not only between main-effect QTLs, but also between QTLs with minor effects. Four pair of epistasis effects between minor QTLs explained about 20 % of the genetic variance observed in the DH population. The results indicated that minor QTLs for flowering time should not be ignored. Significant genotypes × environment interactions were also found for the quantitative traits, and with significant change in the ranking of the DH lines in different environments. The results implied that FQ3 was a non-environment-specific QTL and may control flowering time by autonomous pathway. FQ4 were winter-environment-specific QTL and may control flowering time by photoperiod-pathway. Identification of the chromosomal location and effect of the genes influencing flowering time may hasten the development of canola varieties having an optimal time for flowering in target environments such as for high altitude areas, via marker-assisted selection.     

Phenotypic and seed structural comparison in hybrids of different Brassica rapa and B. oleracea

Brassica napus is an important oil species with short history and narrow genetic background. Interspecific hybrids from crosses between B. oleracea and different B. rapa were obtained. We found the hybrids with white petal resembling B. oleracea, the flavonoid and phenolic content decreased in hybrids, agreeing with the expressional changes of flavonoid biosynthesis genes. Seed coat of hybrids resembled diploid parents, or partly resembled to each parent with a clear outline. The palisade layer in hybrids was thicker than parents, with similar pigment accumulation as B. oleracea but more than B. rapa. Differentially sized protein bodies (PBs) were found in hybrids. The radical and inner cotyledon of all hybrids were identified with larger but less PBs than parents. The average size of PBs in outer cotyledon of resynthesized B. napus was also larger than parents, but the number of PBs was not significantly reduced. The phenotypic and seed structural variations after polyploidization of B. napus would be interesting for genetic broadening and breeding of rapeseed.     

Assessing and broadening genetic diversity of a rapeseed germplasm collection

Assessing the level of genetic diversity within a germplasm collection contributes to evaluating the potential for its utilization as a gene pool to improve the performance of cultivars. In this study, 45 high-quality simple sequence repeat (SSR) markers were screened and used to estimate the genetic base of a world-wide collection of 248 rapeseed (Brassica napus) inbred lines. For the whole collection, the genetic diversity of A genome was higher than that of C genome. The genetic diversity of C genome for the semi-winter type was the lowest among the different germplasm types. Because B. oleracea is usually used to broaden the genetic diversity of C genome in rapeseed, we evaluated the potential of 25 wild B. oleracea lines. More allelic variations and a higher genetic diversity were observed in B. oleracea than in rapeseed. One B. oleracea line and one oilseed B. rapa line were used to generate a resynthesized Brassica napus line, which was then crossed with six semi-winter rapeseed cultivars to produce 7 F₁ hybrids. Not only the allele introgression but also mutations were observed in the hybrids, resulting in significant improvement of the genetic base.     

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.     

Synthesis of somatic hybrids (RCBB) by fusing heat-tolerant Raphanus sativus (RR) and Brassica oleracea (CC) with Brassica nigra (BB)

Brassica carinata (BBCC), a potential oilseed crop for dry land agriculture, is sensitive to high temperatures during germination and early stages of growth, which thereby restricts the possibility of using the residual soil moisture available after the rainy season for its cultivation. To overcome this problem, a three‐genome hybrid, RCBB, was synthesized using Raphanus sativus (RR) and Brassica oleracea (CC) as donor sources for the desired heat tolerance. Protoplasts of RC hybrids obtained through sexual crosses between R. sativus (female) and B. oleracea (male) were fused with protoplasts of Brassica nigra (BB) to produce RCBB somatic hybrids. The hybrid colonies regenerated with an average frequency of 7.6%. Twelve out of 36 hybrids grown to maturity were characterized for their nuclear and organelle genomes. While all the hybrids showed the presence of B. nigra chloroplasts, 10 of the hybrids showed B. nigra‐specific mitochondria and two had Raphanus‐spedfic mitochondria. The somatic hybrids could be backcrossed to B. carinata.